In-house pre-prepared broth microdilution plates: A simple, cheap and pragmatic approach for susceptibility testing of colistin

Abstract

Background: With the upsurge in multi-drug resistant Gram-negative bacilli, it is imperative to optimize and standardize the antibiotic susceptibility testing (AST) methods for polymyxins (colistin & polymyxin B). The long overdue confusion regarding the susceptibility testing of colistin was put to rest in 2016 when the CLSI-EUCAST joint Polymyxin Breakpoint Working Group recommended broth microdilution (BMD) in polystyrene plates as the reference method for AST. Though several commercially-available BMD plates have been assessed by EUCAST, the high cost of such commercial plates during contemporary times hinder their use in routine microbiological laboratories especially in low and middle income countries. Thus, a suitable alternative to commercial BMD plates should be sought for to ensure a cost-effective, rapid and reliable reporting of AST of colistin. Methods and materials: With the aim of reducing this redundancy in the workflow, we designed an experiment to charge several microtiter plates with the drug dilution and the media on day 0, storing them frozen at −200C and then retrieving them sequentially for AST (henceforth called pre-prepared frozen BMD plates (PFBP). The PFBP were charged with same representative organisms on every 2–4 days for two months to check for the reliability of AST reporting. Essential agreement (EA), categorical agreement (CA), very major error (VME) and major errors (ME) were computed in accordance with ISO standard 20776-2. For validation, the criteria of EA3 90%, CA 3 90%, VME £1.5% AND ME£3% was used. Pearson correlation and Cohen's kappa (k) score was calculated using GraphPad (San Diego, CA, USA). Results: For colistin, EA and CA of PFBP was 97.5% and 98.3%, respectively. For polymyxin B, EA and CA of PFBP was 99.1% and 90%. Further, the performance of PFBP for colistin susceptibility testing of A. baumannii in the present study (EA=97.5%, CA=98.3%) was superior to five commercial plates available and evaluated by EUCAST. Conclusion: Based on these observations, we propose that pre-prepared frozen BMD plates can be incorporated in routine workflow of microbiological laboratory as reliable, cheap and rapid method for susceptibility testing of colistin.