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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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 P. 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-DR 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. In conclusion, we developed and validated methods suited to the systematic screening of resistance to colistin in gram negative bacilli.</jats:p><jats:sec><jats:title>Clinical relevance</jats:title><jats:p>colistin continues to be one of the last-line therapeutic options to treat carbapenemase-producing gram negative bacilli. The BMD reference methodology, the only one accepted by current standards for evaluating colistin sensitivity, is difficult to implement in laboratories from low-resource countries. In this work, we propose two simple methodologies 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.</jats:p></jats:sec>

Evaluating the drop test method in measuring colistin susceptibility of Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa

“…However, the present study demonstrated high sensitivity, specificity, PPV and NPV for detecting colistin resistance. Other study has found comparable outcomes for CA (97.4 %), VME (7.7 %) and ME (0%) [15].…”

“…In our study, we evaluated a larger number of strains (n=309) than previous studies, where the number of strains analysed ranged from 75 to 271 isolates [9,11,14,15,20]. Overall, the drop test met the FDA-established criteria for assessing antimicrobial susceptibility [18].…”

Colistin resistance screening by 3 μg/ml colistin agar in Carbapenemase‐producing Enterobacterales

“…They established a CA of 98.5% when compared to broth microdilution for Enterobacterales . 18 CAS has been used in the microbiology laboratory system in Argentina since 2018 with excellent results. Giacoboni et al, and Yauri‐Condor et al, in Peru, reported similar results with CAS for Enterobacterales .…”

“…In 2021, Pasterán et al described the technique known as Colistin Agar Spot (CAS), based on agar dilution using a single colistin sulfate concentration (3 μg/ml) but with a simplified inoculation (20‐mm drop‐spot of a 0.5 McFarland bacterial suspension). They established a CA of 98.5% when compared to broth microdilution for Enterobacterales 18 . CAS has been used in the microbiology laboratory system in Argentina since 2018 with excellent results.…”

How to: screening for mcr-mediated resistance to colistin

“…In the Colistin Agar Spot test, 3.0 μg/mL of colistin is added into melted MHA and subsequently swabbed with a 0.5 McFarland standard suspension of the tested strain within a 1 cm 2 spot. Both methods showed low very major errors rates (0.7%) and major errors rates (~3.7%), but these occurred particularly for the mcr-1 strains [37].…”