Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic

Abstract

Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to lysis and bacterial death. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilised colistin resistance (mcr) genes. Both these colistin resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilisation of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilisation of the outer membrane of colistin resistant isolates by the polymyxin is in turn sufficient to sensitise bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in E. coli is typically due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

Polymyxin and lipopeptide antibiotics: membrane-targeting drugs of last resort

“…Furthermore, it agrees with the finding that some strains of A. baumannii can acquire resistance to very high concentrations of colistin via loss of LPS biosynthesis, resulting in outer and cytoplasmic membranes that consist of phospholipid bilayers [39]. Polymyxin-mediated disruption of the cytoplasmic membrane is sufficient to allow ingress of the fluorescent dye propidium iodide [37,38] and the egress of small molecules such as potassium ions, amino acids and uracil, as well as proteins such as beta-galactosidase [40,41]. However, it is not clear to what extent the release of these molecules is due to the initial interaction of polymyxins with LPS or the subsequent lysis.…”

“…The impact of these differences on colistin resistance is not well understood. However, recent work from our laboratory indicates there may be subtle differences in the susceptibility of strains with different MCR types to colistin-mediated membrane damage [38].…”

“…Permeabilization of the outer bacterial membrane provides colistin molecules with access to the cytoplasmic membrane, the disruption of which is crucial for bactericidal activity [37,38]. However, whilst polymyxin-mediated membrane disruption can cause lysis, this is not required for bacterial killing [37,38].…”

“…Although LPS modified with l-Ara4N and/or PEtN is present at both the outer and cytoplasmic membranes of bacteria with acquired polymyxin resistance, this does not protect both membranes equally [37,38]. Bacteria with resistance via spontaneous mutation or acquisition of an mcr gene are still susceptible to outer membrane damage, as assessed using ingress of fluorescent dyes and antibiotics [37,38]. By contrast, the cytoplasmic membranes of colistin-resistant bacteria are resistant to damage, enabling bacterial survival [37,38].…”

“…Bacteria with resistance via spontaneous mutation or acquisition of an mcr gene are still susceptible to outer membrane damage, as assessed using ingress of fluorescent dyes and antibiotics [37,38]. By contrast, the cytoplasmic membranes of colistin-resistant bacteria are resistant to damage, enabling bacterial survival [37,38]. This is thought to be due to the presence of large concentrations of unmodified LPS in the outer membrane, whereas the abundance of LPS in the cytoplasmic membrane is so low that even if only a fraction of molecules are modified it is sufficient to confer protection [37,38].…”