Identification of Proteus and Providencia · PDF fileIdentification of Proteus and Providencia...
Transcript of Identification of Proteus and Providencia · PDF fileIdentification of Proteus and Providencia...
Identification of Proteus and
Providencia porins
RTN - MTR meeting
Que-Tien Tran, Marseille, April 11th 2008
Dealing with infectious diseases
• Emergence of nosocomial infections
• Decreasing antibiotic susceptibility among Gram-negativebacteria (GNB) in clinic – Multidrug resistant (MDR) isolates frequently observed
• Inefficacity of conventional antibacterial agents - Intrinsicresistance
• Requirement of new alternative efficacious molecules for therapeutic
• Full understanding about resistance mechanisms and theirregulation is difficult but urgently necessary
The bacterial outer membranes
• First barrier of cell defense
• Molecular filter for hydrophilic molecules or permeability barrierfor hydrophobic substances (especially in enteric bacteria).
• Modification of the permeability may concern several antibioticclasses
• Intrinsic resistance of bacteria
Porins
• Pore-forming proteins in outer membrane of GNB, mitochondria, plastids
• Uptake pathway of small molecules (≤600 Da) includingantibiotics: β-lactams, quinolones…
• Modified porin expression (decreasing or mutation) → change of membrane permeability
• Most studied porins: E. coli OmpF, OmpC & PhoE
• Porins classification:
– General (OmpF, OmpC , PhoE) or specific (LamB, ScrY…)
– Trimer (OmpF, OmpC, PhoE) or monomer (OmpG…)
Internal loop3
A) Trimer viewed from the top. B) Monomer viewed from the side.
C) Residues at the constriction zone of the monomer viewed from
the top (Cowan et al., 1992)
E. coli OmpF
It is now required to extend our expertise concerning the E. coli porins to other less-studied Gram-negative pathogens that cause emerging infectious diseases due to their
high resistance level.
Proteus sp. and Providencia sp.
• GNB, Enterobacteriaceae, tribe Proteeae (Proteus, Providencia, Morganella)
• Straight rods, motile, facultative anaerobic
• Environment, gastrointestinal flora
• Opportunistic pathogens, urinary tract infection (bladder stone) → Risk group 2 (lab accessibility in Marseille)
• Hospital-acquired infection
• They are closed in term of DNA relatedness but distant from E. coli
not yetP. mirabilis by Sanger InstituteGenomic
annotation
Aminoglycosides,
fluoroquinolone, C3G,
carbapenem
Aminoglycosides, quinolone,
C3G-C4G, carbapenem,
azthreonam, ticarcillin-
clavulanic acid, trimethoprime-
sulfamethoxazole
Antibiotic
susceptibility
Providencia stuartii
P. heimbachae
P. rettgeri
P. rustigianii
P. alcalifaciens
Proteus vulgaris
P. mirabilis
P. myxofaciens
Species
Providencia sp.Proteus sp.
Proteus sp. and Providencia sp.
Providencia stuartii
• Cutaneous colonisation
• Species with isolates the most resistant towards antibiotics of the family
• Susceptibility : C3G
Carbapenem
Aminoglycoside
Fluoroquinolone
• Antibiotic resistance
– Cephalosporinase (AmpC)
– ESBL: TEM, SHV, CTX-M, VEB…
– Permeability: LPS modification
Outer membrane proteins ?!
ND1621640.125≤0.06≤0.061P. vulgaris 5860
ND644>2>220.125≤0.062P. mirabilis 99594
ND3240.250.250.25≤0.06≤0.062P. mirabilis ATCC 29906 (PR14)
12851264>512>256>2560,514P. stuartii 2636
ND3264>512>256>256114P. stuartii 99645
3225664>512>256>2561-0.548-4P. stuartii NEA16
1282563225664128≤0.060.252P. stuartii 65237
ND3216410.50.250.252P. stuartii 19539
≤0.06322≤0.06≤0.06≤0.06≤0.06≤0.062P. stuartii ATCC 29914
SFXCMFOXCAZCPOFEPMPMEPMIPM
Abbreviations: IPM (imipenem), EPM (ertapenem), MPM (meropenem), FEP (cefepime), CPO (cefpirome),
CAZ (ceftazidime), FOX (cefoxitin), CM (chloramphenicol) , SFX (sparfloxacine), ND (not determined).
Susceptibility to antibiotics by minimal inhibitory
concentration (MIC in µg/ml)
Analysis of outer membrane proteins
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
Negative detection of the antigenic site located inside porin
which is reported as loop 3 marker of enterobacterial porins
In vitro resistance selection with cefepime
1) P. stuartii ATCC 29914 (no
antibiotic treatment)
2) FEP 0.06 µg/ml
3) FEP 0.09 µg/ml
4) FEP 0.25 µg/ml
5) FEP 0.5 µg/ml
The abbreviation means that
the variant was selected at the
concentration of cefepime
indicated.
Same response observed with cefoxitin
Resistance selection with imipenem
1) P. stuartii ATCC 29914 (no
antibiotic treatment),
2) IPM 1 µg/ml,
3) IPM 2 µg/ml,
4) IPM 4 µg/ml,
5) IPM 6 µg/ml,
6) IPM 8 µg/ml.
The abbreviation indicates that the
variants were selected at the
concentration of imipenem
mentioned.
Analysis of outer membrane proteins
in resistant variants
• Several proteins involved in response to antibiotic by losing or decreasing the expression
• Cefepime & cefoxitin variants present a decrease of production of immunorelated porins
Resistance by lowering cephalosporin uptake
• No resistant variant selected with imipenem exhibit a modification of immunorelated porins
Porin expression in response to imipenem is different than to cephalosporins (at least with the concentrations tested)
The internal loop 3 domain
In Proteus, L3 loop length is well conserved with important
modification of residues compared to E. coli general porins
This may explain the negative detection of the enterobacterial
loop 3 marker by F4 antibody
• Semi-conservation of amino acid sequences compared
to E. coli 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
Proteus porin
Special conformation of the eyelet inside porin ?
Different functional and structural organization of
porin channel ?!
Involvement in antibiotic susceptibility ? Role of L3
loop ?
Discussion on OmpPm
Southern blotting
Templates: genomic DNA
Positive controls: ompPm amplicon
Different restrictases used,
Positively clear signal obtained with HindIII
Probe: Proteus mirabilis ompPm
Hybridization t°: 50°C
PCR for Providencia porin amplification
The products were cloned into pGem-T-easy vector for sequencing
1 2 3 4 5 6 7
Providencia porin sequence compared
to Proteus homolog
The protein sequence is bout 50% identity compared to
OmpF, OmpC and PhoE porins
Adaptor PCR in Providencia stuartii
Ta = 50°C Ta = 45°C
Restrictases : NcoI
NcoI_adaptor
Sequencing: in waiting
Future aims
Expression – Regulation of the porins
Physico-chemical characterization
Antibiotic flux through the porins
…
Acknowledgement
Prof. Mathias Winterhalter
Prof. Matthias Ullrich
Dr. Helge Weingart
Dr. Jean-Marie Pagès
Dr. Anne Davin-Régli
Biophysics group
Microbiology group
Laboratory UMR-MD1,
Facultés de Médecine et de
Pharmacie, Marseille