Winter 2016 Biotherapy Journal Review – Anand et. al.

Phage Reviews

Journal Citation:

Anand T, Vaid RK, Bera BC, Singh J, Barua S, Virmani N, K R, Yadav NK, Nagar D, Singh RK, and Tripathi BN. Isolation of a lytic bacteriophage against virulent Aeromonas hydrophila from an organized equine farm. J Basic Microbiol. 2016 (e-published 2015); 55:1-6.

Published Abstract

A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS-PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp23 gene sequence with previously reported phages showed similarity with T4-like phages infecting Enterobacteriaceae instead of Aeromonas spp. Thus, to our knowledge, this report points toward the fact that a novel/evolved phage might exist in equine environment against A. hydrophila, which can be potentially used as a biocontrol agent.

Introduction & Background to Study

Viruses are obligate intracellular parasites, and bacteriophages are obligate parasites of bacteria. Thousands of phages have been identified, but very few infect Aeromonas hydrophila, which is a gram negative bacillus commonly associated with water. It can be the cause of infections in humans, especially in the immunosuppressed host or the recipient of leech therapy. A. hydrophila is also a factor in equine disease, including septic arthritis, enteritis, and abortions.

The authors of this paper isolated, from the sewage of an equine farm, a pathogenic strain of A. hydrophila, which was host to a T4 type bacteriophage.

Materials & Methods

A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm.

Sewage samples were collected from boxstalls and combined drainage/sewage hole at an equine breeding farm. Samples were cultured and individual colonies with distinct morphology were picked and purified. Individual bacteria were used for bacteriophage enrichment, and filtrates were screened for phage activity using a spot assay. The host bacteria showing phage activity was subjected to isolation of genomic DNA.

The bacteriophage was separated by picking a single plaque by double-agar layer technique and purified, concentrated, and characterized by growth curve, adsorption rate, molecular and phylogenetic analysis, and electron microscopy.

Study Results

A total of 69 bacterial isolates were obtained The most abundant bacteria were Bacillus spp., E. coli, and Enterococcus spp.; however, one isolate of A. hydrophila was also obtained from the sewage of one boxstall. No phage activity was seen in the purified isolates of  A. hydrophila, so an isolate was further characterized through biochemical analysis and 16s rRNA sequencing, confirming it to be Aeromonas hydrophila spp. dhakensis (Accession no. VTCCBAA700). The isolate had virulence potential (confirmed by PCR amplification of aerolysin gene lip gene. A bacteriophage (BPA6) which formed plaques of 1–2mm diameter was recovered after plating the enriched filtrate in the presence of host (VTCCBAA700). The plaques appeared translucent with clear centers.

Electron microscopy demonstrated an icosahedral head and a contractile tail with a base plate, consistent with the family Myoviridae. PCR amplification and sequence analysis of gp23 gene, along with protein profiling by SDS-PAGE, indicated phylogenetic resemblance to T4 like virus genus, though it more closely resembled T4-like phages that infect Enterobacteriaceae, not Aeromonas spp.

Author’s Conclusions

Whether the isolated Aeromonas hydrophila phage described in this report is a newly discovered agent or a local mutant of a T4 like Enterobacteriaceae phage, this “novel/evolved phage might exist in equine environment against A. hydrophila, which can be potentially used as a biocontrol agent.”

Reviewer’s Conclusions

The authors have isolated a T4 like phage that infects a virulent strain of A. hydrophila. Their report is a nice example of the methods used to search for phage, many of which can be quite elusive. They describe the importance of their findings as “potentially . . . a biocontrol agent,” but I believe this deserves further elaboration. The more varieties of phage we discover and catalog, the better we will someday be at putting together the ideal therapeutic cocktails for each clinical situation. What works best as a topical agent may be different from what works best intravenously; what works best for A. hydrophila cellulitis (a skin infection) may not be best for osteomyelitis (a bone infection). As I ponder this, I have to wonder if we could add this phage to the leech jar to reduce the frequency of A. hydrophila infections resulting from hirudotherapy? There is a lot that still needs to be investigated. Thankfully researchers such as Anand and colleagues continue to search for more phage, and therefore more potential candidates for biocontrol and biotherapy.