Screening for MCR Genes in Rectal Swabs

Processing of rectal swabs and blood culture have been described in Figure 1 and in supplementary methods. Briefly, rectal swabs were plated on vancomycin (10 mg/L) (MP Biomedicals, California, USA) supplemented chrome agar (CA) (BD BBL, MD, USA) and incubated (37°C, 18–24 h). Cultures from the primary inoculum of each plate and cultured isolates from neonatal blood were screened for mcr genes by polymerase chain reaction (PCR) as described previously [18 (link)]. Sample positive for mcr genes were further enriched in Enterobacteriaceae Enrichment (EE) Mossel broth (37°C, 18–24 h) (BD BBL) and plated on to CA supplemented with/without colistin sulphate (2 mg/L) (MP Biomedicals).
Colonies with different colours were picked from plates with/ without colistin, and again screened for mcr genes. Amplified PCR products were Sanger sequenced and stocked for further analysis [12 (link)]. Any mcr-negative colony of similar colour as the mcr-positive ones were collected from CA plates (without colistin) to check clonality with mcr-positive colonies.