Genome sequencing and comparative genomic analysis of bovine mastitis-associated non-aureus staphylococci and mammaliicocci (NASM) strains from India

Genome sequencing and comparative genomic analysis of bovine mastitis-associated non-aureus staphylococci and mammaliicocci (NASM) strains from India | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genome sequencing and comparative genomic analysis of bovine mastitis-associated non-aureus staphylococci and mammaliicocci (NASM) strains from India Vishnukumar Ramesh, Ramamoorthy Sivakumar, Madhavi Annamanedi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4508846/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Bovine mastitis is a significant issue causing severe economic losses in the global dairy industry, affecting animal well-being and production. Non- aureus staphylococci and mammaliicocci (NASM) are the predominant group of pathogens responsible for mastitis in dairy cattle. Understanding the prevalence of virulence factors and antimicrobial resistance among these pathogens is essential to unravel the molecular epidemiology of mastitis, and it is best accomplished through whole-genome sequencing (WGS). In this study, we describe the WGS and comparative genomic analysis of 22 mastitis-associated NASM strains isolated from India. The mean genome size of the strains was 2.55 Mbp, with an average GC content of 32.2%. We identified 14 different sequence types (STs) among the 22 NASM strains. Of these, ST1 and ST6 of S. chromogenes were exclusively associated with bovine mastitis. Genome-wide SNP-based minimum spanning tree revealed the intricate phylogenetic relationships among NASM strains from India, categorizing them into five major clades. Interestingly, mastitis-associated strains formed separate subclades in all the NASM species studied, indicating distinct host-specific co-evolution. The study identified 32 antimicrobial resistance (AMR) genes and 53 virulence-associated genes, providing insights into the genetic factors which could potentially contribute to the pathogenicity of NASM species. Some virulence and AMR genes were found in the predicted genomic islands, suggesting possible horizontal transfer events. Biological sciences/Immunology Biological sciences/Microbiology Bovine Mastitis Non-aureus Staphylococci and Mammaliicocci MLST Resistome Virulome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Bovine mastitis is an endemic disease affecting dairy cattle worldwide. It causes inflammation of the mammary gland, resulting in economic losses due to decreased milk production, veterinary care costs, and culling of infected animals (Halasa et al., 2007 ; Kovačević et al., 2022 ). Staphylococci are the most common infectious causative agents of bovine mastitis, and were historically divided into two groups. One group includes S. aureus , which is considered more pathogenic, and is a coagulase-positive staphylococcus (CoPS). The other group that is presumed to be less pathogenic, includes other staphylococcal species referred to as the coagulase-negative staphylococci (CoNS) or non-aureus staphylococci (NAS). Some coagulase-positive and coagulase-variable mastitis pathogens (e.g., S. pseudintermedius ) are also included in the CoNS category (De Buck et al., 2021 ). Recently, five Staphylococcus species, i.e., S. sciuri, S. fleurettii, S. lentus, S. stepanovicii , and S. vitulinus , were reclassified into the new genus, namely, Mammaliicoccous (Madhaiyan et al., 2020 ). Together, these organisms, referred to as non-aureus staphylococci and mammaliicocci (NASM), are the most prevalent (ranging from 9.1 to 16.6% of milk samples) agents isolated from intramammary infection (IMI) of dairy cows and the leading cause of subclinical mastitis. Recent studies have identified S. chromogenes , S. epidermidis , S. xylosus , S. vitulinus , S. simulans , and Mammaliicoccus sciuri as the leading causes of IMI in cattle. Among them, S. chromogenes, S. xylosus, and S. haemolyticus are more commonly found in milk samples (Ruiz-Romero & Vargas-Bello-Pérez, 2023 ). Although NASM has become a leading group of pathogens, knowledge of their virulence and antimicrobial resistance mechanisms is still limited. Biofilm formation has been identified as a crucial virulence factor for NASM, particularly in persistent IMI (Simojoki et al., 2012 ), and could potentially account for their heightened antibiotic resistance. The relatively increased capacity of NASM to form biofilm, as compared to other staphylococci, poses challenges in predicting antibiotic efficacy and the likelihood of persistence and recurrence of IMI. It is therefore crucial to understand the factors governing NASM resistance, persistent infections, and recurrent episodes, emphasizing the need for comprehensive research to enhance our knowledge of these intricate dynamics in the context of mastitis (Srednik et al., 2017 ). NASM encompasses a diverse group of species, each with varying pathogenic potentials. Therefore, it is essential to understand the role of individual species on udder health and milk production (Vanderhaeghen et al., 2014 ; De Buck et al., 2021 ). Various molecular subtyping techniques, including pulsed-field gel electrophoresis (PFGE) (Fry et al., 2014 ), random amplification of polymorphic DNA (RAPD) analysis (Petzer et al., 2022 ), multi-locus sequence typing (MLST), and multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) (Dahyot et al., 2018 ), have been used to investigate the molecular epidemiology of NASM. The whole genome sequence (WGS) analysis is expected to provide the best discriminatory power and better insights into the molecular epidemiology and the genetic determinants responsible for the pathogenicity of NASM. Comparative genomic analysis can identify phylogenetic relationships among various species of NASM and distinctions within and between species. This study focused on the comprehensive genomic analysis of 22 NASM strains associated with bovine mastitis isolated in India. These strains were fully sequenced, followed by a comparative analysis of their genetic diversity, virulence factors, antimicrobial resistance (AMR) genes, and sequence types. The study also included a perceptive phylogeny analysis of the previously documented NASM genomes from India, which helped understand the genetic differences between the NASM strains associated with or not associated with bovine mastitis . Methodology Whole genome sequencing and annotation Whole genomes of 22 strains of bovine mastitis-associated NASM were sequenced. These strains were collected between 2009 and 2019 from cows ( Bos taurus ) with mastitis, which included 18 strains from sub-clinical and four from clinical mastitis cases in India. The strains were isolated from three states; 18 from Karnataka, and two each from Gujarat and Meghalaya. The strains were curated at the National Institute of Animal Biotechnology in Hyderabad and the Department of Microbiology at Karnataka Veterinary, Animal & Fisheries Sciences University in Bengaluru. The DNeasy blood and tissue kit (Qiagen) was used to isolate genomic DNA from each strain, following the manufacturer's instructions. The resulting RNA-free DNA was then employed for library preparation and sequenced using the Illumina HiSeq platform at Macrogen, Seoul, South Korea. The process of assembly and analysis of the genome sequences involved multiple steps. First, the Trimmomatic tool was used to eliminate adapters and discard low-quality reads (Bolger et al., 2014 ). Adaptor-trimmed high-quality reads were assembled using SPAdes v 3.11.1 (Zakaria et al., 2020 ). Finally, the assembled genome sequences underwent annotation using the PROKKA (Seemann, 2014 ). Species identification was done using the rMLST (ribosomal multilocus sequence typing) tool available at the PubMLST webserver (Larsen et al., 2014 ). Multilocus sequence typing Multilocus sequence typing (MLST) using respective species-specific housekeeping genes was performed using the PubMLST web server ( https://pubmlst.org/ ) (Aanensen & Spratt, 2005 ). Allelic profiles were compared to the PubMLST database, and sequence types (STs) were identified. Average nucleotide identity (ANI) estimation The genome sequences were analyzed to determine their Average Nucleotide Identity (ANI) values using Jspecies ( https://jspecies.ribohost.com ), which included MUMmer ANIm, ANItetra, and BLAST + ANIb (Richter et al., 2016 ). Each genome was assigned a species name based on an ANI cutoff of > 95% (Colston et al., 2014 ). A heatmap was then generated using the Jspecies data on the Clustviz webserver (Metsalu & Vilo, 2015 ). Phylogeny analysis using genome-wide SNPs We used kSNP v.3 to predict genome-wide SNPs, identifying SNPs without requiring genome alignment (Gardner et al., 2015 ). The k-mer value of 17 was optimal for NASM strains by the KChooser tool of the kSNP v3. Genome-wide SNPs were identified and collected in a data matrix known as the 95% majority SNP matrix. Based on the genome-wide SNPs, a phylogenetic tree was constructed for 22 NASM strains using MEGA 11(Tamura et al., 2021 ). Additionally, a genome-wide SNP-based phylogenetic tree was constructed for 176 genomes of NASM strains reported from India, which includes 22 strains reported in this study, using Grapetree (Zhou et al., 2018 ). Identification of antimicrobial resistance (AMR) genes and virulence factors The resistance gene identifier (RGI) in the comprehensive antibiotic resistance database (CARD) was used to identify the AMR genes using default parameters (Jia et al., 2017 ). The VFanalyzer in the virulence factor database (VFDB) was used to identify the genes associated with virulence factors (VF) (Liu et al., 2019 ). Identification of prophages and genomic islands The presence of prophages in the genome was determined by PHASTER (PHAge Search Tool—Enhanced Release) ( https://phaster.ca/ ) (Naidoo & Zishiri, 2023 ). Based on the scores, prophages were classified into three groups, i.e., intact, questionable, and incomplete, with the corresponding scores > 90, 70–90, and < 70, respectively. Genomic islands (GIs) in each genome were predicted using Island Viewer 4 ( http://www.pathogenomics.sfu.ca/islandviewer/ ) (Bertelli et al., 2017 ). Genome availability NASM genome sequence used in this study has been deposited in NCBI with the following accession numbers: GCA_018986335.1, GCA_018996905.1, GCA_019334185.1, GCA_018997025.1, GCA_018997125.1, GCA_018967705.1, GCA_019149065.1, GCA_019149165.1, GCA_019149045.1, GCA_019193065.1, GCA_019148995.1, GCA_019334235.1, GCA_019429675.1, GCA_019149085.1, GCA_019149255.1, GCA_019149245.1, GCA_019149205.1, GCA_019334165.1, GCA_019100515.1, GCA_019165085.1, GCA_018967945.1, GCA_018968045.1. Results General features of genome sequences of bovine mastitis-associated NASM strains The genomes of 22 strains of NASM associated with bovine mastitis were sequenced using the Illumina platform. These strains belong to nine different species of Staphylococcus and Mammaliicoccus genera (Table 1 ). For each genome, a minimum of 100X mean sequence coverage was obtained. The reads were subjected to quality checks. Adapter removal, and the processed high-quality reads were used for de novo assembly. The draft genomes contained 24 to 150 contigs with a mean genome size of 2.55 Mbp and an average GC content of 32.2%. The rMLST method uses ribosome protein subunit ( rps ) gene sequences for precise taxonomic identification. Of the 22 NASM strains, 21 were identified by exact matches with 56 rps genes. S. chromogenes strain K17 was identified by exact matches with 55 rps genes. The summary of genome sequences is given in Table 2 . Table 1 NASM strains used in this study S. No. Name of the species Number of strains 1 Staphylococcus chromogenes 4 2 Staphylococcus epidermidis 4 3 Staphylococcus gallinarum 1 4 Staphylococcus haemolyticus 4 5 Staphylococcus hominis 1 6 Staphylococcus pseudintermedius 1 7 Staphylococcus xylosus 3 8 Mammaliicoccus lentus 1 9 Mammaliicoccus sciuri 3 Table 2 Summary of NASM genome sequences S.no. Strain Genome size (bp) No. of Contigs Sequence Type (ST) CDS rRNA tRNA Pseudogenes Accession No. 1 M. lentus K169 2848578 54 - 2783 3 29 13 GCA_018967945.1 2 M. sciuri K117.2 2702737 18 ST114 2672 2 14 15 GCA_018967705.1 3 M. sciuri K14 2739519 46 ST115 2723 2 14 18 GCA_019149065.1 4 M. sciuri K91 2715464 150 ST114 2735 6 28 26 GCA_019334185.1 5 S. chromogenes K17 2321223 26 ST1 2225 2 23 31 GCA_019149165.1 6 S. chromogenes K23 2349003 47 ST6 2247 5 42 43 GCA_019334235.1 7 S. chromogenes K26 2340405 55 ST1 2249 6 45 44 GCA_019429675.1 8 S. chromogenes K29 2329474 31 ST6 2231 2 13 35 GCA_019149085.1 9 S. epidermidis K16.1 2407306 76 ST1157 2200 2 9 58 GCA_019149045.1 10 S. epidermidis K3.2 2455169 142 ST1158 2281 5 48 60 GCA_019100515.1 11 S. epidermidis K4.3 2449159 83 ST924 2222 5 37 70 GCA_019165085.1 12 S. epidermidis K60 2460483 67 ST1158 2239 7 44 71 GCA_019149245.1 13 S. gallinarium B13 2935302 55 - 2735 4 40 28 GCA_018997025.1 14 S. haemolyticus A11 2485414 24 - 2336 4 42 78 GCA_018986335.1 15 S. haemolyticus A3.2 2497919 24 - 2355 4 29 80 GCA_018996905.1 16 S. haemolyticus K16.2 2459833 108 ST42 2318 5 33 77 GCA_019193065.1 17 S. haemolyticus K47 2435548 84 ST42 2300 2 19 64 GCA_019149205.1 18 S. hominis K24 2162928 27 ST79 2055 2 48 34 GCA_019149255.1 19 S. pseudintermedius B32 2517961 40 - 2329 4 35 42 GCA_018997125.1 20 S. xylosus K19 2781766 48 - 2575 2 16 20 GCA_019148995.1 21 S. xylosus K46 2801517 88 - 2588 5 46 40 GCA_019334165.1 22 S. xylosus SMG24 2802341 29 - 2585 4 39 34 GCA_018968045.1 Identification of sequence types based on MLST MLST schemes are available for five species of NASM, which were used in this study and can be accessed at the pubMLST server. The sequence types (STs) were determined based on the respective species-specific housekeeping genes (Table S1 ). M. sciuri strains were divided into ST114 (n = 2) and ST115 (n = 1). Similarly, S. chromogenes were represented by ST6 (n = 2) and ST1 (n = 2). Three different STs were identified among S. epidermidis : ST1158 (n = 2), ST1157 (n = 1), and ST924 (n = 1). ST42 (n = 2) was identified in S. haemolyticus ; two other strains representing potential new STs could not be typed. One strain of S. hominis belonged to ST79. Average nucleotide identity analysis The ANI analysis examined the interrelationship among NASM, where species-specific clustering of strains was observed, as shown in (Fig. 1 ). The ANI threshold of 96% was used to establish species delimitation within species-specific boundaries. For instance, S. chromogenes strain K17 was identical to the strains K23, K26, and K29, while S. epidermidis strain K16.1 matched the strains K4.3, K3.2, and K60. Similarly, M. sciuri strain K117.2 shared identity with strains K14 and K9, and S. xylosus strain K19 was similar to the strains K46 and SMG. M. lentus was closer to M. sciuri strains and formed a genus-specific cluster. S. gallinarum strain clustered together with S. xylosus strains. Likewise, S. pseudintermedius strain clustered with S. chromogenes , while S. haemolyticus clustered with S. hominis. Genome-wide SNP-based-phylogeny analysis of NASM We identified 192 core SNPs and 953968 non-core SNPs in 22 NASM strains. Based on the distribution of SNPs, a parsimony phylogenetic tree using MEGA 11 was constructed (Fig. 2 ). The NASM strains grouped into five species-specific clades. S. gallinarum and S. xylosus formed a clade (I) and were divided into species-specific subclades. S. hominis and S. haemolyticus formed a clade (II) and were divided into species-specific subclades. While S. epidermidis formed a separate clade (III). S. pseudintermedius and S. chromogenes formed a clade (IV), and both species were divided into separate subclades. Similarly, Mammaliicoccus formed a separate clade (V), while M. lentus and M. sciuri formed species-specific subclades. In summary, the genome-wide SNP-based phylogeny and ANIb-based phylogeny were similar. In order to further understand the inter-relationship between our strains and other NASM strains from India, a genome-wide SNP-based phylogeny analysis of 176 NASM strains (Table S2 ), including 22 strains from this study, was conducted. The NASM strains could be grouped into five species-specific clades (Fig. 3 A). Clade A consisted of S. epidermidis , which formed a major clade with 71 strains from India, divided into two subclades. Clade B was represented by S. haemolyticus and S. hominis and divided into separate subclades. Clade C comprised S. haemolyticus, S. gallinarum , and S. xylosus . Strains belonging to the species S. haemolyticus were represented in both clades B and C. Clade D was represented by S. chromogenes and S. pseudintermedius . Clade E was represented by the Mammaliicoccus species ( M. lentus and M. sciuri ). We examined the minimum spanning tree in relation to the source of isolation - whether it was from a cow or some other host. The results were interesting as all mastitis-associated NASM strains isolated from cows formed separate subclades or clusters in all clades (Fig. 3 B). On the other hand, NASM strains isolated from other sources, such as humans, plants, dogs, etc. clustered together. This suggests that bovine mastitis-associated strains might have host-specific divergence and evolution. Identification of antimicrobial resistance genes among mastitis-associated NASM strains The 22 NASM strains contained antimicrobial resistance (AMR) genes (AMR genes) against 12 classes of antibiotics (Fig. 4 ). The 22 strains carried 32 AMR genes, while the analysis of all the Indian isolates revealed 57 AMR genes. The number and classes of AMR genes varied significantly among the strains. All of our 22 strains carried the two AMR genes, sdrM and sepA . SdrM is an efflux pump that confers resistance to norfloxacin and ethidium bromide, while SepA is a multidrug efflux pump that confers resistance to disinfectants and antiseptics. The norC , which confers resistance to fluoroquinolone antibiotics, was present in all strains except for S. pseudintermedius strain B32, M. lentus , and M. sciuri species. The vanT , which codes for a membrane-bound serine racemase which confers resistance to vancomycin, was present in multiple strains, except for S. pseudintermedius , S. epidermidis , S. hominis , and S. gallinarum species. The dfrC , responsible for trimethoprim resistance, and the multidrug efflux pump coding gene norA , which confers resistance to fluoroquinolones as well as several antiseptics and disinfectants, were observed in all strains of S. epidermidis but absent in other species. The fusidic acid resistance gene ( fusE ) was detected in all strains of S. chromogenes but not in other species. The genes associated with resistance to penam drugs ( mecl and mecR1 ) were only found in S. epidermidis strain K4.3. The methicillin-resistant gene ( mecA ) was present in S. epidermidis strain K4.3, S. haemolyticus strain K16.2, and S. haemolyticus strain K47. The PC1 beta-lactamase ( blaZ ) gene was detected in all strains of S. haemolyticus , and S. epidermidis strains K3.2 and K4.3. The mgrA , which confers resistance to fluoroquinolone antibiotics, was present in S. haemolyticus strains A11 and A3.2, and all the strains of S. epidermidis . The salB , which confers resistance to lincosamide and class A streptogramins, was only found in M. lentus strain K169. On the other hand the homolog salC , was present in all strains of M. sciuri whereas salE was present in S. xylosus strains K19 and K46. The tetK gene, which is a tetracycline efflux protein, the qacJ gene, which confers resistance to quaternary ammonium compounds, and the SAT-4 gene, which provides resistance to nucleoside antibiotics, were only present in S. haemolyticus strains belonging to ST42. The fosBx 1, which confers resistance to phosphonic acid antibiotics, was found in S. xylosus strains K19 and K46, S. gallinarum strain B13, S. epidermidis strain K16.1, and M. sciuri strains and K117.2. The fexB coding for a phenol antibiotic efflux pump was only present in S. xylosus strain SMG24. The vanW gene, which confers resistance to vancomycin was present only in S. epidermidis strain K60. Among the functional homologs, vanY was present only in M. lentus strain K169, vanY was present in all strains of M. sciuri and vanY was present in S. haemolyticus strains belonging to ST42 and S. xylosus strain SMG24. When the analysis was extended to all 176 NASM isolates from India, 57 antibiotic-resistant genes were identified in all Indian isolates as against 32 genes for our 22 strains. A complete list of AMR genes from 176 NASM strains is shown in Table S3 . Identification of virulence factors among mastitis-associated NASM strains Fifty-two virulence factors were identified in the genomes of NASM strains associated with bovine mastitis. However, none of these genes were present in all the 22 strains. Instead, each strain had a unique set of virulence factors in various combinations. These virulence factors were functionally classified into eight classes, as shown in (Fig. 5 ). A. Adherence-related gene We identified 14 different genes associated with adherence among NASM strains. All three strains of S. xylosus and M. sciuri strain K14 were carried genes coding for intercellular adhesins, icaA , icaB , and icaC . However, the repressor of these genes, icaR , was present only in all S. xylosus strains and not in M. sciuri strain K14. The Staphylococcus protein A ( spa ) gene was only found in S. pseudintermedius strain B32. The autolysin ( atl ) gene was present in all S. epidermidis , S. haemolyticus , S. hominis strains, and S. xylosus strains K19 and K46. Clumping factor genes clfA and clfB were only found in M. lentus . The fibronectin-binding protein gene, fnbA , was present in three strains, namely S. xylosus strains SMG24, S. chromogenes strains, and K17. The gene coding for another fibronectin-binding protein, fnbB , was only present in S. pseudintermedius . The extracellular matrix-binding protein homolog, ebh , was found in all S. epidermidis strains. The gene coding for elastin binding protein, epb , was present in all strains of S. xylosus , S. hominis , S. haemolyticus , and S. epidermidis except strain K3.2. The Ser-Asp-rich fibrinogen-binding proteins coding genes, sdrC and sdrE , were found only in S. haemolyticus strains A11 and A3.2. B. Enzymes coding genes Five enzyme-coding genes associated with virulence were predicted among the 22 NASM genomes. Genes coding for lipases ( lip ) and thermonuclease ( nuc ) were present in all Staphylococcus strains but not Mammaliicoccus species. The lipase-coding gene geh was also found in all S. xylosus , S. pseudintermedius , S. epidermidis strains, and S. chromogenes strains K29 and K26. The gene coding for serine V8 protease, sspA , was present in strains belonging to S. epidermidis , S. xylosus , S. gallinarum , M. lentus , and M. sciuri , while sspB was found only in S. epidermidis strains. C. Immune evasion coding genes Twelve genes associated with immune evasion were identified among the 22 NASM genomes. Of these genes, two species, S. pseudintermedius and S. chromogenes , had gene coding for adenosine synthase A ( adsA ). Except for M. sciuri , M. lentus , and S. pseudintermedius , the capsule protein ( capB and capC ) was found in all genomes. The Bacillus polysaccharide capsule gene ( gtaB ) was present only in M. lentus , and the manA gene was found only in S. gallinarum . The lipopolysaccharide ( Francisella ) wbtP was present in M. lentus and M. gallinarum , while wbtE was found in M. sciuri strains. D. Toxin coding genes Among the 22 NASM strains analyzed, six genes that code for toxins were predicted. The cylR2 gene, which codes for cytolysin, was found in all the strains of S. xylosus , S. hominis , S. haemolyticus , and S. gallinarum . The hib gene coding for b-hemolysin was present in all the strains of S. chromogenes and S. epidermidis . The lukD and lukE genes, which code for leukotoxins D and E, respectively, were found only in the S. pseudintermedius strain B32. The set26 gene, which codes for an exotoxin, was found in all the strains of S. chromogenes . E. Secretion system-related genes The genes coding for the Type VII secretion system (T7SS), esaA, esaB, esaG, essA, essB, essC , and esxA were found only in S. epidermidis strain K4.3. F. Cell surface components Among the 22 NASM strains, three genes coding for cell surface components associated with virulence were identified. The trehalose-recycling ABC transporter ( sugC ) was found in S. pseudintermedius strain B32, M. sciuri strains K117.2 and K91. The lipoprotein diacylglyceryl transferase ( lgt ) was found in all the strains of S. pesudintermedius , M. sciuri , M. lentus , and S. chromogenes . The lipoprotein-specific signal peptidase gene ( lspA ) was found only in S. pseudintermedius strain B32. G. Metal uptake coding gene The gene vctC , responsible for iron uptake, was present in all the strains of S. xylosus . In contrast, the gene ctpV , responsible for copper uptake and export, was only found in S. epidermidis strain K60. H. Intracellular Survival The gene nucleoside diphosphate kinase ( ndk ), responsible for phagosome arrest, was found in all the strains of Mammaliicoccus . The regulational acid resistance gene ( lisR ) was found in all the strains of Mammaliicoccus , S. chromogenes , and S. pseudintermedius . The intracellular survival gene lipoate protein ligase, lplA1 , was found in only M. lentus strain K169. Prophage sequence-associated with NASM We identified nine intact prophage sequences among our NASM strains. All the prophage sequences contain all the genes required for a phage life cycle, as shown in (Fig. 6 ). Two prophage sequences, Staphylococcus phage vB Saus IMEP5 and uncultured caudovirales phage clone 10S.1, were found in S. gallinarum . Staphylococcus phage vB Seps BE21 and Staphylococcus phage S-CoN Ph24 were found in S. epidermidis strains K60 and K16, respectively. Staphylococcus phage SAP3 was found in S. chromogenes strains K29 and K23. Similarly, S. haemolyticus strains A3.2 and A11 harbored Staphylococcus phage IME-SA4. S. xylosus contained sequences the Staphylococcus phage StB20. The presence of prophage sequences is suggestive of their potential role in the pathogenicity of NASM, however further comprehensive investigation is required. Genomic islands of NASM The results of the analysis of genomic islands in the 22 NASM strains are summarized in Table 3 , and a representative image of the predicted genomic island is shown in (Fig. 7 ). The number of GIs in each genome varied from 3 to 9, coding for 85 to 250 genes. These GIs mainly consisted of hypothetical protein-coding genes, transposases, and transcriptional regulators. Some known virulent factors and AMR genes were identified in a few genomic islands. For example, the fosB gene, which encodes metallothiol transferase, leading to fosfomycin resistance, was found in M. lentus . The cadC gene, responsible for cadmium resistance, transcriptional regulatory protein Yyc , which regulates the two-component system WalR/WalK regulatory protein and fosB were found in S. sciuri strains. The lip gene, coding for the virulence-related enzyme lipase, and yyc were found in S. chromogenes strains. S. epidermidis strains carried the ebh gene, coding for extracellular matrix-binding protein related to adherence, blaZ , coding for b-lactamases responsible for penicillin resistance, msr gene, which is an ABC-F subfamily ribosomal protection protein conferring resistance to erythromycin and streptogramin B class of macrolides, qac gene, which is a multidrug efflux pump resistant to fluoroquinolone antibiotics, ssaA , which is staphylococcal secretory antigen, cadC , and yyc . Table 3 Salient features of predicted genomic islands of NASM strains S.No. NASM strains Number of Islands Number of genes in GIs No. of genes coding for hypothetical Proteins Virulence & and antimicrobial resistance genes in genomic islands 1 M. lentus K169 3 90 69 fosB 2 2 M. sciuri K117.2 6 148 79 fosB, cadC, yyc (walR/K) 3 M. sciuri K14 5 100 58 4 M. sciuri K91 5 120 68 fosB, cadC, yyc (walR/K) 5 S. chromogenes K17 4 115 88 yyc (walR/K) 6 S. chromogenes K23 3 85 73 7 S. chromogenes K26 5 132 86 yyc (walR/K) 8 S. chromogenes K29 3 97 81 lip 9 S. epidermidis K16.1 5 88 68 cadC, yyc (walR/K) 10 S. epidermidis K3.2 4 109 56 ebh, blaZ, msr, qacA, qacR, yyc (walR/K) 11 S. epidermidis K4.3 7 150 87 blaZ, msr, ssaA, yyc (walR/K) 12 S. epidermidis K60 8 139 95 cadC, yyc (walR/K) 13 S. gallinarium B13 9 163 103 14 S. haemolyticus A11 5 131 73 blaZ, cadC, farB, yyc (walR/K) 15 S. haemolyticus A3.2 5 132 74 blaZ, cadC, farB, arsR, yyc (walR/K) 16 S. haemolyticus K16.2 7 114 48 blaZ, msr, qacC, cadC, farB, yyc (walR/K) 17 S. haemolyticus K47 7 118 53 blaZ, msr, cadC, yyc (walR/K) 18 S. hominis K24 6 186 115 ebh, yyc (walR/K) 19 S. pseudintermedius B32 7 250 163 spa, lip, msr, ssaA, yyc (walR/K) 20 S. xylosus K19 5 164 83 lip, yyc (walR/K) 21 S. xylosus K46 4 137 70 yyc (walR/K) 22 S. xylosus SMG24 7 193 116 cadC S. haemolyticus strains carried farB , which is the cytoplasmic transporter protein that is part of the farAB efflux pump that confers resistance to fatty acids, arsR , coding for arsenic resistance, and blaZ , cadC , yyc , msr , and qac genes. S. hominis strains contained ebh and yyc genes. The spa , lip , msr , ssaA , and yyc genes were found in S. pseudintermedius strains. S. xylosus strains carried lip , yyc , and cadC . All S. haemolyticus strains consisted of similar islands. S. haemolyticus strains K16 and K47 had similar islands, GI-1, 2, and 3. S. haemolyticus strains A11 and A3 consisted of GI-2 in common. S. epidermidis strains K60 and K16 possessed GI-3 in common. In GI-1 of S. chromogenes strain K29 was similar to the GI-2 of S. chromogenes strain K23. The GI-1 was found in common in M. sciuri strains K117 and K91. A complete list of genomic islands from the 22 NASM strains is shown in Table S4 . Discussion Mastitis significantly threatens the dairy industry, causing substantial revenue losses globally. Other than S. aureus , the non- aureus Staphylococcus and Mammaliicoccus (NASM) are largely responsible for subclinical and clinical mastitis. The disease progression involves a dynamic process shaped by multiple factors, such as the host's genetics, host as well as bacterial resistance mechanisms, host immune response, geographical influences, virulence factors (VFs) and the genetic variability of the bacterium. A comprehensive genome analysis of mastitis-associated NASM strains from diverse geographical regions could enhance our understanding of these factors, aiding in assessing pathogenic potential and infection risk, disease manifestation, and transmission dynamics. We conducted a comparative genomic study on 22 strains of NASM that caused bovine mastitis in three states of India. We sequenced their whole-genomes and identified STs specific to each species, along with the distribution of AMR genes and virulence factors. In previous studies, an ANI threshold of < 96% was suggested for identifying NASM at the species level (Kim et al., 2014 ). By comparing the nucleotide sequences of all the 22 strains, we found that members of the same species had ANI values consistently above 96%, indicating significant genomic similarity. On the other hand, members of different species had less than 96% ANI, indicating a clear genomic distinction between different NASM species. Our study supports the 96% ANI threshold value for species differentiation. This threshold helps understand and classify microbial diversity within this group, making it a valuable criterion for delineating species boundaries. The genome-wide SNP-based phylogeny analysis supported the ANI-based analysis, and a similar phylogenetic classification was observed. The minimum spanning tree (MST) based on SNPs can help us understand the complex relationships among closely related strains. It can reveal patterns of co-evolution, host specialization, and potential transmission pathways. We noticed that strains associated with bovine mastitis clustered together as separate subclades in all the NASM genomes. Previous studies have reported that S. chromogenes ST-1 and ST-6 strains are associated with bovine mastitis (Huebner et al., 2021 ) (Persson Waller et al., 2023 ). Our observation also documents that S. chromogenes ST-1 and ST-6 are associated with bovine mastitis. Additionally, we found that S. epidermidis genotypes ST111 and ST59 are associated with bovine mastitis, which is consistent with another earlier report (Frey et al., 2013 ). Similar sub-clusters unique to particular hosts have been observed in other pathogens (Grillová & Picardeau, 2020 ). In several European countries, S. aureus CC8 strains have been linked with bovine mastitis. Recently, we reported that S. aureus CC97 strains isolated from India were also associated with mastitis (Sivakumar et al., 2023 ). Further research is needed to understand the relevance of subclade-specific SNPs and their possible association with bovine mastitis to better understand their pathogenicity. The increase in drug-resistant strains makes it challenging to treat bovine mastitis with antimicrobial intervention. In our study, we found 32 AMR genes in the 22 NASM genomes. Only one of the 22 strains had mecA and other mec -related genes. Previous studies have documented the presence of mecA -positive S. epidermidis strains in bovine milk samples and the clonal dissemination of multi-drug-resistant S. epidermidis strains carrying mecA within herds (Sawant et al., 2009 ). The emergence of methicillin-resistant S. epidermidis (MRSE) in cattle highlights the need for increased attention, with some researchers suggesting that animals infected with methicillin-resistant NASM should be culled (Gentilini et al., 2002 ). The S. epidermidis strain K4.3 investigated in this study exhibited resistance to methicillin, classifying it as MRSE. However, the other 21 NASM strains analyzed were methicillin-sensitive staphylococcus (MSS) and did not contain mecA , mecR1 , or mec1 genes in their genomes. The blaZ gene was discovered in 6 out of 22 NASM strains. This gene is responsible for the production of penicillinase, which is the primary mechanism of penicillin resistance in NASM (Olsen et al., 2006 ). Multi-drug resistant (MDR) efflux pumps were dispersed throughout the strains and were not associated with specific species or STs. The regulation of MDR efflux pumps is a complex process, requiring multiple regulators to express these elements (Costa et al., 2013 ; Antiabong et al., 2017). Therefore, the presence of these efflux pumps may not necessarily translate to an AMR phenotype. We have identified 53 virulence-associated genes among the 22 NASM genomes. Of these, 15 genes are critical for adherence and biofilm development. The ica operon, which produce polysaccharide intercellular adhesins (PIA) and is a commonly found genetic component in biofilm formation (McKenney et al., 1998 ) stands out as a crucial player. The ica gene was present in S. xylosus and M. sciuri strain K14. The impact of ica genes is particularly noticeable within NASM species linked to the human environment (O’Gara, 2007 ). The diversity of studies examining different variants of the ica genes poses a challenge in understanding the involvement of this gene in biofilm formation (Tremblay et al., 2013 ). All of our NASM strains, excluding Mammaliicoccus spp., have genes that code for lipases. Cell-wall-associated proteins and enzymes play a significant role in the pathogenesis of staphylococci and are essential targets for drug development (Hu et al., 2012 ). Lipases in S. aureus , known as SAL, have been identified in community-associated methicillin-resistant strains (Cadieux et al., 2014 ; Rosenstein, 2000 ). . The spa gene, a major antigen of S. aureus , was exclusively detected in S. pseudintermedius . The sspA gene was found in 12 strains, while the sspB gene was found exclusively in S. epidermidis . In S. epidermidis , the sspA and sspB genes were found to coexist, indicating a potential interplay or cooperative function. The initial player in the staphylococcal proteolytic cascade is aureolysin, a metalloprotease. This enzyme undergoes a rapid process of autocatalytic activation. Subsequently, the activated aureolysin plays a crucial role in activating the sspA serine protease, which in turn, serves as a critical activator for the sspB cysteine protease (Massimi et al., 2002 ; Nickerson et al., 2008 ). Such cascades are often pivotal in regulating various cellular processes and contribute to the pathogenicity and adaptability of members of the genus Staphylococcus . The type VII secretion system (T7SS) has been recently identified and selectively distributed in various pathogens, including Mycobacterium tuberculosis and S. aureus . The T7SS plays an essential role in the virulence of human pathogens. In the case of M. tuberculosis , the T7SS is very important for bacterial access to the host cytosol. In S. aureus , T7SS exports several virulence-associated proteins (Lopez et al., 2017 ). The presence of T7SS cluster was reported in S. lugudensis , in addition to S. aureus (Lebeurre et al., 2019 ). Staphylococci possess genes enabling the production of capsular polysaccharides that form a protective shield against phagocytosis by the host's immune cells. This capsule enhances virulence and bacterial persistence, highlighting encapsulation as a crucial mechanism for evading immune detection and contributing to pathogenicity. The capB and capC genes were detected in all NASM species except Mammaliicoccus spp. and S. pseudintermedius . Prophages are responsible for the horizontal gene transfer, which in turn contributes to virulence (Bae et al., 2006 ; Smirnova et al., 2017 ). The prophage Staphylococcus StB20 was found in S. xylosus strain SMG24. This prophage is reported to exhibit specific proteolytic cleavages in the carboxy-terminal degradation of its tail tape measure proteins (TMP) in S. aureus (Tallent et al., 2007 ). However, the roles of prophages NASM strains are largely unknown. Genomic islands (GIs) are horizontally transferred regions carrying specific genes that confer certain traits to bacteria. These traits include metabolic processes, pathogenicity, antibiotic resistance, and symbiosis. GIs help bacteria establish mutually beneficial relationships with eukaryotic hosts. They often carry genetic material related to virulence or adaptive traits and are commonly located near tRNA or transposase genes at one end of the island. Antibiotic-resistant genes found in GIs make them carriers for the spread of antibiotic resistance, enhancing bacterial species' survival when exposed to antibiotics (Citti et al., 2020 ). Coupling virulence or adaptive traits with antibiotic resistance in GIs plays a significant role in shaping bacterial populations' resilience and adaptability to environmental challenges, especially when exposed to antibiotics. In the 22 NASM that we studied, most islands carried the two-component regulatory system, which includes the walRK gene. This gene plays a crucial role in regulating the expression of genes associated with cell wall metabolism, influencing autolysis, biofilm formation, and virulence. WalK is also involved in sensing the D-Ala-D-Ala moiety of Lipid II, serving as a signal for active cell wall synthesis. The genes yycH and yycI are co-transcribed with walRK and modulate its activity. In S. aureus , disrupting yycH and yycI genes downregulated the walRK regulon (Gajdiss et al., 2020 ). The importance of these regulatory components and their roles in governing cell wall-related processes vary across bacterial species. The fosB , blaZ , and several AMR genes were identified among the GIs, suggesting the possibility of transfer of these traits among the mastitis-associated pathogens. Conclusion Whole genome sequencing and phylogeny analysis of bovine mastitis-associated NASM strains isolated from India revealed species-specific and host-specific clustering. The study identified multiple genes responsible for antibiotic resistance and virulence factors. Certain virulence factors were found to be specific to particular species, and some were specific to particular STs. The analysis also found that some virulence and AMR genes were located in genomic islands, which suggests possible horizontal transfer events. Declarations We declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Funding from the Department of Biotechnology (DBT), New Delhi (BT/PR11245/ADV /90/165/2014) to NRH, SI, and JR is gratefully acknowledged. The MKU-RUSA, UGC-NRCBS, DST-PURSE, DST-FIST Programs of the School of Biological Sciences, Madurai Kamaraj University, are acknowledged. Funding This work was supported by the Department of Biotechnology (DBT), New Delhi, (BT/PR11245/ADV/90/165/2014). Author Contribution N.R.H., J.R., and S.I. designed the experiments; M.A. and S.C. propagated and characterized the NASM strains from the various States of India; V.R. and R.S. performed WGS and analysis; V.R., S.I., J.R., and N.R.H. wrote the paper. 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Journal of Dairy Science, 96 (1), 234–246. https://doi.org/10.3168/jds.2012-5795 Valckenier, D., Piepers, S., De Visscher, A., & De Vliegher, S. (2020). The effect of intramammary infection in early lactation with non-aureus staphylococci in general and Staphylococcus chromogenes specifically on quarter milk somatic cell count and quarter milk yield. Journal of Dairy Science, 103 (1), 768–782. https://doi.org/10.3168/jds.2019-16818 Vanderhaeghen, W., Piepers, S., Leroy, F., Van Coillie, E., Haesebrouck, F., & De Vliegher, S. (2014). Invited review: Effect, persistence, and virulence of coagulase-negative Staphylococcus species associated with ruminant udder health. Journal of Dairy Science, 97 (9), 5275–5293. https://doi.org/10.3168/jds.2013-7775 Zakaria, M. R., Lam, M. Q., Chen, S. J., Abdul Karim, M. H., Tokiman, L., Yahya, A., Shamsir, M. S., & Chong, C. S. (2020). Genome sequence data of Mangrovimonas sp. Strain CR14 isolated from mangrove forest at Tanjung Piai National Park, Malaysia. Data in Brief, 30 , 105658. https://doi.org/10.1016/j.dib.2020.105658 Zhou, Z., Alikhan, N.-F., Sergeant, M. J., Luhmann, N., Vaz, C., Francisco, A. P., Carriço, J. A., & Achtman, M. (2018). GrapeTree: Visualization of core genomic relationships among 100,000 bacterial pathogens. Genome Research, 28 (9), 1395–1404. https://doi.org/10.1101/gr.232397.117 Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx Cite Share Download PDF Status: Published Journal Publication published 22 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Oct, 2024 Reviews received at journal 23 Sep, 2024 Reviewers agreed at journal 19 Aug, 2024 Reviews received at journal 25 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers invited by journal 18 Jul, 2024 Editor assigned by journal 18 Jul, 2024 Editor invited by journal 07 Jul, 2024 Submission checks completed at journal 04 Jul, 2024 First submitted to journal 31 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4508846","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":331792292,"identity":"532ba6f8-5891-4f3a-88e0-445d0f0ebd97","order_by":0,"name":"Vishnukumar Ramesh","email":"","orcid":"","institution":"Madurai Kamaraj University","correspondingAuthor":false,"prefix":"","firstName":"Vishnukumar","middleName":"","lastName":"Ramesh","suffix":""},{"id":331792296,"identity":"60d59712-ad03-44d1-a492-8b5a749d195c","order_by":1,"name":"Ramamoorthy Sivakumar","email":"","orcid":"","institution":"Madurai Kamaraj University","correspondingAuthor":false,"prefix":"","firstName":"Ramamoorthy","middleName":"","lastName":"Sivakumar","suffix":""},{"id":331792297,"identity":"dc1b7ebd-dc45-46c4-bd7c-22e56ef4fea4","order_by":2,"name":"Madhavi Annamanedi","email":"","orcid":"","institution":"National Institute of Animal Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Madhavi","middleName":"","lastName":"Annamanedi","suffix":""},{"id":331792298,"identity":"c64b58a1-d611-4389-8f1f-75d848199c8e","order_by":3,"name":"S. Chandrapriya","email":"","orcid":"","institution":"Karnataka Veterinary Animal and Fisheries Sciences University","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"","lastName":"Chandrapriya","suffix":""},{"id":331792299,"identity":"6735edfe-12a8-4308-90ba-9396c83f9099","order_by":4,"name":"Shrikrishna Isloor","email":"","orcid":"","institution":"Karnataka Veterinary Animal and Fisheries Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Shrikrishna","middleName":"","lastName":"Isloor","suffix":""},{"id":331792300,"identity":"729ca079-cc15-444e-aac9-9cd8a96b4530","order_by":5,"name":"Rajendhran Jeyaprakash","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACxgYGBgMGBuYEEPsBkODhI0ULswFICxuRloG1sEmAmAS1MM8+/KDg4w7rPPP2s8cqv+bYybAxMD98dAOfw/rSDAxnnkkvljmTl3Zbdlsy0GFsxsY5+LT0MBgY87YdTpzBkGN2W3IbM1ALD5s0fi3sH4z/grTwvzErltxWT4wWHgNjRpAWiRwzxo/bDhOlpcCwty0dqOWNsTTjtuM8bMwE/GLYw77N4GebNdBhOYYff26rtudnb374GK+WBgY2AxiHmQdM4lEOAvJAJQ/grvxBQPUoGAWjYBSMTAAAzN1DzYsfgboAAAAASUVORK5CYII=","orcid":"","institution":"Madurai Kamaraj University","correspondingAuthor":true,"prefix":"","firstName":"Rajendhran","middleName":"","lastName":"Jeyaprakash","suffix":""},{"id":331792301,"identity":"d2c6700e-1b29-4edb-bd60-1a15e6d367d8","order_by":6,"name":"Nagendra R Hegde","email":"","orcid":"","institution":"National Institute of Animal Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Nagendra","middleName":"R","lastName":"Hegde","suffix":""}],"badges":[],"createdAt":"2024-05-31 12:10:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4508846/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4508846/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-80533-9","type":"published","date":"2024-11-22T15:57:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61327522,"identity":"51bb9b69-55cd-407e-9bc2-010902f70c67","added_by":"auto","created_at":"2024-07-29 14:12:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap and dendrogram illustrating the phylogenetic relationships based on average nucleotide identity (ANI).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/4fb75552aea2d5800955c669.jpeg"},{"id":61328792,"identity":"516170ba-e23f-4beb-9111-9650d1643201","added_by":"auto","created_at":"2024-07-29 14:28:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":248628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe genome-wide SNP-based parsimony phylogenetic tree of mastitis-associated NASM strains.\u003c/strong\u003e The phylogenetic tree was constructed using kSNP v.3, and visualized using Mega 11. The scale bar indicates 2.00 substitutions per nucleotide position. The strain names, sequence types (STs), clinical (CL) or subclinical (SCL) mastitis, and three different states in India (GJ-Gujarat, KA-Karnataka, and ML-Meghalaya) from where the strains were isolated are indicated, respectively.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/c4969a313c3a9a6e1cefaf55.jpeg"},{"id":61327530,"identity":"0c8d14ca-1970-468c-adc0-c3724fa129c0","added_by":"auto","created_at":"2024-07-29 14:12:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMinimum spanning tree (MST) of 176 NASM genomes reported from India.\u003c/strong\u003eSNPs were predicted using kSNP v.3 and the tree was generated using GrapeTree. A – color codes denote the species names; B – color codes denote the isolation source, cow / other hosts.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/5f089ae075ea3e0f9de6fb95.jpeg"},{"id":61327924,"identity":"3162b60d-1590-41cb-a861-6843129f7b81","added_by":"auto","created_at":"2024-07-29 14:20:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":479448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe resistome of 22 mastitis-associated NASM isolates hierarchically clustered based on the presence (green) or absence (red) of 32 antimicrobial resistance (AMR) genes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/0c16140b4a8485e98f791548.jpeg"},{"id":61327524,"identity":"91110592-0ac3-432f-9a0c-325df2aef435","added_by":"auto","created_at":"2024-07-29 14:12:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":425880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe virulome of 22 mastitis-associated NASM isolates hierarchically clustered based on the presence (green) or absence (red) of 52 virulence genes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/516815449d43835355ffecf2.jpeg"},{"id":61327927,"identity":"8cf2b760-e43e-45a2-a0b9-7ad7d2fc7a94","added_by":"auto","created_at":"2024-07-29 14:20:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":383851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe prophage regions detected in the eight NASM strains.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/f84996a7b2ad92a7e77ff916.jpeg"},{"id":61328791,"identity":"706d1cc5-3738-4653-9ece-7ca205918aa1","added_by":"auto","created_at":"2024-07-29 14:28:38","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":240148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic islands of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. gallinarum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e B13.\u003c/strong\u003e The circle represents a single chromosome, with red bars around the perimeter indicating the locations of GIs. The color codes represent the prediction methods: IslandPath-DIMOB (blue), SIGI-HMM (orange), and Integrated (red).\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/200c7c4685d7260a7e7dbcd6.jpeg"},{"id":69835153,"identity":"672df1a2-118f-4f71-95a4-00aab68a843a","added_by":"auto","created_at":"2024-11-25 16:12:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3710638,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/ae5d58d2-0284-4d0e-a474-b97af6c12f36.pdf"},{"id":61327519,"identity":"1b81f18f-79ac-47db-934c-9299f6efd381","added_by":"auto","created_at":"2024-07-29 14:12:38","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9777,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/dbd629a66e44477c0c811b80.xlsx"},{"id":61327520,"identity":"40d3ff0f-1c30-4a2a-88cb-e9cad421b750","added_by":"auto","created_at":"2024-07-29 14:12:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31794,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/a5ae44260c956e070ea209d3.xlsx"},{"id":61327921,"identity":"949a745e-5a3e-4483-a75a-fdabc1ff29fc","added_by":"auto","created_at":"2024-07-29 14:20:38","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":96803,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/8e8b99b98bf33a4b894d36e6.xlsx"},{"id":61327527,"identity":"0579ddb0-af5a-43fc-b979-a4f7fdd8bcb3","added_by":"auto","created_at":"2024-07-29 14:12:38","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":215671,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4508846/v1/56f0944be3631f6e7c951327.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome sequencing and comparative genomic analysis of bovine mastitis-associated non-aureus staphylococci and mammaliicocci (NASM) strains from India","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBovine mastitis is an endemic disease affecting dairy cattle worldwide. It causes inflammation of the mammary gland, resulting in economic losses due to decreased milk production, veterinary care costs, and culling of infected animals (Halasa et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kovačević et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Staphylococci are the most common infectious causative agents of bovine mastitis, and were historically divided into two groups. One group includes \u003cem\u003eS. aureus\u003c/em\u003e, which is considered more pathogenic, and is a coagulase-positive staphylococcus (CoPS). The other group that is presumed to be less pathogenic, includes other staphylococcal species referred to as the coagulase-negative staphylococci (CoNS) or non-aureus staphylococci (NAS). Some coagulase-positive and coagulase-variable mastitis pathogens (e.g., \u003cem\u003eS. pseudintermedius\u003c/em\u003e) are also included in the CoNS category (De Buck et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently, five \u003cem\u003eStaphylococcus\u003c/em\u003e species, i.e., \u003cem\u003eS. sciuri, S. fleurettii, S. lentus, S. stepanovicii\u003c/em\u003e, and \u003cem\u003eS. vitulinus\u003c/em\u003e, were reclassified into the new genus, namely, \u003cem\u003eMammaliicoccous\u003c/em\u003e (Madhaiyan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Together, these organisms, referred to as non-aureus staphylococci and mammaliicocci (NASM), are the most prevalent (ranging from 9.1 to 16.6% of milk samples) agents isolated from intramammary infection (IMI) of dairy cows and the leading cause of subclinical mastitis. Recent studies have identified \u003cem\u003eS. chromogenes\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. xylosus\u003c/em\u003e, \u003cem\u003eS. vitulinus\u003c/em\u003e, \u003cem\u003eS. simulans\u003c/em\u003e, and \u003cem\u003eMammaliicoccus sciuri\u003c/em\u003e as the leading causes of IMI in cattle. Among them, \u003cem\u003eS. chromogenes, S. xylosus, and S. haemolyticus\u003c/em\u003e are more commonly found in milk samples (Ruiz-Romero \u0026amp; Vargas-Bello-P\u0026eacute;rez, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough NASM has become a leading group of pathogens, knowledge of their virulence and antimicrobial resistance mechanisms is still limited. Biofilm formation has been identified as a crucial virulence factor for NASM, particularly in persistent IMI (Simojoki et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and could potentially account for their heightened antibiotic resistance. The relatively increased capacity of NASM to form biofilm, as compared to other staphylococci, poses challenges in predicting antibiotic efficacy and the likelihood of persistence and recurrence of IMI. It is therefore crucial to understand the factors governing NASM resistance, persistent infections, and recurrent episodes, emphasizing the need for comprehensive research to enhance our knowledge of these intricate dynamics in the context of mastitis (Srednik et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNASM encompasses a diverse group of species, each with varying pathogenic potentials. Therefore, it is essential to understand the role of individual species on udder health and milk production (Vanderhaeghen et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; De Buck et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Various molecular subtyping techniques, including pulsed-field gel electrophoresis (PFGE) (Fry et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), random amplification of polymorphic DNA (RAPD) analysis (Petzer et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), multi-locus sequence typing (MLST), and multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) (Dahyot et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), have been used to investigate the molecular epidemiology of NASM. The whole genome sequence (WGS) analysis is expected to provide the best discriminatory power and better insights into the molecular epidemiology and the genetic determinants responsible for the pathogenicity of NASM. Comparative genomic analysis can identify phylogenetic relationships among various species of NASM and distinctions within and between species.\u003c/p\u003e \u003cp\u003eThis study focused on the comprehensive genomic analysis of 22 NASM strains associated with bovine mastitis isolated in India. These strains were fully sequenced, followed by a comparative analysis of their genetic diversity, virulence factors, antimicrobial resistance (AMR) genes, and sequence types. The study also included a perceptive phylogeny analysis of the previously documented NASM genomes from India, which helped understand the genetic differences between the NASM strains associated with or not associated with bovine mastitis .\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequencing and annotation\u003c/h2\u003e \u003cp\u003eWhole genomes of 22 strains of bovine mastitis-associated NASM were sequenced. These strains were collected between 2009 and 2019 from cows (\u003cem\u003eBos taurus\u003c/em\u003e) with mastitis, which included 18 strains from sub-clinical and four from clinical mastitis cases in India. The strains were isolated from three states; 18 from Karnataka, and two each from Gujarat and Meghalaya. The strains were curated at the National Institute of Animal Biotechnology in Hyderabad and the Department of Microbiology at Karnataka Veterinary, Animal \u0026amp; Fisheries Sciences University in Bengaluru. The DNeasy blood and tissue kit (Qiagen) was used to isolate genomic DNA from each strain, following the manufacturer's instructions. The resulting RNA-free DNA was then employed for library preparation and sequenced using the Illumina HiSeq platform at Macrogen, Seoul, South Korea. The process of assembly and analysis of the genome sequences involved multiple steps. First, the Trimmomatic tool was used to eliminate adapters and discard low-quality reads (Bolger et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Adaptor-trimmed high-quality reads were assembled using SPAdes v 3.11.1 (Zakaria et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Finally, the assembled genome sequences underwent annotation using the PROKKA (Seemann, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Species identification was done using the rMLST (ribosomal multilocus sequence typing) tool available at the PubMLST webserver (Larsen et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMultilocus sequence typing\u003c/h2\u003e \u003cp\u003eMultilocus sequence typing (MLST) using respective species-specific housekeeping genes was performed using the PubMLST web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org/\u003c/span\u003e\u003cspan address=\"https://pubmlst.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Aanensen \u0026amp; Spratt, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Allelic profiles were compared to the PubMLST database, and sequence types (STs) were identified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAverage nucleotide identity (ANI) estimation\u003c/h2\u003e \u003cp\u003eThe genome sequences were analyzed to determine their Average Nucleotide Identity (ANI) values using Jspecies (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jspecies.ribohost.com\u003c/span\u003e\u003cspan address=\"https://jspecies.ribohost.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which included MUMmer ANIm, ANItetra, and BLAST\u0026thinsp;+\u0026thinsp;ANIb (Richter et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Each genome was assigned a species name based on an ANI cutoff of \u0026gt;\u0026thinsp;95% (Colston et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A heatmap was then generated using the Jspecies data on the Clustviz webserver (Metsalu \u0026amp; Vilo, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePhylogeny analysis using genome-wide SNPs\u003c/h2\u003e \u003cp\u003eWe used kSNP v.3 to predict genome-wide SNPs, identifying SNPs without requiring genome alignment (Gardner et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The k-mer value of 17 was optimal for NASM strains by the KChooser tool of the kSNP v3. Genome-wide SNPs were identified and collected in a data matrix known as the 95% majority SNP matrix. Based on the genome-wide SNPs, a phylogenetic tree was constructed for 22 NASM strains using MEGA 11(Tamura et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, a genome-wide SNP-based phylogenetic tree was constructed for 176 genomes of NASM strains reported from India, which includes 22 strains reported in this study, using Grapetree (Zhou et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of antimicrobial resistance (AMR) genes and virulence factors\u003c/h2\u003e \u003cp\u003eThe resistance gene identifier (RGI) in the comprehensive antibiotic resistance database (CARD) was used to identify the AMR genes using default parameters (Jia et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The VFanalyzer in the virulence factor database (VFDB) was used to identify the genes associated with virulence factors (VF) (Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of prophages and genomic islands\u003c/h2\u003e \u003cp\u003eThe presence of prophages in the genome was determined by PHASTER (PHAge Search Tool\u0026mdash;Enhanced Release) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phaster.ca/\u003c/span\u003e\u003cspan address=\"https://phaster.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Naidoo \u0026amp; Zishiri, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Based on the scores, prophages were classified into three groups, i.e., intact, questionable, and incomplete, with the corresponding scores\u0026thinsp;\u0026gt;\u0026thinsp;90, 70\u0026ndash;90, and \u0026lt;\u0026thinsp;70, respectively. Genomic islands (GIs) in each genome were predicted using Island Viewer 4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.pathogenomics.sfu.ca/islandviewer/\u003c/span\u003e\u003cspan address=\"http://www.pathogenomics.sfu.ca/islandviewer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Bertelli et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenome availability\u003c/h2\u003e \u003cp\u003eNASM genome sequence used in this study has been deposited in NCBI with the following accession numbers: GCA_018986335.1, GCA_018996905.1, GCA_019334185.1, GCA_018997025.1, GCA_018997125.1, GCA_018967705.1, GCA_019149065.1, GCA_019149165.1, GCA_019149045.1, GCA_019193065.1, GCA_019148995.1, GCA_019334235.1, GCA_019429675.1, GCA_019149085.1, GCA_019149255.1, GCA_019149245.1, GCA_019149205.1, GCA_019334165.1, GCA_019100515.1, GCA_019165085.1, GCA_018967945.1, GCA_018968045.1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGeneral features of genome sequences of bovine mastitis-associated NASM strains\u003c/h2\u003e \u003cp\u003eThe genomes of 22 strains of NASM associated with bovine mastitis were sequenced using the Illumina platform. These strains belong to nine different species of \u003cem\u003eStaphylococcus\u003c/em\u003e and \u003cem\u003eMammaliicoccus\u003c/em\u003e genera (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For each genome, a minimum of 100X mean sequence coverage was obtained. The reads were subjected to quality checks. Adapter removal, and the processed high-quality reads were used for \u003cem\u003ede novo\u003c/em\u003e assembly. The draft genomes contained 24 to 150 contigs with a mean genome size of 2.55 Mbp and an average GC content of 32.2%. The rMLST method uses ribosome protein subunit (\u003cem\u003erps\u003c/em\u003e) gene sequences for precise taxonomic identification. Of the 22 NASM strains, 21 were identified by exact matches with 56 \u003cem\u003erps\u003c/em\u003e genes. \u003cem\u003eS. chromogenes\u003c/em\u003e strain K17 was identified by exact matches with 55 \u003cem\u003erps\u003c/em\u003e genes. The summary of genome sequences is given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNASM strains used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName of the species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of strains\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus chromogenes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus gallinarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus haemolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus hominis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus pseudintermedius\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus xylosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMammaliicoccus lentus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMammaliicoccus sciuri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of NASM genome sequences\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. of Contigs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSequence Type (ST)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCDS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003erRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003etRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePseudogenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAccession No.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. lentus\u003c/em\u003e K169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2848578\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2783\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_018967945.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. sciuri\u003c/em\u003e K117.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2702737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2672\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_018967705.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. sciuri\u003c/em\u003e K14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2739519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019149065.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. sciuri\u003c/em\u003e K91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2715464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019334185.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2321223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019149165.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2349003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019334235.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2340405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019429675.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2329474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019149085.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2407306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST1157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019149045.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2455169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST1158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019100515.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2449159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019165085.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2460483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST1158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019149245.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. gallinarium\u003c/em\u003e B13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2935302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_018997025.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e A11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2485414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_018986335.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e A3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2497919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_018996905.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e K16.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2459833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019193065.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e K47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2435548\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019149205.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. hominis\u003c/em\u003e K24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2162928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019149255.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. pseudintermedius\u003c/em\u003e B32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2517961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_018997125.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. xylosus\u003c/em\u003e K19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2781766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019148995.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. xylosus\u003c/em\u003e K46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2801517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_019334165.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. xylosus\u003c/em\u003e SMG24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2802341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGCA_018968045.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of sequence types based on MLST\u003c/h2\u003e \u003cp\u003eMLST schemes are available for five species of NASM, which were used in this study and can be accessed at the pubMLST server. The sequence types (STs) were determined based on the respective species-specific housekeeping genes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). \u003cem\u003eM. sciuri\u003c/em\u003e strains were divided into ST114 (n\u0026thinsp;=\u0026thinsp;2) and ST115 (n\u0026thinsp;=\u0026thinsp;1). Similarly, \u003cem\u003eS. chromogenes\u003c/em\u003e were represented by ST6 (n\u0026thinsp;=\u0026thinsp;2) and ST1 (n\u0026thinsp;=\u0026thinsp;2). Three different STs were identified among \u003cem\u003eS. epidermidis\u003c/em\u003e: ST1158 (n\u0026thinsp;=\u0026thinsp;2), ST1157 (n\u0026thinsp;=\u0026thinsp;1), and ST924 (n\u0026thinsp;=\u0026thinsp;1). ST42 (n\u0026thinsp;=\u0026thinsp;2) was identified in \u003cem\u003eS. haemolyticus\u003c/em\u003e; two other strains representing potential new STs could not be typed. One strain of \u003cem\u003eS. hominis\u003c/em\u003e belonged to ST79.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAverage nucleotide identity analysis\u003c/h2\u003e \u003cp\u003eThe ANI analysis examined the interrelationship among NASM, where species-specific clustering of strains was observed, as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The ANI threshold of 96% was used to establish species delimitation within species-specific boundaries. For instance, \u003cem\u003eS. chromogenes\u003c/em\u003e strain K17 was identical to the strains K23, K26, and K29, while \u003cem\u003eS. epidermidis\u003c/em\u003e strain K16.1 matched the strains K4.3, K3.2, and K60. Similarly, \u003cem\u003eM. sciuri\u003c/em\u003e strain K117.2 shared identity with strains K14 and K9, and \u003cem\u003eS. xylosus\u003c/em\u003e strain K19 was similar to the strains K46 and SMG. \u003cem\u003eM. lentus\u003c/em\u003e was closer to \u003cem\u003eM. sciuri\u003c/em\u003e strains and formed a genus-specific cluster. \u003cem\u003eS. gallinarum\u003c/em\u003e strain clustered together with \u003cem\u003eS. xylosus\u003c/em\u003e strains. Likewise, \u003cem\u003eS. pseudintermedius\u003c/em\u003e strain clustered with \u003cem\u003eS. chromogenes\u003c/em\u003e, while \u003cem\u003eS. haemolyticus\u003c/em\u003e clustered with \u003cem\u003eS. hominis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGenome-wide SNP-based-phylogeny analysis of NASM\u003c/h2\u003e \u003cp\u003eWe identified 192 core SNPs and 953968 non-core SNPs in 22 NASM strains. Based on the distribution of SNPs, a parsimony phylogenetic tree using MEGA 11 was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The NASM strains grouped into five species-specific clades. \u003cem\u003eS. gallinarum\u003c/em\u003e and \u003cem\u003eS. xylosus\u003c/em\u003e formed a clade (I) and were divided into species-specific subclades. \u003cem\u003eS. hominis\u003c/em\u003e and \u003cem\u003eS. haemolyticus\u003c/em\u003e formed a clade (II) and were divided into species-specific subclades. While \u003cem\u003eS. epidermidis\u003c/em\u003e formed a separate clade (III). \u003cem\u003eS. pseudintermedius\u003c/em\u003e and \u003cem\u003eS. chromogenes\u003c/em\u003e formed a clade (IV), and both species were divided into separate subclades. Similarly, \u003cem\u003eMammaliicoccus\u003c/em\u003e formed a separate clade (V), while \u003cem\u003eM. lentus\u003c/em\u003e and \u003cem\u003eM. sciuri\u003c/em\u003e formed species-specific subclades. In summary, the genome-wide SNP-based phylogeny and ANIb-based phylogeny were similar.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to further understand the inter-relationship between our strains and other NASM strains from India, a genome-wide SNP-based phylogeny analysis of 176 NASM strains (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), including 22 strains from this study, was conducted. The NASM strains could be grouped into five species-specific clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Clade A consisted of \u003cem\u003eS. epidermidis\u003c/em\u003e, which formed a major clade with 71 strains from India, divided into two subclades. Clade B was represented by \u003cem\u003eS. haemolyticus\u003c/em\u003e and \u003cem\u003eS. hominis\u003c/em\u003e and divided into separate subclades. Clade C comprised \u003cem\u003eS. haemolyticus, S. gallinarum\u003c/em\u003e, and \u003cem\u003eS. xylosus\u003c/em\u003e. Strains belonging to the species \u003cem\u003eS. haemolyticus\u003c/em\u003e were represented in both clades B and C. Clade D was represented by \u003cem\u003eS. chromogenes\u003c/em\u003e and \u003cem\u003eS. pseudintermedius\u003c/em\u003e. Clade E was represented by the \u003cem\u003eMammaliicoccus\u003c/em\u003e species (\u003cem\u003eM. lentus\u003c/em\u003e and \u003cem\u003eM. sciuri\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe examined the minimum spanning tree in relation to the source of isolation - whether it was from a cow or some other host. The results were interesting as all mastitis-associated NASM strains isolated from cows formed separate subclades or clusters in all clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). On the other hand, NASM strains isolated from other sources, such as humans, plants, dogs, etc. clustered together. This suggests that bovine mastitis-associated strains might have host-specific divergence and evolution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of antimicrobial resistance genes among mastitis-associated NASM strains\u003c/h2\u003e \u003cp\u003eThe 22 NASM strains contained antimicrobial resistance (AMR) genes (AMR genes) against 12 classes of antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The 22 strains carried 32 AMR genes, while the analysis of all the Indian isolates revealed 57 AMR genes. The number and classes of AMR genes varied significantly among the strains. All of our 22 strains carried the two AMR genes, \u003cem\u003esdrM\u003c/em\u003e and \u003cem\u003esepA\u003c/em\u003e. \u003cem\u003eSdrM\u003c/em\u003e is an efflux pump that confers resistance to norfloxacin and ethidium bromide, while \u003cem\u003eSepA\u003c/em\u003e is a multidrug efflux pump that confers resistance to disinfectants and antiseptics. The \u003cem\u003enorC\u003c/em\u003e, which confers resistance to fluoroquinolone antibiotics, was present in all strains except for \u003cem\u003eS. pseudintermedius\u003c/em\u003e strain B32, \u003cem\u003eM. lentus\u003c/em\u003e, and \u003cem\u003eM. sciuri\u003c/em\u003e species. The \u003cem\u003evanT\u003c/em\u003e, which codes for a membrane-bound serine racemase which confers resistance to vancomycin, was present in multiple strains, except for \u003cem\u003eS. pseudintermedius\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. hominis\u003c/em\u003e, and \u003cem\u003eS. gallinarum\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003edfrC\u003c/em\u003e, responsible for trimethoprim resistance, and the multidrug efflux pump coding gene \u003cem\u003enorA\u003c/em\u003e, which confers resistance to fluoroquinolones as well as several antiseptics and disinfectants, were observed in all strains of \u003cem\u003eS. epidermidis\u003c/em\u003e but absent in other species. The fusidic acid resistance gene (\u003cem\u003efusE\u003c/em\u003e) was detected in all strains of \u003cem\u003eS. chromogenes\u003c/em\u003e but not in other species. The genes associated with resistance to penam drugs (\u003cem\u003emecl\u003c/em\u003e and \u003cem\u003emecR1\u003c/em\u003e) were only found in \u003cem\u003eS. epidermidis\u003c/em\u003e strain K4.3. The methicillin-resistant gene (\u003cem\u003emecA\u003c/em\u003e) was present in \u003cem\u003eS. epidermidis\u003c/em\u003e strain K4.3, \u003cem\u003eS. haemolyticus\u003c/em\u003e strain K16.2, and \u003cem\u003eS. haemolyticus\u003c/em\u003e strain K47. The PC1 beta-lactamase (\u003cem\u003eblaZ\u003c/em\u003e) gene was detected in all strains of \u003cem\u003eS. haemolyticus\u003c/em\u003e, and \u003cem\u003eS. epidermidis\u003c/em\u003e strains K3.2 and K4.3.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003emgrA\u003c/em\u003e, which confers resistance to fluoroquinolone antibiotics, was present in \u003cem\u003eS. haemolyticus\u003c/em\u003e strains A11 and A3.2, and all the strains of \u003cem\u003eS. epidermidis\u003c/em\u003e. The \u003cem\u003esalB\u003c/em\u003e, which confers resistance to lincosamide and class A streptogramins, was only found in \u003cem\u003eM. lentus\u003c/em\u003e strain K169. On the other hand the homolog \u003cem\u003esalC\u003c/em\u003e, was present in all strains of \u003cem\u003eM. sciuri\u003c/em\u003e whereas \u003cem\u003esalE\u003c/em\u003e was present in \u003cem\u003eS. xylosus\u003c/em\u003e strains K19 and K46. The \u003cem\u003etetK\u003c/em\u003e gene, which is a tetracycline efflux protein, the \u003cem\u003eqacJ\u003c/em\u003e gene, which confers resistance to quaternary ammonium compounds, and the SAT-4 gene, which provides resistance to nucleoside antibiotics, were only present in \u003cem\u003eS. haemolyticus\u003c/em\u003e strains belonging to ST42. The \u003cem\u003efosBx\u003c/em\u003e1, which confers resistance to phosphonic acid antibiotics, was found in \u003cem\u003eS. xylosus\u003c/em\u003e strains K19 and K46, \u003cem\u003eS. gallinarum\u003c/em\u003e strain B13, \u003cem\u003eS. epidermidis\u003c/em\u003e strain K16.1, and \u003cem\u003eM. sciuri\u003c/em\u003e strains and K117.2. The \u003cem\u003efexB\u003c/em\u003e coding for a phenol antibiotic efflux pump was only present in \u003cem\u003eS. xylosus\u003c/em\u003e strain SMG24. The \u003cem\u003evanW\u003c/em\u003e gene, which confers resistance to vancomycin was present only in \u003cem\u003eS. epidermidis\u003c/em\u003e strain K60. Among the functional homologs, \u003cem\u003evanY\u003c/em\u003e was present only in \u003cem\u003eM. lentus\u003c/em\u003e strain K169,\u003cem\u003evanY\u003c/em\u003e was present in all strains of \u003cem\u003eM. sciuri\u003c/em\u003e and \u003cem\u003evanY\u003c/em\u003e was present in \u003cem\u003eS. haemolyticus\u003c/em\u003e strains belonging to ST42 and \u003cem\u003eS. xylosus\u003c/em\u003e strain SMG24.\u003c/p\u003e \u003cp\u003eWhen the analysis was extended to all 176 NASM isolates from India, 57 antibiotic-resistant genes were identified in all Indian isolates as against 32 genes for our 22 strains. A complete list of AMR genes from 176 NASM strains is shown in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of virulence factors among mastitis-associated NASM strains\u003c/h2\u003e \u003cp\u003eFifty-two virulence factors were identified in the genomes of NASM strains associated with bovine mastitis. However, none of these genes were present in all the 22 strains. Instead, each strain had a unique set of virulence factors in various combinations. These virulence factors were functionally classified into eight classes, as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eA. Adherence-related gene\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe identified 14 different genes associated with adherence among NASM strains. All three strains of \u003cem\u003eS. xylosus\u003c/em\u003e and \u003cem\u003eM. sciuri\u003c/em\u003e strain K14 were carried genes coding for intercellular adhesins, \u003cem\u003eicaA\u003c/em\u003e, \u003cem\u003eicaB\u003c/em\u003e, and \u003cem\u003eicaC\u003c/em\u003e. However, the repressor of these genes, \u003cem\u003eicaR\u003c/em\u003e, was present only in all \u003cem\u003eS. xylosus\u003c/em\u003e strains and not in \u003cem\u003eM. sciuri\u003c/em\u003e strain K14. The \u003cem\u003eStaphylococcus\u003c/em\u003e protein A (\u003cem\u003espa\u003c/em\u003e) gene was only found in \u003cem\u003eS. pseudintermedius\u003c/em\u003e strain B32. The autolysin (\u003cem\u003eatl\u003c/em\u003e) gene was present in all \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. haemolyticus\u003c/em\u003e, \u003cem\u003eS. hominis\u003c/em\u003e strains, and \u003cem\u003eS. xylosus\u003c/em\u003e strains K19 and K46. Clumping factor genes \u003cem\u003eclfA\u003c/em\u003e and \u003cem\u003eclfB\u003c/em\u003e were only found in \u003cem\u003eM. lentus\u003c/em\u003e. The fibronectin-binding protein gene, \u003cem\u003efnbA\u003c/em\u003e, was present in three strains, namely \u003cem\u003eS. xylosus\u003c/em\u003e strains SMG24, \u003cem\u003eS. chromogenes\u003c/em\u003e strains, and K17. The gene coding for another fibronectin-binding protein, \u003cem\u003efnbB\u003c/em\u003e, was only present in \u003cem\u003eS. pseudintermedius\u003c/em\u003e. The extracellular matrix-binding protein homolog, \u003cem\u003eebh\u003c/em\u003e, was found in all \u003cem\u003eS. epidermidis\u003c/em\u003e strains. The gene coding for elastin binding protein, \u003cem\u003eepb\u003c/em\u003e, was present in all strains of \u003cem\u003eS. xylosus\u003c/em\u003e, \u003cem\u003eS. hominis\u003c/em\u003e, \u003cem\u003eS. haemolyticus\u003c/em\u003e, and \u003cem\u003eS. epidermidis\u003c/em\u003e except strain K3.2. The Ser-Asp-rich fibrinogen-binding proteins coding genes, \u003cem\u003esdrC\u003c/em\u003e and \u003cem\u003esdrE\u003c/em\u003e, were found only in \u003cem\u003eS. haemolyticus\u003c/em\u003e strains A11 and A3.2.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eB. Enzymes coding genes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFive enzyme-coding genes associated with virulence were predicted among the 22 NASM genomes. Genes coding for lipases (\u003cem\u003elip\u003c/em\u003e) and thermonuclease (\u003cem\u003enuc\u003c/em\u003e) were present in all \u003cem\u003eStaphylococcus\u003c/em\u003e strains but not \u003cem\u003eMammaliicoccus\u003c/em\u003e species. The lipase-coding gene \u003cem\u003egeh\u003c/em\u003e was also found in all \u003cem\u003eS. xylosus\u003c/em\u003e, \u003cem\u003eS. pseudintermedius\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e strains, and \u003cem\u003eS. chromogenes\u003c/em\u003e strains K29 and K26. The gene coding for serine V8 protease, \u003cem\u003esspA\u003c/em\u003e, was present in strains belonging to \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. xylosus\u003c/em\u003e, \u003cem\u003eS. gallinarum\u003c/em\u003e, \u003cem\u003eM. lentus\u003c/em\u003e, and \u003cem\u003eM. sciuri\u003c/em\u003e, while \u003cem\u003esspB\u003c/em\u003e was found only in \u003cem\u003eS. epidermidis\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eC. Immune evasion coding genes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTwelve genes associated with immune evasion were identified among the 22 NASM genomes. Of these genes, two species, \u003cem\u003eS. pseudintermedius\u003c/em\u003e and \u003cem\u003eS. chromogenes\u003c/em\u003e, had gene coding for adenosine synthase A (\u003cem\u003eadsA\u003c/em\u003e). Except for \u003cem\u003eM. sciuri\u003c/em\u003e, \u003cem\u003eM. lentus\u003c/em\u003e, and \u003cem\u003eS. pseudintermedius\u003c/em\u003e, the capsule protein (\u003cem\u003ecapB\u003c/em\u003e and \u003cem\u003ecapC\u003c/em\u003e) was found in all genomes. The \u003cem\u003eBacillus\u003c/em\u003e polysaccharide capsule gene (\u003cem\u003egtaB\u003c/em\u003e) was present only in \u003cem\u003eM. lentus\u003c/em\u003e, and the \u003cem\u003emanA\u003c/em\u003e gene was found only in \u003cem\u003eS. gallinarum\u003c/em\u003e. The lipopolysaccharide (\u003cem\u003eFrancisella\u003c/em\u003e) \u003cem\u003ewbtP\u003c/em\u003e was present in \u003cem\u003eM. lentus\u003c/em\u003e and \u003cem\u003eM. gallinarum\u003c/em\u003e, while \u003cem\u003ewbtE\u003c/em\u003e was found in \u003cem\u003eM. sciuri\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eD. Toxin coding genes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAmong the 22 NASM strains analyzed, six genes that code for toxins were predicted. The \u003cem\u003ecylR2\u003c/em\u003e gene, which codes for cytolysin, was found in all the strains of \u003cem\u003eS. xylosus\u003c/em\u003e, \u003cem\u003eS. hominis\u003c/em\u003e, \u003cem\u003eS. haemolyticus\u003c/em\u003e, and \u003cem\u003eS. gallinarum\u003c/em\u003e. The \u003cem\u003ehib\u003c/em\u003e gene coding for b-hemolysin was present in all the strains of \u003cem\u003eS. chromogenes\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e. The \u003cem\u003elukD\u003c/em\u003e and \u003cem\u003elukE\u003c/em\u003e genes, which code for leukotoxins D and E, respectively, were found only in the \u003cem\u003eS. pseudintermedius\u003c/em\u003e strain B32. The \u003cem\u003eset26\u003c/em\u003e gene, which codes for an exotoxin, was found in all the strains of \u003cem\u003eS. chromogenes\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eE. Secretion system-related genes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe genes coding for the Type VII secretion system (T7SS), \u003cem\u003eesaA, esaB, esaG, essA, essB, essC\u003c/em\u003e, and \u003cem\u003eesxA\u003c/em\u003e were found only in \u003cem\u003eS. epidermidis\u003c/em\u003e strain K4.3.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eF. Cell surface components\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAmong the 22 NASM strains, three genes coding for cell surface components associated with virulence were identified. The trehalose-recycling ABC transporter (\u003cem\u003esugC\u003c/em\u003e) was found in \u003cem\u003eS. pseudintermedius\u003c/em\u003e strain B32, \u003cem\u003eM. sciuri\u003c/em\u003e strains K117.2 and K91. The lipoprotein diacylglyceryl transferase (\u003cem\u003elgt\u003c/em\u003e) was found in all the strains of \u003cem\u003eS. pesudintermedius\u003c/em\u003e, \u003cem\u003eM. sciuri\u003c/em\u003e, \u003cem\u003eM. lentus\u003c/em\u003e, and \u003cem\u003eS. chromogenes\u003c/em\u003e. The lipoprotein-specific signal peptidase gene (\u003cem\u003elspA\u003c/em\u003e) was found only in \u003cem\u003eS. pseudintermedius\u003c/em\u003e strain B32.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eG. Metal uptake coding gene\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe gene \u003cem\u003evctC\u003c/em\u003e, responsible for iron uptake, was present in all the strains of \u003cem\u003eS. xylosus\u003c/em\u003e. In contrast, the gene \u003cem\u003ectpV\u003c/em\u003e, responsible for copper uptake and export, was only found in \u003cem\u003eS. epidermidis\u003c/em\u003e strain K60.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eH. Intracellular Survival\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe gene nucleoside diphosphate kinase (\u003cem\u003endk\u003c/em\u003e), responsible for phagosome arrest, was found in all the strains of \u003cem\u003eMammaliicoccus\u003c/em\u003e. The regulational acid resistance gene (\u003cem\u003elisR\u003c/em\u003e) was found in all the strains of \u003cem\u003eMammaliicoccus\u003c/em\u003e, \u003cem\u003eS. chromogenes\u003c/em\u003e, and \u003cem\u003eS. pseudintermedius\u003c/em\u003e. The intracellular survival gene lipoate protein ligase, \u003cem\u003elplA1\u003c/em\u003e, was found in only \u003cem\u003eM. lentus\u003c/em\u003e strain K169.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eProphage sequence-associated with NASM\u003c/h2\u003e \u003cp\u003eWe identified nine intact prophage sequences among our NASM strains. All the prophage sequences contain all the genes required for a phage life cycle, as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Two prophage sequences, \u003cem\u003eStaphylococcus\u003c/em\u003e phage vB Saus IMEP5 and uncultured caudovirales phage clone 10S.1, were found in \u003cem\u003eS. gallinarum\u003c/em\u003e. \u003cem\u003eStaphylococcus\u003c/em\u003e phage vB Seps BE21 and \u003cem\u003eStaphylococcus\u003c/em\u003e phage S-CoN Ph24 were found in \u003cem\u003eS. epidermidis\u003c/em\u003e strains K60 and K16, respectively. \u003cem\u003eStaphylococcus\u003c/em\u003e phage SAP3 was found in \u003cem\u003eS. chromogenes\u003c/em\u003e strains K29 and K23. Similarly, \u003cem\u003eS. haemolyticus\u003c/em\u003e strains A3.2 and A11 harbored \u003cem\u003eStaphylococcus\u003c/em\u003e phage IME-SA4. \u003cem\u003eS. xylosus\u003c/em\u003e contained sequences the \u003cem\u003eStaphylococcus\u003c/em\u003e phage StB20. The presence of prophage sequences is suggestive of their potential role in the pathogenicity of NASM, however further comprehensive investigation is required.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eGenomic islands of NASM\u003c/h2\u003e \u003cp\u003eThe results of the analysis of genomic islands in the 22 NASM strains are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and a representative image of the predicted genomic island is shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The number of GIs in each genome varied from 3 to 9, coding for 85 to 250 genes. These GIs mainly consisted of hypothetical protein-coding genes, transposases, and transcriptional regulators. Some known virulent factors and AMR genes were identified in a few genomic islands. For example, the \u003cem\u003efosB\u003c/em\u003e gene, which encodes metallothiol transferase, leading to fosfomycin resistance, was found in \u003cem\u003eM. lentus\u003c/em\u003e. The \u003cem\u003ecadC\u003c/em\u003e gene, responsible for cadmium resistance, transcriptional regulatory protein \u003cem\u003eYyc\u003c/em\u003e, which regulates the two-component system WalR/WalK regulatory protein and \u003cem\u003efosB\u003c/em\u003e were found in \u003cem\u003eS. sciuri\u003c/em\u003e strains. The \u003cem\u003elip\u003c/em\u003e gene, coding for the virulence-related enzyme lipase, and \u003cem\u003eyyc\u003c/em\u003e were found in \u003cem\u003eS. chromogenes\u003c/em\u003e strains. \u003cem\u003eS. epidermidis\u003c/em\u003e strains carried the \u003cem\u003eebh\u003c/em\u003e gene, coding for extracellular matrix-binding protein related to adherence, \u003cem\u003eblaZ\u003c/em\u003e, coding for b-lactamases responsible for penicillin resistance, \u003cem\u003emsr\u003c/em\u003e gene, which is an ABC-F subfamily ribosomal protection protein conferring resistance to erythromycin and streptogramin B class of macrolides, \u003cem\u003eqac\u003c/em\u003e gene, which is a multidrug efflux pump resistant to fluoroquinolone antibiotics, \u003cem\u003essaA\u003c/em\u003e, which is staphylococcal secretory antigen, \u003cem\u003ecadC\u003c/em\u003e, and \u003cem\u003eyyc\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSalient features of predicted genomic islands of NASM strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNASM strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of Islands\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of genes in GIs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo. of genes coding for hypothetical Proteins\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVirulence \u0026amp; and antimicrobial resistance genes in genomic islands\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. lentus\u003c/em\u003e K169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003efosB 2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. sciuri\u003c/em\u003e K117.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003efosB, cadC, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. sciuri\u003c/em\u003e K14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. sciuri\u003c/em\u003e K91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003efosB, cadC, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eyyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eyyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. chromogenes\u003c/em\u003e K29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003elip\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ecadC, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eebh, blaZ, msr, qacA, qacR, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eblaZ, msr, ssaA, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e K60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ecadC, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. gallinarium\u003c/em\u003e B13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e A11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eblaZ, cadC, farB, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e A3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eblaZ, cadC, farB, arsR, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e K16.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eblaZ, msr, qacC, cadC, farB, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. haemolyticus\u003c/em\u003e K47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eblaZ, msr, cadC, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. hominis\u003c/em\u003e K24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eebh, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. pseudintermedius\u003c/em\u003e B32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003espa, lip, msr, ssaA, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. xylosus\u003c/em\u003e K19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003elip, yyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. xylosus\u003c/em\u003e K46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eyyc (walR/K)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. xylosus\u003c/em\u003e SMG24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ecadC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eS. haemolyticus\u003c/em\u003e strains carried \u003cem\u003efarB\u003c/em\u003e, which is the cytoplasmic transporter protein that is part of the farAB efflux pump that confers resistance to fatty acids, \u003cem\u003earsR\u003c/em\u003e, coding for arsenic resistance, and \u003cem\u003eblaZ\u003c/em\u003e, \u003cem\u003ecadC\u003c/em\u003e, \u003cem\u003eyyc\u003c/em\u003e, \u003cem\u003emsr\u003c/em\u003e, and \u003cem\u003eqac\u003c/em\u003e genes. \u003cem\u003eS. hominis\u003c/em\u003e strains contained \u003cem\u003eebh\u003c/em\u003e and \u003cem\u003eyyc\u003c/em\u003e genes. The \u003cem\u003espa\u003c/em\u003e, \u003cem\u003elip\u003c/em\u003e, \u003cem\u003emsr\u003c/em\u003e, \u003cem\u003essaA\u003c/em\u003e, and \u003cem\u003eyyc\u003c/em\u003e genes were found in \u003cem\u003eS. pseudintermedius\u003c/em\u003e strains. \u003cem\u003eS. xylosus\u003c/em\u003e strains carried \u003cem\u003elip\u003c/em\u003e, \u003cem\u003eyyc\u003c/em\u003e, and \u003cem\u003ecadC\u003c/em\u003e. All \u003cem\u003eS. haemolyticus\u003c/em\u003e strains consisted of similar islands. \u003cem\u003eS. haemolyticus\u003c/em\u003e strains K16 and K47 had similar islands, GI-1, 2, and 3. \u003cem\u003eS. haemolyticus\u003c/em\u003e strains A11 and A3 consisted of GI-2 in common. \u003cem\u003eS. epidermidis\u003c/em\u003e strains K60 and K16 possessed GI-3 in common. In GI-1 of \u003cem\u003eS. chromogenes\u003c/em\u003e strain K29 was similar to the GI-2 of \u003cem\u003eS. chromogenes\u003c/em\u003e strain K23. The GI-1 was found in common in \u003cem\u003eM. sciuri\u003c/em\u003e strains K117 and K91. A complete list of genomic islands from the 22 NASM strains is shown in Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMastitis significantly threatens the dairy industry, causing substantial revenue losses globally. Other than \u003cem\u003eS. aureus\u003c/em\u003e, the non-\u003cem\u003eaureus Staphylococcus\u003c/em\u003e and \u003cem\u003eMammaliicoccus\u003c/em\u003e (NASM) are largely responsible for subclinical and clinical mastitis. The disease progression involves a dynamic process shaped by multiple factors, such as the host's genetics, host as well as bacterial resistance mechanisms, host immune response, geographical influences, virulence factors (VFs) and the genetic variability of the bacterium. A comprehensive genome analysis of mastitis-associated NASM strains from diverse geographical regions could enhance our understanding of these factors, aiding in assessing pathogenic potential and infection risk, disease manifestation, and transmission dynamics.\u003c/p\u003e \u003cp\u003eWe conducted a comparative genomic study on 22 strains of NASM that caused bovine mastitis in three states of India. We sequenced their whole-genomes and identified STs specific to each species, along with the distribution of AMR genes and virulence factors. In previous studies, an ANI threshold of \u0026lt;\u0026thinsp;96% was suggested for identifying NASM at the species level (Kim et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). By comparing the nucleotide sequences of all the 22 strains, we found that members of the same species had ANI values consistently above 96%, indicating significant genomic similarity. On the other hand, members of different species had less than 96% ANI, indicating a clear genomic distinction between different NASM species. Our study supports the 96% ANI threshold value for species differentiation. This threshold helps understand and classify microbial diversity within this group, making it a valuable criterion for delineating species boundaries. The genome-wide SNP-based phylogeny analysis supported the ANI-based analysis, and a similar phylogenetic classification was observed.\u003c/p\u003e \u003cp\u003eThe minimum spanning tree (MST) based on SNPs can help us understand the complex relationships among closely related strains. It can reveal patterns of co-evolution, host specialization, and potential transmission pathways. We noticed that strains associated with bovine mastitis clustered together as separate subclades in all the NASM genomes. Previous studies have reported that \u003cem\u003eS. chromogenes\u003c/em\u003e ST-1 and ST-6 strains are associated with bovine mastitis (Huebner et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Persson Waller et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our observation also documents that \u003cem\u003eS. chromogenes\u003c/em\u003e ST-1 and ST-6 are associated with bovine mastitis. Additionally, we found that \u003cem\u003eS. epidermidis\u003c/em\u003e genotypes ST111 and ST59 are associated with bovine mastitis, which is consistent with another earlier report (Frey et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similar sub-clusters unique to particular hosts have been observed in other pathogens (Grillov\u0026aacute; \u0026amp; Picardeau, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In several European countries, \u003cem\u003eS. aureus\u003c/em\u003e CC8 strains have been linked with bovine mastitis. Recently, we reported that \u003cem\u003eS. aureus\u003c/em\u003e CC97 strains isolated from India were also associated with mastitis (Sivakumar et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Further research is needed to understand the relevance of subclade-specific SNPs and their possible association with bovine mastitis to better understand their pathogenicity.\u003c/p\u003e \u003cp\u003eThe increase in drug-resistant strains makes it challenging to treat bovine mastitis with antimicrobial intervention. In our study, we found 32 AMR genes in the 22 NASM genomes. Only one of the 22 strains had \u003cem\u003emecA\u003c/em\u003e and other \u003cem\u003emec\u003c/em\u003e-related genes. Previous studies have documented the presence of \u003cem\u003emecA\u003c/em\u003e-positive \u003cem\u003eS. epidermidis\u003c/em\u003e strains in bovine milk samples and the clonal dissemination of multi-drug-resistant \u003cem\u003eS. epidermidis\u003c/em\u003e strains carrying \u003cem\u003emecA\u003c/em\u003e within herds (Sawant et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The emergence of methicillin-resistant \u003cem\u003eS. epidermidis\u003c/em\u003e (MRSE) in cattle highlights the need for increased attention, with some researchers suggesting that animals infected with methicillin-resistant NASM should be culled (Gentilini et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The \u003cem\u003eS. epidermidis\u003c/em\u003e strain K4.3 investigated in this study exhibited resistance to methicillin, classifying it as MRSE. However, the other 21 NASM strains analyzed were methicillin-sensitive staphylococcus (MSS) and did not contain \u003cem\u003emecA\u003c/em\u003e, \u003cem\u003emecR1\u003c/em\u003e, or \u003cem\u003emec1\u003c/em\u003e genes in their genomes.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eblaZ\u003c/em\u003e gene was discovered in 6 out of 22 NASM strains. This gene is responsible for the production of penicillinase, which is the primary mechanism of penicillin resistance in NASM (Olsen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Multi-drug resistant (MDR) efflux pumps were dispersed throughout the strains and were not associated with specific species or STs. The regulation of MDR efflux pumps is a complex process, requiring multiple regulators to express these elements (Costa et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Antiabong et al., 2017). Therefore, the presence of these efflux pumps may not necessarily translate to an AMR phenotype.\u003c/p\u003e \u003cp\u003eWe have identified 53 virulence-associated genes among the 22 NASM genomes. Of these, 15 genes are critical for adherence and biofilm development. The \u003cem\u003eica\u003c/em\u003e operon, which produce polysaccharide intercellular adhesins (PIA) and is a commonly found genetic component in biofilm formation (McKenney et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) stands out as a crucial player. The \u003cem\u003eica\u003c/em\u003e gene was present in \u003cem\u003eS. xylosus\u003c/em\u003e and \u003cem\u003eM. sciuri\u003c/em\u003e strain K14. The impact of \u003cem\u003eica\u003c/em\u003e genes is particularly noticeable within NASM species linked to the human environment (O\u0026rsquo;Gara, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The diversity of studies examining different variants of the \u003cem\u003eica\u003c/em\u003e genes poses a challenge in understanding the involvement of this gene in biofilm formation (Tremblay et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). All of our NASM strains, excluding \u003cem\u003eMammaliicoccus\u003c/em\u003e spp., have genes that code for lipases. Cell-wall-associated proteins and enzymes play a significant role in the pathogenesis of staphylococci and are essential targets for drug development (Hu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Lipases in \u003cem\u003eS. aureus\u003c/em\u003e, known as SAL, have been identified in community-associated methicillin-resistant strains (Cadieux et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rosenstein, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). .\u003c/p\u003e \u003cp\u003eThe \u003cem\u003espa\u003c/em\u003e gene, a major antigen of \u003cem\u003eS. aureus\u003c/em\u003e, was exclusively detected in \u003cem\u003eS. pseudintermedius\u003c/em\u003e. The \u003cem\u003esspA\u003c/em\u003e gene was found in 12 strains, while the \u003cem\u003esspB\u003c/em\u003e gene was found exclusively in \u003cem\u003eS. epidermidis\u003c/em\u003e. In \u003cem\u003eS. epidermidis\u003c/em\u003e, the \u003cem\u003esspA\u003c/em\u003e and \u003cem\u003esspB\u003c/em\u003e genes were found to coexist, indicating a potential interplay or cooperative function. The initial player in the staphylococcal proteolytic cascade is aureolysin, a metalloprotease. This enzyme undergoes a rapid process of autocatalytic activation. Subsequently, the activated aureolysin plays a crucial role in activating the \u003cem\u003esspA\u003c/em\u003e serine protease, which in turn, serves as a critical activator for the \u003cem\u003esspB\u003c/em\u003e cysteine protease (Massimi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Nickerson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Such cascades are often pivotal in regulating various cellular processes and contribute to the pathogenicity and adaptability of members of the genus \u003cem\u003eStaphylococcus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe type VII secretion system (T7SS) has been recently identified and selectively distributed in various pathogens, including \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. The T7SS plays an essential role in the virulence of human pathogens. In the case of \u003cem\u003eM. tuberculosis\u003c/em\u003e, the T7SS is very important for bacterial access to the host cytosol. In \u003cem\u003eS. aureus\u003c/em\u003e, T7SS exports several virulence-associated proteins (Lopez et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The presence of T7SS cluster was reported in \u003cem\u003eS. lugudensis\u003c/em\u003e, in addition to \u003cem\u003eS. aureus\u003c/em\u003e (Lebeurre et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Staphylococci possess genes enabling the production of capsular polysaccharides that form a protective shield against phagocytosis by the host's immune cells. This capsule enhances virulence and bacterial persistence, highlighting encapsulation as a crucial mechanism for evading immune detection and contributing to pathogenicity. The \u003cem\u003ecapB\u003c/em\u003e and \u003cem\u003ecapC\u003c/em\u003e genes were detected in all NASM species except \u003cem\u003eMammaliicoccus\u003c/em\u003e spp. and \u003cem\u003eS. pseudintermedius\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eProphages are responsible for the horizontal gene transfer, which in turn contributes to virulence (Bae et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Smirnova et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The prophage \u003cem\u003eStaphylococcus\u003c/em\u003e StB20 was found in \u003cem\u003eS. xylosus\u003c/em\u003e strain SMG24. This prophage is reported to exhibit specific proteolytic cleavages in the carboxy-terminal degradation of its tail tape measure proteins (TMP) in \u003cem\u003eS. aureus\u003c/em\u003e (Tallent et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, the roles of prophages NASM strains are largely unknown.\u003c/p\u003e \u003cp\u003eGenomic islands (GIs) are horizontally transferred regions carrying specific genes that confer certain traits to bacteria. These traits include metabolic processes, pathogenicity, antibiotic resistance, and symbiosis. GIs help bacteria establish mutually beneficial relationships with eukaryotic hosts. They often carry genetic material related to virulence or adaptive traits and are commonly located near tRNA or transposase genes at one end of the island. Antibiotic-resistant genes found in GIs make them carriers for the spread of antibiotic resistance, enhancing bacterial species' survival when exposed to antibiotics (Citti et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Coupling virulence or adaptive traits with antibiotic resistance in GIs plays a significant role in shaping bacterial populations' resilience and adaptability to environmental challenges, especially when exposed to antibiotics. In the 22 NASM that we studied, most islands carried the two-component regulatory system, which includes the \u003cem\u003ewalRK\u003c/em\u003e gene. This gene plays a crucial role in regulating the expression of genes associated with cell wall metabolism, influencing autolysis, biofilm formation, and virulence. WalK is also involved in sensing the D-Ala-D-Ala moiety of Lipid II, serving as a signal for active cell wall synthesis. The genes \u003cem\u003eyycH\u003c/em\u003e and \u003cem\u003eyycI\u003c/em\u003e are co-transcribed with \u003cem\u003ewalRK\u003c/em\u003e and modulate its activity. In \u003cem\u003eS. aureus\u003c/em\u003e, disrupting \u003cem\u003eyycH\u003c/em\u003e and \u003cem\u003eyycI\u003c/em\u003e genes downregulated the \u003cem\u003ewalRK\u003c/em\u003e regulon (Gajdiss et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The importance of these regulatory components and their roles in governing cell wall-related processes vary across bacterial species. The \u003cem\u003efosB\u003c/em\u003e, \u003cem\u003eblaZ\u003c/em\u003e, and several AMR genes were identified among the GIs, suggesting the possibility of transfer of these traits among the mastitis-associated pathogens.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWhole genome sequencing and phylogeny analysis of bovine mastitis-associated NASM strains isolated from India revealed species-specific and host-specific clustering. The study identified multiple genes responsible for antibiotic resistance and virulence factors. Certain virulence factors were found to be specific to particular species, and some were specific to particular STs. The analysis also found that some virulence and AMR genes were located in genomic islands, which suggests possible horizontal transfer events.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eWe declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements \u003c/h2\u003e\n\u003cp\u003eFunding from the Department of Biotechnology (DBT), New Delhi (BT/PR11245/ADV /90/165/2014) to NRH, SI, and JR is gratefully acknowledged. The MKU-RUSA, UGC-NRCBS, DST-PURSE, DST-FIST Programs of the School of Biological Sciences, Madurai Kamaraj University, are acknowledged.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Department of Biotechnology (DBT), New Delhi, (BT/PR11245/ADV/90/165/2014).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.R.H., J.R., and S.I. designed the experiments; M.A. and S.C. propagated and characterized the NASM strains from the various States of India; V.R. and R.S. performed WGS and analysis; V.R., S.I., J.R., and N.R.H. wrote the paper. The authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAanensen, D. M., \u0026amp; Spratt, B. G. (2005). The multilocus sequence typing network: Mlst.net. 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Genome Research, \u003cem\u003e28\u003c/em\u003e(9), 1395\u0026ndash;1404. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gr.232397.117\u003c/span\u003e\u003cspan address=\"10.1101/gr.232397.117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bovine Mastitis, Non-aureus Staphylococci and Mammaliicocci, MLST, Resistome, Virulome","lastPublishedDoi":"10.21203/rs.3.rs-4508846/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4508846/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBovine mastitis is a significant issue causing severe economic losses in the global dairy industry, affecting animal well-being and production. Non-\u003cem\u003eaureus\u003c/em\u003e staphylococci and mammaliicocci (NASM) are the predominant group of pathogens responsible for mastitis in dairy cattle. Understanding the prevalence of virulence factors and antimicrobial resistance among these pathogens is essential to unravel the molecular epidemiology of mastitis, and it is best accomplished through whole-genome sequencing (WGS). In this study, we describe the WGS and comparative genomic analysis of 22 mastitis-associated NASM strains isolated from India. The mean genome size of the strains was 2.55 Mbp, with an average GC content of 32.2%. We identified 14 different sequence types (STs) among the 22 NASM strains. Of these, ST1 and ST6 of \u003cem\u003eS. chromogenes\u003c/em\u003e were exclusively associated with bovine mastitis. Genome-wide SNP-based minimum spanning tree revealed the intricate phylogenetic relationships among NASM strains from India, categorizing them into five major clades. Interestingly, mastitis-associated strains formed separate subclades in all the NASM species studied, indicating distinct host-specific co-evolution. The study identified 32 antimicrobial resistance (AMR) genes and 53 virulence-associated genes, providing insights into the genetic factors which could potentially contribute to the pathogenicity of NASM species. Some virulence and AMR genes were found in the predicted genomic islands, suggesting possible horizontal transfer events.\u003c/p\u003e","manuscriptTitle":"Genome sequencing and comparative genomic analysis of bovine mastitis-associated non-aureus staphylococci and mammaliicocci (NASM) strains from India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 14:12:32","doi":"10.21203/rs.3.rs-4508846/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-09T08:52:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-24T02:38:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149455058720674306907724642178520216430","date":"2024-08-19T10:10:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-25T13:22:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59042850674053997964340223892208257635","date":"2024-07-19T11:27:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120698604938340478549374145533609982931","date":"2024-07-19T07:23:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-18T21:14:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-18T21:06:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-07T17:43:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-04T11:11:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-31T12:09:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9691ea1f-a1fe-4cf4-adc3-968772d21654","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35130854,"name":"Biological sciences/Immunology"},{"id":35130855,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2024-11-25T16:06:54+00:00","versionOfRecord":{"articleIdentity":"rs-4508846","link":"https://doi.org/10.1038/s41598-024-80533-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-22 15:57:33","publishedOnDateReadable":"November 22nd, 2024"},"versionCreatedAt":"2024-07-29 14:12:32","video":"","vorDoi":"10.1038/s41598-024-80533-9","vorDoiUrl":"https://doi.org/10.1038/s41598-024-80533-9","workflowStages":[]},"version":"v1","identity":"rs-4508846","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4508846","identity":"rs-4508846","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}