‘Breakpoint broth microdilution plate’ for susceptibility testing of Gram negative bacilli against colistin sulfate

The MIC method applicable to Gram negative bacilli including Acinetobacter spp. is broth microdilution (BMD). Cost and/or availability issues limit the use of commercial MIC panels in resource limited settings. Objectives: To design and implement an in-house breakpoint BMD panel (BBMD) for colistin against Gram negative bacilli. Design: BBMD panel was prepared in 96-well plate. MIC concentrations of 1, 2, & 4 ​μg/mL for test, and 0.25, 0.5, 1, 2 & 4 ​μg/mL for control strains were selected to accommodate 19 test and 3 quality control strains per plate. Plates were frozen at −80 ​°C until testing. Validation was performed using strains from a previously published study and compared with freshly prepared MIC panel of 16–0.03 ​μg/mL. Results: Validation showed 100% agreement with the reference method and BBMD was introduced into routine laboratory practice for colistin susceptibility of carbapenem resistant Enterobacterales (CRE), Acinetobacter baumannii complex and Pseudomonas aeruginosa. From 2nd July-16th September 2018, a total of 1294 (mean 16.8 ​± ​5.5 isolates/day) clinical isolates were tested; 1157/1294 were reported (MIC ≤2 ​μg/mL) within 24-h, whereas 133 required resistance confirmation by full-range BMD. Resistance was confirmed for all but 24 isolates. These discrepancies were mostly due to contamination with bacterial genera inherently resistant to colistin. Conclusion: This BBMD plate is a high through-put and practical method that could reliably be utilized in a routine microbiology laboratory for colistin susceptibility testing of CRE, A. bauamanii complex and P. aeruginosa.