Investigation of Colistin Resistance and Heteroresistance in Enterobacterales Isolates from Poultry, Water and Clinical Specimens – Role of mcr-1 genes

Abstract

Background: The mobilized colistin resistance, mcr genes, have attracted global attention, as they confer resistance to polymyxins, one of the last-resort antimicrobials’ family for the treatment of critical infections caused by multidrug-resistant Gram-negative bacteria. This resistance mechanism is plasmid mediated and can be transferred horizontally. Resistance to colistin may go undetected when performing traditional antimicrobial susceptibility testing because of the phenomenon of heteroresistance. This phenotype is denoted by the presence of subpopulations of bacterial cells with higher levels of antimicrobial resistance than those of the rest of the population. Aim: The aim of this study was to determine the prevalence of colistin resistance and to investigate the different mechanisms of colistin resistance and heteroresistance in Escherichia coli and Klebsiella pneumoniae isolates recovered from poultry feces, water samples and clinical specimens in Lebanon. Methods: Colistin resistance was evaluated in 161 E. coli and 92 K. pneumoniae isolates recovered from clinical, poultry feces, and water samples using broth micro-dilution (BMD). Moreover, BMD was done on all the colistin resistant isolates using 19 antimicrobials from different antimicrobial families. The prevalence of mcr genes (mcr-1 to mcr-8) was assessed by multiplex PCR in all the colistin resistant E. coli and K. pneumoniae isolates and subjected to whole genome sequencing. To determine the possible presence of colistin resistant subpopulations in colistin susceptible E. coli and K. pneumoniae isolates, population analysis profiles assay was performed. This was followed by the fitness cost assay to determine the difference in growth rate between the colistin resistant subpopulations and the susceptible ones in the presence and absence of colistin Results: BMD results showed that out of the 253 Enterobacterales isolates, 27 E. coli isolates recovered from poultry feces, 10 E. coli isolates obtained from water samples and 10 K. pneumoniae clinical isolates were colistin resistant. All colistin resistant isolates had also various levels of resistance to a panel of antimicrobial agents. PCR results showed that the mcr-1 gene was harbored in all the E. coli isolates recovered from poultry feces and in 5 out of the 10 E. coli isolates obtained from water samples. Moreover, no mcr gene was detected in any of the colistin resistant clinical K. pneumoniae isolates. WGS results of 18 out of the 27 colistin resistant E. coli isolates recovered from poultry feces showed the presence of 7 different sequence types (ST) with ST1140 being the most prevalent. In addition, the number of antimicrobial resistant determinants detected in these isolates ranged between 4 and 21 with mcr-1 being the most prevalent gene. Furthermore, a total of 15 different plasmids were detected in these isolates with IncX4 being predominant. WGS results of the 5 out of the 10 colistin resistant E. coli isolates obtained from water samples showed that they were distributed into 3 different STs, with ST10773 being the most prevalent. Moreover, a total of 23 different antimicrobial resistant determinants were detected in these 5 isolates with blaTEM-1, tet(A)-1, dfrA-14, floR, and mcr-1 genes being the most predominant. In addition to that, a total of 10 different plasmids were detected among the isolates. WGS results of 2 out of the 10 colistin resistant K. pneumoniae clinical isolates showed that they belonged to ST383 and ST39. Furthermore, a total of 31 antimicrobial resistant determinants were detected, including the carbapenem resistant gene blaNDM-5 (n=1) and blaOXA-48 (n=2). Besides, 3 different plasmids were harbored in each isolate. Population analysis profiles assay showed that none of the 13 colistin susceptible E. coli isolates had a colistin resistant subpopulation. However, 10 out of the 12 colistin susceptible K. pneumoniae isolates showed to have a colistin resistant subpopulation. Fitness cost assay showed that in the absence of colistin, both the colistin susceptible K. pneumoniae isolates and the colistin resistant subpopulations had the same growth rate. However, in the presence of colistin, a significant decrease in the growth rate of the colistin susceptible isolates occurred when compared to the latter in the absence of colistin. In addition to that, similar growth rates were detected between the colistin resistant subpopulation in the absence and presence of colistin. Conclusion: Plasmid mediated colistin resistance, mcr-1 gene, in E. coli isolates recovered from poultry and water poses a serious threat on public health. This gene could be transferred horizontally into colistin susceptible isolates and they could be introduced into the human community if no strict measures are taken. Moreover, the detection of colistin resistant subpopulations among colistin susceptible K. pneumoniae clinical isolates may cause serious complications leading to treatment failure.