Ryotaro Eda

The mobile tmexCD‐toprJ gene clusters encode resistance‐nodulation‐division (RND)‐type multidrug efflux pumps which confer resistance to multiple antimicrobials, including tigecycline. Here we report the first identification of tmexCD‐toprJ‐ harbouring Pseudomonas aeruginosa strain KAM950, isolated from hospital sewage in Japan in 2022. The isolate exhibited reduced susceptibility to tigecycline and carbapenems. Complete genome sequence analysis showed that KAM950 belongs to sequence type 244 (ST244) according to multilocus sequence typing, an internationally recognised epidemic clone, and harbours multiple antimicrobial resistance genes, including the tmexCD‐toprJ variant, tmexC6D6‐toprJ1b . Notably, the tmexC6D6‐toprJ1b gene cluster was located on the chromosome, adjacent to the transcriptional regulator gene tnfxB6 and an IS 5 /IS 1182 family transposase gene. Furthermore, an IS 4 ‐mediated disruption of the porin gene oprD was observed, potentially contributing to carbapenem resistance. BLASTn analysis revealed that the IS 5 /IS 1182‐tnfxB6‐tmexC6D6‐toprJ1b gene cluster present in both chromosomal and plasmid sequences among the order Pseudomonadaceae , indicating potential horizontal gene transfer of tnfxB6‐tmexC6D6‐toprJ1b mediated by IS 5 /IS 1182 . Our findings highlight the ongoing expansion of variant diversity and geographic spread of tmexCD‐toprJ ‐like gene clusters, and underscore the importance of genomic surveillance for emerging antimicrobial resistance determinants in both clinical and environmental settings.

Background: Numerous biomarkers have been identified for the diagnosis of periprosthetic joint infection (PJI), but no single biomarker has been proven to have definitive accuracy. Alpha-defensin is an excellent biomarker that is included as a minor criterion in the 2nd International Consensus Meeting (ICM) PJI diagnostic criteria; however, its high cost limits its accessibility at many general medical facilities. Consequently, alternative biomarkers for PJI diagnosis are under investigation. Myeloperoxidase (MPO), an enzyme with microbicidal properties through the catalysis of hypochlorous acid production, has emerged as a promising alternative. MPO has previously been reported as an effective biomarker for PJI. In the present study, we conducted a rapid MPO test as a point-of-care test (POCT) and evaluated its diagnostic utility. Methods: Patients with a suspected infection after total joint arthroplasty were categorized into the PJI or aseptic failure groups according to the 2nd ICM PJI diagnostic criteria. MPO and alpha-defensin levels in synovial fluid that was collected during surgery were quantified using an enzyme-linked immunosorbent assay (ELISA), and their diagnostic accuracy was compared. Additionally, an MPO POCT was conducted, and its diagnostic accuracy was assessed. Results: The investigation included 23 patients with PJI and 23 with aseptic failure. All were Japanese. MPO and alpha-defensin ELISAs exhibited a strong correlation, and, at the optimal cutoff, both tests demonstrated a sensitivity of 0.957 and a specificity of 0.957. The MPO POCT produced results in just 10 minutes and achieved a sensitivity of 1.00 and a specificity of 0.913. Conclusions: The MPO ELISA demonstrated high diagnostic accuracy for PJI, equivalent to that of the alpha-defensin test. Furthermore, the MPO POCT showed similarly high diagnostic accuracy. The MPO POCT is a promising and rapid tool for diagnosing PJI and could serve as an alternative to the alpha-defensin test. Level of Evidence: Diagnostic Level II . See Instructions for Authors for a complete description of levels of evidence.

Daptomycin (DAP) is one of the most potent antibiotics used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Due to an increase in its administration for combating MRSA infections, DAP non-susceptible (DAP-NS) MRSA strains have recently been reported in clinical settings. The presence of single nucleotide polymorphisms (SNPs) in the multiple peptide resistance factor (mprF) gene is the most frequently reported cause for the evolution of DAP-NS MRSA strains; however, there are some variations of SNPs that could lead to DAP-NS. In this study, we used two clinical MRSA strains, including DAP susceptible (DAP-S) and DAP-NS, isolated from the same patient at different time points. We introduced T345I SNP to mprF of the DAP-S MRSA strain using the gene exchange method with pIMAY vector. Further, we investigated the phenotype of the mutant strain, including drug susceptibility, cell surface positive charge, and growth speed. The mutant strain exhibited (i) resistance to DAP, (ii) up-regulation of positive surface charge, (iii) slower growth speed, and (iv) thickened cell walls. Hence, the SNP in mprF may have caused an up-regulation in MprF function, with a subsequent increase in positive surface charge. Cumulatively, these results demonstrated that the T345I amino acid substitution in mprF represents one of the primary causes of DAP-NS in MRSA strains.

Purpose: New Delhi metallo-β-lactamase 5 (NDM-5) shows stronger resistance to carbapenems and broad-spectrum cephalosporins than NDM-1 because NDM-5 differs from NDM-1 by two amino acid substitutions. In this study, our aim was to characterize a NDM-5-producing Escherichia coli isolate KY1497 from a patient with urinary tract infection in Japan, who had no recent history of overseas travel. Patients and Methods: NDM-5-producing E. coli isolate KY1497 was detected in the urine sample of a patient hospitalized in a tertiary hospital in Japan. The complete genome sequence of isolate KY1497 was determined by short- and long-read sequencing with hybrid assembly, followed by multilocus sequence typing (MLST), core-genome phylogeny analysis, plasmid analysis, and transconjugation experiments. Results: KY1497 was classified as ST405 by MLST, and core-genome phylogeny exhibited the closest lineage to the clinical isolates in Nepal (IOMTU605) and Canada (FDAARGOS_448). KY1497 harbors bla_NDM-5 in the IncFII-IncFIB(pB171) replicon plasmid (pKY1497_1, 123,767 base pairs). Plasmid analysis suggested that the cognate plasmids of pKY1497_1 have a minor plasmid background, rather than the globally disseminated IncX3 plasmid carrying bla_NDM-5. Transconjugation analysis revealed that pKY1497_1 is transmissible to the recipient E. coli J53 strain. Conclusion: We characterized a novel Inc replicon plasmid (IncFII-IncFIB[pB171]) carrying bla_NDM-5 and its host E. coli strain. NDMs are associated with a high risk of infection worldwide because of their antibiotic resistance and untreatable and hard-to-treat infections. Other patients in the hospital showed negative results for carbapenem-resistant Enterobacteriaceae. As NDM-producing strains are only sporadically detected in Japan, attention should be provided to the community prevalence of NDM-producing E. coli strains to prevent nosocomial infections.