A SYBR® Green-based real-time PCR method for improved detection of mcr-1-mediated colistin resistance in human stool samples

J Glob Antimicrob Resist. 2017 Jun:9:57-60. doi: 10.1016/j.jgar.2017.01.007. Epub 2017 Apr 8.

Abstract

Objectives: The aim of this study was to design a rapid and sensitive real-time PCR (rt-PCR) method for colistin resistance mcr-1 gene detection in human faecal samples.

Methods: Stools (n=88) from 36 volunteers were analysed. To isolate mcr-1-producing Enterobacteriaceae, samples were enriched overnight in Luria-Bertani (LB) broth containing 2mg/L colistin and were then plated on selective agar plates with 4mg/L colistin. A SYBR® Green-based rt-PCR targeting mcr-1 was then designed. For method validation and to establish the limit of detection (LOD), total DNA was extracted from mcr-1-negative and mcr-1-positive Escherichia coli. rt-PCR was also performed with DNA extracted from 88 native stools and after enriching them in LB broth containing colistin.

Results: Based on the culture approach, three unique volunteers resulted colonised with mcr-1-harboring E. coli strains. For culture isolates, rt-PCR exhibited a LOD of 10 genomic copies/reaction, with both sensitivity and specificity of 100%. Nevertheless, when testing native stools, only two of the three mcr-1-positive specimens were detected. However, after enrichment in LB broth containing colistin, the rt-PCR was strongly positive for all culture-positive samples. The average cycle threshold was 22, granting rapid and confident detection of positive specimens within 30 cycles. No false positives were observed for the remaining 85 culture-negative specimens.

Conclusions: A rapid rt-PCR for detection of mcr-1 from stool specimens was developed. The detection rate was increased by testing selective broth enrichments. This approach also has the advantage of concomitant isolation of mcr-1-harboring strains for further antimicrobial susceptibility and genetic testing.

Keywords: Colistin; Enterobacteriaceae; Polymyxins; Real-time PCR; Stools; mcr-1.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology*
  • Benzothiazoles
  • Colistin / pharmacology*
  • Diamines
  • Drug Resistance, Bacterial*
  • Escherichia coli Proteins / genetics*
  • Feces / microbiology*
  • Humans
  • Organic Chemicals / metabolism*
  • Quinolines
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Staining and Labeling / methods
  • Volunteers

Substances

  • Anti-Bacterial Agents
  • Benzothiazoles
  • Diamines
  • Escherichia coli Proteins
  • MCR-1 protein, E coli
  • Organic Chemicals
  • Quinolines
  • SYBR Green I
  • Colistin