Simple phenotypic tests to improve accuracy in screening chromosomal and plasmid-mediated colistin resistance in gram-negative bacilli

Abstract

CLSI and EUCAST recommends that only broth microdilution (BMD) should be used for routine colistin susceptibility testing, however, it could be difficult to perform in resource-poor settings. The purpose of this study was to evaluate the accuracy of an agar spot test (COL-AS) and a colistin drop test (COL-DT) as compared to BMD. COL-AS and COL-DT were challenged with a collection of 271 Gram-negative bacilli clinical isolates: 195 Enterobacterales (including 63 mcr-1 positive strains), 37 Acinetobacter spp. and 39 Pseudomonas aeruginosa For COL-AS, 3.0μg/ml (final concentration) of colistin was added to a Mueller-Hinton agar plate and subsequently swabbed with a 0.5 McFarland suspension of tested strain within 1cm2 spot. For COL-DT, 10μl of a 16μg/ml colistin solution was dripped on the surface of a Mueller-Hinton agar plate, previously inoculated with a lawn of tested strain (0.5 McFarland). Colistin solution was made either, by dissolving powder or by disk elution in CA-MHB. Overall, 141/271 (52%) isolates were categorized as colistin resistant by reference BMD. COL-AS yielded a categorical agreement (CA) of 95.5% compared to BMD, with 0.7% very major errors and 3.8% major errors. COL-DT yielded a CA of 96.2% compared to BMD, with 0.7% and 0% very major errors and 3.1% and 3.8% major errors, for colistin powder and disk elution solutions, respectively. Most major errors occurred for mcr-1 producing strains with MICs that fluctuated from 2 to 4 μg/ml according to the method used. In conclusion, we developed and validated methods suited to the systematic screening of resistance to colistin in gram negative bacilli.Clinical relevance: colistin continues to be one of the last-line therapeutic options to treat carbapenemase-producing gram negative bacilli. The BMD reference methodology, recommended by current standards for evaluating colistin sensitivity, is difficult to implement in laboratories from low-resource countries. Recently CLSI endorsed two MIC-based alternative methods for testing colistin in Enterobacterales and P. aeruginosa, a colistin broth disk elution (CBDE) and a colistin agar test (CAT).In this work, we propose two simple methodologies, related to CLSI methods, to screen for colistin resistance, with a performance equivalent to the reference method in detecting resistance to colistin, both of plasmid (mcr) and chromosomal nature. Furthermore, the methods validated here allowed a better identification of those producers of mcr producer with borderline MICs. These screening tests can be routinely performed in addition to the tests currently in use, showed long stability during storage and some of them do not require colistin powder as the source of antibiotic, an important limitation in low-resource countries.