Evaluation of DNA Extraction Methods of Mule Dung

DNA isolation is a critical step in microbial community analysis of animal dung. DNA isolation from mule dung is challenging due to microbial diversity, composition and chemical nature of mule dung. Therefore, selection of an appropriate DNA isolation method is important to analyse the complete microbial diversity. In the current study, we evaluated the DNA isolation from mule dung samples (n=11) using QiAmp Mini stool kit as per manufacturer’s procedure with modifications. The results suggest that modifications in proprietary column based method improved the DNA quality and quantity suitable for mule dung microbial community analyses

Applications of Personalised Phage Therapy highlighting the importance of Bacteriophage Banks against Emerging Antimicrobial Resistance

Emerging antibiotic resistance is one of the most important microbiological issues of the 21st century. This poses a query regarding the future use of antibiotics and availability of other promising therapeutic alternatives. The awareness about antibiotic misuse has improved insufficiently and is evident by the increased incidences of multidrug resistant infections globally. Amongst different antibacterial therapeutic approaches phage therapy has created a niche of its own due to continuous use for treatment of human infections in Eastern Europe. Synergistic compounds along with phages have also been proposed as a better alternative compared to antibiotics or phage alone for treatment of chronic cases and seriously debilitating diseases. As such, why not allow custom made phage therapy for treatment of chronic infections? However, the success of phage therapy will depend upon instant availability of characterised bacteriophages from bacteriophage banks which may serve as the major catalyst in bringing Phage Therapy to main stream treatment alternatives or in combination therapy at least. In the current article we present a glimpse of comprehensive approach about utility of bacteriophage banks and further present personalised phage therapy in a synergistic role with antibiotics to overcome emerging antimicrobial resistance

Comparative Genome Analysis of 19 <i>Trueperella pyogenes</i> Strains Originating from Different Animal Species Reveal a Genetically Diverse Open Pan-Genome

Trueperella pyogenes is a Gram-positive opportunistic pathogen that causes severe cases of mastitis, metritis, and pneumonia in a wide range of animals, resulting in significant economic losses. Although little is known about the virulence factors involved in the disease pathogenesis, a comprehensive comparative genome analysis of T. pyogenes genomes has not been performed till date. Hence, present investigation was carried out to characterize and compare 19 T. pyogenes genomes originating in different geographical origins including the draftgenome of the first Indian origin strain T. pyogenes Bu5. Additionally, candidate virulence determinants that could be crucial for their pathogenesis were also detected and analyzed by using various bioinformatics tools. The pan-genome calculations revealed an open pan-genome of T. pyogenes. In addition, an inventory of virulence related genes, 190 genomic islands, 31 prophage sequences, and 40 antibiotic resistance genes that could play a significant role in organism’s pathogenicity were detected. The core-genome based phylogeny of T. pyogenes demonstrates a polyphyletic, host-associated group with a high degree of genomic diversity. The identified core-genome can be further used for screening of drug and vaccine targets. The investigation has provided unique insights into pan-genome, virulome, mobiliome, and resistome of T. pyogenes genomes and laid the foundation for future investigations

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Not Availablevirulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6–10) and temperatures (4–45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.ICA

Isolation and Characterization of A Bacillus Phage from Equine Carcass Disposal Site

Bacteriophages play an important role in bacterial control in natural niche however a little is known about Bacillus sp. phages prevailing in cadaver affected soils. In the current study, the Bacillus sp. phage was isolated from the equine cadaver disposal site and characterised to gain an insight into the issue of role of phages in biological dynamics of manure thus formed over years. Firstly, the host bacterium was isolated and identified as Bacillus cereus group member as assessed by phylogenetic analysis and secondly it’s corresponding phage from same soil sample was also enriched and characterised. The phage (VTCCBPA38) was found to belong to family Myoviridae and was active within the temperature range of 4°C - 45°C. As assessed by biological sensitivity by spot test, the phage was active against 6/19 (31.6 %) Bacilli tested including Bacillus cereus from goat mastitis. Thus the phagemay find potential use in biocontrol of diseases caused by Bacillus sp. Furthermore, this report is valuable as the first study for investigation of Bacillus sp. phage in carcass burial sites

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Not AvailableKlebsiella pneumoniae is an important emerging pathogen of humans and animals which may lead to major clinical implications including mortality. Moreover the increased use of antibiotics has promoted emergence of carbapenem resistant strains and extended spectrum β-lactamase producers (ESBLs) of K. pneumoniae. Recently, phage therapy has gained momentum as a conceivable alternative against emerging antibiotic resistance. OBJECTIVE: With the aim to explore the efficacy of phage therapy against virulent K. pneumoniae infection, the current study was undertaken to assess the therapeutic effects of a novel lytic phage-VTCCBPA43 in pneumonic mouse model. METHODS: The tailed phage - VTCCBPA43 was assessed for it's growth kinetics, in vitro host range analysis, temperature and pH sensitivity. The protein constituents were analysed by SDS-PAGE and Lc MS/MS and the therapeutic efficacy was observed 2 hr post challenge with virulent K. pneumoniae in BALB/c mouse model. RESULTS: The phage-VTCCBPA43 was found to exhibit high temperature (upto 80 °C) tolerant property. It was most active at pH 5, had a burst size of 172 PFU/ml and exhibited a narrow host range. It was identified as KP36 like phage by shotgun proteomics. Following intranasal application of a single dose of 2 × 109 PFU/mouse post challenge, presence of biologically active phage in vivo and a significant reduction of bacterial load in lungs at all time points was observed which was even more impressive at 96hpi, 6dpi and 10dpi. The loss of severity of lesions suggested overall beneficial effects of phage therapy using BPA43 in the pneumonic mouse model. CONCLUSION: The current research represents first in vivo evidences for effective phage therapy against K. pneumoniae infection by using intranasal route.Not Availabl

Indian Veterinary Research Institute,

Not AvailableA 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.ICA

Development and Evaluation of Bacteriophage Cocktail to Eradicate Biofilms Formed by an Extensively Drug-Resistant (XDR) <i>Pseudomonas aeruginosa</i>

Extensive and multiple drug resistance in P. aeruginosa combined with the formation of biofilms is responsible for its high persistence in nosocomial infections. A sequential method to devise a suitable phage cocktail with a broad host range and high lytic efficiency against a biofilm forming XDR P. aeruginosa strain is presented here. Out of a total thirteen phages isolated against P. aeruginosa, five were selected on the basis of their high lytic spectra assessed using spot assay and productivity by efficiency of plating assay. Phages, after selection, were tested individually and in combinations of two-, three-, four-, and five-phage cocktails using liquid infection model. Out of total 22 combinations tested, the cocktail comprising four phages viz. φPA170, φPA172, φPA177, and φPA180 significantly inhibited the bacterial growth in liquid infection model (p 10 times than the individual dose in the inhibition of XDR P. aeruginosa host. Field emission-scanning electron microscopy was used to visualize phage cocktail mediated eradication of 4-day-old multi-layers of XDR P. aeruginosa biofilms from urinary catheters and glass cover slips, and was confirmed by absence of any viable cells. Differential bacterial inhibition was observed with different phage combinations where multiple phages were found to enhance the cocktail’s lytic range, but the addition of too many phages reduced the overall inhibition. This study elaborates an effective and sequential method for the preparation of a phage cocktail and evaluates its antimicrobial potential against biofilm forming XDR strains of P. aeruginosa

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Not AvailableSwinepox virus (SWPV), a member of the genus Suipoxvirus causes generalized pock-like lesions on the body of domestic and wild pigs. Although outbreak has been reported in India since 1987, virus isolation and genetic characterization remained elusive. In September 2013, an outbreak of acute skin infection occurred in piglets in a commercial piggery unit at Rohtak district in Haryana, India. The presence of SWPV in scab samples collected from piglets succumbed to infection was confirmed by virus isolation, PCR amplification of SWPV-specific gene segments and nucleotide sequencing. Phylogenetic analysis of host-range genes of the SWPV revealed that the Indian isolate is genetically closely related to reference isolate SWPV/pig/U.S.A/1999/Nebraska. To the best of our knowledge this is the first report on isolation and genetic characterization of SWPV from pigs in IndiaNot Availabl

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Not AvailableEquine influenza viruses (EIV)—H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×106.24 EID50 with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.Not Availabl