Miaomiao Xie

Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant

Objectives To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1. Methods Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Results Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216–280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate. Conclusions The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated.

Widespread Dissemination of Carbapenem-Resistant Escherichia coli Sequence Type 167 Strains Harboring blaNDM-5 in Clinical Settings in China.

ABSTRACT This study reports the increasing prevalence of clinical Escherichia coli of sequence type 167 (ST167) carrying both blaNDM-1 and blaNDM-5 on the conjugative IncX3 plasmid in various parts of China. Close surveillance is needed to monitor the future dissemination of ST167 strains that harbor blaNDM-5 or other blaNDM-like genes.

Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

Objectives: To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1. Methods: The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1-bearing plasmid to undergo conjugation was also assessed. The mcr-1-bearing transposon Tn6330 was characterized by PCR and DNA sequencing. Results: Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7-mcr1, was found to harbour the mcr-1-bearing transposon Tn6330, which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the ISApl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1-carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. Conclusions: The Tn6330 element located in the phage-like plasmid pHYEC7-mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the ISApl1 element. This phenomenon indicated that Tn6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.

Molecular Characterization of Escherichia coli Strains Isolated from Retail Meat That Harbor blaCTX-M and fosA3 Genes

ABSTRACT A total of 55 cefotaxime-resistant Escherichia coli isolates were obtained from retail meat products purchased in Shenzhen, China, during the period November 2012 to May 2013. Thirty-seven of these 55 isolates were found to harbor a blaCTX-M gene, with the blaCTX-M-1 group being the most common type. blaCMY-2 was detected in 16 isolates, alone or in combination with other extended-spectrum β-lactamase (ESBL) determinants. Importantly, the fosA3 gene, which encodes fosfomycin resistance, was detected in 12 isolates, with several being found to reside in the conjugative plasmid that harbored the blaCTX-M gene. The insertion sequence IS26 was observed upstream of some of the blaCTX-M-55 and fosA3 genes. Conjugation experiments showed that blaCTX-M genes from 15 isolates were transferrable, with Inc I1 and Inc FII being the most prevalent replicons. High clonal diversity was observed among the blaCTX-M producers, suggesting that horizontal transfer of the blaCTX-M genes among E. coli strains in retail meats is a common event and that such strains may constitute an important reservoir of blaCTX-M genes, which may be readily disseminated to other potential human pathogens.

Widespread distribution of mcr-1-bearing bacteria in the ecosystem, 2015 to 2016

The recently discovered colistin resistance-encoding element, mcr-1, adds to the list of mobile resistance genes whose products rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics, but also the last line agents of carbapenems and colistin. The relative prevalence of mcr-1-bearing strains in various ecological niches including 1,371 food samples, 480 animal faecal samples, 150 human faecal samples and 34 water samples was surveyed using a novel in-house method. Bacteria bearing mcr-1 were commonly detected in water (71% of samples), animal faeces (51%), food products (36%), and exhibited stable carriage in 28% of human subjects surveyed. Such strains, which exhibited variable antibiotic susceptibility profiles, belonged to various Enterobacteriaceae species, with Escherichia coli being the most dominant in each specimen type. The mcr-1 gene was detectable in the chromosome as well as plasmids of various sizes. Among these, two conjugative plasmids of sizes ca 33 and ca 60 kb were found to be the key vectors that mediated mcr-1 transmission in organisms residing in various ecological niches. The high mcr-1 carriage rate in humans found in this study highlights the importance of continued vigilance, careful antibiotic stewardship, and the development of new antimicrobials.

IncFII Conjugative Plasmid-Mediated Transmission of bla NDM-1 Elements among Animal-Borne Escherichia coli Strains

ABSTRACT This study aims to investigate the prevalence and transmission dynamics of the blaNDM-1 gene in animal Escherichia coli strains. Two IncFII blaNDM-1-encoding plasmids with only minor structural variation in the MDR region, pHNEC46-NDM and pHNEC55-NDM, were found to be responsible for the transmission of blaNDM-1 in these strains. The blaNDM-1 gene can be incorporated into plasmids and stably inherited in animal-borne E. coli strains that can be maintained in animal gut microflora even without carbapenem selection pressure.

Efficient generation of complete sequences of MDR-encoding plasmids by rapid assembly of MinION barcoding sequencing data

Abstract Background Multidrug resistance (MDR)–encoding plasmids are considered major molecular vehicles responsible for transmission of antibiotic resistance genes among bacteria of the same or different species. Delineating the complete sequences of such plasmids could provide valuable insight into the evolution and transmission mechanisms underlying bacterial antibiotic resistance development. However, due to the presence of multiple repeats of mobile elements, complete sequencing of MDR plasmids remains technically complicated, expensive, and time-consuming. Results Here, we demonstrate a rapid and efficient approach to obtaining multiple MDR plasmid sequences through the use of the MinION nanopore sequencing platform, which is incorporated in a portable device. By assembling the long sequencing reads generated by a single MinION run according to a rapid barcoding sequencing protocol, we obtained the complete sequences of 20 plasmids harbored by multiple bacterial strains. Importantly, single long reads covering a plasmid end-to-end were recorded, indicating that de novo assembly may be unnecessary if the single reads exhibit high accuracy. Conclusions This workflow represents a convenient and cost-effective approach for systematic assessment of MDR plasmids responsible for treatment failure of bacterial infections, offering the opportunity to perform detailed molecular epidemiological studies to probe the evolutionary and transmission mechanisms of MDR-encoding elements.

Molecular Characterization of Escherichia coli Isolates Carrying mcr-1, fosA3, and Extended-Spectrum-β-Lactamase Genes from Food Samples in China.

ABSTRACT This study surveyed the prevalence of mcr-1 in extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains of food origin in China and identified strains that carried mcr-1, fosA3, and ESBL genes, which were carried in various plasmids. The mcr-1 and ESBL genes could be cotransferred by one or more types of plasmids. The presence of these multidrug-resistant E. coli strains in food products might pose a huge threat to public health.

Complete Sequence of a F33:A-:B- Conjugative Plasmid Carrying the oqxAB, fosA3, and blaCTX-M-55 Elements from a Foodborne Escherichia coli Strain

This study reports the complete sequence of pE80, a conjugative IncFII plasmid recovered from an Escherichia coli strain isolated from chicken meat. This plasmid harbors multiple resistance determinants including oqxAB, fosA3, blaCTX-M-55, and blaTEM-1, and is a close variant of the recently reported p42-2 element, which was recovered from E. coli of veterinary source. Recovery of pE80 constitutes evidence that evolution or genetic re-arrangement of IncFII type plasmids residing in animal-borne organisms is an active event, which involves acquisition and integration of foreign resistance elements into the plasmid backbone. Dissemination of these plasmids may further compromise the effectiveness of current antimicrobial strategies.

Recombination of plasmids in a carbapenem-resistant NDM-5-producing clinical Escherichia coli isolate

Objectives To investigate the genetic features of five plasmids recovered from an NDM-5-producing clinical Escherichia coli strain, BJ114, and to characterize the plasmid recombination event that occurred during the conjugation process. Methods The genetic profiles of the five plasmids were determined by PCR, conjugation, S1-PFGE, Southern hybridization and WGS analysis. Plasmid sequences were analysed with various bioinformatic tools. Results Complete sequences of five plasmids were obtained. Two small plasmids, pBJ114-141 and pBJ114-46, were speculated to have recombined into a large fusion plasmid, pBJ114T-190. When conjugated to other E. coli strains, some of the fusion plasmids were able to be resolved into the original two single plasmids. A non-conjugative plasmid, pBJ114-96, exhibited a high degree of sequence identity with the phage P7-like plasmid as well as an mcr-1-bearing plasmid. Another plasmid, pBJ114-78, was found to contain multidrug resistance genes and various mobile elements. Conclusions The fusion plasmid recoverable from the transconjugant was found to be generated as a result of a recombination event that occurred upon interaction between a blaNDM-5-carrying plasmid and another plasmid present in the parental strain. Such recombination events presumably play a potential role in the dissemination of the blaNDM genes among different plasmids and pathogenic bacterial strains.