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Horizontal gene transfer may occur between distantly related bacteria thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an Enterobacterial species responsible for human infections that may express various acquired ß-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis could be detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobactericeae we used a whole genome sequencing approach that revealed the presence of blaOXA-58 class D carbapenemase gene, so far only identified in Acinetobacter sp.. This gene was located on a 3.1-kb element co-harboring a blaAmpC-like gene. Remarkably these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small and large subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase and the first description of a XerC-XerD dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) represents a major challenge for microbiology laboratories. We evaluated the BYG Carba v2.0 using a simplified protocol, which detects CPE in less than 30 minutes. This new procedure reduces the hands-on-time from 5 to one minute and only requires a limited amount of material (one to three colonies) thereby preventing the need for subculturing bacterial isolates to reach a larger amount of pure biomass. This multicentre study involved four European reference laboratories. For the 1181 isolates tested across four centres, BYG Carba v2.0 yielded overall sensitivity and specificity of 96.3% (CI95: 94.5-97.5) and 99.7% (CI95: 98.6-100) respectively. Considering only the 670 consecutive isolates tested prospectively, the BYG Carba v2.0 displayed overall positive and negative predictive values of 99.7% (CI95: 95.4-98.9) and 97.5% (CI95: 94.9-98.8). Regarding time to positivity, 85% of CPE detected were positive within ten minutes. The BYG Carba v2.0 is a new highly simplified, rapid and accurate electrochemical assay discriminating between CPE and non-CPE in less than 30 min. The real-time quantified signal allows objective and traceable interpretation of the results.

Transpositional DNA rearrangements importantly contribute to the genomic plasticity of bacteria, their viruses and plasmids. Interestingly, enzymatically mediated transposition is not limited to propagating bacteria, but it also occurs in prolonged periods of rest. As revealed with RFLP analysis, genetic polymorphism increases steadily upon storage of bacteria in the resting phase. These results are discussed here in the wider context of accumulating knowledge on molecular mechanisms contributing to overall spontaneous mutagenesis, which is the result of a multitude of specific, often enzyme-mediated processes of variation generation

OXA-48-like beta-lactamase producing bacteria are now endemic in several European and Mediterranean countries. Among this carbapenemase family, the OXA-48 and OXA-181 variants predominate, whereas other variants such as OXA-204 are rarely reported. Here, we report the molecular epidemiology of a collection of OXA-204- positive enterobacterial isolates (n = 29) recovered in France between October 2012 and May 2014. This study describes the first outbreak of OXA-204-producing Enterobacteriaceae in Europe, involving 12 isolates of an ST90 Escherichia coli clone and nine isolates of an ST147 Klebsiella pneumoniae clone. All isolates co-produced the cephalosporinase CMY-4, and 60% of them co-produced the extended-spectrum beta-lactamase CTX-M-15. The blaOXA-204 gene was located on a 150-kb IncA/C plasmid, isolated from various enterobacterial species in the same patient, indicating a high conjugative ability of this genetic vehicle.

Objectives The objective of this study was the evaluation of the performance of two commercially available biochemical tests for the rapid detection of carbapenemase-producing Enterobacteriaceae compared with a home-made technique. Methods A collection of 150 enterobacterial isolates, including 132 isolates with decreased susceptibility to at least one carbapenem molecule, were tested for carbapenemase activity using the RAPIDEC® CARBA NP (bioMérieux), the Rapid CARB Screen® (Rosco Diagnostica) and the home-made Carba NP test. This strain collection included 55 non-carbapenemase producers, 21 KPC producers, 21 NDM producers, 17 VIM producers, 11 IMP producers, 16 OXA-48 producers and 9 OXA-48-like producers (OXA-162, OXA-181, OXA-204, OXA-232 and OXA-244). Results The RAPIDEC® CARBA NP detected all carbapenemase producers except a single OXA-244 producer. Using the Rapid CARB Screen®, one KPC-2, two NDM-1, one OXA-48 and five OXA-48 variant producers gave equivocal results and one OXA-244 producer was not detected. Using the Carba NP test, the same OXA-244 producer was not detected and one OXA-181 producer and one OXA-244 producer gave equivocal results. Sensitivity and specificity were 99% (95% CI 94.3%-99.8%) and 100% (95% CI 93.5%-100%), respectively, for the RAPIDEC® CARBA NP test, 89.5% (95% CI 81.7%-94.2%) and 70.9% (95% CI 57.9%-81.2%) for the Rapid CARB Screen® and 96.8% (95% CI 91.1%-98.9%) and 100% (95% CI 93.5%-100%) for the Carba NP test. The impact of the use of an adequate bacterial inoculum for obtaining the optimal performance with the RAPIDEC® CARBA NP was noted. Conclusions The RAPIDEC® CARBA NP possesses the best performance for rapid and efficient detection of carbapenemase-producing Enterobacteriaceae

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) represents a major challenge for microbiology laboratories. We evaluated the BYG Carba v2.0 using a simplified protocol, which detects CPE in less than 30 minutes. This new procedure reduces the hands-on-time from 5 to one minute and only requires a limited amount of material (one to three colonies) thereby preventing the need for subculturing bacterial isolates to reach a larger amount of pure biomass. This multicentre study involved four European reference laboratories. For the 1181 isolates tested across four centres, BYG Carba v2.0 yielded overall sensitivity and specificity of 96.3% (CI95: 94.5-97.5) and 99.7% (CI95: 98.6-100) respectively. Considering only the 670 consecutive isolates tested prospectively, the BYG Carba v2.0 displayed overall positive and negative predictive values of 99.7% (CI95: 95.4-98.9) and 97.5% (CI95: 94.9-98.8). Regarding time to positivity, 85% of CPE detected were positive within ten minutes. The BYG Carba v2.0 is a new highly simplified, rapid and accurate electrochemical assay discriminating between CPE and non-CPE in less than 30 min. The real-time quantified signal allows objective and traceable interpretation of the results., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: We investigated the molecular mechanism of ß-lactam resistance in extended-spectrum ß-lactamase (ESBL)-producing Enterobacterial strains isolated in neonatal units of different hospitals in Anatnanarivo, Madagascar.Methods: Bacteria were identified by standard biochemical methods, disc diffusion antibiograms and Etest. Resistance genes were sought by PCR. Strains were characterized by Rep- PCR (Diversilab), plasmid analysis and rep-typing.Results: From April 2012 to March 2013, 29 ESBL-producing E. cloacae and 15 K. pneumoniae were isolated from blood culture (n = 32) or gastric samples (n = 12) performed at day 0 or 2 from 39/303 newborns suspected of early neonatal infection. These infants were treated with expanded spectrum cephalosporins, due to lack of carbapenems, leading to a high mortality rate (45 %). Isolates recovered were all, but 4, multidrug resistant, particularly to fluoroquinolones (FQ) except for 21 E. cloacae isolates. Isolates produced TEM-1 and CTX-M-15 ß-lactamases and their genes were located on several self- transferable plasmids of variable sizes sizes that could not be linked to a major plasmid incompatibility group. E. cloacae isolates belonged to 6 Rep-types among which two counted for 11 isolates each. The FQ resistant E. cloacae isolates belonged to one clone, whereas the FQ susceptible E. cloacae isolates belonged to four clones. The K. pneumoniae isolates belonged to 9 Rep-types among which one included five isolates.Conclusion: This study is the first molecular characterization of ESBL- producing isolates from neonatology units in Madagascar, a country with limited epidemiological data. It revealed an important multi-clonal dissemination of CTX-M-15- producing isolates reflecting both the high community carriage and the very early nosocomial contamination of the neonates.