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Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

ABSTRACT: Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections.

SUBMITTER: Chan BK

PROVIDER: S-EPMC4880932 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacteria frequently encounter selection by both phages and antibiotics. However, our knowledge on the evolutionary interactions between phages and antibiotics are still limited. Here, we characterized a phage-resistant Pseudomonas aeruginosa variant PAO1-R1 that shows increased sensitivity to gentamicin and polymyxin B. Using whole genome sequencing, significant genome differences were observed between the reference P. aeruginosa PAO1 and PAO1-R1. Compared to PAO1, 64 gene-encoding proteins with nonsynonymous single nucleotide polymorphisms (SNPs) and 31 genes with insertion/deletion (indel) mutations were found in PAO1-R1. We observed a significant reduction in phage adsorption rate for both phage vB_Pae_QDWS and vB_Pae_W3 against PAO1-R1 and proposed that disruption of phage adsorption is likely the main cause for evolving resistance. Because the majority of spontaneous mutations are closely related to membrane components, alterations in the cell envelope may explain the antibiotic-sensitive phenotype of PAO1-R1. Collectively, we demonstrate that the evolution of phage resistance comes with fitness defects resulting in antibiotic sensitization. Our finding provides new insights into the evolutionary interactions between resistance to the phage and sensitivity to antibiotics, which may have implications for the future clinical use of steering in phage therapies. IMPORTANCE Bacteria frequently encounter the selection pressure from both antibiotics and lytic phages. Little is known about the evolutionary interactions between antibiotics and phages. Our study provides new insights into the trade-off mechanism between resistance to the phage and sensitivity to antibiotics. This evolutionary trade-off is not dependent on the outer membrane proteins (OMPs) of the multidrug efflux pumps. The disruption of phage adsorption that induced phage resistance and the changes in structure or composition of membranes are presumably one of the major causes for antibiotic sensitivity. Our finding may fill some gaps in the field of phage-host interplay and have implications for the future clinical use of steering in phage therapies.

Project description:The increasing ineffectiveness of traditional antibiotics and the rise of multidrug resistant (MDR) bacteria have necessitated the revival of bacteriophage (phage) therapy. However, bacteria might also evolve resistance against phages. Phages and their bacterial hosts coexist in nature, resulting in a continuous coevolutionary competition for survival. We have isolated several clinical strains of Pseudomonas aeruginosa and phages that infect them. Among these, the PIAS (Phage Induced Antibiotic Sensitivity) phage belonging to the Myoviridae family can induce multistep genomic deletion in drug-resistant clinical strains of P. aeruginosa, producing a compromised drug efflux system in the bacterial host. We identified two types of mutant lines in the process: green mutants with SNPs (single nucleotide polymorphisms) and smaller deletions and brown mutants with large (∼250 kbp) genomic deletion. We demonstrated that PIAS used the MexXY-OprM system to initiate the infection. P. aeruginosa clogged PIAS phage infection by either modifying or deleting these receptors. The green mutant gaining phage resistance by SNPs could be overcome by evolved PIASs (E-PIASs) with a mutation in its tail-fiber protein. Characterization of the mutant phages will provide a deeper understanding of phage-host interaction. The coevolutionary process continued with large deletions in the same regions of the bacterial genomes to block the (E-)PIAS infection. These mutants gained phage resistance via either complete loss or substantial modifications of the phage receptor, MexXY-OprM, negating its essential role in antibiotic resistance. In vitro and in vivo studies indicated that combined use of PIAS and antibiotics could effectively inhibit P. aeruginosa growth. The phage can either eradicate bacteria or induce antibiotic sensitivity in MDR-resistant clinical strains. We have explored the potential use of combination therapy as an alternative approach against MDR P. aeruginosa infection.

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.

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Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

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