Evaluation of the feasibility of EUCAST RAST using antimicrobial disks available in Japan.

BACKGROUND: In November 2018, the European Committee for Antimicrobial Susceptibility Testing (EUCAST) established rapid antimicrobial susceptibility testing (RAST), which could be performed directly on positive blood culture samples. Although concentrations of antimicrobial agents in several antimicrobial disks available in Japan are different from those recommended by the EUCAST, the feasibility of EUCAST RAST using antimicrobial disks available in Japan remains to be evaluated. METHODS: Blood culture bottles spiked with 127 clinical isolates (65 Escherichia coli and 62 Klebsiella pneumoniae) were tested by RAST for cefotaxime (CTX), ceftazidime (CAZ), meropenem, and ciprofloxacin using antimicrobial disks available in Japan, and compared with a reference AST method using automated AST instrument (VITEK®2). RESULTS: The overall category agreement (CA) for RAST using antimicrobial disks available in Japan was 96.3%, 96.8%, and 95.6% after 4, 6, and 8 h of incubations, respectively. However, the CAZ RAST for E. coli showed major error of 8.2% (8 h incubation) for the Sensi disk, 14.3% (6 h incubation), and 24.5% (8 h incubation) for the KB disk. The CTX RAST for K. pneumoniae showed 25% (4 h incubation) and 31.3% (4 h incubation) of very major error for the Sensi and KB disks, respectively. CONCLUSIONS: The EUCAST RAST results for E. coli and K. pneumoniae using antimicrobial disks available in Japan suggest their usefulness, although modified RAST breakpoints are required for several antimicrobial agents.

Emergence of an IncX3 plasmid co-harbouring the carbapenemase genes blaNDM-5 and blaOXA-181.

Background: The spread of transmissible plasmids with carbapenemase genes has contributed to a global increase in carbapenemase-producing Enterobacterales over the past two decades, with blaNDM and blaOXA among the most prevalent carbapenemase genes. Objectives: To characterize an Escherichia coli isolate co-carrying blaNDM-5 and blaOXA-181 (JBEHAAB-19-0176) that was isolated in the Japan Antimicrobial Resistant Bacterial Surveillance in 2019-20, and to evaluate the functional advantage of carrying both genes as opposed to only one. Methods: The whole-genome sequence of the isolate was determined using long- and short-read sequencing. Growth assay and co-culture experiments were performed for phenotypic characterization in the presence of different ß-lactam antibiotics. Results: WGS analysis showed that blaNDM-5 and blaOXA-181 were carried by the same IncX3 plasmid, pJBEHAAB-19-0176_NDM-OXA. Genetic characterization of the plasmid suggested that the plasmid emerged through the formation of a co-integrate and resolution of two typical IncX3 plasmids harbouring blaNDM-5 and blaOXA-181, which involved two recombination events at the IS3000 and IS26 sequences. When cultured in the presence of piperacillin or cefpodoxime, the growth rate of the transformant co-harbouring blaNDM-5 and blaOXA-181 was significantly higher than the transformant with only blaNDM-5. Furthermore, in co-culture where the two blaNDM-5-harbouring transformants were allowed to compete directly, the strain additionally harbouring blaOXA-181 showed a marked growth advantage. Conclusions: The additional carriage of blaOXA-181 confers a selective advantage to bacteria in the presence of piperacillin and cefpodoxime. These findings may explain the current epidemiology of carbapenemase-producing Enterobacterales, in which bacteria carrying both blaNDM-5 and blaOXA-48-like genes have emerged independently worldwide.