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

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 bacterial death and lysis. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilized 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 permeabilization of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilization of the outer membrane of colistin-resistant isolates by the polymyxin is in turn sufficient to sensitize bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in these E. coli isolates is due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

Antibiotic Resistance Mechanisms and Their Transmission in Acinetobacter baumannii.

The discovery of penicillin over 90 years ago and its subsequent uptake by healthcare systems around the world revolutionised global health. It marked the beginning of a golden age in antibiotic discovery with new antibiotics readily discovered from natural sources and refined into therapies that saved millions of lives. Towards the end of the last century, the rate of discovery slowed to a near standstill. The lack of discovery is compounded by the rapid emergence and spread of bacterial pathogens that exhibit resistance to multiple antibiotic therapies and threaten the sustainability of global healthcare systems. Acinetobacter baumannii is an opportunistic pathogen whose prevalence and impact has grown significantly over the last 20 years. It is recognised as a barometer of the antibiotic resistance crisis due to the diverse array of mechanisms by which it can become resistant.

Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Antibiotics are life-saving medicines, but many bacteria now have the ability to resist their effects. For some infections, all frontline antibiotics are now ineffective. To treat infections caused by these highly resistant bacteria, clinicians must use so-called 'antibiotics of last resort'. These antibiotics include a drug called colistin, which is moderately effective, but often fails to eradicate the infection. One of the challenges to making colistin more effective is that its mechanism is poorly understood. Bacteria have two layers of protection against the outside world: an outer cell membrane and an inner cell membrane. To kill them, colistin must punch holes in both. First, it disrupts the outer membrane by interacting with molecules called lipopolysaccharides. But how it disrupts the inner membrane was unclear. Bacteria have evolved several different mechanisms that make them resistant to the effects of colistin. Sabnis et al. reasoned that understanding how these mechanisms protected bacteria could reveal how the antibiotic works to damage the inner cell membrane. Sabnis et al. examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that the antibiotic damages both layers of the cell surface in the same way, targeting lipopolysaccharide in the inner membrane as well as the outer membrane. Next, Sabnis et al. used this new information to make colistin work better. They found that the effects of colistin were magnified when it was combined with the experimental antibiotic murepavadin, which caused lipopolysaccharide to build up at the inner membrane. This allowed colistin to punch more holes through the inner membrane, making colistin more effective at killing bacteria. To find out whether this combination of colistin and murepavadin could work as a clinical treatment, Sabnis et al. tested it on mice with Pseudomonas aeruginosa infections in their lungs. Colistin was much better at killing Pseudomonas aeruginosa and treating infections when combined with murepavadin than it was on its own. Pseudomonas aeruginosa bacteria can cause infections in the lungs of people with cystic fibrosis. At the moment, patients receive colistin in an inhaled form to treat these infections, but it is not always successful. The second drug used in this study, murepavadin, is about to enter clinical trials as an inhaled treatment for lung infections too. If the trial is successful, it may be possible to use both drugs in combination to treat lung infections in people with cystic fibrosis.

Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.