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Porins Vs the Unsusceptibility to Imipenem in Providencia Stuartii

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Porins Vs the Unsusceptibility to Imipenem in Providencia Stuartii Porins vs the unsusceptibility to imipenem in Providencia stuartii Que-Tien Tran Cagliari, May 4 th 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 Ps 65237 Ps 2636 Ps NEA16 Ps 99645 Ps 19539 ATCC 29914 (lab coll.) (lab coll.) (clinical) (clinical) (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 4 th generation and unsusceptiblity to carbapenems in clinical isolates Impact of membrane permeability in this multidrug resistance phenotype is not known Negative detection of Metalo-β- lactamases Metalo-beta-lactamase tests: DDST & CDT 6mm Whatman filter paper no. 2 10µg IPM 10mM EDTA (38µg) IPM + EDTA IPM CDT 10µg DDST distance: 1cm Inhibition ring in mm DDST EDTA IPM EDTA 10mM IPM 10mM EDTA IPM + Synergy Strain (10µg) (10µl) EDTA 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 - 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 A. OmpF mutant Gly119Asp with 2 subcompartments of 3-4 Å monomer viewed from the top (Cowan et al. , Nature, 1992) B. Wide type OmpF OmpPst1 K16 L115 W116 A118 E296 Jeanteur et al. , 1994 OmpPst2 Q16 L110 W111 A113 D299 OmpPm K16 L113 W114 G116 S283 OmpF K16 E117 F118 G120 A282 OmpC K16 E109 F110 G112 S288 OmpF OmpC (340 aa) (346 aa) OmpPst1 46.4 53.4 (352 aa) OmpPst2 46.8 54.5 (343 aa) Semi-conservation of amino acid sequences compared to enterobacterial general porins Conservation of typical porin structure with: 16 βββ-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 From the P. stuartii variant selected at 0.5 µg/ml cefepime 1 2 3 4 5 6 7 1) 0.5µg/ml 2) without antibiotic 1 rst 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 + PA βN 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 + PA βN 4 2 8 16 16 64 8 ≤ 0.06 ≤ 0.06 + CLA 6µg/ml 16 16 64 Ps FEP0 – 5 (reversed) 4 2 4 8 16 64 32 ≤ 0.06 ≤ 0.06 + PA βN 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 1) Parental strain 2) 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 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 1 rst 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 + PA βN* 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 + PA βN 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 + PA βN 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 + PA βN 4 1 8 16 16 128 16 ≤ 0.06 ≤ 0.06 + 4µg/ml CLA** 8 8 8 128 FOX0 - 5 4 1 8 16 16 128 64 ≤ 0.06 ≤ 0.06 + PA βN 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 + PA βN* 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 + PA βN 2 0.125 2 4 16 16 32 0.125 ≤ 0.06 IPM2 8 1 8 128 128 32 64 0.25 0.125 + PA βN 8 1 8 128 128 32 32 0.125 0.125 IPM4 8 2 16 128 128 32 64 0.25 0.125 + PA βN 8 2 16 128 128 32 32 0.125 0.125 IPM8 32 8 64 256 512 32 64 0.25 0.125 + PA βN 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.
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