Introduction & Purpose Results Methods Conclusions Acknowledgments

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Introduction & Purpose Results Methods Conclusions Acknowledgments Cooperation between active efflux and porin alteration is sufficient to confer high-level resistance to meropenem in Pseudomonas aeruginosa clinical isolates H. Chalhoub1, Y. Sáenz2, H. Rodriguez-Villalobos3, O. Denis4, B.C. Kahl5, P.M. Tulkens1, F. Van Bambeke1 1 Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium; 2 Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain; 3 Cliniques Universitaires Saint-Luc, Brussels, Belgium; 4 Hôpital Erasme, Brussels, Belgium; 5 University Hospital Münster, Münster, Germany. Françoise Van Bambeke av. Mounier 73, B1.73.05 1200 Brussels - Belgium francoise.vanbambeke@uclouvain.be #P0762 Introduction & Purpose Results Carbapenems are used for treating infections caused by multidrug-resistant Gram-negative bacteria, which may promote the risk of emergence of high-level resistance usually ascribed to carbapenemase expression [1]. Upon screening of a collection of Pseudomonas aeruginosa (Pa) isolates from patients suffering of cystic fibrosis, MICs ≥ 64 mg/L for meropenem were observed in carbapenemase(s) - negative strains. Our aim was to examine whether activity of efflux pumps, alterations of porins and expression of other β-lactamase(s) than carbapenemase(s) could explain the high-level resistance to meropenem in these strains. Figure 1: Phenotypic screening of carbapenemases in meropenem-resistant strains using the Carba NP test [3]. Table 1: Molecular characterization of CF and HAP clinical isolates: expression of efflux systems, porin alterations, expression of -lactamases. Clinical isolates (with patient‘s identification code and date of collection) Expression of genes encoding efflux pumps (relative to PAO1) OprD2 porin sequencing -lactamase mexA mexX mexC no amino acid (WT=443) Amino acid changes in protein sequence Loops affected Insertions/deletions in oprD2 gene CF (DAF69 - 09/09/10) 2.2 0.8 0.0 117 D43N, S57E, S59R, D118STOP L1 Deletion of 2 nt (312-313) AmpC CF (DAF69 – 04/10/10) 3.7 1.7 2.3 CF (DAF69 – 19/10/10) 1.2 0.2 295 D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296STOP L1, L4, L5, L6 - CF (DAF69 – 26/10/10) 14.3 3.1 2.7 CF (DAF69 – 09/11/10) 6.9 2.4 CF (132 - 08/07/12) 0.3 3.5 0.7 228 D43N, S57E, S59R, change in reading frame  229STOP Deletion of 1 nt (410) HAP (DS – 26/12/05) 1.0 441 V127L, E185Q, P186G, V189T, E202Q, I210A, E230K, S240T, N262T, T276A, A281G, K296Q, Q301E, R310E, G312R, A315G, L347M, S403A, R412P* (new mutation in L8), Q424E L2, L3, L4, L5, L6, L7, L8 Shortened loop 7 VIM-2 HAP (DS – 26/01/06) 4.3 2.8 1.4 HAP (DS – 13/02/06) 6.5 13.6 HAP (OG - 08/04/06) 6.4 9.6 5.4 276 V127L, E185Q, P186G, V189T, E202Q, I210A, E230K, S240T, N262T, T276A, W277STOP L2, L3, L4, L5 HAP (OG – 02/05/06) 1.6 0.9 HAP (ND – 10/08/06) 3.4 7.5 HAP (ND – 11/09/06) 4.8 1.3 Non-carbapenemase producer Carbapenemase producer Isolates from HAP (VIM-2) No inoculation Isolates from CF - Imipenem + Methods Carbapenemase phenotypic detection returned negative results for CF strains but positive results for HAP strains (Fig. 1), with presence of blaVIM-2 [metallo-β-lactamase gene] confirmed by PCR. Six meropenem resistant (MEM-R) strains isolated from clinically-confirmed cystic fibrosis (CF) cases were compared to seven MEM-R strains collected from patients suffering from hospital-acquired pneumonia (HAP) (Table 1). Meropenem (MEM) MICs were measured by microdilution in CA-MHB according to CLSI [2] in the absence or presence of the efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN; 20 mg/L [no toxicity at this concentration]). Carbapenemases (VIM, IMP, NDM, OXA-48, KPC), ESBLs blaBEL (BEL-1 to 3), PER (PER-1 to 5, 7), GES (GES-1 to 18), VEB (VEB-1 to 7), CTX-M (1, 2, 9), blaTEM, blaSHV, and blaOXA (1, 2, 9, 10, 18, 20, 23, 24, 30, 58, 198) , and AmpC expression was assessed by molecular techniques (PCR) and/or phenotypic tests (double disk for metallo--lactamases ; ESBL NDP and Carba NP tests [3]). oprD2 gene and its promoter were sequenced. mexA, mexX and mexC transcripts were quantified by qPCR. Figure 2: Influence of the efflux inhibitor (PAβN) on meropenem MIC in CF vs. HAP strains (the horizontal dotted line shows the current meropenem EUCAST susceptibility breakpoint [4]). All clinical isolates showed an increase in transcription levels of mexA, mexX and/or mexC, and mutations in oprD2 gene leading to truncated OprD2 porins. All CF isolates were derepressed for AmpC cephalosporinases. All HAP isolates expressed VIM-2 metallo-β-lactamase. Conclusions Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is as effective as carbapenemases to confer high level resistance to meropenem in strains expressing AmpC. Incomplete restoration of susceptibility upon PAβN addition results from the coexistence of OprD2 mutations, AmpC production and/or possibly also incomplete inhibition of MEM efflux by PAβN at the concentration used. As active efflux can confer cross-resistance to other antipseudomonal agents, i.e. other β-lactams or quinolones for example, determining the mechanism of resistance to meropenem is recommended in clinical settings in order to optimize the antibiotic therapy. References Boyle DP and Zembower TR. Urol Clin North Am. 2015 Nov; 42(4):493-505. Performance Standards for Antimicrobial Susceptibility Testing; 25th Informational Supplement. CLSI document M100-S25. Wayne, PA: Clinical and Laboratory Standards Institute; 2015. Poirel L et al., J Clin Microbiol. 2015 Sep; 53(9):3003-8. European Committee on Antimicrobial Suscpetibility Testing. Breakpoint tables for interpretation of MICs and zone diameters – version 6.0. 2016. H.C. is Boursier of the Belgian Fonds de la recherche dans l’industrie et l’agriculture (FRIA). This work was supported by the Région Wallonne and the Belgian Fonds de la Recherche scientifique. Acknowledgments This poster will be made available after the meeting at http://www.facm.ucl.ac.be/posters.htm Meropenem MICs were decreased of 2 to 4 log2 dilutions in the presence of PAβN for all CF strains but not for HAP strains (Fig. 2).