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First detection of mobilized colistin resistance mcr-1 gene in Escherichia coli isolated from livestock and sewage in Iran.

ABSTRACT: Currently, few studies have investigated the mechanisms of resistance to colistin in Iran. The aim of this study was to investigate mcr-harbouring Escherichia coli dissemination in livestock and sewage in Iran. A total of 115 samples from cows (n = 38), chickens (n = 47) and urban sewage samples (n = 30) were collected. The presence of genes including mcr1-6 and ampC β-lactamase (bla _MOX, bla _CIT, bla _DHA, bla _ACC, bla _EBC, bla _FOX) for colistin-resistant isolates was investigated by multiplex PCR method. Genetic association of colistin-resistant strains was also evaluated by ERIC PCR. Sixty-five isolates were identified as E. coli. Meaningless were resistant to colistin. The highest (26.1%) and lowest (3.07%) resistance were shown to ampicillin and meropenem respectively. Among the three colistin-resistant isolates, 2 (66%) were multidrug resistant, with one of them being mcr-1 positive and the other one positive for DHA ampC β-lactamase gene. No mcr2-6 genes were found. Minimum inhibitory concentration of mcr-producing isolate was 4 mg/L by microbroth dilution. This study reports, first the detection of mcr-1 in E. coli from farm animals in Iran, a finding that is indicative of a global distribution of this plasmidic element and threatning the use of colistin as a last resort antibiotic. No clonal relationship was observed between the colistin-resistant E. coli isolates by ERIC-PCR. Monitoring the presence of these strains in animal sources help as to controlling the spread of resistance genes from animal to human is vital.

SUBMITTER: Nikkhahi F

PROVIDER: S-EPMC8086131 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: The emergence of the plasmid-mediated mcr colistin-resistance gene in bacteria poses a potential threat for treatment of patients, especially when hospitalized. The aims of this study were to search for the presence of mcr-1 and mcr-2 genes among colistin-resistant Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) isolates from clinical specimens and to determine the fingerprint of strains by enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) method. Methods: In this study, 712 nonduplicate Enterobacteriaceae isolates from clinical specimens were examined. All of the isolates were subcultured on suitable media, and the isolated colonies were identified by standard biochemical tests. Antimicrobial susceptibility test on 7 antibiotics was performed by disk diffusion method, and minimal inhibitory concentration (MIC) of isolates to colistin was determined by the E-test method. These isolates were typed by ERIC-PCR method, and the presence of mcr-1 and mcr-2 genes was investigated by PCR method. Results: Out of 712 nonduplicate Enterobacteriaceae, 470 isolates, including 351 (74.7%) E. coli and 119 (25.3%) K. pneumoniae, were detected. The results of antibiogram tests showed that most of the isolates (81.3%) were resistant to ceftazidime; however, the most susceptibility among of E. coli and K. pneumoniae isolates was observed (81.5%) to colistin. The typing results by ERIC-PCR method showed 36 and 23 fingerprint patterns for colistin-resistant E. coli and K. pneumoniae strains, respectively. Among 64 (13.6%) colistin-phenotypically-resistant Enterobacteriaceae, 8 isolates (1.7%) had mcr-1 gene. These 8 isolates were attributed to E. coli and K. pneumoniae with 6 and 2 isolates, respectively. Whereas no isolates carrying the mcr-2 gene was found. These colistin-resistant isolates displayed colistin MIC values >2 μg/ml in the antibiotic concentration by E-test method. Conclusion: Spreading of Enterobacteriaceae strains harboring plasmid-mediated mcr could fail the colistin-included therapy regimen as the last line of treatment against multidrug-resistant bacterial infections.

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:Colistin is one of the last-resort antibiotics for infections caused by multidrug-resistant Gram-negative bacteria. However, the wide spread of novel plasmid-carrying colistin resistance genes mcr-1 and its variants substantially compromise colistin's therapeutic effectiveness and pose a severe danger to public health. To detect colistin-resistant microorganisms induced by mcr genes, rapid and reliable antibiotic susceptibility testing (AST) is imminently needed. In this study, we identified an RNA-based AST (RBAST) to discriminate between colistin-susceptible and mcr-1-mediated colistin-resistant bacteria. After short-time colistin treatment, RBAST can detect differentially expressed RNA biomarkers in bacteria. Those candidate mRNA biomarkers were successfully verified within colistin exposure temporal shifts, concentration shifts, and other mcr-1 variants. Furthermore, a group of clinical strains were effectively distinguished by using the RBAST approach during the 3-h test duration with over 93% accuracy. Taken together, our findings imply that certain mRNA transcripts produced in response to colistin treatment might be useful indicators for the development of fast AST for mcr-positive bacteria. IMPORTANCE The emergence and prevalence of mcr-1 and its variants in humans, animals, and the environment pose a global public health threat. There is a pressing urgency to develop rapid and accurate methods to identify MCR-positive colistin-resistant bacteria in the clinical samples, providing a basis for subsequent effective antibiotic treatment. Using the specific mRNA signatures, we develop an RNA-based antibiotic susceptibility testing (RBAST) for effectively distinguishing colistin-susceptible and mcr-1-mediated colistin-resistant strains. Meanwhile, the detection efficiency of these RNA biomarkers was evidenced in other mcr variants-carrying strains. By comparing with the traditional AST method, the RBAST method was verified to successfully characterize a set of clinical isolates during 3 h assay time with over 93% accuracy. Our study provides a feasible method for the rapid detection of colistin-resistant strains in clinical practice.

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First detection of mobilized colistin resistance mcr-1 gene in Escherichia coli isolated from livestock and sewage in Iran.

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