Extended-spectrum beta-lactamase producing Escherichia coli are one of the most challenging causative agents of bovine mastitis. The emergence of multidrug resistance in ESBL-producing E. coli impedes antibiotic treatment and demands the need for alternative approaches such as phage therapy. In this study, a novel Escherichia phage belonging to Myoviridae family was isolated against multidrug resistant ESBL-producing E. coli. A total of 20 ESBL-producing E. coli were isolated from 84 clinical bovine mastitis milk samples. The 85% of the isolates were phenotypically confirmed as multidrug resistant and 45% of them were carrying antibiotic resistance genes against three or more antibiotics. Phage 92CQ was isolated against multidrug resistant ESBL-producing E. coli 92CQ. The isolated lytic Phage 92CQ displayed a broad host range, large burst size of 150 pfu/cell and short latent period of 30 min. Phage 92CQ was stable in the temperature range of 4°C-50 °C and showed effective lytic activity in the pH range of 5-11. Whole genome sequence analysis identified Phage 92CQ of 129812 bp, with 250 coding sequences, and 16 tRNAs. The lysis spanin complex composed of o-spanin gene embedded within i-spanin was identified in Phage 92CQ. The phylogenetic analysis showed a discrete categorisation of Phage 92CQ as a novel species within genus Justusliebigvirus. These results indicated the absence of antibiotic resistance, virulence and toxic genes in isolated phage with an effective reduction of multidrug resistant E. coli. The study presents Phage 92CQ as a potential candidate for development as a therapeutic agent with additional in-vivo and ex-vivo studies.

CRISPR-Cas9 genome editing offers significant opportunities to improve livestock traits; however, its application in buffalo has been very limited, with no prior reports of live gene-edited animals. Here, we report the successful birth of a buffalo edited in the myostatin (MSTN) gene. To achieve this, five single-guide RNAs (sgRNAs) targeting the buffalo MSTN gene were designed and tested in skin-derived fibroblasts. Among these, sgRNA5 exhibited the highest editing efficiency, approaching ∼50%, as confirmed by T7 Endonuclease I assay, Tracking of Indels by Decomposition, and Inference of CRISPR Edits analyses. Single-cell cloning identified six edited fibroblast clonal populations, including one with a bi-allelic frameshift mutation predicted to severely truncate the MSTN protein. These bi-allelic clonal cells were subsequently used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos, which were transferred into recipient buffaloes (n = 15). This effort established three pregnancies and resulted in the birth of one live MSTN knockout buffalo calf. Phenotypically, the calf displayed accelerated growth and increased muscle fiber number and size while maintaining normal meat composition. In conclusion, this study reports the world's first gene-edited buffalo generated through CRISPR-Cas9-mediated genome editing combined with SCNT. These findings provide a proof-of-concept for genome editing in buffalo and demonstrate that MSTN disruption can effectively enhance muscle growth and meat production traits.

The continuous evolution of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to the misuse of antibiotics lays out the need for the development of new antimicrobials with higher activity and lower resistance. In this study, we have expressed novel chimeric endolysin CHAPk-SH3bk derived from LysK to investigate its antibacterial activity against planktonic and biofilm-forming MRSA. The molecular docking and MD simulation results identified critical amino acids (ASP47, ASP56, ARG71, and Gly74) of CHAPk domain responsible for its catalytic activity. Chimeric endolysin CHAPk-SH3bk showed an effective binding to peptidoglycan fragment using 14 hydrogen bonds. The in-vitro antibacterial assays displayed higher activity of CHAPk against planktonic MRSA with 2-log10 reduction in 2 h. Both CHAPk and CHAPk-SH3bk displayed bactericidal activity against MRSA with ∼4log10 and ∼3.5log10 reduction in 24 h. Biofilm reduction activity displayed CHAPk-SH3bk reduced 33 % and 60 % of hospital-associated ATCC®BAA-44™ and bovine origin SA1 respectively. The CHAPk treatment reduced 47 % of the preformed biofilm formed by bovine-origin MRSA SA1. This study indicates an effective reduction of preformed MRSA biofilms of human and animal origin using novel chimeric construct CHAPk-SH3bk. Stating that the combination and shuffling of different domains of phage endolysin potentially increase its bacteriolytic effectiveness against MRSA.

Interferon lambda (IFN-λ) is an important type III interferon triggered mainly by viral infection. IFN-λ binds to their heterodimeric receptors and signals through JAK-STAT pathways similar to type I IFN. In this study, we deduced the buffalo IFN-λ sequences through the polymerase chain reaction, and then studied IFN-λ’s expression patterns in different tissues, and post induction with poly I:C and live MRSA using RT-qPCR. The full-length sequences of buffalo IFN-λ3, IFN-λ receptors, and a transcript variant of IFN-λ4 were determined. IFN-λ1 is identified as a pseudogene. Virus response elements and a recombination hotspot factor was observed in the regulatory region of IFN-λ. The IFN-λ3 expressed highest in lungs and monocytes but IFN-λ4 did not. The expression of Interferon Lambda Receptor 1 was tissue specific, while Interleukin 10 Receptor subunit beta was ubiquitous. Following poly I:C induction, IFN-λ3 expression was primarily observed in epithelial cells as opposed to fibroblasts, displaying cell type-dependent expression. The cytosolic RNA sensors were expressed highest in endometrial epithelial cells, whereas the endosomal receptor was higher in fibroblasts. 2’,5’-oligoadenylate synthetase expressed higher in fibroblasts, myxoma resistance protein 1 and IFN-stimulated gene 56 in epithelial cells, displaying cell-specific antiviral response of the interferon stimulated genes (ISGs). The endometrial epithelial cells expressed IFN-λ3 after live S. aureus infection indicating its importance in bacterial infection. The induction of IFN-λ3 was S. aureus isolate specific at the same multiplicity of infection (MOI). This study elucidates the IFN-λ sequences, diverse expression patterns revealing tissue specificity, and specificity in response to poly I:C and bacterial stimuli, emphasising its crucial role in innate immune response modulation.

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (−/−) heterozygous, bi-allelic (−/−) homozygous, and mono-allelic (−/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (−/−) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

Type III interferon (IFN), also known as IFN-λ, is an innate antiviral protein. We retrieved the sequences of IFN-λ and their receptors from 42 tetrapod species and conducted a computational evolutionary analysis to understand the diversity of these genes. The copy number variation (CNV) of IFN-λ was determined through qPCR in Indian cattle and buffalo. The tetrapod species feature intron-containing type III IFN genes. Some reptiles and placental mammals have 2 IFN-λ loci, while marsupials, monotremes, and birds have a single IFN-λ locus. Some placental mammals and amphibians exhibit multiple IFN-λ genes, including both intron-less and intron-containing forms. Placental mammals typically possess 3-4 functional IFN-λ genes, some of them lack signal peptides. IFN-λ of these tetrapod species formed 3 major clades. Mammalian IFN-λ4 appears as an ancestral form, with syntenic conservation in most mammalian species. The intron-less IFN-λ1 and both type III IFN receptors have conserved synteny in tetrapod. Purifying selection was noted in their evolutionary analysis that plays a crucial role in minimizing genetic diversity and maintaining the integrity of biological function. This indicates that these proteins have successfully retained their biological function and indispensability, even in the presence of the type I IFNs. The expansion of IFN-λ genes in amphibians and camels have led to the evolution of multiple IFN-λ. The CNV can arise from gene duplication and conversion events. The qPCR-based absolute quantification revealed that IFN-λ3 and IFN-λ4 have more than 1 copy in buffalo (Murrah) and 6 cattle breeds (Sahiwal, Tharparkar, Kankrej, Red Sindhi, Jersey, and Holstein Friesian). Overall, these findings highlight the evolutionary diversity and functional significance of IFN-λ in tetrapod species.

The global rise of antimicrobial resistance (AMR) poses a significant threat to human health. The environment plays an essential role in transmission of antimicrobial resistance genes (ARGs) between human and animal. Bacterial communities harbour diverse ARGs, carried by mobile genetic elements (MGEs) like plasmids and insertion sequences (ISs). Here, a total of 2199 Escherichia coli (E. coli) whole genome sequences from human, animal, bird and environment were retrieved globally to investigate ARG prevalence and assess their genetic framework. The study highlights how the genetic background including plasmids, IS elements and transposons surrounding ARGs influences their transmission potential. The maximum number of ARGs was found in United Kingdom followed by USA, majorly in human hosts. However, IS-associated ARGs were most prevalent in bird hosts. ARGs like aph(6)-ld, aph(3″)-lb, blaCTX-M, blaNDM were widespread across all hosts. Tn2 was the most prevalent, majorly carried by IncFIB plasmids. The IS26 and ISVsa3 carried diverse ARGs, primarily linked to aminoglycoside and β-lactam resistance. The combinations like mph(A)_IS6100 and blaNDM-5_IS5 showed fixed IS-ARG associations. ARGs like blaNDM, blaCTX-M variants displayed strong association with IS elements. The study highlights possible mechanism of transmission due to close proximity of AMR genes to MGEs, offering promising strategies to combat AMR by predicting and addressing future resistance determinants.

Aquaporins (AQPs) are essential membrane proteins facilitating water and small solute transport across cell membranes. Mammals have approximately 13 paralogs of AQPs that may have evolved through gene duplication events. These genes are present in two separate clusters within the genome. In the present study, comprehensive 13 AQP genes (AQP0-12) were cloned and characterized in buffalo and goat. The protein coding region of AQPs in both species ranged from 729 to 990 bps, corresponding to 263-330 amino acid residues. Two important residues including NPA motifs and ar/R selectivity filter were found conserved in all AQPs, except for AQP7, 11 and 12. AQP0, 2, 4, 5, 7, 9, 12 showed tissue-restricted expression, whereas AQP1, 3, 8, and 11 exhibited ubiquitous expression across several tissues. AQP10 was identified as a pseudogene in all artiodactyls. Transcript variants were identified in buffalo and goat, where some variants of goat AQP5 and 6 lacked important motifs. Evolutionary analysis indicated positive selection at or near the NPA motifs and ar/R selectivity filter of AQP0, 3, 6, 7, and 10 that may alter its structure and function. This study is crucial for future investigations aiming to study the molecular mechanisms of AQPs in response to various physiological conditions.

Antimicrobial resistance (AMR) poses a serious threat to human, animal, and plant health on a global scale. Search and elimination techniques should be used to effectively counter the spread of methicillin-resistant Staphylococcus aureus (MRSA) infections. With only a few novel drugs in clinical development, the quest for plant-based alternatives to prevent the spread of antibiotic resistance among bacteria has accelerated. Treatment of MRSA infections is challenging owing to rapidly emerging resistance mechanisms coupled with their protective biofilms. In the present research, we examined the antibacterial properties of ten plant-derived ethanolic leaf extracts. The most effective ethanolic leaf extract against MRSA in decreasing order of zone of inhibition, Cannabis sativa L. > Syzygium cumini > Murraya koenigii > Eucalyptus sp. > while Aloe barbadensis, Azadirachta indica, had very little impact. Mangifera indica, Curcuma longa, Tinospora cordifolia, and Carica papaya did not exhibit inhibitory effects against MRSA; hence, Cannabis was selected for further experimental study. The minimal inhibitory concentration (MIC) of Cannabis sativa L. extract was 0.25 mg ml−1 with 86

Genome editing technology has great potential for precise DNA modification in mammalian cells. The ability to precisely generate the clonal population of CRISPR-edited geno-type is of great importance in gene function/ pathway analysis, drug discovery, and production of genome-edited animals. In the present study, we demonstrated an efficient method to generate CRISPR-edited single-cell clonal populations of farm animals, including buffalo, goats, and sheep. To generate clonal cell populations, the primary fibroblasts were established through explant culture and then electroporated with CRISPR/Cas RNPs targeted for the disrupted MSTN gene. We used a single-cell pickup method in which one cell was picked up using an ultra-fined glass capillary and transferred into each well of a 96-well plate. For promoting the growth of single cells, we used growth factor-supplemented media. After seeding a single cell to each well, the plate was kept undisturbed for 5-7 days, and then cell attachment rates were noted. We reported that the cell attachment rates for buffalo, goat, and sheep cells were 40%, 77.08%, and 83.67%, respectively. The proliferation rates were 70.83%, 75.67%, and 78.05% for buffalo, goat, and sheep cells, respectively. We noticed that cell attachment and proliferation rates were better in the case of goat and sheep cells; also, these cells exhibited less vacuolation compared to buffalo cells. In the present study, we generated 11, 20, and 20 single-cell clones of MSTN-gene-edited buffalo, goat, and sheep cells. In conclusion, our method can be efficiently used to generate genome-edited single-cell clones to harness the potential of CRISPR technologies in farm animals.

In several tropical countries, buffalo milk has a higher-value demand than cow milk due to its nutritional and economic value. In India, the buffalo is the main dairy animal and contributes 45% of the total milk produced in the country. Besides the nutritional value of milk, several allergen proteins such as casein, α-lactalbumin, β- lactoglobulin (BLG), and immunoglobulins have been reported. Breeding strategies, nutritional management, and quantitative genetics have improved milk yield, but these approaches could not lead to significant changes in milk composition. With the development of biotechnology, especially genome editing tools (CRISPRs), it is possible to generate new value-added products such as designer hypoallergenic milk for human health benefits. Keeping this in mind, we planned to utilize the CRISPR tools to disrupt the buffalo β-lactoglobulin (BLG) gene to produce hypoallergenic milk in the long run. In pursuit of our objectives, we designed three single guide RNAs (sgRNAs) targeting the BLG locus in buffalo. Subsequently, we assessed their editing efficiency through a combination of Sanger sequencing, followed by TIDE and ICE analysis. Among three sgRNAs, the most efficient sgRNA was used to generate the clonal population of edited cells. Several single-cell clones were established and screened using the TA cloning (also known as rapid cloning or T cloning) and Sanger sequencing methods. Of 14 single-cell clones screened, eight were found to have BLG gene disruption events (57% editing rates). Using SCNT, we successfully produced cloned blastocyst stage embryos from 4 BLG-gene disrupted clonal cells. The cloned blastocyst production rates (25 to 30%) were similar to non-edited control cells. Efforts are ongoing to establish pregnancies from BLG-KO cloned embryos. This work can lead to the generation of designer buffaloes to produce hypoallergenic milk for human benefit.

The transfer of genome-modification components into farm animal cells is indispensable for the production of genome-modified and transgenic farm animals. Electroporation is a physical transfection method when appropriately used; this technique is safe, simple to use, affordable, and efficient in transfecting cells from several lineages. Electroporation efficiency depends on various physical parameters, of which cell type is considered a major factor for transfection efficiency. Primary cells are generally less susceptible to transfection than other cell types due to their finite lifespan and limited expansion capacity. Previously, we custom-designed a transfection buffer to deliver exogenous genetic components into mammalian cells. In the present study, we examined the effect of the developed buffer on transfection rates and cell viability of primary somatic cells from buffalo, cattle, goats, and sheep. To achieve the aims of this study, t primary somatic cells from skin biopsies were established and were transfected with a Venus-expression vector (pAcGFPs-Venus). We noticed that transfection rates of pAcGFPs-Venus were 22.51%, 17.56%, 22.81%, and 16.16% for buffalo, cattle, goats, and sheep cells, respectively. We also noticed that cell viability and proliferation rates were better in the case of goats, sheep, and cattle cells; also, these cells have less vacuolation than buffalo cells. In addition, we also generated MSTN (myostatin) KO (Knockout) cell clones from these cell populations, in which the efficiency of single-cell clone generation was high for goats and sheep cells. In conclusion, our lab-made transfection buffer can be efficiently used to generate genome-edited or transgenic farm animals for agriculture, biomedical, and veterinary applications.

Aquaporins (AQPs) are integral membrane proteins responsible for water transport across cellular membranes in both prokaryotes and eukaryotes. A subfamily of AQPs, known as aquaglyceroporins (AQGPs), facilitate the transport of small solutes such as glycerol, water, and other solutes across cellular membranes. These proteins are involved in a variety of physiological processes, such as organogenesis, wound healing, and hydration. Although AQPs have been studied extensively in different species, their conservation patterns, phylogenetic relationships, and evolution in mammals remain unexplored. In the present study, 119 AQGP coding sequences from 31 mammalian species were analysed to identify conserved residues, gene organisation, and most importantly, the nature of AQGP gene selection. Repertoire analysis revealed the absence of AQP7, 9, and 10 genes in certain species of Primates, Rodentia, and Diprotodontia, although not all three genes were absent in a single species. Two Asparagine-Proline-Alanine (NPA) motifs located at the N- and C-terminal ends, aspartic acid (D) residues, and the ar/R region were conserved in AQP3, 9, and 10. Six exons encoding the functional MIP domain of AQGP genes were found to be conserved across mammalian species. Evolutionary analysis indicated signatures of positive selection in AQP7, 9, and 10 amongst different mammalian lineages. Furthermore, substitutions of certain amino acids located close to critical residues may alter AQGP functionality, which is crucial for substrate selectivity, pore formation, and transport efficiency required for the maintenance of homeostasis in different mammalian species.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that poses a significant threat in cases of chronic mastitis in dairy animals. The ability of MRSA to persist in the host is attributed to various virulence factors, genes encoding surface adhesins, and determinants of antibiotic resistance, which provide it a survival advantage. This investigation focused to determine the virulence factors, antimicrobial resistance (AMR) profile and biofilm production potential of 46 MRSA isolates from 300 bovine mastitis milk samples. The AMR profile revealed a high level of resistance, with 46 and 42 isolates resistant to cefoxitin and oxacillin, respectively, followed by 24 and 12 isolates resistant to lomefloxacin and erythromycin, respectively. Only 2 isolates resistant to tetracycline and none were resistant to chloramphenicol. The study also evaluated various virulence factors such as coa (n = 46), nuc (n = 35) hlg (n = 36), pvl (n = 14), tsst-1(n = 28) spa (n = 39) and enterotoxin genes sea (n = 12) and seg (n = 28) and identified antibiotic resistance determinants mecA and blaZ in 46 and 27 isolates, respectively. Intercellular adhesion genes icaA and icaD were present in 40 and 43 isolates, respectively and surface adhesion genes ebps, fnbpA, eno, sasG, cna, and bap were found in 43, 40, 38, 26, 21 and 1 isolates, respectively. Microtiter plate (MTP) assay revealed that 29 MRSA isolates were capable of producing biofilms, whereas 17 were not. Biofilms producing MRSA isolates possessed adhesion genes, virulence factors, toxin genes and AMR genes that may act synergistically towards a chronic disease progression, illness and severe damage to the udder, which generally last for several months and very challenging to cure.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.

Bovine mastitis is one of the most expensive diseases that affect cattle. One of the most common Enterobacteriaceae that causes clinical mastitis is Escherichia coli . This study aimed to investigate the prevalence of ESBL-producing E. coli in clinical mastitis, determine the most common phylogenetic E. coli group associated with it, and assess the antibiotic resistance pattern in E. coli isolates. From 550 clinical bovine mastitis milk samples, 112 ESBL-producing E. coli isolates were obtained, with the majority of E. coli isolates belonging to phylogroup A (33%). More than 70% of isolates were resistant to multiple antibiotics, including cefotaxime, ciprofloxacin, streptomycin, and Co-Trimoxazole. In addition, ST131-associated virulence factor fimH30 was found in 73.21% of isolates. The IncF plasmid replicon types were found in 83% of ESBL-producing E. coli isolates. The IncFIB was identified as the most common replicon plasmid associated with ST131 phylogenetic clade C2. The significant prevalence of multidrug resistant and ESBL-producing E. coli in bovine mastitis is revealed in this study, making treatment of affected animals difficult. This prevalence of multidrug-resistant E. coli in food animals poses a serious threat that requires thorough investigation and therapy.

Mobile genetic elements (MGEs) are associated with the emergence of multidrug resistance in extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. This study explores the role of class 1 integrons and IS26 elements in breaching taxonomic barriers. A total of 110 E. coli bacteria were isolated from 300 clinical mastitis milk samples. The 98% E. coli isolates were extended-spectrum beta-lactamase- producers. About 83% of these isolates carried co-resistance for fluoroquinolones. The co-existence of (extended-spectrum beta-lactamase + quinolone resistance determining region mutations) and (extended-spectrum beta-lactamase + plasmid-mediated quinolone resistance genes) was found in 76% and 44% of isolates, respectively. The MGEs were detected in 88% of isolates with IS26 in 82% and class 1 integrase in 40% of isolates. The types of class 1 integron gene cassettes detected includes dfrA7, (dfrA17 + aadA5), and (dfrA1 + aadA1). We discovered 2 and 4 novel variants of the dfrA17 and aadA5 genes, respectively. We report a variant of aadA5 with mutation E235G in the Indian subcontinent earlier reported only in a human clinical isolate from Belgium. About 19 isolates carried IS26 linked to integrase gene intI1 with an internal deletion of 265 bp in the 5`CS of integrase gene intI1, earlier reported only in E. coli ST131 isolates from human clinical, wastewater samples. This study suggests intercontinental dissemination of antibiotic resistant genes (ARGs) across different microbiomes via mobile genetic elements. • The role of mobile genetic elements in the emergence of multidrug-resistant E. coli in bovine mastitis. • Novel variants of the aadA5 (aminoglycoside adenyl transferase) and dfrA17 (dihydrofolate reductase) genes were identified in pathogenic E. coli isolated from bovine mastitis in class 1 integron gene cassette. • Sequence analysis of mobile genetic components revealed the physical connection between IS26 and intI1 genes with an internal deletion in 5'CS of class 1 integrase.

Bovine mastitis is a major infectious disease affecting dairy animals resulting in enormous economic losses, prolonged antibiotic treatment, reduced milk yield and death of livestock. Emergence of Methicillin-resistant Staphylococcus aureus (MRSA) among bovine mastitis is matter of concern for animal health and dairy industry. The present study was conducted to detect the distribution of virulence and enterotoxin genes among MRSA isolates from bovine mastitis. Out of 500 milk samples, 126 isolates were identified as Staphylococcus and from these only 56 were S. aureus. S.aureus were resistant to cefoxitin (75%), ceftazidime (75%), amoxicillin (71.4%), cefodaxime (67.8%), cefepime (66.1%), oxacillin (64.3%), norfloxacin (60.7%) and gentamicin (58.9%). Only 42 isolates were identified as MRSA strains among staphylococci isolates. MRSA were harbouring virulence genes; mecA (100%), coa (100%) and nuc (100%). The other virulence factors such as hlg (80.9%, 34/42), pvl (47.6%, 20/42) and spa (92.8%, 39/42) were also reported. Molecular characterisation of enterotoxin genes revealed that out of 42 tested isolates 11 were found negative (26%) for any enterotoxin gene whereas 7 (16.6%), 6 (14.3%), 18 (42.8%), 1 (2.3%), 26 (61.9%),27(64.2%),3 (7.1%) were found positive for sea, seb, sec, sed, seg, sei, and seq enterotoxin respectively.