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    Antivirulence compounds inhibit pathogen virulence without affecting viability; they can be used in combination with antibiotics to restore efficacy of antibiotics against resistant bacteria and to slow the rate at which bacteria evolve resistance.

    Antimicrobial resistance is one of the most pressing challenges facing the human race; pathogens now exist that are resistant to all known antibiotics. Because of the challenges associated with developing new antibiotics, an alternative method to counteract resistance is to use antivirulence compounds to restore the efficacy of existing antibiotics through combination therapy. Antivirulence compounds inhibit pathogen virulence factors, non-essential molecular determinants of virulence. Whereas antibiotics inhibit bacterial growth or viability, antivirulence compounds inhibit virulence without killing the target. In an unpublished study, Chiara Rezzoagli and colleagues1. report on two antivirulence compounds (furanone C-30 and gallium) and discuss the efficacy of using these compounds in combination with four antibiotics to combat resistance in Pseudomonas aeruginosa, a gram-negative human pathogen. Their findings suggest that antivirulence compounds can indeed restore the efficacy of antibiotics to treat resistant strains and that antivirulence compounds may be useful in slowing the rate at which resistance evolves.

    The problem of antimicrobial resistance has been steadily worsening over the last six decades. When bacteria are exposed to antibiotics, natural selection favours individuals with genetic mutations that enable greater tolerance to antibiotics, meaning these mutations rapidly spread throughout the whole population. The rate at which resistance evolves is exacerbated by bacterial biology: large population size, high mutation rate, and horizontal transfer of resistance genes between individuals.


    Combination therapies of antibiotics and antivirulence compounds may provide a potential solution to antibiotic resistance. Antivirulence compounds do not kill bacteria, so there a lower selection pressure for resistance to these compounds to evolve, meaning resistance evolves more slowly2. However, because antivirulence compounds alone cannot kill bacteria, they must be used in combination with antibiotics to effectively clear infections.

    The current study by Rezzoagli and colleagues explores combination therapies of four antibiotics (colistin, tobramycin, ciprofloxacin, and meropenem) with two antivirulence compounds (gallium and furanone C-30). These antibiotics are all used to treat P. aeruginosa infections so are of clinical relevance. Furanone is an inhibitor of bacterial quorum sensing, the process by which bacterial cells communicate with each other by secreting molecules called N‐acylhomoserine lactones (AHL's). Furanone inhibits AHL's by competitively binding to the LasR receptor. Quorum sensing is used to coordinate bacterial processes such as virulence factor production and the formation of resistant aggregations of bacteria called biofilms. Gallium inhibits a type of siderophore found in Pseudomonas called pyoverdines. Siderophores such as pyoverdines are molecules secreted by bacteria to scavenge ferric iron from their environment; they play an important role in biofilm formation and quorum sensing, facilitating virulence. Gallium inhibits pyoverdine mediated iron uptake by irreversibly binding to pyoverdines.

    First, Rezzoagli and colleagues characterised the interactions between each of the four antibiotics with the two antivirulence compounds (eight combinations in total) at different doses using 9x9 concentration matrices. The extent to which each combination inhibited bacterial growth and virulence factors was recorded. Furanone and gallium displayed varying interactions with the four different antibiotics. The most promising combinations displayed synergy by inhibiting both virulence factors and growth, such as gallium-tobramycin, furanone-tobramycin, and furanone-colistin. Synergy is important for clinical use is as it enables lower concentrations of each drug to be used, reducing patient side effects.

    Further, the interactions between antibiotics and antivirulence compounds were highly concentration-dependent which has consequences for clinical use. This is because drugs accumulate differentially around the patient's body based on their specific chemical properties (pharmacokinetics and pharmacodynamics). Before combination therapies can be used clinically, future research is needed to achieve correct therapeutic levels of both drugs to optimise their synergy3. This may require clinical trials which are expensive to conduct.

    Next, the authors assayed gallium and furanone for their ability to re-potentiate (restore the growth-inhibiting properties) of each antibiotic against strains of P. aeruginosa that had been experimentally selected for antibiotic resistance (AtbR clones). Seven of the eight combinations (all but furanone-ciprofloxacin) re-potentiated antibiotic functionality against AtbR clones in a concentration-dependent manner. This result is promising as re-potentiating existing antibiotics to counter resistance would be much more cost-effective than creating novel antibiotics. However, this experiment was performed in vitro and on lab generated resistant strains. Future research is needed to test if these combinations can re-potentiate antibiotics in vivo against clinical isolates of multi-drug resistant P. aeruginosa from humans. Some studies have indeed demonstrated the efficacy of antibiotic-antivirulence combinations in clearing infections in animal models using gallium-gentamicin4 and furanone-tobramycin5.

    Competition assays were then conducted to assess the effect of each combination on the selection for resistance in a mixed population of two phenotypes of P. aeruginosa: wild-type and resistant clones (AtbR). In the presence of solely antibiotic, selection will favour the resistant clones. Interestingly, three out of eight combinations of antibiotic-antivirulence compound were able to reverse selection for resistance, with tobramycin reversing selection when used in combination with either gallium or furanone. This is promising for fighting antibiotic resistance on a global scale because selection is what underpins the evolution of resistance. Future work is needed to isolate the exact mechanism by which selection for resistance was reversed.

    Finally, the genomes of the AtbR clones were sequenced in order to understand why resistance evolved to some combination treatments but not others. The researchers suggest that the effect of combinatorial treatments on selection for resistance is dependent on the molecular basis of resistance. For example, if antibiotic resistance is conferred by mutations that up-regulate the expression of efflux pumps, this provides cross-resistance against antivirulence compounds like furanone which must enter the cell in order to function. Therefore, there is continued selection in favour of resistance. This mechanism could explain observed patterns with furanone in combination with ciprofloxacin, colistin and meropenem, where mutations in the mexR gene increased expression of a multidrug efflux pump called MexAB–OprM.

    On the other hand, three combinations were able to reverse selection for resistance, meaning the wild-type bacteria out-competed the AtbR strain. The reason why compounds like gallium may cause selection against resistance is due to their iron sequestering target, pyoverdines. The secretion of pyoverdines is a so called cooperative behaviour because they are costly to produce but once secreted, other individuals utilise pyoverdines for iron uptake. Selection, therefore, favours 'cheats' that do not produce their own pyoverdines but benefit from those produced by others. Since gallium inhibits pyoverdine production, being resistant to gallium treatment is individually disadvantageous as the resistant individuals will be the only ones capable of producing costly pyoverdines that benefit the whole population6. Therefore, the wild-type is selected over the resistant clone, despite antibiotics also being present.

    Before antibiotic-antivirulence combinations can be used clinically, further tests on animals are needed to better understand the way in which these compounds spatially accumulate in vivo. This is because different compounds accumulate in different parts of the body due to their pharmacokinetics and pharmacodynamics, making it difficult to predict the therapeutic dose required to achieve optimal internal concentration. Accuraretly predicting these internal concentrations is paramount given the concentration dependency of the synergies demonstrated in this study. It may be possible to overcome the difficulties of achieving optimal concentrations by synthesising single compounds that combine the active domains of both the antivirulence compound and the antibiotic7. However, large hybrid molecules may have difficulty in entering the target bacterial cell3.

    Overall, Rezzoagli and colleagues demonstrated that antivirulence compounds are an extremely promising possibility for extending the life of antibiotics by restoring their efficacy against resistant bacterial strains. Combinations of antibiotic-antivirulence compounds could enable narrow-spectrum therapies that only work against certain species, reducing the rate at which resistance evolves. Further work is needed to optimise the synergy of antivirulence and antibiotic combinations, as well as animal tests better understand toxicity and potential side effects, such as increased expression of virulence factors in response to antivirulence compounds6.


    References[1] Rezzoagli C, Archetti M, Baumgartner M, Kümmerli R. Combining antibiotics with antivirulence compounds is effective and can reverse selection for antibiotic resistance in Pseudomonas aeruginosa.. bioRxiv 861799[2] Laxminarayan, R., Duse, A., Wattal, C., Zaidi, A. K. M., Wertheim, H. F. L., Sumpradit, N., Vlieghe, E., Hara, G. L., Gould, I. M., Goossens, H., Greko, C., So, A. D., Bigdeli, M., Tomson, G., Woodhouse, W., Ombaka, E., Peralta, A. Q., Qamar, F. N., Mir, F., … Cars, O. (2013). Antibiotic resistance—the need for global solutions. The Lancet Infectious Diseases, 13(12), 1057–1098. [3] Tyers, M., Wright, G.D. (2019) Drug combinations: a strategy to extend the life of antibiotics in the 21st century. Nat Rev Microbiol 17, 141–155[4] Banin E, Lozinski A,Brady KM, Berenshtein E,Butterfield PW, Moshe M, Chevion M, Greenberg EP, Banin E (2008). The potential of desferrioxamine-gallium as an anti-Pseudomonas therapeutic agent. Proceedings of the National Academy of Sciences. 105 (43) 16761-16766; [5] Hentzer, M., Wu, H., Andersen, J.B., Riedel, K., Rasmussen, T.B., Bagge, N., Kumar, N., Schembri, M.A., Song, Z., Kristoffersen, P., Manefield, M., Costerton, J.W., Molin, S., Eberl, L., Steinberg, P., Kjelleberg, S., H√∏iby, N. and Givskov, M. (2003), Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. The EMBO Journal, 22: 3803-3815[6] Allen, R., Popat, R., Diggle, S. et al. (2014) Targeting virulence: can we make evolution-proof drugs?. Nat Rev Microbiol 12, 300–308 doi:10.1038/nrmicro3232[7] Parkes, A. L. & Yule, I. A. (2016) Hybrid antibiotics — clinical progress and novel designs. Expert Opin. Drug Discov. 11, 665–680
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