Dataset Information

Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition.

ABSTRACT: IncX4 plasmids are associated with the dissemination of the mcr-1 genes in Enterobacteriaceae. We screened IncX4 plasmids among 2,470 isolates of Enterobacteriaceae and determined the mcr-1 positive isolates. Forty-three isolates were observed to carry IncX4 type plasmid, among which 13 were identified to carry mcr-1 gene. Three representative mcr-1-positive IncX4 plasmids were selected for high-throughput sequencing. Comparative genomics showed that the mcr-1-carrying IncX4 plasmids exhibit remarkable similarity in the backbone, and the major distinction lies in the region containing mcr-1. The major variable regions of all the IncX4 plasmids were fully characterized by PCR-RFLP. The results revealed that the mcr-1 was located on the Variable Region I of IncX4 plasmids in 11 E. coli isolates. Among them, nine E. coli strains possess an epidemic pCSZ4-like IncX4 plasmid containing mcr-1. ISApl1 was presumably involved in the transposition of the mcr-1 cassette and then was lost. Similar genetic contexts were found in different plasmids, even the E. coli chromosome, implying the acquisition of mcr-1 by a unique common mechanism.

SUBMITTER: Sun J

PROVIDER: S-EPMC5428312 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition.

Sun Jian J Fang Liang-Xing LX Wu Zuowei Z Deng Hui H Yang Run-Shi RS Li Xing-Ping XP Li Shu-Min SM Liao Xiao-Ping XP Feng Youjun Y Liu Ya-Hong YH

Scientific reports 20170324 1

IncX4 plasmids are associated with the dissemination of the mcr-1 genes in Enterobacteriaceae. We screened IncX4 plasmids among 2,470 isolates of Enterobacteriaceae and determined the mcr-1 positive isolates. Forty-three isolates were observed to carry IncX4 type plasmid, among which 13 were identified to carry mcr-1 gene. Three representative mcr-1-positive IncX4 plasmids were selected for high-throughput sequencing. Comparative genomics showed that the mcr-1-carrying IncX4 plasmids exhibit rem ...[more]

PMID: 28336940

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Project description:Colistin has become the last-line antimicrobial for the treatment of multidrug resistant (MDR) Enterobacterales in human medicine. To date, several colistin resistance genes have been described. Of them mcr-1 is disseminated worldwide in Escherichia coli of human and animal origin. The aim of this study was to characterize mcr-mediated resistance plasmids from E. coli of animal origin in Spain. From our strain collection, 70 E. coli of pig origin collected between 2005 and 2014 (10 per year, except for years 2009-2010-2013) were randomly selected and screened for the presence of mcr-genes. Additionally, 20 E. coli isolated in 2011 from white storks (Ciconia ciconia) from the same urban household waste landfill associated colony were also included. Whole genome sequencing of mcr-positive isolates was carried out on a MiSeq (Illumina). Hybrid whole genome sequencing strategy combining nanopore and Illumina technologies were performed in a selection of isolates to close the genomes and plasmids and identify the presence of antimicrobial resistance genes. Minimum inhibitory concentration (MIC) was used to assess the susceptibility to colistin. Mating experiments were carried out to evaluate transferability of the mcr-genes. A total of 19 mcr-1 and one mcr-4 positive isolates were detected, 15 from pigs distributed during the study period, and five from storks collected in 2011. No other mcr-variants were found. The MICs for colistin ranged between 4 and >4 mg/L. High diversity of STs were detected among the mcr-1 positive E. coli isolates, with only ST-10 shared between pigs and white storks. Except for one isolate, all were genotypic and phenotypically MDR, and five of them also harbored cephalosporin resistance genes (bla CTX-M- 14, bla SHV- 12, and three bla CMY- 2). mcr-1 genes were mobilizable by conjugation, associated with IncX4, IncHI2, and IncI2 plasmids. In our study, mcr-1 genes have been circulating in pig farms since 2005 harbored by a variety of E. coli clones. Its persistence may be driven by co-selection since plasmids containing mcr-1 also exhibit resistance to multiple drugs used in veterinary medicine. Furthermore, this is the first report of the presence of mcr-1 gene in isolates from white storks in Spain. This finding highlights the potential importance of wildlife that forage at urban household waste landfills in the transmission and spread of colistin resistance genes.

Project description:The emergence of the plasmid-borne colistin resistance gene mcr-1 threatens public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmid conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer. Eighteen essential transfer genes were identified, including five with annotations unrelated to conjugation. Cappable-seq, transcriptome sequencing (RNA-seq), electrophoretic mobility shift assay, and β-galactosidase assay confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. PixR is not active under nonmating conditions, while the expression of the pixR, pilX3-4, and pilX11 genes increased 3- to 6-fold upon contact with recipient Escherichia coli C600. Plasmid invasion and coculture competition assays revealed the essentiality of pixR for spreading and persistence of mcr-1-bearing IncX4 plasmids in bacterial populations. Effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence. IMPORTANCE The spread of clinically relevant antibiotic resistance genes is often linked to the dissemination of epidemic plasmids. However, the underlying molecular mechanisms contributing to the successful spread of epidemic plasmids remain unclear. In this report, we shine a light on the transfer activation of IncX4 plasmids. We show how conjugation promotes the invasion and persistence of IncX4 plasmids within a bacterial population. The dissection of the regulatory network of conjugation helps explain the rapid spread of epidemic plasmids in nature. It also reveals potential targets for the development of conjugation inhibitors.

Project description:Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. IMPORTANCE Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, mcr-1, has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of mcr-1 are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare mcr-1.26 gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of mcr-1.26 and its transmission between different niches.

Project description:The role of plasmids in the complex pandemic of antimicrobial resistance is increasingly being recognized. In this respect, multiple mobile colistin resistance (mcr) gene-carrying plasmids have been described. However, the characteristics and epidemiology of these plasmids within local healthcare settings are largely unknown. We retrospectively characterized the genetic composition and epidemiology of plasmids from mcr-1-positive bacterial isolates identified from patients from a large academic hospital in the Netherlands. Clinical Gram-negative bacteria with an MIC > 2 μg/mL for colistin, obtained from patients hospitalized at the Erasmus MC University Medical Center Rotterdam during the years 2010-2018, were screened for presence of the mcr-1 gene. Extracted plasmids from mcr-1-positive isolates were sequenced using a combination of short- and long-read sequencing platforms, characterized by incompatibility type and genetic composition and compared to publicly available mcr-1-carrying plasmid sequences. In 21 isolates from 14 patients, mcr-1 was located on a plasmid. These plasmids were of diverse genetic background involving Inc types IncX4, IncI2(delta), IncHI2, as well as double Inc types IncHI2/IncN and IncHI2/IncQ. mcr-1-carrying plasmids were found in Escherichia coli, Klebsiella pneumoniae, and Kluyvera georgiana, and within the chromosome of an ST147 K. pneumoniae isolate. In depth analysis indicated intrapatient, interpatient, and interspecies transmission events of mcr-1-carrying plasmids. In addition, our results show that the mcr-1 gene resides in a rich environment full of other (mcr-1 negative) plasmids and of many different Inc types, enabling interplasmidal transfer events and facilitating widespread dissemination of the mcr-1 gene. Multiple mcr-1-carrying plasmid transmission events had likely occurred among isolates from hospitalized patients. Recognition and identification of plasmid transmission events within hospitals is necessary in order to design and implement effective infection control measures.

Dataset Information

Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition.

Publications

Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition.

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