Porins vs the unsusceptibility to imipenem in stuartii

QueTien Tran Cagliari, May 4 th 2009 ; ; ; Enterobacteriales; ; Providencia;

TEM picture by Y. Ramay Providencia stuartii

Gramnegative bacteria, Enterobacteriaceae Tribe Proteeae (Proteus, Providencia, Morganella) Straight rods, motile, facultative anaerobic Environment, gastrointestinal flora Opportunistic , hospitalacquired Urinary tract (e.g. kidney stone) Providencia stuartii is the species of the family with isolates the most resistant towards antibiotics

Negative immunodetection of the antigenic site considered as loop3 marker of enterobacterial porins in Proteus and Providencia strains Multidrug resistance phenotype MIC assays with values in g/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 50g/ml 16 256 256128 128 32 32 CM + Res 32g/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 βnaphthylamide), Res (Reserpine) Multidrug resistance phenotype

 MultiDrug 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 Metaloβ lactamases Metalobetalactamase tests: DDST & CDT

6mm Whatman filter paper no. 2 10g IPM

10mM EDTA (38g) IPM + EDTA IPM CDT 10g DDST distance: 1cm

Inhibition ring in mm DDST EDTA IPM

EDTA 10mM IPM 10mM EDTA IPM + Synergy Strain (10g) (10l) 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 βlactams or floroquinolons using OmpF as the main entrance pathway P. stuartii has OmpF and OmpC immunorelated 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 (βstrand), 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 immunodetection 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 34 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)

 Semiconservation of amino acid sequences compared to enterobacterial general porins  Conservation of typical porin structure with:  16 βββstrands,  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 g/ml; (3) FEP 0.06 g/ml (4) FEP 0.09 g/ml (5) FEP 0.12 g/ml (6) FEP 0.25 g/ml (7) FEP 0.5 g/ml

37 kDa Ab antiOmpF

37 kDa Ab antiOmpC Attempt to restore the antibiotic susceptibility (down regulation or mutation?)

Membrane fraction From the P. stuartii variant selected at 0.5 g/ml cefepime

1 2 3 4 5 6 7 1) 0.5g/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 6g/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 20g/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 g/ml 3) 2 g/ml 4) 4 g/ml 5) 8 g/ml

37 kDa Ab antiOmpF

37 kDa Ab antiOmpC Membrane fraction of FOX mutants

P. stuartii strain selected at 32 g/ml cefoxitin 1 2 3 4 5 6 7 1) 32g/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, 32g/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 + 4g/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 20g/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 g/ml; (3) 2 g/ml; (4) 4 g/ml; (5) 8 g/ml;

37 kDa Ab antiOmpF

37 kDa Ab antiOmpC

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, 8g/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 20g/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 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. 35613566, December 1972 Reconstitution in BLM – Antibiotic translocation

Single channel (trimer) Critical voltage for Channel conductance (nS) in 1M KCl, closure (mV) pH 6 OmpF 4 100150 OmpC 2.5 200250 OmpPm 2.9 ± 0.2 150200 OmpPst1 2.5 ± 0.2 more than 200 mV OmpPst2 3.4 ± 0.2 voltage sensitive (1050mV) OmpPst1 in BLM

P. stuartii ATCC 29914 OmpPst1

Homology modeling based on OmpF and OmpC templates by Eric Hajjar Ps ATCC 29914 OmPst1 Extraction: 20g loaded denatured intact 1M KCl, pH6.0, 200mV, cis side OmpPst1 1M KCl , pH6.0, 50mV, cis side 5mM Ertapenem 5mM Imipenem

No binding effect No translocation?

Strong binding effect 25pA 25pA 100ms 100ms

5mM Cefepime

Good binding effect In correlation with MIC results 25pA

100ms OmpST1 in the presence of Imipenem reduces ion current

No imipenem 150 5mM imipenem

125

100

75 CurrentpA 50

25

0 0.0 0.2 0.4 0.6 0.8 1.0 Time (s) 50mV OmpST1 Imipenem

No antibiotic 2.5mM Imipenem 250 5mM Imipenem 10mM Imipenem

200

150

100 Current,pA

50

0 0.0 0.5 1.0 1.5 2.0

Time,sec 1M KCl, pH 6, 100mV Neurospora crassa mitochondrial porin

OmpPst2 in BLM

Ps ATCC 29914 OmpPst2

Modeled by Eric Hajjar Extraction: 20g loaded OmpPst2 1M KCl, pH6.0, 50mV, denatured intact

Voltage sensitive, closure even at 10 20 mV

Impact of K16Q? 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 !