Isolation, molecular characterization and evaluation of lytic potential of Bacteriophages against multidrug resistant Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is one of the commonly found pathogens and is the most regularly found cause of the morbidity and mortality in different types of infected wounds patients. Different types of antibiotics have been used for the treatment of infection, but it had become resistant to most of these antibiotics. As the antibiotic resistance against P. aeruginosa has increased tremendously and reached to a frightening level, therefore an alternative approach is the need of the hour to control and treat such infections. Bacteriophages are considered to be a very good alternative of antibiotics, and they will be used for the management of infections caused by MDR P. aeruginosa. The specific objective of the current study was to evaluate the lytic potential of indigenous bacteriophages against the Multidrug-Resistant (MDR) P. aeruginosa isolated from various wound infections. A total of (n=532) samples were collected from various infected wounds and processed for isolation of P. aeruginosa. Molecular identification of the P. aeruginosa was conducted through PCR using species-specific primers (OprL, Oprl and PA-SS). Moreover, antibiotic resistance profile of P. aeruginosa was determined against various commonly used antibiotics including cefixime, cefoperazone, ciprofloxacin, polymyxins-B, amikacin, cefepime, aztreonam, amoxicillin-clavulanic acid, imipenem, piperacillin-tazobactam, tigecycline, levofloxacin, doxycycline, meropenem, and ceftriaxone. Furthermore, PCR was conducted to identify the presence of ESBL genes. For the isolation of phages against the resistant P. aeruginosa total (n=40) samples of sewage water was collected from various sites of Faisalabad, Pakistan. Double agar overlay plaque assay, spot test, plaque assay and Transmission electron microscopy was used for the isolation and identification of the bacteriophages. For the characterization of the bacteriophages host range assay, killing assay, pulse field gel electrophoresis, SDS-PAGE and whole genome sequence analysis were conducted. Results of the study revealed that 203/532 (38%) samples were found positive for P. aeruginosa. The susceptibility profile showed that 119/203 (58%) isolates were found as multidrug resistant (MDR) P. aeruginosa. The prevalence of blaNDM was recorded highest (25.9%) while blaKPC was found as least (1.8%). Total 18 (45%) bacteriophages against P. aeruginosa were isolated from 40 different sewage samples. Total 25 different isolates of P. aeruginosa were used to check the host specificity range of all the bacteriophages and phage with the designated name 7A showed the complete lysis against (21) isolates of P. aeruginosa whereas least (03) as were recorded for P21 bacteriophage. Lytic activity xviii of bacteriophages was determined by using the killing assay and all the bacteriophages revealed lytic activity against their host bacterium. Pulse field gel electrophoresis showed different size bands of bacteriophages DNA. Protein analysis using SDS-PAGE exhibited different proteins bands from 20 kDa to 155 kDa. Most prominent bands were of 185 kDa, 140 kDa, 75 kDa and 65 kDa. It was concluded that P. aeruginosa was the pathogen for the infection caused in wound patients and most of the isolates of P. aeruginosa were resistant to multiple antibiotics (MDR). Characterized bacteriophages displayed good lytic activity against indigenous strains of MDR P. aeruginosa. The use of these bacteriophages may be a good alternative to antibiotic therapy for the treatment of infection caused by such MDR P. aeruginosa pathogens in future.