Attenuation of equine herpesvirus 1 through deletion of gE gene and its pathological evaluation in murine model.

Equine herpesvirus 1 (EHV1) infection is a global health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been a continuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. Keywords: quine herpesvirus 1; bacterial artificial chromosome (BAC); mutation; glycoprotein E; vaccine.

Synergy of a virulent phage (φAB182) with antibiotics leading to successful elimination of biofilms formed by MDR Acinetobacter baumannii.

Emergence of multiple drug resistant (MDR) strains of Acinetobacter baumannii and a withering drug discovery pipeline necessitates the search for effective alternatives to replace or synergize with currently used antibiotics. In this report, we have described the synergy assessment of a virulent Acinetobacter baumannii phage φAB182 with a wide range of antibiotics. Myophage φAB182 was isolated from sewage against MDR A. baumannii and exhibited maximum stability at 25 °C and pH 7. It also had a short latent period of 9 min with a large burst size of 287. The phylogenetic analysis of its major capsid protein gene indicated an 84.15% similarity to the lytic A. baumannii phage Acj9. In the presence of antibiotics, phage φAB182 showed the highest synergy (p < 0.0001) with colistin, followed by polymixin B, ceftazidime and cefotaxime and this synergistic effect was further validated by time kill kinetics. The combined action of phage φAB182 with colistin, polymixin B, ceftazidime and cefotaxime was also synergistic for the eradication of biofilms formed by A. baumannii as measured by MBECcombination/MBECantibiotic values (<0.25). We thus propose bacteriophage φAB182 as a potential antibacterial candidate in combination therapy. The findings from this study strongly support the use of phage antibiotic synergy for the successful treatment of biofilm forming MDR A. baumannii infections.

Phage Display Technique as a Tool for Diagnosis and Antibody Selection for Coronaviruses.

Phage display is one of the important and effective molecular biology techniques and has remained indispensable for research community since its discovery in the year 1985. As a large number of nucleotide fragments may be cloned into the phage genome, a phage library may harbour millions or sometimes billions of unique and distinctive displayed peptide ligands. The ligand-receptor interactions forming the basis of phage display have been well utilized in epitope mapping and antigen presentation on the surface of bacteriophages for screening novel vaccine candidates by using affinity selection-based strategy called biopanning. This versatile technique has been modified tremendously over last three decades, leading to generation of different platforms for combinatorial peptide display. The translation of new diagnostic tools thus developed has been used in situations arising due to pathogenic microbes, including bacteria and deadly viruses, such as Zika, Ebola, Hendra, Nipah, Hanta, MERS and SARS. In the current situation of pandemic of Coronavirus disease (COVID-19), a search for neutralizing antibodies is motivating the researchers to find therapeutic candidates against novel SARS-CoV-2. As phage display is an important technique for antibody selection, this review presents a concise summary of the very recent applications of phage display technique with a special reference to progress in diagnostics and therapeutics for coronavirus diseases. Hopefully, this technique can complement studies on host-pathogen interactions and assist novel strategies of drug discovery for coronaviruses.

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.

Characterization of isolates of Bordetella bronchiseptica from horses.

Bordetella bronchiseptica is a well-known Gram-negative bacterial pathogen causing a plethora of diseases in different animals. Although its infection has been reported from pigs and dogs in India, no report of B. bronchiseptica from horses is described. We report for the first time, isolation, identification and characterization of strains of B. bronchiseptica from respiratory infection in horses from different states in India. The antimicrobial susceptibility testing showed resistance to penicillins, ceftazidime, and chloramphanicol. The virulence capability of the strains was confirmed by sequencing genes such as adenylate cyclase toxin (cyaA), bordetella virulence gene (bvgA) and by PCR detection of flagellin gene (fla). We demonstrate the involvement of B. bronchiseptica strains in respiratory tract infection in horses in India.

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.

Genetic characterization of equine herpesvirus 1 isolates from abortion outbreaks in India.

Equine herpesvirus 1 (EHV1) is a common pathogen of horses that causes upper respiratory tract disease, abortion, neonatal death and neurological disease. The neurological form of disease is called equine herpesvirus myeloencephalopathy (EHM). During the past decade, the incidence of EHM has been on the rise in Europe, North America, Australia and Asia. Some EHV1 isolates causing EHM exhibit a single-nucleotide polymorphism (SNP) in the DNA polymerase gene (ORF30) at position 2254 (A2254 to G2254). Further, based on polymorphism in the ORF68, EHV1 isolates have been classified into different groups. The aim of the present study was to estimate the genetic diversity of EHV1 and to determine the prevalence of the neuropathogenic genotype of EHV1 in India. Out of 133 clinical specimens from abortion cases in northern India, 56 were positive for EHV1 infection. Analysis of the A/G SNP by real-time PCR and sequence analysis revealed that 54 of 56 samples (96.43 %) were of the non-neuropathogenic genotype (A2254), while two (3.57 %) had the neuropathogenic marker (G2254). Sequence analysis of the polymorphic region of ORF68 of EHV1 isolates (n = 9) from India indicated that the Delhi/1998, Tohana-2/2013, Hisar-2/2014 and Hisar-15/1990 isolates belonged to group 4, while the Jind/1996, Rajasthan/1998, Delhi-3/2007 and Tohana-5/1996 isolates clustered within group 5. One isolate (Hisar-7/1990) exhibited SNPs at positions C710 and C713, forming a separate group. Here, we report for the first time the detection of neuropathogenic genotypes of EHV1 in India and show that Indian EHV1 isolates cluster within groups 4 and 5.

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.

Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Modulation of mammary tumour progression using murine model by ethanol root extract of Saussurea costus (falc.) lipsch.

ETHNOPHARMACOLOGICAL RELEVANCE: Breast cancer is a major cause of death among human females across the globe. The anti-neoplastic agents or therapies used for the treatment of cancers can enhance longevity but are subsequently observed to deteriorate the quality of life due to the extensive side effects produced. Saussurea costus is a potential medicinal plant of the Himalayas with noticeable ethnopharmacological properties. The phytochemicals present in Saussurea costus are responsible for anti-carcinogenic potential and warranted nil or minimal side effects of Saussurea costus and directed to use this plant as a preventive or therapeutic drug candidate against cancers. AIM OF THE STUDY: The present study was planned to evaluate the anti-neoplastic activity of Saussurea costus root extract (SL) in rat mammary tumour model. MATERIALS AND METHODS: The anti-neoplastic activity of SL root extract at 3 different doses (100, 250 and 500 mg/kg BW) for 18 weeks against 12-dimethylbenz (a) anthracene (DMBA)-induced mammary tumours in Sprague Dawley (SD) female rats was analyzed through serum biochemistry (ALT, AST, ALP, Total protein, Creatinine and BUN), oxidative stress parameters (Lipid peroxidation, Catalase and Reduced glutathione), pro-inflammatory cytokines (TNF-α and NF-κB), immunohistochemical markers (Ki-67, MMP-9 and VEGF), real-time PCR (PCNA, p53, bax, bcl-2 and caspase-3, genes) and molecular docking. RESULTS: Inhibition of tumour parameters, minimal alteration in the liver (ALT, AST and ALP) and kidney enzymes (Creatinine and BUN), decreased activity of MDA, elevated levels of GSH and catalase, reduction in the levels of pro-inflammatory cytokines i.e. TNF-α and NF-κB, reduced gross and histomorphological changes, declined expression of Ki-67, MMP-9 and VEGF in vivo rat model, mRNA expression of cancer-related genes and docking of dehydrocostus lactone and costunolide with NF-κB and TNF-α demonstrated the chemopreventive action of SL root extract. CONCLUSIONS: The in-vivo trial elucidates anti-neoplastic activity of Saussurea costus root extract as demonstrated through the reduction of biochemical indices, oxidative stress parameters, histological changes, pro-inflammatory cytokines (NF-κB and TNF-α), cellular proliferation (Ki-67), metastases (MMP-9) and neovascularization (VEGF) markers with highest anti-neoplastic effect of SL extract at the dose of 500 mg/kg body weight. Therefore, the present study signifies the need to use the active principles present in the root extract of Saussurea costus against breast cancer as a therapeutic regimen.

Complete genome sequence analysis of Japanese encephalitis virus isolated from a horse in India.

The complete genome of the Japanese encephalitis virus (JEV) strain JEV/eq/India/H225/2009(H225), isolated from an infected horse in India, was sequenced and compared to previously published JEV genomes. H225 genome was 10,977-nucleotides long, comprising a single ORF of 10,299-nucleotides, a 5'-UTR of 95 nucleotides and a 3'-UTR of 582 nucleotides. The H225 genome showed high levels of sequence identity with 47 fully sequenced JEV genomes, ranging from 99.3 % to 75.5 % for nucleotides and 99.2 % to 91.5 % for amino acid sequences. Phylogenetic analysis of the full-length sequence indicated that the H225 strain belongs to genotype III and is closely related to the Indian JEV strain Vellore P20778. A comparison of amino acids associated with neurovirulence in the E proteins and non-structural proteins of known virulent and attenuated JEV strains suggested H225 to be a highly virulent strain. This is the first report of whole-genome sequencing of a genotype III JEV genome isolated from equines.

Intracellular ROS production and apoptotic effect of quinoline and isoquinoline alkaloids on the growth of Trypanosoma evansi.

Trypanosoma evansi, a hemoflagellate poses huge economic threat to the livestock industry of several countries of Asia, Africa, South America and Europe continents of the world. Limited number of available chemical drugs, incidents of growing drug resistance, and related side effects encouraged the use of herbal substitutes. In the present investigation, the impact of six alkaloids of quinoline and isoquinoline group was evaluated on the growth and multiplication of Trypanosoma evansi and their cytotoxic effect was examined on horse peripheral blood mononuclear cells in an in vitro system. Quinine, quinindine, cinchonine, cinchonidine, berbamine and emetine showed potent trypanocidal activities with IC50/24 h values 6.631 ± 0.244, 8.718 ± 0.081, 16.96 ± 0.816, 33.38 ± 0.653, 2.85 ± 0.065, and 3.12 ± 0.367 µM, respectively, which was comparable to the standard anti-trypanosomal drug, quinapyramine sulfate (20 µM). However, in the cytotoxicity assay, all the drugs showed dose dependent cytotoxic effect and quinine, berbamine and emetine showed selectivity index more than 5, based of ration of CC50 to IC50. Among the selected alkaloids, quinidine, berbamine and emetine exhibited higher apoptotic effects in T. evansi. Likewise, drug treated parasites showed a dose-dependent and time-dependent increase in reactive oxygen species (ROS) production. Therefore, increased apoptosis in combination with ROS generation could be responsible for the observed trypanocidal effect which could be further evaluated in T. evansi-infected mice model.

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

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 < 0.0001). The minimal inhibitory dose of each phage in a cocktail was effectively reduced to >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.

Double and quadruple deletion mutant of EHV-1 is highly attenuated and induces optimal immune response.

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge.

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.

Antibiotics targeting bacterial protein synthesis reduce the lytic activity of bacteriophages.

Combination therapy of bacteriophages and antibiotics requires careful selection of specific antibiotics as it is crucial towards determining the success of phage therapy to treat multiple drug-resistant bacterial infections. So, we examined how different antibiotics can affect phage lytic activity when used in combination against targeted bacteria. Various antibiotics targeting bacterial protein synthesis pathways were tested for their bactericidal action in combination with bacteriophages of Acinetobacter baumannii (φAB145, φAB182), Staphylococcus aureus (φSA115, φSA116) and Salmonella Typhimurium (φST143, φST188). The phages displayed highly significant antagonism with most of the protein/ribosomal machinery targeting antibiotics: φSA115 (13/13); φSA116 (13/13); φST143 (11/13); φAB145 (11/13); φST188 (9/13); φAB182 (7/13). To validate this antagonistic effect, synergy assessment of these phages with gentamicin (GEN) and tetracycline (TE) was performed using time kill curve assays and counting the remaining viable bacterial cells at the end of the experiment. An increase in bacterial turbidity in phage-antibiotic combination groups was observed as compared to the treatment with phages individually. Also, GEN exhibited 4.22, 5.90, 2.02, 3.15, 2.68, and 2.60 log proliferation in viable cell count, respectively, for φSA115, φSA116, φST145, φAB182, φST143 and φAB188 in combination group in comparison to their individual actions. TE supplementation also led to 2.40, 4.90, 1.61, 2.73, 3.93, and 1.81 log increments in viable bacterial count when combined with φSA115, φSA116, φST145, φAB182, φST143 and φAB188, respectively. This study concludes that antibiotics targeting the bacterial protein biosynthetic machinery may lead to a reduction in the lytic activity of bacteriophages, thus lowering their therapeutic potential. Hence, such compounds must be carefully screened before their employment in combination treatment regimens.

Tracking the phage trends: A comprehensive review of applications in therapy and food production.

In the present scenario, the challenge of emerging antimicrobial resistance is affecting human health globally. The increasing incidences of multidrug-resistant infections have become harder to treat, causing high morbidity, and mortality, and are posing extensive financial loss. Limited discovery of new antibiotic molecules has further complicated the situation and has forced researchers to think and explore alternatives to antibiotics. This has led to the resurgence of the bacteriophages as an effective alternative as they have a proven history in the Eastern world where lytic bacteriophages have been used since their first implementation over a century ago. To help researchers and clinicians towards strengthening bacteriophages as a more effective, safe, and economical therapeutic alternative, the present review provides an elaborate narrative about the important aspects of bacteriophages. It abridges the prerequisite essential requirements of phage therapy, the role of phage biobank, and the details of immune responses reported while using bacteriophages in the clinical trials/compassionate grounds by examining the up-to-date case reports and their effects on the human gut microbiome. This review also discusses the potential of bacteriophages as a biocontrol agent against food-borne diseases in the food industry and aquaculture, in addition to clinical therapy. It finishes with a discussion of the major challenges, as well as phage therapy and phage-mediated biocontrols future prospects.

Detection and genetic characterization of porcine circovirus 3 (PCV3) in pigs in India.

Porcine circovirus type 3 (PCV3), a novel circovirus, has been reported recently from major swine growing countries globally, and the virus is associated with diseases like porcine dermatitis, nephropathy syndrome and reproductive failure. This report describes the identification of PCV3 associated with reproductive failure in sows and piglet mortality and circulation of the virus in healthy pigs in India. The pathological changes in various tissues from stillborn piglet and characterization of the virus genomes were reported. The genome sequences of Indian PCV3 strains showed 91.4%-99.8% nucleotide identity with other sequences of PCV3 strains circulating worldwide. The phylogenetic analysis showed clustering of Indian strains into a separate group with the isolate from USA (MN/2016) under PCV3a genotype. The results confirmed the circulation of PCV3 in Indian pigs and its association with clinical cases. This study speculates emergence of PCV3 as an important pig pathogen in the country, which warrants the thorough investigation on PCV3 epidemiology, pathogenesis and to implement the control measures.

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.