Multidrug-resistant community-acquired infections caused by the opportunistic human pathogen Pseudomonas aeruginosa are increasingly reported in India and other locations globally. Since this organism is ubiquitous in the environment, samples such as sewage and wastewater are rich reservoirs of P. aeruginosa bacteriophages. In this study, we report the isolation and characterization of a novel P. aeruginosa N4-like lytic bacteriophage, vB_Pae_AM.P2 (AM.P2), from wastewater in Kerala, India. AM.P2 is a double-stranded DNA podovirus that efficiently lyses the model strain, PAO1, at a multiplicity of infection as low as 0.1 phage per bacterium and resistance frequency of 6.59 × 10⁻⁴. Synergy in bactericidal activity was observed between AM.P2 and subinhibitory concentrations of the antibiotic ciprofloxacin. Genome sequencing of AM.P2 revealed features similar to those of the N4-like P. aeruginosa phages LUZ7 and KPP21. As judged by two independent assay methods, spot tests and growth inhibition, AM.P2 successfully inhibited the growth of almost 30% of strains from a contemporary collection of multidrug-resistant P. aeruginosa clinical isolates from South India. Thus, AM.P2 may represent an intriguing candidate for inclusion in bacteriophage cocktails developed for various applications, including water decontamination and clinical bacteriophage therapy. IMPORTANCE In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen P. aeruginosa in sewage and water bodies in India is well documented. The isolation and characterization of bacteriophages that could target emerging P. aeruginosa strains, representing possible epicenters for community-acquired infections, could serve as a useful alternative tool for various applications, such as phage therapy and environmental treatment. Continuing to supplement the repertoire of broad-spectrum bacteriophages is an essential tool in confronting this problem.

We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (bla_NDM-1) within our sample. This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.

Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa . Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM ( PA0718 ), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans . We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages. IMPORTANCE: Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM ( PA0718 ) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Earlier studies from our laboratory have demonstrated that clove bud oil (CBO) attenuates expression of certain virulence factors of Pseudomonas aeruginosa PAO1. Here, we probe more deeply into the effect of CBO on four pseudomonal proteases - elastase A, elastase B, protease IV and alkaline protease - each known to play key roles in disease pathogenesis. CBO inhibited the activity of these proteases present in the bacterial culture supernatant. Zymography studies indicated that these proteases can activate host matrix metalloproteases (MMPs) to establish infection, through conversion of pro-MMP-2 to active MMP-2. PAO1 is a predominant pathogen in burn wound infections and we show the modulatory effect of CBO on MMPs in an in vitro model of burn injury. Furthermore, CBO induced dose-dependent neutrophil extracellular trap formation in human neutrophils. CBO also increased the survival of C. elegans infected with PAO1, establishing an anti-infective role in a whole animal model of pathogenesis. LC-MS/MS analysis indicated that CBO treatment elicited a significant reduction of signalling molecules (Acyl-Homoserine-Lactone) involved in quorum sensing regulation. Our observations demonstrate that CBO attenuates key virulence mechanisms of this important human pathogen, while concomitantly enhancing host innate immunomodulatory functions, with potential implications for topical therapy against antibiotic-resistant infections.