Colistin susceptibility testing: time for a review

Abstract

Sir, Colistin has re-emerged as an important antimicrobial in recent times owing to limited therapeutic options against carbapenemresistant Gram-negative bacteria. Current guidelines (BSAC, CLSI and EUCAST) recommend routine colistin susceptibility testing by estimation of MIC because the disc diffusion test does not reliably detect low-level resistance. – 5 Broth microdilution (BMD) is widely used as a method of MIC estimation in Europe and the USA. Colistin exhibits a varying degree of adherence to organic and inorganic materials due to its polycationic nature, resulting in loss during experimental conditions. Also, polysorbate 80 (P-80), a surfactant widely used as a dispersing agent in BMD panels, may influence the free drug concentration of colistin and hence MIC results. We evaluated the impact of the use of different BMD panels and the presence of P-80 on colistin MIC estimation. A total of 146 clinical isolates collected from a variety of sources and stored at 2708C were evaluated in this study. The isolates included 56 Pseudomonas aeruginosa, 29 Acinetobacter spp. and 61 Enterobacteriaceae. The MIC testing was carried out on two different types of polystyrene microtitre trays (MTTs), namely non-coated V-bottom MTTs (NMTTs; costar 3896; Corning, NY, USA) and tissue-culture-coated round-bottom MTTs (TCMTTs; costar 3799; Corning). The MICs of colistin for the isolates were determined using the CLSI broth dilution method using colistin sulphate. MIC determination was carried out by using an initial bacterial inoculum of 5×10 cfu/mL in Mueller–Hinton broth with or without P-80 (final P-80 concentration of 0.002%) on both types of MTT. The experiments were done in triplicate, and quality control was assured by concurrent testing of P. aeruginosa ATCC 27853 as a control, with all results within the range published by the CLSI. MICs for the isolates in both types of MTT with or without P-80 are shown in Table 1. The NMTT MICs (mean 0.54+0.58) were significantly lower than the TCMTT MICs (mean 2.84+1.93) (P,0.0001; 95% CI –2.5 to –2.1). The tissue coating on MTTs, achieved by means of excess negative electric charge, resulted in an overall 5.3-fold increase in MIC value, probably due to decreased free colistin concentration within the microwells. The differences in MIC results were seen among all types/groups of isolates (3.2, 5.5 and 9.4, respectively, for P. aeruginosa, Enterobacteriaceae and Acinetobacter spp.). The addition of P-80 to NMTTs significantly decreased the colistin MIC (mean 0.09+0.09) by 6-fold (P,0.0001; 95% CI 0.4 –0.5). Although there was a relatively smaller decrease (1.24-fold) in the mean MIC determined using TCMTTs with added P-80 (mean 2.3+1.5), this was also statistically significant (P,0.001; 95% CI 20.31 to 20.75). Comparing the MICs determined using NMTTs and