Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model.

OBJECTIVES: Klebsiella pneumoniae is an important emerging pathogen of humans and animals leading to serious clinical consequences. Increased antibiotic use has promoted the emergence of carbapenem-resistant and extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae strains. Recently, phage therapy has gained momentum as a possible alternative against emerging antimicrobial resistance. This study was performed to assess the therapeutic effects of a novel lytic phage (VTCCBPA43) in a pneumonic mouse model in order to explore the efficacy of phage therapy against virulent K. pneumoniae infection. METHODS: The tailed phage VTCCBPA43 was assessed for its growth kinetics, in vitro host range, and temperature and pH sensitivity. Protein constituents were analysed by SDS-PAGE and nLC-MS/MS. Therapeutic efficacy was observed 2 h post-challenge with virulent K. pneumoniae in a BALB/c mouse model. RESULTS: Phage VTCCBPA43 was found to be highly temperature-tolerant (up to 80 °C). 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 a KP36-like phage by shotgun proteomics. Following intranasal application of a single dose (2 × 109 PFU/mouse) post-challenge with virulent K. pneumoniae, the presence of biologically active phage in vivo and a significant reduction in the lung bacterial load at all time points was observed. A reduction in lesion severity suggested overall beneficial effects of VTCCBPA43 phage therapy in the pneumonic mouse model. CONCLUSION: This research represents the first in vivo evidence of effective phage therapy against K. pneumoniae infection by the intranasal route.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

A virulent 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.

Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution.

BACKGROUND: Equine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective. RESULTS: The group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2. CONCLUSIONS: Various lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs.

Abundance of antibiotic resistance genes in environmental bacteriophages.

The ecosystem is continuously exposed to a wide variety of antimicrobials through waste effluents, agricultural run-offs and animal-related and anthropogenic activities, which contribute to the spread of antibiotic resistance genes (ARGs). The contamination of ecosystems with ARGs may create increased opportunities for their transfer to naive microbes and eventually lead to entry into the human food chain. Transduction is a significant mechanism of horizontal gene transfer in natural environments, which has traditionally been underestimated as compared to transformation. We explored the presence of ARGs in environmental bacteriophages in order to recognize their contribution in the spread of ARGs in environmental settings. Bacteriophages were isolated against environmental bacterial isolates, purified and bulk cultured. They were characterized, and detection of ARG and intI genes including blaTEM, blaOXA-2, intI1, intI2, intI3, tetA and tetW was carried out by PCR. This study revealed the presence of various genes [tetA (12.7 %), intI1 (10.9 %), intI2 (10.9 %), intI3 (9.1 %), tetW (9.1 %) and blaOXA-2 (3.6 %)] and blaTEM in a significantly higher proportion (30.9 %). blaSHV, blaOXA-1, tetO, tetB, tetG, tetM and tetS were not detected in any of the phages. Soil phages were the most versatile in terms of ARG carriage. Also, the relative abundance of tetA differed significantly vis-à-vis source. The phages from organized farms showed varied ARGs as compared to the unorganized sector, although blaTEM ARG incidences did not differ significantly. The study reflects on the role of phages in dissemination of ARGs in environmental reservoirs, which may provide an early warning system for future clinically relevant resistance mechanisms.

Isolation of a lytic bacteriophage against virulent Aeromonas hydrophila from an organized equine farm.

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.

Isolation and characterization of a bacteriophage with broad host range, displaying potential in preventing bovine diarrhoea.

Phage therapy has been previously tried for treatment of diarrhoea in calves, pigs and lambs but those trials were conducted without any detailed information of used phages. Here, we report isolation of a broad-spectrum phage which showed bactericidal activity against 47.3 % of calf diarrhoeal isolates of Escherichia coli, in vitro. The isolated phage resembled the characteristics of Myoviridae family and showed ~97 % similarity with earlier reported bacteriophages of sub family-Tevenvirinae, genus-T4-like virus, based on nucleotide sequence of major head protein-gp23 gene. The phage exhibits the potential to be used as drug substitute tool against E. coli causing diarrhoea in cattle in farm environments.

Sequence and phylogenetic analysis of host-range (E3L, K3L, and C7L) and structural protein (B5R) genes of buffalopox virus isolates from buffalo, cattle, and human in India.

Buffalopox virus (BPXV), a close variant of vaccinia virus (VACV) has emerged as a zoonotic pathogen. The host tropism of poxviruses is governed by host-range genes. Among the host-range genes: E3L, K3L, and C7L are essential for virus replication by preventing interferon resistance, whereas B5R is essential for spread of the virus and evasion from the host's immune response as in VACV. We report sequence analysis of host-range genes: E3L, K3L, C7L, and membrane protein gene (B5R) of BPXVs from buffalo, cattle, and human from recent outbreaks in India-their phylogenetic relationship with reference strain (BP4) and other Orthopoxviruses. BPXVs revealed a sequence homology with VACVs including zoonotic Brazilian VACV-like viruses. The aa sequences of E3L and K3L genes were 100 % similar in buffalo, cattle, and human isolates. However, four significant point mutations (I11K; N12K and S36F in C7L gene and D249G in B5R gene) were observed specific to buffalo isolate only. This signifies that different strains of BPXV were circulated during the outbreak. The mutations in C7L and B5R could play an important role in adaptation of BPXV in human and cattle which needs further functional studies. The strain of BPXV isolated from buffalo may not be adopted in human and cow. Various point mutations were observed in the host-range genes of reference strain (BPXV-BP4) which may be due to several passages of virus in cell culture. The phylogeny constructed based on concatenated gene sequences revealed that BPXVs are not as closely related to vaccine strain (Lister and Lister-derived strain-LC16m8), as hypothesized earlier, rather they are more closely related to reference strain (BPXV-BP4) and other vaccinia and vaccinia-like viruses such as Passatempo and Aracatuba viruses. The availability of information regarding host tropism determinants would allow us to understand molecular mechanism of species tropism of poxviruses which would be useful in unveiling new strategies to control zoonotic poxviral infections.