Although KPC enzymes are most common among carbapenemases produced by Enterobacter cloacae complex globally, the epidemiology varies from one country to another. While previous studies have suggested that IMP enzymes are most common in Japan, detailed analysis has been scarce thus far. Here, we carried out a molecular epidemiological study and plasmid analysis of IMP-1-producing E. cloacae complex isolates collected from three hospitals in central Tokyo using whole-genome sequencing. Seventy-one isolates were classified into several sequence types (STs), and 49 isolates were identified as Enterobacter hormaechei ST78. Isolates of ST78 were divided into three clades by core-genome single nucleotide polymorphism (SNP)-based phylogenetic analysis. Whereas isolates of clade 3 were isolated from only one hospital, isolates of clade 1 and 2 were identified from multiple hospitals. Ten of 12 clade 1 isolates and 1 of 4 clade 2 isolates carried bla _IMP-1 on IncHI2 plasmids, with high similarity of genetic structures. In addition, these plasmids shared backbone structures with IncHI2 plasmids carrying bla _IMP reported from other countries of the Asia-Pacific region. All isolates of clade 3 except one carried bla _IMP-1 in In1426 on IncW plasmids. An isolate of clade 3, which lacked IncW plasmids, carried bla _IMP-1 in In1426 on an IncFIB plasmid. These observations suggest that IMP-producing E. cloacae complex isolates with a diversity of host genomic backgrounds have spread in central Tokyo, and they indicate the possible contribution of IncHI2 plasmids toward this phenomenon.

In this study, we have evaluated the efficacy of calcium-EDTA (Ca-EDTA) as an inhibitor of bacterial metalloenzymes, such as metallo-β-lactamase (MBL) and other proteases, in a mouse model of Pseudomonas aeruginosa pneumonia. The simultaneous presence of Ca-EDTA (32 μg/ml) reduced the MICs of imipenem (IPM) in all MBL-producing P. aeruginosa isolates (IMP-1, -2, -7, and -10 and VIM-2) but not non-MBL-producing strains. In the pneumonia model, mice were intranasally infected with MBL-producing P. aeruginosa and then kept under conditions of hyperoxia to mimic ventilator-associated pneumonia. With both intranasal and subcutaneous administrations, Ca-EDTA significantly potentiated survival benefits of IPM compared to those of IPM alone. Ca-EDTA combination therapy induced a significant reduction of the bacterial burden in the lungs ( P < 0.05). Furthermore, the inhibition activity of Ca-EDTA against MBL activity was confirmed by using the purified IMP-1 enzyme, which was characterized by a 50% inhibitory concentration (IC ₅₀ ) of 55 ± 8.2 μM. Finally, the protective effects of Ca-EDTA were demonstrated by culture supernatant-induced epithelial cell damage and acute lung injury in mice. These data suggest the therapeutic potential of Ca-EDTA not only by the blocking of MBLs but also by neutralizing tissue-damaging metalloproteases in P. aeruginosa infections.

Integrating conjugative elements (ICEs) are mobile genetic elements that can transfer from the chromosome of a host to the chromosome of a new host through the process of excision, conjugation, and integration. Although SXT/R391-related ICEs, originally demonstrated in Vibrio cholerae O139 isolates, have become prevalent among V. cholerae isolates in Asia, the prevalence of the ICEs among Gram-negative bacteria other than Vibrio spp. remains unknown. In addition, SXT/R391-related ICEs carrying genes conferring resistance to extended-spectrum cephalosporins have never been described. Here we carried out a genetic analysis of a cefoxitin-resistant Proteus mirabilis clinical isolate, TUM4660, which revealed the presence of a novel SXT/R391-related ICE, ICE Pmi Jpn1. ICE Pmi Jpn1 had a core genetic structure showing high similarity to that of R391 and carried xis and int genes completely identical to those of R391, while an IS 10 -mediated composite transposon carrying bla _CMY-2 was integrated into the ICE. A nucleotide sequence identical to the 3′ part of IS Ecp1 was located upstream of the bla _CMY-2 gene, and other genes observed around bla _CMY-2 in earlier studies were also present. Furthermore, the nucleotide sequences of hot spot 2 and hot spot 4 in ICE Pmi Jpn1 showed high similarity to that of hot spot 2 in SXT ^MO10 and with a part of the nucleotide sequence found in P. mirabilis ATCC 29906, respectively. ICE Pmi Jpn1 was successfully transferred to Escherichia coli , Klebsiella pneumoniae , Salmonella enterica serovar Typhimurium, and Citrobacter koseri in conjugation experiments. These observations suggest that ICEs may contribute to the dissemination of antimicrobial resistance genes among clinically relevant Enterobacteriaceae , which warrants careful observation of the prevalence of ICEs, including SXT/R391-related ICEs.

ME1071, a maleic acid derivative, is a novel specific inhibitor for metallo-beta-lactamases (MBL). In this study, the potentiation of ME1071 in combination with several beta-lactams was evaluated using MBL-producing Pseudomonas aeruginosa isolates. The rates of susceptibility of MBL producers to carbapenems (imipenem, biapenem, and doripenem) and ceftazidime were increased by 8 to 27% in the presence of 32 microg/ml of ME1071. The corresponding resistance rates were decreased by 13 to 46%, respectively. On the other hand, ME1071 showed weaker or no potentiation with non-MBL producers. The K(i) value of ME1071 for IMP-1 was 0.4 microM, significantly lower than the K(m) values of carbapenems for the IMP-1 enzyme. On the other hand, the K(i) value of ME1071 for VIM-2 was 120 microM, higher than the K(m) values of carbapenems for the VIM-2 enzyme. Results of this study indicate that ME1071 can potentiate the activity of ceftazidime and carbapenems against MBL-producing strains of P. aeruginosa.

Community-associated MRSA, which is a multidrug-resistant organism and can cause infections in otherwise-healthy individuals, has become a global problem. This paper describes a nationwide surveillance conducted in Japan to investigate changes in molecular epidemiological characteristics of CA-MRSA over the past decade and provides a detailed review of the characteristics of Panton-Valentine leucocidin (PVL)-positive strains isolated in 2018.