Modifications of mgrB instigate colistin resistance in poultry gut-isolates in Bangladesh

Abstract

Background: Acquisition of colistin resistance by a plasmid-mediated gene, mcr-1, was described for Enterobacteriaceae in many countries. We identified mcr-1-carrying colistin-resistant poultry gut-bacteria in Bangladesh and observed a group of colistin-resistant isolates without harboring mcr-1. We sought to investigate the genetic factor conferring colistin-resistance in those mcr-1-naive isolates. Several recent studies have reported inactivation of a chromosomal gene, mgrB, to be responsible for acquired colistin-resistance in absence of mcr-1. In this study, we investigated any potential deletion or mutation in mgrB gene to link the colistin-resistance phenotype. Methods and materials: Different selective culture media were used to isolate various bacteria from chicken dropping. Conventional biochemical procedures were followed by API 20E kit (BioMèrieux, Durham, NC) for identification of bacteria. A part of bacterial identification was validated further by genotyping using 16S rDNA analyses. Disc-diffusion and minimal inhibitory concentration (MIC) measurement were performed to determine the colistin susceptibility of the isolates. mgrB gene was amplified by polymerase chain reactions (PCR). Any alterations of nucleotides and/or amino acids in the mgrB gene clones were checked primarily by single sequence blasting with online database and multiple sequence alignment analysis by ClustalW program. Results: Among 99 isolates 87.5% showed phenotypic colistin-resistant by disc-diffusion assessment. All the resistant isolates showed MIC level, between >8 μg/mL to >256 μg/mL. Among these resistant isolates only 36.2% were harboring mcr-1 gene. The identified mcr-1 gene showed complete harmony to phenotypic colistin-resistance. To determine the mgrB alteration, 15 isolates were analyzed in three groups: group-1 was colistin-resistant carrying mcr-1 gene; group-2 was colistin-resistant without mcr-1 gene; and group-3 was colistin-susceptible isolates. Analyses of nucleotide and amino acid sequences revealed deletion mutation and shortening of MgrB protein in all the colistin-resistant isolates. In addition, a novel single amino acid substitution at 28 position of MgrB from phenylalanine (F) to cysteine (C) was observed associating colistin-resistance. Conclusion: Modulation of mgrB gene expression was the key factor for the acquired resistance to colistin. The deletion or non-synonymous substitution of the mgrB gene was shown to associate colistin resistance in gram negative bacteria. These findings attested the regulatory role of mgrB leading to phenotypic polymyxins-resistance.