Antimicrobial susceptibility analysis

In an effort to assess the role of colistin resistance in strains positive for the mcr gene all strains were subjected to antimicrobial susceptibility analysis to colistin sulfate (Alfa Aesar, Ward Hill, MA) using broth microdilution and agar dilution assays. Overnight cultures of each strain were grown on Tryptone Soya Agar (TSA) plates and colonies selected and adjusted to a OD 0.5 McFarland in sterile water using an nephelometer (Sensititre); then 10ul of the suspension was added to 11 mls of Mueller Hinton (MH) broth which was used to inoculate the broth microdilution plates and the agar dilution plates.

The broth microdlution and agar dilution plates tested antimicrobial resistance to colistin at the following dilution range 0.5 to 32 ug/ml. Once all plates were inoculated as appropriate they were incubated at 37°C for 18h. Plates were observed for growth and minimum inhibitory concentrations (MIC’s) were defined as the lowest concentration of antimicrobial to inhibit growth of the test strains.