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ABSTRACT: Objectives: KHM-1-metallo-β-lactamase-producing Enterobacterales strains, of which only a few have been found, were isolated from four inpatients in Osaka, Japan during 2016 to 2020. We compared whole genomes of the four KHM-1-producing isolates, including one Enterobacter hormaechei subsp. hoffmannii, one Escherichia coli, and two Citrobacter freundii. Methods: These isolates were characterized by whole-genome sequencing, comparative analysis of blaKHM-1-encoding plasmids with earlier reported plasmids, and antimicrobial susceptibility tests. Results: Multilocus sequence typing classified the E. hormaechei subsp. hoffmannii isolate to ST78, the E. coli isolate to ST354, and the two C. freundii isolates to ST95. These isolates harboured various antimicrobial resistance genes aside from blaKHM-1 on their chromosomes and plasmids. In all four isolates, blaKHM-1 was located on 137 kbp to 213 kbp plasmids of IncC replicon type. Although there were common resistance genes such as blaKHM-1-ISEc68, class I integron cassette, and fosG, the four blaKHM-1-encoding plasmids were distinguishable into two lineages based on differences of the resistance gene components and their surrounding regions. Conclusion: Because no epidemiological contact was observed among the inpatients, the blaKHM-1-encoding IncC plasmids might have spread horizontally to multiple bacterial species through repeated recombination and insertion.

ABSTRACT: Objectives: The extensive spread of colistin resistance represents an enormous concern to infectious disease treatment, because colistin is one of the few effective antibiotics against multidrug-resistant bacterial infections, including carbapenem-resistant bacteria. This dissemination can be caused by plasmid transfer containing the colistin resistance gene mcr. Therefore, the plasmid host range affects horizontal gene transfer. This study reports a fusion plasmid of different incompatibility types, which could easily expand the plasmid host range, allowing widespread mcr prevalence in the microbial community. Methods: Genome sequences of colistin-resistant Escherichia coli isolates from stool specimens of healthy human residents in Ecuador were determined using the DNBSEQ and MinION platforms. Hybrid genome assembly was performed using Unicycler, and the genomes were annotated using DFAST. Genome analysis was performed using the Geneious Prime software. Results: Two colistin-resistant E. coli strains isolated separately from different residents presented mcr-carrying plasmids with fused different incompatibility types, IncFIA, IncHIIA, and IncHIIB. The phylogenies of these host bacteria were different. The sizes of the mcr-carrying fusion plasmids pLR-06 and pLR-50 with the full Tn6330 mcr-transposon were 260 Kbp and 198 Kbp, respectively. Both fusion plasmids possessed other resistance genes, including tet(B), tet(M), blaTEM-1b, sul3, cmlA1, aadA1, aadA2, fosA3, and dfrA12. Conclusion: This is the first report of a fusion plasmid comprising different incompatibility types with mcr from colistin-resistant E. coli strains isolated from community residents. The mcr fusion plasmid may play a crucial role in achieving horizontal mcr transmission and the evolution of the multidrug resistance plasmid among hosts.

Objectives: The spread of carbapenemase-producing Enterobacterales (CPE) with colistin resistance is a critical public health issue. We genetically characterized the clinical isolate Enterobacter roggenkampii OIPH-N260, which harboured carbapenemase genes blaIMP-1 and blaGES-5 with multiple resistance genes, including mcr-9 and blaCTX-M-9. Methods: This isolate was characterized by whole-genome sequencing, comparative analysis of resistance plasmids, susceptibility tests, bacterial conjugation, S1-nuclease digested pulsed-field-gel electrophoresis, and Southern blot hybridization. Results: The OIPH-N260 isolate exhibited resistance to most β-lactams and colistin. It co-harboured two resistance plasmids, the blaIMP-1- and blaGES-5-encoding IncP6 plasmid pN260-3 and mcr-9- and blaCTX-M-9-encoding IncHI2 plasmid pN260-1. The comparative analysis of pN260-3 indicated that a unique blaIMP-1-surrounding region was inserted into the blaGES-5-encoding plasmid with the mobile element IS26, which plays an important role in the spread of resistance genes. pN260-1 did not possess the mcr-9 expression regulative gene qseBC. Both plasmids were transferable into other bacterial species via conjugation. Conclusions: This is the first study to report not only a blaIMP-1 and blaGES-5 co-encoding plasmid, but also the co-harbouring of another plasmid carrying mcr-9 and blaCTX-M-9 in Enterobacter cloacae complex. The development of advanced resistance via IS26-mediated insertion and the co-harbouring of resistance plasmids highlights the need to monitor for resistance genes in CPE.

Objectives: To analyse plasmids carrying blaIMP-6 in Klebsiella pneumoniae isolates obtained from multicentre carbapenem-resistant Enterobacteriaceae surveillance. Methods: Plasmids harbouring blaIMP-6 were characterised by the whole-genome sequencing of four Klebsiella pneumoniae isolates carrying blaIMP-6, and compared with the pKPI-6 plasmid, which is widespread in western Japan, through pulsed-field gel electrophoresis, Southern blotting, bacterial conjugation, and qPCR. Results: Whole-genome sequencing analysis revealed that three of the four isolates carried approximately 50 kbp plasmids similar to the pKPI-6 plasmid; however, one isolate carried a 250 kbp plasmid harbouring blaIMP-6 (pE196_IMP6). So far, all of the reported plasmids carrying blaIMP-6 were similar to the pKPI-6 plasmid, and this plasmid was a novel blaIMP6-carrier. The size and transferability of this plasmid was confirmed by Southern hybridisation and conjugation experiments. It was demonstrated that the generation of plasmid pE196_IMP6 was due to an intramolecular transposition mediated by IS26, and a homologous recombination between plasmids pKPI-6 and pE013 that was obtained from another carbapenem-resistant Enterobacteriaceae isolate in this analysis. As a result of co-integration with pE013, pE196_IMP6 acquired six additional pairs of type II toxin-antitoxin systems that pKPI-6 does not carry. Transcription of all of the toxin-antitoxin systems were confirmed in an isolate carrying pE196_IMP6 by qPCR. Conclusions: This study detected a novel plasmid carrying blaIMP-6, and revealed the origin of this plasmid. Toxin-antitoxin system acquisition could enable pE196_IMP6 maintenance persistently through successions, even without selection pressure by the clinical usage of antimicrobials, generating broad dissemination and longer carbapenem-resistant Enterobacteriaceae colonisation duration in patients.

Objectives: Extended-spectrum β-lactamase/AmpC β-lactamase-producing Escherichia coli (ESBL/AmpC-E. coli) are common in developing countries and travellers represent potential reservoirs for their dissemination. The aim of this study was to quantify cefotaxime (CTX)-resistant E. coli harboured by travellers to Vietnam and to conduct a follow-up study. Methods: In total, 19 travellers and 34 travel events were investigated. After confirming that travellers were not colonised with CTX-resistant E. coli before travel, 15 travellers and 20 travel events were studied to quantify travellers harbouring CTX-resistant E. coli after travel. A stool sample (0.1 g) in 10 mL of PBS was diluted and plated. Selected colonies were identified, genotyped and further verified by pulsed-field gel electrophoresis (PFGE). Results: After travel, travellers harboured an average of 3.6 × 107 CFU/g faeces of CTX-resistant E. coli, which continued to be detected for an average of 14 weeks. In the follow-up study, travellers from two travel events had ESBL-E. coli for 12 months. Multiplex PCR showed blaCTX-M-55 and blaCTX-M-27, with replicon types FIA and F, and FIB and F, respectively. PFGE showed that two travellers harboured a single clonal ESBL-E. coli strain for 12 months. By PFGE, the follow-up study showed that a single type of ESBL/AmpC-E. coli was only detected in individuals living in Japan. Moreover, the same clonal ESBL-E. coli isolate was detected in a group that travelled to Ho Chi Minh City. Conclusion: ESBL/AmpC-E. coli colonised the gut of travellers to Vietnam with CTX-resistant E. coli (>107 CFU/g faeces) lasting for 14 weeks.

Objectives: The aim of this study was to report the draft genome sequences of two multidrug-resistant bacteria (Bacteroides thetaiotaomicron F9-2 and Escherichia coli 09-02E) isolated from stool samples of a healthy resident in Vietnam. Methods: Genome sequences were determined using MiSeq and MinION platforms. Genome assembly was performed using Platanus Assembler v.1.2.4 and Canu v.1.7. The DDBJ Fast Annotation and Submission Tool were used for genome annotation. Results: The genome of B. thetaiotaomicron F9-2 comprised 6 283 774 bp with a GC content of 42.7% and 4802 protein coding sequences (CDS), whereas the genome of E. coli 09-02E comprised 5 246 320 bp with a GC content of 50.6% and 4991 protein CDS. Both strains harboured common antimicrobial resistance genes, such as those for sulfonamides (sul2) and aminoglycosides (strA, strB). However, the sul2–strA–strB cassette was located on the chromosome of B. thetaiotaomicron F9-2, whereas it was located on a plasmid in E. coli 09-02E. These genes were flanked by different insertion sequences. Conclusion: Considering their diversities in the human gut resistome, these strains would be of considerable interest for detailed comparative genomic analysis. Notably, the same sul2 cassette was found in facultative and obligate anaerobic bacterial isolates (resident in humans). However, the different location of the cassette indicates a possible mechanism of gene transfer among gut microbes.