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We modified rapid polymyxin Nordmann-Poirel (RPNP) test, called rapid colistin disk elution (RCDE) test, for detecting colistin resistance in Gram-negative bacilli and evaluated its performance compared with colistin broth disk elution (CBDE) test recommended by Clinical and Laboratory Standards Institute (CLSI). The RCDE test was performed by using a 10-μg colistin disk in 2.7 mL volume (final colistin concentration of 3.7 μg/mL) of either cation-adjusted Mueller-Hinton broth or phenol red broth base media with bacterial inoculum of 1-μL loop, and 1-4 and 16-20 hr incubation for Enterobacteriaceae and Acinetobacter baumannii isolates, respectively. Both tests were evaluated in 236 Enterobacteriaceae and 49 A. baumannii isolates using broth microdilution as reference method. Among the Enterobacteriaceae isolates, categorical agreement and very major error (VME or false intermediate susceptibility) rate were 98.3% and 5.4%, respectively, for the RCDE test, compared with 97.9% and 7.1%, respectively, for the CBDE test. Both tests had major error (ME or false resistance) rate of 0.6%. For the A. baumannii isolates, the RCDE and CBDE tests gave high VME rates of 8.3% and 16.7%, respectively. The RCDE test showed good performance comparable with the CBDE test but is cheaper and more rapid (3 hr) and convenient, thus suggesting as an alternative for detecting colistin resistance among Enterobacteriaceae in low-income countries.

The global emergence of colistin resistance in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates is a serious public health concern. We therefore aimed to investigate colistin resistance mechanisms in 5 colistin-resistant (COL-R) CRAB isolates collected from Thai patients in 2016 by whole genome sequencing (WGS) compared with those of 5 colistin-intermediate (COL-I) CRAB isolates from the same period. All isolates were subjected to antimicrobial susceptibility testing, efflux pump inhibitor-based test and WGS. Mutations in known genes associated with colistin resistance were analyzed and deleterious mutations were then predicted by PROVEAN tool. The 10 CRAB isolates carried bla OXA-23 with the addition of bla OXA-58 in 1 isolate. All COL-R isolates exhibited colistin MICs of 4 μg/mL except for 1 isolate with that of 16 μg/mL. They belonged to ST2, ST16, ST23, ST164 and ST215, whereas the COL-I isolates with colistin MICs of ≤0.25-1 μg/mL were ST2, ST164 and ST215. Neither increased efflux pump activity nor mcr gene was found in any COL-R isolate. Three COL-R isolates contained different PmrB variants: a novel 10-amino acid (aa) repeat sequence insertion, VILGCILIFS between positions 27 and 28 (S27_A28insVILGCILIFS) in transmembrane domain (TM); a 1-aa insertion, alanine between positions 162 and 163 (A162_I163insA) in TM; and a 1-aa substitution, A226T in histidine kinase domain. One COL-R isolate possessed PmrA variant with A80V substitution. These alterations were predicted as deleterious. Mechanisms of colistin resistance in the remaining COL-R isolate were still unknown. In conclusion, the alterations in both PmrB and PmrA were predicted and suggested as initial mutations responsible for low-level colistin resistance in our CRAB isolates. Under selective pressure, these isolates may exhibit higher level colistin resistance by the additional mutations, leading to more therapeutic difficulties., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5-2 µg/mL, with MIC 50 and MIC 90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3-8 μg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC 50 and MIC 90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried bla OXA-23-like . The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.