The isolation and characterisation of a temperate phage ɸPh_AJ01 against Acinetobacter junii

Acinetobacter junii is an emerging opportunistic human and animal pathogen, fast gaining antimicrobial resistance. As conventional treatment becomes ineffective, alternatives are needed to address this challenge. Bacteriophages offer a promising yet underexplored solution in combating A. junii infections. Here, we report the isolation and characterisation of a temperate phage, ɸPh_AJ01, effective against a carbapenem-resistant A. junii. Moreover, the study adopts a technique for biasing the temperate phage to the lytic life cycle to address the issues of lysogeny. The efficacy of the phage in rescuing A. junii-infected Caenorhabditis elegans is also demonstrated.

Phage ɸPh_AJ01 was isolated and purified from the Cooum River in Chennai, India. Host range analysis, adsorption assay, growth curve, and in vitro bacteriolytic test were performed. Transmission electron microscopy and whole-genome sequencing were carried out to understand its morphological and genetic features. Phage stability was tested in different temperatures and pH conditions. Antibiofilm activity of the phage was studied using inhibition and disruption assays. Bacteriophage-insensitive mutation frequency of A. junii was evaluated by the patch screen test. Experiment to bias the temperate phage towards lytic life cycle and, survival assay with A. junii-infected C. elegans were also performed.

ɸPh_AJ01 is a temperate, narrow-spectrum, icosahedral, tailed phage that efficiently infected A. junii, yielding a burst size of 74 ± 8 virions per cell with good temperature and pH stability. ɸPh_AJ01 inhibited biofilm formation by 92%, and disruption by 73.8%. A. junii gained resistance to ɸPh_AJ01 at a frequency of 6.88E-06 CFU/mL. The double-stranded DNA genome of 44,561 bp and GC content of 38.62%, encodes 65 open reading frames. The phage is novel, with only 80% similarity to other Acinetobacter phages. The genome is devoid of antibiotic-resistant and virulence genes. The temperate phage, when biased with ciprofloxacin (50 ng/mL), resulted in ≥ 5 log reduction of A. junii, with an associated 6-log reduction in lysogens and 5-fold increase in phage titre. The in vivo study yielded a 65% increase in survival of A. junii-infected C. elegans with the phage and ciprofloxacin when compared to 28% with phage alone.

The study describes the isolation and characterisation of a temperate phage ɸPh_AJ01 against A. junii. A lysogeny biasing strategy was additionally evaluated under both in vitro and in vivo conditions to address the limitations of the temperate phage.