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The worldwide dissemination of carbapenem-resistant Enterobacteriaceae (CRE) poses a critical human health issue by limiting the range of antibiotics that are usable in the treatment of common bacterial infections. Along with CRE, carbapenem heteroresistance has disseminated worldwide, which is described as different levels of carbapenem resistance within a seemingly isogenic bacterial population. Unstable carbapenem resistance will likely lead to unexpected treatment failure due to the enhanced resistance after initiation of treatment, contradicting antimicrobial susceptibility test results. Porin mutation and tandem amplification of the carbapenemase gene have been reported as mechanisms underlying enhanced carbapenem resistance. In this study, we identified multimerization of plasmids carrying carbapenemase genes, by using Southern blotting, whole-genome sequencing, and quantitative PCR (qPCR) analysis for the CRE isolates obtained in our previous surveillance in Osaka, Japan. Plasmids harboring a carbapenemase gene were multimerized by recA , likely through recombination at two consecutive sets of transposase genes of the IS 91 family, thereby producing various plasmids of discrete sizes in a single bacterial cell of an Escherichia coli isolate. This multimerization resulted in increased copy numbers of carbapenemase genes, leading to enhanced gene transcription as well as carbapenem resistance. Prior exposure to meropenem further increased the copy number of carbapenemase genes, readily resulting in enhancement of carbapenem resistance. This mechanism may lead to clinical treatment failure by sifting antimicrobial resistance after the treatment initiation. IMPORTANCE We demonstrated the multimerization of plasmids harboring carbapenemase genes, and multimeric plasmids of various discrete sizes existed in a host bacterial cell of Escherichia coli. Plasmid multimerization along with increased copy numbers of carbapenemase genes resulted in enhanced carbapenemase resistance, which was readily accelerated by an overnight preexposure to meropenem. This mechanism may lead to treatment failure in clinical settings after the initiation of antimicrobial therapy.

The emergence and global spread of extended-spectrum or AmpC β-lactamase (ESBL/AmpC)-producing Enterobacteriaceae in companion animals have led to the hypothesis that companion animals might be reservoirs for cross-species transmission because of their close contact with humans. However, current knowledge in this field is limited; therefore, the role of companion animals in cross-species transmission remains to be elucidated. Herein, we studied ESBL/AmpC-producing Enterobacteriaceae, Escherichia coli in particular, isolated from extraintestinal sites and feces of companion dogs. Whole-genome sequencing analysis revealed that (i) extraintestinal E. coli isolates were most closely related to those isolated from feces from the same dog, (ii) chromosomal sequences in the ST131/C1-M27 clade isolated from companion dogs were highly similar to those in the ST131/C1-M27 clade of human origin, (iii) certain plasmids, such as IncFII/pMLST F1:A2:B20/blaCTX-M-27, IncI1/pMLST16/blaCTX-M-15, or IncI1/blaCMY-2 from dog-derived E. coli isolates, shared high homology with those from several human-derived Enterobacteriaceae, (iv) chromosomal blaCTX-M-14 was identified in the ST38 isolate from a companion dog, and (v) eight out of 14 tested ESBL/AmpC-producing E. coli isolates (i.e., ST131, ST68, ST405, and ST998) belonged to the human extraintestinal pathogenic E. coli (ExPEC) group. All of the bla-coding plasmids that were sequenced genome-wide were capable of horizontal transfer. These results suggest that companion dogs can spread ESBL/AmpC-producing ExPEC via their feces. Furthermore, at least some ESBL/AmpC-producing ExPECs and bla-coding plasmids can be transmitted between humans and companion dogs. Thus, companion dogs can act as an important reservoir for ESBL/AmpC-producing E. coli in the community., Competing Interests: The authors have declared that no competing interests exist.

KHM-1 was first reported in 1997 in Japan as a novel metallo-β-lactamase mediated by Citrobacter freundii carrying pKHM-1 plasmid. There have been few reports in the clinical field since then. A blaKHM-1-positive Enterobacter hormaechei subsp. hoffmannii in E. cloacae complex, isolate OIPH-N069 was isolated from an inpatient blood culture in 2016. The isolate was characterized by whole-genome sequencing, comparative analysis of the blaKHM-1 encoding plasmid, antimicrobial susceptibility tests, and bacterial conjugation. OIPH-N069 was classified into ST78 of E. cloacae complex, and was multidrug resistant because of the presence of antimicrobial resistance genes in addition to blaKHM-1 on its chromosome and plasmids. blaKHM-1 was located on 136,816 bp of the IncA/C2 plasmid pN069-1, which could be transferred to different bacterial species. The backbone structure, genetic arrangement of the class 1 integron cassette, and the blaKHM-1 gene located downstream of the IncA/C2 antibiotic resistance island, ARI-A, in pN069-1 and pKHM-1 were identical. Horizontal gene transfer of the blaCTX-M-2-ISEcp1 resistance gene module only occurred with pN069-1. The study findings indicate not only the structural conservation of blaKHM-1 encoding plasmids over time and across species, but also the risk of the spread of blaKHM-1 encoding plasmids to other bacterial species and the accumulation of additional resistance genes., Competing Interests: The authors have declared that no competing interests exist.

Background: BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. Results: In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1–3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. Conclusion: This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains. [ABSTRACT FROM AUTHOR]

KHM-1 was first reported in 1997 in Japan as a novel metallo-β-lactamase mediated by Citrobacter freundii carrying pKHM-1 plasmid. There have been few reports in the clinical field since then. A blaKHM-1–positive Enterobacter hormaechei subsp. hoffmannii in E. cloacae complex, isolate OIPH-N069 was isolated from an inpatient blood culture in 2016. The isolate was characterized by whole-genome sequencing, comparative analysis of the blaKHM-1 encoding plasmid, antimicrobial susceptibility tests, and bacterial conjugation. OIPH-N069 was classified into ST78 of E. cloacae complex, and was multidrug resistant because of the presence of antimicrobial resistance genes in addition to blaKHM-1 on its chromosome and plasmids. blaKHM-1 was located on 136,816 bp of the IncA/C2 plasmid pN069-1, which could be transferred to different bacterial species. The backbone structure, genetic arrangement of the class 1 integron cassette, and the blaKHM-1 gene located downstream of the IncA/C2 antibiotic resistance island, ARI-A, in pN069-1 and pKHM-1 were identical. Horizontal gene transfer of the blaCTX-M-2–ISEcp1 resistance gene module only occurred with pN069-1. The study findings indicate not only the structural conservation of blaKHM-1 encoding plasmids over time and across species, but also the risk of the spread of blaKHM-1 encoding plasmids to other bacterial species and the accumulation of additional resistance genes. [ABSTRACT FROM AUTHOR]

In this study, we characterized the first clinical Klebsiella pneumoniae strain co- harboring mcr-1 and blaNDM-4 genes in Vietnam, which was recovered from a patient admitted to hospital in 2015. This strain demonstrated nonsusceptible to all tested antibiotics, including last-line antibiotics such as carbapenems (MICs ≥128 μg/mL) and colistin (MIC =32 μg/mL), except tigecycline (MIC =1 μg/mL). Whole-genome analysis using both MinION and MiSeq data revealed that the strain carried 29 resistance genes. Particularly, mcr-1 and blaNDM-4 genes were carried by different self-conjugative plasmids and able to be transferred to a recipient by conjugation. The colistin resistance of this strain was conferred by mcr-1 and additional chromosomal resistance determinants. Eight amino acid substitutions found in PmrA, PmrB, PmrC, PmrI, and PmrJ, all proteins that are involved in lipopolysaccharide modifications, may be associated with chromosomal colistin resistance. The accumulation of multiple antibiotic resistance mechanisms in this clinical isolate raises alarm on potential spread of extensively drug-resistant K. pneumoniae in healthcare settings. [ABSTRACT FROM AUTHOR]

The co-production of MCR and carbapenemase in Enterobacteriaceae has been previously reported. Here, we describe a clinical strain of Escherichia coli from Vietnam carrying both mcr-1 and bla NDM–1. Whole-genome sequencing showed that the genome of this strain consists of a 4,975,832-bp chromosome and four plasmids. The mcr-1 and bla NDM–1 genes are located on IncI2 and IncA/C2-type plasmids, respectively. Genetic analysis revealed the presence of a multidrug-resistant region with the structure of a novel complex class 1 integron including a class 1 integron region bearing two 5′ conserved segments and one 3′ conserved segment and two complete structures of IS CR1. The complex integron contains aminoglycoside resistance genes aadA2 , aadB , strA , strB , and aphA6 , quinolone resistance gene qnrA1 , extended-spectrum β-lactamase gene bla OXA–4, and a Tn 125 -like transposon bearing bla NDM–1. In addition, the dfrA12 - gcuF - aadA2 - cmlA1 - aadA1-qacH gene cassette array belonging to the sul3 -type integron was also identified, but the region found downstream of the gene cassette array is the IS 440 - tet(M) -IS 26 element instead of the sul3 gene. The results further support that Enterobacteriaceae isolates co-harboring mcr and bla NDM are widely being distributed. The structural characteristics of the complex integron reveal that IS CR1 elements play an important role in the mobilization of bla NDM–1 and the development of multidrug-resistant regions. [ABSTRACT FROM AUTHOR]