Temperate Bacteriophages from Chronic Pseudomonas aeruginosa Lung Infections Show Disease-Specific Changes in Host Range and Modulate Antimicrobial Susceptibility

Abstract

Temperate bacteriophages are a common feature of Pseudomonas aeruginosa genomes, but their role in chronic lung infections is poorly understood. This study was designed to identify the diverse communities of mobile P. aeruginosa phages by employing novel metagenomic methods, to determine cross infectivity, and to demonstrate the influence of phage infection on antimicrobial susceptibility. Mixed temperate phage populations were chemically mobilized from individual P. aeruginosa, isolated from patients with cystic fibrosis (CF) or bronchiectasis (BR). The infectivity phenotype of each temperate phage lysate was evaluated by performing a cross-infection screen against all bacterial isolates and tested for associations with clinical variables. We utilized metagenomic sequencing data generated for each phage lysate and developed a novel bioinformatic approach allowing resolution of individual temperate phage genomes. Finally, we used a subset of the temperate phages to infect P. aeruginosa PAO1 and tested the resulting lysogens for their susceptibility to antibiotics. Here, we resolved 105 temperate phage genomes from 94 lysates that phylogenetically clustered into 8 groups. We observed disease-specific phage infectivity profiles and found that phages induced from bacteria isolated from more advanced disease infected broader ranges of P. aeruginosa isolates. Importantly, when infecting PAO1 in vitro with 20 different phages, 8 influenced antimicrobial susceptibility. This study shows that P. aeruginosa isolated from CF and BR patients harbors diverse communities of inducible phages, with hierarchical infectivity profiles that relate to the progression of the disease. Temperate phage infection altered the antimicrobial susceptibility of PAO1 at subinhibitory concentrations of antibiotics, suggesting they may be precursory to antimicrobial resistance. IMPORTANCE Pseudomonas aeruginosa is a key opportunistic respiratory pathogen in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. The genomes of these pathogens are enriched with mobile genetic elements including diverse temperate phages. While the temperate phages of the Liverpool epidemic strain have been shown to be active in the human lung and enhance fitness in a rat lung infection model, little is known about their mobilization more broadly across P. aeruginosa in chronic respiratory infection. Using a novel metagenomic approach, we identified eight groups of temperate phages that were mobilized from 94 clinical P. aeruginosa isolates. Temperate phages from P. aeruginosa isolated from more advanced disease showed high infectivity rates across a wide range of P. aeruginosa genotypes. Furthermore, we showed that multiple phages altered the susceptibility of PAO1 to antibiotics at subinhibitory concentrations.

The population and evolutionary dynamics of bacteriophage: Why be temperate revisited

“…When serial transfer cultures are initiated with mixtures of sensitive E. coli, virulent and temperate phage, l VIR and l + resistant mutants emerge and ascend to dominate the bacterial population, but both temperate and lytic phage continue to persist along with a minority population of newly formed lysogens. The frequency of these newly formed lysogens is markedly increased if, the addition of low concentrations of kanamycin, which selects for the carriage the l KAN prophage [19,43,44] A recent study showed that temperate phage of P. aeruginosa in the lungs of CF patients carries antibiotic resistance genes and relates to the progression of the disease by providing a selective advantage to their host bacteria [45]. Most importantly, the results of these experiments with combinations of lytic and temperate l indicate that a substantial fraction of the resistant bacteria evolving in these populations are lysogens.…”

CRISPR-Cas systems restrict horizontal gene transfer inPseudomonas aeruginosa

“…Mobile genetic elements, on the other hand, can be either parasitic or beneficial, depending on conditions. For example, the acquisition of prophage can alter improve Pseudomonas metabolism and increase competitive ability (16,44), but prophage entry into the lytic cycle leads to cell lysis and death. Similarly, ICE and plasmids carry genes that can allow Pseudomonas to exploit new niches, such as novel metabolites or eukaryotic hosts, or resist stresses, such as heavy metals and antibiotics, but the acquisition of these elements also tends to be associated with costs that can generate selection against carriage (32,(45)(46)(47).…”

Temperate Bacteriophages (Prophages) in Pseudomonas aeruginosa Isolates Belonging to the International Cystic Fibrosis Clone (CC274)

“…Virulence factors as pyocyanin production, biofilm and motility are regulated by QS and also influenced by the phage infections (Morkunas et al, 2012;Hosseinidoust et al, 2013;Latino et al, 2014;Castañeda-Tamez et al, 2018;Tariq et al, 2019). The infection with the wild type phage, AUS531phi, carrying the bci gene, increased the production of virulence factors, pyocyanin and biofilm, whose presence is characteristic in the lung of CF patients (Castañeda-Tamez et al, 2018).…”

“…The higher pyocyanin production may be due to a protective response to a higher infectivity capacity of the phage AUS531phi. Temperate phages could help P. aeruginosa select for bacterial characteristics that favor persistence of bacteria in the lung (Latino et al, 2014;Tariq et al, 2019). Thus, the bci gene may help clinical isolates of P. aeruginosa to survive in lung infections, increasing their chance of being infected by temperate phages.…”