The superbug Pseudomonas aeruginosa, which can cause lung infections in people on ventilators in intensive care units. [Imperial College London]

Work by scientists at Imperial College London and the University of Texas has revealed how a key “last resort” antibiotic kills bacteria. The research has revealed how the antibiotic, colistin, punches holes in both of outer and inner membranes of Gram-negative bacteria, causing them to pop like balloons. The new insights could help researchers develop more powerful and effective forms of antibiotic against some of the deadliest superbugs, and improve therapeutic outcomes for patients. Initial experiments in mice showed how combining colistin with an experimental antibiotic boosted the effectiveness of colistin against a bacterium that can cause devastating lung infections.

Akshay Sabnis, PhD, at Imperial’s Department of Infectious Disease, explained, “As the global crisis of antibiotic resistance continues to accelerate, colistin is becoming more and more important as the very last option to save the lives of patients infected with superbugs. By revealing how this old antibiotic works, we could come up with new ways to make it kill bacteria even more effectively, boosting our arsenal of weapons against the world’s superbugs.” Sabnis is lead author of the team’s published study, in eLife, which is titled, “Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.”

First described in 1947, Colistin is a polymyxin antibiotic that is one of the very few antibiotics that is active against some of the most deadly superbugs, including Escherichia coli, which causes potentially lethal infections of the bloodstream, and Pseudomonas aeruginosa and Acinetobacter baumannii, which frequently infect the lungs of people receiving mechanical ventilation in intensive care units. “The emergence of multi-drug-resistant Gram-negative pathogens such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa has led to the increased use of polymyxin antibiotics, which are often the only viable last-resort therapeutic option,” the team wrote.

However, while colistin is effective at killing Gram-negative bacteria in vitro, the antibiotic is less effective in vivo, and up to 70% of patients may fail to respond to colistin treatment, the researchers continued. And despite being discovered over 70 years ago, the process by which this antibiotic kills bacteria has, until now, remained unclear.

The bacteria targeted by colistin have both an outer cell membrane and an inner cell membrane, and to kill these organisms colistin must breach both of these membranes. However, whilst it was known that colistin damages the outer membrane (OM) by targeting a chemical called lipopolysaccharide (LPS), how the inner, cytoplasmic membrane (CM) is pierced wasn’t known.

“Barriers to increasing polymyxin efficacy include the significant gaps in our understanding of their mode of action,” the investigators noted. “Whilst it is well established that the binding of polymyxins to lipopolysaccharide (LPS) on the surface of Gram-negative bacteria leads to disruption of the outer membrane (OM), it is unclear how this results in cell lysis and bacterial death.”

Led by Andrew Edwards, PhD, from Imperial’s Department of Infectious Disease, the investigators examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that as well as targeting lipopolysaccharides in the outer membrane, the antibiotic also targets lipopolysaccharide in the inner, cytoplasmic membrane, even though the cytoplasmic membrane contains very little LPS. It effectively damages both layers of the cell surface in the same way. The authors further stated, “In this work, we demonstrate that colistin targets LPS in the CM, resulting in membrane permeabilization, bacterial lysis, and killing.”

The superbug Pseudomonas aeruginosa after being “popped” by the antibiotic colistin. [Imperial College London]

Edwards said: “It sounds obvious that colistin would damage both membranes in the same way, but it was always assumed colistin damaged the two membranes in different ways. There’s so little LPS in the inner membrane that it just didn’t seem possible, and we were very sceptical at first. However, by changing the amount of LPS in the inner membrane in the laboratory, and also by chemically modifying it, we were able to show that colistin really does puncture both bacterial skins in the same way— and that this kills the superbug.”

Next, the team investigated whether they could use this new information to find ways of making colistin more effective at killing bacteria. They focused on Pseudomonas aeruginosa, which also causes serious lung infections in people with cystic fibrosis. They found that a new experimental antibiotic, called murepavadin, caused a build up of LPS in the bacterium’s inner membrane, making it much easier for colistin to puncture this CM and kill the bacteria.

Experiments in a mouse model of P. aeruginosa lung infection confirmed that while using either colistin or murepavadin alone wasn’t very effective against the bacteria, the antibacterial effects of colistin were magnified when it was combined with the murepavadin. “Mono-therapy with colistin alone or murepavadin alone had very little effect on the bacterial load assessed after 3 hr treatment compared with the no-treatment control,” the team noted. “By contrast, combination therapy with colistin and murepavadin caused a ~500-fold reduction in c.f.u. [colony forming unit] counts relative to the no-treatment control.”

The studies provide new insights into the mechanism of action of colistin by demonstrating that polymyxin antibiotics target LPS in both the OM and the CM, and that this leads to the disruption of both membranes, resulting in the bactericidal and lytic activities of the antibiotic,” the team concluded. “ … murepavadin synergises with colistin both in vitro and in vivo, suggesting it may be useful as a combination therapeutic approach for lung infections caused by P. aeruginosa … Modulation of LPS levels in the CM can enhance colistin activity, providing the foundations for new approaches to enhance the efficacy of this antibiotic of last resort.”

The team acknowledged that as murepavadin is an experimental antibiotic, it can’t be used routinely in patients yet, but clinical trials are expected to start soon. If these trials are successful, it may be possible to combine murepavadin with colistin to make a potent treatment for a vast range of bacterial infections.

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