Complete sequence of carbapenem-resistant clinical isolate co-producing novel class D β-lactamase OXA-1176 and OXA-1177 in Japan.

In 2020, the strain JARB-RN-0044 was isolated from a midstream urine sample of an elderly hospitalized patient in Japan and was highly resistant to carbapenem (i.e., imipenem, meropenem, and doripenem). Whole-genome sequencing revealed that the complete genome consists of two replicons, a 3.5-Mb chromosome and a 1.5-Mb large non-chromosomal replicon which has not been reported in and referred to as the "megaplasmid" in this study based on Cluster of Orthologous Group of proteins functional analysis. The strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D β-lactamase genes and on the megaplasmid. Cloning experiments indicated that recombinant clone expressing gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that gene encodes carbapenemase. In contrast, recombinant clone expressing gene showed increased MICs of piperacillin and cefazolin, but not of carbapenem. Interestingly, the 44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of gene, and comparative analysis with some previously reported isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant isolate may raise human health concerns in Japan, where the population is rapidly aging.IMPORTANCE is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil. The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients and it has been recently recognized as an emerging opportunistic human pathogen. Furthermore, some isolates are resistant to various antimicrobial agents, including β-lactams and aminoglycosides, making antimicrobial therapy challenging and clinically problematic. However, clinical awareness of this pathogen is limited. To our knowledge, in Japan, there has been only one report of a carbapenem-resistant clinical isolate from urine by Suzuki et al. in 2015. In this study, whole-genome sequencing analysis revealed the presence and genetic context of novel and genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to isolate in clinical settings.