Genome sequencing and comparative genomic analysis of bovine mastitis-associated Staphylococcus aureus strains from India.

BACKGROUND: Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India. RESULTS: The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes. CONCLUSION: This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

First complete genome sequence of lumpy skin disease virus directly from a clinical sample in South India.

Lumpy skin disease (LSD), a notifiable disease listed by the World Organization for Animal Health and a fast fast-moving transboundary viral disease infecting cattle and buffaloes, was reported in India in 2019 and has since rapidly spread across the country. This study reports the first complete genome sequence and analysis of a pathogenic LSD virus (LSDV) from India (LSDV/208/PVNRTVU/2020) obtained by direct sequencing of a suspected clinical sample using Illumina and Nanopore sequencing technologies. The complete genome sequence of LSDV/208/PVNRTVU/2020 is 150445 bp long, codes for 156 putative genes and carries identical 2254 bp inverted terminal repeats at either ends. The unique features reported in the LSDV isolates from the recent outbreaks in Asia, namely, the insertions of 12 nucleotides in the viral G-protein coupled receptor (GPCR) and 27 nucleotides leading to duplication of 9 aminoacids in the extracellular enveloped virus-specific (EEV) genes were also conserved in LSDV/208/PVNRTVU/2020. Phylogenetic analysis of the complete genome sequence of LSDV/208/PVNRTVU/2020 revealed its close relation with Kenyan strains and clustered away from vaccine strains. Further analysis showed evidence of strong purifying selection without any recombination events. The data presented in this study could be useful for designing effective strategies such as developing rapid diagnostics and vaccines to control LSD.

A Japanese Encephalitis Vaccine From India Induces Durable and Cross-protective Immunity Against Temporally and Spatially Wide-ranging Global Field Strains.

BACKGROUND: Japanese encephalitis (JE) is a vaccine-preventable acute disease. We report the results of a phase 2/3 trial of JENVAC, a Vero cell-derived vaccine developed using an Indian strain of JE virus (JEV). METHODS: JENVAC was administered in 2 doses 28 days apart, and immunogenicity was compared to that from a single dose of SA-14-14-2, the only approved JE vaccine and regimen at the time in India. RESULTS: After both the doses, seroconversion and seroprotection were >90% for JENVAC. For SA-14-14-2, seroconversion and seroprotection were 57.69% and 77.56%, respectively, on day 28 and 39.74% and 60.26%, respectively, on day 56. The geometric mean titers at day 28 and day 56 were 145.04 and 460.53, respectively, for JENVAC and 38.56 and 25.29, respectively, for SA-14-14-2. With a single dose of JENVAC, seroprotection titers lasted at least 12 months in >80% of the subjects. Following receipt of 2 doses, 61.17% of subjects retained seroprotection titers at 24 months, and immunogenicity criteria were higher than that for SA-14-14-2 at 12, 18, and 24 months each. Sera from JENVAC subjects neutralized JEV genotypes I, II, III, and IV equally well. Adverse events were not significantly different between the 2 vaccines. CONCLUSIONS: JENVAC elicits long-lasting, broadly protective immunity. CLINICAL TRIALS REGISTRATION: CTRI/2011/07/001855.

Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens.

The development of persistent antibiotic resistance by human methicillin-sensitive Staphylococcus aureus (MSSA) strains and substantial association with poly-N-acetyl glucosamine (PNAG) in biofilms is reported in this investigation. Sixteen of 31 MSSA strains under study were found to have developed resistance to one or more antibiotics, with four strains, two of which did not produce biofilms, showing resistance to cefoxitin, undetectable by mecA amplification. Antibiotic resistance displayed by 13/14 biofilm-forming S. aureus isolates remained persistent for 4 weeks prior to reverting back to the original antibiotic susceptibility, prompting a suggestion of determining antibiograms for clinical S. aureus isolates subcultured from biofilms developed in vitro as well as planktonic subcultures prepared from the site of infection. While there was correlation of antibiotic resistance with biofilm formation confirming previous reports, this is the first time that persistence of the biofilm-associated antibiotic resistance by S. aureus as planktonic cells is reported. Among the two methods used for assessment of biofilm formation, the tissue culture plate (TCP) method revealed that almost all strains were strong or moderate biofilm producers whereas only 19/31 strains were biofilm producers using the Congo Red agar (CRA) method indicating the superiority of the TCP method in detecting biofilm producers. We also observed no association between biofilm formation and major capsule types. However, substantial, although not absolute, association of biofilm formation with PNAG was observed, warranting continued identification of additional surface-associated polysaccharide and/or protein antigens associated with biofilm formation for development of an effective vaccine against S. aureus infections regardless of capsular phenotype.

Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus.

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.

Complete genome sequence of bluetongue virus serotype 9: implications for serotyping.

The second complete genome of bluetongue virus serotype 9 (BTV-9) is presented in this report. The sequence analysis points to continued circulation in India of a mixed topotype virus apparently belonging to the BTV-9 serotype, and it raises questions about approaches for serotyping bluetongue viruses.

Incidence of subclinical mastitis and prevalence of major mastitis pathogens in organized farms and unorganized sectors.

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10(5)/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.

Assessment of Immune Responses to Rabies Vaccination in Free-Ranging Dogs in Bengaluru, India.

Rabies is a fatal encephalomyelitis mainly transmitted to humans and other animals by rabid dog bites. Hence, vaccination programs are being instituted for the control of rabies in dogs. Though stray dogs have been vaccinated for years under various programs initiated for control of the disease, the effectiveness of these programs can be ascertained only by assessing the immunity of these dogs. With this in view, a study was conducted to assess the effectiveness of the ongoing mass dog vaccination (MDV) program by the Bengaluru City Municipal Corporation, Bengaluru, India. Whole blood and serum samples (n = 260) from vaccinated stray dogs in 26 wards of 8 corporation zones were tested by rapid fluorescent focus inhibition test (RFFIT) as well as an in-house quantitative indirect enzyme-linked immunosorbent assay (iELISA) for a humoral response and by interferon-gamma (IFN-γ) ELISA for a cellular response. As determined by the cut-off value of 0.5 IU/mL of serum, 71% and 87% of the samples from vaccinated dogs revealed adequate levels of antibodies presumed to confer protection by RFFIT and iELISA, respectively. The sensitivity and specificity of the iELISA were 100% and 63.3%, respectively. The IFN-γ ELISA revealed adequate cellular response in 50% of the samples. The quantitative iELISA was found to be useful in large-scale seromonitoring of MDV programs to aid in the elimination of dog-mediated rabies.

Evidence of bluetongue virus serotype 21 (BTV-21) divergence.

Bluetongue virus serotype 21 (BTV-21) was originally isolated from Australia, but has now been reported from India, Indonesia, China and Japan. We report the isolation, and sequencing of BTV-21 from India. The complete ORF sequence of VP2 gene of this isolate showed that it is closely related to recent BTV-21 isolates from Japan (93-94% identity), and distantly related to BTV-21 reference strain (86% identity). Our results, along with the available sequences of Japanese isolates, suggest that the currently circulating BTV-21 strains from India and Japan are divergent from the original strain(s) from Australia and shed light on designing molecular diagnostics for the detection of BTV.

Genetic characterization of bluetongue virus serotype 9 isolates from India.

Recent incursions of bluetongue virus (BTV) into previously naive geographical areas have emphasised the need to better understand virus movement and epidemiology. Several bluetongue virus (BTV) serotypes are known to exist in India, and some serotype viruses have been isolated. However, the complete genome of not a single isolate is available to date. We report the complete genome sequence of one, and partial sequences of three other Indian isolates of BTV-9. Evolutionary relationships with segment-2 and -6 sequences of BTV isolates around the world, deduced using four different phylogenetic analyses and a similarity programme, show that BTV-9 (Eastern), BTV-9 (Western), and BTV-5 form a triad of equidistant, genetically distinct groups of viruses. The Indian BTV-9 isolates were closely related to Mediterranean and European BTV-9 isolates (Eastern topotype) based on segment-2 and -6 sequences. By contrast, segment-5 analyses clustered the Indian BTV-9 isolates with South African BTV-3 reference strain (98% identity), which belongs to one of the Western types. These results have implications on BTV origin and movement, genotyping, serotyping, and vaccine design.

COVID-19: A Veterinary and One Health Perspective.

Interface with animals has been responsible for the occurrence of a major proportion of human diseases for the past several decades. Recent outbreaks of respiratory, haemorrhagic, encephalitic, arthropod-borne and other viral diseases have underlined the role of animals in the transmission of pathogens to humans. The on-going coronavirus disease-2019 (COVID-19) pandemic is one among them and is thought to have originated from bats and jumped to humans through an intermediate animal host. Indeed, the aetiology, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect and cause disease in cats, ferrets and minks, as well as be transmitted from one animal to another. The seriousness of the pandemic along with the zoonotic origin of the virus has been a red alert on the critical need for collaboration and cooperation among human and animal health professionals, as well as stakeholders from various other disciplines that study planetary health parameters and the well-being of the biosphere. It is therefore imminent that One Health principles are applied across the board for human infectious diseases so that we can be better prepared for future zoonotic disease outbreaks and pandemics.

Effects of Dietary Antimicrobial Growth Promoters on Performance Parameters and Abundance and Diversity of Broiler Chicken Gut Microbiome and Selection of Antibiotic Resistance Genes.

Antimicrobial growth promoters (AGPs) are commonly used in broiler production. There is a huge societal concern around their use and their contribution to the proliferation of antimicrobial resistance (AMR) in food-producing animals and dissemination to humans or the environment. However, there is a paucity of comprehensive experimental data on their impact on poultry production and the AMR resistome. Here, we investigated the effect of five antimicrobial growth promoters (virginiamycin, chlortetracycline, bacitracin methyl disalicylate, lincomycin, and tylosin) used in the commercial broiler production in the Indian subcontinent and in the different parts of the world for three consecutive production cycles on performance variables and also the impact on gut bacteria, bacteriophage, and resistome profile using culture-independent approaches. There was no significant effect of AGPs on the cumulative growth or feed efficiency parameters at the end of the production cycles and cumulative mortality rates were also similar across groups. Many antibiotic resistance genes (ARGs) were ubiquitous in the chicken gut irrespective of AGP supplementation. In total, 62 ARGs from 15 antimicrobial classes were detected. Supplementation of AGPs influenced the selection of several classes of ARGs; however, this was not correlated necessarily with genes relevant to the AGP drug class; some AGPs favored the selection of ARGs related to antimicrobials not structurally related to the AGP. AGPs did not impact the gut bacterial community structure, including alpha or beta diversity significantly, with only 16-20 operational taxonomic units (OTUs) of bacteria being altered significantly. However, several AGPs significantly reduced the population density of some of the potential pathogenic genera of bacteria, such as Escherichia coli. Chlortetracycline increased the abundance of Escherichia phage, whereas other AGPs did not influence the abundance of bacteriophage significantly. Considering the evidence that AGPs used in poultry production can select for resistance to more than one class of antimicrobial resistance, and the fact that their effect on performance is not significant, their use needs to be reduced and there is a need to monitor the spread of ARGs in broiler chicken farms.

Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4+ CTL.

Human cytomegalovirus (HCMV) infects endothelial, epithelial, and glial cells in vivo. These cells can express MHC class II proteins, but are unlikely to play important roles in priming host immunity. Instead, it seems that class II presentation of endogenous HCMV antigens in these cells allows recognition of virus infection. We characterized class II presentation of HCMV glycoprotein B (gB), a membrane protein that accumulates extensively in endosomes during virus assembly. Human CD4+ T cells specific for gB were both highly abundant in blood and cytolytic in vivo. gB-specific CD4+ T cell clones recognized gB that was expressed in glial, endothelial, and epithelial cells, but not exogenous gB that was fed to these cells. Glial cells efficiently presented extremely low levels of endogenous gB--expressed by adenovirus vectors or after HCMV infection--and stimulated CD4+ T cells better than DCs that were incubated with exogenous gB. Presentation of endogenous gB required sorting of gB to endosomal compartments and processing by acidic proteases. Although presentation of cellular proteins that traffic into endosomes is well known, our observations demonstrate for the first time that a viral protein sorted to endosomes is presented exceptionally well, and can promote CD4+ T cell recognition and killing of biologically important host cells.

Complete genome sequencing and assessment of mutation-associated protein dynamics of the first Indian bovine ephemeral fever virus (BEFV) isolate.

BACKGROUND: Bovine ephemeral fever (BEF) is a re-emerging disease caused by bovine ephemeral fever virus (BEFV). Although it poses a huge economic threat to the livestock sector, complete viral genome information from any South Asian country, including India, lacks. AIM: Genome characterization of the first Indian BEFV isolate and to evaluate its genetic diversity by characterizing genomic mutations and their associated protein dynamics. MATERIALS AND METHODS: Of the nineteen positive blood samples collected from BEF symptomatic animals during the 2018-19 outbreaks in India, one random sample was used to amplify the entire viral genome by RT-PCR. Utilizing Sanger sequencing and NGS technology, a complete genome was determined. Genome characterization, genetic diversity and phylogenetic analyses were explored by comparing the results with available global isolates. Additionally, unique genomic mutations within the Indian isolate were investigated, followed by in-silico assessment of non-synonymous (NS) mutations impacts on corresponding proteins' secondary structure, solvent accessibility and dynamics. RESULTS: The complete genome of Indian BEFV has 14,903 nucleotides with 33% GC with considerable genetic diversity. Its sequence comparison and phylogenetic analysis revealed a close relatedness to the Middle Eastern lineage. Genome-wide scanning elucidated 30 unique mutations, including 10 NS mutations in the P, L and GNS proteins. The mutational impact evaluation confirmed alterations in protein structure and dynamics, with minimal effect on solvent accessibility. Additionally, alteration in the interatomic interactions was compared against the wild type. CONCLUSION: These findings extend our understanding of the BEFV epidemiological and pathogenic potential, aiding in developing better therapeutic and preventive interventions.

Antibody Therapy: From Diphtheria to Cancer, COVID-19, and Beyond.

The dawn of the 20th century saw the formative years of developments in immunology. In particular, immunochemistry, specifically pertaining to antibodies, was extensively studied. These studies laid the foundations for employing antibodies in a variety of ways. Not surprisingly, antibodies have been used for applications ranging from biomedical research to disease diagnostics and therapeutics to evaluation of immune responses during natural infection and those elicited by vaccines. Despite recent advancements in cellular immunology and the excitement of T cell therapy, use of antibodies represents a large proportion of immunotherapeutic approaches as well as clinical interventions. Polyclonal antibodies in the form of plasma or sera continue to be used to treat a number of diseases, including autoimmune disorders, cancers, and infectious diseases. Historically, antisera to toxins have been the longest serving biotherapeutics. In addition, intravenous immunoglobulins (IVIg) have been extensively used to treat not only immunodeficiency conditions but also autoimmune disorders. Beyond the simplistic suppositions of their action, the IVIg have also unraveled the immune regulatory and homeostatic ramifications of their use. The advent of monoclonal antibodies (MAbs), on the other hand, has provided a clear pathway for their development as drug molecules. MAbs have found a clear place in the treatment of cancers and extending lives and have been used in a variety of other conditions. In this review, we capture the important developments in the therapeutic applications of antibodies to alleviate disease, with a focus on some of the recent developments.

Randomly amplified polymorphic DNA analysis of Staphylococcus chromogenes isolated from bovine and bubaline mastitis in Karnataka.

BACKGROUND AND AIM: In recent times, non-aureus staphylococci (NAS) have emerged as the major organisms isolated from mastitis cases in dairy animals, with a predominance of Staphylococcus epidermidis and Staphylococcus chromogenes. As compared to Staphylococcus aureus, much less is known about the molecular types or the spatiotemporal epidemiology of these NAS species. In the present study, randomly amplified polymorphic DNA (RAPD) was employed to detect genetic polymorphisms, intraspecies diversity, and epidemiology of S. chromogenes strains (n=37) isolated from bovine and bubaline mastitis cases in the state of Karnataka. MATERIALS AND METHODS: Thirty-seven S. chromogenes isolates (14 from bovines and 23 from bubaline) isolated from subclinical mastitis cases, from organized and unorganized sectors, were subjected to RAPD typing. Further, methicillin resistance was determined by cefoxitin disk diffusion method. RESULTS: The amplified DNA fragments ranged from 150 to 3000 base pairs and yielded several RAPD profiles. Further analysis using Digital Image Correlation Engine correlation coefficient and UPGMA method showed that the 37 isolates could be classified into 12 distinct RAPD types (A to L) at 62% similarity (D=0.889). Four of the most predominant RAPD types, B, A, C, and E, in that order, and together, represented 65% of the isolates. High diversity was observed among the isolates both within farms and between geographic locations. Most of the isolates exhibited methicillin resistance. This is the first such report from India. CONCLUSION: In the absence of defined multilocus sequence type protocols or sufficient sequences available in the public domain, RAPD can be employed to determine genetic diversity of S. chromogenes isolates.

Molecular fingerprinting of bovine mastitis-associated Staphylococcus aureus isolates from India.

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. Owing to the mostly backyard dairy practices, we hypothesized that genetic diversity among mastitis-associated S. aureus from India would be high, and investigated 166 isolates obtained mostly from the Southern State of Karnataka, but also from a few other states. The results revealed (a) 8 to 13 fragments in pulsed-field gel electrophoresis (PFGE), forming 31 distinct patterns, and (b) 34 spa types, of which three (t17680, t18314, and t18320) were newly identified. Multi-locus sequencing typing (MLST) identified 39 sequence types (STs), with ST2454 (34.4%) and ST2459 (24%) being the most commonly represented, which clustered to clonal complexes (CC) CC9 and CC97, respectively; 12 STs were newly identified. Thirty-four (20.5%) of the 166 isolates displayed oxacillin resistance. On the other hand, whereas none were mecC+, 44 (26.5%) isolates were mecA+, with a predominance of SCCmecIVb (26/32 isolates, others being untypeable); 24 isolates (14.46%) were oxacillin-susceptible methicillin-resistant S. aureus (OS-MRSA; mecA+ but OS). Integrated analysis revealed that CC9-ST2454- and CC97-ST2459-SCCmecIVb were the predominant MRSA, although the distribution of CC9 and CC97 was similar between methicillin-resistant and -susceptible isolates. By PCR, 56.25%, 28.75% and 47.5% of the 166 isolates were positive for hlg, tsst and pvl genes, respectively. Our results, for the first time describe the application of a combination of various molecular methods to bovine mastitis-associated S. aureus isolates from India, corroborate the worldwide distribution of CC97 and CC9, and suggest pathogenic potential of the isolates.

Adenovirus vectors expressing hantavirus proteins protect hamsters against lethal challenge with andes virus.

Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdG(N) and AdG(C)) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infection that mimics the pulmonary disease seen in humans. When administered once, all three Ad vectors, individually or in combination, elicited a robust immune response that protected hamsters. No vaccinated animal died, and there were no obvious clinical signs of disease. Further, hantavirus RNA was not detected by sensitive reverse transcription-PCR in tissues and blood of hamsters immunized with both AdG(N) and AdG(C). Cellular immunity appeared to be important for protection because the AdN vector completely protected animals. All three Ad vectors produced strong cytotoxic T-lymphocyte responses directed to hantavirus proteins in mice. Moreover, hamsters vaccinated with AdN, AdG(N), or AdG(C) produced no detectable neutralizing antibodies yet were protected. These Ad vectors represent the first vaccines that prevent lethal hantavirus disease and, in some instances (AdG(N) and AdG(C)), provide sterile immunity. These observations set the stage for a more detailed characterization of the types of immunity required to protect humans from hantavirus infections.

Molecular characterisation of methicillin-resistant Staphylococcus aureus isolated from patients at a tertiary care hospital in Hyderabad, South India.

Context: Infections with methicillin-resistant Staphylococcus aureus (MRSA) greatly influence clinical outcome. Molecular characterisation of MRSA can help to predict their spread and to institute treatment and hospital protocols. Aim: The aim of this study is to understand the diversity of MRSA in a tertiary care hospital in Hyderabad, India. Settings and Design: Samples collected at Gandhi Medical College, Hyderabad, and designed to assess hospital-or community-associated MRSA (HA-MRSA or CA-MRSA). Subjects and Methods: MRSA were subjected to antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and staphylococcal cassette chromosome-mec (SCCmec) typing. Statistical Analysis Used: Discriminatory index and 95% confidence interval. Results: Of the 30 MRSA, (a) 18 and 12 were HA-MRSA and CA-MRSA, respectively, and (b) 23.3% and 6.6% displayed induced clindamycin and intermediate vancomycin resistance, respectively. Genetic diversity was evident from the presence of (a) 20 pulsotypes, (b) eight spa types, with the predominance of t064 (n = 9) and (c) seven sequence types (ST), with the preponderance of ST22 and ST8 (9 each). ST22 and ST8 were the most prevalent among HA-MRSA and CA-MRSA, respectively. SCCmec type IV was the most frequent (n = 8). 44.4% of HA-MRSA belonged to SCCmec IV and V, whereas 33.3% of CA-MRSA belonged to SCCmec I and III; 33.3% (5/15) of the isolates harbouring the pvl gene belonged to SCCmec IVC/H. Conclusions: ST8 was a dominant type along with other previously reported types ST22, ST239, and ST772 from India. The observations highlight the prevalence of genetically diverse clonal populations of MRSA, suggesting potential multiple origins.

Contrasting selective patterns across the segmented genome of bluetongue virus in a global reassortment hotspot.

For segmented viruses, rapid genomic and phenotypic changes can occur through the process of reassortment, whereby co-infecting strains exchange entire segments creating novel progeny virus genotypes. However, for many viruses with segmented genomes, this process and its effect on transmission dynamics remain poorly understood. Here, we assessed the consequences of reassortment for selection on viral diversity through time using bluetongue virus (BTV), a segmented arbovirus that is the causative agent of a major disease of ruminants. We analysed ninety-two BTV genomes isolated across four decades from India, where BTV diversity, and thus opportunities for reassortment, are among the highest in the world. Our results point to frequent reassortment and segment turnover, some of which appear to be driven by selective sweeps and serial hitchhiking. Particularly, we found evidence for a recent selective sweep affecting segment 5 and its encoded NS1 protein that has allowed a single variant to essentially invade the full range of BTV genomic backgrounds and serotypes currently circulating in India. In contrast, diversifying selection was found to play an important role in maintaining genetic diversity in genes encoding outer surface proteins involved in virus interactions (VP2 and VP5, encoded by segments 2 and 6, respectively). Our results support the role of reassortment in driving rapid phenotypic change in segmented viruses and generate testable hypotheses for in vitro experiments aiming at understanding the specific mechanisms underlying differences in fitness and selection across viral genomes.