In vitro activity of methylene blue and EMB agar on colistin-resistant A.baumannii: An experimental study

Abstract

Background Colistin is one of the last resort antibiotics used against carbapenem-resistant Acinetobacter baumannii (AB); however, colistin resistance has been reported recently. Methylene blue (MB) is used in microbiology for staining, and in medicine as an antidote drug. Here, we investigated antimicrobial effects of MB and Eosin Methylene blue (EMB) agar against colistin-resistant AB strains. Methods The AB ATCC 19606 strain and 31 AB clinical isolates were included in the study. In the first round, ATCC strain and a clinical isolate were transformed into colistin-resistant forms, using Li's method, with increasing colistin concentrations. At each step, new MICs were determined and subcultures were inoculated to EMB and sheep blood agar (SBA). The colistin MIC values of the subcultures were also determined using Mueller Hinton Agar (MHA) containing 14 µg/mL MB. In the second round, colistin resistant clones of all collected clinical isolates (n=31) were obtained and screened to investigate their susceptibility to EMB agar by inoculating on SBA and EMB agar. Results At the beginning, the MICs of two strains were 0.5 µg/mL. At the last stage, both MICs had risen to 64 µg/mL. Subpopulations with high colistin resistance (>=32 µg/mL) were inhibited by MB and EMB agar, but could grow well on SBA. In MHA plates containing MB, the MICs decreased to the 0.5 µg/mL level for colistin-susceptible or moderately resistant clones. Additionally, clones with high colistin resistance showed atypical colony morphology on SBA. In the second round, MICs of the colistin resistant clones of all clinical isolates rose to 8 µg/mL after colistin exposure and 35% of those clinical isolates were inhibited by EMB agar while they could grow on SBA. Conclusion Highly resistant strains were totally inhibited by the effect of MB and EMB agar, while the MICs of the susceptible and moderate resistant clones decreased. EMB agar and MB may have inhibitory effects against colistin-resistant AB strains and MB may have a potential to be used as an antimicrobial drug. Secondly, using only EMB agar for subculturing may cause missing of colistin-resistant strains and giving incorrect identification or antibiogram reports in clinical microbiology laboratories.