Isolation and characterization of bacteriophages from water sources effective against carbapenem resistant acinetobacter baumannii isolates

Acinetobacter baumannii is a Gram-negative non-fermentative bacterial pathogen responsible for many hospital infections. Since its discovery, A. baumannii has become resistant to several antibiotics due to its intrinsic and acquired resistance mechanisms. The increasing prevalence of carbapenem-resistant A. baumannii (CRAB) has led to a search for alternative antimicrobial therapy and to the development of new therapeutic strategies. Bacteriophage (phage) therapy is a potential candidate for the treatment of multidrug- resistant (MDR) bacterial infections. In this study, it is aimed to isolate and characterize phages effective against CRAB from various water sources such as wastewater and lake and to investigate the lytic activity of these phages in clinical isolates. A. baumannii isolates were collected from different clinical specimens in Baskent University Ankara Hospital Infectious Diseases and Clinical Microbiology Laboratory. Carbapenem susceptibility was determined by disc diffusion method and Polymerase Chain Reaction (PCR) was used to detect blaOXA- 23, blaOXA-24 and blaOXA-58 ve chromosomal blaOXA-51 carbapenem-resistance genes. The genetic relation between clinical A. baumannii isolates was determined by Pulsed-Field gel electrophoresis (PFGE). Water samples were taken periodically from various water sources for phage isolation. After the isolated phages were enriched and purified, their lytic activity was determined by a spot test. One-step growth curve, the multiplicity of infection (MOI), and thermal and pH stability of phages were evaluated. Transmission electron microscopy (TEM) was used to determine the morphologies and taxonomic families of phages. Restriction digestion and Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-Page) were applied for the molecular characterization and proteome analysis of phages. Chromosomal blaOXA-51 gene was detected in all of the clinical CRAB isolates, while blaOXA-23 was found in 73 of the isolates (n=73/96, 70%) and blaOXA-24 gene in 12 (=12/96, 12.5%). The blaOXA-58 gene was not detected in any of the isolates. PFGE revealed that the bacterial isolates were divided into 5 pulsotypes, including one major pulsotype. Four phages having lytic activity against CRAB A. baumannii were isolated from Baskent University Ankara Hospital wastewater, Kütahya wastewater, Kızılırmak and Kırıkkale horse stud wastewater. For φAb65, the latent period was 5 min, while for φAb31 and φAb69, it was 10 min. The latent period of φAb59 was 15 min. The burst size of φAb31, φAb59, φAb65 and φAb69 was 3500, 1699, 2600 and 118 PFU/CFU, respectively. All phages were thermally stable and tolerant to a wide pH range. TEM analysis revealed that phages belonged to Myoviridae family. Restriction analysis demonstrated that isolated phages had distinct digestion profiles. Evaluated phages in this study were found to be promising for further in vivo experiments and for usage as therapeutic agents to treat MDR bacterial infections.