Transmission of global clones of NDM-producing Enterobacterales and interspecies spread of IncX3 plasmid harbouring blaNDM-5 in Tokyo.

OBJECTIVE: The aim of this study is to characterise the molecular characteristics of NDM-producing Enterobacterales, which have been on the increase in recent years in Japan, where IMP-producing bacteria are dominant among carbapenemase-producing Enterobacterales. METHODS: We collected 21 strains of NDM-producing Enterobacterales detected between 2015 and 2022 at five hospitals in Tokyo and performed illumina whole genome sequencing. For the seven selected strains, nanopore long-read sequencing was also performed to characterise the plasmids harbouring blaNDM. RESULTS: Fourteen strains were Escherichia coli and all carried blaNDM-5. Among these strains, eight and three were sequence type (ST) 410 and ST167, respectively, and both groups of strains were spread clonally in different hospitals. Two strains of Klebsiella pneumoniae ST147 carrying blaNDM-1 were detected in a hospital, and these strains had also spread clonally. The remainder included Enterobacter hormaechei, Klebsiella quasipneumoniae, Citrobacter amalonaticus, and Klebsiella michiganensis. Plasmid analysis revealed that an identical IncX3 plasmid harbouring blaNDM-5 was shared among four strains of different bacterial species (E. coli, C. amalonaticus, K. michiganensis, and E. hormaechei) detected at the same hospital. In addition, a Klebsiella quasipneumoniae strain detected at a different hospital also carried an IncX3 plasmid with a similar genetic structure. CONCLUSIONS: Nosocomial spread of multiple multidrug-resistant global clones and transmission of IncX3 plasmids harbouring blaNDM-5 among multiple species were detected as the major pathways of spread of NDM-producing Enterobacterales in Tokyo. Early detection of carriers and measures to prevent nosocomial spread are important to prevent further spread of NDM-producing organisms.

Porin deficiency or plasmid copy number increase mediated carbapenem-resistant Escherichia coli resistance evolution.

This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 µg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 µg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

IncX3 plasmid-mediated spread of blaNDM gene in Enterobacteriaceae among children in China.

OBJECTIVES: The blaNDM gene was prevalent among children and became the predominant cause of severe infection in infants and children. This study aimed to investigate the epidemiology and molecular characteristics of blaNDM in Enterobacteriaceae among children in China. METHODS: Carbapenem-resistant Enterobacteriaceae (CRE) were collected in the Children's Hospital of Fudan University from January 2016 to December 2022. Five carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48) were screened by PCR method. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses. blaNDM-carrying plasmids were typed by PCR-based Incompatibility (Inc) typing method. Moreover, plasmid comparison was performed with 213 publicly available IncX3 plasmids. RESULTS: A total of 330 CRE strains were enrolled, 96.4% of which carried carbapenemase genes. blaNDM gene accounted for 64.8% (214 strains) and included four variants, including blaNDM-1 (59.8%), blaNDM-5 (39.3%), blaNDM-7 (0.5%), and blaNDM-9 (0.5%). There were no predominant MLST lineages of blaNDM carrying strains. IncX3 was the major plasmid carrying blaNDM-1 (68.0%) and blaNDM-5 (72.6%) and was dominant in blaNDM-Klebsiella penumoniae (79.8%), blaNDM-Escherichia coli (58.2%), and blaNDM-Enterobacter cloacae (61.0%), respectively. Most (79.0%) clinical IncX3 plasmids in the world carried blaNDM, and the prevalence of blaNDM in IncX3 plasmids was more common in China (95.8%) than other countries (58.1%, P <0.01). CONCLUSION: blaNDM is highly prevalent in CRE among children in China. The spread of blaNDM was mainly mediated by IncX3 plasmids. Surveillance and infection control on the spread of blaNDM among children are important.

Clone dissemination of IncX3 plasmid carrying blaNDM-1 in ST76 carbapenem resistance Klebsiella pneumoniae and bactericidal efficiency of aztreonam combined with avibactam in vitro and in vivo.

OBJECTIVES: The rapid spread of the New Delhi Metal-ß-lactamase-1 (NDM-1) gene in Klebsiella pneumoniae poses a substantial challenge to pediatric therapeutic care. Here, we aimed to characterise the IncX3-type plasmid carrying the blaNDM-1 gene in ST76 carbapenem resistance K. pneumoniae (CRKP) strains and assess the in vitro and in vivo bactericidal efficacy of Aztreonam (ATM) combined with Avibactam (AVI) (ATM+AVI) against CRKP. METHODS: The broth microdilution method and PCR were used to detect antimicrobial susceptibility and antibiotic resistance genes. Genetic relatedness was determined using Pulsed-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). The plasmid conjugation assay was used to verify the transmissibility of drug-resistant plasmids. Whole-Genome Sequencing (WGS) was employed to elucidate the genomic attributes of the genes. The Fractional Inhibitory Concentration (FIC) was calculated based on the checkerboard titration assay to determine the antimicrobial effect of ATM+AVI. The time-kill curve assay and a mouse anti-infection model were used to investigate the in vitro and in vivo bactericidal efficiency of ATM+AVI. RESULTS: Seven blaNDM-1-producing strains were found to be highly resistant to carbapenems, and they all belonged to the same sequence type (ST76) and were classified into the same PFGE clusters with an 89.1% similarity. The conjugation assay showed that the blaNDM-1-carrying plasmid was successfully transferred to Escherichia coli 600, resulting in transconjugants with carbapenem antibiotic resistance. A 54-kb IncX3 plasmid (pNDM-XZA88) carried the blaNDM-1 gene located on a Tn125 transposon-like element structure, demonstrating the transferability of resistance genes. Genome comparative analysis revealed that pNDM-XZA88 was highly similar to pCQ17 × 3 and pRor-30818cz and had relatively conserved backbones and variable accessory regions compared to the other four plasmids (pC39-334 kb, pNDM-1-DY1928, pNDM-K725, and pNDM-Z244). The checkerboard titration and time-kill curve assays revealed that the ATM+AVI combination therapy exerted significant bactericidal efficacy against the blaNDM-1-producing strains in vitro. The ATM+AVI combination also significantly reduced the bacterial burden in a mouse infection model constructed using the blaNDM-1-producing K. pneumoniae. CONCLUSION: This study demonstrated the clone dissemination of blaNDM-1-harboring IncX3 plasmids among the ST76 K. pneumoniae isolated from pediatric patients. Therefore, more attention should be paid to preventing this high-risk clone from harming pediatric patients. Moreover, we deduced that the ATM+AVI combination therapy is an effective strategy for treating blaNDM-1-producing K. pneumoniae.

First Report of OXA-181-Producing Enterobacterales Isolates in Latin America.

We characterized five carbapenemase-producing Enterobacterales (CPE) isolates from two health care institutions in Lima, Peru. The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). All were identified as blaOXA-48-like gene carriers using conventional PCR. Whole-genome sequencing found the presence of the blaOXA-181 gene as the only carbapenemase gene in all isolates. Genes associated with resistance to aminoglycosides, quinolones, amphenicols, fosfomycins, macrolides, tetracyclines, sulfonamides, and trimethoprim were also found. The plasmid incompatibility group IncX3 was identified in all genomes in a truncated Tn6361 transposon flanked by ΔIS26 insertion sequences. The qnrS1 gene was also found downstream of blaOXA-181, conferring fluoroquinolone resistance to all isolates. CPE isolates harboring blaOXA-like genes are an increasing public health problem in health care settings worldwide. The IncX3 plasmid is involved in the worldwide dissemination of blaOXA-181, and its presence in these CPE isolates suggests the wide dissemination of blaOXA-181 in Peru. IMPORTANCE Reports of carbapenemase-producing Enterobacterales (CPE) isolates are increasing worldwide. Accurate detection of the ß-lactamase OXA-181 (a variant of OXA-48) is important to initiate therapy and preventive measures in the clinic. OXA-181 has been described in CPE isolates in many countries, often associated with nosocomial outbreaks. However, the circulation of this carbapenemase has yet to be reported in Peru. Here, we report the detection of five multidrug-resistant CPE clinical isolates harboring blaOXA-181 in the IncX3-type plasmid, a potential driver of dissemination in Peru.

Adaptive Evolution Compensated for the Plasmid Fitness Costs Brought by Specific Genetic Conflicts.

New Delhi metallo-ß-lactamase (NDM)-carrying IncX3 plasmids is important in the transmission of carbapenem resistance in Escherichia coli. Fitness costs related to plasmid carriage are expected to limit gene exchange; however, the causes of these fitness costs are poorly understood. Compensatory mutations are believed to ameliorate plasmid fitness costs and enable the plasmid's wide spread, suggesting that such costs are caused by specific plasmid-host genetic conflicts. By combining conjugation tests and experimental evolution with comparative genetic analysis, we showed here that the fitness costs related to ndm/IncX3 plasmids in E. coli C600 are caused by co-mutations of multiple host chromosomal genes related to sugar metabolism and cell membrane function. Adaptive evolution revealed that mutations in genes associated with oxidative stress, nucleotide and short-chain fatty acid metabolism, and cell membranes ameliorated the costs associated with plasmid carriage. Specific genetic conflicts associated with the ndm/IncX3 plasmid in E. coli C600 involve metabolism and cell-membrane-related genes, which could be ameliorated by compensatory mutations. Collectively, our findings could explain the wide spread of IncX3 plasmids in bacterial genomes, despite their potential cost.

Case Report of bla NDM-7-Harboring IncX3 Plasmid in ST196 Klebsiella quasipneumoniae in China.

Klebsiella quasipneumoniae isolate SBH035 was recovered from a patient in Jiangsu Province, China. The isolate showed resistance to ampicillin, cefazolin, cefotaxime, meropenem, ceftazidime-avibactam, and fosfomycin. The carbapenemase-encoding gene bla NDM-7 was identified, and whole genome sequencing analysis indicated that bla NDM-7 was located in an IncX3 plasmid with a conserved structure of IS26-ΔcutA-tat-trpF-ble MBL -bla NDM-7-ISAba125-IS3000-ΔTn2. To date, this is the first identification of a bla NDM-7-harboring IncX3 plasmid in ST196 K. quasipneumoniae from a patient in China. Greater attention to controlling the dissemination of IncX3 plasmids is needed owing to potential horizontal transfer via mobile genetic elements.

Whole-Genome Sequencing Analysis of blaNDM-5/IncX3 Plasmid Estimated to Be Conjugative-Transferred in the Gut.

We characterized plasmids carrying blaNDM-5 detected in Escherichia coli isolated from the infection site and stool sample of a Japanese patient, with no international travel history, by whole-genome sequencing (WGS). WGS was performed using MiSeq and MinlON sequencer followed by hybrid de novo assembly. blaNDM-5 was detected on IncX3 (blaNDM-5/IncX3) plasmids; pMTY18530-4_IncX3 in E. coli TUM18530 isolated from a wound above the pubis; pMTY18780-5_IncX3 and pMTY18781-1_IncX3 in E. coli TUM18780 and TUM18781, respectively, isolated from stool. These three plasmids resembled each other and pGSH8M-2-4, previously detected in E. coli isolated from a Tokyo Bay water sample. E. coli TUM18530 and TUM18780 belonged to sequence type (ST) 1011 and had only two single nucleotide polymorphisms on the core-genome, whereas TUM18781 belonged to ST2040. Three blaNDM-5/IncX3 plasmids (pMTY18530-4_IncX3, pMTY18780-5_IncX3, and pMTY18781-1_IncX3) exhibited conjugative transfer in vitro at an average frequency of 1.71 × 10-3 per donor cell. The transconjugant was resistant to only ß-lactams, including carbapenem, except aztreonam. Similarity of the blaNDM-5/IncX3 plasmids isolated from our patient compared with that isolated from the Tokyo bay water sample suggested that the plasmids may have already spread throughout the Japanese community. The blaNDM-5/IncX3 plasmid exhibited potential for easy transmission to different strains in the patient's intestine.

Clonal and plasmid-mediated dissemination of environmental carbapenem-resistant Enterobacteriaceae in large animal breeding areas in northern China.

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) constitute a major global health problem. The environment plays an important role in the dissemination of CRE, but large-scale studies on CRE in groundwater environments in animal breeding areas are scarce. The aim of this study was to investigate CRE occurrence and environmental transmission of carbapenem resistance genes in large animal breeding areas in northern China. In total, 280 well water and 102 animal feces samples in large animal breeding areas in six counties from the two provinces Inner Mongolia and Shandong in China, were screened for CRE. A total of 39 CRE were isolated and characterized with next-generation sequencing. 5.3% of well water samples were contaminated with CRE. The well water in chicken farms had the highest number of detections of CRE (15.9%). More than half of the isolates carried closely related, conjugative IncX3 plasmids with blaNDM-genes from multiple geographic areas, indicating that this kind of plasmid plays an important role in dissemination of carbapenem resistance determinants. The clonal expansion of various CRE isolates in well water and animal feces were demonstrated; clonally related CRE were isolated from different wells within the same county, from different counties in the same province, and even from different provinces. In addition to harboring various ARGs, two closely related K. pneumoniae belonging to ST11 isolated from well water carried genetic hypervirulence determinants on a virulence plasmid, highlighting the potential health risk posed by further dissemination of this strain. These findings suggest that groundwater may be an underappreciated reservoir and source of dissemination of CRE, from which resistance genes may disseminate among different bacterial strains and over large geographic distances. Further research and multi-sectorial monitoring, with a "One health" perspective, is urgently needed to investigate the need for interventions aimed at preventing CRE dissemination.

Emergence of IncX3 Plasmid-Harboring blaNDM-5 in a Citrobacter sedlakii Isolated from Outdoor Aerosol in Wastewater Treatment Plant.

A carbapenem-resistant Citrobacter sedlakii strain AA2CS carrying blaNDM-5 was detected in outdoor aerosols of a wastewater treatment plant (WWTP) in China and the whole genome was sequenced subsequently. AA2CS was captured in an aerobic tank with aerosol particles of sizes ranging from 4.7 to 7.0 µm. Besides blaNDM-5, AA2CS also harbored 21 other antibiotic resistance genes and displayed a high level of resistance to ampicillin, cefotaxime, ceftazidime, tetracycline, and meropenem. BlaNDM-5 was located on the IncX3 plasmid (pCSNDM-5) with an IS3000-IS5-blaNDM-5-bleMBL-trpF-dsbD-IS26 structure. pCSNDM-5 was highly homologous to other blaNDM-5-carrying IncX3 plasmids in China and can be transferred to the Escherichia coli recipient J53. To our knowledge, this is the first report of carbapenem-resistant Enterobacteriaceae in outdoor aerosols in WWTPs.

In silico Evolution and Comparative Genomic Analysis of IncX3 Plasmids Isolated From China Over Ten Years.

IncX3 plasmids are correlated with the dissemination and acquisition of carbapenem resistance in Enterobacteriaceae and have been prevalent in China over the last 10 years. Since the distribution characteristics of IncX3 plasmids across China as well as their evolutionary traits for 10 years remain unclear, here we conducted a retrospective literature review and in silico comparative analysis of IncX3 plasmids in publicly available IncX3 plasmid genomes. IncX3 plasmids distributed in 17 provinces or cities were extracted for analysis, which tend to be specifically associated with hospital-isolated Escherichia coli ST410 from phylogroup A. Although the backbones of IncX3 plasmids have remained highly conservative over the last 10 years, the bla NDM resistance genetic contexts on these plasmids could fall into five subtypes, among which AR_N1_I has been identified in Enterobacter cloacae174 chromosome and AR_N5_I was simultaneously located on IncF and IncA/C plasmids. This suggests that the bla NDM resistance gene environment can spread between different plasmids, between different bacterial genera, or between strains and plasmids, highlighting that it is imperative to adopt more stringent infection control measures targeting IncX3 plasmid spread.

First Report of blaNDM-1 Bearing IncX3 Plasmid in Clinically Isolated ST11 Klebsiella pneumoniae from Pakistan.

The New Delhi Metallo-ß-lactamase (NDM) is among the most threatening forms of carbapenemases produced by K. pneumoniae, well-known to cause severe worldwide infections. The molecular epidemiology of blaNDM-1-harboring K. pneumoniae is not well elucidated in Pakistan. Herein, we aim to determine the antibiotics-resistance profile, genes type, molecular type, and plasmid analysis of 125 clinically isolated K. pneumoniae strains from urine samples during July 2018 to January 2019 in Pakistan. A total of 34 (27.2%) K. pneumoniae isolates were carbapenemases producers, and 23 (18.4%) harbored the blaNDM-1 gene. The other carbapenemases encoding genes, i.e., blaIMP-1 (7.2%), blaVIM-1 (3.2%), and blaOXA-48 (2.4%) were also detected. The Multi Locus Sequence Typing (MLST) results revealed that all blaNDM-1-harboring isolates were ST11. The other sequence types detected were ST1, ST37, and ST105. The cluster analysis of Xbal Pulsed Field Gel Electrophoresis (PFGE) revealed variation amongst the clusters of the identical sequence type isolates. The blaNDM-1 gene in all of the isolates was located on a 45-kb IncX3 plasmid, successfully transconjugated. For the first time, blaNDM-1-bearing IncX3 plasmids were identified from Pakistan, and this might be a new primary vehicle for disseminating blaNDM-1 in Enterobacteriaceae as it has a high rate of transferability.

Dissemination and Stability of the blaNDM-5-Carrying IncX3-Type Plasmid among Multiclonal Klebsiella pneumoniae Isolates.

NDM-5 carbapenemase was mainly identified in Escherichia coli, while the rapid transmission of blaNDM-5 among Enterobacteriaceae has raised serious public attention. This study identified 14 NDM-5-producing Klebsiella pneumoniae isolates from 107 carbapenem-resistant K. pneumoniae isolates, recovered from blood, urine, and normally sterile body fluids of pediatric patients from January 2016 to December 2018. All NDM-5-producing isolates were highly resistant to ß-lactams, while tigecycline and polymyxin B exhibited excellent antimicrobial activity. These 14 strains belonged to 9 different sequence types (STs) and displayed various pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they were not clonally related. S1-PFGE followed by Southern blotting showed that the blaNDM-5 gene was located on an ∼46-kb IncX3 plasmid in all strains. All blaNDM-5-carrying plasmids were successfully transferred into recipient E. coli J53. PCR-based sequencing demonstrated that all of the blaNDM-5-carrying plasmids shared highly similar backbones, with nucleotide sequence identity of >99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 blaNDM-5-carrying plasmids, with dynamic changes observed only in blaNDM-5-surrounding elements. Interestingly, the IncX3 blaNDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% blaNDM-5-carrying plasmid loss after coculture with linoleic acid for 5 days. These results showed that the IncX3 plasmid facilitated the dissemination of blaNDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniaeIMPORTANCE The emergence and spread of New Delhi metallo-ß-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 blaNDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of blaNDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 blaNDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 blaNDM-5-carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 blaNDM-5-carrying plasmids.

First Report of OXA-181-Producing Klebsiella pneumoniae in China.

We present here the first report of an OXA-181-producing Klebsiella pneumoniae isolated from the fecal specimen of a patient in China. The OXA-181-encoding gene bla OXA-181 was located on a 51 kb IncX3-type plasmid. Conjugation assay and whole-genome sequencing analysis revealed that this transferrable plasmid in the K. pneumoniae isolate might have originated from Escherichia coli and have the potential to mediate the spread of bla OXA-181.

Impact of Plasmid-Encoded H-NS-like Protein on blaNDM-1-Bearing IncX3 Plasmid in Escherichia coli.

BACKGROUND: This study was performed to assess the role of the histone-like nucleoid-structuring (H-NS)-like protein, carried by blaNDM-1-encoding IncX3-type plasmids, in the dissemination of IncX3 plasmids. METHODS: The blaNDM-1-encoding IncX3 plasmids were analyzed using southern blot, conjugation, and competition assays. Virulence was evaluated with a Galleria mellonella infection model. An hns-knockout IncX3 plasmid was also constructed to identify the functions of plasmid-borne H-NS-like protein in Escherichia coli. RESULTS: The assasys detected blaNDM-1-encoding IncX3-type plasmids with similar fingerprint patterns in all New Delhi metallo-ß-lactamase (NDM) 1-producing carbapenem-resistant Enterobacteriaceae. The IncX3 plasmid conferred a fitness advantage to E. coli J53 but had no effect on host virulence. Moreover, the transconjugation frequency of the hns-null IncX3 plasmid pHN330-△hns was increased by 2.5-fold compared with the wild type. This was caused by up-regulation of conjugation-related plasmid-borne genes and the partition-related gene, in the J330-pHN330-△hns strain. In addition, decreased virulence was detected with this variant. CONCLUSIONS: Our results highlight the important role of IncX3 plasmids in the dissemination of blaNDM-1 in south China. Plasmid-encoded H-NS-like protein can inhibit plasmid conjugation, partition, and the expression of related genes, in addition to promoting virulence in the host.

New Delhi Metallo-ß-Lactamase 1-Producing Klebsiella pneumoniae ST719 Isolated from a Neonate in China.

The spread of carbapenem-resistant Klebsiella pneumoniae has become a significant problem for public health in recent years. In this study, we reported a New Delhi metallo-ß-lactamase 1 (NDM-1)-producing K. pneumoniae strain KP14003 from a neonate in Beijing, China. Whole-genome sequencing was performed. The strain belonged to sequence type ST719. Coexistence of blaNDM-1 and blaSHV-12 was found on a self-transferable plasmid, which had a typical IncX3 backbone. The horizontal transfer of blaNDM-1 was associated with Tn125 followed by possible transposition events. Other class A extended-spectrum ß-lactamase genes (blaSHV-27 and blaTEM-1) were also identified on chromosome or plasmid. The dissemination of NDM-1-producing K. pneumoniae causes great challenges to the treatment of clinical infections. Effective actions need to be taken to control the further spread of this pathogen.

Epidemic IncX3 plasmids spreading carbapenemase genes in the United Arab Emirates and worldwide.

PURPOSE: Plasmids of the incompatibility group X type 3 (IncX3) were described carrying various carbapenemase genes in carbapenemase-producing Enterobacteriaceae (CPE) worldwide and in the United Arab Emirates (UAE), as well. To understand the driving force behind the emergence of such plasmids in the UAE, the relationship between IncX3 plasmids encountered locally and globally was investigated. METHODS: CPE strains isolated in the UAE during 2009-2014 were screened by X3 PCR-based replicon typing. The clonal relationship of CPE carrying IncX3 plasmids was determined by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Complete sequence of selected IncX3 plasmids was determined. Phylogenetic relationship between the carbapenemase carrying IncX3 plasmids from the UAE and of those reported worldwide was established by comparing the plasmid backbones. RESULTS: 10.2% of the 295 CPE tested were identified to carry IncX3 plasmids: 13 Escherichia coli, 13 Klebsiella pneumoniae, two Enterobacter cloacae, one Citrobacter freundii and one Morganella morganii isolate, respectively. Most of them were non-clonal; with small clusters of triplets and pairs of E. coli and K. pneumoniae, and a cluster of five K. pneumoniae ST11 exhibiting >90% similar PFGE patterns, respectively. The 30 isolates harbored either bla NDM-1, bla NDM-4, bla NDM-5, bla NDM-7, bla OXA-181 or bla KPC-2 carbapenemase genes on IncX3 plasmids. Phylogenetic analysis of the backbone region of IncX3 plasmids carrying various beta-lactamase genes from the UAE (n=23) and that of North-America, Europe, Asia and Australia (n=35) revealed three clusters based on the carbapenemase genes carried: plasmids harboring bla OXA-181 and bla NDM-5 formed two distinct groups, whereas backbones of plasmids with bla NDM-1, bla NDM-4 and bla NDM-7 clustered together. Each cluster contained plasmids of diverse geographical origin. CONCLUSION: The findings suggest that different carbapenemase gene carrying IncX3 plasmids encountered in the UAE do not evolve locally, rather are subtypes of this epidemic plasmid emerging in this country due to international transfer.

First Detection of New Delhi Metallo-ß-Lactamase-5-Producing Escherichia coli from Companion Animals in Korea.

The gene for New Delhi metallo-ß-lactamase-5 (NDM-5) in Escherichia coli has been identified in many countries mainly from human clinical specimens. The isolates carrying this gene are even more rarely isolated from companion animals. In this study, four carbapenem-resistant isolates were recovered from four dogs in Korea. All isolates carried blaNDM-5 and exhibited resistance to meropenem and imipenem, and were susceptible to colistin. Epidemiological analysis showed that all four isolates were sequence type 410 (ST410) and shared 99% similarity as determined by pulsed-field gel electrophoresis analysis. Among the four isolates, the Z0117EC0033 strain was randomly selected for whole-genome sequencing, composed of a 4.7Mb circularized chromosome carrying the CMY-2 gene and two plasmids. The first plasmid of the IncFIB type had 83 coding sequences (CDS) in ca. 74 kb. The second smaller plasmid of the IncX3 type had 57 CDS and carried only the blaNDM-5 gene in ca. 46 kb. The plasmid structures were highly similar (> 99%) to those of the NDM-5 human-like IncX3 plasmid. This is the first report of carbapenemase-producing Enterobacteriaceae from companion animals in Korea. The human-like blaNDM-5 IncX3 plasmid identified in this study suggests a potential transmission route of the NDM-5 plasmid between humans and companion animals.

Various Sequence Types of Enterobacteriaceae Isolated from Commercial Chicken Farms in China and Carrying the blaNDM-5 Gene.

A total of 108 meropenem-resistant Enterobacteriaceae isolates were obtained from 1,658 rectal swabs collected from 15 unrelated commercial chicken farms in China between 2014 and 2016. These samples yielded 16 Escherichia coli and 2 Klebsiella pneumoniae isolates of diverse sequence types carrying a blaNDM-5-bearing IncX3 plasmid. K. pneumoniae strain sequence type 709 (ST709) has two blaNDM-5-carrying plasmids that were transferred together to E.coli.