C-158 Genetic and Biochemical Characterisation of OXA-405, a New Extended Spectrum Class D -Lactamase from Serratia marcescens L. Dortet1,2, S. Oueslati1, K. Jeannot2,3, D. Tandé4, F. Deschamps5, T. Naas1,2 and P. Nordmann1,2,6 1INSERM U914, 2Antibiotic Resistance Reference Center, 3Besançon hospital, 4Brest hospital, 5Reims hospital, France, 6Med Microbiol Unit, Dept Medicine, University Fribourg, Switzerland Background Results Antibiograms of the two S. marcescens isolates Negative Carba NP test Positive Carba NP test AMX, Amoxicillin; AMC, Amoxicillin + clavulanate; TIC, Ticarcillin; TCC, Ticarcillin + clavulanate; PIP, Piperacillin; PTZ, Piperacillin + tazobactam; TEM, Temocillin; FOX, Cefoxitin; CTX, Cefotaxime; CAZ, Ceftazidime; FEP, Cefepime; ATM, aztréonam; IMP, Impenem; MEM Meropenem; ETP, Ertapenem; AKM, Amikacin, TMN, Tobramycin; GMI, Gentamicin; NET Netilmicin; TET, Tetracycline; TGC Tigecycline; CHL, Chloramphenicol; FTN, Nitrofurane; CST, Colistin; RAM, Rifampicin; FSF, Fosfomycin; OFX, Ofloxacin; LVX, Levofloxacin; CIP, Ciprofloxacin Protein alignment of OXA-163, OXA-405, OXA-247 and OXA-48 The aim of this study was to investigate the mechanisms responsible for extended-spectrum generation cephalosporins and carbapenem resistance from two Serratia marcescens isolates recovered from the same patient. S. marcescens OXA-405 PTZ PIP TIC AMX ETP TCC CAZ TEM FOX IMP AMC CTX MOX MEM ATM FEP TET TMN AKM GMI NET CHL SXT FTN CST TGC RAM SUL FSF OFX LVX NOR Methods Susceptibility testing Antibiotic susceptibilities were determined by the disk diffusion method and Etest. Results were interpreted according to CLSI guidelines updated in 2014. Strain comparison Genotypic comparison of the two S. marcescens was done by rep-PCR technology using Diversilab (bioMérieux) Carbapenemase detection Carbapenemase production was assessed using the Carba NP test, MALDI-TOF mass spectrometry and UV-spectrophotometry. Plasmid content and conjugation assays Plasmid DNA obtained from the clinical isolates were extracted by using Kieser method. Whole plasmid preparations were electroporated into E. coli TOP10. E. coli NCTC50192, harboring plasmids of 154,66,48 and 7 kb was usued as size marker. Direct ransfert of plasmids in E. coli J53 were attempted using matting out assays at 37°C. Plasmid characterization Plasmid DNA was obtained using a Maxiprep plasmid purification kit (Qiagen). Plasmid incompatibility groups were determined by PCR-based replicon typing scheme. Genetic environment The genetic surrounding structures of -lactamase encoding genes were characterized by PCR mapping and DNA sequencing. Cloning experiments In order to express the different blaOXA-48-like genes in an isogenic background, cloning of the blaOXA-48, blaOXA-405, and blaOXA-163 was performed using a PCR-BluntII-TOPO cloning kit (Invitrogens, followed by selection of clones on plates containing 100mg/L of ticarcillin and 30 mg/L of kanamycin. The PCR fragments comprising the entire blaOXA-48-like genes were obtained with primers preOXA48A (5’-TATATTGCATTAAGCAAGGG-3’) and preOXA48B (5’-CACACAAATACGCGCTAACC3’) Kinetic studies Biochemical characterization was performed using UV spectrophotometry on the supernatant of a whole cell crude extract obtained from an overnight culture of the different clones. Amino-acids involved in the active site of the carbapenemase OXA-48 Antibiotic susceptibility of clinical isolates of S. marcescens and blaOXA-48-like clones in E. coli Specific enzymatic activities of OXA-48, OXA-405 and OXA-163 S. marcescens OXA-48 PTZ PIP TIC AMX ETP TCC CAZ TEM FOX IMP AMC CTX MOX MEM ATM FEP TET TMN AKM GMI NET CHL SXT FTN CST TGC RAM SUL FSF OFX LVX NOR β-Lactams MIC (mg/L) S. marcescens   E.coli TOP10 OXA-48 OXA-405 pTOPO-OXA-48 pTOPO-OXA-405 pTOPO-OXA-163 no plasmid Amoxicillin >256 2 Amoxicillin+ Ac clav 192 96 Piperacillin 128 1.5 Piperacillin + Tazo 12 24 32 1 Temocillin 8 4 Ticarcillin Cefepime 0,25 3 0,032 0.5 0.023 Cefotaxime 1,5 6 0.19 0.06 Ceftazidime 0.25 16 0.12 Imipenem 0,5 Meropenem 0,19 0.094 0,023 0.01 Ertapenem >32 0,75 0.032 Doripenem 0,125 0.064 Aztreonam 0.047 β-Lactams Specific Activity (mU/mg of protein) OXA-48 OXA-405 OXA-163 Amoxicillin 981 485 795 Amoxicillin+ Ac clav 364 643 Piperacillin 450 436 214 Piperacillin + Tazo 168 149 39 Temocillin 10,6 5 4,8 Ticarcillin 647 63 80 Cefepime 4,6 27 30 Cefotaxime 60 117 167 Cefoxitine 1,9 1,2 1,1 Ceftazidime 1,8 9,2 53 Cephalotine 75,4 139,5 130 Imipenem 57 2,6 2 Meropenem 2,8 1,6 Ertapenem 2,2 1,4 Doripenem 1,3 Aztreonam 4,7 13,8 18 Genetic environment of blaOXA-48 and blaOXA-405 Conclusions IncL/M pOXA-48 This is the very first example of successive identification in a same patient of an OXA-48 enzyme with carbapenemase activity (without extended-spectrum activity) followed by identification of a novel OXA-48 variant, OXA-405, losing its carbapenemase activity but gaining a hydrolysis activity against extended-spectrum cephalosporins blaOXA-48 Genomic comparison of the two S. marcescens isolates using rep-PCR IncL/M pOXA-405 S. marcescens OXA-405 S. marcescens OXA-48 Unrelated S. marcescens blaOXA-405