title: A SYBR®Green-based real-time PCR method for improved detection of mcr-1-mediated colistin resistance in human stool samples.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3001 Bern, Switzerland.
authors.forename: Valentina
authors.initials: V
authors.lastname: Donà
authors.affiliation: Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3001 Bern, Switzerland; Graduate School of Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
authors.forename: Odette J
authors.initials: OJ
authors.lastname: Bernasconi
authors.affiliation: Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3001 Bern, Switzerland.
authors.forename: Sara
authors.initials: S
authors.lastname: Kasraian
authors.affiliation: Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3001 Bern, Switzerland.
authors.forename: Regula
authors.initials: R
authors.lastname: Tinguely
authors.affiliation: Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3001 Bern, Switzerland. Electronic address: andrea.endimiani@ifik.unibe.ch.
authors.forename: Andrea
authors.initials: A
authors.lastname: Endimiani
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D052160:Benzothiazoles; D003091:Colistin / Q000494:pharmacology*; D003959:Diamines; D024881:Drug Resistance, Bacterial; D029968:Escherichia coli Proteins / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014838:Volunteers
chemical_list: D000900:Anti-Bacterial Agents; D052160:Benzothiazoles; D003959:Diamines; D029968:Escherichia coli Proteins; C000614037:MCR-1 protein, E coli; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D003091:Colistin
keywords: Colistin; Enterobacteriaceae; Polymyxins; Real-time PCR; Stools; mcr-1