A SYBRGreen-Based Real-time PCR Method for Improved Detection of Mcr-1-mediated Colistin Resistance in Human Stool Samples

Background: 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 fecal samples.
Methods: Stools (n=88) from 36 volunteers were analyzed. To isolate mcr-1-producing Enterobacteriaceae samples were enriched overnight in LB broth containing 2 mg/L colistin and then plated in selective agar plates with 4 mg/L colistin. We then designed a SYBRGreen-based RT-PCR targeting the mcr-1. For method validation and to establish the limit of detection (LOD), total DNA was extracted from mcr-1-negative and mcr-1-positive E. coli. RT-PCR was also performed with DNA extracted from the 88 native stools and after enriching them in LB broth containing colistin.
Results: Based on the culture approach, 3 unique volunteers resulted colonized with mcr-1-harboring E. coli strains. For culture isolates, our RT-PCR exhibited a LOD of 10 genomic copies/reaction with both sensitivity and specificity of 100%. Nevertheless, when testing native stools, only two out of the three mcr-1-positive specimens were detected. However, after enrichment in LB 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: We developed a rapid RT-PCR for the detection of mcr-1 from stool specimens. We increased the detection rate by testing selective broth enrichments. This approach has also the advantage of concomitant isolation of the mcr-1-harboring strains for further antimicrobial susceptibility and genetic tests.