What is the problem?

Gram negative bacteria include Escherichia coli, Klebsiella spp., Citrobacter spp., Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii, which collectively cause more than two thirds of healthcare-associated infections and resulting deaths and a wide range of community infections in the UK. This group of bacteria is frequently antibiotic resistant, and it is very difficult to identify new antimicrobials that kill them. The reason for this is that each bacterium is surrounded by an outer membrane, which acts as a permeability barrier, blocking the entry of antibiotics and other antimicrobials. To enter the cell, antimicrobials need to use protein channels, called porins. Bacteria can control the number of porins, reducing – or increasing – entry in a controlled manner.

Even when antimicrobials gain access via porins, they are frequently pumped back out of the cell by efflux pumps. Again, the cell can control which pumps are produced and under which circumstances. The combination of reduced entry and increased efflux is referred to as “reduced permeability” and is usually caused by mutations that affect the regulation of porin and efflux pump protein production. Reduced permeability can cause antibiotic resistance on its own, but it can also increase the ability of other resistance mechanisms to cause resistance.

Accordingly, studying the control of antibiotic permeability, and understanding the regulatory systems involved, and how different mutations can affect permeability in different ways, is beneficial for two main reasons. It helps researchers predict whether a bacterium will be antibiotic resistant or not – i.e. it improves the ability of whole genome sequencing technologies to predict resistance. Understanding these regulators and the permeability factors that are being regulated may also pave the way to developing chemicals that increase antibiotic permeability – by reducing efflux or increasing penetration through porins.

A potential solution 

We have identified the effects of numerous efflux pumps and their regulators. For example, we have characterized the relative importance of mutations in the OqxR and RamR regulators in K. pneumoniae in causing antibiotic resistance, with and without other mechanisms. We have identified several novel permeability mediated mechanisms of antibiotic resistance, including resistance to fluoroquinolones, aminoglycosides (Dulyayangkul et al, 2020 Antimicrob Agents Chemother 65:e01284-20) and beta-lactam/beta-lactamase combinations (Satapoomin et al, 2022 Antimicrob Agents Chemother 66: e0217921), predominantly in E. coli, Klebsiella pneumoniae and S. maltophilia. So for example a novel mechanism of resistance to the key community pyelonephritis drug cefalexin in E. coli (Alzayn et al 2021 Antimicrob Agents Chemother 65:e0100421) and the first example of TonB dependent uptake of beta-lactam antibiotics, demonstrated in S. maltophilia (Calvopina et al, 2020 Mol Microbiol 113:492-503). We have also identified novel ways of facilitating the penetration of antibiotics into bacteria, for example using a “trojan horse” method to increase the penetration of the failed antibiotic lactivicin into S. maltophilia.

Next steps

The team are currently identifying additional permeability mediated mechanisms of resistance to beta-lactam/beta-lactamase combinations in the Enterobacteriales, including those that work in combination with beta-lactamase production. We are also studying permeability mechanisms of aminoglycoside resistance in E. coli and we are using genomic surveillance to rank the importance of different antibiotic exclusion strategies in bacteria from clinical infections. We are also following up key findings that manipulation of two regulators in K. pneumoniae increases the permeability of the cell to all antimicrobials, heralding the potential for new “resistance-busting” strategies.