Gram Positive Rods: Identification beyond `Diphtheroids`

Transcription

Gram Positive Rods:
Identification beyond ‘Diphtheroids’
Kathryn Bernard
National Microbiology Laboratory
Winnipeg, Manitoba Canada
kathy.bernard @phac-aspc.gc.ca
APHL lecture April 16, 2013
Faculty Disclosure
The Association of Public Health Laboratories adheres to established standards
regarding industry support of continuing education for healthcare professionals.
The following disclosures of personal financial relationships with commercial
interests within the last 12 months as relative to this presentation have been
made by the speaker(s):
Kathryn Bernard
“Nothing to disclose”
NML-Public Health Agency of Canada
LMN -Agence de la Santé Publique du Canada
2
Objectives
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WHEN isolates should be identified to
genus and species
Review of commonly-used test methods;
caveats
Review of current taxonomy of the genus
Corynebacterium & various medicallyrelevant coryneforms
Review of some diseases & typing
methods for these agents
Antimicrobial susceptibility testing [AST]:
multidrug resistance trends
3
When to Characterize Isolate Further?
1. Recovered from sterile body fluids, 2 or
more blood cultures or from sterile sites
2. Predominant organism from aseptically
collected specimens (e.g. deep tissues,
implanted prosthetic devices, other)
3. Predominant (>105 CFUs /ml or greater)
organism or sole organism (>104 CFUs /ml
from urine
4
Also consider characterization/Ref centre if …
4. Significant pathogen, from
immunocompromised, special patient
population [i.e. IV drug users, homeless]
5. Suspected to be ‘novel taxon’ or ‘rare’
pathogen [  reference centre]
6. Prelim characterization of a sterile-site
derived organism suggests unusual taxon
[  reference centre ]
7. Molecular methods required for outbreak
tracking [  reference centre ]
5
Identification Methods commonly used
Hospital Level Lab (Canada/US):
• Colony morphology
• Gram stain [these taxa LACK spores]
• Phenotypic (biochemical) reactions
[nearly all taxa in this review are
catalase positive]
• Rxs of detectable enzymes associated
with panel
• AST considered for clinically relevant
isolates
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Larger Hospital/ Reference Centre Methods
Genetic characterization e.g., using
• 16S rRNA gene sequencing (partial/full)
• rpo B or sequencing of other relevant gene
targets
• MALDI- TOF
Specifically for diph toxin producing species:
C. diphtheriae/ C. ulcerans / C. pseudotuberculosis
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Growth, appearance on selective agars
[e.g. Tinsdale Agar]
Toxin assay (diphtheria toxin) using ELEK,
other methods (PCR targets)
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C. diphtheriae on Tinsdale Medium: colonies are both brown-black and
have halo around each colony (Photo courtesy of C. Hinnebusch, UCLA Hosp Los Angeles)
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Initial Workup-Observations of Colonies
24h, 37C, air/5% CO2, blood agar, establish:
• Colony size, shape, edge
• Relative amount of growth [NG to 4+]
• Pigment if present
• Texture: smooth, mucoid, dry adherent
[note: aerial hyphae [nocardia like] forms
NOT found among these taxa]
• Haemolysis: beta, alpha, none
• Optimal Atmosphere, Temperature
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1mm- >3mm dry, ‘sugary’ grey-white
C. amycolatum, 24h
All NML photos
C. ulcerans 24h ATCC 9015T
C. canis 24h NML 090322
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1mm- >3mm, sticky, yellowish
Microbacterium sp, 24h
1mm- >3mm, smooth, lemon yellow
All NML photos
Arthrobacter species , 24h
Auritidibacter sp. 48h (close to Rothia, Micrococcus)
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Establish if Colony is Good Grower (or NOT)
If you observe poor growth on SBA in
24h, consider if strain:
• requires different atmosphere
• requires lipid supplement to grow at all
or to grow well (e.g., is lipophile)
• is inherently fastidious, that is, simply
takes longer to grow i.e. 48-72h
with/without supplements
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Demonstrating Lipophilia - Corynebacterium
1. Subculture organism in BHIB and BHIB +
Tween 80 (0.1%- 1%, v/v)
• Lipophilic species will have NG or 1+ in BHIB
but ++++ (4+) growth in BHIB+TWEEN
2. SBA and SBA +Tween 80 (0.1-1%):
lipophiles have larger colonies, luxuriant
growth on enriched SBA
3. Add 1 drop of sterile Tween 80 [or other lipid]
to lawn of fine growth: colonies closest to
drop grow larger [can get messy…]
Lipophilia thought to be due to the absence of a
fatty acid synthase gene
[Tauch et al, 2005 J. Bact. 187:4671-82 & J. Biotech. 136:22-30]
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Some lipophilic Corynebacterium spp (all NML photos)
C. afermentans ss lipophilum
CCUG 32105, 48h
C. jeikeium 72h
C. kroppenstedtii CCUG
37717T 48h
C tuberculostearicum
[CDC group G] 48h
C. lipophiloflavum
CCUG 37338T 48h
C.macginleyi 48h,
CCUG 32361T
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A good Gram stain is critical …
L: C. pseudodiphtheriticum- pneumonia
(photo courtesy of Dr. J. Nigrin, Dynacare-Kasper Lab, Edmonton AB)
R: C. diphtheriae -skin scraping
(photo courtesy of Dr. J. Nigrin, Dynacare-Kasper Lab, Edmonton AB)
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‘Straight to slightly curved rods with tapered ends. Short or
medium length. Club-shaped forms may be observed ‘
[Bergeys 2012 2nd ed The Actinobacteria, Part A, pg 245 re: Corynebacterium spp]
C. diphtheriae NML 090283
NML photo, 48h, 1000x
C. striatum NML 83-0629
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‘Sometimes
ellipsoidal, ovoid, or rarely , ‘whip
handles’ or thinner rods with bulges observed’‘
[Bergeys 2012 2nd ed The Actinobacteria, Part A, pg 245: re Corynebacterium spp]
C. atypicum CCUG 45804T
C. durum CCUG 37331T
C. matruchotii
NML 060816 NML photo,
48h, 1000x
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Phenotypic Testing If Done
Establish: metabolic process [oxidizer,
fermenter, assimilates, none of these]
• Catalase and oxidase reactions
Sugars: acidifies /utilizes: glucose, ribose,
xylose, mannitol, maltose, lactose,
sucrose, glycogen (+ other)
Other: urease, NO3 (NO2) reduction,
esculin hydrolysis, CAMP / reverse CAMP
reaction; motility
Enzymes: PYZ, PAL others (strips /stand alone)
Identification Schemes: MCM 10th, Bergeys
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NML photo
CAMP Test
St. aureus ATCC 25923
C. diphtheriae
CAMP+ve Coryne
glucuronolyticum
Rev CAMP +ve
(=CAMP inhibition)
Coryne ulcerans
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Biochemical Methods
• Macro vol (‘conventional’) sugars
• Manual Kits: API Coryne,
RapidID CB Plus, Crystal Gram Positive, etc
[some authors incubate strips beyond 48h
for slower growers]
• Automated
VITEK 2, MICROSCAN, BIOLOG
NML photo: Code 7001004, 99.9% C pseudodiphtheriticum
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API Coryne Pros / Cons
Rapid, convenient; can be ACCURATE if:
• Good grower, reactive in 24-48h
• Has somewhat unique codes
• Strain does NOT require supplements
CAVEATS:
• Biomérieux not updated codes or
augmented API Coryne database with
new species for many years, even on
‘API WEB’;
• Strips/panels generate similar codes for
species which react slowly or not at all
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Caution re: use of rapid ID strips
API Coryne codes 2100004 / 2100104
=C. jeikeium [corroborated by 16S seq]
SAME API CODE can come up for other
taxa which are relatively inert:
• some strains of C. auris,
• C. coyleae,
• C. mucifaciens,
• C. tuberculostearicum [CDC group G]
• Turicella otitidis
(species ID’ed using sequence based methods)
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No Matter WHICH method used for Biochemicals
• if non-reactive / poorly reactive with
substrates in 24-48h < OR >
• If identical or similar codes generated
for > 1 species then …
An accurate final ID will NOT BE achieved
by phenotypic means alone….
Suggest using additional approaches, if
definitive ID required..
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Genetic Identification Methods
• 16S rRNA gene sequencing, obtain
nearly full seq [1400-1500 bps]
• Partial sequence [400-500 bps] used by
some, either by in house or commercial
[Microseq, MIDI] methods
• In our experience, we apply 16S for all
genera described in this talk for
definitive ID
• Interpretation should be done by those
with taxonomy expertise
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Usefulness of 16S as ID method
Corynebacterium spp: most species
1.
2.
3.
4.
5.
6.
7.
separated by >2% identity EXCEPT….
C. afermentans, coyleae, mucifaciens (<2%)
C. aurimucosum, minutissimum, singulare (<2%)
C. propinquum and pseudodiphtheriticum (<1%)
C. sundsvallense and C. thomssenii (<1.5%)
C. ulcerans and C. pseudotuberculosis (<2%)
C. xerosis, C. hansenii, C. freneyi (<2%)
C. ureicelerivorans and C. mucifaciens (<1.5%)
Resolved Using: secondary gene target
• rpoB [full or partial] widely used
[Khamis et al 2004 JCM 42:3925-3931]
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Use of MALDI-TOF for Corynebacterium spp or Coryneforms
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Bruker & Biomérieux systems mostly used
Preparatory methods, instrument costs similar
Because of cell wall structures, Bruker has
recommended a Mayo clinic-based amendment to
extraction in January 2013 update:
BUG
Maldi plate
1ul 70% FA matrix Run
(or)1ul 70% FA Maldi plate
BUG matrix
Run
• Recent publications
MALDI very promising tool
for Corynebacterium spp (little data-other genera)
• Species close by 16S/rpoB also close by MALDI;
correct ID may come in 2ND, 3rd
[Alatoom et al JCM 50:160-163; Theel et al JCM 50:3093-3095]
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Antimicrobial Susceptibility Methods
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CLSI guidelines, breakpoints for AST of
Corynebacterium and Coryneforms, last
updated Aug 2010 [Table M45-A2]
Broth microdilution method recommended by
CLSI
E Test or Kirby Bauer: although used by some,
not recommended by CLSI
Method: we use commercial Sensititre 
panels, cation-adjusted Mueller Hinton broth
with lysed horse blood (2-5%v/v)
EUCAST has some data re: Corynebacterium
spp
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Antimicrobial Susceptibility - Corynebacterium
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Corynebacterium (medically relevant)
Canadian strains still susceptible to
vancomycin, linezolid, daptomycin
many clinically-relevant species found to
be fully susceptible to wide variety of drug
classes
For some species, nearly all strains are
multidrug resistant (MDR) BUT occasional
strain found to be fully susceptible (e.g. C.
amycolatum)
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Species occasionally or usually MDR
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C. afermentans ss afermentans
C. amycolatum
C. aurimucosum
C. confusum
Ref: MDR defined as
C. coyleae
strains of species
C. glucuronolyticum
expressing resistance to
3 or more drug classes
C. jeikeium
- Species which bear
C. macginleyi
ermX are likely resistant
C. minutissimum
to erythromycin and
clindamycin, possibly
C. propinquum
multiple classes
C. pseudodiphtheriticum
C. resistens (resistant to most drug classes)
C. striatum
C. tuberculostearicum (includes many CDC group G)
C. urealyticum
C. ureicelerivorans
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Unusual resistance in C. diphtheriae strain
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NML 090066 strain from 38y male (lives near
Vancouver) with abscess on left toe
Infection started while on trip to India
No trauma, no injuries to foot noted
Strain ID’ed as a toxigenic strain of C.
diphtheriae biovar mitis; MLST ST 136 (unique)
Resistant to erythromycin, clindamycin (ermX
gene detected), tetracycline,
trimethoprim/sulfamethoxazole (4 drug classes)
Intermediate to ceftriaxone and cefotaxime
(many Canadian strains have this trait)
tested for tet genes described in literature- all
negative
[Mina et al 2011 JCM 49:4003-05]
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Validly-named Corynebacterium species Total N=88 [Euzeby LPSN]
Group1. Found to date only in HUMANS (N=42) (as of March 26 2013)
A-C C. accolens *, C.afermentans ss afermentans; ss
lipophilum *, C. ammoniagenes, C. appendicis *,
C. aquatimens*, C. argentoratense, C. atypicum, C.
aurimucosum, C. auris, C. confusum, C. coyleae
D-I C. durum, C. falsenii, C. freneyi, C. hansenii, C. imitans
J-O C. kroppenstedtii (*), C. lipophiloflavum (*),C.macginleyi*,
C. massiliense, C. matruchotii, C. minutissimum, C.
mucifaciens, C. mycetoides
P-S C. pilbarense, C. propinquum, C. pseudodiphtheriticum, C.
pyruviciproducens, C. resistens *, C. riegelii, C.simulans, C.
singulare, C. sputi *, C. stationis, C. striatum, C.
sundsvallense
T- Z C. thomssenii, C. timonense, C. tuberculostearicum *
contains many CDC group G strains), C. tuscaniense, C.
ureicelerivorans*, C. xerosis
Species marked with “ * “ are lipophilic or can be lipophilic (*)
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Group 2. Found to date in BOTH HUMANS AND ANIMALS, with or
without evidence of zoonotic transmission
(N=12 as of March 26 2013)
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C. amycolatum
C. bovis *
C. canis@
C. diphtheriae biovars gravis, mitis, intermedius *,
belfanti
C. freiburgense@
Ref: 2, 6;
C. glucuronolyticum
- species “ * “ are lipophilic;
C. jeikeium *
- “@“ , presumed to be transmitted
from animal to human, but not as yet
C. kutscheri
recovered from animal, only from
C. mastitidis*
human after dog bite
- Evidence of infection of human from
C. pseudotuberculosis
animal by handling of infected animals
C. ulcerans
occupationally or as companion pet
C. urealyticum *
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Group 3. Validly-named Corynebacterium species recovered to date ONLY from
animals, birds, or the environment N=34 (as of March 26, 2013)
A-L C. aquilae, C. auriscanis, C. callunae, C.
camporealensis, C. capitovis, C. casei, C.
caspium, C. ciconiae, C. cystidis, C. doosanense,
C. efficiens, C. epidermicanis, C. felinum, C.
flavescens, C. glaucum, C. glutamicum, C.
halotolerans, C. humireducens , C. lubricantis
M- Z: C. marinum, C. maris, C. mustelae, C. nuruki,
C. phocae, C. pilosum, C. renale, C. sphenisci, C.
spheniscorum, C. suicordis,
C. terpenotabidum, C. testudinoris, C. ulceribovis C.
variable, C. vitaeruminis
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C. diphtheriae
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Most important GPR discussed here today
‘Biotype’ term still being used; strains grow
well in 24h, SBA except intermedius
‘gravis’: Nitrate reduced; glycogen +ve (starch
+ve)
‘mitis’: nitrate reduced, glycogen -ve (starch -ve)
‘belfanti’ nitrate NOT reduced, glycogen –ve,
starch –ve; genetically distinct
‘intermedius’ poor grower in 24h; “lipophilic”;
nitrate +ve, very rarely isolated today
Molecular typing: An MLST method described
recently [Bolt et al 2010 JCM 48:4177-4185]
- otherwise, ribotyping historically used
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Diphtheria- esp Diphtheria toxin (DT) producing strains
•
Developed countries: immunized as children;
Canada & US adults boosted every 10y
US, Canada <= 1 case of ‘classic’
nasopharyngeal diphtheria per year
Similar to US, Canadian case definition includes
C. ulcerans, C. pseudotuberculosis & sites not
throat etc in 2009 http://198.103.98.171/publicat/ccdr-rmtc/09vol35/35s2/Diphter-
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eng.php
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Serious disease including death can occur:
•
Patient has no access to vaccine (esp immigrants) or
chooses NOT to get immunized (occasional case in
Canada of this type)
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Diphtheria- esp Diphtheria toxin (DT) producing strains
•
Non-toxigenic strains CAN and HAVE
caused systemic disease and death, esp
among:
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very poor, living in crowded conditions
homeless
IV drug users
immunocompromised
C. diph can also cause endocarditis,
pharyngitis
[De Winter et al 2005 43:3447-3449; Lowe et al 2011 JCM 49:2664-2666]
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Diphtheria Cases among poor in downtown Vancouver
N= 33 cases [1996-2007], C. diph infections
review among patients from poor area in
Vancouver, linked with:
• IV drug use (67%)
• Co-morbidities included hepatitis B
(18%), hepatitis C (63%), HIV (33%)
• Recurrent ulcers (24%)
• Psychiatric illnesses (21%)
[Lowe et al 2011 JCM 49:2664-2666]
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MLST data from among strains sent to the NML
•
N= 175 strains tested, 8 ref, 163 WS
received over 14y (4 from cats [ST 74]
and 4 bv intermedius (3 Ref & 1 WS- all
ST=38)
Prevalence by ST (only 6/36 STs in Bolt paper)





ST 76 (34%), not cited in Bolt paper
ST 32 (23%)
ST 50 (14%)
ST 5 (5%)
1-2 strains of 30 additional STs
[Bolt et al JCM 48:2010 4177-4185; Bernard et al. Abstract, ASM 2011 New Orleans]
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C. ulcerans and C. pseudotuberculosis
•
C. diphtheriae, C. ulcerans and C
pseudotuberculosis, can all harbour diphtheria
tox gene & cause diphtheria-like disease
•
API Coryne ID pretty good [corrob with genetic
/other methods; rev CAMP+]
C. ulcerans emerging as zoonotic agent
transmitted from infected animals, including
companion pets, to humans
Animal handlers should keep vaccinations
current
~50% of Canadian C. ulcerans strains toxigenic
•
•
•
[Wagner et al Diphtheria in the UK 1986-2008: Increasing role of C. ulcerans Epi Inf.
138:1519-1530]
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Commonly Recovered Species
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C. amycolatum, C jeikeium: most
common Corynebacterium spp to cause
serious disease;
These can be MDR; usual habitat ? skin
C urealyticum: UTI – associated with
alkaline pH, struvite crystals; can cause
infections at other sites; can be MDR
C. tuberculostearicum: can be MDR,
various infection sites.
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Commonly Recovered Species
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C. striatum, C. pseudodiphtheriticum & C.
propinquum can cause pneumonia and
infections at other sites;
often MDR
Some strains C. propinquum urease +
[Diez-Aquilar 2012 EJCMID in press; Bernard et al 2012 IJSEM in press]
Site specific species:
• EAR: C. auris, C. afermentans, other
• EYE: C. macginleyi, C. accolens, C.
mastitidis-like, other
[Eguchi et al 2008 JCM 46:527-532]
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Not lipophilic- utilize sugars
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C. amycolatum; C. argentoratense,
C. atypicum, C. aurimucosum
C. confusum, C. coyleae
C. falsenii, C. freneyi
C. glucuronolyticum, C hansenii
C. imitans
C. minutissimum, C. mucifaciens
Ref: some of
C. riegelii
these species
may not be in
C. singulare, C. simulans
API Coryne
database
C. striatum, C. sundsvallense
C. thomssenii ,C. xerosis
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Species Not lipophilic & non-utilizers
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C. afermentans ss afermentans
C. auris
C. propinquum
C. pseudodiphtheriticum
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Other CoryneformTaxa
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Generally opportunistic, very rare
pathogens;
To date, associated with:
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prosthetic device infections;
immunocompromised state;
nosocomial & soft tissue infections, other
Taxonomy understudied evolving
Microbiology community slow to
acknowledge any role in human disease
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Medically Relevant
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Coryneforms- seen at NML
Arthrobacter(O,rod-
coccus cycle)
Auritidibacter (O)
Brevibacterium (O)
Cellulomonas (F)
Cellulosimicrobium(F)
Curtobacterium (O)
Dermabacter (F)
Dermatophilus (F)
Exiguobacterium (F)
All (except Exiguobacterium) in
suborder Micrococcineae, order
Actinomycetales;
-other genera in suborder not yet
associated with human disease
- Most appear to have usual niche
= the environment (some
exceptions, ie Rothia)
- Often yellow or yellowish pigment
- Many poorly reactive
biochemically
- Most are NOT INCLUDED nor well
discerned by phenotypicallybased, rapid ID systems [some
exceptions (i.e. Rothia,
Dermabacter]
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Medically Relevant
•
•
•
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Janibacter (O)
Leifsonia (O)
Microbacterium
Oerskovia
(F &
Coryneforms
All (except Exiguobacterium) in
suborder Micrococcineae, order
Actinomycetales;
O) -other genera in suborder not yet
associated with human disease
- Most appear to have usual niche
= the environment (some
exceptions, ie Rothia)
- Often yellow or yellowish pigment
- Many poorly reactive
biochemically
- Most are NOT INCLUDED nor well
discerned by phenotypicallybased, rapid ID systems [some
exceptions (i.e. Rothia,
Dermabacter]
(F,pseudohyphae)
Rothia (F, rod coccus
cycle)
Turicella (O)
NML, unpublished cases:
Brachybacterium (O)
Knoellia (O);
O, oxidizer; F, fermenter
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Some Commonalities
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Nearly all are catalase positive;
Most are strict aerobes;
Motility varies
‘Irregular shaped’ Gram Pos Rods, also
coccoidal or coccobacillary forms ie Rothia
Most grow well in 24h, usual conditions
Many are pigmented
Have ‘branched chain type’ CFAs, which is
qualitatively not like Corynebacterium spp
Definitive ID should include genetic
analyses [16S]
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AST For Various Taxa
Taxa susceptible to most antimicrobics BUT:
• Arthrobacter  resistance or diminished susc.
• Auritidibacter  resistant to 3 drug classes (NML
data)
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Brevibacterium  resistance or diminished susc.
Cellulomonas  resistance or diminished susc.
Cellulosimicrobium  res. or diminished susc.
Curtobacterium  usually susc.; some res. to 1-
•
Dermabacter  usually susc.; some res. to 1-2
•
•
Dermatophilus  usually susceptible (?)
Exiguobacterium  ??? (BMD recommended for some of these genera in
2 classes
classes
Health Agency
Canada
M45-2A; Susc.,NML-Public
sucsceptible,
res., ofresistant)
48
Coryneforms AST to various drug classes
•
•
Janibacter  usually susc.; reduced susc. to 1-2
classes
Leifsonia  usually susc.; some res. to 1-2
classes
•
Microbacterium  usually susc.; some res. to 12 classes; M. resistans resistant to vancomycin
•
Oerskovia  resistance or diminished susc.
•
Rothia  resistance or diminished susc. to
vanco has been rarely reported
(BMD recommended for many of these genera in M45-2A; Susc., sucsceptible, res., resistant)
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The Genus Rothia- Opportunistic Pathogens
Most important species are:
• R. dentocariosa: oral cavity but also recovered
from blood cultures, respiratory specimens,
abscesses, endocarditis, ocular infections, sinus
– Selective agar for this recently described
[2012 Uchibori et al J Micro
Meth 91:205-207]
• R. aeria: blood cultures, respiratory specimens,
abscesses [2012 Falcone et al J. Clin. Imm. 32:1400-1403]
• R. mucilaginosa (formerly Stomatococcus
mucilaginosus) oral cavity, but also blood cultures,
respiratory, CNS infections, peritonitis,
osteomyelitis, necrotizing fasciitis, endophthalmitis
and endocarditis [Cho et al 2013 Ann. Lab Med 33:145-149]
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Rothia dentocariosa 95-0154, with
typical ‘spoke wheel’ colony
Cellulomonas hominis NML 78-0402 from CSF
Auritidibacter sp. NML 100628
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Rothia aeria NML 090190, 24h
Arthrobacter gandavensis NML 00-0005, 48h
Rothia dentocariosa from abscess
Auritidibacter species NML
100628, 48h
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NML photos
Dermabacter hominis 24h ATCC 51325
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Brachybacterium
rhamnosus 48h, NML 03-0005
Br. rhamnosus 5day, 03-0005
Below, Gram at 48h
Janibacter spp
48h,NML 02-0242-2
Janibacter 5day, 02-0241-2
Below, Gram at 48h
NML-Public Health Agency of
Canada
LMN -Agence de
All
NML
photos
du Canada
la Santé Publique
Knoellia spp 72h,NML 03-0172
Knoellia 5day, 03-0172
Below, Gram at 72h
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Oerskovia sp, demonstrating pseudohyphae (NML photo)
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Relevant Reviews
1. Bernard, K. 2012. The Genus Corynebacterium
and other Medically Relevant Coryneform-like
Bacteria. J. Clin. Micro. 50:3152-3158
2. Bernard, K. A. and G. Funke. 2012. The Genus
Corynebacterium. Bergey’s Manual of
Systematic Bacteriology, 2nd ed. In Vol 5, Part
A, The Actinobacteria pg 245-289
3. Funke, G. , and Bernard, K., 2011 Coryneform
Gram-positive rods Manual of Clinical
Microbiology, ASM Press, Washington DC, 10th
ed. Pg 413-442 [11th ed writing underway]
4. Wagner et al 2012 Diphtheria in the
postepidemic period, Europe, 2000-2009. EID
18:217-225
56
Thank you!
Any Questions??
kathy.bernard @phac-aspc.gc.ca
57

Gram Positive Rods: Identification beyond `Diphtheroids`

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