Exploiting lung adaptation and phage steering to clear pan-resistant Pseudomonas aeruginosa infections in vivo.

Pseudomonas aeruginosa is a major nosocomial pathogen that causes severe disease including sepsis. Carbapenem-resistant P. aeruginosa is recognised by the World Health Organisation as a priority 1 pathogen, with urgent need for new therapeutics. As such, there is renewed interest in using bacteriophages as a therapeutic. However, the dynamics of treating pan-resistant P. aeruginosa with phage in vivo are poorly understood. Using a pan-resistant P. aeruginosa in vivo infection model, phage therapy displays strong therapeutic potential, clearing infection from the blood, kidneys, and spleen. Remaining bacteria in the lungs and liver displays phage resistance due to limiting phage adsorption. Yet, resistance to phage results in re-sensitisation to a wide range of antibiotics. In this work, we use phage steering in vivo, pre-exposing a pan resistant P. aeruginosa infection with a phage cocktail to re-sensitise bacteria to antibiotics, clearing the infection from all organs.

Antibiotics Limit Adaptation of Drug-Resistant Staphylococcus aureus to Hypoxia.

Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how the subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known to vary extensively at sites of infection. We show that acquiring tetracycline resistance was costly, reducing competitive growth against the isogenic strain without the resistance gene in the absence of the antibiotic, for S. aureus under hypoxic but not normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein-encoding gene and duplications of tetL, encoding the acquired tetracycline-specific efflux pump. In contrast, evolutionary adaptation by S. aureus to hypoxic conditions, which evolved in the absence of antibiotics through mutations affecting gyrB, was impeded by antibiotic treatment. Together, these data suggest that the horizontal acquisition of a new resistance mechanism is merely a starting point for the emergence of high-level resistance under antibiotic selection but that antibiotic treatment constrains pathogen adaptation to other important environmental selective forces such as hypoxia, which in turn could limit the survival of these highly resistant but poorly adapted genotypes after antibiotic treatment is ended.

Functional diversity increases the efficacy of phage combinations.

Phage therapy is a promising alternative to traditional antibiotics for treating bacterial infections. Such phage-based therapeutics typically contain multiple phages, but how the efficacy of phage combinations scales with phage richness, identity and functional traits is unclear. Here, we experimentally tested the efficacy of 827 unique phage combinations ranging in phage richness from one to 12 phages. The efficacy of phage combinations increased with phage richness. However, complementarity between functionally diverse phages allowed efficacy to be maximized at lower levels of phage richness in functionally diverse combinations. These findings suggest that phage functional diversity is the key property of effective phage combinations, enabling the design of simple but effective phage therapies that overcome the practical and regulatory hurdles that limit development of more diverse phage therapy cocktails.

Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation.

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.

Eco-evolutionary Dynamics Set the Tempo and Trajectory of Metabolic Evolution in Multispecies Communities.

The eco-evolutionary dynamics of microbial communities are predicted to affect both the tempo and trajectory of evolution in constituent species [1]. While community composition determines available niche space, species sorting dynamically alters composition, changing over time the distribution of vacant niches to which species adapt [2], altering evolutionary trajectories [3, 4]. Competition for the same niche can limit evolutionary potential if population size and mutation supply are reduced [5, 6] but, alternatively, could stimulate evolutionary divergence to exploit vacant niches if character displacement results from the coevolution of competitors [7, 8]. Under more complex ecological scenarios, species can create new niches through their exploitation of complex resources, enabling others to adapt to occupy these newly formed niches [9, 10]. Disentangling the drivers of natural selection within such communities is extremely challenging, and it is thus unclear how eco-evolutionary dynamics drive the evolution of constituent taxa. We tracked the metabolic evolution of a focal species during adaptation to wheat straw as a resource both in monoculture and in polycultures wherein on-going eco-evolutionary community dynamics were either permitted or prevented. Species interactions accelerated metabolic evolution. Eco-evolutionary dynamics drove increased use of recalcitrant substrates by the focal species, whereas greater exploitation of readily digested substrate niches created by other species evolved if on-going eco-evolutionary dynamics were prevented. Increased use of recalcitrant substrates was associated with parallel evolution of tctE, encoding a carbon metabolism regulator. Species interactions and species sorting set, respectively, the tempo and trajectory of evolutionary divergence among communities, selecting distinct ecological functions in otherwise equivalent ecosystems.

Extremely fast amelioration of plasmid fitness costs by multiple functionally diverse pathways.

The acquisition of plasmids is often accompanied by fitness costs such that compensatory evolution is required to allow plasmid survival, but it is unclear whether compensatory evolution can be extensive or rapid enough to maintain plasmids when they are very costly. The mercury-resistance plasmid pQBR55 drastically reduced the growth of its host, Pseudomonas fluorescens SBW25, immediately after acquisition, causing a small colony phenotype. However, within 48 h of growth on agar plates we observed restoration of the ancestral large colony morphology, suggesting that compensatory mutations had occurred. Relative fitness of these evolved strains, in lab media and in soil microcosms, varied between replicates, indicating different mutational mechanisms. Using genome sequencing we identified that restoration was associated with chromosomal mutations in either a hypothetical DNA-binding protein PFLU4242, RNA polymerase or the GacA/S two-component system. Targeted deletions in PFLU4242, gacA or gacS recapitulated the ameliorated phenotype upon plasmid acquisition, indicating three distinct mutational pathways to compensation. Our data shows that plasmid compensatory evolution is fast enough to allow survival of a plasmid despite it imposing very high fitness costs upon its host, and indeed may regularly occur during the process of isolating and selecting individual plasmid-containing clones.

Resistance Evolution against Phage Combinations Depends on the Timing and Order of Exposure.

Phage therapy is a promising alternative to chemotherapeutic antibiotics for the treatment of bacterial infections. However, despite recent clinical uses of combinations of phages to treat multidrug-resistant infections, a mechanistic understanding of how bacteria evolve resistance against multiple phages is lacking, limiting our ability to deploy phage combinations optimally. Here, we show, using Pseudomonas aeruginosa and pairs of phages targeting shared or distinct surface receptors, that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance. Whereas sequential exposure allowed bacteria to acquire multiple resistance mutations effective against both phages, this evolutionary trajectory was prevented by simultaneous exposure, resulting in quantitatively weaker resistance. The order of phage exposure determined the fitness costs of sequential resistance, such that certain sequential orders imposed much higher fitness costs than the same phage pair in the reverse order. Together, these data suggest that phage combinations can be optimized to limit the strength of evolved resistances while maximizing their associated fitness costs to promote the long-term efficacy of phage therapy.IMPORTANCE Globally rising rates of antibiotic resistance have renewed interest in phage therapy where combinations of phages have been successfully used to treat multidrug-resistant infections. To optimize phage therapy, we first need to understand how bacteria evolve resistance against combinations of multiple phages. Here, we use simple laboratory experiments and genome sequencing to show that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance evolution in the opportunistic pathogen Pseudomonas aeruginosa These findings suggest that phage combinations can be optimized to limit the emergence and persistence of resistance, thereby promoting the long-term usefulness of phage therapy.

Cross-resistance is modular in bacteria-phage interactions.

Phages shape the structure of natural bacterial communities and can be effective therapeutic agents. Bacterial resistance to phage infection, however, limits the usefulness of phage therapies and could destabilise community structures, especially if individual resistance mutations provide cross-resistance against multiple phages. We currently understand very little about the evolution of cross-resistance in bacteria-phage interactions. Here we show that the network structure of cross-resistance among spontaneous resistance mutants of Pseudomonas aeruginosa evolved against each of 27 phages is highly modular. The cross-resistance network contained both symmetric (reciprocal) and asymmetric (nonreciprocal) cross-resistance, forming two cross-resistance modules defined by high within- but low between-module cross-resistance. Mutations conferring cross-resistance within modules targeted either lipopolysaccharide or type IV pilus biosynthesis, suggesting that the modularity of cross-resistance was structured by distinct phage receptors. In contrast, between-module cross-resistance was provided by mutations affecting the alternative sigma factor, RpoN, which controls many lifestyle-associated functions, including motility, biofilm formation, and quorum sensing. Broader cross-resistance range was not associated with higher fitness costs or weaker resistance against the focal phage used to select resistance. However, mutations in rpoN, providing between-module cross-resistance, were associated with higher fitness costs than mutations associated with within-module cross-resistance, i.e., in genes encoding either lipopolysaccharide or type IV pilus biosynthesis. The observed structure of cross-resistance predicted both the frequency of resistance mutations and the ability of phage combinations to suppress bacterial growth. These findings suggest that the evolution of cross-resistance is common, is likely to play an important role in the dynamic structure of bacteria-phage communities, and could inform the design principles for phage therapy treatments.

Ecological conditions determine extinction risk in co-evolving bacteria-phage populations.

BACKGROUND: Antagonistic coevolution between bacteria and their viral parasites, phage, drives continual evolution of resistance and infectivity traits through recurrent cycles of adaptation and counter-adaptation. Both partners are vulnerable to extinction through failure of adaptation. Environmental conditions may impose unequal abiotic selection pressures on each partner, destabilising the coevolutionary relationship and increasing the extinction risk of one partner. In this study we explore how the degree of population mixing and resource supply affect coevolution-induced extinction risk by coevolving replicate populations of Pseudomonas fluorescens SBW25 with its associated lytic phage SBW25Ф2 under four treatment regimens incorporating low and high resource availability with mixed or static growth conditions. RESULTS: We observed an increased risk of phage extinction under population mixing, and in low resource conditions. High levels of evolved bacterial resistance promoted phage extinction at low resources under both mixed and static conditions, whereas phage populations could survive when phage susceptible bacterial genotypes rose to high frequency. CONCLUSIONS: These findings demonstrate that phage extinction risk is influenced by multiple abiotic conditions, which together act to destabilise the bacteria-phage coevolutionary relationship. The risk of coevolution-induced extinction is therefore dependent on the ecological context.