Porins vs the unsusceptibility to imipenem in Providencia stuartii

Porins vs the unsusceptibility to
imipenem in Providencia stuartii
Que-Tien Tran
Cagliari, May 4th 2009
Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales;
Enterobacteriaceae; Providencia; Providencia stuartii
TEM picture by Y. Ramay
Providencia stuartii
- Gram-negative bacteria, Enterobacteriaceae
- Tribe Proteeae (Proteus, Providencia, Morganella)
- Straight rods, motile, facultative anaerobic
- Environment, gastrointestinal flora
- Opportunistic pathogens, hospital-acquired infection
- Urinary tract infections (e.g. kidney stone)
- Providencia stuartii is the species of the family with
isolates the most resistant towards antibiotics
Negative immuno-detection of the antigenic site considered as loop3
marker of enterobacterial porins in Proteus and Providencia strains
Multidrug resistance phenotype - MIC assays with values in µg/ml
P. stuartii
ATCC 29914
Ps 65237
(lab coll.)
Ps 2636
(lab coll.)
Ps NEA16
(clinical)
Ps 99645
(clinical)
Ps 19539
(clinical)
ESBL
-
+
+
+
+
-
IPM
2
2
4
8
4
2
EPM
≤0.06
0.25
1
4
1
0.25
MPM
≤0.06
≤0.06
0.5
1
1
0.25
FEP
≤0.06
128
>256
>256
>256
0.5
CPO
≤0.06
64
>256
>256
>256
1
CAZ
≤0.06
256
>512
>512
>512
4
FOX
2
32
64
64
64
16
CM
32
256
512
256
32
32
CM + PAβN 50µg/ml
16
256
256-128
128
32
32
CM + Res 32µg/ml
nd
256
nd
128
nd
nd
SFX
≤0.06
128
128
32
nd
nd
SFX + PAβN
≤0,06
128
128
32
nd
nd
Abbreviations: IPM (imipenem), EPM (ertapenem), MPM (meropenem), FEP (cefepime), CPO (cefpirome), CAZ (ceftazidime), FOX (cefoxitin),
CM (chloramphenicol) , SFX (sparfloxacine), ND (not determined), PAβN (Phenylalanine arginine β-naphthylamide), Res (Reserpine)
Multidrug resistance phenotype
Multi-Drug Resistance phenotype towards different structurally
unrelated antibiotics in P. stuartii isolates
High level of resistance to cephalosporins of the 4th generation and
unsusceptiblity to carbapenems in clinical isolates
Impact of membrane permeability in this multidrug resistance
phenotype is not known
Negative detection of Metalo-βlactamases
Metalo-beta-lactamase tests: DDST & CDT
6mm Whatman filter paper no. 2
10µg IPM
10mM EDTA (38µg)
IPM +
EDTA
CDT
DDST distance: 1cm
Inhibition ring in mm
DDST
EDTA
EDTA
10mM
Strain
IPM
(10µg)
10mM EDTA
(10µl)
IPM +
EDTA
Synergy
IPM/EDTA
ATCC
22
0
23
-
65237
23
0
23
-
2636
22
0
22
-
NEA16
21
0
21
-
99645
21
0
21
-
19539
20
0
20
-
IPM
10µg
IPM
Outer membrane of Gram Negative bacteria
as a barrier to hydrophilic molecules such as β-lactams or floroquinolons using
OmpF as the main entrance pathway
P. stuartii has OmpF- and
OmpC immuno-related porins
Membrane fraction
1. P. stuartii ATCC 29914
2. P. stuartii 65237
3. P. stuartii 19539
4. P. stuartii NEA16
5. P. mirabilis ATCC 29906
6. P. vulgaris 5860
P. stuartii has two porins sharing 76% identity to each other and
about 50% to E. coli OmpF and OmpC
OmpPst1
OmpPst2
OmpF
OmpC
Alignment of porin sequences from P. stuartii ATCC 29914 (OmpPst1 and OmpPst2) with E. coli general porins
OmpF and OmpC. Abbreviations: β (β-strand), L (loop), T (turn). Residues are colored according to their hydropathy.
The internal loop 3 domain
OmpPst1
OmpPst2
OmpPm
OmpF
OmpC
OmpN
PhoE
In Proteus and Providencia, L3 loop length is well conserved with
important modification of residues compared to E. coli general porins
That may explain
Negative immuno-detection of the antigenic site considered as loop3
marker of enterobacterial porins in Proteus and Providencia strains
Key residues
Important residue modifications compared to OmpF
Residues at the constriction zone of
monomer viewed from the top (Cowan et
al., Nature, 1992)
OmpPst1
K16
L115
W116
A118
E296
OmpPst2
Q16
L110
W111
A113
D299
OmpPm
K16
L113
W114
G116
S283
OmpF
K16
E117
F118
G120
A282
OmpC
K16
E109
F110
G112
S288
A.
OmpF mutant Gly119Asp with 2
subcompartments of 3-4 Å
B.
Wide type OmpF
Jeanteur et al., 1994
OmpF
(340 aa)
OmpC
(346 aa)
OmpPst1
(352 aa)
46.4
53.4
OmpPst2
(343 aa)
46.8
54.5
Semi-conservation of amino acid sequences compared to
enterobacterial general porins
Conservation of typical porin structure with:
16 β-strands,
8 periplasmic turns.
8 extracellular loops
Several porin key residues conserved
Important modification in the internal L3 loop
Alignment of consensus sequences
between P. stuartii isolates
OmpPst1
OmpPst2
Ps ATCC 29914
Ps ATCC 29914
Ps 65237
100%
Ps 65237
100%
Ps 2636
100%
Ps 2636
100%
Ps NEA16
89.2%
Ps NEA16
100%
Ps 99645
88.1%
Ps 99645
100%
Ps 19539
87.9%
Ps 19539
100%
OmpPst1 alignment
ATCC
NEA16
99645
19539
Porin investigation
Bacterial cells were trained for resistance to cefepime (FEP) and imipenem (IPM).
With FEP: the mechanism of porin loss to reduce the intracellular uptake of the
antibiotic was detected.
(1) P. stuartii ATCC 29914
1
2
3
4
5
6
7
(2) P. stuartii ATCC 29914 selected in
FEP at 0.03 µg/ml;
(3) FEP 0.06 µg/ml
(4) FEP 0.09 µg/ml
(5) FEP 0.12 µg/ml
(6) FEP 0.25 µg/ml
(7) FEP 0.5 µg/ml
37 kDa
Ab anti-OmpF
37 kDa
Ab anti-OmpC
Attempt to restore the antibiotic susceptibility
(down regulation or mutation?)
Membrane fraction
1
2
3
4
From the P. stuartii variant selected at 0.5 µg/ml cefepime
5
6
7
1)
0.5µg/ml
2)
without antibiotic 1rst streak
3)
2nd streak
4)
3rd streak
5)
4st streak
6)
5st streak
7)
wild type P. stuartii ATCC 29914
MIC of Cefepim mutants
IPM
ERT
FEP
CPO
CAZ
FOX
CM
SFX
CFX
Ps ATCC 29914
2
≤ 0.06
≤ 0.06
0.125
0.125
4
32
≤ 0.06
≤ 0.06
+ PAβN
2
≤ 0.06
≤ 0.06
0.125
0.125
4
16
≤ 0.06
≤ 0.06
Ps FEP 0.06
4
0.125
0.5
2
8
16
64
≤ 0.06
≤ 0.06
+ PAβN
4
0.125
0.5
2
8
8
16
≤ 0.06
≤ 0.06
Ps FEP 0.5
4
4
8
16
16
64
32
≤ 0.06
≤ 0.06
+ PAβN
4
2
8
16
16
64
8
≤ 0.06
≤ 0.06
16
16
64
+ CLA 6µg/ml
Ps FEP0 – 5 (reversed)
4
2
4
8
16
64
32
≤ 0.06
≤ 0.06
+ PAβN
4
1
4
8
16
64
16
≤ 0.06
≤ 0.06
* The concentration of PAβN used was 20µg/ml
** CLA : clavulanic acid
Membrane fraction of FOX variant strains
selected for MIC test
P. stuartii ATCC 29914 parental
strain selected in cefoxitin
1
2
3
4
5
37 kDa
Ab anti-OmpF
37 kDa
Ab anti-OmpC
1)
Parental strain
2)
1 µg/ml
3)
2 µg/ml
4)
4 µg/ml
5)
8 µg/ml
Membrane fraction of FOX mutants
P. stuartii strain selected at 32 µg/ml cefoxitin
1
2
3
4
5
6
7
1)
32µg/ml
2)
without antibiotic 1rst streak
3)
2nd streak
4)
3rd streak
5)
4st streak
6)
5st streak
7)
wild type P. stuartii ATCC 29914
MIC of FOX variant strains
P. stuartii ATCC 29914 parental strain, resistant to cefoxitin at 1, 2, 4,
32µg/ml
IPM
ERT
FEP
CPO
CAZ
FOX
CM
SFX
CFX
ATCC 29914
2
≤ 0.06
≤ 0.06
0.125
0.125
4
32
≤ 0.06
≤ 0.06
+ PAβN*
2
≤ 0.06
≤ 0.06
0.125
0.125
4
16
≤ 0.06
≤ 0.06
FOX 1
4
≤ 0.06
0.25
0.5
0.5
32
64
≤ 0.06
≤ 0.06
+ PAβN
4
≤ 0.06
0.25
0.5
0.5
32
32
≤ 0.06
≤ 0.06
FOX 4
4
0.5
1
2
2
64
32
≤ 0.06
≤ 0.06
+ PAβN
4
0.5
1
2
2
64
16
≤ 0.06
≤ 0.06
FOX 32
4
1
8
16
16
128
32
≤ 0.06
≤ 0.06
+ PAβN
4
1
8
16
16
128
16
≤ 0.06
≤ 0.06
8
8
8
128
+ 4µg/ml CLA**
FOX0 - 5
4
1
8
16
16
128
64
≤ 0.06
≤ 0.06
+ PAβN
4
1
8
16
16
128
16
≤ 0.06
≤ 0.06
* The concentration of PAβN used was 20µg/ml
** CLA : clavulanic acid
nd: not deternimed
IPM variants
1
2
3
4
5
(1) P. stuartii ATCC 29914;
(2) selected in IPM at 1 µg/ml;
(3) 2 µg/ml;
(4) 4 µg/ml;
(5) 8 µg/ml;
37 kDa
Ab anti-OmpF
37 kDa
Ab anti-OmpC
With IPM: the porin expression level was maintained.
Functional conformation mechanism?
MIC of Imipenem variant strains
P. stuartii ATCC 29914 parental strain, resistant to IPM at 1, 2, 4, 8µg/ml
IPM
ERT
FEP
CPO
CAZ
FOX
CM
SFX
CFX
ATCC 29914
2
≤ 0.06
≤ 0.06
0.125
0.125
4
32
≤ 0.06
≤ 0.06
+ PAβN*
2
≤ 0.06
≤ 0.06
0.125
0.125
4
16
≤ 0.06
≤ 0.06
IPM1
2
0.125
2
4
16
16
64
0.25
≤ 0.06
+ PAβN
2
0.125
2
4
16
16
32
0.125
≤ 0.06
IPM2
8
1
8
128
128
32
64
0.25
0.125
+ PAβN
8
1
8
128
128
32
32
0.125
0.125
IPM4
8
2
16
128
128
32
64
0.25
0.125
+ PAβN
8
2
16
128
128
32
32
0.125
0.125
IPM8
32
8
64
256
512
32
64
0.25
0.125
+ PAβN
32
8
64
256
512
32
32
0.125
0.125
* The concentration of PAβN used was 20µg/ml
No modification of the porin production
Multidrug resistance mechanisms associated
with porin modification.
Pagès JM., James CE. and Winterhalter M.,
Nature Reviews Microbiology, 2008
Loss of porin(s) seems to be the efficient mechanism
for P. stuartii to resist to cephalosporins but NOT for
imipenem in in vitro
Porin natural structure mechanism for imipenem?
Overexpression & extraction of porins
Proteus mirabilis ATCC 29906
Providencia stuartii ATCC 29914 & clinical strains
• Overexpression vector: pGOmpF
• Host strain: E. coli BL21(DE3)omp8, ∆lamB
ompF::Tn5 ∆ompA ∆ompC (Prilipov et al., 1998)
• IPTG induction
• French press
Black lipid bilayer
M. Montal and P. Muellert, Proc. Nat. Acad. Sci. USA,
Vol. 69, No. 12, pp. 3561-3566, December 1972
Reconstitution in BLM – Antibiotic translocation
Single channel (trimer)
conductance (nS) in 1M KCl,
pH 6
Critical voltage for Channel
closure (mV)
OmpF
4
100-150
OmpC
2.5
200-250
OmpPm
2.9 ± 0.2
150-200
OmpPst1
2.5 ± 0.2
more than 200 mV
OmpPst2
3.4 ± 0.2
voltage sensitive (10-50mV)
OmpPst1 in BLM
P. stuartii ATCC 29914
OmpPst1
Homology modeling based on OmpF and OmpC
templates
by Eric Hajjar
Ps ATCC 29914 OmPst1
Extraction: 20µg loaded
denatured
- 1M KCl, pH6.0, 200mV, cis side
intact
OmpPst1 - 1M KCl, pH6.0, 50mV, cis side
5mM Ertapenem
5mM Imipenem
Strong binding effect
25pA
25pA
No binding effect
No translocation?
100ms
100ms
5mM Cefepime
25pA
Good binding effect
100ms
In correlation with MIC results
OmpST1 in the presence of Imipenem reduces ion current
No imipenem
150
5mM imipenem
Current pA
125
100
75
50
25
0
0.0
0.2
0.4
Time (s)
50mV
0.6
0.8
1.0
OmpST1- Imipenem
No antibiotic
2.5mM Imipenem
250
5mM Imipenem
10mM Imipenem
Current,pA
200
150
100
50
0
0.0
0.5
1.0
Time,sec
1.5
2.0
1M KCl, pH 6, 100mV
Neurospora crassa
mitochondrial porin
OmpPst2 in BLM
Ps ATCC 29914
OmpPst2
Modeled by Eric Hajjar
OmpPst2 -
Extraction: 20µg loaded
1M KCl, pH6.0, 50mV,
denatured
Voltage sensitive, closure even at 10 - 20 mV
Impact of K16Q?
intact
OmpST2 5mM Ertapenem
OmpST2 5mM Imipenem
OmpST2 5mM cefepime
75mV
Conclusions
• The unsusceptibility to carbapenems in Providencia stuartii is
hereby reported the first time in molecular level.
• Impermeability is suggested to be the main mechanism of
resistance to carbapenems and/or cephalosporins.
• Decreasing of porin expression is efficient mechanism of
resistance to cephalsporins
• Modification of functional conformation of porins seems to
concern the carbapenems
• The data obtained underlines the flexibility of the bacterial porins
as the gateway in response to antibiotics such as cephalosporins
and carbapenems due to structural nature of porin channel
Acknowledgements
Thank you for your attention !