Isolation and Characterization of Lytic Bacteriophage(s) against Bovine Mastitic Pathogen(s)
Abstract
Bovine mastitis in dairy animals is one of the important production diseases causing an adverse economic impact on a global scale. The disease is mostly of infectious nature with predominant causal organisms being cell-walled bacteria mainly S. aureus and E. coli. The indiscriminate use of antibiotics for treatment has led to the development of antibiotic resistance. To address this problem, alternate means of treatment are being explored like phage therapy. Therefore, in the present investigation, a lytic bacteriophage against mastitis causing E. coli has been isolated and characterized to check for its therapeutic application. E. coli was isolated from milk samples of a buffalo suffering from mastitis and characterized using Gram‟s staining, automated VITEK® compact system, biochemical assays and PCR. The antibiotic sensitivity test revealed that the isolated E. coli was resistant to most of the drugs. A lytic bacteriophage was isolated against E. coli from waste water sample of LUVAS Buffalo farm. The bacteriophage was enriched to 7x108 PFU/mL after observing clear plaques against E. coli on LB agar plates. The isolated phage exhibited a narrow host range and specificity by showing positive Spot test against E. coli only and negative against Pseudomonas, Klebsiella, Streptococcus and Staphylococcus spp. The phage was classified under order Caudavirales and family Myoviridae on the basis of icosahedral head and tail observed after TEM (EMNL, PAU, Ludhiana). For genomic characterization, the extracted DNA was subjected to PCR using different phage (T4, T7 and Lambda) gene specific primers. PCR products revealed that the isolated phage is T4 like, hence, was given the nomenclature „Escherichia phage BoMT4LUVAS‟ as per the guidelines. Nucleotide sequence alignment using BLAST and CLUSTALW of 305 bp and 435 bp sequences of the isolated phage revealed a homology of 98% to 99% with T4 phage of different hosts. A 435 bp nucleotide sequence submitted to GenBank was assigned the Accession No. MK787183. The results of Restriction Endonuclease Profile Analysis of the PCR product of 1345 bp of Escherichia phage BoMT4LUVAS showed that only EcoRI, BseGI, HindIII were able to cut the PCR product (1345 bp) while there was no site for XapI, XhoI, KpnI, NotI, SchI and XbaI enzymes. One step growth curve experiment showed that the phage exhibited a latent period of 30 min with a burst size of 44 phages per bacterial cell. The kinetic of bacterial growth reduction assay demonstrated that the phage inhibited bacterial growth to ~OD 0.1 upto 48 h post infection. In vitro phage activity test revealed that phage was equally effective in milk as in LB broth. This is the first report of phage isolation and characterization against mastitis causing E. coli from Haryana. The applications of these lytic phages could be an asset for biocontrolling of pathogenic agents in medical and veterinary sciences.
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