EVALUATION OF LYTIC BACTERIOPHAGES FOR MULTI-DRUG RESISTANT Pseudomonas aeruginosa ISOLATED FROM SELECTED CLINICAL SAMPLES

Abstract

Pseudomonas aeruginosa is one of the Gram-negative pathogens most difficult to isolate clinically because of its propensity to acquire resistance determinants against almost all antibiotics currently in use. The goal of this study was to define the spectrum of resistance in clinical isolates from a local region and to examine the hypothesis that indigenous phages obtained from the environment might serve as a biological control for these isolates. Specimens (wound, urine, high vaginal, urethral, anus and ear swabs) were plated onto selective and general growth media and the isolates presumptive to be Pseudomonas were confirmed by microscopical and biochemical tests (gram stain, oxidase, catalase, TSI). The confirmed isolates were tested for sensitivity to twelve antibiotic agents by the Kirby-Bauer disc diffusion method, and in some cases further confirmed using 16S rRNA gene sequencing. Raw sewage was screened as a potential phage source by enrichment using a clinical strain, filtration, double-layer agar plating, and Transmission electron microscopy to visualise the phages present in the environment. We collected 12 Pseudomonas strains from 36 clinical samples; it was evident that all the isolates were multi-resistant to almost every tested β-lactam, fluoroquinolone, macrolide and aminoglycoside drugs; isolates are sensitive to levofloxacin and gentamicin, respectively. The 16S rRNA sequences showed not only Pseudomonas aeruginosa but also three related species: Pseudomonas putida, Pseudomonas mendocina, Pseudomonas stutzeri. The later information suggests that after culturing and biochemical testing a wider range of species can be identified from a bacterial population. The enrichment allowed us to obtain lytic phages able to lyse all of the multi-resistant Pseudomonas strains: the zones of lysis were round, distinct and 1-3 mm in diameter; the particles identified via electron microscopy were tailed icosahedral phages of approx. 20 nm and with short, non-contractile tails. In conclusion, multi-resistant Pseudomonas isolates do remain present in the investigated population of Nigeria, and the sewage is an easily and accessible natural source of Pseudomonas-active lytic phages. This study gives useful data specific for Nigeria to be further investigated on the lytic activity of phages against Pseudomonas aeruginosa.