Mechanism and biological cost of MCR-1 mediated colistin resistance in enterobacteriaceae

Colistin is one of the key antibiotics to treat infections caused by multi-drug resistant (MDR) Gram-negative bacteria. However; in 2015, plasmid mediated colistin resistance, designated mcr-1, was first reported in China. MCR-1 was unusually found in Escherichia coli and conferred only low levels of resistance to colistin. Soon mcr-1 was found worldwide and caused great concern in public health. \nA small number of studies have shown that acquisition of mcr-1 plasmid is associated with no or only a slightly decrease in bacterial fitness. In order to assess the capacity to develop high colistin resistance in mcr-1 harbouring E. coli and its effect on bacterial fitness and virulence, seven wild-type E. coli strains (PN16, PN21, PN23, PN24, PN25, PN42, PN43) from Phitsanulok, Thailand were selected and challenged with increased concentration of colistin for 14 days. All isolates showed an increase in colistin resistance (4- to 64- fold increase in colistin MIC up to 256mg/L), and subsequently, designated high level colistin resistant mutants (HLCRMs). In all seven HLCRMs, two showed 11- and 3- fold increase in mcr-1 expression (PN21 [showed 11-fold] and PN25). No increase in mcr-1 copy number or mutations in the immediate genetic context of mcr-1 was detected in all HLCRMs. Interestingly, in PN25 and PN42 HLCRMs, amino acid mutations in PmrA and PmrB were identified, respectively. \nThose HLCRMs were associated with significant either fitness burden or reduction in virulence, or both. In-vitro fitness was measured by growth rate. Compared with wild-type isolates, HLCRMs showed slower growth in colistin-free medium (p <0.01). Competition assays showed relative fitness compared HLCRMs with parental strains which ranged for 0.4-0.7 (p=**) (except for PN16 [relative fitness 0.9]). \nA Galleria pathogenicity model was used to measure the virulence of wild-type strains and mutants. In every case, the death rate of Galleria for HLCRMs was lower than that for wild-type strains. Significant difference in bacterial mortality were identified in PN16, PN21, PN23 (p=**/***). \nDue to its high expression of mcr-1 (11-fold), cellular morphology by transmission electron microscopy (TEM) was undertaken on PN21 and its HLCRM to further understand how MCR-1 has an effect on bacterial cell outer-membrane. However, no obvious difference on outer-membrane between PN21 and mutant was identified. \nThe study shows that HLCRMs from wild-type strains are associated with significant fitness burden and decrease in virulence. These data will contribute to our understanding of mcr-1 and its impact on bacterial fitness, and the emergence and management of colistin resistance.