An investigation of Mobilized Colistin-Resistance (MCR) gene and underlying dissemination mechanism using clinical, poultry and environmental samples in Bangladesh

Abstract

Colistin, widely considered as the last-resort antibiotic for the treatment against multidrug-resistant (MDR) gram-negative bacteria worldwide, is being used indiscriminately in the poultry, livestock and fishery farms of Bangladesh as a growth promoter and an antimicrobial agent. Although the presence of mobilized colistin-resistance (mcr) gene in human, poultry and environmental samples has been reported in E.coli in Bangladesh recently, there is no report of mcr gene in other gram-negative bacteria. Moreover, the presence of colistin-resistant gene and their minimum inhibitory concentration was also analyzed and coherence was found. Therefore, a study was carried out to scrutinized mcr gene(s) from above mentioned all the sources in order to investigate those mcr possessing isolates which are intimately related to some hospitalacquired infections (HAIs). This study also aims to investigate clinical as well as poultry and environmental samples in order to identify the mechanism of dissemination of mcr genes in gram negative bacteria and assess the risk of transmitting resistant genes from poultry, livestock to human gut bacteria through the environment in Bangladesh. A total of 450 clinical isolates (acute respiratory infection, n=212; wound sample, n=150; diarrheal disease, n=88), Poultry (n=100), cow fecal (n=60) and water (n=65) were analyzed. All the isolates were cultured and confirmed using standard microbiological and biochemical tests. Kirby-Bauer disk diffusion method was used to screen colistin-resistant isolates and the DNA templates were subjected to PCR using mcr gene specific primers (mcr-1 to mcr-7). Furthermore, the minimum inhibitory concentration (MIC) was determined using broth micro-dilution method and MIC was found higher for those isolates which possess mcr-2 than those, possess mcr-1. Next, the plasmid DNA was extracted and the PCR was performed again in order to differentiate between plasmid-mediated andchromosome-mediated resistance of colistin. Overall, 97 colistin-resistant (mcr-1 and mcr-2 only) bacteria were isolated, of which, 13.80% E.coli, 37.93% Klebsiella pneumoniae, 31.03% Acinetobacter baumanii, 3.45% Pseudomonas aeruginosa, 3.45% Enterobacter spp. Our study also revealed that mcr-1 gene was present in all type of sample and mcr-2 was detected in clinical and cow fecal samples only. We provide new evidence that besides E.coli, four other gram-negative bacteria in Bangladesh have become colistin resistant and also guidelines for relevant authorities to tackle the extensive and uncontrolled use of colistin in Bangladesh