April 21, 2020  |  

Conjugal Transfer, Whole-Genome Sequencing, and Plasmid Analysis of Four mcr-1-Bearing Isolates from U.S. Patients.

Four Enterobacteriaceae clinical isolates bearing mcr-1 gene-harboring plasmids were characterized. All isolates demonstrated the ability to transfer colistin resistance to Escherichia coli; plasmids were stable in conjugants after multiple passages on nonselective media. mcr-1 was located on an IncX4 (n?=?3) or IncN (n?=?1) plasmid. The IncN plasmid harbored 13 additional antimicrobial resistance genes. Results indicate that the mcr-1-bearing plasmids in this study were highly transferable in vitro and stable in the recipients.This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.


April 21, 2020  |  

Diverse Commensal Escherichia coli Clones and Plasmids Disseminate Antimicrobial Resistance Genes in Domestic Animals and Children in a Semirural Community in Ecuador.

The increased prevalence of antimicrobial resistance (AMR) among Enterobacteriaceae has had major clinical and economic impacts on human medicine. Many of the multidrug-resistant (multiresistant) Enterobacteriaceae found in humans are community acquired, and some of them are possibly linked to food animals (i.e., livestock raised for meat and dairy products). In this study, we examined whether numerically dominant commensal Escherichia coli strains from humans (n?=?63 isolates) and domestic animals (n?=?174 isolates) in the same community and with matching phenotypic AMR patterns were clonally related or shared the same plasmids. We identified 25 multiresistant isolates (i.e., isolates resistant to more than one antimicrobial) that shared identical phenotypic resistance patterns. We then investigated the diversity of E. coli clones, AMR genes, and plasmids carrying the AMR genes using conjugation, replicon typing, and whole-genome sequencing. All of the multiresistant E. coli isolates (from children and domestic animals) analyzed had at least 90 or more whole-genome SNP differences between one another, suggesting that none of the strains was recently transferred. While the majority of isolates shared the same antimicrobial resistance genes and replicons, DNA sequencing indicated that these genes and replicons were found on different plasmid structures. We did not find evidence of the clonal spread of AMR in this community: instead, AMR genes were carried on diverse clones and plasmids. This presents a significant challenge for understanding the movement of AMR in a community.IMPORTANCE Even though Escherichia coli strains may share nearly identical phenotypic AMR profiles and AMR genes and overlap in space and time, the diversity of clones and plasmids challenges research that aims to identify sources of AMR. Horizontal gene transfer appears to play a more significant role than clonal expansion in the spread of AMR in this community.Copyright © 2019 Salinas et al.


April 21, 2020  |  

Spreading Patterns of NDM-Producing Enterobacteriaceae in Clinical and Environmental Settings in Yangon, Myanmar.

The spread of carbapenemase-producing Enterobacteriaceae (CPE), contributing to widespread carbapenem resistance, has become a global concern. However, the specific dissemination patterns of carbapenemase genes have not been intensively investigated in developing countries, including Myanmar, where NDM-type carbapenemases are spreading in clinical settings. In the present study, we phenotypically and genetically characterized 91 CPE isolates obtained from clinical (n = 77) and environmental (n = 14) samples in Yangon, Myanmar. We determined the dissemination of plasmids harboring genes encoding NDM-1 and its variants using whole-genome sequencing and plasmid analysis. IncFII plasmids harboring blaNDM-5 and IncX3 plasmids harboring blaNDM-4 or blaNDM-7 were the most prevalent plasmid types identified among the isolates. The IncFII plasmids were predominantly carried by clinical isolates of Escherichia coli, and their clonal expansion was observed within the same ward of a hospital. In contrast, the IncX3 plasmids were found in phylogenetically divergent isolates from clinical and environmental samples classified into nine species, suggesting widespread dissemination of plasmids via horizontal transfer. Half of the environmental isolates were found to possess IncX3 plasmids, and this type of plasmid was confirmed to transfer more effectively to recipient organisms at a relatively low temperature (25°C) compared to the IncFII plasmid. Moreover, various other plasmid types were identified harboring blaNDM-1, including IncFIB, IncFII, IncL/M, and IncA/C2, among clinical isolates of Klebsiella pneumoniae or Enterobacter cloacae complex. Overall, our results highlight three distinct patterns of the dissemination of blaNDM-harboring plasmids among CPE isolates in Myanmar, contributing to a better understanding of their molecular epidemiology and dissemination in a setting of endemicity.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Complete Sequence of a Novel Multidrug-Resistant Pseudomonas putida Strain Carrying Two Copies of qnrVC6.

This study aimed at identification and characterization of a novel multidrug-resistant Pseudomonas putida strain Guangzhou-Ppu420 carrying two copies of qnrVC6 isolated from a hospital in Guangzhou, China, in 2012. Antimicrobial susceptibility was tested by Vitek2™ Automated Susceptibility System and Etest™ strips, and whole-genome sequencing facilitated analysis of its multidrug resistance. The genome has a length of 6,031,212?bp and an average G?+?C content of 62.01%. A total of 5,421 open reading frames were identified, including eight 5S rRNA, seven 16S rRNA, and seven 23S rRNA, and 76 tRNA genes. Importantly, two copies of qnrVC6 gene with three ISCR1 around, a blaVIM-2 carrying integron In528, a novel gcu173 carrying integron In1348, and six antibiotic resistance genes were identified. This is the first identification of two copies of the qnrVC6 gene in a single P. putida isolate and a class 1 integron In1348.


April 21, 2020  |  

One Health Genomic Surveillance of Escherichia coli Demonstrates Distinct Lineages and Mobile Genetic Elements in Isolates from Humans versus Livestock.

Livestock have been proposed as a reservoir for drug-resistant Escherichia coli that infect humans. We isolated and sequenced 431 E. coli isolates (including 155 extended-spectrum ß-lactamase [ESBL]-producing isolates) from cross-sectional surveys of livestock farms and retail meat in the East of England. These were compared with the genomes of 1,517 E. coli bacteria associated with bloodstream infection in the United Kingdom. Phylogenetic core genome comparisons demonstrated that livestock and patient isolates were genetically distinct, suggesting that E. coli causing serious human infection had not directly originated from livestock. In contrast, we observed highly related isolates from the same animal species on different farms. Screening all 1,948 isolates for accessory genes encoding antibiotic resistance revealed 41 different genes present in variable proportions in human and livestock isolates. Overall, we identified a low prevalence of shared antimicrobial resistance genes between livestock and humans based on analysis of mobile genetic elements and long-read sequencing. We conclude that within the confines of our sampling framework, there was limited evidence that antimicrobial-resistant pathogens associated with serious human infection had originated from livestock in our region. IMPORTANCE The increasing prevalence of E. coli bloodstream infections is a serious public health problem. We used genomic epidemiology in a One Health study conducted in the East of England to examine putative sources of E. coli associated with serious human disease. E. coli from 1,517 patients with bloodstream infections were compared with 431 isolates from livestock farms and meat. Livestock-associated and bloodstream isolates were genetically distinct populations based on core genome and accessory genome analyses. Identical antimicrobial resistance genes were found in livestock and human isolates, but there was limited overlap in the mobile elements carrying these genes. Within the limitations of sampling, our findings do not support the idea that E. coli causing invasive disease or their resistance genes are commonly acquired from livestock in our region. Copyright © 2019 Ludden et al.


April 21, 2020  |  

Reconstruction of the genomes of drug-resistant pathogens for outbreak investigation through metagenomic sequencing

Culture-independent methods that target genome fragments have shown promise in identifying certain pathogens, but the holy grail of comprehensive pathogen genome detection from microbiologically complex samples for subsequent forensic analyses remains a challenge. In the context of an investigation of a nosocomial outbreak, we used shotgun metagenomic sequencing of a human fecal sample and a neural network algorithm based on tetranucleotide frequency profiling to reconstruct microbial genomes and tested the same approach using rectal swabs from a second patient. The approach rapidly and readily detected the genome of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in the patient fecal specimen and in the rectal swab sample, achieving a level of strain resolution that was sufficient for confident transmission inference during a highly clonal outbreak. The analysis also detected previously unrecognized colonization of the patient by vancomycin-resistant Enterococcus faecium, another multidrug-resistant bacterium.IMPORTANCE The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.


April 21, 2020  |  

Emergence of a ST2570 Klebsiella pneumoniae isolate carrying mcr-1 and blaCTX-M-14 recovered from a bloodstream infection in China.

The worldwide emergence of the plasmid-borne colistin resistance mediated by mcr-1 gene not only extended our knowledge on colistin resistance, but also poses a serious threat to clinical and public health [1, 2]. Since its first discovery, mcr-1-carrying Enterobacteriaceae from human, animal, food, and environmental origins have been widely identified, but few mcr-1-positive clinical strains of Klebsiella pneumoniae have been reported so far, especially when associated with community-acquired infections [3, 4]. Here, we report the emergence of a colistin-resistant K. pneumoniae isolate, which belonged to a rare sporadic clone, co-carrying mcr-1 and blaCTX-M-14 genes simultaneous recovered from a community-acquired bloodstream infection in China. Whole-genome sequencing and microbiological analysis were performed to elucidate its antimicrobial resistance mechanisms.


April 21, 2020  |  

Whole genome sequencing of NDM-1-producing serotype K1 ST23 hypervirulent Klebsiella pneumoniae in China.

The emergence and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is causing worldwide concern, whereas NDM-producing hvKP is still rare. Here we report the complete genome sequence characteristics of an NDM-1-producing ST23 type clinical hvKP in PR China.Capsular polysaccharide serotyping was performed by PCR. The complete genome sequence of isolate 3214 was obtained using both the Illumina Hiseq platform and Pacbio RS platform. Multilocus sequence type was identified by submitting the genome sequence to mlst 2.0 and the antimicrobial resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder, respectively. Transferability of the blaNDM-1-bearing plasmid was determined by conjugation experiment, S1 pulsed-field gel electrophoresis and Southern hybridization.Isolate 3214 was classified to ST23 and belonged to the K1 capsular serotype. The isolate’s total genome size was 6 171 644?bp with a G+C content of 56.39 %, consisting of a 5 448 209?bp chromosome and seven plasmids. The resistome included 18 types of antibiotic resistance genes. Fourteen resistance genes including blaNDM-1 and blaCTX-M-14 were located on plasmids and five also including blaCTX-M-14 were in the chromosome. Plasmid pNDM_3214 carrying blaNDM-1 harboured six types of resistance genes surrounded by insertion sequences and was conjugative. The worldwide pLVPK-like virulence plasmid harbouring rmpA2 and rmpA was also found in this isolate.This study provides basic information of phenotypic and genomic features of ST23 CR-hvKP isolate 3214. Our data highlights the potential risk of spread of NDM-1-producing ST23 hvKP.


April 21, 2020  |  

Transmission of ciprofloxacin resistance in Salmonella mediated by a novel type of conjugative helper plasmids.

Ciprofloxacin resistance in Salmonella has been increasingly reported due to the emergence and dissemination of multiple Plasmid-Mediated Quinolone Resistance (PMQR) determinants, which are mainly located in non-conjugative plasmids or chromosome. In this study, we aimed to depict the molecular mechanisms underlying the rare phenomenon of horizontal transfer of ciprofloxacin resistance phenotype in Salmonella by conjugation experiments, S1-PFGE and complete plasmid sequencing. Two types of non-conjugative plasmids, namely an IncX1 type carrying a qnrS1 gene, and an IncH1 plasmid carrying the oqxAB-qnrS gene, both ciprofloxacin resistance determinants in Salmonella, were recovered from two Salmonella strains. Importantly, these non-conjugative plasmids could be fused with a novel Incl1 type conjugative helper plasmid, which could target insertion sequence (IS) elements located in the non-conjugative, ciprofloxacin-resistance-encoding plasmid through replicative transcription, eventually forming a hybrid conjugative plasmid transmissible among members of Enterobacteriaceae. Since our data showed that such conjugative helper plasmids are commonly detectable among clinical Salmonella strains, particularly S. Typhimurium, fusion events leading to generation and enhanced dissemination of conjugative ciprofloxacin resistance-encoding plasmids in Salmonella are expected to result in a sharp increase in the incidence of resistance to fluoroquinolone, the key choice for treating life-threatening Salmonella infections, thereby posing a serious public health threat.


April 21, 2020  |  

First report of isolation and complete genome of Vibrio rotiferianus strain SSVR1601 from cage-cultured black rockfish (Sebastes schlegelii) associated with skin ulcer.

Vibrio rotiferianus is an important marine pathogen of various aquatic organisms and can be found widely distributed in the marine environment. To further characterize this pathogen, the pathogenic properties and genome of V. rotiferianus SSVR1601 isolated from Sebastes schlegelii with skin ulcer were analysed. SSVR1601 was shown to be short rod-shaped cell with a single polar flagellum. Different degrees of pathological changes in fish kidney, intestine, gills and liver were observed after SSVR1601 challenge. The SSVR1601 genome consists of two chromosomes and two plasmids with a total of 5,717,113 bp, 42.04%-44.93% GC content, 5,269 predicted CDSs, 134 tRNAs and 40 rRNAs. The common virulence factors including OMPs, haemolysin, flagellin, DNase, entF, algU, tcpI, acfB and rfaD were found in strain SSVR1601. Furthermore, factors responsible for iron uptake (fur, fepC and ccmC) and types II, IV and VI secretion systems were detected, which are likely responsible for the pathogenicity of SSVR1601. The antimicrobial resistance genes, bacA, tet34 and norM, were detected based on Antibiotic Resistance Genes Database. The phylogenetic analysis revealed SSVR1601 to be most closely related to V. rotiferianus strains CAIM577 and B64D1. © 2019 John Wiley & Sons Ltd.


April 21, 2020  |  

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial-resistant (AMR) Aeromonas strains (A. hydrophila GSH8-2 and A. caviae GSH8M-1) carrying the carabapenemase blaKPC-2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8-2 and GSH8M-1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired blaKPC-2 -positive IncP-6-relative plasmids (pGSH8-2 and pGSH8M-1-2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to blaKPC-2 , pGSH8M-1-2 carries an IS26-mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities. © 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.


April 21, 2020  |  

Complete genome sequence of an IMP-8, CTX-M-14, CTX-M-3 and QnrS1 co-producing Enterobacter asburiae isolate from a patient with wound infection.

The aim of this study was to investigate the characteristics and complete genome sequence of an IMP-8, CTX-M-14, CTX-M-3 and QnrS1 co-producing multidrug-resistant Enterobacter asburiae isolate (EN3600) from a patient with wound infection.Species identification was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Carbapenemase genes were identified by PCR and Sanger sequencing. The complete genome sequence of E. asburiae EN3600 was obtained using a PacBio RS II platform. Genome annotation was done by Rapid Annotation using Subsystem Technology (RAST) server. Acquired antimicrobial resistance genes (ARGs) and plasmid replicons were detected using ResFinder 2.1 and PlasmidFinder 1.3, respectively.The genome of E. asburiae EN3600 consists of a 4.8-Mbp chromosome and five plasmids. The annotated genome contains various ARGs conferring resistance to aminoglycosides, ß-lactams, fluoroquinolones, fosfomycin, macrolides, phenicols, rifampicin and sulfonamides. In addition, plasmids of incompatibility (Inc) groups IncHI2A, IncFIB(pECLA), IncFIB(pQil) and IncP1 were identified. The genes blaIMP-8, blaCTX-M-14 and blaCTX-M-3 were located on different plasmids. The blaIMP-8 gene was carried by an 86-kb IncFIB(pQil) plasmid. The blaCTX-M-3 and qnrS1 genes were co-harboured by an IncP1 plasmid. In addition, blaCTX-M-14 was associated with blaTEM-1B, blaOXA-1, catB3 and sul1 genes in a 116-kb non-typeable plasmid.To our knowledge, this is the first complete genome sequence of an E. asburiae isolate co-producing IMP-8, CTX-M-14, CTX-M-3 and QnrS1. This genome may facilitate the understanding of the resistome, pathogenesis and genomic features of Enterobacter cloacae complex (ECC) and will provide valuable information for accurate identification of ECC.Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Emergence of an Escherichia coli strain co-harbouring mcr-1 and blaNDM-9 from a urinary tract infection in Taiwan.

Multidrug-resistant bacteria have become a serious threat worldwide. In particular, the coexistence of carbapenemase genes and mcr-1 leaves few available treatment options. Here we report a multidrug-resistant Escherichia coli isolate harbouring both mcr-1 and blaNDM-9 from a patient with a urinary tract infection.Antimicrobial susceptibility and resistance genes of the E. coli isolate were characterised. Furthermore, the assembled genome sequences of mcr-1- and blaNDM-9-carrying plasmids were determined and comparative genetic analysis with closely related plasmids was carried out.Three contigs were assembled comprising the E. coli chromosome and two plasmids harbouring mcr-1 (p5CRE51-MCR-1) and blaNDM-9 (p5CRE51-NDM-9), respectively. Whole-genome sequencing revealed that the two antimicrobial resistance genes are located on individual plasmids.The emergence of coexistence of carbapenemase genes and mcr-1 in Enterobacteriaceae highlights a serious threat to antimicrobial therapy.Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Retrospective whole-genome sequencing analysis distinguished PFGE and drug-resistance-matched retail meat and clinical Salmonella isolates.

Non-typhoidal Salmonella is a leading cause of outbreak and sporadic-associated foodborne illnesses in the United States. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibility testing (AST) have been used to facilitate public health investigations of Salmonella infections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, USA, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historic Salmonella isolates, subdivided into 3 subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed that the retail meat isolates within S. Heidelberg, S. Typhimurium var. O5- subset 1 and S. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6-12, 41-96 and 21-81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, including fosA7, a gene only recently found in a limited number of Salmonella and a =95?%?phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated in S. Heidelberg and S. Typhimurium var. O5- subset 2, whereas AMR was chromosomally carried in S. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance for Salmonella from multiple sources.


April 21, 2020  |  

Characterization of NDM-5- and CTX-M-55-coproducing Escherichia coli GSH8M-2 isolated from the effluent of a wastewater treatment plant in Tokyo Bay.

New Delhi metallo-ß-lactamase (NDM)-5-producing Enterobacteriaceae have been detected in rivers, sewage, and effluents from wastewater treatment plants (WWTPs). Environmental contamination due to discharged effluents is of particular concern as NDM variants may be released into waterways, thereby posing a risk to humans. In this study, we collected effluent samples from a WWTP discharged into a canal in Tokyo Bay, Japan.Testing included the complete genome sequencing of Escherichia coli GSH8M-2 isolated from the effluent as well as a gene network analysis.The complete genome sequencing of GSH8M-2 revealed that it was an NDM-5-producing E. coli strain sequence type ST542, which carries multiple antimicrobial resistance genes for ß-lactams, quinolone, tetracycline, trimethoprim-sulfamethoxazole, florfenicol/chloramphenicol, kanamycin, and fosfomycin. The blaNDM-5 gene was found in the IncX3 replicon plasmid pGSH8M-2-4. Gene network analysis using 142 IncX3 plasmid sequences suggested that pGSH8M-2-4 is related to both clinical isolates of  E. coli and Klebsiella species in Eastern Asia. GSH8M-2 also carries the blaCTX-M-55 gene in IncX1 plasmid pGSH8M-2-3.This is the first report of environmental NDM-5-producing E. coli isolated from a WWTP in Japan. NDM-5 detection is markedly increasing in veterinary and clinical settings, suggesting that dual ß-lactamases, such as NDM-5 and CTX-M-55, might be acquired through multiple steps in environment settings. Environmental contamination through WWTP effluents that contain producers of NDM variants could be an emerging potential health hazard. Thus, regular monitoring of WWTP effluents is important for the detection of antimicrobial-resistant bacteria that may be released into the waterways and nearby communities.


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