Search

ABSTRACT: Objectives: KLUC β-lactamase is a minor extended-spectrum β-lactamase (ESBL) derived from chromosome-encoded cefotaximase in Kluyvera cryocrescens. This study aimed to characterize the genetic context of KLUC-3-producing Escherichia coli and blaKLUC-3-harboring plasmids and assess nosocomial transmission. Methods: In a national genomic surveillance conducted in 2019 and 2020, KLUC-3-producing E. coli strains (JBEAACH-19-0093 and JBEAACH-19-0210) were recovered from two pediatric inpatients in a Japanese hospital. Short- and long-read sequencing analyses using the HiSeq X Five and GridION were performed to determine the complete genome sequences. Results: JBEAACH-19-0093 and JBEAACH-19-0210 belong to the B2-O1:K1:H7-ST95-fimH41 global high-risk clones and carry virulence genes related to extraintestinal pathogenic and uropathogenic E. coli. Single nucleotide polymorphism analysis showed high homology (13 SNPs) between the strains, suggesting nosocomial transmission. The blaKLUC-3 gene was flanked by ISEcp1 and Δorf477 on 100-kb IncB/O/K/Z plasmids with a complete sequence identity. Comparative analysis revealed ISEcp1-mediated transposition of blaKLUC-3 into the IncB/O/K/Z plasmid; the complete plasmid sequence was highly similar to blaCTX-M- and blaCMY-2-harboring plasmids from E. coli ST131 isolates. In GenBank database, 26 Enterobacterales harbored blaKLUC-1 to blaKLUC-7, particularly in Asia. Among them, the genomic structure of blaKLUC-orf477/Δorf477 is conserved in 23 strains. In 13 Enterobacterales, except K. cryocrescens, ISEcp1 was inserted upstream of blaKLUC and 10 strains carried blaKLUC on the plasmids. Conclusion: This is the first case of nosocomial transmission of KLUC-3 producers outside China. The blaKLUC-3 emergence in the virulent pandemic lineage ST95 is a public health problem highlighting the need for further investigations to prevent its potential dissemination.

Objectives: The treatment options available for infections caused by multidrug-resistant Gram-negative pathogens are often limited. Cefiderocol (CFDC) is a novel siderophore cephalosporin that exhibits activity against these pathogens. Several studies have reported the in vitro activity of CFDC against isolates from Europe, the United States, and China, but the activity against carbapenem-resistant bacteria with IMP-type carbapenemase has not been extensively studied. We, therefore, studied the in vitro activities of CFDC against carbapenem-resistant bacteria with available genomic backgrounds based on whole-genome sequencing (WGS) in Japan, where the IMP-type is the predominant carbapenemase produced by Gram-negative rods. Methods: We selected 603 isolates (528 Enterobacterales, 18 Pseudomonas aeruginosa, and 57 Acinetobacter spp.) from a collection of Gram-negative clinical isolates collected during a Japan Antimicrobial Resistance Bacterial Surveillance program and evaluated the antimicrobial activities of CFDC, ceftolozane/tazobactam (CTLZ/TAZ), imipenem-relebactam (IPM/REL), and ceftazidime/avibactam (CAZ/AVI) against carbapenemase-producing Enterobacterales, carbapenemase-non-producing meropenem-non-susceptible Enterobacterales, and carbapenemase-producing nonfermentative bacteria. Results: Among these, 97.7% of carbapenemase-producing Enterobacterales (99.2% of IMP-type carbapenemase-producing Enterobacterales), 100% of carbapenemase-producing P. aeruginosa, and 91.2% of carbapenemase-producing Acinetobacter spp. were susceptible to CFDC, showing better antimicrobial activity than the other antimicrobial agents evaluated in this study. CFDC was highly effective against class A-, B-, and D β-lactamase-harbouring isolates when compared to the other antimicrobial agents. In addition, the relationship between CFDC resistance and three genetic factors involved in resistance was discussed. Conclusions: This is the first large-scale study to systematically demonstrate the efficacy of CFDC against IMP-type carbapenemase-producing strains with known genomic backgrounds.

ABSTRACT The extended-spectrum β-lactamase (ESBL) gene, blaVEB-1, was identified for the first time in an Escherichia coli clinical isolate, JARB-RN-0061, from blood cultures in a Japanese general hospital in 2021. The isolate exhibited high resistance to broad-spectrum cephalosporins, including ceftazidime (MIC >128 mg/L) and cefepime (MIC = 16 mg/L). blaVEB-1 was identified during whole-genome sequencing and characterization of the isolate. JARB-RN-0061 belonged to the B2-O2:K1:H7-ST95-fimH41 lineage and was classified as presumptive extraintestinal pathogenic E. coli (ExPEC) and uropathogenic E. coli (UPEC). Moreover, the strain harbored multiple virulence genes on the chromosome. The Col156/IncFIB(AP001918)/IncFII(29)-type plasmid (114,216 bp), with clbB and tcpC genes involved in bacteremia, was unique to the fimH41 subclone. The blaVEB-1 gene was located on a non-typeable and non-conjugative plasmid, pJARB-RN-0061_VEB-1 (17,093 bp). It was embedded in the class 1 integron In1883-like, with multidrug resistance gene cassettes for aacA4, aadB, cmlA5, qnrVC4, and dfrA14. Notably, comparative analysis of the complete sequence of plasmid pJARB-RN-0061_VEB-1 revealed that it was highly homologous to the blaVEB-1-harboring plasmid, pMS2H7VEB-1 (100% coverage and 99.99% identity), except for the Tn3 family transposon (4,931 bp) and the plasmid pRHBSTW-00138_5 (97% coverage and 100% identity) harbored by Klebsiella quasipneumoniae subsp. similipneumoniae strains from hospital sewage in Japan and wastewater influent in the United Kingdom, respectively. The emergence of a human pathogenic E. coli clinical isolate with the blaVEB-1-carrying plasmid in the B2-ST95 worldwide pandemic lineage, characterized by the virulence potential of ExPEC or UPEC but a low prevalence of antimicrobial resistance, would raise public health concerns.IMPORTANCEESBLs are plasmid-mediated enzymes that confer resistance to clinically significant antimicrobial agents, such as broad-spectrum cephalosporins. Recently, the rapid spread of CTX-M-type ESBL-producing E. coli has become a global issue, including in Japan, where ESBL production in human pathogenic E. coli, such as the ExPEC and UPEC lineages, which typically harbor several virulence genes, is a severe public health concern. To date, VEB (Vietnamese extended-spectrum β-lactamase) producers have been found only in hospital wastewater and rivers in Japan. Thus, we describe the first detection of a very rare human-derived blaVEB-1 gene in the E. coli B2-ST95 pandemic clonal lineage that is highly associated with ExPEC and UPEC in a Japanese clinical setting. Furthermore, we characterized the genomic features of plasmids harboring the class 1 integron-borne blaVEB-1. Our findings highlight the significance of closely monitoring ESBL-producing E. coli isolates to prevent the potential dissemination of this resistance determinant in Japan.

Cloning and transfer of long-stranded DNA in the size of a bacterial whole genome has become possible by recent advancements in synthetic biology. For the whole genome cloning and whole genome transplantation, bacteria with small genomes have been mainly used, such as mycoplasmas and related species. The key benefits of whole genome cloning include the effective maintenance and preservation of an organism's complete genome within a yeast host, the capability to modify these genome sequences through yeast-based genetic engineering systems, and the subsequent use of these cloned genomes for further experiments. This approach provides a versatile platform for in-depth genomic studies and applications in synthetic biology. Here, we cloned an entire genome of an insect-associated bacterium, Spiroplasma chrysopicola, in yeast. The 1.12 Mbp whole genome was successfully cloned in yeast, and sequences of several clones were confirmed by Illumina sequencing. The cloning efficiency was high, and the clones contained only a few mutations, averaging 1.2 nucleotides per clone with a mutation rate of 4 × 10−6. The cloned genomes could be distributed and used for further research. This study serves as an initial step in the synthetic biology approach to Spiroplasma.

ABSTRACTIn 2020, the Ralstonia mannitolilytica 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 R. mannitolilytica, 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 blaOXA-1176 and blaOXA-1177 on the megaplasmid. Cloning experiments indicated that Escherichia coli recombinant clone expressing blaOXA-1176 gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that blaOXA-1176 gene encodes carbapenemase. In contrast, E. coli recombinant clone expressing blaOXA-1177 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 blaOXA-1176 gene, and comparative analysis with some previously reported R. mannitolilytica isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging.IMPORTANCERalstonia mannitolilytica 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 R. mannitolilytica 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 R. mannitolilytica 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 blaOXA-1176 and blaOXA-1177 genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant R. mannitolilytica isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to R. mannitolilytica isolate in clinical settings.

ABSTRACT: Objectives: Linezolid is an antibiotic used to treat infectious diseases caused by vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. Recently, Enterococcus Spp.–carrying mobile linezolid resistance genes were reported. Herein, we report the complete genome sequence of Enterococcus raffinosus JARB-HU0741, which was isolated from a bile sample of a patient in Japan on May 5, 2021, and carries a linezolid resistance gene, cfr(B). Nevertheless, this isolate was susceptible to linezolid. Methods: Whole-genome sequencing was performed using HiSeq X FIVE (Illumina) and GridION (Oxford Nanopore Technologies). The sequence reads were assembled using Unicycler v0.4.8, and the complete genome was annotated using DFAST v1.2.18. Antimicrobial resistance genes were detected with Abricate v1.0.1, using the ResFinder database. The minimum inhibitory concentrations (MICs) were determined using broth microdilution and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. Results: E. raffinosus JARB-HU0741 contained a 3 248 808-bp chromosome and a 1 156 277-bp megaplasmid. cfr(B) was present in the Tn6218-like transposon, which was inserted into a gene encoding a PRD domain–containing protein present in the megaplasmid, but the isolate was susceptible to linezolid (MIC, 0.5 µg/mL). The Tn6218-like transposon was similar to the Tn6218 of Clostridioides difficile Ox3196 and the Tn6218-like transposon of Enterococcus faecium UW11733; however, three genes encoding a topoisomerase, an S-adenosylmethionine-dependent methyltransferase, and a TetR family transcriptional regulator were present in the previous Tn6218- or Tn6218-like transposon. Conclusion: This is the first report of the complete genome sequence of E. raffinosus carrying cfr(B). E. raffinosus carrying cfr(B) without linezolid resistance poses a threat, as it could serve as a reservoir for mobile linezolid resistance genes.

ABSTRACT Colistin, a last-line antimicrobial agent, is recommended for the treatment of severe infections caused by multidrug-resistant (MDR) microorganisms. However, reports on plasmid-mediated mobilized colistin resistance (mcr) genes have prompted the importance of routine colistin resistance surveillance. Herein, we report the emergence of Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1 carrying an mcr-9.1 gene in vegetables from Japan. A total of 308 colistin-resistant isolates were retrieved from 200 fresh vegetables in Hiroshima Prefecture, Japan. PCR detection of mcr-1 to mcr-9 was conducted. While none of the isolates detected positive for mcr-1 to mcr-8 genes, we found two (0.65%) positive strains, E. cloacae CST17-2 and R. ornithinolytica CST129-1, that harbored mcr-9.1 allele. These isolates were subjected to phenotypic susceptibility testing, whole-genome sequencing (WGS), PCR-based replicon typing, and conjugation experiment. We found that both isolates had high colistin resistance [minimum inhibitory concentration (MIC) 16 or >64 µg/mL] and showed MDR phenotypes. WGS of both isolates revealed mcr-9 on a plasmid of the IncHI2/HI2A backbone. The mcr-9-bearing plasmid, pCST17-2_1, was self-transferable, although the pCST129-1_1 plasmid was not. Despite being colistin-resistant, the so-called two-component regulatory operon, qseBC, which induces polymyxin resistance, was absent from the genetic arrangements downstream of mcr-9 in R. ornithinolytica CST129-1. Nonetheless, a conjugation experiment demonstrated that mcr-9 in a Raoultella-type background is capable of mediating colistin resistance. In silico genomic analysis and comparison revealed distinct genetic structures surrounding mcr-9, especially in the downstream vicinities. The E. cloacae CST17-2 strain is of sequence-type ST738, a sequence type that has emerged in mcr-9.1-containing E. cloacae. Remarkably, we report the first mcr-9-carrying colistin-resistant Enterobacteriaceae isolated from Japanese vegetables, which is a grave public health concern. Our findings highlight the importance of strict epidemiological monitoring to track and/or prevent further dissemination of mcr homologs across the vegetable industry. IMPORTANCE Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

ABSTRACT The spread of the gene encoding the NDM-type carbapenemase, bla NDM, poses a serious threat to clinical practice and public health. This gene is usually carried by transferable plasmids, which facilitates its rapid spread among various species of Enterobacterales. Plasmids with the IncX3 replicon play a major role in the spread of bla NDM in Asian countries. In this study, we conducted genomic sequencing and a comparative analysis of the IncX3-type plasmids harboring bla NDM-5 and bla NDM-16b, a single-nucleotide variant of bla NDM-5, isolated in a national antimicrobial-resistant bacterial genomic surveillance in Japan (Japan Antimicrobial Resistant Bacterial Surveillance), which was carried out during 2019–2020. We compared five plasmids harboring bla NDM-5 and one plasmid harboring bla NDM-16b. They were highly identical and differed in several nucleotide substitutions or variations in insertion sequences, although they were carried by different species or genotypes. Notably, bla NDM-16b-carrying pJBBDACG-19-0070 was identical to a plasmid carrying bla NDM-5 except for one nucleotide substitution in the bla NDM gene throughout the 46 kb sequences. Transformants carrying these two plasmids showed comparative resistance to carbapenems, indicating that a substitution on the NDM enzyme (Ala233 to Val) did not affect carbapenem resistance, at least under normal experimental conditions. A database search revealed that several identical plasmids carrying bla NDM-5 were reported from neighboring countries, mainly China, before our surveillance period. Our analysis suggested that the IncX3 plasmid harboring bla NDM-5 is spreading in Japan, and bla NDM-16b emerged from a single nucleotide substitution on the plasmid, with widespread dissemination in Asian countries. IMPORTANCE IncX3 plasmids harboring bla NDM-5 play a major role in the spread of carbapenem resistance in Asia, particularly in China, in clinical and environmental settings. In this study, we present that Enterobacterales isolates carrying IncX3 plasmids harboring bla NDM-5 have been disseminated in Japan, where their identification was previously rare. In addition, bla NDM-16b, a single-nucleotide variant of bla NDM-5, was found to be carried by an identical IncX3 plasmid. A comparative sequence analysis revealed that the bla NDM-16b gene emerged from a single nucleotide substitution on an IncX3 plasmid harboring bla NDM-5. The bla NDM-16b gene did not confer elevated carbapenem resistance compared to bla NDM-5 in our assay using transformants carrying the plasmid harboring either of these genes, although the A233V substitution was reported to confer stability to the enzyme in ion-depleted conditions. Nevertheless, vigilance regarding the emergence of novel variants is required.

ABSTRACT We characterized carbapenemase-producing organism (CPO) detected in municipal wastewater to better understand the epidemiology of CPOs in the community. In total, 36 samples were collected at six sampling sites every other month from December 2020 to October 2021. CPOs were not recovered from influent taken from inlet A receiving separated sewer line, treated effluents, and river water samples upstream and downstream of the effluent outlet. By contrast, 75 CPOs were detected in all influent samples taken from inlets B and C receiving combined sewer lines collecting both domestic/industrial wastewater and rainwater runoff. Aeromonas caviae was the dominant species (25/75, 33.3%), and the other 11 Aeromonas spp. together accounted for 48% of CPOs. The remaining 39 Enterobacterales strains mainly comprised 17 Klebsiella spp. and 10 Raoultella spp. CPOs carrying bla GES carbapenemase genes were overwhelmingly dominant, accounting for 72 of 75 isolates, including two isolates harboring both bla GES-24 and bla IMP-1 (96%), followed by three bla IMPs-positive isolates, where those carbapenemase genes were mainly carried in diverse class 1 integrons. Among bla GES variants, including six new variants (bla GES-47, bla GES-48, bla GES-49, bla GES-50, bla GES-51, and bla GES-54), bla GES-5 was detected in 28 CPOs, with Aeromonas spp. accounting for 53.6% of these organisms. Quantitative analysis revealed that the repeated detection of bla GES-48-positive A. caviae ST1056 from both inlets B and C ranked the total number of this bacterial clone highest in the wastewater influent. In summary, our study revealed the high prevalence and persistence of diverse bla GES carbapenemase genes among CPOs isolated from influent inlets connected to combined sewer systems. IMPORTANCE The emergence and spread of carbapenemase-producing organisms (CPOs) represent a global health threat because they are associated with limited treatment options and poor clinical outcomes. Wastewater is considered a hotspot for the evolution and dissemination of antimicrobial resistance. Thus, analyses of municipal wastewater are critical for understanding the circulation of these CPOs and carbapenemase genes in local communities, which remains scarcely known in Japan. This study resulted in several key observations: (i) the vast majority of bla GES genes, including six new bla GES variants, and less frequent bla IMP genes were carbapenemase genes encountered exclusively in wastewater influent; (ii) the most dominant CPO species were Aeromonas spp., in which a remarkable diversity of new sequence types was observed; and (iii) CPOs were detected from combined sewer wastewater, but not from separate sewer wastewater, suggesting that the load of CPOs from unrecognized environmental sources could greatly contribute to their detection in influent wastewater.

ABSTRACT: Objectives: The occurrence of linezolid resistance in enterococci has recently increased. Here, we report the genomic characterization of Enterococcus faecalis strain JARB-HU0796—isolated from the open pus of a patient in Hiroshima, Japan—which shows nonsusceptibility to linezolid (MIC of 4 µg/mL). Methods: JARB-HU0796 whole-genome sequencing was performed using short-read sequencing with Illumina Hiseq X Five and long-read sequencing using GridION. These reads were collected using the assembly pipeline Unicycler and annotated with DFAST. Antimicrobial resistance genes were detected using the Abricate and ResFinder databases, and the sequence type identified using PubMLST. The antimicrobial susceptibility of JARB-HU0796 was determined with the Eiken dry-plate QH02 system. Results: The JARB-HU0796 complete genome contained a circular chromosome (2 722 585 bp) and two circular plasmids (85 996 bp and 58 872 bp). The chromosome harbours the optrA gene, which confers resistance to oxazolidinones and phenicols. JARB-HU0796 showed nonsusceptibility to linezolid and multidrug resistance to other antibiotics. MLST analysis identified JARB-HU0796 as ST476, similar to the optrA-positive E. faecalis ST476 isolates from swine (South Korea, 2020) and pet food (Switzerland, 2022). The optrA region of JARB-HU0796 is nearly identical to that of ST476 E. faecalis strain TZ2, isolated from humans (China, 2013). Conclusions: To the best of our knowledge, this is the first report of the complete genome sequence of E. faecalis ST476 carrying optrA on a chromosome isolated from a patient in Japan. The strain may have originated in animals, suggesting that the organisms acquired resistance to linezolid because the optrA gene may be closely spread between animals and humans.

ABSTRACT: Objectives: This study aimed to characterize Gram negative bacteria carrying blaGES carbapenemase genes detected in wastewater from a hospital with no history of detection of clinical isolates producing GES carbapenemases. Methods: Six hospital effluent samples were screened for carbapenemase-producing organisms (CPO) using CHROMagar mSuperCARBA and MacConkey agar with 1 µg/mL imipenem. Polymerase chain reaction (PCR) amplification and sequencing of carbapenemase genes, multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing were performed. Results: Among 21 CPO isolates, 11 Klebsiella spp. and 5 Enterobacter kobei isolates carried blaGES-24, and 4 E. roggenkampii and 1 Pseudomonas aeruginosa isolates carried blaGES-5. Genomic analysis of 8 representative isolates comprising 6 blaGES-24-positive and 2 blaGES-5-positive revealed that class 3 integrons with complete or defective Tn402-like transposition modules were predominantly associated with two tandem copies of blaGES-24. Furthermore, a total of 5 new class 3 integrons, In3-18 to In3-22, were identified among 5 blaGES-24 and 1 blaGES-5 plasmids. One strain each of K. pneumoniae subsp. pneumoniae and K. quasipneumoniae subsp. similipneumoniae harboring blaGES-24 plasmids also carried a rare blaVEB-1-positive class 1 integron on a non-typeable plasmid, where these blaVEB-1 plasmids had high sequence similarity. Virulence gene profiles differed between Klebsiella spp. and Enterobacter spp.; the former harbored type III fimbriae cluster, salmochelin, and T6SS type i2 gene clusters, while the latter had curli pili operon, aerobactin, T2SS gene clusters, and T6SS type i3 gene clusters. Conclusion: Our findings confirmed the linkage of blaGES-24 with rare Tn402-like class 3 integrons and the structural diversity of their gene cassette arrays.

ABSTRACT Carbapenemase-producing Enterobacteriaceae (CPE) are one of the most detrimental species of antibiotic-resistant bacteria globally. Phage therapy has emerged as an effective strategy for the treatment of CPE infections. In western Japan, the rise of Klebsiella pneumoniae strains harboring the pKPI-6 plasmid encoding bla IMP-6 is of increasing concern. To address this challenge, we isolated 29 phages from Japanese sewage, specifically targeting 31 K. pneumoniae strains and one Escherichia coli strain harboring the pKPI-6 plasmid. Electron microscopy analysis revealed that among the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) phages belonged to myovirus, siphovirus, and podovirus morphotypes, respectively. Host range analysis showed that 18 Slopekvirus strains within the isolated phages infected 25–26 K. pneumoniae strains, indicating that most of the isolated phages have a broad host range. Notably, K. pneumoniae strain Kp21 was exclusively susceptible to phage øKp_21, whereas Kp22 exhibited susceptibility to over 20 phages. Upon administering a phage cocktail composed of 10 phages, we observed delayed emergence of phage-resistant bacteria in Kp21 but not in Kp22. Intriguingly, phage-resistant Kp21 exhibited heightened sensitivity to other bacteriophages, indicating a “trade-off” for resistance to phage øKp_21. Our proposed phage set has an adequate number of phages to combat the K. pneumoniae strain prevalent in Japan, underscoring the potential of a well-designed phage cocktail in mitigating the occurrence of phage-resistant bacteria. IMPORTANCE The emergence of Klebsiella pneumoniae harboring the bla IMP-6 plasmid poses an escalating threat in Japan. In this study, we found 29 newly isolated bacteriophages that infect K. pneumoniae strains carrying the pKPI-6 plasmid from clinical settings in western Japan. Our phages exhibited a broad host range. We applied a phage cocktail treatment composed of 10 phages against two host strains, Kp21 and Kp22, which displayed varying phage susceptibility patterns. Although the phage cocktail delayed the emergence of phage-resistant Kp21, it was unable to hinder the emergence of phage-resistant Kp22. Moreover, the phage-resistant Kp21 became sensitive to other phages that were originally non-infective to the wild-type Kp21 strains. Our study highlights the potential of a well-tailored phage cocktail in reducing the occurrence of phage-resistant bacteria.

ABSTRACT Epidemiological surveys have shown that carbapenem resistance is mainly transmitted across species by carbapenemase genes located on conjugative plasmids. As chromosomal integration of carbapenemase genes has rarely been identified, only a few studies have investigated their advantages to the carbapenem-resistant bacterial community. Here, we confirmed the increased stability of blaIMP-6 on a chromosome-integrated plasmid in an Escherichia coli isolate compared with that on original plasmids in the absence of antibiotic pressure. Although plasmids carrying carbapenemase genes are supposedly lost in successive generations, we found that the complete plasmid backbone was retained in bacterial cells even after the occasional loss of their antibiotic-resistance cassettes. This backbone structure has been observed worldwide to carry various antimicrobial resistance genes. Although the chromosomally integrated plasmid carrying blaIMP-6 could not be transmitted by conjugation, we found that meropenem treatment for 1 wk allowed the plasmid to be released from the chromosome and spread among E. coli strains that were susceptible to meropenem. The copy number of blaIMP-6 on the plasmid was amplified eight times, resulting in enhanced resistance. Although the carbapenemase producers that carry chromosomal carbapenemase genes comprised of small subpopulations, they functioned as stable, long-term reservoirs of carbapenem resistance that could be disseminated via plasmids with amplified resistance upon meropenem stimulation. Although plasmids occasionally lose their resistance cassettes as a scaffold for the acquisition of another resistance gene, chromosomal integration may contribute to the effective sharing of carbapenem resistance within a population, complicating the development of a strategy to avoid the dissemination of antimicrobial resistance. IMPORTANCE Although carbapenem antibiotics are the last resort for combating multidrug-resistant organisms, global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens public health. Carbapenemases, which are enzymes responsible for carbapenem resistance, are mainly encoded by genes on plasmids that can be transmitted across bacterial species. Owing to the rarity of chromosomally encoded carbapenemase genes, studies investigating their properties in bacterial communities are lacking. In our study, we revealed the stability of carbapenemase genes on chromosomes compared with those on plasmids, which can be lost through the loss of antimicrobial resistance cassettes despite robust retention of plasmid backbones. Following exposure to meropenem, the carbapenemase gene integrated into the chromosome was released as a plasmid, restarting the dissemination of enhanced carbapenem resistance through amplified copy numbers of carbapenemase genes. Chromosomally encoded carbapenemase genes may function as a reservoir of resistance genes within the bacterial community and challenge infection control against CRE dissemination.

ABSTRACT The spread of New Delhi metallo-β-lactamase (NDM)-producing Enterobacterales represents a public health risk. The horizontal transfer of plasmids encoding the NDM gene, blaNDM, usually mediates its spread to other bacteria within the family. In contrast, Enterobacterales with a chromosome-located blaNDM is rarely reported. The phenotypic differences between chromosome- and plasmid-located carbapenemase genes are poorly understood. To determine the significance in terms of the location of drug resistance genes, we examined carbapenemase activity and stability of chromosome- and plasmid-located blaNDM. Escherichia coli M719 possessing both chromosomes- and plasmid-located blaNDM genes was used as a wild-type strain (WT) for the construction of mutants, ΔpblaNDM and ΔcblaNDM, wherein chromosome- or plasmid-located blaNDM, was knocked out, respectively. The mutant ΔpblaNDM showed lower hydrolyzing activity against imipenem and gene expression than the WT or ΔcblaNDM mutant. The MICs of both mutant strains were still above the breakpoint of imipenem and meropenem. Moreover, the chromosome-located blaNDM gene was stable for at least 30 days in the absence of antimicrobial pressure, whereas the ΔcblaNDM mutant lost blaNDM to 87% at 30 days compared to that of the initial inoculum. Organisms harboring the plasmid-located carbapenemase genes were found to provide a higher level of carbapenem resistance than those with chromosome-located genes. However, the latter organisms with chromosomal carbapenemase genes exhibited more stable carbapenem resistance than did the former ones. In summary, chromosomally located carbapenemase genes require further monitoring and more attention should be paid to them. IMPORTANCE Carbapenem-resistant Enterobacterales (CRE) carrying blaNDM have spread worldwide since they were first reported in 2009. Many studies using whole-genome sequencing have identified the genetic structures, plasmid scaffolds of blaNDM, and mechanisms of spread via horizontal transfer. Chromosome-located blaNDM and integration mechanisms from plasmids have rarely been reported, and their significance is not fully understood. Here, we showed that the chromosome-located blaNDM was associated with lower levels of carbapenem resistance and carbapenemase activity than the plasmid-located blaNDM. However, it conferred carbapenem resistance above the breakpoints and the loss of chromosome-located blaNDM was not observed in the absence of antibiotic pressure. This study suggests that CRE strains carrying chromosome-located blaNDM may persist in clinical and environmental settings for a long period even without antibiotic pressure and need to be monitored along with plasmid-located blaNDM.

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.

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

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) infections, high in morbidity and mortality, pose serious clinical challenges due to limited treatment options. A previous CRE surveillance study on 1,507 patients from 43 hospitals in Osaka, Japan, revealed that 12% of patients carried CRE and that 95% of the CRE isolates were IMP-type carbapenemase producers. Here, the mechanisms for this regional dissemination of a single carbapenemase gene were investigated. Since the dissemination of CRE is primarily due to the transmission of carbapenemase genes located on plasmids, we analyzed the plasmidome of 230 CRE isolates carrying blaIMP by whole-genome sequencing and Southern blotting. blaIMP-6 was found to be predominantly disseminated among chromosomally distinct isolates through the pKPI-6 plasmid. Underlying the vast clonal dissemination of pKPI-6, various subpopulations deriving from pKPI-6 were identified, which had acquired advantages for the dissemination of CRE isolates. A cluster exhibiting heteroresistance against meropenem by the transcriptional regulation of blaIMP-6 caused an outbreak likely through covert transmission of blaIMP-6. For stable carriage of blaIMP-6, they occasionally integrated blaIMP-6 on their chromosomes. In addition, we detected one isolate that broadened the range of antimicrobial resistance through a single point mutation in blaIMP-6 on pKPI-6. Multifaceted analysis of the plasmidome granted us more accurate perspectives on the horizontal spread of CRE isolates, which is difficult to trace only by comparing the whole genomes. This study revealed the predominant spread of a specific carbapenemase-encoding plasmid accompanying the emergence of phenotypically diverse derivatives, which may facilitate further dissemination of CRE in various environments. IMPORTANCE Global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens human health by limiting the efficacy of antibiotics even against common bacterial infections. Carbapenem resistance, mainly due to carbapenemase, is generally encoded on plasmids and is spread across bacterial species by conjugation. Most CRE epidemiological studies have analyzed whole genomes or only contigs of CRE isolates. Here, plasmidome analysis on 230 CRE isolates carrying blaIMP was performed to shed light into the dissemination of a single carbapenemase gene in Osaka, Japan. The predominant dissemination of blaIMP-6 by the pKPI-6 plasmid among genetically distinct isolates was revealed, as well as the emergences of pKPI-6 derivatives that acquired advantages for further disseminations. Underlying vast clonal dissemination of a carbapenemase-encoding plasmid, heteroresistance was found in CRE offspring, which was generated by the transcriptional regulation of blaIMP-6, stabilization of blaIMP-6 through chromosomal integration, or broadened antimicrobial resistance due to a single point mutation in blaIMP-6.

Colistin is used as an alternative therapeutic for carbapenemase-producing Enterobacteriaceae (CPE) infections which are spreading at a very high rate due to the transfer of carbapenemase genes through mobile genetic elements. Due to the emergence of mcr-1, the plasmid-mediated colistin resistance gene, mcr-1-positive Enterobacteriaceae (MCRPEn) pose a high risk for the transfer of mcr-1-carrying plasmid to CPE, leading to a situation with no treatment alternatives for infections caused by Enterobacteriaceae possessing both mcr-1 and carbapenemase genes. Here, we report the application of PCR-dipstick-oriented surveillance strategy to control MCRPEn and CPE by conducting the PCR-dipstick technique for the detection of MCRPEn and CPE in a tertiary care hospital in Thailand and comparing its efficacy with conventional surveillance method. Our surveillance results showed a high MCRPEn (5.9%) and CPE (8.7%) carriage rate among the 219 rectal swab specimens examined. Three different CPE clones were determined by pulsed-field gel electrophoresis (PFGE) whereas only two MCRPEn isolates were found to be closely related as shown by single nucleotide polymorphism-based phylogenetic analysis. Whole genome sequencing (WGS) and plasmid analysis showed that MCRPEn carried mcr-1 in two plasmids types—IncX4 and IncI2 with ~99% identity to the previously reported mcr-1-carrying plasmids. The identification of both MCRPEn and CPE in the same specimen indicates the plausibility of plasmid-mediated transfer of mcr-1 genes leading to the emergence of colistin- and carbapenem-resistant Enterobacteriaceae. The rapidity (