A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates

Abstract

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.

Keywords: N4-like viruses; Pseudomonas aeruginosa; antibiotic resistance; bacteriophage therapy; bacteriophages; clinical isolates; community-acquired infection; phage therapy.

FIG 1

AM.P2, a Podoviridae phage isolated from wastewater, produces plaques against PAO1. (A) Isolated phage AM.P2 against host strain PAO1, plated by a double-layer agar method to visualize individual plaques. AM.P2 produces large plaques (5-mm diameter) against PAO1 with clear centers and turbid edges when plated with 0.7% soft agar. (B) Cryo-EM raw image of the AM.P2 phage with the capsid and the tail. (C) Image of several AM.P2 phage particles attached to a bacterial cell-like structure. (D) Restriction digestion of AM.P2 nucleic acid with EcoRI, BamHI, and HindIII.

FIG 2

AM.P2 inhibits PAO1 growth and maintains decreased cell viability at MOIs as low as 0.1. (A) AM.P2 one-step growth curve suggests a latent phase of 20 min for PAO1 infectivity. (B) Bacterial growth curve of PAO1 treated with AM.P2 at MOIs of 10, 1, and 0.1. The OD₆₀₀ was measured every minute for 3 h. (C and D) The effect of AM.P2 treatment at the same MOIs after 24 h on OD₆₀₀ (C) and on number of CFU/ml (D). PAO1 without phage was considered the control. Average values of triplicate readings from three independent experiments are plotted with SEM. Significance was determined using t test (*, P  < 0.05). (E) Resistance frequency of PAO1 to AM.P2 at 24 and 48 h.

FIG 3

Effect of combinatorial AM.P2 and subinhibitory antibiotic concentrations on PAO1. The effect of PAO1 treated with AM.P2, antibiotics (colistin [COL], gentamicin [GEN], and ciprofloxacin [CIP]) at 1/2×MIC and 1/4×MIC, and combinations of AM.P2 and sub-MIC levels of each antibiotic was assessed by measuring the OD₆₀₀ over the first 3 h of treatment (A), OD₆₀₀ after 24 h of treatment (B), and number of CFU/ml after 24 h of treatment (C). Average values of triplicate readings from three independent experiments are plotted with SEM. Significance was determined using a one-way ANOVA (*, P  < 0.05).

FIG 4

AM.P2 genome map. AM.P2 has a genome of 73,308 bp with a GC content of 53.54%, 110 coding DNA sequences, and 10 rho-independent terminators. Gene calling was done using Glimmer3, MetaGeneAnnotator, and Sixpack, and functional annotation was done using BLASTP with the NCBI nonredundant database and the Swiss-Prot and TrEMBL databases. Terminators were identified using TransTermHP.

FIG 5

Comparative genomic analysis of AM.P2 and other Pseudomonas luszeptimaviruses and litunaviruses. A BLASTN search of the AM.P2 genome against the NCBI representative RefSeq database resulted in 18 phages with high percent identity to the AM.P2 genome. Whole genomes were aligned using ClustalW, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA X. Each phage is labeled with the percent query cover followed by the percent identity to AM.P2 in parentheses.

FIG 6

Phylogenetic analysis of AM.P2-sensitive and -resistant clinical strains of Pseudomonas aeruginosa. Spot tests of AM.P2 against 41 clinical MDR and seven antibiotic-susceptible P. aeruginosa strains were done as three independent experiments. Circles indicate strain AM.P2 sensitivity (blue) and resistance (yellow), while squares indicate strain type, such as clinical MDR (red), clinical non-MDR (purple), and laboratory (green) strain.

FIG 7

AM.P2 inhibits the growth of MDR P. aeruginosa clinical strains. Bacterial growth curves of the 46 clinical P. aeruginosa strains treated with AM.P2 at an MOI of 10 over a period of 3 h. Average values of triplicate readings from two independent experiments are plotted with SEM. A t test was used to determine significant differences in the levels of OD₆₀₀ at t = 3 h (*, P  < 0.05).