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Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage-antibiotic treatments.

ABSTRACT: Antimicrobial resistance has been estimated to be responsible for over 700,000 deaths per year; therefore, new antimicrobial therapies are urgently needed. One way to increase the efficiency of antibiotics is to use them in combination with bacteria-specific parasitic viruses, phages, which have been shown to exert additive or synergistic effects in controlling bacteria. However, it is still unclear to what extent these combinatory effects are limited by rapid evolution of resistance, especially when the pathogen grows as biofilm on surfaces typical for many persistent and chronic infections. To study this, we used a microcosm system, where genetically isogenic populations of Pseudomonas aeruginosa PAO1 bacterial pathogen were exposed to a phage 14/1, gentamycin or a combination of them both in a spatially structured environment. We found that even though antibiotic and phage-antibiotic treatments were equally effective at controlling bacteria in the beginning of the experiment, combination treatment rapidly lost its efficacy in both planktonic and biofilm populations. In a mechanistic manner, this was due to rapid resistance evolution: While both antibiotic and phage selected for increased resistance on their own, phage selection correlated positively with increase in antibiotic resistance, while biofilm growth, which provided generalized resistance mechanism, was favoured most in the combination treatment. Only relatively small cost of resistance and weak evidence for coevolutionary dynamics were observed. Together, these results suggest that spatial heterogeneity can promote rapid evolution of generalized resistance mechanisms without corresponding increase in phage infectivity, which could potentially limit the effectiveness of phage-antibiotic treatments in the evolutionary timescale.

SUBMITTER: Moulton-Brown CE

PROVIDER: S-EPMC6183449 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage-antibiotic treatments.

Moulton-Brown Claire E CE Friman Ville-Petri VP

Evolutionary applications 20180621 9

Antimicrobial resistance has been estimated to be responsible for over 700,000 deaths per year; therefore, new antimicrobial therapies are urgently needed. One way to increase the efficiency of antibiotics is to use them in combination with bacteria-specific parasitic viruses, phages, which have been shown to exert additive or synergistic effects in controlling bacteria. However, it is still unclear to what extent these combinatory effects are limited by rapid evolution of resistance, especially ...[more]

PMID: 30344632

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Project description:Background and objectivesAntimicrobial resistance is a growing global concern and has spurred increasing efforts to find alternative therapeutics. Bacteriophage therapy has seen near constant use in Eastern Europe since its discovery over a century ago. One promising approach is to use phages that not only reduce bacterial pathogen loads but also select for phage resistance mechanisms that trade-off with antibiotic resistance-so called 'phage steering'.MethodologyRecent work has shown that the phage OMKO1 can interact with efflux pumps and in so doing select for both phage resistance and antibiotic sensitivity of the pathogenic bacterium Pseudomonas aeruginosa. We tested the robustness of this approach to three different antibiotics in vitro (tetracycline, erythromycin and ciprofloxacin) and one in vivo (erythromycin).ResultsWe show that in vitro OMKO1 can reduce antibiotic resistance of P. aeruginosa (Washington PAO1) even in the presence of antibiotics, an effect still detectable after ca.70 bacterial generations in continuous culture with phage. Our in vivo experiment showed that phage both increased the survival times of wax moth larvae (Galleria mellonella) and increased bacterial sensitivity to erythromycin. This increased antibiotic sensitivity occurred both in lines with and without the antibiotic.Conclusions and implicationsOur study supports a trade-off between antibiotic resistance and phage sensitivity. This trade-off was maintained over co-evolutionary time scales even under combined phage and antibiotic pressure. Similarly, OMKO1 maintained this trade-off in vivo, again under dual phage/antibiotic pressure. Our findings have implications for the future clinical use of steering in phage therapies. Lay Summary: Given the rise of antibiotic-resistant bacterial infection, new approaches to treatment are urgently needed. Bacteriophages (phages) are bacterial viruses. The use of such viruses to treat infections has been in near-continuous use in several countries since the early 1900s. Recent developments have shown that these viruses are not only effective against routine infections but can also target antibiotic resistant bacteria in a novel, unexpected way. Similar to other lytic phages, these so-called 'steering phages' kill the majority of bacteria directly. However, steering phages also leave behind bacterial variants that resist the phages, but are now sensitive to antibiotics. Treatment combinations of these phages and antibiotics can now be used to greater effect than either one independently. We evaluated the impact of steering using phage OMKO1 and a panel of three antibiotics on Pseudomonas aeruginosa, an important pathogen in hospital settings and in people with cystic fibrosis. Our findings indicate that OMKO1, either alone or in combination with antibiotics, maintains antibiotic sensitivity both in vitro and in vivo, giving hope that phage steering will be an effective treatment option against antibiotic-resistant bacteria.

Project description:Antibiotic resistance has become a major public health concern as bacteria evolve to evade drugs, leading to recurring infections and a decrease in antibiotic efficacy. Systematic efforts have revealed mechanisms involved in resistance. Yet, in many cases, how these specific mechanisms accelerate or slow the evolution of resistance remains unclear. Here, we conducted a systematic study of the impact of the AcrAB-TolC efflux pump on the evolution of antibiotic resistance. We mapped how population growth rate and resistance change over time as a function of both the antibiotic concentration and the parent strain's genetic background. We compared the wild-type strain to a strain overexpressing AcrAB-TolC pumps and a strain lacking functional pumps. In all cases, resistance emerged when cultures were treated with chloramphenicol concentrations near the MIC of their respective parent strain. The genetic background of the parent strain also influenced resistance acquisition. The wild-type strain evolved resistance within 24 h through mutations in the acrAB operon and its associated regulators. Meanwhile, the strain overexpressing AcrAB-TolC evolved resistance more slowly than the wild-type strain; this strain achieved resistance in part through point mutations in acrB and the acrAB promoter. Surprisingly, the strain without functional AcrAB-TolC efflux pumps still gained resistance, which it achieved through upregulation of redundant efflux pumps. Overall, our results suggest that treatment conditions just above the MIC pose the largest risk for the evolution of resistance and that AcrAB-TolC efflux pumps impact the pathway by which chloramphenicol resistance is achieved.IMPORTANCE Combatting the rise of antibiotic resistance is a significant challenge. Efflux pumps are an important contributor to drug resistance; they exist across many cell types and can export numerous classes of antibiotics. Cells can regulate pump expression to maintain low intracellular drug concentrations. Here, we explored how resistance emerged depending on the antibiotic concentration, as well as the presence of efflux pumps and their regulators. We found that treatments near antibiotic concentrations that inhibit the parent strain's growth were most likely to promote resistance. While wild-type, pump overexpression, and pump knockout strains were all able to evolve resistance, they differed in the absolute level of resistance evolved, the speed at which they achieved resistance, and the genetic pathways involved. These results indicate that specific treatment regimens may be especially problematic for the evolution of resistance and that the strain background can influence how resistance is achieved.

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Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage-antibiotic treatments.

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Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage-antibiotic treatments.

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