this PDF file - Microbial Ecology in Health and Disease

MICROBIAL ECOLOGY IN HEALTH AND DISEASE
VOL.
8: i-xxxiv (1995)
Abstracts of the IX International Congress on Microbial
Ecology and Disease, Rome (Italy), 1g-21 September,
1994
The Society for Microbial Ecology and Disease
(SOMED) organises an International Congress
every year, held alternately in the US and in a
European country. The 1994 Congress in Rome
was organised jointly by the Italian National
Institute of Health and SOMED. The scientific
programme includes invited lectures, oral communications and poster discussions covering the
following topics:
Microflora in the immunocomprised host
Chairmen: A Cassone and G Panichi . .
......
....
.
Oral and upper respiratory tract microflora
Chairmen: C. Grassi and M. Kilian . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
V
Biomaterials related infections
Chairmen: P. Speziale and T. Wadstrom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vi
Gastrointestinal microflora
Chairmen: L. C. Hoskins and M. G. Menozzi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
viii
Milk and intestinal flora
Chairmen: S. Gorbach and S. Guandalini. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
x
Bacterial toxins and receptors
Chairmen: G. Donelli and T. D. Wilkins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xii
Strategies in prophylaxis of bacterial infections
Chairmen: P. Mastrantonio and S. Gorbach. . . . . . . . . . .
xiv
...
......
Antibiotics and bacterial resistance
Chairmen: F. Baquero and G. Privitera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xvii
Vaginal microflora
Chairmen: G . Botta and A. B. Onderdonk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xviii
Molecular approaches to the study of microbial ecosystems
Chairmen: P. Borriello and R. Rappuoli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xix
Poster session . . . . . . . .
.......
.......
.....
..
xxi
The abstracts have been photo-reproduced as received. The Journal editors therefore have had no
involvement in presentation or English usage.
089 1-060X/95/01000i-34
(rI 1995 by John Wiley & Sons, Ltd
11
MECHANISMS INVOLVED I N THE
TRANSLOCATION
OF INTESTINAL
BACTERIA-A MAJOR SOURCE OF COMPLICATING INFECTIONS I N 1I'lMUNOSUPPRESSED PATIENTS.
C a r o l L. Wells
U n i v e r s i t y of Minnesota, M i n n e a p o l i s , USA.
B a c t e r i a l t r a n s l o c a t i o n can be d e f i n e d as t h e p a s s a g e of
b a c t e r i a ( l i v e and d e a d ) , and b a c t e r i a l p r o d u c t s ( e x o t o x i n s ,
e n d o t o x i n s , c e l l w a l l f r a g m e n t s ) , from a s i t e n o r m a l l y c o l o
n i z e d by m i c r o b e s t o a s t e r i l e d i s t a l s i t e .
Translocating
i n t e s t i n a l b a c t e r i a c a u s e a s i g n i f i c a n t p r o p o r t i o n of nosocomial i n f e c t i o n s ( i n c l u d i n g s e p s i s ) i n
postsurgical,
trauma, and immunosuppressed p a t i e n t s . I n i t i a l
research
documented t h e c l i n i c a l c o n d i t i o n s a s s o c i a t e d w i t h b a c t e r i a l t r a n s l o c a t i o n , e . g . , i n f l a m m a t o r y bowel d i s e a s e . e n d 2
toxemia, c o l o - r e c t a l and g y n e c o l o g i c a l c a r c i n o m a , i n t e s t i n a l o b s t r u c t i o n , b u r n wounds and o t h e r trauma, mesen
t e r i c i s c h e m i a and hemorrhagic s h o c k , o b s t r u c t i v e j a u n d i c e , p a n c r e a t i t i s , hepatectomy, o r g a n t r a n s p l a n t .
and
antibiotic-induced
e n t e r i c overgrowth. Due t o t h e c o g
p l e x dynamics of t h e i n t e s t i n a l t r a c t , t h e mechanisms
involved i n b a c t e r i a l
translocation
remain
elusive,
although r e c e n t r e s e a r c h has c l a r i f i e d
some
factors
whereby t r a n s l o c a t i o n can be f a c i l i t a t e d o r c o n t r o l l e d .
For example, t r a n s l o c a t i o n f o l l o w i n g b u r n
i n j u r y may
be r e l a t e d t o m e s e n t e r i c
vasoconstriction
and
gut
a t r o p h y , and my be modulated w i t h i n s u l i n - l i k e growth
factor-1,
f i b r o b l a s t growth f a c t o r
plus
sulcralfate,
and
p r o s t a g l a n d i n E a n a l o g s , h e p a r i n , thromboxane A2.
e a r l y e n t e r a l feeding. Considerable e f f o r t
has
been
devoted t o t h e e f f e c t of n u t r i t i o n on b a c t e r i a l t r a c
s l o c a t i o n , i n c l u d i n g t h e p o t e n t i a l b e n e f i t s of d i e t a r y
supplements. Although much of t h i s r e s e a r c h is clouded
is
that
by c o n f l i c t i n g d a t a , t h e c l e a r e s t e v i d e n c e
e n t e r a l n u t r i t i o n is p r e f e r a b l e t o p a r e n t e r a l
nutrit i o n , and t h a t e a r l y post-trauma e n t e r a l f e e d i n g may be b e
n e f i c i a l . There i s a l s o e v i d e n c e t h a t t h e macrophageand t h e
e n t e r o c y t e may p l a y p i v o t a l r o l e s i n t h e
p a t h o g e n e s i s of
b a c t e r i a l t r a n s l o c a t i o n . S p e c i f i c a l l y , i t - h a s been shown t h a t
including
t i s s u e macrophages can t r a n s p o r t ly p a r t i c l e s ,
b a c t e r i a , from t h e i n t e s t i n a l lumen t o t h e d r a i n i n g m e s e n t e
r i c lymph n o d e . However, r e c e n t e x p e r i m e n t s i n macrophaged e p l e t e d and g e n e t i c a l l y m a c r o p h a g e - d e f i c i e n t a n i m a l s i n d i c a t e t h a t , macrophage t r a n s p o r t may n o t be a primary mechanism of b a c t e r i a l t r a n s l o c a t i o n . E x p e r i m e n t a l a n i m a l s have
been used t o l o c a l i z e t r a n s l o c a t i n g b a c t e r i a w i t h i n e n t e r o c y t e s , and c u l t u r e d e n t e r o c y t e s have been used t o show t h a t
normal e n t e r i c b a c t e r i a (such a s E. c o l i and e n t e r o c o c c u s )
c l o s e l y a s s o c i a t e with a p i c a l m i c r o v i l l i , a r e i n t e r n a l i z e d
i n low numbers compared t o more p a t h o g e n i c e n t e r i c s p e c i e s ,
and e n t e r e n t e r o c y t e c y t o p l a s m i c , membrane-bound v a c u o l e s .
C l a r i f i c a t i o n of mechanisms i n v o l v e d i n t r a n s l o c a t i o n w i l l
l i k e l y s u g g e s t new t r e a t m e n t r e g i m e n s t o t r e a t t h e c o s t l y
m o r b i d i t y a s s o c i a t e d w i t h i n f e c t i o n s caused by t r a n s l o c a t i n g
intestinal bacteria.
ABSTRACTS
ENTEROTOXIC ACTIVITY DETECTED IN AiDS
PATIENTS WITH ENTERIC CRYPTOSPORiDlOSlS
Alfred0 Guarino
Department of Pediatrics, University "Federico 1111 of
Naples, Italy.
Cryptosporidium parvum is a protozoan pathogen capable of
inducing severe diarrhea in immunocompromised patients,
namely in those with HIV infection. The loss of large volumes of
watery stools suggests enterotoxic mechanism. Enterotoxic
effect was detected in the fecal supernatant of diarrheai calves
infected with Cryptosporidium parvum. using human jejunal
specimens. The experimental system to detect the effect
consisted in the recording the electrical parameters of intestinal
epithelium mounted in Ussing chambers. The addition of fecal
aiiquots to the mucosal ,side induced an increase in
transepithelial short circuit current, with no effect on tissue
conductance. The enterotoxic effect was time and dose
dependent, saturable and reversible. Furthermore it was heatsensitive and dependent on the presence of calcium and
chloride. An enterotoxic effectsimilar to that observed in human
jejunum was subsequently obtained in Caw-2 cells, which were
used to screen fecal samples of HIV-infected patients with
cryptosporidial diarrhea. An enterotoxic activity was found in 9
out of 11 samples. Fecal osmotic gap was consistent with
secretory diarrhea in all enterotoxln-positive samples, but it was
not so in the enterotoxin-negative samples.
These findings suggest that cryptosporidial diarrhea in most HIVinfected patients is of secretory type and is mediated by an
enterotoxic activity.
MYCOBACTERIA INFECTIONS AMONG SAROlNlAN HIV+ PATIENTS
G.Panichi*, F.Saba*, S.Babudieri*, S.ZanUtti*,
G.Fadda*, R . O i Rosa**.
* I s t i t u t o d i M a l a t t i e I n f e t t i v e , Universith d i
Sassari, I t a l y ; * * I s t i t u t o Medicina Interna I l l ,
Universitk "La Sapienza", Roma, I t a l y .
From March 1986 t o March 1994 the I.M.I.
(Istituto
M a l a t t i e I n f e t t i v e ) o f Sassari (North Sardinia) has
treated about 1000 H I V + p a t i e n t s (25% of which Mantou+)
and has reported 187 cases o f AIDS. S i x t y s i x (35.2%)
o f these were caused by mycobacterial i n f e c t i o n s : 53
(28.3%) by M. tubercolosis (MT), (2.8
f o l d greater
than I t a l i a n MT/AIOS incidence and 7 f o l d greater than
USA M T / A I O S incidence), and 13 (16.9%) by Non M. tubercol o s i s (NUT).
Typical forms were 15% and a t y p i c a l 85%.
The mean s u r v i v a l period from the moment of the i n f e c t i o n
was 20.7 months for MT, and 6.25 for NMT. Seventyfive %
of MT i n f e c t i o n s revealed a NP of CO4+>100/mm , 100%
of NMT i n f e c t i o n s a No o f CD4+<70.
Mycobacteria i s o l a t i o n (66%) and antimicrobial susceptibil i t y studies were performed by radiometric Bactec 460.
MT and NMT i s o l a t e s were i d e n t i f i e d by Accuprobe. Two
s t r a i n s o f MT were multiple-drug-resistant (MOR).
ABSTRACTS
CiRCULATlNG EACTEt7IAL LIPOPOLYSACCHARIDES (LPSJ I N P A T I E N T S
WITH INFLAMMATORY BOllEL DISEASE ( I B D ) .
i.Jirillo,L.Caradonna,B.Greco,R.Satalino,V.Pugliese,~.Sisto~,
:.Laxa",I.Giorgio".
1;limunopathol ogy. "Surgery, 1. R .C . C . S. " S . de Be1 1 i s " ,CASTELLANA
GROTTE ( B A R I ) , I t a l y .
B a c t e r i a l LPS or e n d o t o x i n s r e p r e s e n t the major c o s t i t u e n t s o f
t h e c e l l w a l l f r o m t h e o u t e r membrane of gram-negative b a c t e ria.Among t h e i r b i o l o g i c a l e f f e c t s , t h e r e l e a s e of i n f l a m m a t o r y
c y t o k i n e s (C<i may p l a y a paramount r o l e i n t h e paLhogenesis
of a v a r i e t y o f d i s e a s e s . I n t h i s framework,we have taken i n t o
c o n s i d e r a t i o n t h e p o s s i b l e r o l e o f LPS i n t h e e v o l u t i o n o f
iBD.This s t u d y i s based on t h e immunological m o n i t o r i n g , LPS
d e t e c t i o n and CK a s s a y , r e s p e c t i v e l y i n 10 p a t i e n t s with Cmhn's
Disease ( C u , and i n 15 p a t i e n t s w i t h u l c e r a t i v e c o l i t i s (UC).
O f n o t e , a l l p a t i e n t s were under s t e r o i d t r e a t m e n t at t k m m e n t
o f e n t e r i n g t h e z l i n i c a l p r o t o c o l . B o t h t y p e s of p a t i e n t s were
c h a r a c t e r i z e d sy t h e f o l l o w i n g immune a l t e r a t i o n s :
1 ) r e d u c t i o n o f phagocytosis and k i l l i n g of Candida a l b i c a n s
oy polynorphonuclear c e l Is and monocytes.
2 ; reduced f u n c t i o n of t h e a n t i b a c t e r i a l a c t i v i t y e x e r t e d by
CD4t and CD8t c e l l s , u s i n g Salmonella t h y p h i as t a r g e t .
3 ) I n c r e a s e d r e l e a s e of t h e s o l u b l e r e c e p t o r f o r i n t e r l e u k i n
(IL)-2.
LPS determined by a s p e c t r o p h o t o m e t r l c Limulus Assay were pr'
sent i n d e t e c t a b l e amounts i n t h e 60% o f b o t h p a t i e n t s . A s f a r
as CK assay i n sera and s t o o l i s concerned, d a t a demonstrate
t h e presence o f s i g n i f i c a n t ainounts o f i L - 1 0 i n UC p a t i e n t s '
s t o o l . G i t h r e g a r d t o t h e o r i g i n o f LPS t h e y may d e r i v e from
e i t h e r exogenous o r endogenous sources b u t t h e reduced immune
c l e a r a n c e c o u l d be r e s p o n s i b l e f o r t h e i r spreading i n t o t h e
host.The d e t e c t i o n o f f e c a l i i - 1 0 inay s u p p o r t t h e r o l e of g r a y
n e g a t i v e m i c r o f l o r a and/or t h e i r p r o d u c t s i n t h e maintenance
o f an i n f l a n , , i a t o i y process i n iinmunocompromised p a t i e n t s w i t h
IBD.
SELECTIVE TRANSLOCATION OF CAECAL COLtFORh4S B Y
STRAlNS ADHERING TO THE CECAL EPITHELIUM IN A
STRESSED RAT MODEL
M. Katouli', V. Muratov', C G. Nettebladtz, T. Bark?, T. Svenbergz
and R.Mo1lby' 'Microbiology and Tumorbiology Centre, Karolinska
Institute, and 2Department of surgery, Karolinska Hospital,
Stockholm, Sweden.
Starvation for 48 hours and hemorrhage to 5 5 m . H g mean arterial
blood pressure (MAP) resulted in translocation of the cecal coliforms
to mesenteric lymph nodes (MLNs) of rats (3/11 and 6/11
respectively). In contrast, only one out of 13 not treated control rats
showed translocation. The mean number of colifonns in starved rats
(4.8 x 1O1O cWgr cecal content) and haemorrhaged rats (1.6 x 1011
cwgr) was significantly higher than that of the control group (1.7 x
lo7 cfdgr) This was associated with an increase in number of
coliforms closely attached to the cecal epithelium in treated rats (4.5 x
lo6 cfuicm' cecal epithelium for starved rats and 2 0 x los cfdcm2 for
haemorrhaged rats vs controls 61 cfu/cm2). A biochemical
fingerprinting method (the PhF' system) was used to characterise
coliform bacteria in cecal content, on the epithelium and in MLNs.
Coliforms were divided based on their biochemical phenotypes (BPTs)
and their diversity in each sample was calculated by Simpson's index of
diversity. Out of several different BPTs of coliforms found in cecum,
few were found closely adhering to the cecal epithelium and yet only
some of these adhering coliforms were found in MLNs. There was no
correlation between the proportion of adhering phenotypes in cecal
content or on the cecal epithelium and their rate of translocation Rats
which did not show translocation had a high diversity of coliforms in
cecum and on the cecal epithelium. In all cases diversity of the
adhering colifonn on the cecal epithelium was more important for
translocation These results suggest that starvation for 48 h. and
hemorrhage results in an increase of cecal coliforms which is
associated with an increase of certain adhering strains to the cecal
epithelium. However, only few of these adhering coliorms were able
to translocate to MLNs. Furthermore, a high diversity of colifonns in
cecum andor a high diversity of adhering coliForms on the epithelium
is reversibly effecting translocation of these bactcria under the stress
conditions used.
iii
EFFECT OF LACTOBACILLI ON CRYPTOSPORlDIUM PARVUM
INFECTION I N MAN AND ANIMALS
6 . Famularo, J.A. Harp, C. De Simone, 5. Tzantzoglou,
F. S o r i c e , R . Vesely, w. Chen.
-
US Dept o f
I n f e c t i o u s Diseases L ' A q u i l a and Rome, I t a l y
A g r i c u l t u r e , Ames, Iowa, USA - Trudeau I n s t . , Saranac Lake USA
Although many GI i n f e c t i o n s i n AIDS p a t i e n t s do respond t o
s p e c i f i c a n t i - i n f e c t i v e therapy, t r e a t m e p t o f p a r a s i t e - a n d
protozoan- induced diseases i s poor. Most p r o b l e m a t i c i s
c r y p t o s p o r i d i o s i s.
I n o u r s t u d y 6 of 7 p a t i e n t s w i t h c r y p t o s p o r i d i o s i s t r e a t e d
w i t h l a c t o b a c i l l i ( 5 . t h e r m o p h i l u s , L. a c i d o p h i l u s , L. bulgar i c u s , L. c a s e i , L. p l a n t a r u m and a m i x t u r e o f b i f i d o b a c t e r i a )
n o t e d s u b s t a n t i a l improvement o r disappearance o f d i a r r h e a .
Notably, t h e same p a t i e n t s had been t r e a t e d w i t h n o n - s p e c i f i c
a n t i - d i a r r h e a l p r e p a r a t i o n s w i t h o u t major improvement o f t h e i r
symptoms. The r e s u l t s o f t h e study conducted on germ-free severe combined immunodeficient (SCID) mice s u p p o r t t h e hypothes i s t h a t t h e c o l o n i z a t i o n o f t h e g u t w i t h l a c t o b a c i l l i can
p r o t e c t f r o m C. parvum i n f e c t i o n . The animals c o l o n i z e d w i t h
l a c t o b a c i l l i were c l e a r l y l e s s i n f e c t e d w i t h C. parvum t h a n
c o n t r o l s . T h i s p r o t e c t i o n was n o t c o r r e l a t e d w i t h i n d u c t i o n
o f IFN-gamma by l a c t o b a c i l l i , s i n c e t h e two groups c h a l l e n g e d
w i t h C . parvum b o t h produced mRNA f o r IFN-gamma d e s p i t e t h e
f a c t t h a t mice c o l o n i z e d w i t h l a c t o b a c i l l i were p r o t e c t e d
f r o m i n f e c t i o n and t h e n o n - c o l o n i z e d group was n o t . I n a d d i t i o n , animals t r e a t e d w i t h l a c t o b a c i l l i b u t n o t w i t h C. p a r vum d i d n o t produce mRNA f o r IFN-gamma.
PHARHACOKINETIC STUDIEB OF
SACCHAROXCYEB BOULARDII (SB) IN
PATIENTS WITH HIV-RELATED CHRONIC
DIARREEA L IN HEALTHY VOLUNTEERS.
Elmer (I, noyer K, vega R, Burawics c,
Collier A, Hooton X, c XcFarland L.
Dept. Xed. Cham. t School of Wed.,
Univ. of Washington, t Xadison clinic,
Seattle, Washington, USA.
Eleven HIV positive patients with chronic
diarrhea were entered into a study to determine an
effective anti-diarrheal dose of BE. Patients had
a mean CD4 count of 185 f 221, a mean duration
of diarrhea of 27 f 2 5 months, stool frequency of
7.9 f 4.l/day and a mean loss of 8.1 2 8 . 7 lbs in
the prior month. Patients were started on 3 9 BE/d
for one week, then decreased to 2g/d for one
week and then lg/d for another week. Seven
(64%) reported an improvement of diarrhea: 3
while on 3g/d, 4 while on 2g/d and none during
lg/d. Of the 7 improving while on 2-3g/d, 6 (86%)
lost the response when the dosewas decreasedto
lg/d. S i x stayed on BB (2-3g/d for a mean of 5.5
months) to control diarrheal symptoms and no
serious adverse reactions were noted.
since many HIV infected patients take
fluconasole prophylactally, the influence of this
highly absorbable antifungal and of the nonabsorbable nystatin on steady state levels of SB
in healthy volunteers was determined. A crossover
design was used and the dose of SB ( 3 9 ) and the
antifungals were staggered by 4-6 h. Nystatin
(l.5n unita/d) oompletely eliminated S B from the
stool. However, there was no effeat of fluconaeole
(50 or 1 0 0 mg/d) cgmpared wit# maan CPU of
controls: 3 . 1 x 10 , 3.7 x 10 and 2 . 6 x 10';
raspactively. The effective dose for HIV diarrhea
was found to be 2-3 g of BB/d and was unaffeated
by staggered oral doses of fluconasole.
ABSTRACTS
iv
ENTEROCOCCAL INFECTIONS OF RESPIRATORY TRACT
HIV PATIENTS
R . D i Rosa*, E . D i Rosa", I.Mezzaroma*,
S.Babudieri**,
G.Panichi**.
IN
A.Prozzo*,
*Dept. o f I n t e r n a l Medicine Ill,U n i v e r s i t y "La Sapienza",
Rome, **Dept. o f I n f e c t i o u s Diseases, U n i v e r s i t y o f Sassari
Italy
The range o f o p p o r t u n i s t i c pathogens
i d e n t i f i e d as
causing i n f e c t i o n s i n H I V i n f e c t e d p a t i e n t s continues
t o increase. Among b a c t e r i a l i n f e c t i o n s , gram p o s i t i v e
environmental
organisms are
frequently
encountered:
e s p e c i a l l y Enterococci, derived from endegenous f l o r a ,
appear t o be an i n c r e a s i n g l y s i g n i f i c a n t cause o f i n f e c t i o n s i n r e c e n t years, a l t h o g h t h e y a r e r e p o r t e d t o
be uncommon causative agents o f pneumonia. This study
was performed t o assess the e t i o l o g y of b a c t e r i a l acute
r e s p i r a t o r y t r a c t i n f e c t i o n s i n HlV p a t i e n t s ; the r e s u l t s
o f m i c r o b i o l o g i c a l examination o f 76 sputum samples,
obtained from 60 HlV i n f e c t e d o u t p a t i e n t s i n a 18 month
period, are reported and revealed an important r o l e
for Enterococci.
S i x t y f o u r of 76 samples (84%) revealed a s i n g l e i n f e c t i o n
and 12 (16%) a mixed i n f e c t i o n .
Among s i n g l e i n f e c t i o n s , 43 cases (67%) were from Gram
p o s i t i v e b a c t e r i a . Enterococci were l a r g e l y p r e v a l e n t
among t h e i s o l a t e s , w i t h 31 p o s i t i v e samples
(40%)
(25 i n s i n g l e and 6 i n mixed i n f e c t i o n s ) . Risk f a c t o r s
f o r enterococcal i n f e c t i o n i n these
p a t i e n t s appear
t o be p r i o r a n t i b i o t i c therapy or p r o p h i l a x i s and the
s e v e r i t y of underlying disease. Also f o r the m u l t i p l e
a n t i b i o t i c resistances, r a p i d i d e n t i f i c a t i o n o f p a t i e n t s
i n f e c t e d by Enterococci seems necessary t o c o n t r o l
and prevent i n f e c t i o n s i n H I V p a t i e n t s .
V
ABSTRACTS
Host-parasite relationships on rnucosal membranes of the oral and
upper respiratory tract.
M. Kilian', J. Hohwy', H. Lomholt', J. Reinholdt' & K. Poulsen'
'Institute of Medical Microbiology and 'Department of Oral Biology,
University of Aarhus, The Bartholin Building, DK-8000 Aarhus C,
Denmark
To be able to persist on mucosal membranes bacteria must evade the local
immune factors, of which IgA is the principal mediator of specific humoral
immunity. Certain of the bacteria that inhabit the upper respiratory tract
including the oral cavity have developed socalled IgAl proteases. These
proteases induce specific cleavage of human IgA subclass 1 in the hinge
region. While the monomeric Fab fragments released by IgAl proteases
retain their antigen-binding capacity, the enzyme effectively eliminates
secondary effector mechanisms associated with the Fc part of the antibody
molecule. IgAl proteases are produced by the three principal causes of
bacterial meningitis, Neisseria meningitidis, Haemop/cilus influeme
(including non-capsulated forms), and Streptococcus pneumoniae and by
important members of the oral microflora. The latter include Streptococcus
sanguis, S. oralis, S. mitis, and all oral Prevotella and Capnocytophaga
species. While IgAl proteases from the pathogens show a high degree of
antigenic diversity resulting from recombination events involving part of
IgAl protease genes (iga) no variation is seen within the species belonging
in the oral cavity. Neutralizing antibodies against IgAl proteases are
induced as a response to infection or colonization and conceivably play a
part in the intricate host-parasite relationships involving IgAl proteaseproducing bacteria. Thus, neutralizing antibodies against H.influenme IgAl
proteases appear to play a role in the frequent clonal exchange of
noncapsulated forms of this species in the pharynx particularly during
childhood. Oral streptococcal species also show significant clonal diversity
and clonal exchange during childhood but antibodies to IgAl proteases
seem to be unrelated to this process.
CORYNEBACTERIUM DIPHTHERIAE:
MOLECULAR EPIDEMIOLOGY AND CHARACTERISATION
STUDIES ON EPIDEMIC AND ATYPICAL ISOLATES
Androulln Efstratiou
Respiratory and Systemic Infection Laboratory
Central Public Health Leboralory, London, UK
Diphtheria is one of the most important communicable diseases of bscterial etiology;
the causative organism beiig ~orvnebscteriumdwhtheriae. Within the UK, the
pattern of infection is mainly of small outbre&s and occasional sporsdrc cpses. the
majority of which are imported. The disesse is endemic or epidemic in some
regions of the world, for example, Eastern Europe, Russia, the ULmine. Turkey,
Vieloam. B~gladesh. Africa, the tropics and area within South America
(Ecuador). Since 1989, there has been a major resurgence of diphthem in Europe
with 19,046 registered cpses in 1993 alone. 80% of the.?^, wem reported from
Russia. These epidemics, therefore, pose a potential Lhnat of introduction of
laxi~cnrcC.dinhtheriae to ofher countries. Imported cases associated with the
Russian epidemics have already been reported fromGermany, Finland, Noway and
other countries in Eastern Europe. Documented imported cases from other
countnes includc a recent fatal case in Italy originating from South Amenca (Peru).
Within the UK. the majonty of imparted C.divhtheriaa isolates are from Africa and
Asia. The application of molecular typing (ribtyping and pulsed field gel
electrophoresis) to expiore the epidemiology of C.diuhtherie has proved useful in
tracing the spread and source of the organism. In current and recent snrdies from
epidemics in Russia and Eastern Europe, one clonal group appears to predormnnte.
This presentation will discuss the emergence of lhis clonal group, the extent of its
spread and its genetic relatedness to isolates from other parts of the world. Further,
the examination of other epidemic and sporadic isolates confirms tbnt different
molecular patterns are generated by strains from different geqraphical locations.
The clarity and polymorphisms within patterns suggest the usefulness of molecular
typing for the charactensation and identification of new clones in the 'clonal
evolution' of this i f e c t i o u disease.
J3xmmA pEI(NssIs AM, EaTEmIA PAR4mRNSIS INmmms :
mE op BPCIFRIAZ. IumnAE ticks? IN ME IN"(R ?HE DIs'4!3
N. GUISO AND N. KHELEF
INSTITUT PASTEUR
-
PARIS
- FRANCE
Bordetella p e r t u s s i s and Bordetella parapertussis a r e clos e l y r e l a t e d species. Although both a r e responsible of
whooping cough in humans and produce s i m i l a r virulence
f a c t o r s including filamentous hemagglutinin, fimbriae,
p e r t a c t i n , t r a c h e a l cytotoxin and adenylate cyclase-hemolys i n (AC-Hly) o n l y B
e r t u s s i s produces the well known
Dertussis to& (PTX-esesDiratorv
model we showed
;hat B. parapertussis a s i. p e r t u s s i s mutants d e f i c i e n t
i n PTX expression were able t o adhere and colonize the
lungs of infected mice but were cleared f a s t e r than 8p e r t u s s i s v i r u l e n t s t r a i n s whereas B. p e r t u s s i s mutant
d e f i c i e n t i n AC-Hly expression multiply much more slowly
the f i r s t 5 days of i n f e c t i o n . Furthemore. w e showed t h a t
i ) B. p e r t u s s i s is cytotoxic f o r murine alveolar macrophages ii) c e l l cytotoxicity occured through apoptosis iii)
induction of apoptosls is dependent on the expression of
AC-Hly. Finally. we demonstrated t h a t AC-Hly, unlike PTX,
is a protective antigen against b a c t e r i a l colonization.
I n conclusion, 8. p e r t u s s i s expresses an AC-Hly which might
a c t a t the beginning of the i n f e c t i o n by k i l l i n g the f i r s t
l i n e of host defenses and a PTX which may play a r o l e in
b a c t e r i a l persistence.
vi
ABSTFL4cT.S
MICROBIAL COLONIZATION OF PROSTHETIC MATERIALS
Gordon D. Christensen
Harry $3. Truman Memorial Veterans' Hospital and the Universi
ty of Missouri, Columbia, Missouri, United States of America
More than any other factor, infection limits the use of mechanical devices for the support of life functions. Staphylg
cocci cause most biomaterial associated infections, but the
pattern of disease is different between :he "virulen:" &
aureus (SA) and the "avirulent" S . epidermidis (SE). SA is
associated with an early abrupt onset while SE is associated
with a late indolent onset. Despite the less toxic presentation, SE causes most biomaterial related infections. Pathogg
nic strains of SA and SE have the capacity to colonize
smooth surfaces by forming a biofilm. Colonization is a complex, multifactorial, multiphasic process; SA appears to form
thin films while SE appears to form thick films ("slime")
due to the elaboration of copious amounts of extracellular
polysaccharides (EP). EP have yet to be identified, but Eurc
pean +nd American investigators have made progress. Phase vi
riation and phenotypic modulation, processes which are integral to the ecology of the organism, complicate the study of
slime and bacterial pathogenesis.
PREVENTION OF POLYMER-ASSOCIATED STAPHYLOCOCCAL INFECTIONS
G. Peters, Institute o f Medical Microbiology.
University of Muenster, Germany
The pathogenesis of staphylococcal polymer-associated
infections is characterized by the ability of the staphylococci to adhere to and grow on polymer surfaces. The
molecular pathogenesis csn be dissected in the early phase
of adhesion and the late phase of accumulation and slime
production. The result is the establishment of a thick
matrix on the polymer surface containing multiple staphylococcal cell layers embedded in extracellular slime substance and host proteins. In this matrix the staphylococci are
protected against host response mechanisms as well as
antibiotic therapy. Therefore prevention is very crucial.
Optimal surgery and strict aseptic procedures during
surgery and catheter insertion are essential. Also special
measures such as the use of lamina air flow can be important. Perioperattve antibiotic prophylaxis is also recommended although it is very difficult to substantiate
efficacy. Modern strategies in the prevention of polymerassociated infections are directed by the goal to obtain
anti-infective foreign body surfaces. i.e. to avoid either
adhesion OK the following proliferation. This is based on
the two following concepts: 1. Phyaico-chemical modification of polymer surfaces. Such modifications may lead to a
primarily anti-infective surface O K may create a reactive
surface to which antiadhesive substances, antiinfective
substances OK antibodies may be bound which then result in
an infection-resistant surface. 2 . Coafing or incorporating
of antimicrobial substances on or into polymer materials to
prevent adherence or colonization. Examples for both
concepts are given, also with special regard to their
r
.._
L,11>._
.
I __
L^
c__-
PATRXENESIS IN B I C M A r r a u I G A s ~ T E ZMFECTIONS
Asa Ljmm
Dept.of Medical Microbiology,
Sweden
University of Lurd,
Lurd,
During the l a s t decade significant prcyress has been made
i n the d e v e l o p n t of plywrs, mtals and term for
t m p r a x y or p e m e n t inplantation i n h m s . Infections
are a m j o r ccmplication in the use of these S.C. biaraterials, e-ially
with skin-penetrating devices. The mst
comoon etiologic agents are coagulase-negative s t a p h y l m c i (CXS), Propionibacterium acnes and C a n d i d a albicans,
i.e. lay virulent mmhers of the normal skin flora. After
adhesion to a solid surface microbes turn dawn t h e i r
mtabolism and @we
a plysaccharide substance. 'Ihis
qlycocalyx protects the microbes fron host defense mechanisms and f r a n antimicrobial agents. Expression of birdinq
of host proteins like f i b r i x q e n (Fq) and fibronectin (m)
by Staphylococcus aureus has been studied i n detail, and fn
and Fg have heen proposed as mdiatoris of adhesion by
microbes to p o l p r s . More recently, it has been shown t h a t
CNS s t r a i n bind h b i l i z e d fn, vitronectin, collagens,
thmnbqmndin and laminin to the same or higher extent
than S. Aureus strains.
Shortly a f t e r implantation, host proteins and qlycosarninoglycans adsorb to the b i m t e r i a l surface. The canposition
of t h i s layer varies w i t h the boay localization and with
properties of the bicmaterial. After i n i t i a l adsorption
proteins undergo changes of configuration to w i n g extent
which are likely to influence exposure or potentially
microbe b i d i n g sites, Several protein-protein interactions
have been descrited. Hence, proteins may fonn a layer on
the surface. We have used Atanic Force Microsccpy and
Scannrng Electron MicroscGpy to s W y luninal 7of
host proteins on central venous catheters, Cerebrospinal
shunts and peritoneal dialysis catheters extirpated fran
patients. Protein adsorption a d subsequent bacterial
adhesion has been studied in a f l m perfusion rcdel with
different boay rluids, usinq different plymrs with and
without end-point attached heparin. The latter surface is
antithrombogenic, induces low complement activation, and
has a low incidence of infections.
vii
ABSTRACTS
IS SLIME THE VIRULENCE FACTOR IN STAPHYLOCOCCALBIOMATERIALINFECTIONS?
Lucilla Baldassarri and Gianfranco Donelli
Laboratorio di Ultrastrutture, Istituto Superiore di Saniti,
Rome Italy
-
In the last decade coagulase-negative staphylococci(CoagNS)
have emerged as the most common etiologic agents in the set
ting of prosthetic device-associated infections. The phenomenon of slime production, first observed in 1972, has been
since regarded as a potential virulence factor i n the path2
genesis o f staphylococcal diseases. Several epidemiologic
studies have confirmed the association of slime production
with clinically significant strains, while a number of
others did not. The unavailability of a chemical or antigenic marker for slime has until now hampered the clarification of its exact role i n virulence. Also, the lack of a
standardized method for measuring slime production actually
invalidated the comparison of results between studies that
used different procedures. Detailed studies performed by
Pier and co-workers have described a polysaccharide adhesin
(PS/A) inhibiting the first step of CoagNS adhesion
they
speculated was a slime antigen. It seems however
unlikely
to actually identify such structure with slime. A more suitable candidate might result the slime-associated
antigen
(SAA) first identified by Christensen. While the exact role
of this material i n the colonization process has not yet
been established, preliminary data suggest it may fit better
with the generally accepted definition of slime. The comple
te chemical characterization of this structure might provide the tool to definitely assign a role to staphylococcal
slime i n thb pathogenesis of CoagNS infections.
ISOLATION OF A COLLAGEN RECEPTOR FROM S.PYOGENES.
P.Speziale, L.Visai, S.Bozzini, G.Raucci, A.Toniol0
Department of Biochemistry,University of PaviaJtaly
In this rewrt we have analyzed the binding of collagen to
Streptoc&us
pyoqenes strain 6414. This binding was rapid,
specific, essentially irreversible and involved cell surface
receptors. By quantitating the maximal m u n t of collagen
bound the receptor copy n
&
r
was calculated to be an
average of 11,603 copies per cell. Wnen the proteins in a
streptoccccal lysate were fractionated by plyacrylamide gel
electrophoresis, blotted into a nitrocellulose filter, and
probed with
125 I-labelled Collagen, a prominent
collagen-binding (CBP) protein of 57 -kDa was identified as
well as minor 130-150 -kDa caqmnents. The mjor 57-kDa
protein was isolated by affinity chromatography on a
collagen
Sepharose
followed
by
gel
filtration
chromatography. Addition of purified 57-kDa (Cap) to
streptococcal cells
strongly
inhibited binding
of
radiolabelled collagen to bacteria. The 57-kDa protein
purified fran S.pycgenes was used to raise a monospecific
antibody which also reacted with a CBP of similar mlecular
size isolated fran Streptococcus zooepidemicus. The two CBPs
fran streptococci have a similar amino acid capxition and
iscelectric pints. Native type I1 collagen effectively
inhibited the binding ofl2?-1abelled collagen to cells of
S.pycgenes whereas denatured type I1 collagen were not
effective indicating that the collagen confomtion may play
an inprtant role for recognition by the receptor molecule.
Isolated CBP was specifically recognized by
I-collagen in
a solid-phase binding assay and displayed an affinity for
the ligand quite similar to that exibited by intact bacteria
(K d = 4.87 versus 3.5 x 1 0 - 9 ~ . respectively). surface
labelled bacterial cells attached to microtiter well coated
with different collagen types and the 57-kDa protein blockeo
the adhesion to collagen substrate. We propose that the
57-kDa protein is a bacterial adhesin involved in the
attachnt of streptococci to host tissues.
...
Vlll
ABSTRACTS
.MUCOSAL RESPONSE TO
H.
w,R. Fiocca. L. Villani and 0. Luineni.
PYLORI INFECTION
Department of Pathology University of Pavla and IRCCS Policlinico San Malteo,
27100 Pavia, Italy
Purpose: To clarify the cellular mechanisms of gastric mucosa
immune-inflammatory response to H. pylori infection and their
contribution to mucosal damage.
Methods. Endoscopic biopsies were stained with Giemsa for H
pylori and immunoperoxidase for H. pylori, cathepsin E, HLA-DR,
IgA or IgG immunoglobulins, CD20 antigen of B lymphocytes,
CD43 or CD45RO antigens of T lymphocytes, S-100 protein of
dendritic cells and cathepsin D of macrophages. Conventional
electron microscopy and ultrastructural immunolocalization of H.
pylori antigens and cathepsin E were also performed.
Results. Activation and dilation of the endocytic-endosomal
system, up to vacuolization and endoluminal bulging of the
supranuclear cytoplasm was seen in H. pylori colonized sufacefoveolar epithelium. Bacterial antigens and the antigenprocessing enzyme cathepsin E were detected in endosomal
vesicles, together with expression of HLA-DR antigen. B and T
lymphocytes were prominent in the deep, IgA plasma cells and
activated macrophages and dendritic cells (some harboring H.
pylori antigens) in the superficial to mid, and IgG plasma cells
and granulocytes in the mid, renewal zone of the mucosa.
Conclusions. H. pylori antigens transport and, likely,
processation and presentation by gastric epithelium has a role in
B and T cell mediated mucosal immune response. Direct
bacterial cytotoxicity and granulocyte and macrophage infiltration
contribute to the epithelial erosion and impaired regeneration
which predispose to peptic ulcer, as confirmed by their rapid
suppression in H. pylori eradicated patients cured of their ulcer
disease.
MICROORGANISMS OF THE GASTRIC ANTRUM
IN HELICOBACTER PYLORI INFECTION
Eve Kriisk, Marje Oona,Tiina Riigo, Heidi Maaroos,
Marika Mikelsaar, Hannu Korhonen, Seppa Salminen
Tartu University, Estonia; Turku University and
Institute of Food Research, Finland
IMKINOGENICITY OF PURIFIED AND RECOMBINANT
ANTIGENS OF HELICOBACTER PYLORI I N THE MOUSE
Oderda
G, *
Burroni D,
Bugnoli M,
* Ghiara
P.
P e d i a t r i c Gastroenterology, University of Torino
and t Biocine SPA, Siena, I t a l y
H p y l o r i (HP) Urease i s an important virulence f a c t o r f o r
b a c t e r i a l c o l o n i z a t i o n , and some s t r a i n s a r e known t o
produce a vacuolating c y t o t o x i n which is more commonly
found i n p a t i e n t s with p e p t i c u l c e r .
AIM: To study i m u n o g e n i c i t y of p u r i f i e d Urease and
recombinant Vacuolating-associated-cytotoxin-A (VacA) i n
mice used as animal model f o r HP i n f e c t i o n .
MATERIAL b METHODS: Twelve 6 wk-old BALB/c mice were
immunized a t day 1, 14 and 28, e i t h e r p a r e n t e r a l l y
(subcutaneously) or o r a l l y with 100 pg/dose/mouse of
e i t h e r p u r i f i e d recombinant VacA expressed i n E.coli o r
Urease p u r i f i e d from water e x t r a c t of H Pylori grown i n
l i q u i d medium. P a r e n t e r a l immunization was accomplished
using conventional CFA adjuvant while o r a l immunization
was performed on 24hr f a s t i n g animals using LT of
e n t e r o t o x i g e n i c E . c o l i a s adjuvant. Sera were c o l l e c t e d
a t days 0 , 12, and 4 1 and t h e i r r e a c t i v i t y a g a i n s t H
Pylori was assessed by immunoblot using a whole b a c t e r i a l
s o n i c a t e a s a n t i g e n and an anti-mouse-Ig a s second
antibody
RESULTS: Both VacA and Urease were found t o be s t r o n g l y
imunogenic when i n j e c t e d p a r e n t e r a l l y . Sera from
p a r e n t e r a l l y immunized mice with VacA s t r o n g l y r e a c t e d i n
a VacA ELISA a s s a y (mean t i t e r = 1 : 1 0 , 0 0 0 ) . On t h e o t h e r
hand when administered o r a l l y n e i t h e r antigen induced a
serum response.
CONCLUSION: Both Urease and VacA can be used t o immunize
mice. A challenge of crude b a c t e r i a l e x t r a c t t o a s s e s s
t h e i r p r o t e c t i o n from g a s t r i c damage induced i n vivo is
i n progress. The r e s u l t s of t h e s e i n vivo experiment w i l l
a l s o be presented.
.
WLICOBACTER PYLORI PUTATIVE GASTRIC TISSUE ADHESINS
2
3
4
T. Wadstr5m‘. F. Ascencio , S . Hirmo , 0. Larm , M. U t t 3
1
2Dept.Med.Microhiology, Univ.of Lund, Lund, Sweden
gCentro 1nvestig.Biologicas. La Paz, Mexico
qDept.Biochemistry, Univ.of Tartu. T a r t u , Estonia
Dept.Exp.Surg.Karolfneka I n s t i t u t e , Stockholm, Sweden.
Helicobacter pylori (HP) is recognised as the major causative agent
of chronic gastritis. However, little information is available about
other types of bacteria in the gastric epithelium of children.
Aim: To compare the frequency of occurrence of HP with the frequency of other bacteria in histologically documented gastritis of
children.
Patients and methods: From 32 children (9-16y) with gastritis
mucosal specimens were taken from the antrum for histological and
bacteriological investigation. Paraffin sections of the biopsy samples
were stained by Giemsa method, with haematoxylin eosin for light
microscopy, and with acridine orange for fluorescence microscopy.
HP and several microorganisms were searched for by seeding the
homogenate of the specimens on 4 different media.
Results: HP could he found in the histological sections of the
mucosa in 12 cases. Fluorescence microscopy allowed us to
distinguish between HP, cocci, bacteria and yeasts. Only 8 samples
failed to show any bacteriologically detectable microorganisms.
Aerotjic microorganisms were revealed in 16, anaerobes in 4
patients. In 4 cases HP remained the only isolated microorganism.
From 12 children with HP colonization other microbes were
revealed in 8 children (76%) and in 20 HP-negative cases from 12
(60%) patients. The spectrum of the revealed microorganisms was
significantly larger in patients no[ colonised with HP. Staphylococci
and streptococci occurred more frequently in association with HP.
Thus, the antral mucosa of children with gastritis is colonised with
different aerobic and anaerobic microorganisms and HP seems not
tn
intervm+ ?he more<<
Relicobacter p y l o r i , u n l i k e A f e l i s and some o t h e r animal
R e l i c o b a c t r r s p e c i e s express two c l a s s e s of c e l l s u r f a c e
haemagglutinins. one recognizing v a r i o u s sialoglycoconjugat e s , and t h e o t h e r n o n s i a l i c a c i d compounds. I n t e r e s t i n g l y ,
l i k e some o t h e r e x t r a c e l l u l a r pathogens, H
lor1 strains
r e g u l a r l y d s o bind sulphated g l y c o c o n j u g a t ~ o s a m i n o glycans, GAG:s) such a s heparan sulphate (Ascencio e t 81..
J. Med. Microhiol. 1993; 38: 240-4). Binding of H pyosurface-immobilized heparin i n t e r f e r e s with i n t e r a c t i o n of
iodine-labelled h e p a r i n dependent growth f n c t o r s such as
b a s i c f i b r o b l a s t growth f a c t o r (hFGF). A model t o study
i n t e r a c t i o n s of GAG molecules and sialoglycoconjugates with
various chemical analogues in m i c r o t i t r e p l a t e s has been
developed. In t h i s model s p i r a l and coccoidal forms have
been compared. In c o n t r a s t t o f i n d i n g s with Bordetella
e r t u s s i s , heparinoids and o t h e r GAG molecules do not
Pinhibit haemagglutination by H. p y l o r i .
ix
ABSTRACTS
IIYDROPHILICAND IIYUKOPIiORIC FORMS O F A BLOOD GROUP A-DEGRADING o-hACEIYLOAY\CIOMMIStDASE I'URIFIED FROM CULTURFS O F ' M E FECAL
PRESENCE O F ENTEROTOXICENIC EACTEROIDES FRAGILIS
(ETBF) STRAINS IN ITALY
A. Pantostil, A. Fratel, M.G. Menozzi2, L. Sanfilippo2, C.Piersimoni3.
R. Di Rosa4
Laboratono di Batteriologia e Micologia Medica, Istituto Superiore di
Sanita. Romal; Istitulo di Microbiologia, Universita di Parma2; Ospedale
"Le Torrette", Ancona3: Dipartimento di Medicina Clinica, Policlinico
Umberto 1. Roma4. Italy.
Strains of B.frugilis with the property to produce a n enterotoxin (ETBF
strains) have been identified in recent years and have been associated with
diarrheal diseases in young farm animals. Their role in intestinal
disturbances in humans has been investigated with non-univocal results.
Aim of this research was to study the circulation of ETBF strains in Italy,
and to define their prevalence among B.Pugilis strains both from feces of
individuals with and without diarrhea and from extra-intestinal infections.
ETBF were detected using the cytotoxicity assay for HT-29 cells and
positivity was confirmed by a cytotoxicity neutralization assay using
antiserum produced to the enterotoxin.
Our results showed that ETBF strains represented approximately 10% of a11
Blfrogilih examined independently of their source (whether feces or extraintestinal). Among E.frugilis isolated from feces, two F T B F were from
children with diarrhea, but none from healthy children. In one of the two
positive children, free enterotoxin was also detected in feces by the HT-29
assay. ETBF were also obtained from the feces of adults, both with and
without intestinal disturbances: in three cases from hospitalized patients and
in two cases from healthy subjects. An healthy adult subject c a m e d two
different ETBF strains as determined by plasrnid fingerprinting.
Among extra-intestinal isolates, several ETBF strains were identified: no
particular source seemed related to ETBF, as the strains were isolated from
various types of infections.
In conclusion, ETBFcan be detected among E.frogili.$ isolated in Italy, with
a frequency similar among intestinal and extra-intestinal strains. However,
our data suggest that there could be an association between f T B F strains
and diarrheal diseases in particular groups of population. The significance
of ETBF strains from extra-intestinal infections deserves further evaluation.
~
HoskinrLC., Bou1ding.E T., & 'Larson,G
Dcpl Medicinc, VA Medical Center and Case Weslcm Rcscrvc Univcrsily, and
'Dcpt. Clin. Chem. UNV of Gothenburg, Sweden.
R. lorques str. IX-70 ic one of a small number of commensal human cnleric
bacteria which constitutively produce extracellular glycosidases that degrade
oiigosaccharidc chains or gut mucus glycoproteins and cell membrane glycolipids.
In ordcr to study its action on epilopes of blood group antigen A on erythrocyler
wc purified an cxo-a-N-acctylgalactosaminidase ('a-GalNAc'as") from ccll-free
culture supernates ofthis strain by ammonium srilhte lracliunalion and sequeiitial
column chromatographyon Phenyl Scpharose CUB, hydroqapatite (HA), and
Sephacryl S-300.The kcy stcp was hydrophobic interaclinn chromatography on
Phcnyl Scph. in which Lhc samplc in 4M NaCI-O.l% viv Triton X-100 was applicd
to and eluted from the column during a decreasing 4.0 M 0.1 M NaCl conc.
gradient a-GalNAc'ase activity clutcd m 3 main fractions: F-I, the niosl
hydrophilic, clulcd i n 3.2 M NiCl ahcad d 5 01hcr glycosidasc activitics, lhc loss
hydrophilic F-I1 cluting at 2.9 M NaCl wilh p-galactosidasc, and thc most
hydrophobic F-I11 eluting at d 1.0 M NaCI. HA chromatography resolved F-I
inio 2 active peaks, IA and IB, with M. = 265 and 417 kDa on Seph S-3M) and
submits of M, = 158 and 173 Kda, raspectivcly, on denaturing SDS-PAGE. F111 cluied anomalously from Scph. S-300, with 52% eluting in a pcak at Knva =
0 23 (M,= 530 kDa and the rest eluting over 2 column volumes (subunit M, =
205 kDa on SDS-PAGE) On non-denaturing PAGE F-IA migratcd as 5 active
hands w h weight-nve M, = 136 kDa, sugestmg that F-IA,-IB, and 111 wcre di-.
IF,- and tctr.imcric rums ~Clhis
b~sics~ruclurc.
Althuugh similai in pH optinwm
(6 0-6 2), pH rangc (5.0-8.9, and kinetics ony-nilrophenyl-a-GaiNAc (K, = 2.24 8 mhl, V,,
= 7X-115 rrrnollrndmg), F-I11 diffcicd from F-IA!B in i1s clutiiin
kom Phcnyl Scph. and Scpb. 300, and m no1 binding to a-GalNAc-ligandcd
agarose. F-IA/B a n d F-Ill clcavcd lcrrninal a-GalNAc from A + gut iniULm
glycoproteins aud from mono- and di-fucosyl A and Forssman glycolipids wilh
uncovering of underlying H ( 0 ) antigen epitope whcn present. Cunclusirm
production 01 a-GaWAc'ase isoenzymcs with ddfcrenl hydrophobicity may
racililatc cleavage of utilizable monosaccharidesfrom walcr iolublc glycoprolcins
and water insoluble membrane glycolipids in gut lumen contents
-
X
ABSTRACTS
HUMAIi MILK CONSWTION AND PROTECTION
AGAINST
ENTERIC
INFECTION.
David J . H e n t g e s l , Wallace W. Marsh1, Mohammad E. Rahmanl,
Bryon W. Petschowz, and C a t h e r i n e S. McVayl.
lDept.of Microbiol.and Immunol., Texas T e c h U n i v e r s i t y H e a l t h
*Bristol-Myers Squibb Corn
S c i e n c e s C e n t e r , Lubbock, Texas
pany, Mead Johnson R e s e a r c h C e n t e r , E v a n s v i l l e , I n d i a n a .
-
P r e v i o u s s t u d i e s i n O U K l a b o r a t o r y showed t h a t g n o t o b i o t i c
mice a s s o c i a t e d w i t h human i n f a n t i n t e s t i n a l f l o r a were mor e r e s i s t a n t t o c o l o n i z a t i o n w i t h S a l m o n e l l a typhimurium
when consuming human m i l k t h a n when consuming b o v i n e m i l k ,
cows' m i l k based formula (CMBF) o r f o r m u l a m o d i f i c a t i o n .
To determine mechanisms of p r o t e c t i o n a f f o r d e d byhuman m i l k
consumption, we examined t h e p o s s i b l e involvement o f a n t i s. typhimurium a n t i b o d i e s in t h e m i l k , g a s t r i c a c i d i t y , and
c e c a l pH and v o l a t i l e f a t t y a c i d (VFA) l e v e l s . Consumption
of human m i l k of r e l a t i v e l y h i g h a n t i b o d y t i t e r by f l o r a - a s
s o c i a t e d mice d i d n o t p r o v i d e b e t t e r p r o t e c t i o n a g a i n s t
of
c h a l l e n g e w i t h S. typhimurium t h a n consumption of m i l k
lower a n t i b o d y t i t e r . When consuming human m i l k , t h e pH of
t h e stomach c o n t e n t s o f t h e mice was s i g n i f i c a n t l y lower
(2.18) t h a n when consuming CMBF ( 3 . 2 4 ) . S. typhimurium s u r v i v e d f o r l e s s t h a n 30 min. i n T r y p t i c s o y b r o t h a d j u s t e d
mice
t o pH 3.34. C e c a l VFA c o n c e n t r a t i o n s were g r e a t e r i n
consuming CMBF ( 1 9 6 8 u g / g ) t h a n i n mice consuming human m i l k
( 1 4 0 4 u g l g ) , however, t h e pH of c e c a l c o n t e n t s of mice consuming human m i l k ( 5 . 6 9 ) was s i g n i f i c a n t l y lower t h a n t h a t
of mice consuming CMBF ( 6 . 4 4 ) . C e c a l c o n t e n t s , o b t a i n e d from
g e r m f r e e mice, were a d j u s t e d t o s i m u l a t e t h e VFA and pX l e v e l s measured in t h e c e c a of t e s t mice, and were t h e n i n o c g
l a t e d w i t h S . typhimurium. While VFA and pH a t l e v e l s pres e n t i n c e c a l c o n t e n t s of human milk-consuming mice
were
b a c t e r i c i d a l f o r S. typhimurium, m u l t i p l i c a t i o n of t h e o r g s
n i s m o c c u r r e d a t l e v e l s p r e s e n t in c o n t e n t s of CMBF-consuming mice. Lowering t h e pH l e v e l of c e c a l c o n t e n t s o b t a i n e d
from g e r m f r e e mice from 6 . 6 9 t o 5 . 6 9 , w i t h o u t
concomitant
VFA a d d i t i o n , d i d n o t r e t a r d t h e m u l t i p l i c a t i o n of S. t y p h i
i n d i c a t i n g t h a t hydrogen-ion a c t i v i t y a l o n e was not
r e s p o n s i b l e f o r i n h i b i t i o n . R a i s i n g t h e pH l e v e l of c e c a l
c o n t e n t s o b t a i n e d from human m i l k consuming mice c o n t a i n i n g
VFA t o 6 . 4 4 , t h e l e v e l determined in t h e c o n t e n t s o f CMBF
consuming m i c e , O K t o 7.50 r e v e r s e d i n h i b i t i o n . The r e s u l t s
i n d i c a t e t h a t t h e low g a s t r i c pH and t h e n o n i o n i z e d VFA pr:
s e n t in t h e a c i d i c environment of t h e cecum d u r i n g human
m i l k consumption impede c o l o n i z a t i o n of t h e g a s t r o i n t e s t i n a l
t r a c t of f l o r a a s s o c i a t e d mice w i t h S . typhimurium.
e,
PROBIOTICS I N PAEDIATRICS.
S. Guandalini
C a t t e d r a d i P e d i a t r i a U n i v e r s i t y of Reggio C a l a b r i a , School
of Medicine in C a t a n z a r o .
Oral b a c t e r i o t h e r a p y in p e d i a t r i c s h a s been u t i l i z e d s i n c e
a l o n g t i m e in a s e r i e s of c o n d i t i o n s , b u t on a r a t h e r empi
r i c b a s i s . In t h e l a s t few y e a r s , however, some new evidenc e h a s been produced t h a t might p r o v i d e a s c i e n t i f i c r a t i o n a l e f o r i t s u s e , and namely:
- The u s e of y o g u r t in l a c t o s e i n t o l e r a n t s u b j e c t s ;
- L a c t o b a c i l l u s GG in t h e t h e r a p y o f a c u t e e n t e r i t i s due t o
Rotavirus;
- Saccharomyces b o u l a r d i i in p e r s i s t e n t d i a r r h e a s due t o
Clostridium d i f f i c i l e .
Yogurt in l a c t o s e i n t o l e r a n c e . A sound e v i d e n c e is by now
a v a i l a b l e t h a t l a c t o s e i n t o l e r a n t s u b j e c t s may d i g e s t
and
a b s o r b l a c t o s e from y o g u r t much b e t t e r t h a n f r o m o t h e r d a i r y
p r o d u c t s . T h i s h a s been shown c o n c l u s i v e l y b o t h in a d u l t s
( s u b j e c t s w i t h a d u l t t y p e h y p o l a c t a s i a ) a n d , from o u r group,
i n c h i l d r e n w i t h s e c o n d a r y i n t o l e r a n c e . Not o n l y t h e v a l u e s
show no malabo b t a i n e d w i t h t h e "hydrogen b r e a t h test"
s o r p t i o n when t h e s o u r c e o f l a c t o s e is y o g u r t , b u t a l s o t h e
a f t e r l a c t o s e adminisatration
c l i n i c a l signs t h a t e n s u e
( d i a r r h e a , abdominal d i s t e n s i o n a n d cramps) a r e l a c k i n g a f t e r
y o g u r t . T h i s phenomenon, whose o b s e r v a t i o n a l l o w s t o
feed
w i t h y o g u r t s u b j e c t s t h a t would o t h e r w i s e had t o e x c l u d e
m i l k p r o t e i n s o r a t least l a c t o s e from t h e i r d i e t , is l i k e l y due t o t h e B - g l i c o s i d a s e a c t i v i t y of t h e l a c t i c b a c t e r i a .
L a c t o b a c i l l u s GG i n R o t a v i r a l d i a r r h o e a . T h i s is p r o b a b l y
t h e s i n g l e most c o n v i n c i n g e v i d e n c e of t h e r a p e u t i c e f f i c a c y
of o r a l b a c t e r i o t h e r a p y . It h a s in f a c t been shown, by Isol a u r i and co-workers, t h a t t h e a d m i n i s t r a t i o n of such
a
L a c t o b a c i l l u s , c e r t a i n l y a b l e t o f u l l y c o l o n i z e t h e human
i n t e s t i n e , r e s u l t s in a c l e a r improvement of t h e d i a r r h e a l
outcome in i n f a n t s . The d i a r r h e a , as w e l l a s t h e
hospital
s t a y o f a f f e c t e d c h i l d r e n , a p p e a r s s h o r t e n e d by 1-2 d a y s .
i
T h i s e f f e c t is l i k e l y due t o t h e s t i m u l a t i o n of a humoral !
munity b o t h n o n - s p e c i f i c and s p e c i f i c , a s s u g g e s t e d by t h e
i n c r e a s e d number o f plasma c e l l s s e c r e t i n g IgG, I@ and IgM
and of t h e plasma c e l l s secretingspecificallyanti-Rotavirus
immunoglobulins.
T h i s s p e c i f i c i t y a c t u a l l y may r i s e the q u e s t i o n of t h e e f f i c a c y of t h e p r o b i o t i c in the.'treatment
of o t h e r t y p e s
of
d i a r r h e a , due t o d i f f e r e n t e t i o l o g i e s and, c o n s e q u e n t l y , t o
d i f f e r e n t p a t h o g e n e t i c mechanisms. To answer t h i s q u e s t i o n ,
we have r e c e n t l y s t a r t e d a p e r s p e c t i v e d o u b l e - b l i n d c l i n i c a l
trial, administering, along with the o r a l rehydration solumonths
t i o n , 109 u n i t s o f L a c t o b a c i l l u s o r p l a c e b o t o 1-36
o l d c h i l d r e n , a d m i t t e d f o r a c u t e d i a r r h e a . In e a c h c h i l d a
c o m p l e t e e t i o l o g i c a l s e a r e c h is performed, i n c l u d i n g R o t a v i K U S , e n t e r i c t y p e A d e n o v i r u s , Campylobacter, Y e r s i n i a , Salm:
t h e l a t t e r s by s p e c i f i c DNA pron e l l a , S h i g e l l a , E. c o l i
, and P r o t o z o a . The
b e s f o r ETEC, EPEC, EAggEC, EIEC, EIEC
r e s u l t s , t o be c o n s i d e r e d o n l y a s v e r y p r e l i m i n a r y a s t h e y
o n l y r e f e r t o 12 t r e a t e e i n f a n t s (2 R o t a v i r u s , 3 S a l m o n e l l a ,
1 Campylobacter and 6 u n i d e n t i f i e d ) and 9 c o n t r o l s ( 2 R o t a v i
r u s , 2 S a l m o n e l l a , 1 EPEC and 4 u n i d e n t i f i e d ) , show a non si
g n i f i c a n t r e d u c t i o n of d i a r r h e a l d u r a t i o n in t h e t r e a t e d patients.
Saccharomyces B o u l a r d i i . In t h e l a s t y e a r s new d a t a h a v e b e e n
g e n e r a t e d b o t h in t h e a n i m a l and in humans, on t h e e f f e c t i v e n e s s of t h e mycete Saccharomyces b o u l a r d i i in p r e v e n t i n g
a s w e l l a s in t r e a t i n g a number of d i a r r h e o g e n i c c o n d i t i o n s .
In p a r t i c u l a r , the mycete was found a b l e t o i n h i b i t t h e b i n
d i n g and t h e s u b s e q u e n t e n t e r o t o x i g e n i c i t y of t h e A Toxin
of C l o s t r i d i u m d i f f i c i l e and of C h o l e r a Toxin (CT). Subseq u e n t l y , i t h a s a l s o been found a b l e t o r e d u c e t h e r a t e s of
r e l a p s e s in c h i l d r e n w i t h C l o s t r i d i u m d i f f i c i l e e n t e r o p a t h y .
S t i l l missing is however, any e v i d e n c e of e f f i c a c y in t h e
most f r e q u e n t l y s e t t i n g of Saccharomyces b o u l a r d i i u s e , 1 . e .
a c u t e d i a r r h e a in c h i l d r e n . Only an a d e q u a t e l y planned pers p e c t i v e s t u d y , as t h e one under p r e p a r a t i o n by OUT group,
w i l l d e f i n i t e l y be a b l e t o provide conclusive evidence.
-
-
ABSTRACTS
IMMUNOGLOBULIN AND NON-IMMUNOGLOBULIN
COMPONENTS OF HUMAN MILK INHIBIT CLOSTRIDIUM
DIFFICILE TOXIN A-RECEPTOR BINDING. Rial D. Rolfe and
Woosun Song. Dept. of Microbiology and Immunology, Texas
Tech Univ. Hlth. Sci. Ctr., Lubbock, Texas, USA.
Clostridium dijficile is isolated from the intestinal
tracts of greater than 50% of healthy infants. The mechanism
of asymptomatic C. difficile intestinal colonization of infants is
unknown but may reflect the presence in maternal milk of
neutralizing activity against C . dificile toxin A. O n this basis,
we analyzed the ability of human milk to inhibit toxin A
receptor binding. Ten human milk samples inhibited the
binding of toxin A to a purified hamster brush border
membrane receptor by an average of 90%. Heating ( 1 O K for 5
min) and dialysis did not significantly alter the inhibitory
activity in any of the milk samples.
Ultrafiltration
demonstrated that the inhibitory factors in human milk had
molecular weights >100,000 kD. Gel filtration and native PAGE
suggested that the factors present in human milk which inhibit
toxin A binding are complexing with toxin A rather than
breaking the toxin into inactive subunits. SDS-PAGE and
ligand blot analyses showed that there were a t least four
distinct factors in human milk that specifically bound toxin A.
These factors had molecular weights of >200,000, 110,000, 75,000
and 55,000 kD. Thiophilic adsorption chromatography was
used to separate immunoglobulin from non-immunoglobulin
IgA was t he only
components of h uman milk.
immunoglobulin detected in human milk and greater than
90% of this immunoglobulin was recovered following
purification by thiophilic adsorption. Both the unbound nonimmunoglobulin and bound immunoglobulin fractions of
human milk inhibited the binding of toxin A to the purified
receptor. These results suggest that human milk may be
important in protecting infants against C. digicile-associated
intestinal disease.
FAECAL ENZYMES AND DETECTION OF Lactobacillus GG
AFTER ORAL FERMENTED MILK INTAKE IN JAPANESE
AND FINNISH VOLUNTEERS
S.
Salminen, Y. Benno, H. Mykkanen, H. Iino, M.
Hosoda, F. He, M. Hiramatsu, H. Hashimoto. Department
of Biochemistry and Food Chemistry, University of
Turku, Japanese Collection of Microorganisms, Tokyo,
Department of Clinical Nutrition, University of Kuopio,
Finland
The influence of oral Lactobacillus GG yoghurt intake
on the detection of Lactobacilfus GG in faeces and on
faecal bacterial enzymes and was determined i n
h ea lt h y Ja p a n e se and Finnish
volunteers.
A
Lactobacillus GG (ATCC 53103) fermented yoghurt was
given at levels of 108 and 109 cfu/d to healthy
volunteers. Faecal detection of LGG was significantly
higher in Japanese subjects than Finnish subjects.
Activities of faecal bacterial urease, 13 -g 1uc u r on i d a se
and iS-glucosidase levels were higher in Finnish than
Japanese subjects and urease and 5 - g l u c u r o n i d a s e
levels were decreased during Lactobacillus GG intake.
In Japanese subjects the average activities of these
enzymes were significantly lower. No changes in faecal
enzyme activities were observed during Lactobacillus
GG intake. These results indicate that Lactobacillus GG
is able to survive the passage through the intestinal
tract. The changes observed in intestinal microecology
and metabolism appear to be related to the initial
status as well as prevailing dietary factors.
xi
INTESTINAL MICROFLORA COMPOSlTlON 1N PREMENSTRUAL
SYNDROME (PMS) AND EFFECT OF LACTOBACILLUSBIFIDOBACTERIUM ADMINISTRATION.
Min Ili , Benini A, Vicentini L. *Grussu P, *Cerutti R.
Institute of Pharmacology and *Department of Obstetrics and
Gynaecology, Universities of Verona and *Padova, Italy.
PMS consists of emotional, behavioural and physical symptoms that recur
regularly during the second half of each menstrual cycle. In our preliminary
study, intestinal microflora of women with PMS showed microbial
fluctuations with increase of different enterobacteria. We can hypotize that
the microbial flora may be an additional factor involvied in the PMS with
other phoenomena such as the modifications of sex steroid hormones
pattern. In order to correct gastrointestinal symptoms (constipation,
diarrhoea, abdominal pain and microflora alterations) we administered to
24 young women (range 28-35 yrs) with severe PMS a sup lement of
Lactobacillus acidoohilu and Bifidobacterium bifidum (4xIOE + 4x106
bacteridg) three capsules a day for 2 months and SAMe (S-adenosylmethionine), 800 mg twice a day for a week before menstruation. SAMe is
an antidepressant drug; moreover is a methyl donor which panecipate to
hepatic biochemical reactions and should favour the metabolism of sexsteroid hormones during entero-hepatic cycle. 8 healthy young women
wer included as controls. Faecal samples were collected on the 1 lth and
2 d d a y of cycle before the starting therapy and on 23'd d during therapy.
Microflora composition was performed by standard methods. Women with
PMS showed a low bacterial counts in comparison to the heaIthy controls.
Ldi 2, E. cloacae, Hafnia. mrobacte r freundii were frequentely isolated
in the majority of PMS women before treatment, but decreased or
disappeared after 2 months of therapy. Aerobic lactobacilli increased 1 log.
The mean count of closmdia and bacteroides was not significantly altered.
Different closnidia species appeared during treatment. Anaerobic gram
positive non-spore-forming rods increased in species number, E.lrmosum,
Proo.acnesappeared as new species.One month after the end of treatment
2. During
appeared B cloacae, C. freundii and K. wneumoniz, not
treatment the following faecal enzymes leucine-arylamidase, acid
phosphatase, esterase-lipase and P-glucuronidase achieved levels and
frequency comparable to those determined in healthy women. Moreover
we noted a relief of some symptoms of PMS in studied women, i.e. minor
abdominal distress and pain, no constipation and better psycological
behaviour.
xii
ABSTRACTS
THE BACTEROIDES FRAGILdS E N T E R O ~ X I NIs
A ZINC METALLOPROTEASE.
B. L. Van
J. S. Moncrief, D. M. Lyerly and T. D. Wilkins.
Department of Biochemisay and Anaerobic Microhiology, VPI & SU,
Blacksburg, ViNa. USA
INTERACTION OF CYTOTOXIC NECROTiZtNG FACTOR TYPE 1 (CNF1) FROM
PATHOGENIC E. COL/ WITH MAMMALIAN CELLS
u,
Carla Fiorentini, Patrice Boquet' and Gianfranco Donelli
Department of Ultrastructures, Istituto Superiore di Sanitd, Rome ITALY:
+Unit&des Toxines Microbiennes, lnstitut Pasteur, Paris FRANCE
Within the past decade, certain strains of Bncreroides frugilis
which comprises 1-2% of the colonic microflora have been associated
with diarrhea in domestic animals and humans. These enterotoxigenic
strains produce a toxin which induces a fluid response in ligated ileal
loops and a cytotoxic response in certain colon carcinoma cell lines. The
toxin is produced in viuo as an extracellular protein (-20,000 Mr) which
has been purified to homogeneity and characterized by a toxin specific
enzyme immunoassay (EM). PCR cloning and sequencing studies have
revealed an amino acid consensus motif for zinc-binding proteases within
the toxin gene. Sequence comparisons with other zinc proteases (e.g.,
collagenases, gelatinases, elastases, etc.) indicated that theB.fragi1i.s toxin
is in the 'metzincins' class of zinc endopeptidases. Substrate screening
studies revealed that the toxin has gelatinase activity and cleaves actins.
In addition, the toxin is capable of autodigestion resulting in the loss of
cytotoxic activity while the EIAresponse remains unchanged. T h e proteolytic activity is inhibited by metal chelators hut not by inhibitors of
other classes of proteases. Analysis of bacterial cell fractions by EIA
indicates that larger immunoreactive, non-cytotoxic forms of the toxin
may be integrally associated with the cell membranes of the enterotoxigenic mains. Ongoing studies are focusing on further characterizing this
newly identified mamx metalloprotease and determining if the membraneassociated forms are toxin complexes, protoxin (proenzyme) or other
antigenically related proteins.
-
~
Certain E. coli strains of clinical relevance have been reported ( 1 ) to elaborate
a toxic factor termed cytotoxic necrotizing factor 1 (CNFl). This factor, a
monomeric protein of about 110 kDa, causes necrosis in rabbit skin and the
formation of giant multinucleated cells in cultures. Most CNFl-produclng
strains are haemolytic and the two genes encoding CNFl and hemolysin are
located in close proximity on the bacterial chromosome ( 2 ) . CNFl has been
described as interacting with the microfilament system promoting cell
spreading and probably interfering with the cytokinesis (3). The main effect of
the toxin in cultured epithelial cells is a time- and dose-dependent increase in
actin assembly characterized by the formation of prominent ruffles and stress
fibers. It Is known t h a t , in higher mammalian cells. actin
assembly/disassembly is controled by the small GTP-binding ras-related rho
proteins. Our results show that CNFl covalently modify the rho protein
thereby activating permanently this molecule. Actin assembly is a process
required for bacterial entry into cells. Interestingly. CNF1-producing €.colt
strains have been isolated, not only from humans with diarrhea but also to a
greater extent from cases of urinary tract infections and often from
bactedemia. The spreading of such intestinal bacteria from the lumenal side of
the gut t o the urinary tract or to blood stream, might be explained by the
potent phagocytic-like activity that CNFl induces in epithelial cells (4. 5 )
which allows non-invasive bacteria t o enter cells and to probably multiply
intracellularly. The various steps of this CNF1-driven phagocytic behaviour
have been explored by electron microscopy. Thus.actin assembly via a Gprotein induced by CNFl might represent a key mechanism for this novel type
of host cell-pathogen interactions.
1.
2.
3.
4.
5.
STLDY OF THE D P H T H W A TOXIK ENDOCYTIC PATHWAY USLhG
EPITOPETAGGED TOXIN .4W EPITOPE TAGGED TOXIN G3.L
RECEPTOR
Patrice BOQUET and Ernmanuel LEMICHEZ
STUDIES ON THE CELLULAR MECHANISM OF ACTION OF CLOSTRIDIUM
DIFFICILE TOXINS A Ah9 B
T. Baldwin. E. Chippeck, S. Hyde and S.P. B o r r i e l l o
Dept.Nicrobiology and I n s t i c u t e of I n f e c t i o n s and Immunity,
QMC. Nottingham, U.K.
The a c t i o n of C. d i f f i c i l e t o x i n s A and B on h o s t c e l l s
promotes d i s r u p t i o n of t h e c e l l c y t o s k e l r t o n and changes
c e l l morphology. The mechanism f o r e i t h e r t o x i n i s s t i l l
u n c l e a r . On2 approach t o d e l i n e a t e t h e p r o c e s s is t o
i n h i b i t v a r i o u s c e l l f u n c t i o n s t o determine how t h e t o x i n
i s processed. Use of chemicals and changes i n t e m p e r a t u r e
demonstrated t h a t t h e r e i s no requirement f o r t h e f u s i o n of
phagosomes or endosomes t o lysosomes, o r p r o c e s s i n g by
lysosomes. F u r t h e r , a low pH compartmenr i s a l s o unnecess a r y , a s confirmed using a mutant CHO c e l l l i n e d e f i c i e n t
in i n t r d c e l l u l a r a c i d i f i c a t i o n . These f i n d i n g s c o n t r a d i c t
t h o s e of o t h e r s u s i n g s i m i l a r methods. However, we d i d
confirm t h a t lanthanum, which i n t e r f e r e s w i t h c a l c i u m
pathways, i n h i b i t s t o x i n a c t i v i t y , b u t showed t h a t t h i s i s
due t o an e f f e c t on t h e t o x i n s , n o t on t h e c e l l . We have
also attempted t o a n a l y s e c e l l u l a r changes due t o t o x i n A
by f o l l o w i n g changes
c e l l p r o t e i n phosphorylation
f o l l o w i n g l a b e l l i n g w i t h 'P.
P r o t e i n s of i n t o x i c a t e d c e l l s
were s e p a r a t e d by SDS-PAGE, t o t a l p r o t e i n s v i s u a l i s e d w i t h
coomassie b l u e and phosphorylated p r o t e i n s (PP's) by
autoradiography. There were t h r e e main PP's of t h e f o l l o wing molecular w e i g h t s , 21 kDa (myosin l i g h t c h a i n ) , 2 9 kDa
and c 380 kDa. I n a d d i t i o n f o u r t o h e r p r o t e i n s appeared,
t h r e e a t 100-120 kDa (rwo of them a t 2 min and one a t l h )
and one at 70 kDa (3h a f t e r t o x i n t r e a t m e n t ) . Ic i s concluded t h a c t h e r e i s no c l a s s i c a l i n t r a c e l l u l a r p r o c e s s i n g of
t h e t o x i n s and t h a t t h e i r mechanism of a c t i o n i n v o l v e s
p h o s p h o r y l a t i o n of c e r t a i n p r o t e i n s .
Caprioli et al., J. Clin. Microbiol. 25. 146-149. 1987
Falbo et a/., Infect. Immun. 60, 2182-2187. 1992
Fiorentini etal., Toxicon 26. 1047-1056, 1988
Falzano e t a / . . Mol. Microbiol. 9, 1247-1254, 1993
Donelli eta/., Zbl. Bakt., Suppl. 24, 60-71. 1994
Instirut Pasteur. Unite des Toxincs Microbienncs.
28, m c du Dr Roux 75724 Paris Ccdcx IS France.
Diphtheria
toxin
(DT)
is
synthesized
by
toxigenic
Corynebocrerium
diphrheriae as a single chain protein. DT is
ihen rapidly cleaved into a two ciiains molecule (DTA and DTB)
still hold by a disulfidc bridgc. DT action on sensitive cells is
class,caly divided into three steps uhich involve each. a
particular domain of [he toxin molecule. Binding of DT to its cell
receptor is accompl:shcd h y the receptor binding dom3in
localized at the C-terminus end of thc toxin molccule. After
binding the toxin is rapidl) cndocytosed by clallirin coated
vesicles and transportcd to an iniraccIIu1ar acidic compartmeni
in which the transiocntion domairi localizcd at m e aminoterminus end of DTB a l l o w ihe transport of the enzymatic DTA
fragment into the cytosol. DTA in thc cytosol inhibits protein
qnthcsis by ADP ribos)lalion of Elongation Factor 2 (EF2). Due to
the particular antigenic naturc of diphiheria toxin and the low
rcccpior number of scnsitive CCIIS. the study of the DT endocytic
pithway Sy j~.im163:Iuo:csccrcc
c:icroscopy has n e v c i 5 x 2
s u c c c s s i u l l ~ acconiplishco. \Vc have thus study the cndoc) iosis.
o f I / a recombinani epiiopc.tiggcd diphthcria t o x , n niolcculc
inio Cor cells transfected rbith ihc cDNA coding for tnc native DT
receptor. 21 a recombinant epitopc-tagged diphthcrin toxin
receptor cDNA ~ransfcctco into Cos cells. Wiih this techniquc we
~ c r e able
to
follou.
by
c I ~ s s i c a 1 and
confocal
nim uno fl uo resccncc studies, Ihe d i phi hcr i a lox in endoc y t ic
p3thway. Within 5 m.n. of internalization. the coniplcx between
receptor and toxin reached the early endosomnl coiiipartnient
\rere translocation of DTA x r o s s !lie nicnioranc t o the cytosol
occurs The DT niolecules whicn do not entcr liic c)tosol, irc ihcn
transfcrrcd togc.hcr \ v i i i i DT rcccpior iiiio \ , c s ~ c l c s o l llic 13tc
ciido sum 3l/pr e I) sosoni 21 c oni p3rtnicni
by
3
ni ic r o t J b L l c
u:pcndciit
rncc.ian.siii 3110 cciiceniritcd i i i tlicsc \c,icIcs
,probnbl) b) J nicziijnisni siniilir to i n a t dcicribcd in tlic t:isc
o i EGF) Dcgradat.on of diplitlicrta m x i n is f i n J i l y ocliic\cd
3.most
c x c I JFI v e I y
i ii thc
I ~ t c c i i d o s o i : i ~ l / ~I rycs o so11 JI
.~iiip~r:i!ic:it IIius bciorc ::.!c.ii:i?
I .soFoni:5
.
...
ABSTRACTS
GARDNERELLA VAGINALIS CYTOLYSIN AND
EFFECT
Xlll
CHOLESTEROL ANCHORING
Sabina Cauci
Dip.Scienze e Tecnologie Biomediche, Fac.di Medicina,
Univ. di Udine, Italy.
Gardnerella vaginalis, previously classified as Haemophilus
vaginalis or Corynebacterium vaginale, is a pleomorphic,
gram-positive bacterium implicated in gynaecological and
urological disorders. Although Gardnerella vaginalis is
currently recovered as the prevalent organism in 98% to
100): of women with bacterial vaginosis its pathogenic role
has been controversial because of the frequent aesociation
with mycoplasmas and variuus anaerobic bacteria.
We purified to homogeneity from the culture broth supernat a m of Gardnerella vaginalis a haemolytic toxin (Gvh)
which likely constitutes a virulence determinant of this
unique bacterium ( 1 ) . The cytolysin shows perforin-like
behaviour being able to provoke che formarion of a 2 . 4 nm
radius pore o n human erythrocytes.
Gardnerella vaginalis is p-haemolytic on human but not on
sheep blood agar plates. We studied the species specificity
of the purified Gvh on different red blood cells. We found
a remarkable specificity towards human erythrocytes with
the following order of reactivity: human>> cat> pig> cow>
dog> rabbit> horse> sheep.
The Gvh membrane damaging activity on human erythrocytes i s
selectively inhibited by the presence of competing cholrsterol. The inhibitory effect i s greatly enhanced (up to
4000 times) when cholesterol i s mixed with phospolipids and
incorporated into small double-layered unilamellar vesicles. Moreover cholesterol i s two orders of magnitude more
effective when mixed with negatively charged rather than
with zwitterionic phospholipids. Lipid vesicles are able to
prevmt red blood cells haemolysis by binding the toxin
faster and more avidly. It results that even subtle variations in the lipid composition of the target membrane can
produce dramatic changes in the binding affinity, suggesting a possible reason €OK the species specificity connected to a modulated cholesterol-anchoring effect similar to
that postulated for che family of the thiol-activated
coxins produced by Gram- obitive bacteria. Although Gvh
shares seeera1 fs-iituies wfth this family of cytolysins it
does not truly belong to it being antigenically distinct.
This demonstrate the peculiarity of Gardnerella vaginalis,
which developed its toxin a s a tool for finding a specific
niche in the vaginal environment. The lysis of human red
blood cells i s markely affected by temperature being
optimal at 37°C. Moreover the hemolytic activity of Gvh i s
modulated by pH and low pH enhanced the damaging activity
of the toxin ( 2 ) . These findings are not general among
hemolysins and suggest that the cytolysin may be an important factor in the invasiveness of Gardnerella vaginalis at
an early stage. In fact, the normal pH of the vagina
(around 4 . 5 ) is unfavourable for the bacterial proliferation but potentiates the Gvh action. The involvement of Gvh
in che in vivo pathogenicity is documented by the detection
of high specific IgA levels in the vaginal fluid of patients with acute bacterial vaginosis.
1) S. Cauci, R. Monte, M. Ropele, C. Missero, T. Not, F.
Quadrifoglio, G. Menestrlna. “Mol. Microbiol.” 2, 11431 1 5 5 , (1993).
2) S . Cauci, R. Monte, F. Quadrifoglio, M. Ropele. G. Men:
strina. “Biochim. Biophys. Acta” 1153,53-58. ( 1 9 9 3 ) .
BINDING AND PORE-FORMING ACTIVITY OF BORDETELLA PERUSSIS
ADENYLATE CYCLASE TOXIN
Brotherston, C., Westrop. G., Parton, R., Coote, J . and
Freer, J.H.
Department of Microbiology, University of Glasgow, Glasgow,
G 1 2 BQQ, U.K.
Adenylate cyclase toxin (CyaA) of Bordetella pertussis i s a
177 kDa bifunctional protein with adenylate cyclase and
haemolytic activities. It is capable of binding to and
penetrating eukaryotic cells whereupon it is stimulated by
calmodulin to catalyse the formation of intoxicating levels
of cyclic 3 ’ : 5 ‘ AMP. The toxicity of CyaA requires posttranslational activation by an accessory cyaC gene product.
The C-terminal portion of the CyaA protein has homology
with members of the RTX family of toxins including the
Pasteurella haemolytica leukotoxin (LktA), which is activated in a similar manner by the LktC gene product. However,
whereas CyaA is toxic to a wide range of eukaryotic cell
types, the toxicity of LktA is limited to leukocytes and
platelets of ruminant origin.
Activated and non-activated forms of adenylate cyclase
toxin were produced using recombinant E. coli strains
expressing either (i) CyaA alone, (11) CyaA and CyaC OK
(111) CyaA and LktC. These toxins were used to investigate
the role of activation in target cell specificity and pore
formation. The specificity of binding of these recombinant
proteins was assayed by immunoblotting of toxin treated
membranes from nucleated and non-nucleated cell types. The
toxins were also assayed for their pore forming ability in
membranes by the measurement of the release of a fluorescent marker from liposomes. Whereas the activated and
non-activated forms of CyaA bound to sheep erythrocytes
there were differences in their binding to the nucleated
cell types used in this study. The activated form of CyaA
elicited marker release from liposomes, while the non-activated form did not. These results suggest that toxin
activation is important in modulating CyaA binding to and
the subsequent pore formation in cell mebranes.
DAMAGE TO THE GASTROINTESTINAL EPITHELIUM
CAUSED BY PURIFIED BACTERIAL LIPOPOLYSACCHARDEq
R.H. Hill and H.M Cowley
Department of Anatomy and Human Biology, University of the
Wihvatersrand, Johannesburg, South Africa.
An extensive literature over nearly a decade has clearly shown that
lippolysaccharide (LPS) has far-reaching pathological consequences
when administred parentally. Yet the enormous challenge of LPS to
which the colonic epithelium is continuosly exposed, has never been
considered. To examine the hypothesis that LPS also exerts an
adverse effect on the intestinal epithelium, we purified
lipplysaccharides of wild strains of ficherichia coli and Shigella
J7exneri from bulk laboratory cultures. The sterile LPS preparations
were administered to germ-free BALBc mice by intermittent
intragastric and intrarectal instillation over three days. Histological
studies and mucin histochemistry, together with measurements of
changes in the structure of the cryptslvillus unit showed that the E.
coli LPS caused only minor damage to the intestinal epithelium
compared to control tissues instilled with physiological saline solution.
The LPS from S. Jexneri, caused extensive necrosis of the mucosae
of the duodenum and ileum with up to 50%loss of lamina propria and
epithelial cells from the apical part of the villi, although the deep
crypts were free of any observable damage.
There was no cell or tissue damage seen in the large intestine, but
mucin histochemistry with alcian blue and the High Iron Diamine
techniques showed that both LPS treatments had inhibited sulphation
of goblet cell mucin in the colonic crypts.
This study has shown that LPS from the microflora of the gut,
applied to the lumenal surface of the gastrointestinal epithelium in
vivo, may be responsible for a low grade inflammatory response in the
normal gastrointestinal tract.
xiv
ABSTRACTS
ANTIMICROBIAL PRoPnYLmIs FOR INTESTINAL SURGERY
S.L. Gorbach.
RATIONAL DESlGN OF VACCINES AGAINST
POLYSACCHARIDES
Rino Rappuoli
IRIS, Biocine Research Center, Sienn. ITALY
TOXINS
AND
Tufts U n i v e r s i t y School o f Medicine, Boston,MA, USA
Prevention of postoperative infection in elective colorectal
surgery is based on a proven microbiologic principle: reduction in the
inoculum size of the infecting microflora. This is partially performed
by mechanical flushing of the bowel prior to surgery to reduce fecal
mass. Additional suppression of the microflora is optionally achieved
by using oral antibiotics. For anaerobes, erythromycin or
metronidazole is chosen, and for aerobidfacultative bacteria,
neomycin. Controlled trials have shown superiority of this oral
antimicrobial regimen over parenteral antibiotics. Penetrating
abdominal trauma caused by a gunshot or knife injury is associated
with contamination of the peritoneal cavity by the microflora. Early
use of antimicrobial drugs with activity against anaerobic and
aerobic/facultative bacteria is recommended. A short duration of
treatment, 1-2 days, appears sufficient. Appendicitis is another
infectious process associated with contamination by the microflora.
All patients undergoing appendectomy should receive preoperative
preventive antimicrobial drugs that suppress anaerobic and
aerobiclfacultative pathogens. Failure to use appropriate antimicrobial
drugs in appendicitis produces poor clinical outcomes. The overall
objective in prophylaxis of abdominal surgical procedures is
suppression of the intestinal microflora leading to a reduction in the
inoculum of potential pathogens.
THE ROLE OF PROBIOTICS IN ENHANCING
INTESTINAL IMMUNITY
M. Kaila, E,Isolauri
Department of Clinical Medicine,
University of Tampere, Finland
In the gut significant defense mechanisms have evolved to
provide selective exclusion and immune elimination of
potentially harmful substances. Gut microflora is considered a
central component of this barrier, interacting closely with the
elaborate immune system of the intestinal tract.
Probiotic bacteria are being actively studied as a means to
reinforce the mucosal barrier and to stimulate the local immune
system. Lactic acid bacteria have been shown to have such
beneficial properties, shortening the course of acute rotavirus
diarrhea a n d protecting against increased intestinal
permeability.
A study was designed to compare the immunological effects
of viable and heat inactivated lactic acid bacteria. Lactobacillus
casei strain G G (LGG) administered as a viable preparation
during acute rotavirus diarrhea resulted in a significant
rotavirus specific IgA response by convalescence. The heat
inactivated LGG was clinically as efficient, but the IgA response
was not seen.
It therefore appears that in order to induce immune
stimulation, the lactic acid bacteria need to b e viable: able to
survive the passage through the gastrointestinal tract, to adhere
to the mucosa a n d to colonise. Preliminary experience indicates
that viable lactic acid bacteria can be also used to improve
immune responses evoked by peroral vaccines.
A new generation of vaccines has been recently developed whcre antigens have been
manipulated in order to maximize their immune response and miniize the risks
associated with immunization. The best examples of this rational design of new
vaccines are the genetic detoxificationof backrial toxins, such as pertussis and cholera
toxins, and the development of glycoconjugate vaccines against Haemophilus
influenzae, MeningococcusA and MeningocoecusC.
Genetic detoxification of toxins
Pahrssis is the major virulence factor ofSordeteNa pertussis and the main component
of acellular vaccines against whooping cough. Most of the new vaccines contain a
form ofpertussis toxin which has been detoxified by treatment with formaldehydeor
other chemical agents. This treatment abolishes the toxicity, but also decreases the
immunogenicity of the molecule, most likely by altering the conformation of the
prolein We have usedsite-directedmutagenesis to change the two aminoacids that are
responsiblefor the toxicity of patussis toxin and generated a mutant B.pertussu strnn
that produces a non toxic (i.e., devoid of enzymatic activity) PT molecule. Ths
molecule can be included in vaccines without further modification. Indeed, vaccines
containing this genetically detoxified PT have been extensively tested in preclinical,
phase I and phase Il studies. In all instances, they were much more immunogenic than
vaccines containing chemically detoxified forms of PT. One formulation of thts
vaccine has been introduced for infant vaccination in Italy.
Using a similar approach, we took advantage of the knowledge of the threcdimensional shllcture of Ecali LT to design and gcnerate non toxic mutants of LT and
of the related cholera toxin. Such mutants have becn produced in large scale and
shorn toinduce excellent titers of toxinneutralizing antibodies both against the A and
the B subunit of the toxin These mutanb may be used to improve live and non living
vaccines against traveller's diarrhea and may be uscd as adjuvants for mucosal
immunization.
Coniueate vaccines
Immunity against capsular polysaccharides of Hemophilus influenzae, and
Meningococcus is h o w lo confer protection against diseasc. Unfortunately, the
capsular plysaccharides are T cell-independent antigens and are not immunogenic in
infants To make them T cell-dependent. polysaccliandes have been chemically
conjugated to carrier proteins. The vaccincs obtained arc very immunogenic in infants.
A vaccine against H influenzar is already being used for large scale vaccmabon. A
vaccine against Meningococcus C is in advanced clinical testing
The I t a l i a n c l i n i c a l t r i a l of a c e l l u l a r p e r t u s s i s
vaccines
.P_. H a s t r a n t o n i o . H. G i u l i a n o , S . S a l m a s o , D.Greco.
S.Wassilak:
I s t i t u t o S u p e r i o r e d i S a n i t A . Rome, I t a l y a n d
' N I A I D N a t i o n a l I n s t i t u t e o f H e a l t h , B e t h e s d a . USA
The N a t i o n a l I n s t i t u t e o f H e a l t h i n I t a l y i s c o o r d i n a t i n g a randomized double blind.
placeboc o n t r o l l e d t r i a l of p e r t u s s i s v a c c i n e s i n an
i n f a n t p o p u l a t i o n . I n our c o u n t r y a n a v e r a g e o f
a b o u t 1 2 , 0 0 0 c a s e s a r e a n n u a l l y r e p o r t e d and t h e
a c t u a l number i s l i k e l y t e n - f o l d h i g h e r . The
vaccine coverage f o r p e r t u s s i s i s about 40% and
t h e common r e a s o n f o r n o t u s i n g t h e a v a i l a b l e
w h o l e - c e l l v a c c i n e s is t h e f e a r o f a d v e r s e e v e n t s .
The p r i m a r y a i m o f t h e i t u d y is t o e s t i m a t e t h e
a b s o l u t e e f f i c a c y of two a c e l l u l a r a n d o n e w h o l e c e l l v a c c i n e a g a i n s t p e r t u s s i s d e f i n e d by c l i n i c a l
and l a b o r a t o r y c r i t e r i a .
1 4 . 7 4 7 c h i l d r e n were r a n d o m i z e d i n t o a f o u r - a r m e d
trial.
After
parental
consent,
participants
r e c e i v e d o n e o f two a c e l l u l a r p e r t u s s i s v a c c i n e s
i n combination w i t h d i p h t e r i a and t e t a n u s t o x o i d s .
a DT w h o l e - c e l l p e r t u s s i s v a c c i n e o r DT o n l y ( i n a
r a t i o of 313,321). The t r i a l s t a r t e d i n S e p t e m b e r
' 9 2 . p r i m a r y i m m u n i z a t i o n s w e r e c o m p l e t e d in midFebruary
'94
and t h e c l i n i c a l
follow-up
of
c h i l d r e n f o r d i a g n o s i s of p e r t u s s i s w i l l c o n t i n u e
up t o A p r i l ' 9 5 . A d v e r s e e v e n t s h a v e b e e n c l o s e l y
m o n i t o r e d . F o r t h e s u r v e i l l a n c e of s u s p e c t e d cases
a n y c o u g h i l l n e s s o f more t h a n 7 d a y s d u r a t i o n
t r i g g e r e d t h e c o l l e c t i o n o f a mucus a s p i r a t e f o r
c u l t u r e a n d PCR a n a l y s e s a n d o f b l o o d s a m p l e s f o r
a n t i body
determination
by
ELISA.
xv
ABSTRACTS
I M M U N I Z A T I O N WITH THE B SUBUNIT OF SHIGA-LIKE TOXIN1 (SLT-I)
PROTECTS I N A MODEL OF HEMORRHAGIC COLITIS.
E . B o e d e k e r l , C. M u l l e t t l , D. Achesonz, W a l t e r Reed Army
PREVENTION OF STAPHYLOCOCCAL INFECTIONS BY
TREATING NASAL CARRIERS OF STAPHYLOCOCCUS AUREUS
P i o t r B.
I n s t i t u t e o f Research1, New England Medical C e n t e r ,
MA2
Heczko and Malgorzata Bulanda
Boston,
E . c o l i s t r a i n RDEC-H19A i s a n a t t a c h i n g and e f f a c i n g r h b b i t
pathogen produced by t h e t r a n s f e r of t h e t o x i n - c o n v e r t i n g
e n t e r o p a t h o g e n i c E. c o l i
phage H19A of EHEC t o t h e r a b b i t
RDEC-1
which p r o d u c e s h i g h l e v e l s o f S h i g a - l i k e t o x i n I
(SLT-I). We have p r e v i o u s l y shown t h a t t h i s s t r a i n c o l o n i z e s
cecum and c o l o n , and i n d u c e s d i s e a s e i n r a b b i t s
resembling
human EHEC i n f e c t i o n which is m a n i f e s t by w e i g h t l o s s , submu
c o s a l edema, h e t e r o p h i l i c i n f i l t r a t e o f t h e l a m i n a p r o p r i a ,
and e n d o t h e l i a l c h a n g e s , and i s accompanied by serum and b i l i a r y immune r e s p o n s e s t o SLT-I B s u b u n i t ( G a s t r o e n t e r o l o g y
92:1643 6 104:A751). To d e t e r m i n e w h e t h e r p a r e n t e r a l immuniz a t i o n w i t h SLT-I B s u b u n i t r e s u l t i n g in h i g h t i t e r serum
IgG r e s p o n s e s would p r o t e c t r a b b i t s a g a i n s t d i s e a s e induced
by RDEC-HI9A; we hyperimmunized r a b b i t s
with
recombinant
SLT-I B s u b u n i t i n c o m p l e t e F r e u n d ' s a d j u v a n t (CFA). measured t h e i r anti-SLT-I B s u b u n i t IgG t i t e r s by ELISA and t h e i r
serum SLT-I n e u t r a l i z i n g c a p a c i t y i n a c y t o t o x i c i t y
assay,
and then c h a l l e n g e d them w i t h 5x102 RDEC-H19A by o r o g a s t r i c
i n o c u l a t i o n . The SLT-I B used f o r immunization was a recombi
n a n t p r o d u c t , p r e v i o u s l y shown t o be p e n t a m e r i c .
Responses
of I 0 immunized r a b b i t s were compared t o t h o s e of 10 r a b b i t s
who r e c e i v e d o n l y CFA. F o l l o w i n g o r o g a s t r i c c h a l l e n g e , rabb i t s were ohserved f o r w t . l o s s , e v i d e n c e of i l l n e s s and f e c a l s h e d d i n g , t h e n s a c r i f i c e d on day 7 f o r d e t e r m i n a t i o n o f
c o l o n i z a t i o n of i n t e s t i n a l segments, and f o r h i s t o p a t h o l o g i c a l a s s e s s m e n t of e n t e r o a d h e r e n c e , submucosal edema and i n flammatory i n f i l t r a t e s . R e s u l t s : Immunized a n i m a i s developed
h i g h t i t e r serum IgG r e s p o n s e s t o SLT-I B s u b u n i t and t h e i r
s e r a were a b l e t o n e u t r a l i z e t h e i n v i t r o t o x i c i t y of SLT-I
f o r HeLa c e l l s . Only t h r e e immunized r a b b i t s had d i a r r h e a ( 1 )
o r l o o s e s t o o l s ( 2 ) . whereas 8 c o n t r o l r a b b i t s were a f f e c t e d
( 4 d i a r r h e a : 4 loose s t o o l s ) . Immunized r a b b i t s
were prot e c t e d a g a i n s t t h e m a j o r m a n i f e s t a t i o n of RDEC-Hl9A d i s e a s e .
w t l o s s . The non-immunized group l o s t w t ( 5 / 1 0 a n i m a l s ; a v g
-38 gm) whereas t h e immunized group g a i n e d w t ( 9 / 1 0 a n i m a l s ;
a v g 80.8 gm) d u r i n g t h e week a f t e r c h a l l e n g e , (P-05 Wilcoaon
s i g n e d r a n k t e s t ) . There was a p o s i t i v e c o r r e l a t i o n between
c o n t i n u e d w t . g a i n a f t e r c h a l l e n g e and t h e
toxin-neutraliz i n g c a p a c i t i e s ( r = 3 6 9 ) and anti-SLT-I
IgG ELISA
titers
(r=281) of t h e s e r a . P r o t e c t i o n was n o t a b s o l u t e , s i n c e t h e
o n l y d e a t h o c c u r r e d i n t h e s i n g l e immunized r a b b i t which dev e l o p e d d i a r r h e a and l o s t w e i g h t . There was n o d i f f e r e n c e
between t h e g r o u p s in t h e d e g r e e of RDEC-H19A e n t e r o a d h e r e n c e t o r a b b i t cecum, i n t e s t i n a l c o l o n i z a t i o n w i t h , o r f e c a l
s h e d d i n g o f , RDEC-Hl9A. t l i s t o p a t h o l o g i c a l a n a l y s e s a r e pend i n g . These r e s u l t s i n d i c a t e t h a t serum a n t i c y t o x i c IgG a n t l
protective
body d i r e c t e d a g a i n s t t h e B s u b u n i t of SLT-I is
a g a i n s t t h e major s y s t e m i c m a n i f e s t a t i o n of RDEC-H19A d i s e a s e , w i t h o u t p r e v e n t i n g c o l o n i z a t i o n o r a d h e r e n c e . The r e s u l t s
s u g g e s t t h a t a s i m i l a r s t r a t e g y of immunization w i t h SLT B
s u b u n i t may l i m i t t h e t o x i n - r e l a t e d m a n i f e s t a t i o n s of e n t e r ?
hemorrhagic Escherichia c o l i i n f e c t i o n .
I n s t i t u t e of M i c r o b i o l o g y ,
S c h o o l , Cracow, P o l a n d
University
Medical
A r e v i e w of t h e l i t e r a t u r e on t h e a c t u a l e p i d e m i o l o g i c a l s i t u a t i o n r e l a t e d t o nosocomial
i n f e c t i o n s c a u s e d b y S t a p h y l o c c o c u s a u r e u s and
t h e i m p o r t a n c e of t h e n a s a l c a r r i a g e of t h i s
pathogen i s g i v e n together w i t h selected d a t a
d e r i v e d f r o m our own s t u d i e s on p o s s i b i l i t i e s t o
i n h i b i t t h e c a r r i a g e w i t h t h e m e a n s of t o p i c a l
a n t i - s t a p h y l o c o c c a l agents.
NO DEGRADATION OF INTESTINAL MUCUS GLYCO
PROTEINS BY LdCTOBACILLUS CASH STRAIN GG.
Jonannoke GH RLseler-van EmbdRn and Leo MC wan
L ashout
Dept. of lmmuiio ogy E r s s m d Un.versity Rotterdam.
P.O. Box 1738, 3000 DR ROTTERDAM, Tne Netherlands
Lactic acid bacteria have been used to re-establish the normal human
intestinal microflora for the last decades. Recently therapeutic significance to mucosal diseases such as ulcerative colitis has become under
investigation. During our studies on bacteria-mucosa interactions in ileal
reservoirs we developed the conception that pouchitis might be prevented by lactic acid bacteria. Pouchitis is a complication of ileal reservoir
surgey and is confined to patients who underwent protocolectomy for
ulcerative colitis. The antibiotic rnetronidazole has proved to be very
effective in the treatment of pouchitis. However. when this therapy is
interrupted more than half of the patients relapse and the incidence of
relapse is rising with longer duration of follow-up. A prerequisite for
successful treatment of pouchitis with lactobacilli is that the strains
leave the protective intsstinal mucusglycoproteins intact. This was
tested for Lactobacillus casei strain GG. The strain was cultured in vitro
on hog gastric mucine and on human intestinal glycoproteins. Furthermore germ-free rats were mono-associated with Lactobacillus GG. The
intestinal mucus was colonized, but bacteria were not found in the deep
crypts. Glycoproteins wwre isolated from rat distal ileum, caecum and
colon. Mucus degradation was established by assaying carbohydrates
ihexosamlnes. hexoses. pentosed. proteins and bloodgroup antigenicity.
Degradation of mucusglycoproteins was observed neither in vitro nor in
vivo. It is concluded that Lactobacillus GO does not damage the intestinal mucus layer and thus far is safe to usw for therapy.
xvi
ABSTRACTS
IS TRANSLOCATION PREVENTABLE? LESSONS PROM AN ANIMAL HODEL
OF SMALL BOWEL TRANSPLANTATION (SET).
,-
Biffi R, Matinato C, Nicastro 0 , Andreoni B.
Meso-substitutedcationic porphyrins as
efficient photosensitizersof Grampositive and Gram-negatlve bacteria.
University of Milan Medical School, nilan, Italy.
Compared to transplantation of other organs, SBT is
complicated by a higher rate of sepsis secondary to
bacterial translocation. Aim of our study was to evaluate
the efficacy of a selective intestinal dscontamination
regimen ( s m ) (colistin 1,500,000 U, tobramycin 100 mg,
vancomycin 1 g and nystatin 500.000 U daily) or of
parenteral cefazolin (CEZ) ( 2 g im daily) in preventing
intestinal bacterial overgrowth and translocation using an
animal model of SBT. 3 1 pig6 weighing 3 0 2 2 kg were
assigned to 6 groups, receiving respectively the following
procedures and drugs: A (n.6). which acted an control, no
operation or antibiotics i B (n.7) orthotopic homologous
SBT, cyclosporin A 25 mg/kg sc (CsA) and CEZ; C (n.6) SBT,
CsA and SXD ; D Ln.7) SBT. CsA, SID and liquid f o m u l a defined enteral feeding ( 3 5 kcal/kg body weight daily), E
(n.5) autologous SBT and STD. Upon sacrifice at day 2 9 .
proximal, middle and distal graft segments were removed and
the counts of l w i n a l aerobic and anaerobic bacteria
determined. Adjacent mesenteric lymph nodes and fragments
of spleen, liver and lungs were also excised, homogenized
and cultured. Random amplified polymorphic DNA analysis was
used to assess the identity of strains isolated from the
gut
and
parenchimal
organs.
Compared
to
controls,
eignificant increases in the counts of total aerobes and
anaerobes were found in the proximal jejunum of all groups.
Translocation
also occurred
in all grafted
animals
regardless of aurgical procedure, immunosuppression or
antibiotic treatment, but not in controls. The addition of
enteral feeding as a supportive measure did not influence
the microbiological paraeters of translocation, but
significantly lowered the rate of pneumonia (p <0.01).
We conclude that bacterial overgrowth and translocation
were largely dependent by surgical trauma and nutritianal
status of the animals and that prophylactic antibiotics
were not effective in preventing their occurrence.
Michele Merchat f-2, Giulio Berloloni 2, Paolo
Giacomoni I,Angeles Villanueva and Giulio Jori 4.
1 bbcntdre dn Rochenhes COrbi, P a h (France)
2 l m o dl M k r ~ h , Udi n
PaJbn.
~ psdwa (llaly)
3 Dsprtamsntade Eblogia. U n M Alltowru do Wnd. M s d l
(Spin)
4 DIpartimenlO dl B w m , Univerutddi padova, Padova (Ilaly)
Previous studies on the photosensitizationof bacterial
cells with different neutral or negatively charged
porphyrins and phthaiocyaninesdemonstratedthat while
Gram positive bacteria are efflciently photoinactlvated,
Gram-negative bacteria become photosensilive only
after modiflcation of the permeability of their outer
membrane.
The results described in this paper show that two mesasubstituted cationic porphyrins. namely tetra(4N-methylpyridy1)porphine
tetraiodide
and
tetre(4N,N,N,trimethylanilinlum)prphine , effluently
photosensitizethe inactivationof Gram (-) bacteria, such
as Vibrio anguillarum and Escherichia coli. A negatively
charged
mesa-substituted
porphyrin,
letra(4sulphonat0phenyl)porphlne
has
no
appreciable
photosensitizing activity toward Gram (-) bacteria,
although all three porphyrins exhibit a similar subcellular
distribution pattern being mainly localized in the
cytoplasmic membrane. Moreover, the three porphyrins
show similar efficiency In the photoinactivation of the
Gram (+) bacterium Ent8rococcusseriolicide.
ABSTRACTS
POPULATION SELECTIVE FIIIPLIFICFITION BY
F I N T I B I O T I C S . F . Baquero*, N.C. Negri, M.I.
Morosini, J.Blazquez. Department o f
Microbiology, Rambn y Cajal Hospital, Natl.
Inst. o f Health (INSFILLID), 28034 Madrid, Spain.
The
evolution
of
bacterial
antibiotic
resistance may depends on the consequences of
an antibiotic dependent concentration-specific
punctuated selection ( C S P S ) , resulting in the
amplification of particular resistant variant.
Being a wild population highly predominant on
a
variant
population, physical/mechanical
models predict that a precise concentration of
an inhibitor exert a punctuate selection of a
particular variant. Once this concentration is
surpassed the selection is not more exerced.
Based on two models using bacterial mixtures
of
predominant
"wild"
and
"variant"
(resistant)
populations
of Streptococcus
pneumoniae
(showing
susceptibility, intermediate
or
high-level
resistance
to
penicillin) and E s c h e r i r h i a coli, harbouring
TEM-1 O-lactamase,
or
TEN-12
and TEN-10
in
vitro
by directed
variants
obtained
mutagenesis, and producing different levels o f
resistance to cefotaxime, the selection of
particular bacterial variant populations a t
particular
antibiotic
concentrations
is
showed.
Pharmacokinetic
curves
could be
converted
into
selective
curves.
The
ecological
implications of this result may be
of interest. Punctuated selective phenomena
have been proposed to explain species sorting
in macroevolution (Gould (Ir Eldredge, Nature
366:223, 1993).
but concentration -specific
selection
within
gradients
in bacterial
evolution -for instance towards resistancewas not previously explored.
EMERGING PATHOGENS AND BACTERIAL RESISTANCE PATTERNS
I N LOWER RESPIRATORY TRACT INFECTIONS.
G . G i a l d r o n i Grassi
C h a i r of Chemotnerapy, U n i v e r s i t y o f P a v i a , I t a i y
The w i d e s p r e a d u s e o f a n t i b i o t i c s and t h e p r e s e n c e o f
larger p o p u l a t i o n o f "new" p a t i e n t s , p a r t i c u l a r l y t h o s e
with more or l e s s pronounced d e f e c t s i n t h e d e f e n s e s y s t e m .
h a s l e d t o profound c h a n g e s i n t h e e t i o l o g y o f lower
r e s p i r a t o r y tract i n f e c t i o n s ( L R T I ) .
Among emerging p a t h o g e n s , p r e v i o u s l y unknown a g e n t s have
been i d e n t i f i e d , s u c h a s L e g i o n e l l a pneumophila and
a
new
pneumonis,
while
known a g e n t s have assumed
epideniiological impact ( M . c a t a r r h a l i s . Acinetobacter s p p . ,
M y c o b a c t e r i a o t h e r t h a n t u b e r c u l o s i s ( M O T T ) , P. c a r i n i i ,
Rhodococcus
equi,
etc.).
In
addition
resistance
to
a n t i b i o t i c s h a s been s p r e a d i n g among common r e s p i r a t o r y
path0gens.H. i n f l u e n z a ? , K . pneumoniae, E n t e r o b a c t e r i a c e a e ,
S t a p h . a u r e u s , p a r t i c u l a r l y if h o s p i t a l - a c q u i r e d , produce
beta-lactamases
and
are
often
multiresistant.
M e t h i c i l l i n - r e s i s t a n t Staph. a u r e u s , even i f n o t a f r e q u e n t
r e s p i r a t o r y pathogen, causes d i f f i c u l t - t o t r e a t i n f e c t i o n s .
due t o i t s s c a r c e s u s c e p t i b i l i t y t o a n t i b i o t i c s . &
pneumoniae is s t i l l t h e most f r e q u e n t c a u s e of pneumonia.
I t s r e s i s t a n c e t o p e n i c i l l i n , due t o a l t e r e d forms of PEPS
( P e n i c i l l i n Binding P r o t e i n s ) w i t h decreased a f f i n i t y f o r
b e t a - l a c t a m a n t i b i o t i c s , i s becoming a s e r i o u s worldwide
problem. H i g h l y r e s i s t a n t i s o l a t e s i n f a c t are p r a c t i c a l l y
a n t i b i o t i c s . e x c e p t vancomycin and
resistant t o all
teicoplanin.
xvii
GENETICS AND BIOLOGICAL ASPECTS OF 5-NITROIMIDAZOLE R E S I STANCE I N BACTEROIDES
S c c p h a n i r Z r l n h , A b d d i l d r i f Urpgcud, J e a n - p h i l i p p e C a r l i e r
and G i l l e s R e y s s e t
1nstlCuL P d b t e u r , P a r i s , F r a n c e
S i n c e 1989, s p e c i f i c c r a n s f e r a b l r g e n e t i c d e t e r m i n a n t s ,
c o n f e r r i n g low l e v e l of r e s i s t a n c e t o 5 - n i t r o i m i d a z o l e
( N i r ) h a v e been i d e n t i f i e d i n s t r a i n s of B d c t r r o i d e s
i s o l a t e d i n v a r i o u s h o s p i c a l s i n F r a n c e . The c o r r e s p o n d i n g
r e s i s c a n c e d e c e r m i n a n t s were shown t o b e l o c a c e d e i t h e r o n
p l a s m i d s O K on t h e chromosome. Amongst t h e N I T s t r a i n s ,
t h r e e m o b i l i z a b l e p l a s m i d s of small s i z e , pIP417 (7.7 k b ) ,
pIP419 (IOkb) and pIP421 ( 7 . 3 k b ) , have b e e n shown L O c a r r y
a N i r d e t e r m i n a n c . Although d i f f e r r n c by t h e i r r e s t r i c t i o n
maps, t h e p l a s m i d s s h a r e t h e same g e n e t i c o r g a n i z a t i o n ,
w i t h t h r e e main r e g i o n s i n v o l v e d i n (i) p l a s m i d r e p l i c a t i o n
(XI, (ii) p l a s m i d m o b i l i z a t i o n (mob) and (iii) r e s i s t a n c e
r e g i o n s of p l a s m i d pIP417 have
phenotype. The 3 and
b e e n c h a r a c t e r i z e d by g e n e c l o n i n g and s e q u e n c i n g , and t h e
r e l a c i o n s h i p amongst t h e c h r r e p l a s m i d s was made by Sout h e r n b l o c s a n a l y s i s using s p e c i f i c i o t r a g e n i c p r o b e s of
t h e d i f f e r e n t r e g i o n s . The t h r e e p l a s m i d i c N i l drcerminanc:
have a l s o been sequenced, a s w e l l a s che chromosomal Ni
d s r e r m i n a n t of B. f r a g i l i s s c r a i n BF8. The c o r r e s p o n d i n g
p e n e s were named L . 4 t o D_. The f o u r n u c l e o t i d i c s e q u e n c e s
s h a r e o n l y 49% of i d e n c i c y . The @ g e n e s d i r e c t e d t h e
s y n t h e s i s of p o l y p e p c i d e s of 18 L O 20 kDa s h a r i n g 57.6% of
s t r i c t i d e n t l r y b u t 78.5% of S i m i l a K i t y . A s a g e n e r a l r u l e ,
e a c h & gene is p r e c e d e d by one i n s e r t i o n s e q u e n c e e l e mant(1S) which p r o v i d e s t h e t r a n s c r i p t i o n S t d C t s l g n a l . A C
ledst c h r e a d i f f e r e n t I S b s l o n g i n g to two d i f f e r e n t famil i e s a r e a b l e t o a c t i v a t e t h e p& gene. Moreover, one of
chem, I S 1168, was shown by t h e C o l l a t z group t o a c t i v a t e a
carbdpenemase gene ( c f l A ) also p r e s e n r i n s t r a i n BF8.
On che o c h e r hand, b i o c h e m i c a l e x p e r i m e n t s performed e i t h e r
with " r e s t i n g c e l l s " OT c r u d e e x t r a c t s of r e s i s t a n t vs.
sensitive s t r d i n s , l e d U S L O h y p o t h e c i z e t h a c t h e N i m
p r o t e i n s a r e involved i n enzymatic i n a c t i v a t i o n o f t h e
5-nitroimidazole molecule.
xviii
APPLICATION OF IN VITRO AND I N VIVO TO THE STUDY
MICROFLOFA
Andrew B. Onderdonk
ABsTRAtTs
OF
HUMAN
m a n n i n g L a b o r a t o r y . Department of P a t h o l o g y , Brigham and
Women's H o s p i t a l , Harvard Medical School. Boston, Massachus e t t s , U.S.A.
P r e v i o u s s t u d i e s in t h i s l a b o r a t o r y have been d i r e c t e d a t
t h e development of methods f o r modeling human v a g i n a l
m i c r o f l o r a . Using a d a t a b a s e c o n s t r u c t e d from q u a n t i t a t i v e
and q u a l i t a t i v e o b s e r v a t i o n s of v a g i n a l m i c r o f l a r a . i t h a s
been shown t h a t a p r e d i c t i v e m a t h e m a t i c a l model can b e
a p p l i e d . It h a s f u r t h e r been s h o r n t h a t a s i m p l i f i e d
system based on c o n t i n u o u s c u l t u r e c a n s i m u l a t e many
of t h e a a p e c t s of t h e v a g i n a l m i c r o f l o r a .
C u r r e n t r e s e a r c h i s f o c u s e d on a p p l y i n g t h e s e methods t o
t h e s t u d y of p o t e n t i a l l y abnormal m i c r o f l o r a . i n c l u d i n g
v a g i n a l i n f e c t i o n s and inflammatory bowel d i s e a s e . The
r e s u l t s of these s t u d i e s w i l l be discussed.
INVESTIGATION OF FACTORS WHICH MAY HAVE AN
INFLUENCE ON THE VAGINAL ECOSYSTEM
Elisabeth Nagy
Department of Clinical Microbiology of Albert Szent-GyOrgyi
Medical University, Szeged, Hungary
Vagina harbours a complex microflora the composition of which is
dynamic and is influenced by several factors, such as glycogen
content of epithelial cells in different ages, hormone status. sexual
activity, pregnancy, birth control methods etc. Lactobacillus
dominance in the normal vaginal flora present in premenopausal
women seems to be an important factor in the maintenance of the
ecological balance of the vagina. During bacterial vaginosis (BV)
decrease of the lactobacilli and overgrowth of mixed anaerobic
bacteria can bee seen. Different factors may influence this flora
change. Antibiosis (growth inhibition) between members of the
indigenous flora of the vagina and those characteristically present
in BV was investigated Both lactobacilli producing or not
prcducing H 2 Q effectively inhibited the growth of
Peptostreplococcusand Mobiluncus swains in an in vitro test. This
inhibition was influenced by the iron content of the medium, but
changes in rhe pH between 5.0 and 6.5 of the medium did not have
any effect. Fibronectin binding of lactobacilli isolated from women
with or wirhout BV as well as different anaerobic strains
characteristic for BV w a ~tested using labelled fibronectin and its
29 kDa domain. Only lactobacilli bind to fibronectin. The binding
proved to be specific and time dependent. It was more expressed at
pH 4.0 than at pH 7.0. Bacteriocin production and lysogeny of
lactobacilli isolated from women with or without BV was also
tested. No differences in the species distribution or antibiotic
resistance was observed between lactobacilli with different origin.
Unusual morphological structure among lactobacilli isolated form
BV was seen.
YEAST KILLER TOXIN MIMICRY OF HUMAN NATURAL SECRETORY ANTIBODIES
P o l o n e l l i L., De B e r n a r d i s F.", C o n t i S., G y l o n i M., C a n t e l
li C., F i s i c a r o P., M a g l i a n i W., Cassone A.
I n s t i t u t e of Microbiology. U n i v e r s i t y of Parma, Parma, I t a l y .
* I s t i t u t o S u p e r i o r e d i S a n i t B , Roma.
Based o n t h e network t h e o r y , an i d i o t y p e v a c c i n e c o n s i t u t e d
by a monoclonal a n t i b o d y n e u t r a l i z i n g , i n v i t r o , t h e c a n d i d 5
c i d a l a c t i v i t y of a y e a s t k i l l e r t o x i n from P i c h i a anomala
was used t o immunize a g a i n s t c a n d i d o s i s i n a r a t v a g i n i t i s
model. I d i o t y p i c v a c c i n a t i o n e l i c i t e d in t h e rat v a g i n a p r o
t e c t i v e s e c r e t o r y y e a s t k i l l e r t o x i n - l i k e a n t i i d i o t y p i c ant i b o d i e s t h a t were a b l e t o k i l l , i n v i t r o , t h e c h a l l e n g i n g
y e a s t c e l l s and t o p a s s i v e l y t r a n s f e r i m u n o p r o t e c t i o n t o
nonimmunized a n i m a l s . Candida a l b i c a n s c e l l s b e a r i n g i d i o t y
pe-like c e l l w a l l recepcors s p e c i f i c f o r the yeast k i l l e r
t o x i n a c t e d , in e x p e r i m e n t a l i n f e c t i o n s , a s t h e i d i o t y p e
v a c c i n e in e l i c i t i n g o r r e c a l l i n g y e a s t k i l l e r t o x i n - l i k e
a n t i b o d i e s . C a n d i d a c i d a l a n t i b o d i e s were a l s o c o n s i s t e n t l y
found in t h e v a g i n a l f l u i d from v a g i n i t i s p a t i e n t s who were
i n f e c t e d by C. a l b i c a n s b u t had n e v e r been exposed t o t h e
i d i o t y p e v a c c i n e . Thus, t h e h o s t ' s s e c r e t o r y
immune r e sponse may, i n t h e c o u r s e of n a t u r a l i n f e c t i o n s ,
exploit
s p e c i f i c r e c e p t o r s of p a t h o g e n i c y e a s t s t o mimick candidacf
d a l a n t i b o d i e s r e p r e s e n t i n g t h e i n t e r n a l image of
killer
t o x i n from o t h e r microorganisms s u g g e s t i n g a new r o l e f o r
i d i o t i p y i n t h e c o n t r o l of f u n g a l i n f e c t i o n s .
W D K J A ALBKXNS IN AN IN VITRO MODEL OF THE VAGINAL
ECOSYSTEM.
Robin A. ROSS, Maw L. oelaney, and Andrew E. onderdonk.
The Channlng Laboratory, BWH& HMS, Boston, MA, USA.
The effect of challenging an in vitro model of the vaglnal
ecosystem with C. ablcans (Calwas studied. in vltro studles were
conducted using a growth system model simulating t h e healthy
vaginal ecosystem. The status of the model, or t h e probabillW that
It was either in a normal or an abnormal state, was evaluated uslng
a generalized estimatlon equatlon formulated from In vlvo human
data obtained from healthy subjects. Using a continuous culture
growth system, a chemlcallv defined medlum, and a dilution rate of
0.051-0.055/h, a stable mixed culture of Lactobaclllus acldophllus
(La),StJphylOCOCCUSepldermldls (Se),Prevotella blvia (Pbl, and group
D Streptococcus SD. (GDS) was tested at pH 4.8-5.0. By 24 h after Ca
challenge, the concentration ICFUlmU of Pb had decreased >I
to 1.8 logloand ca had Increased 0.5 log,, from a challenge
concentration of 2.1 log,,. All other mlcroflora remalned
unchanged. The probabllltv of abnormaiiW (PA) for t h e model,
whlch began at a pre-challenge value In t h e normal range (<O.I.
reached a value slightly above normal (0.131. By 5 days after
challenge. La was a t a concentration of 5.1
(a decrease of 0.8
log,. units,, PD was at a concentration of 0.04 iog,, and the
concentrattons of CDS and se were similar to their values a t ca
challenge. The PA value for the model had increased to an
abnormal value of 0.17. Durlng the next 7 days, se began to
decrease In concentratlon from 3.1 to 2.0 log,o,Pb was ellmlnated
from t h e model, and the concentrations of CDS and La remained
stable. The PA value over this same perlod contlnued to Increase
and at experiment termination was 0.36, a clearly abnormal value.
We conclude that lnfectlon of the vaglnal ecosystem wlth Ca hgs a
deleterlous effect on members of the mlcroflora, affectlng Pb and
se to a greater degree than La or CDS. Challenge wlth ca had an
lmmedlate effect on the model causlng a shift from a normal to
abnormal state. We feel this In vitro model will be vew useful in
future studles of lnfectlon of the vaglnal ecosystem.
xix
ABSTRACTS
Uses of molecular typing for fecal bacteria.
Antoine Andremont, Service de Microbiologie,
Institut Gustave-Roussy, 94800 Villejuif.
Numerous molecular typing techniques have been
developped
to
caracterize
and
compare
microorganisms. They are based either on DNA
restriction fragment analysis associated or not
with Southern hybridization, or on amplication o f
various DNA targets. They are very discriminant
and easy to perform and compete favorably with the
t r a d i t i o n a l t y p i n g t e c h n i q u e s (serotyping,
biotyping o r lysotyping), as well as with
techniques based on enzymatic polymorphisms.
In the field of microbial ecology and disease we
a p p l i e d t h e m t h r e e instances. F i r s t , w e
investigated t h e epidemiological relationship
between strains of C l o s t r i d i u m difficile which
colonized the intestinal tract of subjects taking
oral beta-lactam antimicrobial agents. We have
used both techniques based of analysis of DNA
fragments after restriction with rare cutting
endonucleases and techniques based on DNA
amplification with randomly choosen primers.
Second, we h a v e u s e t h e m t o study t h e
physiopathology of intestinal colonization and
infection caused by vancomycin-resistant strains
of Enterococcus faecium in hospitalized patients
with cancer. We showed that cases of such overt
infections originated often in the intestinal
tract and that dissemination o f vancomycin
resistance genes was caused either by strains,
plasmids, o r transposons mobilisation.
Third, we investigated wether translocation of
i n t e s t i n a l E n t e r o b a c t e r i a c e a e was a major
mechanism in the pathophysiology of gram-negative
bacteremia in malnourished children. We showed
that translocation could occur but was not
frequent and was unrelated to the severity of
malnutrition.
I t i s e s t i m a t e d t h a t half ofthemicro-organismspresentin dent a l p l a q u e a r e u n c u l t u r a b l e . T h i s i s a m a j o r o b s t a c l e t o t h e und e r s t a n d i n g of t h e a e t i o l o g y of o r a l i n f e c t i o n . 165 r i b o s o m a l
RNA(I6sRNA) s e q u e n c e i n f o r m a t i o n h a s b e e n o f enormous v a l u e in
the phylogenetic a n a l y s i s o f b o t h c u l t u r a b l e a n d n o n s u l t u r a b l e
b a c t e r i a . I n t h i s study16SrRNAgeneswere r e t r i e v e d d i r e c t l y
f r o m p l a q u e s a m p l e s b y t h e polymerase c h a i n r e a c t i o n
(PCR),
u s i n g p r i m e r s u n i v e r s a l f o r t h e e u b a c t e r i a kingdom.PCR p r o d u c t s
E. c o l i
were d i r e c t i o n a l l y c l o n e d i n t o v e c t o r pUCBMLIwith
JM107 s e r v i n g a s h o s t . Recombinant p l a s m i d s were s e l e c t e d
on
t h e b a s i s o f a m p i c i l l i n r e s i s t a n c e and alpha-complementaion
of the beta-galactosidase gene. I n s e r t s of t h e a p p r o p r i a t e s i z e
( ~ 1 5 0 0 b p were
)
sequenced u s i n g automated methods and f l u o r e s c e n t s e q u e n c i n g p r i m e r s . Seven c l o n e s were sequenced and corn
pared t o 16SrRNA d a t a b a s e s . R e t r i e v e d s e q u e n c e s show high lev e l s o f h o m o l o g y b o t h t o w e l l known o r a l b a c t e r i a e . g . S t r e p t o
c a c c u s s a n g u i s a n d t o b a c t e r i a a b o u t which l i t t l e i s known-=piouigeniummodestum. O t h e r r e t r i e v e d s e q u e n c e s s h o w o n l y low
t o m o d e r a t e l e v e l s of homologytomembers of theLowG+C d i v i s i o n of Gram-positive e u b a c t e r i a . S p e c i f i c o l i g o n u c l e o t i d e s
have been d e s i g n e d on t h e b a s i s o f t h e s e q u e n c e s a n d a r e b e i n g
u s e d t o s c r e e n p l a q u e samples f r o m b o t h h e a l t h y and d i s e a s e d
m o u t h s t o a s s e s t h e s i g n i f i c a n c e of t h e r e t r i e v e d sequences
which may r e p r e s e n t p r e v i o u s l y u n r e c o g n i s e d , p o s s i b l y u n c u l t u r e d micro-organisms. I n c o n c l u s i o n , sequence a n a l y s i s of
1 6 s r R N A g e n e s r e t r i e v e d from d e n t a l p l a q u e by PCR h a s enab l e d t h e m i c r o b i a l c o m p o s i t i o n o f p l a q u e t o be s t u d i e d
without t h e biases of t r a d i t i o n a l c u l t u r a l techniques.
DETECTION OF VIRULENCE FACTORS I N CLINICAL AND ENVIRONMENTAL
VIBRO CHOLERAE AND ESCHERICHIA COLI USING PCR.
P. C a p p u c c i n e l l i * . P. R a p p e l l i * , G. Maddau*, A. Santona*, S.
Mastrandrea*, R. Calundungo*, M. Colombo**.
I s t i t u t o d i M i c r o b i o l o g i a , U n i v e r s i t i d i S a s s a r i . **Dlpt. d i
Biologia C e l l u l a r e e d e l l o Svlluppo, UniversitP"La Sapienza"
Roma
Italy.
-
V i r u l e n c e f a c t o r s i n V . c h o l e r a e and E. c o l i a r e n o t u b i q u i t a
r i o u s and i n some c a s e s can be l o s t O K a c q u i r e d u r i r e g a r d i n g
t h e i r d i f f e r e n t s e r o t y p e s . T h e r e f o r e i t is p o s s i b l e t o i s o l a
t e n o n - t o x i g e n i c s t r a i n s of p o t e n t i a l p a t h o g e n i c
serotypes
b o t h in p a t i e n t s and i n t h e environment. To d e f i n e t h e path?
g e n i c i t y of i s o l a t e s we have a p p l i e d PCR t o d i r e c t l y d e t e c t
t o x i g e n i c d e t e r m i n a n t s i n E . c o l i ETEC ( s t I and It l a . 1t
I b ) , EPEC (bfpA and e a e ) , E I E C ( i n v ) , EHEC (sltl and s l t 2 )
and V. c h o l e r a e (ctxA, a c e , Z o t ) . A t o t a l of 174 E. c u l i and
76 V i b r i o spp. from c l i n i c a l and e n v i r o n m e n t a l s a m p l e s is012
t e d i n Luanda (Angola) were t e s t e d f o r v i r u l e r c e f a c t o r s by
PCR. We u t i l i z e d p u b l i s h e d p r i m e r s f o r s t , s l t l a n d c t x A ; new
p r i m e r s were d e s i g n e d f o r t h e o t h e r g e n e s (as shown by u s i n g
s t a n d a r d s t r a i n s ) , a l l o w i n g t h e s t u d y of t h e d i s t r i b u t i o n o f
t h e v i r u l e n c e f a c t o r s i n t h e i s o l a t e s . Among clinical-,
ETEC s t r a i n s were i d e n t i f i e d s i n c e s t and I t g e n e s were amp l i f i e d ; o t h e r s t r a i n s r e s u l t e d i n v p o s i t i v e and were d e f i ned as b e l o n g i n g t o E I E C group. A l l c l i n i c a l V. c h o l e r a e 01
i s o l a t e s gave t h e e x p e c t e d a m p l i c o n s of t h e 3 t o x i n g e n e s ;
2 e n v i r o n m e n t a l s t r i a n s r e s u l t e d n e g a t i v e . S i n c e b o t h E.coli
and V i b r l o s p p . a r e t y p i c a l e n v i r o n m e n t a l b a c t e r i a found i n
for
w a t e r r e i i i l - v o i r s , PCR d e t e c t i o n may be e a s i l y a p p l i e d
t h e i d e n t i f i c a t i o n o f p a t h o g e n i c v a r i a n t s i n p c l l u t i o n cont r o l procedures.
RAPD FINGERPRINT MAY BE A POWERFUL TOOL TO INVESTIGATE
OUTBREAKS OF NOSOCOMIAL INFECTIONS.
E . C a r r e t t o , S. T e l e c c o , D. B a r b a r i n i , P . Marone.
Dept.of I n f e c t i o u s D i s e a s e s , IRCCS P o l . " S . Matteo", UniveF
s i t y of P a v i a
The a p p l i c a t i o n o f n u c l e i c a c i d a n a l y s i s a p p e a r s v e r y u s e f u l
t o c h a r a c t e r i z e b a c t e r i a l s p e c i e s and s t r a i n s , f o r example
d u r i n g n o s o c o m i a l o u t b r e a k s caused by s t a p h y l o c o c c i , gener a l l y o c c u r r i n g a s "exploding" e p i d e m i c s . Random a m p l i f i e d
polymorphic DNA (RAPD) f i n g e r p r i n t i s based on t h e a b i l i t y
of a s i n g l e p r i m e r of a r b i t r a r y n u c l e o t i d e sequence t o g e n e
Kate, u n d e r a d e g u a r e c o n d i t i o n s , polymorphic a m p l i f i c a t i o n s
p r o d u c t s f o r any a u c a r y o t i c and p r o c a r y o t i c
genome. T h i s
t e c h n i q u e d i f f e r s from a s t a n d a r d PCR i n t h a t o n l y a s i n g l e
and
o l i g o n u c l e o t i d e primer a r b i t r a r i l y chosen i s employed
n o p r i o r knowledge of t h e genome t o a n a l y z e i s r e q u i r e d . A t
low a n n e a l i n g t e m p e r a t u r e , t h e p r i m e r a n n e a l s t o DNA a t d i i
f e r e n t s i t e s whose s e q u e n c e s a r e n o t e x a c t l y complementary
t o t h e p r i m e r sequence. A d i s c r e t DNA segment w i l l be amp l i f i e d when two primer a n n e a l i n g s i t e s a r e a r r a n g e d i n i n verted orientatian a t a distance suitable for amplification.
With t h i s method i d e n t i c a l genomes w i l l g i v e on g e l e l e c t r g
p h o r e s i s i d e n t i c a l p a t t e r n s of a m p l i f i c a t i o n p r o d u c t s w i t h
t h e same p r i m e r . I n o u r s t u d y we compared b i o t y p i n g ,
antibiograms, plasmid p r o f i l e s , chromosomal DNA r e s t r i c t i o n end o n u c l e a s e a n a l y s i s (REA) and RAPD f i n g e r p r i n t i n o r d e r t o
c h a r a c t e r i z e staphylococcal s t r a i n s i s o l a t e d i n an i n t e n s i ve c a r e u n i t of o u r h o s p i t a l . R e s u l t s were a n a l y z e d
using
t h e n u m e r i c a l taxonomy methods, and dendrograms
generated
by a s t a t i s t i c a l method were e l a b o r a t e d u s i n g a
computer
s o f t w a r e . B i o t y p i n g , a n t i b i o g r a m s and plasmid p r o f i l e s a r e
n o t s u f f i c i e n t l y d i s c r i m i n a t i n g t o type t h e d i f f e r e n t
p h y l o c o c c u s s p p . i s o l a t e s . RAPD and REA p a t t e r n s a r e s i m i l a r and t h e y were a b l e t o d i s c r i m i n a t e t h e d i f f e r e n t i s o l a t e s . S i n c e t h e RAPD t e c h n i q u e i s e a s i e r and f a s t e r t h a n REA,
i t a p p e a r s a v e r y u s e f u l t o o l i n e p i d e m i o l o g y of nosocomial
outbreaks.
e-
xx
DETECTION OF SALMONELLA SPP. BY NESTED
POLYMERASE CHAIN REACTION
-~
I. Maffezzoli, A. Galli, R. Caldarelli, E. Omodeo
Zorini, P. Ferrante.
DISTAM, M.A.A.E. Section and Inst. Med Mierobiol,
Milan University
Biology Lab IRCCS Fondazione Don C. Gnocehi,Milan.
Salmonella is a commonly occurring pathogen and is
detected almost exclusively by colture methods, that are
quite time-consuming; there is then a need for rapid,
specific and sensitive methods to detect Salmonella spp.,
both for infections diagnosis and to study any possible
source of infection, like food.
For this reason we have set up a nested Polymerase Chain
Reaction (n-PCR) assay amplifying the invA gene, whch
is essential for invasion of epithelial cells by Salmonella
and has proved to contain sequences unique to Salmonella
and therefore can be a suitable PCR target.
In order to evaluate the sensitivity of our n-PCR assay,
different kinds of food and whole blood samples were
spiked with serial dilution of salmonellas culture. Dilution
range was 103- 100 cells per ml of blood and 106- 100
CFU125 g of food sample. In all were tested 37 different
strains of Salmonella and 10 other bacteria.
We found that the n-PCR assay was able to amplify all the
Salmonella strains, while the other bacteria used as
control always gave negative results.
With regard to sensitivity, our dilution tests proved that nPCR can detect as few as 1 Salmonella celVml of blood
and lcelV25 g of food sample.
On the whole OUT results suggest that the n-PCR assay is
rapid (6 hours), sensitive and specific; its application may
open new prospects in studies on Salmonella diffusion
both in human pathology and in the environment.
ABSTRACT'S
xxi
ABSTRACTS
A NOVEL PARASITE ASSOCIATED WITH CHRONIC DIARRHEA
IN AN AIDS PATIENT7
Daniele Dionisio. S. Di Lollo*, P.G. Rogasi. M M e 4 G.Sterrantino,
S. carbonai, M Pozzi, F. Leaiicini
U.O. Malanic lnfenive .Prcsid~oOspedaliero Careggi. Firem
* Istihltodi Anatomia Patologica - Universita di Firenze
QBIECTTVE: To pnsent the submicroscopic morphology of an uncommon parasite
found in lhe duodenum of an AIDS patient.
_CASE HISTORY AND METHODS: A 32 years-old AIDS italian woman was
hospitalized in October 1991 in the Infectious Diseases D e p m e n t of Careggi
Hospital in Florence because of relapsing chronic watery W h e a substained by
Isospora belli. She was a drug abuser until four years before. No travels in tropical
countries were referred. She was hospitalized five months before baaux of fever,
weight loss and watay diarrhea with 10-20 discharges daily. Isospora belli was
diagnosed in her noOls and the therapy was performed with oral c o t r i m o m l e for
14 days (80+400 mg: 2 tablets b.i.d): the remission was obtained but continuous
early relapses implied m e n 1 cotrimoxazole or metronidazole or furazolidoiie
treatments. The samples of her stools collected during four months were negative for
occult b l m 4 bacterial or viral pathogens, Myoobacteria, parasires other than
Isospora belli and Clostridium dificile toxin. At the new admission in October 1991
the patient showed oral candidiasis and oral ulcerations c a d by HSV1, CMV
retinitis, recurrent urticdlial-like rash. She was cachectic, febrile and alert with mild
awominal pain W 4 + cells were 28/mm3.Upper endoscopy was performed
showing aesophagcal candidiasis. Some biopsies were taken from the third p n i o n of
duodenum and processed by light minoscopy (LM) and transmission electron
microscopy ITEM). For LM the specimens were embedded in paraBm and rmned
with hemal~xylin-easin, Giemsa, acid fast, periodic acid-ScW and Grccon
methenamine silver Stains. For E M the specimens were f k d in 2.5%
glutaraldeide, sodium cacodylate buffer, post-fixed with 1% osmium tetroxide,
stained with 2% uranyl acetate, dehydrated in graaed alcohol and propylene oxide
and embedded in Epon resin. The polymerisation was at 8 0 T . Semi-thin (0.5-1
mm) sections were cut and stained with toluidine blue stain, .Thin sections (40-60
nm) were slained with uranyl acelate and lead citrate and e m n e d with Plulips 4 10
LS electron microscope.
RESULTS: On LM only mild non specific phlogosis, oedema and telan@iectasiesof
duodenal muwsa were observed in p d i n sections. No additional details nor
parasites were demonstrated in sem-lhin plastic sections. On TEM. besides the
presence of Isospora belli developing stages, another parasite was revealed i n the
enterocytes. "his paraslte (Fig.) measured 2.8 x 2.3 p,it was at direct coutact wlth
the host cell cyloplasm and showed an oval shape and a central nucleous without
nuclear membraae. The microrganism had a single layer contour membrane. profiles
of endoplasmatic remJlum,a few nuthocondria, free ribsomes and a paranuclear
clustcr of dense core irregular structures viral inclusion-like. Other unwmmon
structures were also present mto the near enterat dirm contact with the host
cells cytoplasm. Neverlheless, we pointed out two additional parasitic morphologies
both into vacuoles: 1) a collected ring-like inclusion (2.1 x 1.6 pm) and 2) bodies of
the same dunensions shovmg "umbilical cords" between the parasite and the vacuole
outline. "disk-like" structures at a parasite end and, in one picture, a perhaps
incipient &vision by binary fission.
DISCUSSIm: To the best of our knowledge the observed piclures arc no1
compatible with any known human inuaenterocytic parasite. To an anempt of
identification we are actually in contact with dr. R. Weber (Infectious Diseases
DMsio4 Universitasspital, Zurich, Swiss), dr. G.S. Visvesvara (parasitic Diseases
Branch, CDC, Atlanta, Geargia, USA), dr. I. Paperne (Museum National d'fistoire
NaIurelle, Paris, France), dr. A. Aeschlimann (Institut de zoologic, Universilh de
Neuchatel. Swiss), dr. R P . Bendall (Department of Clinical Parasitology, London,
UK). dr. P.F.L. Eloreham (Queensland Institute of Medical Research, Bnsbane,
Australia). dr. J.P. Dubey (Laboratory of h n o t i c Disease, Beltsville, Maryland,
USA). Drs. R Wckr, G.S. Visvesvara, R P . Bendall and I. Paperne suggest that,
baause of irmfiiciat details, this parasite cannot be classfied at this time. However
t h 9 think that it is neither a microsporidium (because of the presence of
mithocondria) nor a uxcidium (because of the absence of characteristic apical
organelles such as the wnoid or the rhoptries). Dr. 1. Paperne alm suggests that it
should be a fungal organism. Moreover dr. RP. Bendall Ihmks the elemon dense
inclusions and adjacent circular structures in the above-said main picture do not
resemble stnrctures of other recognized inIracellular parasites. Drs. A. AeschlimaM,
P.F.L. Boreham and J.P. Dubey have yet sent us their comments
MYCOBACTERIUM OENAVENSE INFECTON IN AIDS QUANTITATIVE
SENSITIVTR TEST3 TO 14 AMlBlOnCS As PRELLMWARY OULDE TO THE
THERAPY
pvuslc W a i o . E ToMIP. M T S u n m . . M Mob. 0
haruymo. S
C&MI.
M
Inhibition o f bacterial translocation from
the gastrointestinal tract to the mesenteric lymph nodes by cyclophosphamide injection
S U Z U K I , T ~,. ITOH K Z . , H A G I W A R A . T ~ N A K A Y A M % H ~ ,
K A N E K O , T .4,
~ suzi~r,
H I . H O N J Y O ,~3 ' H I A O T A Y,
tfITSUOKA,.T .
3Meiji Milk Products, 'The University of4 Tokyo,
National Institute o f Animal Health, NiPPOn
Veterinary and Animal Science University, JAPAN
Bacterial translocation(BT1) is defined as
the passage of viable bacteria from the
gastrointestinal(G1) tract through the mucosal
e p i t h e l i u m to other s i t e s . We studied the
e f f e c t s of intraperitoneal injection with
cyclophosphamide, an immunosuppressive agent, on
the d e g r e e of bacterial translocation and
morphology of Peyer's patch(PP) using SPF mice
monoassociated with Escherichia
C25. I t has
been
reported(Berg,
R.O.,
1983)
that
immunosuppressive agents promoted BTL.
In our
studies, however, treatment with cyclophosphamide
inhibited E . coli C25 translocation from the
gastrointestinal tract to the mesenteric lymph
nodes(MLN). although the numbers of lymphoid
cells, especially B cells, i n P P , M L N and the
s p l e e n w e r e s i g n i f i c a n t l y reduced.
Four
injections o f cyclophosphamide at a dose o f 100
mg/Kg inhibited BTL more than one injection at a
dose of 200 mg/Kg.
Germfree m i c e , however,
treated with one dose of 2 0 0 mg/Kg were inhibited
BTL the same level as those with four times of
100 mg/Kg.
In c y c l o p h o s p h a m i d e - t r e a t e d mice, PP
was definitely smaller than that in control mice,
M-cells were similar in appearance of absorptive
epithelial c e l l s except f o r their Short
microvilli and immune cells among the
M-cells
disappeared. These results suggest that the
inhibition
of
BTL
in
mice
receiving
cyclophosphamide may be due to morphological and
physiological changes of epithelial cells in the
GI tract, especially M-cells, as a point of entry
o f invading bacteria, independent of the changes
in immunological function.
xxii
ABSTRACTS
MICROBIOLOGIC EVALUATION OF NOSOCOMIAL INFECTIONS
OF THE LOWER RESPIRATORY TRACT IN INTENSIVE CARE
UNITS.
A. Lippolis. V. Giannuni, M. Minoia, G. De Michele, G. Micelli', M
Coviello', M. Quaranta
Laboratorio Analisi IRCCS "S. de Bellis". Castellana Grotie Bari,
and 'Ospedale Oncologiu, IRCCS. Bari. Italy
Patients admitted to medical intensive care units are al risk of
hospital-acquired infections which result in higher risk of death.
longer hospitalization and increase in hospital costs. In parlicular
the lower respiratory trad is a common site of nosocomial infection
in the intensive care units because of the extrinsic risk factors
largely represented in those units (i-e. endotracheal tube, mechanical ventilation, nasogastric tube). In order to ascertain the
incidence of hospital-acquired infections of the lower respiratory
tract we evaluated 33 patients who developed bronchopulmonary
infection afier their admission to the intensive care unit. IRCCS "S.
de Bellis", Castellana Grotte, Earl, from January 1990 to April 1994.
Microbiologic studies were performed on the malerial obtained by
bronchoalveolar aspiration. Either microscopic examination or
cul!ure of the purulent respiratory secretions were positive in 25/33
cases (75%). 12/25 were monomicrobial cultures and 13/25
exhibited more than one bacterium. Microorganisms most frequently
(36%).
isolated are shown: Pseudomonas ae '
Staohvlococcus aureus (20%), Cendida a l b i m w k ) . These
results are in agreement with those shown by other authors and
further suggest that Pseudomonasa e r w is the most important
organism in producing dinically important nosominial infedions of
the lower respiratory trad.
BACTERW INFECTION IN PATIENTS WITH CHRONIC LIVER
DISEASE
A. Lippolis, V. Giannuui. M. Minoia, G. De Michele. G.
MiregliOtt8*, A Mosw', R. Del Prete', M. Quaranta
Laboratorio Analisi IRCCS "S. de Bellis", Castellana Grotte Bari,
and *Istituto di Microbiologia Medica, Universita di Bari. Bari, Italy
Bacteriuria very often occurs among patients with different types of
liver disease. Allhough the reasons for their susceptibility to
bacleriuria is not completely understood, disturbances of both
cellular and humoral immunity might play an important role.
In order lo evaluate the prevalence of baderiuria and the type(s) of
organism(s) involved we have examined 40(3 patients (182 males,
218 females) with chronic liver disease admitted to the IRCCS "S.
de Bellis' during the period January 1989 December 1992.
Ninetyeight patients (24.5%) had siQnificantbacteriuria. Of them 15
-re males (15.4%) and 83 were females (84.6%).
was
cultured in 70 (71.4%) of the specimens. Other organisms were
a oneurnoniae (4,08%), Citrobacter freuopu
.' (3.06%).
Eoteroc4aus (2.04%). Fnterobacter- a
(4.08%).
PPP.
omonas a e r uIn ' m, proteus mirabik,
ella -0
Aeromonas
and Wobade
r i' di
S!QLWS Klebslelle
Qi%&!Z
represented 9.22%. In
addition the following bacterial associations were observed:
terocom& with
(2.04%). m a c t e r f reundti' (1.02%).
(1.02%) and Enterobacter SOD with m , i e l l a spp
(2.04%).
Our results confirm that the infection of the urinary tract is more
common in female patients with chronic liver disease and thal
WeriChia coli is the most involved microorganism.
-
w
%A
-
MICROBIOLOGICLL ASPECTS OF DISSEMINATED
FUNGAL INFECTIONS
A.Samet , W.Fi$tkowska, A. Wnorowska
Dept . C l i n . B a c t e r i o l o g y , Medical School i n
G d d s k and Dept.Tropica1 Med., I n s t i t u t e of Marit i m e and T r o p i c a l Medicine i n Foland
The i n c i d e n c e of f u n g i a s i n f e c t i v e a g e n t s , e s p e c i a l
l y i n o p p o r t u n i s t i c s y s t e m i c i n f e c t i o n s , i s a growi
problem i n r e c e n t ye a rs . The most importa nt problem
i s growth o f f u n g a l i n f e c t i o n s i n immunocompromised
p a t i e n t s . I n our s t u d y we wanted t o answere two
q u e s t i o n s : are t h e r e a ny symptoms c h a r a c t e r i s t i c
f o r d i s s e m i n a t e d f u n g a l i n f e c t i o n s and a r e t h e r e
any m i c r o b i o l o g i c a l o r immunological t e s t s correlat e d t o c l i n i c a l signs.V!e ha ve a na l$s e d a l l c a s e s
of d i s s e m i n a t e d f u n g a l i n f e c t i o n s i n o u r h o s p i t a l
f o r r e c e n t 10 y e a r s . Ve emphasize r o l e of mycologic a l c u l t u r e s , e s p e c i a l l y from b l o o d , u r i n e and s i t e
of prima ry i n f e c t i o n , f o r e a r l y d i a g n o s i s of f u n g a l
i n f e c t i o n . We a l s o performed immunological t e s t s
f o r l e v e l of a n t i b o d i e s t o Candada and A s p e r g i l l u s
w ith mgthods o f h e m a g l u t i n a t i o n and g e l p r e c i p i t a t i o n . We f i n d t h a t u r i n e c u l t u r e s wene p o s i t i v e i n
about 70% of our p a t i e n t z , b u t o n l y 10% of them had
p o s i t i v e blood c u l t u r e s , Immunological t e s t s were
n o t c h a r a c t e r i s t i c . # a n y C a t i e n t s c olonis e d w i t h fun
g i had F o s i t i v e t e s t s f o r m t i b o d i e s and immunolog i c a l t e s t s were n e g a t i v e i n immunocompromised
p a t i e n t s w i t h fungemia i n many c a s e s . C l i n i c a l s i g n
were n o t s p e c i f i c . I n 40% p a t i e n t s we observed cons t a n t high body te mpe ra ture . O t h e r symptoms resemble d b a c t e r i a l s e p s i s . Ye c onc lude , t h a t t h e r e a r e
not any c h a r a c t e r i s t i c symptoms and l a b o r a t o r y t e s t
f o r d i a g n o s i s of dis s e mina te d f u n g a l i n f e c t i o n s .
M o r t a l i t y i n t h i s i n f e c t i o n s i s v e r y h i g h (70%) ir.
s p i t e o f a p p r o p r i a t e t h e r a p y , which i s c o n t r i b u t e d
t o u n d e r l y i n g d e f e c t s of t h e h o s t d e f e n s e system.
A MODEL OF MICROBIOLOGICAL MONITORING FOR PREVENTIONOFH.A.1.
IN A BURNS CENTRE
V. Torregrossa, L. Valentine, M. Masellis*, M. Sucameli*, P.
Cucchiara*.
Policlinic "P. Giaccone"-University of Palermo-Italy
*Dept.Plastic Surgery and Burn Therapy-Palermo-Italy
It's very useful to have a model of microbiological monitoring for prevention of H.A.I. i n a Burn Centre where there
is a very high risk of infections.
Very dangerous infections are often expected and sometimes
they can compromise the outcome of the best reconstructive
treatments and the course of the illness too.
A mierobiologica~surveillance programme for patients, medical and nursing staff, and the environment is used i n the Ci
vic Hospital's Burn Centre in Palermo to propose suitable
measures of prevention.
The survey of the microbiological degree of pollution i n the
environment . i s carried out either after measures to saniti
ze the environment or during the routine work i n the B u m
Centre.
The L A . use the system plate's sedimentation, exposing plates with different medium all over the B u m Centre.
Every month medical and nursing staff i s subjected to nasal
and oro-pharyngeal buffers to search f o r pathogenes.
The A.A. carry out smears cultures and haemocultures on patients i n case of suspicious sepsis. The A.H.
carried out
three hundred and nlnetysix microbiological controls during
the period from November 1993 to February 1 9 9 4 . After saniti
zation a considerable decline of microbiological environmental strength was observed but when patients and staff are
admitted to the Burn Centre, values of CFUs are sometimes in
compatible with an environment at high risk. The /,.A.
have
found some broods of Staphylococcus Aureus Methicillin resistent i n the Burn Centre during the period of observation.
The same microorganism was found i n eight patients and i n
four people of the staff. The survey of environment and
staff i s very important i n aprogramme for prevention of H.A.I.
The A.A. emphasize the importance of frequent controls on
staff that can be an important reservoir for H.A.I., considering the correctness of the measures to sanitize the environment.
xxiii
ABSTRACTS
EVALUATION OF THE AMPLICOR MYCOBACERlUM PCR TEST FOR
DIRECT DETECTION OF AflCOBACTERIUW TUBERCULOSISCOMPLEX
A MULllCENTE?STUDY Oh Ah71MnC7061ALS.SCi?Ila L iY Ah0 TYP NG OF GROUP A
SmEPTOCOCCl (GAS)FROM CASES OF PMRVNGOIOhS-nlS AhQ SCARLR FEVER.
F. Mandler. A.Guarino, D. Stangalini'
Cenlro Cardiologico .Fondazione "I. Monzino" IRCCS,Milan. Italy.
'Sczione di Biologia Molecolare, Lalmiatono "Reming".Abbiatcgrasso.Milan. l i a l i
A lotal OT I068 respiralon. specimens irom 540 patienls were lested for
dircci dctccuon of A<wobacrenum rubemdons comples by
AMPLICOR MTB PCR direct detection method. Resulls were
comparcd uilh those of conventional methods of fluorescence
microscopy and coILurcs. The specimens were esnminated after
homogenization with dithiotiireitol.
Rcsulls ofearly date. AMF'LICOR MTB and microscop!, are rcponed
Onc hundred and liilcen specimens were AMPLICOR MTB and
microscop! posili!'~. 68 specimens were AMPLICOR MTB positn c
and microscop!. negaln'e. and 9 specimens were AMPLlCOR MTB
incgali~eand microscopy positive. From 68 specimens AMFLICOR
MTB positire and iiiicroscopy negative 82% tiere from 7b patienis
treated continuously more than six months; Trom 9 specimens
AMPLICOR MTB negative and microscop!, posilire a suspccted
diagnosis ofMOTT inkclion was supposed in 8 cases.
Final interprclalion of the test should al\\'a?s be discussed I n
conjunction \\ith colture, but M L I C O R MTB PCR is an useiul
technique Tor Lhe rapid diagnosis of tuberculosis also bcfore obtaining
col~urcsdata.
RECOVERY IN SALIVA AND DENTAL EFFECTS OF
LACTOBACILLUS GG (ATCC 53103) IN HUMAN
SUBJECTS
H. Antila, S. Salrninen, J . Meurman
Department of Dentistry and Cariology, University of
Kuopio and Department of Biochemistry and Food
Chemistry, University of Turku, Finland.
Lactobacillus GG has been shown to exert inhibitory
activity against a variety of bacterial species. It has
also been shown to increase mucosal immunity in the
intestine. We studied the colonization of this sucrosenegative strain in human volunteers. The recovery of
L a c f o b a c i l l u s GG was studied in nine subjects after
consuming twice daily for seven days fermented milk
produced with the strain. The results indicated that
Lactobacillus GG was detected in the saliva samples of
all subjects one week after discontinuing the
treatment. In 8/9 subjects saliva samples were
positive after two weeks. It is concluded that
L a c t o b a c i l l u s GG colonizes the oral cavity at least
temporarily for periods up to two weeks in human
volunteers consuming Lactobacillus GG products. The
complex microecology in the oral cavity may benefit
from the colonization with this probiotic strain which
does not ferment sucrose or lactose. Further studies
are needed to clarify the dental effects and potential
long-term benefits.
Nve hundred and nlnetv-six strains of GAS malnlv from ohorvnaeolswabs
of Datients with phoryngc-tonsillitis and scarlet fever were isdoted from
hospnals In Faenza. Regglo Emilio, Ozierl and Rome.
The strains were tyoed by T oratein sera and assaved far sensltlvltv
against O-lactams and macrolldes by disk diffusion method onto Mueller
Hlnton agar plotes. odditloned with 5% sheep blood. The following
antlmicroblals were used: Azithromvcin (AZM). Ciorithromvcin (CLR).
Erythromycin (E): Josarnycin (JM). kiocamycin (MIO). Penicillin (P).
Amoxlclllm-clovulonlcacid ( A m 0 ond Cefoclor (CEO.
Three hundred and two strains (51.18%) were typed by a monovalent
serum: 35 (5.8%) were untypoble ond the remaining 259 (43.02%)
showed a pattern of reactions. Types T 4 and T 1 were the prevalent
serotypes and accounted for 119 (19.97%) and 95 (15.94%) of the
stralns respectively. Among 157 coses of scorlet fever serotype T 4 was
responslble for 42 cases (27.39%).T 1 far 26 cases (16.56%). A pattern of
reactlons with T 5, 11, 27 sero wos found In 19 cases (12.10%).
Onlv 337 strains were examined for antlmicroblal susceDtibllitv. 100% was
found to be susceptible to P and AmC and 99,06%'to CEC Among
mocrolldes 98.22% of the strains was susceptible to MIO. 97.62%to JM,
86.65% to CLR. 85.46% to E ond 84.27% to AZM About 73.17% of the
stralns resistant to E. CLR ond AZM wos T 4 and 14.63% T 6 No other
correlations between antlmicrobiol resistonce and T tyDinQ were found
In conciusion: Bloctams ore still very active against GAS; MI0 and JM
were shown ta be more active than E or other mocrolides compounds.
Whlie both T 4 and T 1 types are highly prevalent among the cases of
streptococcal diseose, In particular omong scarlet fever cases, T 4
seems to be asswioted with a higher resistance to mocrolldes.
BIFIDOBACTERIA AS COMPONENTS OF DENTAL CARIES HTCROFLORA
Crociani F.1.Chiarini C.2. Biavati
Scardovi '3.1
I Istituto Citomorfologia lonuale e Patologica-CNR-Bologna
2 Istituto Microbiologia Agraria. Universit& di Bologna
Bifidobacteria commonly inhabit the digestive tract of man
and animals; of the 29 listed species, Bifidobacteri-t
dAnLi2 was found frequently only in dental caries. In the
present study we isolated and characterized two new species
of bifidobacteria associated with thise habitat, we
determined their frequency and that of B.dentium in dental
caries and some characters that may be used for a rapid
identification of the three species. Several years ago from
a hundred samples of dental caries 240 strains assigned to
the species B.dentium were isolated. With the purpose to
determine the isolation frequency of this species we
examined again 19 samples of dental caries and from 14 we
isolated 70 strains of bifidobacteria. All the isolates were
grouped on the basis of their morphological and biochemical
characters and of electrophoretic protein profiles. Of the
three groups found it has been tested their DNA-DNA homology
with B.dentium and the other 28 species of the genus.
Twenty-two strains derived from 1 samples with an isolation
frequency of 50% were classified as B
d
e
n
s
. and like this
species shoed the unique ability among the bifidobacteria to
ferment locust beans and w a r gums. The other 48 strains all
fermenting dextran. unique character among bifidibacteria.
were referred t o two groups. The first named "Homology Group
I" (32 strains isolated from 8 samples with an isolation
frequency of 57%) showed extremely small coccoid cells. a
very low GC% value of 45 and the phenomenon of phase
variation with two types of opaque and transparent colonies
characterized respectively by long and coccoid cells. The
other group named "Homology Group 11" (16 strains isolated
from 5 samples and with a frequency value of 36%) exhibited
small rods occasionally arranged in a '2-shape with a GC%
value of 57. The fermentation characteristics of the three
species and the unusual morphology of "Homology Group I" may
h% useful for their rapid identification.
xxiv
ABSTRACTS
IN VITRO PROTEIN MEDIATED BIOFILM FORMATION ON THE
SURFACE OF ORpL TITANIUM IPPLANTS.
Resperini, G., Haldoni, M., Salvato A.! Calleqari, M.L.?
Morelli, L?
'Istituto di Discipline Odontostomatologiche, H. San
Raffaele, Universit.3 deyli Studi di Milano.
21stitnto di Microbioloqia, Facolt.3 di Agraria, UCSC,
Piacenza
MIcroSCop~C analysis of events occuring at the interfaces of
various types of implants are of relevant interest to
understand interactions occuring between biomaterials and
I
bacteria. Aim of the present study is to investigate the &
vitro formation of a bacterial biofilm on the surface of
oral titanium implants using as a model system of well
characterized strain of one Lactobacillus plantarum strain,
able to adhere to several surfaces by means of hydrophobic
interactions mediated by a secreted, wall associated 32 Kda
protein. Adhesion on titanium cover screw was assayed by
means of Scanning Electron Microscopy and the role of
hydrophobic reactions was established. A stable biofilm was
obtained when resting bacterial cells were overlayed on
these titanium surfaces togheter with the 32 Kda protein;
adhesion was greatly reduced when this protein was omitted
from the mixture and completely absent when the protein was
inactivated by the presence of specific polyclonal
antibodies. It was then possible to show that one
Lactobacillus strain is able to stick themselves on surfaces
of oral titanium implants and that this adhesion is due to
the hydrophobic reaction of the bacterial cell surface.
Moreover, cornpetion experiments have positely identified in
a 3 2 Kda secreted protein one of the molecules conferring
to the used strain its hydrophobicity: this in vitro model
system may could be used f o r a beter understanding of
biomaterials colonization mechanisms.
INFLUENCE OF ROXITROEMCIN ON U K I N DEGWUY\TION IN RATS.
E. Bezirtzoaloy', E. Norin*, J. Chen' and '1. Midtvedt'.
1
2
Oept of Microbiology, University of loannina,
Medical School, 45110 - Ioannina, Greece.
Lab of Medical Microbial Ecology, Karolinska
Institute, 5-171 77 Stockholm, Sweden.
One obvious feature of a germfree (GF) rat is the large
amount of mucin excreted in the f a y s . Presence of
faecal mucin can be defined as a germfree animal
characteristic" (GAC). In conventional (CONV) rats, no
mucin can be demonstrated, and this can be defined as a
"microflora associated characteristic" (MAC). By giving
antimicrobial drugs to CONV rats, various MAC/GAC
switches may be anticipated. In order to evaluate the
influence of the macrolide roxitromycin (Roussel) on one
MAC - the microbial degradation of mucin, 8 CONV rats of
the AGUS strain were given roxitromycin (125 mg/kg bdw)
intragastrically once/day for 5 days.Faeca1 samples were
collected every second hour during daytime 3 days prior
to, during treatment period and five times within three
weeks after the drug was given. Thereafter, in order to
ensure that the intestinal function was reestabllshed,
an, enema of 10% dilution of cecum contents from CONV
rats were given to all rats 18 days after antimicrobial
treatment was completed. Investigation of the mucin
pattern was performed by agar gel high voltage
electrophoresis on faecal supernatants (homogenated and
centrifuged). A specific band pattern was obtained after
colouring with toloudine blue (TB), typical staining
method for glucoproteins. Another band pattern was
obtained after staining with the protein stain Coomassie
Brilliant Blue (CBB). Concerning the TB parameter, the
MAC turned to GAC during the treatment period and all
rats had returned to MAC before the full flora enema
were given. With regard to the CBB, MAC turned t o GAC
during the treatment, but not all animals had returned
to MAC even three days after full flora enema was given;
3 of 8 animals retained their GAC within the study time.
To conclude, roxitromycin caused significant alterations
with regard t o the mucin parameter.
LACTIC ACID BACTERIA (LAB) AND INTESTINAL EICROFLORA
B. Bianchi
-
Salvadori
Centro Sperimentale del Latte, Milano, Italy
Health benefits of LAB (especially yoghurt LAB) and Bifidobacteria on the stabilization of the intestinal microflora
have been demonstrated by a number of studies carried out
by a group of researchers of Centro Sperimentale del Lattet
Yomo since 1966, managed by Dr. R. Vesely.
Experimental evidence i n man and animals have shown a protection from gastrointestinal disorders due to an increase
of autocrone LAB and Bifidobacteria and to stimulation of
the immune response; a cancer-preventing action in the production of gamma-interferon and in Peyer cells was also noted. The antagonistic role of LAB against Escherichia coli
in germ-free animals has been observed, as well a6
their
inhibiting action against the translocation of Escherichia
coli through the lymphatic system. There is also evidence
that LAB do not cause changes in bile salts and exert a hypocolesterolemic effect. LAB are proved to improve lactose
intolerant subjects and f o r this
digestion in lactose
reason particular LAB have been selected for their high prc
duction of beta-galactosidase.
-
-
DIFFERENTIAL EFFECTS OF SHORT CHAIN FA"Y
ACIDS
ON MUCOSAL STRUCTURE OF THE GASTROINTESTINAL
TRACT OF GNOTOBIOTIC M I C E
H.M.Cowley and R.H.HilI
Department of Anatomy and Human Biology, University of the
Witwatersrand,7 York Road, Johannesburg, 2193, South Africa.
The effect of bacterial metabolic-end products on the homeostasis of
the gastrointestinal epithelium of gnotobiotic mice was investigated.
Bacterial fermentationof undigested starch and fibrein the hindgut of
conventional animals, results in the production of short chain fatty
acids, the most predominant being acetate, butyrate and propionate
which are normally present at concentrationsin the mMolar range. In
this study, w e exposed the gastrointestinaltract of germfree mice to
salts of each of these fatty acids by intermittent intragastric and
intrarectal instillationover a 48 hour period. Four hours after the last
instillation, the mice were killed, weighed and the entire
gastrointestinal tract fixed for histological processing. Results
indicated that with the exception of the propionate-!xated mice, all
experimental mice were 2-3gheavier than their control countepaxts,
suggesting that acetate and butyrate could be used as a preferred
energy source. Histological,histochemicaland imageanalysisof crypt
parameters indicated that acetate, propionate and the mixture, all
affected gastrointestinal integrity and induced apical villus necrosis.
In contrast, the epithelium of butyrate treated mice was pristine and
duodenal villi were significantly longer. All treatments reduced the
number of goblet cells in the crypt/villusunit, suggesting that they
either acted as secretagogues or inhibited goblet cell differentiation.
In addition,much in the fatty acid treated mice was more sulphated
than in saline-instilledcontrol mice. From these results we conclude
that the large bowel may be intrinsically resistant to the damaging
effects of bacterially produced metabolites,while this protection may
not occur in the more proximal regionsof the intestine.Furthermore,
these findings suggest that bacterial metabolites may affect the
physiology and metabolism of gastrointestinalepithelial cells.
xxv
ABSTRACTS
MEMBRANE MOLECULES OF NORMAL HUMAN FIBROBLASTS:
INFLUENCE OF CYTOKINES AND OF HIV-I INFECTION
A.,C. Serra',
F. Tilocca*. P. Marongtu', S. Currelt', A. Modesti@, A Toniolop & F. Ameglio#. *In% Microbiology & Virology, Univ. of Sassari; @Chair of General Pathology, University of Chietl; glnst. of Medicine and Public Health, Pavia
Univ. at Varese; # San Galltcano Institute, Rome ITALY.
Obiective: Human normal fibroblasts from lung, breast and gingiva were analyzed with respeci to plasmamembrane molecules and sensthvity to HIV adsorpiion and
replication.
Methods: By the use of a panel of monoclonal antibodies, we evaluated the
expression and modulanon by HIV infechon, of molecules involved tn HIV adsorption,
such as CD4, galactosylcsrehroside (GalC) and CD26, and molecules lhat can wntnbute to HIV unspecific binding to cells, such as HLA Class I and I1 and several adhesion
molecules (VCAM, ICAM, E-SELECTIN, CD44, CD49d, etc.). HIV-1 adsorpiron and
replication (tntra- and extracellular virus) was evaluated as ability to form syncytia in
the C8166 T cell line. and as production of p24 antigen In ELISA.
Results: Normal fibroblasts bind HIV-1, and withm 2-3 weeks release COIISIsten1 amounts of progeny virus. Acute infection by HIV-1 causes a transient dcoavo
induction of DR expression and increases that of CD26, whereas the expression of
CD44 is decreased, as detecled by bindtng of specific monoclonal antibody to the suriacc of living cells. Stimulatlon of DR and CD26 and downregulation of CD44 are iindings observed also in othercells from solid tissues, such as epithelial Hcl,-T4 and
MEC-2 mammary cells, and in HUVE-CST endolhelial cells.
The effects observed in infected cells can be due etlher to HIV directly,
through a taf-mediated acttvanonofgene expression, or they may be mediated by some of the cytokines induced by virus infection. Among those produced by HIVinfected fibroblasts are mainly IL-6 (released In ng amounts), IL-la, IL-lp and TNFa
@g amounts). Production of IFN is around detection Iinufs. When exogenous cytokines
are given to umnfected fibroblasts, some of these cytokincs increase the expression of
some membrane molecules, among which are CD4 and CD26 and the adsorption of
HIV-1, as alieady observed in HeLa-T4 cells (Dolei er d.,Arch Virol 134:157, 1994).
When infected cells are mantalned in culture, this leads regularly to the establishment
of persistently infected fibroblasts, capable of cnntinuom virus producnon (Dole, el ni.,
AIDS Res Human Retroviruses, In press). Among the factors that stlmulaie HIV production by these cultures are exposure to IL-6 and TNFa, as 11 occurs on circulating
cells. Treatment with rffNgamma does reduce virus release, but this effeci is reversed
by treatment with exogenous taf. The fact that in normal fibroblasts HIV adsorption and
spread can be mediated by HIV-induced cytokines may be Elevant in the pathogenesis
of AIDS disease, as 11 may wnstitute a possible self enhancing model of HIV infection. Work supported by 1.S.S-Progctto AIDS 1994 n"9208.46
ANTIBACTERIAL SUBSTANCES IN GNOTOBIOTIC RAT
C B Z A : ROLES OF THE EXOCRINE PANCREAS A N D A
PEPTOSTREPTOCOCCUS STRAIN
F.RAMARE.
J.DABARD,
I.HAUTEFORT,
T.CORRING,
M.LADIRE, P.RAIBAUD
lnstitut National de la Recherche Agronomique, Jouy-enJosas, France
Diffusible antibacterial substances appeared within one day
in faeces of gnotobiotic rats harboring a human intestinal
strain o f Peptosfreptococcus I P.productus ). They were
active in vitro against several gram-positive bacteria
including potentially pathogenic Clostridium such as
Cperfringens, C.difficile, Cburyricurn. A sensitive strain of
Cperfringens type A was eliminated within one day from
the
intestine
of
rats
monoassociated
with
Peptostreptococcus strain.
These antibacterial substances disappeared when the bile
pancreatic
duct
of
rats
monoassociated
with
Peptostreptococcus strain was ligatured. The efficiency of
the ligature was demonstrated by a significant decrease in
all the enzymatic activities of the small intestine and a
concomitant disappearance of tryptic activity in caecum.
Likewise, they disappeared from faeces when the
monoassociated rats ingested a trypsin inhibitor, allowing
the sensitive strain of Cperfringens type A to became
established when inoculated in these rats.
Anaerobic cultures of the Pepotsrfeprococcus strain
exhibited an antibacterial activity against the same bacteria
only when media were supplemented with trypsin. The
presence of trypsin was necessary during the growth
exponential phase. Trypsin inhibitor blocked the appearance
of this activity.
Several other strains of Peptostreptococcus isolated from
faeces of child and adult were able to produce antibacterial
substance in the same conditions.
These findings demonstrate for the first time that very
potent antibacterial substances can be produced through B
mechnism Involving both intestinal bacteria and exocrine
pancreatic secretions.
INTEMCTIONS BETWEEN INTESTINAL IMMUNE CELL POPULATIONS AND
THE AUTOCHTHONOUS MICROBIOTA I N ANTIBIOTIC-COMPROMISED M I C E .
H . R . G a s k i n s l , R . I . M a c k i e l , T. May1 a n d K . A . G a r l e b Z . l D i v i
s i o n o f N u t r i t i o n a l S c i e n c e s and D e p a r t m e n t o f A n i m a l S c i e o
c e s , l l n i v . o f I l l i n o i s , U r b a n a , I L 61801 and 2 R o s s P r o d u c t s DL
v i s i o n o f A b b o t t L a b o r a t o r i e s , Columbus, O h i o 4 3 1 2 5 .
I n f e c t i o n w i t h t h e nosocomial pathogen, C l o s t r i d i u m d i f f i c i d e p e n d e n t on a n t i b i o t i c c o m p r o m i s e . A c c o r d i n g l y , i t i s
presumed t h a t a u t o c h t h o n o u s m i c r o o r g a n i s m s i m p a r t c o l o n i z a t i o n
r e s i s t a n c e t o t h i s and o t h e r i n f e c t i o u s o r g a n i s m s . G I h i l e d e v e
l o p i n g a m o u s e model t o understand
specific interactions
between t h e i n d i g e n o u s m i c r o b i o t a a n d C. d i f f i c i l e s u s c e p t i b i l i t y , w e o b t a i n e d e v i d e n c e t h a t i n t e s t i n a l immune c e l l p o p u l a
t i o n s a r e a l t e r e d by o r a l a n t i b i o t i c t r e a t m e n t . P r e s u m a b l y ,
s u c h c h a n g e s i n h o s t c e l l compartments a r e mediated v i a e c o l g
study
g i c a l c h a n g e s i n t h e r e s i d e n t m i c r o b i o t a . The p r e s e n t
f u r t h e r d e s c r i b e s i n t e r a c t i o n s between t h e i n t e s t i n a l microb i o t a and r e s i d e n t immune c e l l p a p u l a t i o n s i n a n t i b i o t i c - c a m
p r o m i s e d mice. C57BLI6NHsd m i c e were f e d a l o w r e s i d u e d i e t
an
( E n s u r e R ) +/- f r u c t o o l i g o s a c c h a r i d e (FOS), t r e a t e d w i t h
o r a l a n t i b i o t i c ( c e f o x i t i n ; 100 ug/g BW), a n d s u b s e q u e n t l y
c h a l l e n g e d w i t h C. d i f f i c i l e ( s t r a i n VPI 1 0 4 6 3 ) . The f e r m e n
t a b l e f i b e r , FOS, was i n c l u d e d t o p r o m o t e r e - e s t a b l i s h m e n t
of a b a l a n c e d p o p u l a t i o n o f i n t e s t i n a l o r g a n i s m s a f t e r a n t i
b i o t i c c h a l l e n g e . Animals w e r e s a c r i f i c e d a f t e r C . d i f f i c i
l e c h a l l e n g e a n d i n t e s t i n a l ( s m a l l i n t e s t i n e , cecum, c o l o n )
s a m p l e s o b t a i n e d f o r immunocytochemical d e t e c t i o n o f n e u t r g
p h i l s , a n t i g e n p r e s e n t i n g c e l l s (APC: m a c r o p h a g e s , d e n t r i t i c
cells), a n d f L t T - c e l l s . F e c a l s a m p l e s were c o l l e c t e d a f t e r
for
a n t i b i o t i c t r e a t m e n t a n d a f t e r C. d i f f i c i l e c h a l l e n g e
enumeration o f t o t a l c u l t u r a b l e b a c t e r i a , c l o s t r i d i a and &
d i f f i c i l e . T o t a l c u l t u r a b l r b a c t e r i a l and c l o s t r i d a l c o u n t s
w e r e i n c r e a s e d a f t e r a n t i b i o t i c t r e a t m e n t o n l y in mice supp l e m e n t e d w i t h FOS; b a c t e r i a l r e g r o w t h was
not observed
in a n i m a l s r e v e i v i n g E n s u r e a l o n e . I n t e r e s t i n g l y , a n t i b i o t i c
e f f e c t s on i n t e s t i n a l immune c e l l t y p e s were a l s o d e p e n d e n t
on d i e t . D e n d r i t i c a n d T - c e l l n u m b e r s i n t h e l a m i n a p r o p r i a
of t h e cecum a n d c o l o n w e r e i n c r e a s e d b y a n t i b i o t i c t r e a t ment o n l y in m i c e f e d E n s u r e a l o n e . A l t e r n a t i v e l y , a n t i b i o t i c t r e a t m e n t i n c r e a s e d m a c r o p h a g e n u m b e r s in t h e cecum and
c o l o n i n FOS-supplemented mice. N o t a b l y , immune c e l l r e s p o n
ses t o a n t i b i o t i c were o b s e r v e d i n t h e l a r g e i n t e s t i n e wher e b a c t e r i a l d e n s i t y i s h i g h e s t and where C . d i f f i c i l e - i n d u
ced l e s i o n s a r e l o c a l i z e d . Moreover, t h e changes n o t e d c o r diarrhea
r e l a t e w i t h p r o t e c t i o n f r o m C. d i f f i c i l e - i n d u c e d
and d e a t h i n t h i s m o d e l . T h e s e d a t a i n d i c a t e t h a t i n t e r a c autot i o n s b e t w e e n r e s i d e n t i n t e s t i n a l immune c e l l s a n d
c h t h o n o u s b a c t e r i a l p o p u l a t i o n s may c o n t r i b u t e t o c o l o n i z a t i o n r e s i s t a n c e i n antibiotic-compromisedmiee.
l e is
XXVl
ABSTRACTS
THE INFLUENCE OF ANTIMICROBIAL AGENTS O N
MICROFLORA AND COLONIZATION RESISTANCE OF T H E
HOST.
- .
B.A. Shenderoy
Gabrichevsky Institute of Epidemiology and
Microbiology, Moscow, Russia.
SURVEY OF HLlMAN MICROSPORIDIOSIS IN ITALY BETWEEN 1992-1994
Voglino M.C. ( 1 ) . Rossi P. ( 2 ) , Pozio E. ( 2 ) . DonelliG. (1)
(1) Dept.of Ultrastructures and (2) Dept.of Parasitology,
Istituto Superiore di Sanith, Viale Regina Elena, 299-00161
Rome, Italy.
Based on t h e scientific literature and t h e
materials received in the author's laboratory it
was shown that many antimicrobial preparations
can induce disbalance in the microbial ecology
of the intestine. I t is accompanied by reduction
of colonization resistance which leads to t h e
change in time of persistence in the intestine
of the potentially pathogenic microrganisms. The
data prove that children whose mothers had
disbalanced intestinal microflora due to
chemotherapy had also reduced colonization
resistance. During the experiments o n animals it
was proved that different antibiotics,
administered orally, changed the feces excretion
of protein, polysaccharides. amino acids,
organic acids. cations and other compounds. T h e
correlation was discovered between t h e quantity
of some carbonic acids and phenol compounds,
excreted with feces and t h e feces antagonistic
activity against potentially pathogenic
bacteria. Use of scanning electron microscopy
has permitted to study the changes i n the
biofilm of the cecum of the rats w h o received
chemical preparations. The factors which
determine the possibility and the expression of
changes in the microbial ecology of the host
under influence of the antimicrobic agents are
also given. A list of compounds. which together
with chemical preparations are potentially
capable of interfering with the host microflora.
i s provided. The mean8 of therapy are considered
to decrease most effectively the possibility of
microbial disfunctionsz use of eubiotics.
selective decontamination, treatment of patients
in the conditions of gnotobiological department
and their combinations.
The prevalence of microsporidiosis in Italy is yet unknown,
due to difficulties mostly related to diagnostic procedures.
Definitive diagnosis of microsporidiosis depends, because
of the size of the parasite, upon its detection in clinical
samples by trasmission electron microscopy, moreover the ui
trastructural observation allow to define the parasite gene
ra. We report the total cases of human microsporidiosis recorded in Italy between 1992-1994.
One case of nasal and corneal infection due to Encephalitozoon sp. in an AIDS patient suffering from chronic sinusitis and keratoconjunctivitis was recorded. The parasite was
found in nasal mucus smears, in the epithelial cells of nasal mucosa and in corneal scrapings. This is the first case
diagnosed in Italy and the fifth recorded i n the world.
The first case of Enterocytozoon bieneusi infection was reported in 1992, in a homosexual 44-years-old man, suffering
a severe watery diarrhea and 15 kg weight loss. Up to now
18 cases of intestinal microsporidiosis have been diagnosed
i n our country: 13 from northern, 4 from central and 1 from
southern Italy. Only two of them were recorded in women.
Among male patients, 6 (33%) were omosexual, 2
(11x1 bisexual and 8 ( 4 4 % ) drug abusers. All cases were detected in
patients suffering chronic diarrhea.
In conclusion, among the five genera infecting humans only
two, Encephalitozoon and Enterocytozoon, have been identified i n Italy so far.
___
BIFIDOBACTERIA USED IN FERMENTED MILK PRODUCTS
Biavati B . , l Mattarelli P.,i Alessandrini A . , I
Crociani F..2 Guerrini M..1
IIstituto d i Microbiologia Agraria. UniversltA di
Bologna. *Istituto di Citonorfologia Normale e
Patologica, CNR Bologna.
Bifodobacteria have long been recognized as bacteria with
probiotic nutritive and therapeutic prope~%ies. The
ingestion of fermented milk containing bifidobacteria MY
help to maintain a normal intestinal microflora balance, to
improve lactose tolerance and digestibility of milk
to prevent
products, to reduce plasma cholestorol.
carcinogenic activity and to provide B-complex vitamins.
This report concerns I the examination of samples of
fermented
milk
preparations for
the presence
of
bifidobacteria; their identification based on the w s t
reliable methods employed in the taxonomic study of the
genus Bifidobacteriumi the determination of the intestinal
origin of the strains isolated by means of the comparison of
electrophoretograw of cellular soluble proteinsr the
survival in acidic environment of bifidobacteria from human
habitat and the fecal recovery in human of Bifidobacterium
animalis ingested with fermented milk. He obtained the
following results, the bifid strains added to the fermented
milk were viable; seven of the eight samples examined
collected in different countries were shown to contain
strains identified as B.animalis a species found only in
animal habitats; the isolates compared by electrophoretic
patterns proteins with strains of B.animalis f r m known
origins were recognized to be either of rabbit or chicken
gastrointestinal tract origin; of the one hundred and ten
bifidobacteria strains from human habitat tested. more than
10% of the start population survived in 10 strains after 15
days and the same percentage of survived cells WIU present
in three atrains after 30 daysi finally the integration of
the normal diet with a commercialized fermented milk
containing B.anhalls proved that this species w i v e s the
passage through the gastrointestinal tract and 1. present at
the level of other bifidobacteria as long as the yogurt is
consumd. while if the ingestion stops the exogenous
Bifidobacterium gradually decreases and is no longer
detectable.
__I__--
MICROECOLOGICAL ASPECTS OF FUNCTIONAL FOOD.
B.A. Shenderov
-______
G.P. Gabrichevsky Institute of Epidemiology and
Microbiology. MOSCOW. Russia.
Information on scientific investigations in the
field of medical microbial ecology in Russia
over the last 20 years i s briefly given. It's
stressed that traditional explanations of t h e
developmental mechanisms of many diseases based
on the primary or exclusive role o f the host
organs and tissues have not yet provided any
constructive suggestions as to the development
of efficient measures for prophylaxis and
treatment of atheroslerosis. spondyloarthrosis,
urolithiasis. allergy and other diseases in man.
In some respect it is due t o ignoring or
underestimating the role o f numerous
microrganisms permanently or transitorily
present on the host mucosa i n the
etiopathogenesis of the diseases. Extension and
intensification of researches in t h e field o f
biochemical, molecular and evolutionary ecology
of microrganisms in Russia allowed t o work o u t a
new technology for receiving pharmacologically
active compounds and functional food on the
basis o f t h e normal living microrganisms, their
complexes o r component o f microbial cell,
regulating the composition o f host microflora
increasing colonization resistance, normalizing
t h e level o f serum cholesterol, urine oxalate.
pool of histamine, liver functions both in vitro
and in vivo. Special attention has been paid to
B3fidumbacteria and Lactobacilluq and their
complex with oligosaccharides, diet fiber and
unsaturated fatty acids. T h e author supposes
that the development of functional food industry
in t h e 21st century will determine t h e health of
humans.
xxvii
ABSTRACTS
PROTEIN SUBUNITS COMPOSING SURFACE
LAYER OF C.
. DIFFICILE
M. Cerauettil, A. Sebastianellil, A. Molinariz, A. Gelosiaz. G. Donelli2.
P. Mastrantoniol.
~
~~~
~
Laboratorio di Batteriologia e Micologia Medical, Laboratorio di
Ultrastrutture2, lstituto Superiore di SanitP, Roma, Italy.
A surface protein layer (Slayer) is found in various microrganisms
including C. difficile, the etiological agent of pseudomembranous colitis
(PMC) and most cases of antibiotic associated dirrhoea (AAD).
Our previous studies regarding electrophoretic profiles of EDTA-extracted
proteins of C. dificile isolates showed that a major protein of 36kDa was
present only in strains associated with the disease and that this protein was
an antigen expressed in "vivo" in patients with AAD.
Immuno-electron microscopy and immunofluorescence revealed that the 36
kDa protein was exposed on the cell surface of our prototype strain (~2.53)
of clinical isolates from cases of PMC and AAD..The presence of an
arranged regular array was demonstrated in the outer wall layer of C .
difficile ~2.53.The isolation of the S-layer proteins was obtained from the
cell wall fragments by treatment with 8M urea. The SDS-PAGE of the ureaextracted proteins showed the presence of two proteins with molecular
weights of 36 and 47
Similar regular arrays were also observed on the
cell walls of other C. difficile strains isolated from various sources
(neonates, healthy children, healthy carriers etc.). All regular arrays studied
were composed of two proteins with different molecular weights ranging
from 3 6 to 56 kDa.
These results suggest that the 36 kDa protein is a component subunit of the
S-layer of some C.dificile strains; the presence of an S-layer is not a unique
characteristic of C. difficile associated-disease strains; the C. diflcile Slayer is generally composed of two distinct proteins.
ma.
FLUORIMETRIC DETECTION OF FAECAL BACTERIAL
ENZYMES
Cenci G., Caldini G., Braganti S., lsidori C.
Deparfmenf of Cellular and Molecular Biology
Universify of Perugia (lfaly)
Much attention is being devoted to the possible
relationships between diet a n d gut microflora a n d their related
role in the pathogenesis of colon cancer. Epidemiological a n d
experimental studies suggest a relationship between some dietary
components a n d c h a n g e s in intestinal bacteria. Microflora enzyme
expression may h a v e a marker significance both in defining diet
modified ecosystems, a n d at same time may be able to evaluate
risk factors related to t h e s e modifications.
T h e aim of this study was to evaluate the expression of
faecal bacterial hydrolytic activities in rats, fed on experimental
diets containing different sources a n d amounts of fermentable
carbohydrates (sucrose,starch, resistant starch). Representative
activities were c h o s e n from different hydrolases s u b c l a s s e s
(estefases, arylamidases, glycosidases, proteases), and their
levels were tested by fluorimetric 4-methylumbelliferyl-conjugates
(MU-Cs), if availables, or chromogenic substrates.
Results obtained from different experiments indicate
that 9/19 tested activities @mol/h x mg protein) have a role for
assessing the influence of diet on microflora. Particular
importance may be attributed to deconjugant (pg/ucurunidase,
pglucosidase)
and
mucolytic glycosidases (N-acetil-8-Dglucosamiflidase,Kacefil-~-Dgalacfosaminidase,&ga/actosidase).
Good agreement was found between MU-Cs assay a n d
semiquantitative micromethod (API N M ) , but a greater
discriminatory power was obtained by t h e fluorimetric method.
EVALUATION OF FAECAL FLORA IN CROHN'S
DISEASE AND JUVENILECHRONIC ARTHRITIS
M.lvlalin, E&L&ULI
'
H. Mykkinen
Deparment of Clinical Medicine,
University of Tampere, Finland
The human indigenous intestinal microflora has an important protective
function for the host. Impaired mucoaal barrier is supposed to play an
important role in the pathogenesis of Crohn's disease, and association of
arthrihk and various intestinal disorders is weU known. Specifics h a h s of the
species of Lactobacillus are able to stabilize the intestinal mucosal barrier.
Our a m was to evaluate faecal flora in patients with Crohris disease ,
juvenile chronic arthritis (JCA) and controls. Specifically, we studied the
effect of human Lactobacillus casei strain GG on the intestinal microecology
in these patients.
Face1 samples were obiamed from 24 patients with Crobn's disease (5 to
51 years), 25 patients with JCA (2 to 14 years) and 15 controls (3 to 52 years).
Changes in gut microecology were assessed by measuring bacterial enzyme
activities (P-glucosidase. P-glucuronidase and urease) in faeces. Paediatric
patients from these study groups were allocated to a trial with Lactobacillus
GG, l O l o cfu twice daily for ten days. Faecal samples were analyzed before
Lactobacillus GG immediately after and one month later.
The mean (SD)urease activity was increased in patients with JCA. 32.6
(14.8)m o l x min-l x mg-* , compared to controls; 18.1 (13.0). while in
patients with Crohn's disease the urease 6.1 (7.6)as all enzyme levels were
remarlrably low, F = 29.23, p = O.wO1. After oral bacteriotherapy the enzyme
activitiesin patients with Crows disease approached the controlsvalues.
Our results indicate that in Crohn's disease and JCA the intestinal
micrmology balance is disturbed, and Lactobacillus GG may provide and
adjunct therapy for clinical conditions associated with impaired mucosal
barrier function.
xxviii
ABSTRACTS
LACTIC ACID BACTERIA IN ROTAVIRUS DIARRHEA
INTESTINAL MICROBIAL ECOLOGY IN
Clostridium difficile INFECTION
H. Majamaa, E. Isolauri, M. Saxelin, T. Vesikari
Department of Clinical Medicine,
University of Tampere, Finland
Different lactic acid bacteria were compared for their effects on
the immune response to rotavirus in children with acute
rotavirus gastroenteritis. After initial oral rehydration, 49
children aged 6 to 35 months with rotavirus gastroenteritis
randomly received either Lactobacillu & subsp. & strain
GG (LGG), M subsp. r h a m n o m (LactophilusR) or the
combination of Streutococcus thermophilus and L. delbruckii
subsp. bulgarim (YalactaR) twice daily for five days. Serum
antibodies to rotavirus, total number of immunoglobulinsecreting cells (ISC) and specific antibody-secreting cells (sASC)
to rotavirus were measured at the acute stage and at
convalescence. The mean (SD) duration of diarrhea was 1.8 (0.8)
days in children who received LGG, 2.8 (1.2) days in those
receiving LactophilusR, 2.6 (1.4) days in those receiving YalactaR
(F= 3.3, p = 0.04). The duration of diarrhea in patients not
receiving lactobacilli was 2.6 (1.3) days. The ISC response was
comparable in the thrcc study groups, while the rotavirusspecific immune responses were different. LGG therapy was
associated with an enhancement of IgA sASC to rotavirus and
serum IgA antibody level at convalescent stage. It is suggested
that certain strains of lactic acid bacteria, paricularly LGG,
promote serum and mtestinal immune responses to rotavirus,
which may be of importance for protective immunity against
rotavirus reinfections.
C. DIFFICILE VIRULENCE: COWELATION BETWEEN TOXINOGENICITY,
ADHERENCE, ENZYME PRODUCTION AND SEROGROUP
Karjalainen T., Collignon A . , Barc M.-C, Bouredu H. G m e z
M.. Poilane I, and Bourlioux P.
Universiti de Paris-Sud, Faculte de Pharmacie, Chatenay-Malabry, France
Previous studies using animal models concerning the virulence of C. difficile-associated disease have suggested a
relationship between toxin production, mucosal association,
and proteolytic enzyme synthesis; strains that are highly
virulent are more apt tn produce tissue-degrading enzymes
and adhere to intestinal mucosa. Likewise, it has been
s h a m that the pathogenicity i s linked to the serogroup.
The aim of our study was to examine the relationship
between toxin A and B production, cellular and mucus
association and enzyme (collagenase, gelatinase, hyaluronidase) production by strains of C. difficilerepresentmg
various serogroups.
Toxin A was assayed by an immunoenzymatic assay and toxin B
by a cytotixicity assay. Cell adherence onto Vero cells was
investigated by light microscopy, attachment to mucus by an
immunoenzymatic assay developed by us. Collagen degradation
was studied by spectrophotometry, azocoll assay and by
SDS-PAGE; gelatine degradation by spertrophotometry and
SDS-PAGE.
Ayaluronidase production was investigated by
spectrophotometry. The serogroups of the strains were
determined according to DelmCe.
Virulent strains, such 7 9 6 8 5 . isolated from pseudomembranous colitis and belonging K O the serogroup 53 adhere at a
high level after heat shock to Vero cells and to mucus.
This isolate degrades collagen and gelatine. In contrast,
strains isolated from healthy infanfs and belonging to Khe
non pathogenic, non toxinogenic serogroup D adhere after
heat shock to a lesser degree to Vero cells and to mucus.
These isolates have neither gelatinase nor collagenase
activity. The hyaluronidase activity is identical regardless of the origin of che isolates. Work is in progress on
the isolates of other serogroups.
Pa ul Naaber, Marika M i k e l s a a r
I n s t i t u t e of Mic robiology, T a r t u U n i v e r s i t y , T a r t u ,
Estonia
C l o s t r i d i u m d i f f i c i l e c a us e d i n f e c t i o n ( C D I )
d e v e l o p e s i n p a t i e n t s whose i n t e s t i n a l t r a c t ' s
is
weakened
due
to
colonization
resistance
a n t i b a c t e r i a l t r e a t m e n t . W e i n v e s t i g a t e d i n two
s t u d i e s w he the r t h e numbers of a na e robe s and
l a c t o b a c i l l i w e r e i n f l u e n c i n g t h e c o l o n i z a t i o n of
i n t e s t i n a l t r a c t by C l o s t r i d i u m d i f f i c i l e . In the
first study of 18 random h o s p i t a l i s e d p a t i e n t s
43
y)
with
clinically
and
(median
age
cases
of
CDI
the
microbiologically
prove n
anaerobic/aerobic
ratio
a nd
the
number
of
l a c t o b a c i l l i a n d C. d i f f i c i l e i n f a e c a l samples was
examined. C. d i f f i c i l e w a s i s o l a t e d from a l l t h e
p e r s o n s s t u d i e d . The c o u n t s of C. d i f f i c i l e varied
between 2 . 3 a n d 8 . 4 l o g CFU/g, t h e t o t a l c o u n t s of
anaerobes
a nd l a c t o b a c i l l i were decreased i n
comparison t o o u r p r e v i o u s s t u d i e s of h e a l t h y
p e r s o n s . I n 1 3 samples t h e c o u n t s of a e r o b e s exceeded
t h o s e of a n a e r o b e s a n d l a c t o b a c i l l i . I n 7 cases C.
d i f f i c i l e w a s t h e predominant mic robe , r e a c h i n g even
up t o 1 0 0 % i n 5 p a t i e n t s .
In the second study of t h e BAlb/c m i c e dosed
w i t h c e f o x i t i n and c h a l l e n g e d w i t h a t o x i n produc ing
s t r a i n of C. d i f f i c i l e , we found s i m i l a r changes
c o n c e r n i n g a e r o b e s / a n a e r o b e s r a t i o in t h e c o n t e n t of
c o l o n ; i n t h e mucosa only t h e a e r o b e s c o u l d be
r e v e a l e d . C . d i f f i c i l e c o l o n i z e d t h e c o l o n of a l l
mice.
However, no p a t h o l o g i c a l
l e s i o n s except
diarrhea
c o u l d be
revealed.
The
c ounts
of
l a c t o b a c i l l i were kept h i g h b o t h i n c o l o n i c c o n t e n t s
a nd mucosa. T h i s s t u d y shows t h a t n o t o n l y t h e
aerobeslanaerobes
ratio
but
also
some o t h e r
mic roorga nis ms ,
l i k e l a c t o b a c i l l i seem t o be
i m p o r t a n t i n g u a r a n t e e i n g of c o l o n i z a t i o n r e s i s t a n c e
a g a i n s t t h e development of C D I .
VIRIJLENLE
FACTOI;!
C'F YEFISINIAC I i O L A T E G FFOM
VARIGI~IC. MATCRIALS I N TIHE FESARO-IJRBINO AREA
P i a n e t t i A.,6affone
W.,Ei'uscolini F . , * B i f f i
M . R . ,Albano V. ,**Salvaagio L.
Institute
of
Toxicologic,Hygienic
and
E t i v it'oiime t i t a 1
Sc ie n c e - IJ n ive I , s it y
o f 1-1 I b ino- I t a 1 y
*Institute
o f V i r o l o g y snd * * I n s t i t u t e o f H y g i e n e
a n d P i ' e v e n t iv e M e d ic in e - ?in i vi. r 5 it y o f M i1 a n - I t a 1 y
Ver's i n ia
e i n t i . r o c 0 1 it i c.3 ii a p a t h o g e i i t ' e s p o n s ib 1 i
for
3
vai.iety
of
clisorclsi's,
although
acute,
noncumpl i c a t e c l
entai.itii
man? F c s t a t i o n . O f
t h
ciii' i i i n.stui'.:,
only
The pui'poze
of
this
st
virulence
c h s r a c t c i is t i c
Y.eiitet.ocolitics
anti
7
isolated
fi,om
various
m.
IJt,bino a r e a .
T h e 4 1 isolates w s i ' e t e s t s - i l f o i , i n v a s i o n o f t i s j u c
cu1tui.e c e l l s , s a l i c i n FsrmenCation-ssculin hydro1yzis,
p y i ' a z i n a m i c l a s ~a c t i v i t y , c a l c i u m d i p e n d e n ce,
c1,istal
v i o l e t : b i n d i n g , u p t a t ( + o f congo r e d ,
O:7,8)
isolated
On
the
basis
of
t h e j e r e s t i l t s we c a n c o n c l u d e :
1)tlis
source of i s o l a t i o n o f t e n i s not p r e d i c t i v e
of t h e p a t h o g e n i c i t y of t h e y e r s i n i a s t r a i n ; 2)foi.
a d i a g n o s t i c p u ~ ~ p o st eh e o c c u r r e n c e o f a d s f i n i t i ve
combination
o f r l i s p o n s e s to t h e v a r i o u s t e s t 5
c a n set've a s a i r e l i a b l e m a r k e r of v i r u l e n c e .
xxix
ABSTRACTS
I N H I B I T I O N OF B A C T E R I A L COLONIZATION OF THE G U T BY
HUMAN AND BOVINE C A S E I N
C . A . Guzrnanl. G . P i a t t i z C . P r o n z a t o z . A . C r i p p a , .
C . G u a r d a t i z and C . P r u z z o 3
GBF-National C e n t r e for B i o t e c h n o l o g y .
Bra uns chw ei g. Germany, 1 I n s t i t u t e o f
M i c r o b i o l o g y . U n i v e r s i t y of Genova. I t a l y and
3 I n s t i t u t e of H i c r o b i o l o g y . U n i v e r s i t y of Ancona.
Italy
I
artaat-feeding is associated with a redneed incidtnce of itreral inftctious dlatasts.
SecrCtOry illUlOglObulins
and OtbCr factor8 anch aa lyIOZJiC, h C t O f t r r h , tbe
laCtOperOlida8~ sr8ttl and C t l h l a r C O l P O ~ e ~ t aIC
8 reIpOnlibh €01 milk pIOtCCti011
against Infectioni. Pecently, a protective role 111 aicribed to component8 that
ioterferr vitb bacterial adherence to intestinal surfacei. Ibe iain protein preient
in bovine and human 1111: is casein, represantlnq EOI and 101 of the total protein
content, rc~)ectively. Ie analyzed the ability of huian and bovine c a i t i n to Inhibit
attacbieot to cultured bunan inteatinil cells [Inteitine 40T and taco) of bacteria
that are part of the norial huian intertinal flora 01 could infect tbe huian
intestinal tract causing diacase. & itraini carrying CIAI, U A I I and type I
fiibrlae, and strains belonglop to tbc rpeciea froteui iirabllls, Erlionells Q h J ,
lntlrobacter FkiEe,
[email protected], &IEI!j CnttrOCOllC?.
SICUdOlOPa8 8trUplOQ#a, BaCtIIOldCI
p W d O t ~ b e r C O l O ! ~ , CaipglObtCtO:
trafiiljs, Clostrldiun plrflhgtn!, StaPhY1OCCOCUl a U I C U I . 106 @terOCOCCOB f N C l l i l
werr tested.. A decreaie i n adherence efficacy la1 obirrvrd for all itrains. Ibi
labibition varied froi 901 (P.airabilis and C . l U f r h g c n l ) LO 10-151
(!.CnterOcO~itiCa, C
C
i. m and a r $ E l . A Iililtr rtdUCtIOU YaI Qb8trVld Up LO 1
l h h a l CQnCeOtratiOl Of Of 1.15PJ 11’. BOliOt C l S C h a d d and W J l a t h hidroralates
inhibited adberenee of all tested strains at a sliqbtly lover degree tban that
observed vitb vbolt catein. 91111ar results were obtained with a, B
and k fractions of bovine castln. Ihue rerolts sugqeit that In rlro caaein l a y have
1 significant protective effect igainit Intestinal hfCCtiO01. A1 hotb h u l u ind
borine cairins eIbibit slillar activitlri, tbry la1 affect gut ColOniZatlon during
the lifetime.
u,
REDUCTIOK OF AZO DY€S BY ANAEROBIC BACTERIA
ISOLATED FROM HUMAN INTESTINAL TRACT.
Fnvmeh RaW. Carl E. Csrnigld and
I . G . H. Ruwler-Van Embdeo’
.Divioioo of Microbiology, National Center for
Toxicological Rerearch, FDA, Jefferson. Arkansas 72079,
U S A. ’Dept of Immunology, Er~rmus Uoivcnity.
RotierdYrn, The Ndherlands
Azo dyes are extensively used in the textile, pharmaceutical, food and
cosmetic industries. Seven water-soluble dyes, Fuchsin, Deep
Maroon, Ponceau SX, Alura Red, Orange 11, Tartrazine and Sunset
Yellow, and four water-insoluble dyes, Flaming Red, Toney Red,
Lithol Rubin B. and Solvent Red, which are used in foods, drugs and
cosmetics, were rested with azoreductase-producing bacteria isolated
from the human intestinal tract. All of these dyes contain one am
group, except for Toney Red, with two am groups. Fifty pl of a
water-soluble dye, dissolved in water, or 50 pl of a water-insoluble
dye, dissolved in dimethyl sulfoxide. was added to Brain Heart
Infusion broth inoculated with 0.1 ml of an overnight culture of
Closmridium pefringens. C.purapurnpcwn and Closmridium sp. under
anaerobic conditions. The cultures were overlaid with oil and
incubated at 37°C overnight. All three of the bacteria completely
decolorized the water-soluble dyes plus Solvent Red; they panially
decolorized Flaming Red, Toney Red, and Lithol Rubin B. These
results indicate that Ciosrndium spp. isolated from human intestinal
flora are capable of cleaving am bonds in a wide spectrum of a20
dyes.
M E T B M O G E N I C BACTERIA IN HUMAN GASTROMTESTMAL TRACT
Ferrari A,, Brusa T.,
a.
and E. Canzi
Department of Food Science and Microbiology, University of Milan,
Milan, Italy.
Methanogens inhabit human colon of most adults although the
concentrations are extremely variable. We first observed the presence
of methanobacteria in subgingival plaque and characterized the strains
isolated 60m the oral cavity as a new species, Methanobrevibacter
oralis sp. nov. There is little knowledge on methanogens colonization
of digestive tract. The aim of the present study was to evaluate the
incidence and concentration of methanobacteria in the feces and in the
oral cavity in a healthy population on a Mediterranean diet. Adults
unaffected by periodontal diseases and children 3 and 5 years of age
were tested. Bacteriological examinations were performed in anaerobic
glove cabinet. Methanogenic cultures in modified Balch medium 1
were tested for fluorescence and methane production by
gaschromatographic analysis. Methanobacteria were observed in the
feces of 90% of the adults, whereas only 45% of the subjects harbored
methanobacteria in the plaque and saliva. The incidence of
methanogenic bacteria in the feces and saliva of the 3-year-old children
were 40% and 20% respectively. Methanobacteria were observed in
the feces of 60% of the 5-year-old children, whereas only 30% of them
harbored methanobacteria in the saliva. It is noteworthy that all the
subjects harboring methanobacteria in the subgingival plaque also
harbored such bacteria in saliva and feces Of interest is the finding of
methanobacteria in the saliva; in fact, even though saliva cannot be
considered an ideal anoxic ecosystem for the growth of methanogens,
such microorganisms could come from the periodontal pockets because
of the washing action of saliva. However, the existence of anoxic
microniches in the oral cavity, not necessarily in the subgingival
pockets but in close relation with the saliva, cannot be excluded.
The study was supported by CNR, project RAISA, subproject n.4.
SAFETY AND TOLERANCE OF LACTOBACILLUS REUTFRI
IN HEALTHY ADULT MALE SUBJECTS
A. Cases
Ross Products Division of Abbott Laboratories, Columbus, OH,USA and
BioGaia Biologics, Inc., Raleigh, NC. USA
B. W. Wolf, K. A. Garleb, and I.
Thirty healthy, adult mala subjects were used in a two-group, doubleblind, parallel design to evaluate the safety and tolerance Of a potential
probiotic organism, Lactobacillus reuteri. Subjects (1Bltreatment)
consumed two gelatin capsules per day for 21 days (dl containing either
a freeze dried L. reuteripreparation in a cryoprotectant or a placebo (just
cryoprotectantl. The concentration of L . reuteriwas such that subjects
consumed 1 x 10” CFU per day. The study was 28 d in length with
daily documentation of the presence of gastrointestinal symptoms
[nausea, diarrhea, cramping, distention, flatulence, vomiting,
constipation, burping and rafluxl. In addition, serum chemistries,
hematology, urinalysis, urinary indican excretion, and fecal microbiota
(L. reutsri and total Lactobacillus spp. enumeration) were measured
weekly (d 0, 7, 14, 21, and 28) while a physical exam was given on d
0. 21, and 28. An additional fecal sample was obtained on d 77 for
microbial enumeration. Subjects could consume their regular diets;
however, alcohol was not allowed. Physical exam and urinalysis
parameters were not clinically different between treatments.
Supplemental L. reuteri reduced (P < .05) urinary indican excretion at
d 7, but had no effect (P > ,051 on subsequent urine collections.
Although significant differences were observed for a few of the serum
chemistry and hematology variables measured, all of the analyses
remained within the expected ranges for healthy, male adults. Subjects
consuming supplemental L. reuteri had increased (P < .051 levels of L.
reuteriin their feces on d 7 , 1 4 , 21, and 28. However, colonization was
lost within two months of washout (d 771. Level of total Lactobacillus
spp. was not different between treatments throughout the trial:
however, the ratio of L. reuterktotal Lactobacillus increased (P < .05)
for subjects consuming supplemental L. reuteri. Occurrence of
gastrointestinal symptoms was infrequent and similar for both
treatments. In zonclusion, supplemental L. reuteri may be fed et 1 x
10” CFU/d without any clinically significant safety or tolerance
problems. Intake of L. reuteri ( 1 x 10” CFU/dl results in colonization
(as measured by fecal level1 within 7 d of consumption end is
maintained for at least 7 d post consumption; however, colonization is
lost within two months of washout.
xxx
ABSTRACTS
VIER10 CHOLERA€ ZONULA OCCLUDENS TOXiN ROT) REORGANIZES ACTIN IN
CULTURED EPITHELIAL CELLS
Fabbri, A., Fiorentini, C., Fasano, A.', Kaper, J. 6:. and Doneiii, G.
Department of Ultrastructures. lstituto Superiore di SanitP. Rome ITALY;
'University of Maryland at Balto., School of Medicine, Baltimore, Maryland,
USA
Attenuated Vibrro cholerae vaccine strains, specifically mutated in genes
encoding cholera toxin still cause mild diarrhea. In fact a second toxin, named
ZOT for Zonula Occludens Toxin, has been recently identified. This toxin
increases intestinal tissue permeability by regulating the structure of
intraceilular tight junctions (tj). The ZOT activity is reversible. heat-labile,
sensitive to protease digestion and found in coiture supernatant fractions
containing molecules beetwen 10 and 30 kDa in size. Since this new toxin
affects intestinal tj we studied whether the cytoskeleton. and in particular the
F-actin organization. was altered by the ZOT activity. For this purpouse iEC-6
cells, an epithelial cell line derived from crypt cells of rat small intestine,
were exposed to colture supernatant containing ZOT for 3 and 24 hours. After
3 h of treatment no modifications of F-actin could be detected while after 24 h
a percentage of IEC-6 cells showed a rearrangement of the stress fibers,
mainly consisting in a redistribution of F-actin to the subcortical compartment
of the cells. Similar results were obtained with the rabbit ileum. where ZOTexposed tissues showed an irregular F-actin staining within the brush border
associated with actin redistribution in the underlying enterocytes. The action
of ZOT appeared to be selective in that LLCPKl cells, derived from pig kidney
cortex, showed no significant changes in the F-actin cytoskeieton when
exposed to the toxin. This is in agreement with the observation that ZOT
increases tissue permeability only in the ileum but not in the colon. This
modification of the F-actin distribution could be responsable for the modulation
of tj in the ZOT-induced pathogenesis.
1.
2.
Fasano et a/.. Proc. Natl. Acad. Sci. 8 8 5242-5246, 1991
Baudry ef al., Infect. immun. 6 0 428-434. 1992
STABILITY AND ACTIVATION OF THE PRO-TOXIN FORM OF CLOSTRIDlUN DIFFICILE TOXIN A
S . Hyde and S.P. B o r r i e l l o
Dept.Microbio1ogy and I n s t i t u t e o f I n f e c t i o n s and
QMC, Nottingham, U.K.
Immunity
There is a 'pro-coxin' form of C. d i f f i c i l e t o x i n A which
f a i l s t o haemagglutinate r a b b i t e r y t h r o c y t e s . T h i s is
s t a b l e a c 4"C, room temperature and 37°C b u t a c t i v a t e d by
h e a t i n g t o 45°C (1-4h) and freeze-thawing from -20°C. The
pro-toxin i s a l s o a c t i v a t e d by s o n i c a t e s o f a non-toxigenic
s t r a i n of C. d i f f i c i l e . E a r l y s t u d i h s m d l c r r c d t h a t t h i s
was due t o a t h i o l p r o t e a s e . However, i t was n o t p o s s i b l e
t o p u r i f y such an enzyme from C. d i f f i c i l e by a r g i n i n e
a f f i n i t y chromatography ( s p e c i f i c f o r t h i o l p r o t e a s e s ) , O K
t o a c t i v a t e t h e pro-toxin w i t h c l o s t r i p e i r . (a Lhiol p r o t e e se from C.
histolyticum) o r the plane chiol-protease
papain. Use of s p e c i f i c i n h i b i t o r s i n d l c a r e t h a t t h e
p r o t e a s e i s one of t h e f a m i l y of a c i d p r o t e a s e s , a s t h e C,
d i f f i c i l e i i z ~ w ";.is
. ~ :&ihitrd
by p c p s t a t i n .
PLASMATIC LIPOPOLYSACCHARIDES (LPS) AND CD14+ CELL FREQUENCY I N CElRONIC EEPATITIS C VIRUS (cRCV) PATIENTS.
E. J i r i l l o , B. Greco, L. Caradonna, R. S a t a l i n o . V. Pugliese, 'R. Cozeolongo, 'R. Cuppone. *F. Rosina. '0.G. Mannhisi
Immunopathology ,- 'Med.Gas&enterology,
I .R. C.C. S "i.d e
Bellis" C a s t e l l a n a G r o t t e ( B a r i ) ; *Med.Gastroenterology
"Le Molinette" Rosp., T o r i n o , I t a l y .
.
According t o r e c e n t d a t a , c i r c u l a t i n g LPS or endotoxins
form complexes w i t h t h e LPS-Binding P r o t e i n (LBP), which i n
t u r n a d h e r e t o t h e CD14 molecule on monocytes. These
i n d e x e s were i n v e s t i g a t e d i n 47 p a t i e n t s w i t h CECV. A l l
subjects before entering a clinical trial with interferonwere f e a t u r e d by profound d e f i c i t s of b o t h phsgocyte and
lymphocyte f u n c t i o n s . I n p a r t i c u l a r , frequency o f T c e l l
s u b s e t s and CD14+ cells were reduced i n a s i g n i f i c a n t
manner. The p r e s e n c e of immune a l t e r a t i o n s coupled t o the
e s t a b l i s h e d concept of l i v e r d e t o x i f i c a t i o n of LPS, prompt e d u s t o s c r e e n p a t i e n t s f o r t h e p r e s e n c e of endotoxine.
LPS d e t e c t e d by a s p e c t r o p h o t o m a t r i c Limulus Assay were
p r e s e n t i n 7 o u t 33 p a t i e n t s . With the a i m t o c o r r e l a t e a
s t a t u s o f endotoxaemia with t h e frequency of CD14+ c e l l s ,
we d i v i d e d up t h e above s c r e e n e d s u b j e c t s i n t o a Limulus
p o s i t i v e and a Limulus n e g a t i v e group, r e s p e c t i v e l y . Data
i n d i c a t e a reduced frequency of CD14+ c e l l s i n t h e Limulus
p o s i t i v e subset.
Taken t o g e t h e r , t h e s e f i n d i n g s s u g g e s t t h a t LPS may a f f e c t
t h e count o f CD14+ c e l l s i n t h e f o l l o w i n g ways:
1 ) t h e complex LPS-LBP c a n mask t h e CD14 molecule on monocytes;
2) CD14 t o g e t h e r w i t h t h e above complex c a n b e shed from mc
nocyte surfaces;
3) monocytes complexed t o LPS-LBP c a n m i g r a t e from c i r c u l a t i o n t o d i s t a n t organs.
F i n a l l y , c i r c u l a t i n g LPS may o r i g i n a t e from b o t h exogenous
and/or endogenous s o u r c e s , owing t h e impaired k n e and
h e p a t i c c l e a r a n c e s . I n c o n c l u s i o n , d e t e c t i o n of LPS and
count of CD14+ c e l l s seem t o r e p r e s e n t a d d i t i o n a l markers
eo b e monitored i n terms of inflammatory c y t o k i n e s r e l e a s e
and s y s t e m i c changes.
A ROLE OF PERTUSSIS TOXIN SUBUNITS I N MOUSEENCEPHALOPATHY
Hiroko S a t 0 & Yuji S a t o ,
-N a t i o n a l I n s t i t u t e of Health,
Tokyo Japan
P e r t u s s i s t o x i n (PT) i s t h e main v i r u l e n c e f a c t o r produced by B o r d e t e l l a p e r t u s s i s and a s p r o t e c t i v e a n t i g e n
i s t h e most i m p o r t a n t component of t h e p e r t u s s i s vaccine.
One of t h e most a d v e r s e r e a c t i o n s t o t h e p e r t u s s i s vacc i n e i s c o n s i d e r e d t o be encephalopathy. Although a
d i r e c t r e l a t i o n of t h e v a c c i n e t o encephalopathy h a s n o t
been proved, w e have a t t e m p t e d t o i n d u c e encephalopathy
i n mice by i n t r a c e r e b r a l (IC) i n j e c t i o n of PT. The mice
developed encephalopathy; #en s t i m u l a t e d b y t a p p i n g on
t h e c a g e or t o u c h i n g t h e body t h e mice responded with
abnormal movement such as jumping and c i r c l i n g i n t h e i r
cage. The area around t h e i r e y e s became i r r i t a t e d and
b l e d a f t e r r e p e t i t i v e s e l f s c r a t c h i n g . A t 60 ng and 200
ng dosages of PT, t h e encephalopathy and d e a t h o c c u r r e d ,
r e s p e c t i v e l y , i n moat o f t h e mice. The encephalopathy was
always observed b e f o r e d e a t h by IC i n j e c t i o n w i t h PT. The
main p a t h o l o g i c a l change i n t h e b r a i n s of t h e PT-injected
mice was v e n t r i c u l o - m e n i n g i t i s w i t h o u t s i g n i f i c a n t c e l l u lar d e g e n e r a t i o n or n e u r o n a l damage. I n d u c t i o n o f t h e s e
p a t h o l o g i c a l changes by PT c o u l d be n e u t r a l i z e d by a n t i PT monoclonal a n t i b o d y (MAb). When t h e anti-FT MAb was
g i v e n i n t r a v e n o u s l y t o t h e mice I C - i n j e c t e d w i t h FT,
a n t i - S 1 , S2 and 54 a n t i b o d i e s could i n h i b i t t h e encephalopathy c o m p l e t e l y b u t a n t i - S 3 MAbs showed a l i t t l e i n h i b i t i o n i n s p i t e of t h e i r s t r o n g i n h i b i t i o n t o t h e leukoc y t o s i s . The n e u t r a l i z i n g a c t i v i t y of MAbs t o t h e leukoc y t o s i s was n o t always p a r a l l e l t o t h a t t o t h e encephalop a t h i c d e a t h . T h i s r e s u l t s u g g e s t s t h a t S2 and s3 may
play a d i f f e r e n t r o l e i n t h e i n d u c t i o n of t h e encephalopa t h y , l e u k o c y t o s i s or o t h e r b i o l o g i c a l a c t i v i t i e s and
t h a t mechanisms of i n d u c t i o n of t h e encephalopathy and
l e u k o c y t o s i s i n mice may be d i f f e r e n t . The mouse-encephalopathy caused by I C i n j e c t i o n of FT must become a
u s e f u l a n i m a l model t o a n a l y z e a role of s u b u n i t s i n
d i f f e r e n t b i o l o g i c a l a c t i v i t i e s Of PT.
xxxi
ABSTRACTS
INFLUENCE OF STAF'HYLOCOCCUS AUREUS ALPHA-TOXIN
ON RABBIT BLOOD-BRAIN BARRER PERMEABILIIY
-1,
R Gadamslu2, W Hrymewczl, J Albrecht*
'Nauonal Institute of Hygiene, Warsaw, Poland
*Medical Research Center Polish Academy of Science,
Warsaw, Poland
The am of this study was to get inside into the neurotoxicity of S aureus
haemolysin - alpha-toxin, which was preliminanly indicated in earlier
studies
Alpha-toxm was obtained from S aureus Wood 46 strain Puntication
was performed with the application of ion-exchange chromatography
followed by preparauve isoelectric focusing Such prepared,
electrophoreticaly pure t o m was tnjected into foot vam of wistar rats
No general blood-bran bamer changes for at least 24 hours were
observed when a dose slightly below LD50 was apphed Separated rat
brains were cut into slices of 40 and 70 micrometer thin, for Evans blue
and horse radish peroxidase staining, respectively Immunostaining
specific for an astroglia specific protein - GFAP revealed an elevated
reaction in a number of cerebral structures including corpus callosum,
stna terminalis and nucleus reticulans thalanu Morphometnc analysis
revealed a marked (approximately 50%) increase of small vessel volume
in cerebral cortex and hippocampus, which was associated wth changes
in erythrocytes shape In the control rats they were elongated, while in
capillaries of alpha-toxln-treated animals, erythrocytes were round This
phenomenon was associated with decrease of erythrocyte surface, what in
consequence could interfere with blood-brain tissue exchange of
metabolites
The above presented study may indicate that staphylococcal alpha-toxin
induced neurotoxicity secondary to capillary and erythrocyte changes
I N V I T R O I N T E R A C T I O N S O F VAGINAL ISOLATES OF
LACTOBACILLI W I T H OTHER MEMBERS OF VAGINAL FLORA
Maria B a s t a , A l i c j a Budak. M a l g o r z a t a Bula nda
a n d P i o t r 8 . Heczko
._
I n s t i t u t e of M i c r o b i o l o g y , U n i v e r s i t y Me dic a l
S c h o o l , Cracow. P o l a n d
S t r a i n s of L a c t o b a c i l l u s i s o l a t e d from t h e
v a g i n a of h e a l t h y women a nd t h o s e w i t h v a g i n i t i s
were c h a r a c t e r i z e d b i o c h e m i c a l l y a n d c l a s s i f i e d
on t h i s basis i n t o s e v e r a l s p e c i e s . T h e i r
I n h i b i t o r y p r o p e r t i e s were f u r t h e r t e s t e d i n
v i t r o a g a i n s t v a g i n a l s t r a i n s of Ca ndida
b e l o n g i n g t o d i f f e r e n t s p e c i e s a nd i s o l a t e d from
v a g i n a l c a n d i d i o s i s . Some L a c t o b a c i l l i showed
stable inhibitory properties against the yeast.
The same s t r a i n s of L a c t o b a c i l l u s were a g a i n
t e s t e d a g a i n s t c l i n i c a l i s o l a t e s of
S t a p h y l o c p c c u s a u r e u s . A s i n t h e c a s e of
C a n d i d a , some L a c t o b a c i l l i were a b l e t o i n h i b i t
t h e gr ow t h of S . a u r e u s i n v i t r o . The o b s e r v e d
a c t i v i t y may e x p l a i n some mechanisms l e a d i h g t o
b a c t e r i a l v a q l n o s i s a nd c a n d i d a l v a g i n i t i s .
CLUSTER ANALYSIS O F VAGINAL MICROBIAL DATA
M.-L.
T. Le e , R . A .
ROSS,
A.B.
Onderdonk
Channing L a b o r a t o r y , Harvard Medical School
and Brigham & Women's H o s p i t a l
W e conducted c l u s t e r a n a l y s i s on a l a r g e d a t a s e t
assembled from i n v i v o s t u d i e s d e s c r i b i n g t h e
h e a l t h y v a g i n a l e n v i r o n m e n t . R e s u l t s show t h a t
v a r i a b l e s were c l a s s i f i e d i n t o s e v e r a l g r o u p s .
C o r y n e b a c t e r i u m , S t a p h y l o c o c c u s , STPl c t ,
PREVl-ct, a n d PSCl-ct w e r e c o n s i d e r e d - a s h i g h l y
correlated variables.
Lactobacillus, Total
A e r o b i c and T o t a l Anaerobic were a s a n o t h e r g r o u p
of v a r i a b l e s . A c c o r d i n g l y , normal v a g i n a l
microflora iteractions were classified into several
g r o u p s . We are c u r r e n t l y u s i n g t h e i d e n t i f i e d
c l u s t e r s t o determine whether they r e p r e s e n t
g r o u p s more or less l i k e l y t o d e v e l o p v a g i n a l
infections.
INTERFERENCE OF SHORT CHAIN PATTY ACIDS PRODUCED
BY ANAEROBES WITIt ENDOCELLULAR PATHOGENS: A N
EXPERIMENTAL MODEL.
BiuLL. AnaD A.. Tranl (1.. Boiu O.A.
laititule of Microbiology . Medlul School and University Hospital
33100 Udine . IUly
Short chain hUy acids (SCFA) produced by maaobic bacteria are not to be
considered inen meUbolic sad product:. but active rnolcculer that might play in
humans a very relevant role both In manhining homcostarii of various
ecoiysamr and In plrlecipaling to the infectious process as pathogenetic
factors.
The cffecu of SCPA on several functions of suciryotic cells (such as on
chcmouxis Md phagocyrorir by human grmulocyias) and of procaryotic cells
(growth rate and toxin production by enteromxigenic Closuidia) have been
already documented. In this study the attention was focused on the role played
by SCFA in the vaginal ceosyrlem. both in physlolopiul and In pathological
conditions. The effeci of hclic. bulyrlc and ruccinic acids on the infecting
pmsors by endocellular palhogen&w u urayed by M ia vilro modsl. McCoy cell
cullurcs. gmwa on confluent m n o h y a . wore Infectad with C. rrachomrit ATCC
VR902 strain (ratio 195 . 150 Chlanydi. w. cocaryollc cells). suspended in
0.5% ~ILICOK D-MBM cell cullurc medium. The mdiurn w u modified by adding
diffcreni concenuatlonr of each SCPA under invcriigation (3-13-30-50 mM,
final pH= 7.2): the infected cell cullurcs were incubatad at 37T. 3% '2%. for 72
his. Chlmydial reticulale and elementary bodies were ravcalcd on fixed cells
(msthmol: scetone 5 0 5 0 . -20.C for IOmin) by wing nuorcrcenl monoclonal
lntibodiea agllast C. rrnchornalis major ooier membrenc proroin (MOW. 40 kD).
A aisnificicmt reduelion of ralicul.ia body numb- -.ersus sonuals (f<O.OOl)
was
obmved in infected eulturcs incubstad in the presence of 3050 mM Issiic wid,
sugge*linp In intufsrsncc by lhlr SCFA in tho infesting process by the
prhogen. No reduction w u obacrved in the presence of butyrlc and succinlc
acids st the conoantrationr Lolled. but other affsctr were vliible: incubation of
cellr with 30 mM buiyrlc acid determined an Incrcuo of the rsilsulats body
numbs (P<O.OS). w h u u s c d h incuhued with SOmM rucclnic acid were ahown
to be lnlcclal by high numbsr of elcmcnury bodiol. Wid- rcticul.te bodies.
These resulU might indluta an interference exerted by SCFA in tho infection:
inraraningly. lactic scid is normally presmi in &he vaginal ecosystem In
physiological conditlons. thus reasiuring ihe protective mlc of the rssidcnt
mlcmhlou. On he conusry. butyric and ruwinic acids. whose consenaatioor in
the vagina significantly Incrmrc when I dlsmlcroblsm occurs (0.g. vrglnosis).
mighl exst M adversa affecr UI the host, predbpoiing to infection.
The work w u supportad by G m t CNR FATMA PPII; tho authan thank Dz. M.
Zonmnon fm prscbus a d v b lad discussion.
xxxii
ABSTRACTS
1N VlVO EFFICACY OF A NEW FLUOROQUINOLONE,
FOR
THE
TREATMENT
OF
BAYy3118,
EXI’ERIMENTAL INTIUABDOMINAL SEI’SIS DUE TO
CHALLENGE
WITH A MIXED MICROBIAL
POPULATION.
Ronald L. Cisneros and Andrew B. Ondcrdonk.
Brigham & Women’s Hospital and Channing Laboratory,
Harvard Mcdicnl School, Boston
Previous studies using an experimental model for
intraabdominal sepsis have documented the efficacy of several
therapeutic regimens for this serious and complex infectious
process. Antimicrobial agents with activity versus coliforms,
which reduce mortality in this model system, and agents effective against obligate anaerobes including Bacteroides fraoilis,
which reduce the incidence of abscesses, have been shown to
be an effective therapeutic cornbination.
Therapy with
singie-drug, broad spectrum agents have aiso been evaluated
for their effect on mortality and the development of intraab
dominal abscesses during this mixed aerobelanaerobe experimental infection. This study reports t h e w effectiveness of
BAYy3118, a new, highly active fluoroquinolone. Rats were surgically implanted with a mixed microbial inoculum, into the peritoneal cavity, followed by appropriate antimicrobial therapy.
Three groups of animals were included in this trial. Group 1
received BAYy3118,15mg/dose: group 2 received a combination
of gentamicin, 2 mgldose, plus clindamycin, 15 mgldose: group
3 served as untreated controls The results of this study indicate
that BAYy3118 therapy compares favorably with established
treatment regimens in this model system simulating
intraabdominal sepsis.
TISSUE PENETRATlON OF CIPiOFLOXAClN (CP) 1N DIABETIC ( D )
FCOT ( F ) INFECTION ( 1 ) .
F.de L a l l a , A.Nwelli*, G.Erle, G.Pellizzer, I.Piva, hl.Strazzabosco, P.Bendetti, S.Fallani*, and il.Mingardi.
S.Bortolo Hospital, Vicenza, and University of Florence*,
Italy.
The polymicrdial nature of OBF and the poor local penetra t i c a of drugs i n t o t i s s u e s c q r a n i s e d by vasculopathy
and neuropathy o r both, require appropriate s e l e c t i m .
dosage and r m t e of administration of antimicrobial agent s . We evaluated the concentrations (C) of CP, a b r d - s p
e c t w fluoroquinolone, i n s e w (Se) and skin-subcutanee
u s t i s s u e (!Xi) biopsies (Biopsy punch, 2 mn diameter)
f r m t h e viable m r g i n s of F lesicns, i n 17 p t s with
s e r i m s DFI ( f e r a l e s : 3; nales: 14; mean age: 60 y; a l l
with n o m l renal function). 1 2 p t s (grcup A ) received CP
200 ng BID (loading dose 400 n g ) i.v. over a 60 nlin
infusion; 5 p t s (grmp B) were given oral CP 750 q BID.
At day 5th of treatment, Se and 5Ti were sarrpled a t 1 and
12 h m r s a f t e r the a n t i b i o t i c adrninistraticn. C [ng/l-kg
CiSD)] of CP i n Se and !Xi were microbiologically assayed
( t e s t organism: E.coli X121553 and a r e show in the
table.
C(fSDI
Se(ng/l)
tiw
CP:ZO&rg
l.V.
75Qrg P.O.
lh
12h
0.81(0.14)
O.ZO(0.17)
2.41(1.00)
O.SG(0.29)
S i ( n g / k g ) lh
12h
0.52(0.35)
0.26(0.28)
1.17(0.90)
0.84( 0.60)
In DFl oral CP 750 ng BID s e w t o provide nure adequate
Se and STi C against susceptible organisnis (p ranging frun
0.01 t o 0,059).
M O N I T O R I N G OF RESISTANCE I N H E N I N G O C O C C I
H.
E.
Congiu. P. H a s t r a n t o n i o
L a b o r a t o r y of B a c t e r i o l o g y a n d M e d i c a l
Mycology. I s t i t u t o S u p e r i o r e d i S a n i t A .
Rome, I t a l y
The I s t i t u t o S u p e r i o r e d i S a n i t A h a s b e e n
c a r r y i n g o u t a n a c t i v e s u r v e i l l a n c e of
meningococcal m e n i n g i t i s i n I t a l y s i n c e 1984.
Isolated strains are sent t o the Central
B a c t e r i o l o g y L a b o r a t o r y i n ISS on a v o l u n t a r y
basis. Besides typing t h e s t r a i n s t h e c e n t r a l
l a b o r a t o r y monitors t h e H I C s f o r p e n i c i l l i n ,
c e f t r i a x o n e . r i f a m p i c i n . s u l p h a d i a z i n e and
tetracycline t o control t h e i r efficacy i n
t h e r a p y o r p r o p h y l a x i s . O b s e r v a t i o n of s t r a i n s
collected over t h e l a s t years has enabled us t o
s e t up g u i d e l i n e s which recommend t h e u s e o f
r i f a m p i c i n . i n s t e a d of s u l p h a d i a z i n e . t o t r e a t
c o n t a c t s and a v o i d s e c o n d a r y c a s e s . A h i g h
p e r c e n t a g e of L - m e n i n g i t i d i s s t r a i n s ( 7 0 9 )
c i r c u l a t i n g i n I t a l y have i n f a c t proved h i g h l y
r e s i s t a n t t o s u l p h a d i a z i n e . No s t r a i n s w i t h a
lower s e n s i t i v i t y t o p e n i c i l l i n ( 2 0.12uglml)
have y e t been observed as opposed t o t h e t r e n d
r e p o r t e d i n o t h e r European c o u n t r i e s . I n r e c e n t
y e a r s , however, t h e r e h a s b e e n a n i n c r e a s e i n
s t r a i n s with H I C s f o r rifampicin 2 0.25uglnl.
which s u g g e s t s t h a t g r e a t e r a t t e n t i o n s h o u l d b e
p a i d t o m o n i t o r t h i s t r e n d in t h e f u t u r e . Agar
d i l u t i o n t e s t and t h e more r a p i d E - T e s t h a v e
b e e n used t o d e t e r m i n e t h e H I C s and a
s a t i s f a c t o r y c o r r e l a t i o n was o b s e r v e d when
r e s u l t s from t h e two t e s t s ware compared w i t h
each o t h e r .
SEROTYPING OF BACTEROIDES FRAGILIS S T R A I N S
ISOLATED FROM FECAL AND CLLNICAL SAMPLES IN ITALY
USING MONOCLONAL ANTIBODIES DIRECTED TO CAPSULAR
ANTIGENS
R. Colaneelil. A. Pantostil, F. DAmbrosiol. A. O.Tzianabos2,
D.L. Kasperz
Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di
SanitA, Roma. Italy I ; Channing Laboratory, Harvard Medical School.
Boston, U.S.A.
The polysaccbaride capsule of Bacreroides fragilis represents the
principal virulence factor of this microrganism. It is composed of two
distinct polysaccharides (PS A and PS B) and appears to be
antigenically heterogeneous among strains. We have produced
monoclonal antibodies (MAbs) to the capsular polysaccharides of three
B.fragilis reference strains (NCTC 9343,NCTC 2429 and ATCC
23745)and have used them to type B.fragilis on the basis of capsular
composition.With a panel of 10 MAbs we have obtained 32 different
serotypes comprising over 3%of the strains examined.
Aim of the present study was to compare typing characteristicsof fecal
and extra-intestinal (clinical) B.frugilis isolates from Italy. The strains
were examined by a dot-immunobindiogassay; after growing in broth,
the strains were spotted onto nitrocellulose sheets and probed with the
MAbs. 41% and 57% of the fecal and extra-intestinal isolates
respectively, were typable by the MAbs used. The typable isolates from
feces belonged to 10 different serotypes,while the typable isolates from
clinical samples were distributed into 22 serotypes. The serotypes of
clinical strains encompassed those of fecal isolates. One serotype
(sentype 18)was prevalent among clinical isolates (26% of the typable
strains),but not among intestinal isolates.
We also examined enterotoxigenic B.Jrqilis strains (ETBF) isolated
from both fecal and clinical samples. The typable strains (13out of 24)
belonged to 8 different serotypes. A new MAb directed to the
polysaccharidecapsule of an ETBF strain recognized only 4 out of 24
eutentoxigenic strains (17%).
Our results show that B.fragilis strains from feces are slightly less
typahle than clinical isolates. Moreover it is possible that some
serotypes are characteristicof clinical (extra-intestinal)isolates. As for
ETBF strains, they do not appear to belong to the same or to a limited
number of serotypes as it happens for other toxigenic bacteria a8 ETEC.
Serotyping of B.fragiilis strains by MAbs has the potentiality to generate
new information about the virulence and the epidemiology of this
important human pathogen.
xxxiii
ABSTRACTS
"GENETIC DETERMINANTS FOR ANTIBIOTIC
RESISTANCE AND AEROBACTIN PRODUCXION OF
EFFECTS
ON
RAT
INTESTINAL
FLORA
CY CLOPHOSPHAMIDE AND ANIYMICllOBIAL DRUGS
OF
SALMONEUA W E N ISOLATES".
Paolo V i s c a l , Vincenzo Sanfilippol. Emma
Filetici2 and Mirella Fantasia2.
'Istituto Microbiologia Universiti "La Sapienza",
and 2lstituto Superiore di Saniti, Rome, Italy.
Salmonella
w i e n represented one of the
major agents of human salmonellosis in
Mediterranean and Middle Est countries during
the decade 1969-1979. In Italy the epidemic
outburst of S . w i e n was confined to years 19741978. After this period such a serotype was
isolated at endemic level in the south of Italy.
The epidemic spread of S . w i e n was associated to
the acquisition of large plasrnids belonging 10
t h e incompatibility group FI
encoding for
multiple antibiotic resistance.
One hundred and nine strains of S w i e n were
examined for antibiotic susceptibility. plasmid
profile and an aerobactin-mediated iron uptake
system expression. The strains were isolated 79
in Italy during years 1974-1989 and 30 in Tunisia
during years 1980-1990.
FI plasmid were found in 73.4% and 46,6% of
strains isolated in Italy and Tunisia respectively.
Loss of the FI plasmid was shown by strains
isolated in Italy after 1987, and has been
associated with the loss of the capacity to
produce aerobactin.
All S . w i e n strains isolated in Tunisia did not
syntetize aerobactin, independently of the
presence of FI plasmid.
In recent years strains circulating i n the
southern Italy show an antibiotic resistence
profile closely related to that shown by strains
isolated in Tunisia.
SURVEY ON OXACILLIN RESISTANT STAPHYLOCOCCUS
AUREUS (ORSA) IN ROME: A PRELIMINARY ANALYSIS.
Baiocchi P., Capone A., Santini C., Di Rosa
R., Venditti M. and the Roman study group
on ORSA.
Dip. Medicina Clinica - Universith "La Sapienza", Roma.
From January 1994 to May 1994 we have evaluated
OK by standard agar screen method among consecutive SA isolates from two University hospitals
(UH), 6 comunity hospitals (CH) and 3 private laboratories (PL). At a preliminary analysis OR was
found in 160 (55.9%) of 286 isolates from hospitalized patients and in 32 (7.7%) of 414 isolates
from non hospitalized patients (pto.0001). ORSA
were 79 (66.9%) of 118 isolates from UH and 81
(48.2%) from CH ( p = 0 . 0 0 3 ) . Among extranosocomial
isolates, OKSA were 26 (12.7%) of 204 isolates
from non hospitalized patients attending hospital laboratories, versus 6 (2.8%) of 210 isolates from patients attendlng PL.
In Rome, OR seems to represent not only a problem among nosocomial isolates but also an increasing phenomenon among extranosocornial isolates.
k m z z o n i Minelli E,, Uenini A.
Chair of Chemotherapy, University of Verona, Italy.
Selective decontamination of the gastrointestinal tract may reduce the
incidence of infections due to gram-negative bacteria or cocci in patients
with severely impaired host defences. The possible effects of interference
between the immunosuppressive treatment and the antimicrobial
chemotherapy on the intestinal microflora were studied in an experimental
animal model (50 Wistar rats). We studied the effects of treatment with
high doses of Cyclophosphamide (Cy, 100 mg/kg/die, orally for 4 days) on
the intestinal flora composition alone (group I) and in combination with
antimicrobial treatment. Ciprofloxacin (Cp, 8 mg/kg/die, 0 s ) and
aztreonam (Ar, 100 mg/kg/die, i.m.) were given for 10 days before
immunosuppression (groups IV and V, respectively) and for 4 days after
immunosuppression (groups I! and 111. respectively). Immunosuppression
caused an increase in mean counts of
E, faecium. fungi and
clostridia and ;I decrease of bacteroides and lactobacillli. Antimicrobid
chemotherapy ;tnd immunosuppressive treatment reduced the overgrowth
of intestinal micro-organisms caused by Cy. The inhibitory effects of Cp
and Az in immunosuppressed rats were lower than those obtained with
drugs alone. Prophylaxis with antimicrobial drugs followed by
immunosuppression exerted a marked inhibition on intestinal microflora.
while the administration of Az and Cp to immunosuppressed rats produced
a lower inhibitoly effect. Az and Cp were effective for elimination of
potentially pithogenic gram-negative micro-organisms in the gut;
moreover, they exerted only limited activity against anaerobic bacteria.
Thc rats immunosuppressed and treated with Cp and Az died after the
week of treatment: [hose pretreated with antibiotics survived more than 15
days after the end of treatment. These changes in rat are comparable to
those observed in humans. Early selective decontamination of gut may
prove more effective for the prevention of severe infections in subjects
with impaired host defences.
u,
ANTIBIOTICS AND INTESTlNAL CONTENTS INTERACTION.
Bertazzoni Minelli E , Benini A , Vicentini L., Muner A
Chair of Chemotherapy, University of Verona, Italy
Faeces can inhibit the antimicrobial activity of some antibiotics in the gut.
The aim of our study was to evaluate 1) the antimicrobial activity of
Cefetamet Pivoxil (CP, 50% bioavailability) and Ramoplanin (RP, not
absorbed in the gut) in presence of faeces and 2) factors affecting their
activity in intestinal content. We studied RP an CP antimicrobial activity in
presence of human fresh faeces in relation to bacterial flora composition
and its enzymatic activity. Faecal samples were obtained from 66 healthy
subjects. CP (10 mgA, final concentration) and RP (100 mg/l, final
concentration) were incubated at 37°C for 1 and 4 hours with fresh and
sterilised faeces The antimicrobial activity of supernatant was determined
by microbiological agar-well-diffusion method Pellets were resuspended
in saline in order to evaluate the drug binding capacity to faecal material,
than supernatant assayed. RP pellets were resuspended several times with
guanidine chloride solution The CP antimicrobial activity was reduced to
60% by sterile faeces and this effect became more evident in presence of
microbial flora (50% activity); the prolonged incubation (4 h) reduced the
CP activity to 30%. Anaerobic incubation gave similar results The high
rate of inactivation of CP was directly related to faecal concentrations of
E. limosum and clostridia and to enzymatic activity of microflora
(esterase-lipase) Faecal components bind RP in high percentage (8090%) Free RP in aqueous supernatant was 4-7% at time 0; the recovery
after seven serial extractions achieved 73.9%. Incubation of RP with
faeces for 4h increased RP-faeces binding . RP recovery from sterile faeces
was similar RP seems not t o be inactivated by intestinal bacterial flora.
Binding and subsequent release of RP from faeces can presumably
maintain adequate long-lasting levels in G.I. tract against enterococci and
clostridia. RP offers a great potential for oral use in selective gut
decontamination. CP should be considered an "ecological" antibiotic
sparing the intestinal microflora
xxxiv
GENOME CHARACTERIZATION OF LACTOBACILLUS ENTERIC
STRAINS.
Morelli, L., Cesena, C., Campominosi, E., Bottazzi, V.
Istituto di Microbiologia, Facolta di Agraria, UCSC,
Piacenza.
The idea that lactic acid bacteria contribute to the
health and well-being of humans is one which goes back to
the beginning of bacteriology. Strains taxonomically
identified as Lactobacillus acidophilus are generally
believed to be the most represented in the human intestine.
This species, however, was recently splitted into at least
6 different recognized species: moreover, variable results
reported in the scientific literature on intestinal
colonization ability of lactobacilli could be related to
an to a poor knowledge of the Lactobacillus species realy
present in the human intestine. Aim of this study is the
characterization of the genomic structure of strains
belonging to species previously recognized as “acidouhilus?
and the detection, by means of Pulse Field Gel
Electrophoresis (PFGEI of bacterial biotypes related to
specific ecological niches. PFGE restriction patterns
obtained using rare cutting enzymes have allowed us to
differentiate strains belonging to the different species
derived from Lb. acidouhilus. Moreover, Southern
hybridizations have showed the presence on specific DNA
fragments of highly conserved genes, useful for species
differentiation. Improvement of the ecological
characterization of human enteric lactobacilli is
relevant for a better comprehension of the role played by
this group of bacteria in human intestine.
ABsTRAas
USE OF POLYMERASE CHAIN REACTION TO DETECT
B.PERTUSSIS ON NASOPHARYNGEAL ASPIRATES.
P.Stefane1li.H.Giuliano.T.Sofia.H.Bottone.
P.Mastrantonio.
Istituto Superiore di SanitA.Rone.Italy.
DNA probes technology proved particularly
advantageous improving sensitivity and
shortening tine-requirements for the identification of B.pertussis.In our study to assess the efficacy of new acellular pertussis
vaccines.the PCR is used t o detect B.pertussis
on nasopharyngeal aspirates collected from
children with suspected pertuss1s.A 191bp
sequence in the pertussis toxin promoter region
was amplified using primers pPTOXl(26bp) and
pPTOX2(29bp).The results were determined by
ethidium bromide staining of agarose gels.After
electrophoresis the sensitivity of PCR can be
increased by using Southern-blot analysis with
11-dUTP labelling probe.An internal DNA control
was included in the PCR assay t o avoid false
negative results.
In this study PCR assay was performed on 509
nasopharyngeal aspirates 90 of which oulture
positive for B.pertussis and 419 culture
negative.The 191bp fragment was detected in all
aspirates culture-positive for B.pertussis
except five.in none culture-positive for
B.parapertussis and in 18 culturenegative.Among these PCR-positive culture
negative cases. 12 (70%) showed typical symptoms
of whooping cough and 5 ( 3 0 \ ) had mild
synptoms.The increase in identification of
positive cases with PCR(6.QI) compared with
culture, showed that this assay could detect
B.pertussis overcoming the difficulties of the
bacterial isolation due to different causes such
as antibiotic treatment or vaccination status of
the patient.