Genome based Investigation of AMR Determinants in High-Priority Bacterial Pathogens Circulating in India

Genome based Investigation of AMR Determinants in High-Priority Bacterial Pathogens Circulating in 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 Research Article Genome based Investigation of AMR Determinants in High-Priority Bacterial Pathogens Circulating in India Paparaidu Sanapala, Suraj Shukla, Santosh Pagare, Geeti Maheshwari, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9677975/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antimicrobial resistance (AMR) has emerged as a key focus of research in infectious diseases, owing to the severity of infections and elevated worldwide death rates. Pathogens such as Salmonella spp., S. aureus , and Enterococcus spp. have recently attracted attention because of their escalating resistance; therefore, they have been classified as high-priority pathogens by the WHO. This study aimed to obtain a comprehensive understanding of the AMR patterns among these pathogens Eighteen clinical isolates, comprising Salmonella spp. ( n = 10), S. aureus ( n = 5), and Enterococcus spp. ( n = 3), were collected and tested for antibiotic susceptibility, subsequently subjected to whole-genome sequencing. Additionally, 535 genome submissions including Salmonella spp. ( n = 344), S. aureus ( n = 142), and Enterococcus spp. ( n = 49) from India were retrieved from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) for bioinformatics analysis and were annotated for sequence types (STs), AMR genes, their locations, and plasmids. Multidrug resistance (MDR) was identified in 50% of the Salmonella isolates, all S. aureus , and a single E. faecium isolate; conversely, the remaining Salmonella and both E. faecalis isolates were susceptible. Genomic analysis identified the dominant STs: ST1 in both clinical isolates and retrieved genomes of Salmonella ; ST22 in clinical isolates, and ST772 in retrieved S. aureus , while ST80 was observed in retrieved E. faecium . All dominant STs were highly resistant, however, ST1 in Salmonella retrieved genomes exhibited reduced resistance. Overall, among high-priority pathogens, Salmonella remains manageable due to lower resistance; however, S. aureus , particularly ST772 and ST239, as well as E. faecium , mainly ST80, exhibits a significant risk for severe infections due to their extensive gene reservoirs and high prevalence. Molecular Epidemiology Infectious Diseases Bioinformatics Antimicrobial resistance Salmonella spp. Staphylococcus aureus Enterococcus spp. High priority pathogens Whole genome sequencing Figures Figure 1 Figure 2 Figure 3 1. Introduction The upsurge of antibiotic-resistant bacteria signifies a major global health threat. It challenges many of the lifesaving drugs and puts millions of us at risk of incurable infections. Failure to address this challenge puts every individual at risk. The Global Research on Antimicrobial Resistance (GRAM), a collaborative project between University of Oxford and Institute for Health Metrics and Evaluation, provides the first comprehensive analysis of global antimicrobial resistance (AMR) trends, reported one million people died each year due to AMR between 1990–2021. It approximates 1.91 million potential deaths by 2050 as result of AMR, an increase of 75% over 2022 (Naghavi et al., 2024 ). In response to the antimicrobial resistance (AMR) challenges, the WHO Country Office for India, in partnership with the Department of Biotechnology, Government of India, announced a list of Indian Priority Pathogens, categorizing them into three distinct priority tiers depending on resistance: critical, high, and medium (WHO (INDIA) & DBT, GOI, 2021). Critical-priority pathogens are antibiotic-resistant (ABR) bacteria posing the greatest public health threat due to limited treatment options, high morbidity and mortality, increasing resistance, and a lack of effective drugs in development. High-priority pathogens are ABR bacteria that are difficult to treat, with significant disease burden, show rising resistance, and may be highly transmissible; while not globally critical, they can be severe in specific populations or regions (WHO (INDIA) & DBT, GOI, 2021). The Indian high-priority bacterial pathogens encompass gram-negative Salmonella species as well as the gram-positive pathogens Staphylococcus aureus and Enterococcus species. Salmonella is a rod-shaped microorganism that is part of the Enterobacteriaceae family. These pathogens are non-sporulating facultative anaerobes with significant zoonotic potential. They are the primary etiological agents of numerous gastrointestinal and systemic infectious diseases in both mammals and avians. S. aureus is a clinically significant species, flexible, facultative aero-anaerobic coccus, serves as both a common commensal and a significant opportunistic pathogen (Tigabu & Getaneh, 2021 ). S. aureus causes a wide array of diseases, from localized skin and soft tissue infections (SSTIs) to severe systemic illnesses, such as pneumonia, septic arthritis, osteomyelitis, endocarditis, and bacteremia (Taylor et al., 2026 ). Enterococcus faecium and Enterococcus faecalis are facultative anaerobic cocci and the principal etiological agents of human enterococcal diseases. Major enterococcal infections are caused by E. faecalis , however, in clinical setting E. faecium cases are increasing (Sangiorgio et al., 2024 ). Therapeutic management is increasingly compromised by the emergence of resistance phenotypes against last resort antibiotics. This includes alarming increase in azithromycin and third generation cephalosporins resistance in Salmonella , vancomycin-resistance in Enterococci (VRE), and methicillin-resistance in S. aureus (MRSA). The clinical challenge is further compounded by delayed diagnostic turnaround times, which prevent the timely initiation of targeted antimicrobial therapy. The mph(A) and erm genes together with AcrB efflux pump protein mutations and ribosomal protein 23S (rRNA) mutations function as essential factors that contribute to azithromycin resistance and extended spectrum beta-lactamases (ESBLs) genes such as bla CTX−M−15 , bla TEM−1 and bla SHV−12 in Salmonella (Argimón et al., 2022 ; Chavan & Angadi, 2025 ). MRSA is characterized by the incorporation of the SCCmec genetic element, which harbors the mecA or mecC genes that provide resistance to methicillin. Although medicines such as Linezolid are often successful against these infections, the introduction of the cfr gene is progressively enabling bacteria to circumvent this treatment (Suleiman et al., 2025 ). Vancomycin-Resistant Enterococcus (VRE) evades antibiotics by modifying its cell wall precursors through the vanA and vanB genes. Collectively, these genetic adaptations pose considerable obstacles in the treatment of contemporary gram-positive bacterial illnesses (Pidgeon & Pires, 2017 ). Detecting AMR genes with high accuracy is crucial for managing infections, typically through Whole-genome sequencing (WGS) followed by bioinformatics analysis using tools like the Comprehensive Antibiotic Resistance Database (CARD) and Resistance Gene Identifier (RGI). These tools classify detected genes as perfect (100% match), strict (high sequence similarity to curated reference model, but not 100% match) and loose (variants with low similarity and below the threshold) hits to distinguish known resistance markers from distant homologs, new or unknown variants (Alcock et al., 2019 ). Whole-genome sequencing augments antimicrobial resistance surveillance by offering extensive insights into AMR mechanisms, gene profiling, transmission dynamics, facilitating prompt investigation and implementation of infection control measures (Oniciuc et al., 2018 ; Matsumura et al., 2025 ). A combined data for high-priority pathogens Salmonella species, Staphylococcus aureus and Enterococcus species from India is limited. Further, to our knowledge, this is first such attempt from India utilizing publicly accessible whole genome sequencing data and clinical isolates for all three high-priority pathogens to elucidate the circulating antimicrobial resistance genes in the country. This study will aid us to understand the current AMR patterns in high-priority pathogens across India, rather than relying on global data, and address the challenges associated with locating fragmented data. This will further assist in diagnostics and healthcare management to combat antimicrobial resistance. 2. Methodology 2.1 Sample collection & Identification : Clinical specimens of Salmonella enterica ( n = 10), Staphylococcus aureus ( n = 5), Enterococcus faecalis ( n = 2), and Enterococcus faecium ( n = 1) were obtained from Toprani Advanced Lab Systems, Vadodara. All isolates were subcultured and reidentified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) prior to sending for whole genome sequencing. 2.2 Antibiotic susceptibility testing: Antibiotic susceptibility testing was conducted with the VITEK® system (bioMérieux, Marcy-l'Étoile, France) and the disc diffusion method in accordance with M02, Clinical and Laboratory Standards Institute (CLSI) M100 standards. Antibiotics were selected for various species as delineated by (Magiorakos et al., 2012 ). Isolates were further classified as sensitive, multidrug-resistant (MDR) and extensively drug-resistant (XDR), based on the AST profile for respective isolates. 2.3 DNA extraction and Whole genome sequencing: Genomic DNA was extracted from 18 clinical isolates by using the XpressDNA Bacterial DNA extraction kit (MagGenome, Chennai, India) in accordance with the manufacturer's protocol. All isolates subjected to sequencing by Illumina MiSeq platform (Illumina, CA, USA) using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) to prepare NGS libraries according to the directions provided in the manual. All 18 sequences submitted to NCBI (BioProject no.: PRJNA1431137), with each sequence's accession number provided in Table 1 . Table 1 Genomic accession number of clinical isolates Sr. no. Sample name Strain name Genome accession no. Species 1 PSMSU_001 MSUH228 JBVVVM000000000 Staphylococcus aureus 2 PSMSU_002 MSUH231 JBVVVL000000000 Staphylococcus aureus 3 PSMSU_003 MSUH256 JBVVVK000000000 Staphylococcus aureus 4 PSMSU_004 MSUH257 JBVVVJ000000000 Staphylococcus aureus 5 PSMSU_005 MSUH258 JBVVVI000000000 Staphylococcus aureus 6 PSMSU_006 MSUH241 JBVVVH000000000 Salmonella enterica 7 PSMSU_007 MSUH242 JBVVVG000000000 Salmonella enterica 8 PSMSU_008 MSUH245 JBVVVF000000000 Salmonella enterica 9 PSMSU_009 MSUH246 JBVVVE000000000 Salmonella enterica 10 PSMSU_010 MSUH247 JBVVVD000000000 Salmonella enterica 11 PSMSU_011 TPS71 JBVVVC000000000 Salmonella enterica 12 PSMSU_012 TPS72 JBVVVB000000000 Salmonella enterica 13 PSMSU_013 TPS73 JBVVVA000000000 Salmonella enterica 14 PSMSU_014 TPS74 JBVVUZ000000000 Salmonella enterica 15 PSMSU_015 TPS75 JBVVUY000000000 Salmonella enterica 16 PSMSU_016 MSUH265 JBVVUX000000000 Enterococcus faecium 17 PSMSU_017 MSUH331 JBVVUW000000000 Enterococcus faecalis 18 PSMSU_018 MSUH333 JBVVUV000000000 Enterococcus faecalis 2.4 NGS Data analysis: The raw data were assessed in the Galaxy web server ( https://usegalaxy.eu/ ). FastQC tool v0.74 + galaxy1 was utilized for quality assessment of raw reads, Trimmomatic v0.39 + galaxy2 was employed for filtering and trimming adaptors of low-quality reads, followed by De novo assembly preparation using the Unicycler tool v0.5.1 + galaxy0. Assembly quality was assessed using the QUAST tool v5.3.0 + galaxy1. The genome annotation was conducted using the Prokka tool v1.14.6 + galaxy1. 2.5 Genome retrieval: Since we had limited numbers of clinical isolates for genomic analysis, to define the clear picture of AMR gene pattern in India, we obtained the extensive genomes of these bacterial species from the bacterial and viral bioinformatics resource center (BV-BRC) tool v3.42.3, which included Salmonella spp. ( n = 344), S. aureus ( n = 142), and Enterococcus spp. ( n = 49) which included E. faecium ( n = 36), E. faecalis ( n = 10), E. lactis ( n = 2), and E. casseliflavus ( n = 1) till December 2024. We employed the filters "Genome Quality: Good, Genome Status: Complete and/or WGS, Isolation Country: India, Host Group: Human" for genome retrieval. 2.6 Bioinformatics analysis: To identify the antimicrobial resistance genes, the Resistance Gene Identifier (RGI) v6.0.5 from the Comprehensive Antibiotic Resistance Database (CARD) v4.0.1 ( https://card.mcmaster.ca/ ) and the ResFinder tool v4.7.2 from the Centre for Genomic Epidemiology ( https://www.genomicepidemiology.org/ ) were utilized, while their genomic locations (either on chromosomes or plasmids) were determined using PlasFlow Galaxy v1.1.0 + galaxy0 ( https://github.com/smaegol/PlasFlow ). Multi-locus sequence typing (MLST) was ascertained using organism-specific MLST from BIGSdb ( https://pubmlst.org/software/bigsdb ). Plasmid replicons were identified using the Plasmidfinder tool v2.1 ( https://cge.food.dtu.dk/services/PlasmidFinder/ ). A phylogenetic tree based on single nucleotide polymorphisms (SNPs) was constructed using CSI Phylogeny v1.4 software ( https://cge.food.dtu.dk/services/CSIPhylogeny/ ). SNP calling and filtering were performed using the default CSI Phylogeny pipeline with the following thresholds: minimum depth at SNP positions of 10×, minimum relative depth of 10%, minimum SNP quality score of 30, minimum read mapping quality of 25, and a minimum Z-score of 1.96. These filtering criteria were applied to ensure high-confidence SNP detection across the analyzed genomes. The reference strain LT2 (GenBank no.: GCA_000006945.2) for S. enterica , strain NCTC 8325 (GenBank no.: GCA_000013425.1) for S. aureus , and strain T5 (GenBank no.: GCA_000393015.1) for Enterococcus spp. were selected due to their well-annotated and widely used complete genome for comparative analyses in respective species. Subsequently, the obtained data was annotated and compiled using the iTOL tool v7.5.1, accessible at ( https://itol.embl.de/itol.cgi ). 3. Result 3.1 MALDI- TOF identification All 18 clinical isolates grown overnight on Luria-Bertani (LB) and subjected to identification by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) showed a score greater than 2 with a confidence range (2.08 to 2.42), except 4 TPS (TPS71, TPS72, TPS73, and TPS74) isolates showed a range between 1.6 and 1.98 as listed in Table 2 and supplementary sheet 1. 3.2 Antibiotic susceptibility testing (AST) In drug susceptibility testing for Salmonella , all five MSUH isolates exhibited sensitivity to antibiotics, except for ciprofloxacin. On the other hand, the five TPS isolates were resistant to multiple antibiotics, demonstrating non-susceptibility to extended-spectrum cephalosporins (third and fourth generation: cefepime, cefotaxime, ceftazidime), fluoroquinolones (ciprofloxacin), folate pathway inhibitors (trimethoprim-sulfamethoxazole), monobactams (aztreonam), non-extended spectrum cephalosporins (first and second generation: cefazolin, cefuroxime, ampicillin), and tetracyclines (doxycycline, tetracycline). In case of S. aureus , all five isolates exhibited lower multidrug resistance and demonstrated non-susceptibility to anti-staphylococcal β-lactams (oxacillin), fluoroquinolones (ciprofloxacin, moxifloxacin), lincosamides (clindamycin), macrolides (erythromycin), and streptogramins (quinupristin-dalfopristin). All five isolates were classified as MRSA, as they exhibited an intermediate level of resistance to Methicillin, except for isolate MSUH256, which showed full resistance. However, they exhibited susceptibility to major antibiotics such as oxazolidinone (linezolid), cyclic lipopeptide (daptomycin), and glycylcyclines (tigecycline). In Enterococcus spp., the E. faecium isolate MSUH265 exhibited non-susceptibility to carbapenems (doripenem and imipenem), fluoroquinolones (ciprofloxacin, levofloxacin), streptogramins (quinupristin-dalfopristin), tetracycline (doxycycline), and penicillins (ampicillin); it demonstrated intermediate susceptibility to tetracycline (minocycline) and aminoglycoside (gentamicin). Although both E. faecalis isolates (MSUH331 and MSUH333) were classified as sensitive, they exhibited non-susceptibility to gentamicin, ciprofloxacin, levofloxacin and quinupristin-dalfopristin, as well as intermediate susceptibility to imipenem. However, all three isolates exhibited susceptibility to glycopeptide (vancomycin), tigecycline, daptomycin, and linezolid. The phenotypic analysis of 18 clinical isolates revealed (provided in supplementary file 1) a notable occurrence of multidrug resistance (MDR) across different bacterial species, with 11 isolates (61.1%) classified as MDR and 7 (38.9%) as susceptible listed in Table 2 . Salmonella enterica isolates ( n = 10), there was a split in the resistance profile: 50% were multidrug-resistant (TPS71, TPS72, TPS73, TPS74, and TPS75), and the other 50% were susceptible (MSUH241, MSUH242, MSUH245, MSUH246, and MSUH247). All five S. aureus isolates (MSUH228, MSUH231, MSUH256, MSUH257, and MSUH258) were found to be MDR. Among the Enterococcus species, the single E. faecium isolate (MSUH265) displayed MDR, while both E. faecalis isolates (MSUH331 and MSUH333) showed susceptibility. This data highlights a significant burden of multidrug resistance, particularly in S. aureus and specific strains of S. enterica , however the overall number of isolates was less. Table 2 Identification and resistance categorization of clinical isolates Isolates MALDI Identification Resistance Category Isolates MALDI Identification Resistance Category MSUH241 Salmonella sp. Susceptible TPS75 Salmonella sp. MDR MSUH242 Salmonella sp. Susceptible MSUH228 S. aureus MDR MSUH245 Salmonella sp. Susceptible MSUH231 S. aureus MDR MSUH246 Salmonella sp. Susceptible MSUH256 S. aureus MDR MSUH247 Salmonella sp. Susceptible MSUH257 S. aureus MDR TPS71 Salmonella sp. MDR MSUH258 S. aureus MDR TPS72 Salmonella sp. MDR MSUH265 E. faecium MDR TPS73 Salmonella sp. MDR MSUH331 E. faecalis Susceptible TPS74 Salmonella sp. MDR MSUH333 E. faecalis Susceptible 3.3 Whole genome sequence data statistics The whole-genome sequencing and de novo assembly of the 18 clinical isolates produced high-quality genomic blueprints, revealing species-specific characteristics and assembly inconsistencies as shown in Table 3 . The Salmonella enterica isolates ( n = 10) had the largest genomes, which ranged from 4.55 to 4.98 Mb, and their GC content was consistent at around 52%. They also had the most coding sequences (average 4,630 CDSs) and tRNA ranged from 67 to 75 in number. The TPS series has bigger genomes (4.96 Mb on an average) than the MSUH series (4.63 Mb on an average) in this species. On the other hand, the S. aureus isolates ( n = 5) had smaller genomes (2.75 to 2.83 Mb) and a much lower GC content (average 32.73%). The number of CDSs ranged from 2,537 to 2,638. Except MSUH228 with 54 tRNA, all four isolates had 58 tRNA in number. The three Enterococcus species had genomic sizes between 2.72 and 2.81 Mb and GC contents of about 37.6%. E. faecium had 2656 CDSs along with 54 tRNA, whereas in both the E. faecalis isolates the number of CDSs were 2517 and 2634 respectively, with 48 and 44 tRNA. The S. enterica TPS series demonstrated more fragmentation, encompassing up to 289 contigs. S. aureus showed moderate level of assembly contiguity with number of contigs in range of 51 to 65; whereas the assembly contiguity for E. faecalis MSUH331 was high with 12 contigs and N50 of 1,410,860 bp. Across the 18 isolates tmRNA was confined to 1 in number, while the rRNA counts ranged from 2 to 10, which highlights the structural diversity among the clinical isolates. Table 3 Whole genome sequencing data statistics of clinical isolates Isolates No. of contigs Largest contig GC (%) N50 Genome Size CDS rRNA tRNA tmRNA Repeat region MSUH241 64 466235 52.11 171823 4669728 4498 4 69 1 1 MSUH242 37 436308 52.16 241014 4559165 4357 3 75 1 2 MSUH245 73 261464 52.08 131909 4551637 4373 4 67 1 1 MSUH246 68 339749 52.04 143220 4706362 4532 4 70 1 1 MSUH247 81 392766 52.07 110055 4694109 4522 4 70 1 1 TPS71 105 217626 51.95 101842 4948661 4801 9 73 1 1 TPS72 214 163791 51.95 49279 4960659 4835 10 73 1 1 TPS73 270 123415 51.95 33210 4967293 4844 9 72 1 1 TPS74 289 143581 52 33697 4977480 4848 9 73 1 1 TPS75 167 163354 51.94 57652 4955158 4819 8 73 1 1 MSUH228 61 188020 32.72 120466 2762224 2557 2 54 1 - MSUH231 65 188019 32.73 109426 2762699 2558 4 58 1 - MSUH256 57 321337 32.71 156267 2833727 2638 4 58 1 - MSUH257 51 188019 32.75 135952 2799556 2618 2 58 1 - MSUH258 52 310602 32.73 156282 2749925 2537 4 58 1 - MSUH265 171 173501 37.79 55833 2813165 2656 4 54 1 - MSUH331 12 1410860 37.63 1410860 2721729 2517 3 48 1 2 MSUH333 21 569421 37.36 292188 2796204 2634 3 44 1 1 3.4 Genome analysis for MLST, AMR genes & their location, Plasmids, and their association. 3.4.1 MLST : In the clinical isolates shown in Fig. 1 (a) , six isolates of Salmonella enterica were categorized as ST1, two were previously undetected ST6142, the remaining two isolates one categorized as ST2 and the other one ST129. All S. aureus isolates were confined to a unique ST22 shown in Fig. 2 (a) . Whereas the three Enterococcus isolates exhibited distinct STs; E . faecium was defined as ST117, and E. faecalis isolates were designated as ST394 and ST134 shown in Fig. 3 (a) . MLST analysis of retrieved genomes as shown in Figure 1 (b) , 23 distinct STs identified within Salmonella genomes ( n = 344), with ST1 ( n = 196, 56.98%) being the predominant type, succeeded by a varied array of other STs: ST2 & ST19 ( n = 50, 14.53% each), ST198 ( n = 7, 2.03%), ST13 ( n = 5, 1.45%), and both ST36 and ST2344 at n = 4, 1.16% each, while the remaining 16 different STs accounted for less than 1% each. The analysis of 142 S. aureus genomes, 31 different STs were identified shown in Figure 2 (b) . The predominant ST was ST772, accounting for 34.51% ( n = 49), followed by ST239 at 9.86% ( n = 14), ST22 at 8.45% ( n = 12), ST672 at 7.75% ( n = 11), ST1 at 4.93% ( n = 7), ST1482 at 3.52% ( n = 5), ST6 at 2.82% ( n = 4), and ST121 at 2.11% ( n = 3). The remaining 27 STs each exhibited a frequency of less than 2%. Whereas in Enterococcus spp. ( n = 49), among four identified species, E. faecium ( n = 36) represented across seven different STs as shown in Figure 3 (b) . Notably, ST80 accounted for 80.55% ( n = 29), followed by ST17 at 5.55% ( n = 2), while the remaining isolates confined to individual ST. Remarkably, none of the STs identified in the E. faecalis genomes ( n = 10) were repetitive, each genome exhibited distinct STs. In contrast, both genomes of E. lactis were classified as ST798, while E. casseliflavus ( n = 1) could not be assigned any ST. 3.4.2 Association of AMR genes with plasmids and STs 3.4.2.1 Perfect Hits: In clinical isolates of Salmonella ( n = 10) collected in the present work as shown in Fig. 1 (a) , three distinct AMR genes, bla CTX−M−15 (ESBLs), sul2 (sulfonamide resistance), and qnrS1 (fluoroquinolone resistance), were identified exclusively on plasmids, surprisingly all AMR genes were found only in ST1 except for the MSUH247 isolate that does not carry AMR genes. Genes bla CTX−M−15 were identified in 66.66% isolates, whereas sul2 , and qnrS1 were detected in 83.33% isolates of ST1. Interestingly both plasmids IncFIB (pHCM2) and IncFIB(K) were confined to ST1 only. The analysis of retrieved Salmonella genomes ( n = 344) as shown in Fig. 1 (b) for AMR genes revealed that the beta-lactamase gene bla Tem−1 had the highest prevalence at 13.95%, followed by sul1 (sulfonamide resistance) at 12.50%, catA1 (phenicol resistance) at 10.76%, bla CMY (cephalosporin resistance) and AAC(6')-type (aminoglycoside resistance), each at 3.20%. Other resistance genes exhibited frequencies below 3% each. Fortunately, in Indian isolates, the occurrence of carbapenem resistance genes remains rare, with the bla NDM (carbapenem resistance) gene identified solely in isolate P5558. Additionally, the prevalence of the ESBL gene bla CTX−M was also below 3%. All the perfect hits genes among the isolates were located on plasmids, with a few exceptions like AAC(6')-type which was found primarily on chromosomes. Further, efflux genes were associated with resistance to specific antibiotic classes, such as armA and rmtC (aminoglycoside resistance) and qacEdelta1 (disinfectant and antiseptic resistance), as well as broader antibiotic classes, golS , mdsA , mdsB , msrE , and sdiA were identified across the genomes of Salmonella , with golS, mdsA, mdsB , and sdiA exhibiting the highest frequencies at 22.0%, 15.40%, 15.40%, and 16.86%, respectively. The number of efflux genes ranged from 0 to 5 across genomes, however, the instance of efflux genes of 5 and 3 were detected only in Bareilly_strain_FC745 and AMRIR00166 strains. Further, 15.40% of isolates possessed four efflux genes, 1.74% had two efflux genes, and 15.98% exhibited just one efflux gene. Interestingly, the bulk of isolates, 66.27%, exhibited no efflux genes. Most of the AMR genes were confined to a limited number of ST groups, such as ST1, ST13, ST198, and ST36. Genes bla TEM−1 , sul1 , and catA1 were predominantly associated with ST1 and ST13. Particularly, ST13 had several additional genes, namely bla CTX−M−15 and qnrB1 (fluoroquinolone resistance), which were present in all instances except one, whereas bla OXA−1 (beta-lactam resistance) was found in approximately half of the ST13. ST198 and ST36 possessed distinct gene sets: ST198 included bla TEM−1 , AAC(3)-Id (aminoglycoside resistance), and sul1 , whereas ST36 contained bla CMY and AAC(6')-type . Interestingly, neither of the second and third most prevalent sequence types, ST2 and ST19, exhibited any perfect gene hits, except two genomes of ST2 which contained a limited number of AMR genes, and each ST19 genome contained four efflux genes. Nonetheless, the quantity of the bla SHV−12 (beta-lactam resistance) gene was confined to only five genomes, yet all bla SHV−12 genes coexisted in five instances of the IncX3 plasmid. The majority of ST36 isolates possessed the bla CMY gene and harbored the IncI1-I(Alpha) plasmid. A significant association was observed, where all ST19 isolates had four efflux genes, and most genomes exhibited from the highly prevalent IncFIB(S) and IncFII(S) plasmids. In the clinical isolates of S. aureus (n = 5) collected in the present work as shown in Fig. 2 (a) , only AAC(6')-Ie-APH(2'')-Ia (aminoglycoside resistance) gene was found across all five genomes and harbored four efflux genes. A plasmid, rep10 was identified across all genomes, rep22 along with ANT(4')-la (aminoglycoside resistance) gene was also observed in 60% of isolates. The AMR investigation of retrieved S. aureus genomes ( n = 142), as shown in Fig. 2 (b) , indicated the presence of 10 distinct AMR genes, including aminoglycoside resistance genes APH(3')-IIIa (56.33%) and AAC(6')-Ie-APH(2'')-Ia (45.77%), the fosfomycin resistance gene fosB (48.59%), the diaminopyrimidine resistance gene dfrG (45.07%), the penicillin beta-lactam resistance gene blaPC1 (10.56%), and the macrolide resistance gene ermA (9.15%). Based on the genomic analysis, the large pool of genotypic resistance was noted with the predominant ST772, with 91.83% of genomes containing the APH(3')-IIIa gene, followed by 89.79% each for fosB and dfrG genes, and 67.34% for AAC(6')-Ie-APH(2'')-Ia (aminoglycoside resistance) gene. Out of which 65.30% of ST772 isolates co-harbored APH(3')-IIIa and AAC(6')-Ie-APH(2'')-Ia genes. Furthermore, ST772 also included efflux genes in a range of four to six. Except for a few genomes, all dfrG genes were located on plasmids, while all fosB genes (100%) were identified on chromosomes. Unfortunately, the locations of AAC(6')-Ie-APH(2'')-Ia and the bifunctional protein APH(3')-IIIa could not be discovered by the employed tool. The second most prevalent ST239 exhibited the highest degree of resistance. Some of the isolates within this group possessed almost all varieties of AMR genes, except the fusC gene. 78.57% of isolates contained both bla PC1 and ermA genes, APH(3')-IIIa and fosB genes in 64.28%. The methicillin resistance gene mecR1 was detected with frequency of 57.1%. ST239 also contained the highest number of efflux genes, varying from 6 to 8. Only in five instances tet(K) (tetracycline resistance) genes were recorded and located on plasmids only. In a single incidence it was detected in ST789 and ST2371. Additionally, it was associated with rep7a plasmids in 80% of occurrences. The third most prevalent ST22 possessed only AAC(6')-Ie-APH(2'')-Ia gene in 83.33% of this cohort, in conjunction with 3–4 efflux genes. Unfortunately, the gene's location was not detected by tool. All ST672 (100%) isolates possessed the fosB gene, which were located on the chromosome; whereas 81.81% of isolates in this group also contained the APH(3')-IIIa gene, which were located on plasmids, additionally 3 to 4 efflux genes also detected. Across genomes, ST1 exclusively harbored fusc gene except in one isolate 331100. The ST1 additionally possessed the AAC(6')-Ie-APH(2'')-Ia gene in 71.42%, followed by the APH(3')-IIIa gene in 57.14% of the group, apart from that efflux genes were also detected in a range of 4–5 in number. Certain ST groups, including ST6, ST291, ST1004, and ST1079 were devoid of all AMR genes, except for ST6, which had only efflux genes within a range of 5–6. Notably, S. aureus genomes exclusively contained only rep type plasmids, which includes rep5a (28.87%), succeeded by rep16 (27.46%), rep21 (17.61%), rep10 (14.79%), rep7c (7.75%), rep20 (4.93%), and rep7a (4.23%). Among clinical isolates of Enterococcus spp. ( n = 3) collected in the present work as shown in Fig. 3 (a) , only the E. faecium isolate (MSUH265) exhibited the perfect hits of AMR genes, such as dfrG and efflux pump efmA (macrolide resistance), both located on the chromosome. In contrast, neither of the E. faecalis isolates (MSUH331 and MSUH333) possessed any AMR genes. Moreover, none of the isolates carried any plasmids. Whereas retrieved data from Enterococcus spp. ( n = 49) as shown in Fig. 3 (b) , E. faecium exhibited the more genotypic resistance than other species in the group. All AMR genes identified within Enterococcus spp. were confined to E. faecium , except for two genomes of E. faecalis . Both the E. lactis and E. casseliflavus , exhibited no genetic presence for AMR genes; nevertheless, two genomes of E. faecalis , specifically LREF-1, harbored the AAC(6')-Ie-APH(2'')-Ia, ANT(9)-Ia (aminoglycoside resistance), and ermA genes, while the SVJ-EF01 isolate included the lnuG (lincosamide resistance) gene. Among the E. faecium isolates, the majority of AMR genes were identified in ST80, gene APH(3')-IIIa was predominant with 93.10%, followed by the vancomycin resistance gene cluster, including the vanR gene in the vanA cluster (86.20%), the vanZ gene in the vanA cluster (82.75%), and the vanH gene in the vanA cluster (79.31%); notably, all genes were located exclusively on plasmids. Genes such as cfr(D ) (multiclass resistance) and dfrG are occasionally detected only in ST80. However, few exceptions were observed in four isolates: BA3972, which exhibited a complete absence of all genes, while BA17063, BA7523, and A710, which were deficient in the vanH, vanR , and vanZ clusters of the vanA gene, they possessed the APH(3')-IIIa gene along with efflux genes. The plasmid analysis indicated that a col type plasmid ColKP3 was only detected in BA7523 of E. faecium , which was linked to the carbapenem resistance gene bla OXA−232 . The remaining plasmids were of rep type, which included repUS15 (69.39%) present in E. faecium and E. lactis , whereas it was absent in both E. faecalis and E. casseliflavus . Followed by rep2 (59.18%), rep17 (38.78%), rep18a (36.73%), rep11a (32.65%), rep14a (28.57%), repUS43 (24.49%), rep1 (14.29%), repUS57 (14.29%), and rep11C (10.20%). Except for repUS43 which was present in 30% of E. faecalis , the other plasmids were not identified. Across Enterococcus spp., only in E. faecalis strain LREF-1, genes AAC(6')-Ie-APH(2'')-Ia, ANT(9)-Ia and ermA , and plasmid rep9b were found. The AAC(6')-Ie-APH(2'')-Ia gene was detected on plasmid, while both ANT(9)-Ia and ermA genes were on chromosome. 3.4.2.2 Strict Hits: In clinical isolates, the analysis of strict hits to AMR genes in Salmonella , genes conferring resistance to aminoglycosides such as AAC(6')-Iy; to peptide resistance arnT, bacA, pmrF; to β-lactam resistance PBP3 seem to be intrinsic as detected throughout the genomes. Other genes, vanG (90%), and fosfomycin gene uhpT (80%) were also distributed across isolates. Majority of gyrA with mutations, and all five instances of both aminoglycoside resistance gene APH(6)-Id , and tetracycline resistance gene tet(A) were detected in ST1 isolates. Most of the APH(6)-Id gene and some of tet(A) genes were on plasmids, rest all other genes were detected on chromosomes. All isolates had large pool of efflux genes ranged between 17 to 19. In retrieved genomes of Salmonella , a total of 33 distinct types of AMR genes were identified. Genes such as PBP3 , arnT , bacA , pmrF ; glpT and uhpT (fosfomycin resistance); and vanG were predominant and detected in at least 95% of isolates, irrespective of their STs. Further, genes conferring resistance to aminoglycosides, such as AAC(6')-Iy (80.23%), AAC(6')-Iaa (17.73%), and APH(6)-Id (10.75%); fluoroquinolone resistance genes, including gyrA (48.54%) and parC (17.44%); and the pulvomycin resistance gene Ef-Tu (14.82%) were detected across isolates irrespective of STs. While the remaining genes were observed in less than 10% of isolates. Nearly all genes, except for vanG , were situated on chromosomes. The AAC(6')-Iaa gene had a strong association with the ST19 isolates. A significant association was seen with the sul2 gene, it exhibited a 100% occurrence with IncQ1 plasmid, regardless of STs. In clinical isolates, all five S. aureus genomes were identified as MRSA and were found to harbor the methicillin resistance gene mecA . Other genes ermC (multiclass resistance), parC, gyrA , and vanT gene in vanG cluster were also detected across the isolates. Genes PC1 beta-lactamase ( blaZ ) and dfrC (diaminopyrimidine resistance) were detected in all isolates except in MSUH231 and MSUH257 respectively. However, another gene ANT(4')-Ia was only detected in isolates that carried rep22 plasmid. All five genomes carried seven efflux genes. In retrieved genome of S. aureus , the vanT gene of vanG cluster was most prevalent, which was found in 97.18% of isolates regardless of STs, and exclusively located on chromosomes. This was followed by mecA , present in 64.08% of isolates, predominantly associated with major STs such as ST772, ST239, ST22, and ST1, also located solely on chromosomes. The mphC (macrolide resistance) gene was identified in 49.30% of isolates, distributed among ST772, ST672, and ST1482, and located on plasmids. The bla PC1 gene was found in 47.89% isolates, primarily in ST772, ST671, ST1, and ST1482, mostly on plasmids except four isolates which were on chromosomes. The PC1 beta-lactamase ( blaZ ) gene was present in 42.96% of isolates, specifically in ST772, ST22, and ST121, all were located on chromosomes. Gene conferring resistance to lincosamide lnuA (42.25%) was predominantly identified in ST239, whereas all ST1 isolates possessing the lnuA gene, the gene was also detected in few isolates of ST672 and ST1482. ST772, despite being the most widespread, lnuA genes were accounted only in 16.33% of the genomes and primarily situated on the plasmids. The msrA (streptogramin resistance) gene (35.92%) was exclusively confined to ST772, ST672, and ST1482, and situated on plasmids. Nucleoside resistance gene SAT-4 (25.35%) was distributed among ST239, ST672, ST1482, and ST1. The genes were situated on plasmids in all STs, except for ST239, which had on chromosome. It is noteworthy that the most prevalent ST772 and ST22 completely lacked the SAT-4 gene. Gene fosB (21.83%) was identified in less prevalent STs, such as ST1482, ST121, occasionally in ST239 & ST772, all were located exclusively on the chromosome. Gene ermA/C (14.08%) identified in ST22, ST121, and ST6 exclusively on plasmids, and associated with the rep10 plasmid. Gene dfrC/G (12.68%) is confined to ST239 and ST22, whereas AAC(6')-Ie-APH(2'')-Ia (9.15%) was restricted to ST239; unfortunately, the location of genes could not be discovered by the employed tool. In clinical isolates of Enterococcus genomes, different sets of genes were identified in different species. Both the genomes of E. faecalis (MSUH331 and MSUH33) carried dfrE (diaminopyrimidine resistance), vanT gene in vanG cluster, and multiclass resistance gene efrA , whereas E. faecium (MSUH265) carried AAC(6')-Ii, ermT (multiclass resistance), and tet(45) genes. In retrieved genome of Enterococcus spp. , a distinctive pattern was identified, wherein the vanT gene in vanG cluster and dfrG were present in 100% of all E. faecalis isolates situated on the chromosome. Efflux genes were identified in 90% of E. faecalis genomes, they were also infrequently identified in two less prevalent ST17. The AAC(6')-Ii gene was consistently seen throughout the genomes of E. faecium and E. lactis , regardless of the sequence types. The vanY gene in vanB cluster is predominantly found in E. faecium (89.65%) and less frequently (20%) in E. faecalis isolates, with both residing only on the chromosome. Additional genes, ermB (89.65%), vanY gene in vanA cluster (82.75%), both genes vanA, and vanX gene in vanA cluster (75.86% each), were confined to ST80 in E. faecium isolates, and only found on plasmids. Additionally, few genes, including aad(6), SAT-4 , and tet(S) , were also identified, which were classified inside cluster 1 of the phylogenetic tree. 4. Discussion This study provides the inaugural comprehensive analysis of high-priority Indian bacterial pathogens, encompassing a collection of 18 clinical samples of Salmonella ( n = 10), S. aureus ( n = 5), and Enterococcus spp. ( n = 3), alongside 535 retrieved genomes, which include Salmonella ( n = 344), S. aureus ( n = 142), and Enterococcus spp. ( n = 49), with respect to whole genome sequencing-based antimicrobial resistance profiling. The comprehensive genomic data from the clinical isolates of Salmonella revealed two distinct sets of genomic statistics between susceptible (MSUH) and MDR (TPS), wherein all MSUH isolates exhibited a reduced number of contigs, an elevated N50 value, fewer coding sequences, and lower rRNA counts compared to the TPS isolates. This discrepancy is attributed to the fact that all TPS isolates were MDR and likely acquired antimicrobial resistance genes on plasmids. A comparable pattern was also seen in Enterococcus spp. between susceptible strains (MSUH331 and MSUH333) and a MDR strain (MSUH265), despite the absence of plasmids among the isolates. This implies that the resistance may be associated with the acquisition of more rRNA. This observation contradicts previous findings, indicating that resistance is linked to a reduction in rRNA operon copies rather than an increase in S. aureus isolates (Fluit et al., 2016 ). Nonetheless, the uniformity observed across all S. aureus genomes, characterized by a lower number of contigs and greater N50 values, substantiates the quality standards of the assembly. In the present study, the MLST results for both clinical and retrieved genomes of Salmonella were anchored to ST1 with more than 50% frequency. This is due to specific evolutionary advantages such as enhanced virulence, augmented antibiotic resistance, or greater adaptation to human hosts. This study from retrieved genomes reveals that only a limited number of ST1 isolates contained AMR genes and plasmids, whereas the majority of ST2 isolates completely lacked both AMR genes and plasmids except for few isolates. These findings aligns with the currently available Indian as well as global data (Sharma et al., 2016 ). Additionally, one study ( n = 122) on extensive drug-resistant Salmonella Typhi during 2022–23 in Gujarat indicated a prevalence of ST1 at 98.36%, which was higher than the percentage of clinical isolates from this study (Akshay et al., 2025 ). Studies from India further reveals that Salmonella resistance to carbapenem and azithromycin was infrequently identified at the genetic level across genomes. It is significant that approximately 15% of ST1 isolates exhibited positivity for the bla TEM−1 , sul1 , and catA1 genes, like our findings. The heterogeneous distribution of antimicrobial resistance genes within identical Salmonella ST1 is driven by the interplay of strain-specific genetic backgrounds, the presence of integrons or plasmid replicons, and localized evolutionary pressures (Liao et al., 2019 ; Hernández-Díaz et al., 2022 ). Notably, it was observed that there were no perfect hits AMR genes, other than efflux in ST19, and a significant association was observed with IncFIB(S) & IncFII(S) plasmids, which aligns with earlier global reports (Elnekave et al., 2019 ; Harmer & Hall, 2020 ). Despite the less number of genomes, ST13 appears to be challenging, since large numbers of perfect hits genes particularly sul2 , and the ESBL gene bla CTX−M−15 a well-known genetic marker for 3rd generation cephalosporin resistance, along with strict hits genes especially APH(3')-IIIa, APH(6')-ld, fosA7 , and dfrA14 , with most types detected on plasmids, hence potential to transmit to other strains/species. Similar to perfect hits gene pattern, some of the ST1 also harbored exclusive strict hits genes APH(6)-Id, sul2 , and dfrA7 , out of which sul2 showed a significant association with IncQ1. Nevertheless, certain strict hits genes, including PBP3, arnT, bacA, pmrF, vanG, glpT , and uhpT , appear to be intrinsic across the genomes of Salmonella . The phenotypic expression of these genes can be challenging, as it may confer resistance to specific antibiotics in bacteria. A comparable trend was noted in clinical isolates of ST1, the genetic pattern for perfect hits AMR genes bla CTX−M−15 , sul1 , and catA1 , differs from the retrieved Indian data. Despite the number of clinical isolates being less, we cannot ignore the possible threat of emergence of 3rd generation cephalosporin resistant Salmonella , which warrants a large-scale genomic analysis to confirm or deny the facts. Almost similar patterns of strict hits genes, as seen in retrieved genomes, were also noted in clinical isolates except for additional gene tet(A) . In retrieved genomes of S. aureus , the findings from present study showed ST772 as predominant strain, which diverge from prior reports from India, that indicated ST22 was the most prevalent, surpassing ST772 (Abrudan et al., 2023 ). In worldwide statistics, ST5 serves as a significant international representative of the epidemic MRSA clone (Chen et al., 2024 ). In contrast, across retrieved genomes ST772 identified as the predominant MRSA lineage, followed by ST239 and ST22. The second most prevalent ST239 carried the large pool of AMR genes both from perfect and strict hits, which could emerge as future challenge. This conclusion aligns with other Indian studies that reported MRSA prevalence of 27% in ST772 (Bakthavatchalam et al., 2022 ), 15.48% in ST239 and 29.7% in ST22 (Abrudan et al., 2023 ). The AMR pattern of S. aureus differed from Salmonella . The predominant STs in S. aureus , such as ST772 and ST239, appear to be more detrimental as they possessed many AMR genes from both the perfect and strict hits, on the other hand the top three STs in Salmonella showed less genotypic resistance. Surprisingly, there was no association identified between AMR genes and plasmids in S. aureus . This could be likely arises from the limitations of short-read sequencing technologies like Illumina, which frequently encounter difficulties in accurately assembling plasmids because of their repetitive structures (Berbers et al., 2020 ), which may lead to partial or complete loss of plasmid in assembly. In ST22, we found similar patterns of perfect and strict hits of AMR genes in both retrieved and clinical isolates. However, a distinct plasmid profile was observed. All clinical isolates possessed rep10, whereas it was absent in some of the isolates from retrieved genomes. In retrieved genomes of Enterococcus , the importance of examining E. faecium is more than other species in this genus. This work's genomic analysis reveals that E. faecium , primarily in dominant ST80, identified nearly all perfect hits of AMR genes. Prevalence of sequence types observed in this investigation exhibited a consistent trend with earlier Indian studies (Rao et al., 2021 ), wherein ST80 was dominant strain followed by ST17. Another Indian publication which showed ST1643 was a predominant strain exceeded ST80, that contradicted with our findings (Bakthavatchalam et al., 2022 ). However, in global data, similar to this study ST80 was dominant, followed by ST117, whereas the ST17 was the 3rd most prevalent ST which differs from our findings (Tedim et al., 2017 ; Bezdicek et al., 2023 ). Unfortunately, majority of ST80 genomes harbored the vanA gene cluster linked to vancomycin resistance, a similar trend was documented in other regions of the world, including China (Deng et al., 2025 ), Ireland (Egan et al., 2022 ), and Russia (Fedorova et al., 2026 ). In E. faecalis , the resistance was minimal, as there were rarely perfect hits and the strict hits genes were remote. Despite the limited number of clinical samples, there was similar pattern observed against the retrieved genomes. Notably, Salmonella genomes exhibited Col type and Inc type plasmids, while the S. aureus and Enterococcus harbored Rep type plasmids. Overall, all high-priority pathogens possess distinct plasmid types, however they confer the antibiotic resistance. The existence of various plasmid types among these bacterial species highlights the intricacy of AMR gene transfer, which warrants the necessity for thorough monitoring of these mobile genetic elements. Comprehending these distinctions can facilitate the formulation of targeted methods to mitigate the proliferation of resistance in diverse bacterial species. An advantage of this study, which included "Strict hits" eliminates the likelihood of excluding AMR genes with high-confidence results of ≥ 95% similarity. Moreover, there are limited number of publications that incorporate the strict hits genes in the research (Katiyar et al., 2020 ; Rose et al., 2023 ; Wu et al., 2023 ). Additionally, a multispecies study from India also included strict hits in the surveillance of AMR genes; however, the study primarily concentrated on beta-lactamase genes (Gheewalla et al., 2026 ).The importance of including strict hits genes has highlighted in a study, the presence of the tet(B) gene can augment the minimum inhibitory concentration (MIC) of minocycline in carbapenem-resistant A. baumannii (Yang et al., 2022 ). A similar pattern was observed in clinical isolates of Salmonella (TPS) for tet(A) gene; however, the ineffective agents were doxycycline and tetracycline. It was also observed in S. aureus as well; MRSA was identified in clinical isolates, when the detection criteria for AMR genes broadened to strict hits. We analyzed n = 553 genomes of Indian high priority pathogens including retrieved ( n = 535) and clinical isolates ( n = 18), for AMR profiling and their association with STs, and plasmids. Further the genomic data was correlated with phenotypic data with 18 isolates. The only limitation of the study is that we could not derive more samples to validate correlation of genomic data with phenotypic characteristics. Although the number of clinical isolates are less, it paves way for future studies with larger sample size to observe latest trends and predict emergence of new resistance variants. 5. Conclusion To best of our knowledge, this is the first attempt from India to comprehensively analyze three high-priority AMR pathogens using both perfect (100% match) and strict hits (with mutations) criteria for gene identification. The findings indicate that Salmonella sequence types ST1, ST2, and ST19 are predominant but exhibit relatively low levels of genotypic resistance. In contrast, ST13, despite its lower prevalence, harbors a greater number of AMR genes, underscoring the need for continued genomic surveillance. Among S. aureus , ST772 and ST239 are the dominant lineages in retrieved genomes, whereas ST22 in clinical isolates, accounting for most MRSA. In Enterococcus , E. faecium ST80 emerged as the most prevalent lineage, and it was associated with key resistance determinants, including van gene clusters. The study further emphasizes the importance of applying strict hits thresholds for AMR gene detection, particularly for tet and mec genes, to ensure accurate resistance profiling. Fortunately, the bla CTX-M-15 gene conferring resistance to third-generation cephalosporins was detected only in a limited number of Salmonella isolates, which keeps these stains manageable with available antibiotics. Declarations Ethics declarations : Competing interests The authors declare no competing interests. Funding: This research was funded by the Gujarat State Biotechnology Mission (GSBTM), Department of Science and Technology (DST), Government of Gujarat, under grant ID GSBTM/0018/09/2025. Acknowledgements: We acknowledge Ms. Heli Upadhyaya and Mr. Jigresh Gohil for their support in sample collection. We would like to thank Mr. Raj Topagi for his assistance in experiments. We thank Dr. Tushar Toprani, Toprani Advanced Lab Systems, Vadodara for providing samples. We also thank Gujarat Biotechnology Research Centre, Gandhinagar for whole genome sequencing. Contributions : P.S. and D.G. designed the study and contributed to the revision of the manuscript. P.S., and S.S. performed wet lab experiments, bioinformatical analysis and data management. G.M and D.G. helped to collect clinical bacterial samples. P.S. and S.S. drafted the first version of the manuscript, while P.S., S.S., S.P., and D.G participated in the discussion and manuscript revision. All authors read and approved the final manuscript. 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ACS Chem Biol 12(7):1913–1918. https://doi.org/10.1021/acschembio.7b00412 Rao C, Dhawan B, Vishnubhatla S, Kapil A, Das B, Sood S (2021) Clinical and molecular epidemiology of vancomycin-resistant Enterococcus faecium bacteremia from an Indian tertiary hospital. Eur J Clin Microbiol Infect Dis 40(2):303–314. https://doi.org/10.1007/s10096-020-04030-3 Rose TFA, Kannan P, Ruban SW, Srinivas K, Milton AAP, Ghatak S, Elango A, Rajalakshmi S, Sundaram S (2023) Isolation, susceptibility profiles and genomic analysis of a colistin-resistant Salmonella enterica serovar Kentucky strain COL-R. 3 Biotech 13(5):140. https://doi.org/10.1007/s13205-023-03559-2 Sangiorgio G, Calvo M, Migliorisi G, Campanile F, Stefani S (2024) The Impact of Enterococcus spp. in the Immunocompromised Host: A Comprehensive Review. Pathogens 13(5):409. https://doi.org/10.3390/pathogens13050409 Sharma P, Dahiya S, Balaji V, Kanga A, Panda P, Das R, Dhanraju A, Mendiratta DK, Sood S, Das BK, Kapil A (2016) Typhoidal Salmonellae: Use of Multi-Locus Sequence Typing to Determine Population Structure. PLoS ONE 11(9):e0162530. https://doi.org/10.1371/journal.pone.0162530 Suleiman AS, Bhattacharya P, Islam MA (2025) Global prevalence and dynamics of mecA and mecC genes in MRSA: Meta-meta-analysis, meta-regression, and temporal investigation. J Infect Public Health 18(7):102802. https://doi.org/10.1016/j.jiph.2025.102802 Taylor TA, Tobin EH, Unakal CG (2026) Staphylococcus aureus Infection. In StatPearls . StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK441868/ Tedim AP, Lanza VF, Manrique M, Pareja E, Ruiz-Garbajosa P, Cantón R, Baquero F, Coque TM, Tobes R (2017) Complete Genome Sequences of Isolates of Enterococcus faecium Sequence Type 117, a Globally Disseminated Multidrug-Resistant Clone. Genome Announcements 5(13):e01553–e01516. https://doi.org/10.1128/genomeA.01553-16 Tigabu A, Getaneh A (2021) Staphylococcus aureus, ESKAPE Bacteria Challenging Current Health Care and Community Settings: A Literature Review. Clinical Laboratory , 67 (07/2021). https://doi.org/10.7754/Clin.Lab.2020.200930 WHO (INDIA), &, DBT GOI (2021) New Indian Priority Pathogen List to guide discovery of effective antibiotics . https://www.who.int/india/news-room/detail/09-03-2021-new-indian-priority-pathogen-list-to-guide-discovery-of-effective-and-affordable-antibiotics Wu X, Luo H, Ge C, Xu F, Deng X, Wiedmann M, Baker RC, Stevenson AE, Zhang G, Tang S (2023) Evaluation of multiplex nanopore sequencing for Salmonella serotype prediction and antimicrobial resistance gene and virulence gene detection. Front Microbiol 13:1073057. https://doi.org/10.3389/fmicb.2022.1073057 Yang J-L, Yang C-J, Chuang Y-C, Sheng W-H, Chen Y-C, Chang S-C (2022) Minocycline Susceptibility and tetB Gene in Carbapenem-Resistant Acinetobacter baumannii in Taiwan. Infect Drug Resist 15:2401–2408. https://doi.org/10.2147/IDR.S357344 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementarySheet1.xlsx Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-9677975","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638153867,"identity":"45ab47fe-99e2-4a16-bb87-686a4e63f1b6","order_by":0,"name":"Paparaidu Sanapala","email":"","orcid":"https://orcid.org/0009-0009-6884-9493","institution":"Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara- 390 002, Gujarat, India","correspondingAuthor":false,"prefix":"","firstName":"Paparaidu","middleName":"","lastName":"Sanapala","suffix":""},{"id":638153868,"identity":"c7a46021-e9f6-4422-8cb9-d705e997ef3e","order_by":1,"name":"Suraj Shukla","email":"","orcid":"","institution":"Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara- 390 002, Gujarat, India","correspondingAuthor":false,"prefix":"","firstName":"Suraj","middleName":"","lastName":"Shukla","suffix":""},{"id":638153869,"identity":"f0552824-2238-459a-9946-68807424d351","order_by":2,"name":"Santosh Pagare","email":"","orcid":"","institution":"Molecular Diagnostics (MDx), Siemens Healthcare Pvt. Ltd., Vadodara- 391 775, Gujarat, India","correspondingAuthor":false,"prefix":"","firstName":"Santosh","middleName":"","lastName":"Pagare","suffix":""},{"id":638153870,"identity":"0ecf1e86-36df-4553-ac7e-877f6315453d","order_by":3,"name":"Geeti Maheshwari","email":"","orcid":"","institution":"Toprani Advanced Lab Systems, Vadodara- 390 020, Gujarat, India","correspondingAuthor":false,"prefix":"","firstName":"Geeti","middleName":"","lastName":"Maheshwari","suffix":""},{"id":638153871,"identity":"4852addd-c188-420e-86d8-e9a127f17151","order_by":4,"name":"Ekadashi Rajni","email":"","orcid":"","institution":"Department of Microbiology, Mahatma Gandhi University of Medical Sciences \u0026 Technology, Jaipur 302 015, Rajasthan, India","correspondingAuthor":false,"prefix":"","firstName":"Ekadashi","middleName":"","lastName":"Rajni","suffix":""},{"id":638153872,"identity":"5f920ecd-65af-4e5d-880c-02948f174fa8","order_by":5,"name":"Devarshi Gajjar","email":"data:image/png;base64,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","orcid":"","institution":"Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda","correspondingAuthor":true,"prefix":"","firstName":"Devarshi","middleName":"","lastName":"Gajjar","suffix":""}],"badges":[],"createdAt":"2026-05-11 10:06:25","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9677975/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9677975/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109170246,"identity":"995fee15-4f39-4f78-b078-3733f14b9720","added_by":"auto","created_at":"2026-05-13 08:46:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2555175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic analysis of clinical and retrieved genomes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e spp. \u003c/strong\u003e1(a) shows the clinical isolates (\u003cem\u003en\u003c/em\u003e = 10), and (1b) shows the retrieved \u003cem\u003eSalmonella\u003c/em\u003egenomes (\u003cem\u003en\u003c/em\u003e = 344) for the sequence types (STs), AMR genes (perfect and strict hits) and their location denoted with C (chromosome), P (plasmid), U (uncharacterized), and plasmid replicons. The colour indicated in the legend denotes STs. Circle/Square filled with colours indicate the presence of genes/plasmids. Isolates with red colour in fig. 1(a) indicate MDR phenotype.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9677975/v1/df6d4018022869d2c6359cf2.jpg"},{"id":109170279,"identity":"01b6c7b7-c658-4337-876e-cb3f05efb073","added_by":"auto","created_at":"2026-05-13 08:47:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1149569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic analysis of clinical and retrieved genomes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (\u003c/strong\u003e2a) shows the clinical isolates (\u003cem\u003en\u003c/em\u003e = 5), and (2b) shows the retrieved\u003cem\u003e S. aureus\u003c/em\u003e genomes (\u003cem\u003en\u003c/em\u003e = 142) for the sequence types (STs), AMR genes (perfect and strict hits) and their location denoted with C (chromosome), P (plasmid), U (uncharacterized), and plasmid replicons. The colour indicated in the legend denotes STs. Circle/Square filled with colours indicate the presence of genes/plasmids. Isolates with red colour in fig. 2(a) indicate MDR phenotype.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9677975/v1/0bc59aa6f52fd48563d9d18a.jpg"},{"id":109170337,"identity":"2af0c15b-b47f-4831-8df2-6b8f507f7357","added_by":"auto","created_at":"2026-05-13 08:47:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":967007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic analysis of clinical and retrieved genomes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003espp. \u003c/strong\u003e(3a) shows the clinical isolates (\u003cem\u003en\u003c/em\u003e = 3), and (3b) shows the retrieved \u003cem\u003eEnterococcus\u003c/em\u003espp. (\u003cem\u003en\u003c/em\u003e = 49) for the sequence types (STs), AMR genes (perfect and strict hits) and their location denoted with C (chromosome), P (plasmid), U (uncharacterized), and plasmid replicons. The legend in square indicates for the respective STs, whereas triangle indicates species with different colours. Circle/Square filled with colours indicates the presence of genes/plasmids. Isolates with red colour in fig. 3(a) indicate MDR phenotype.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9677975/v1/a5fa823e2a9bb71d02e200f8.jpg"},{"id":109205344,"identity":"0381b369-d8c5-48eb-98f0-ce453eb881f4","added_by":"auto","created_at":"2026-05-13 15:04:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5241131,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9677975/v1/e5a09622-ba68-4942-9296-edb15472764c.pdf"},{"id":109170336,"identity":"d099f101-bf18-41c3-ac21-fd917fd026b8","added_by":"auto","created_at":"2026-05-13 08:47:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15751,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarySheet1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9677975/v1/4e87d0e98fae16f7eb7e2880.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eGenome based Investigation of AMR Determinants in High-Priority Bacterial Pathogens \u003c/p\u003e\n\u003cp\u003eCirculating in India \u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe upsurge of antibiotic-resistant bacteria signifies a major global health threat. It challenges many of the lifesaving drugs and puts millions of us at risk of incurable infections. Failure to address this challenge puts every individual at risk. The Global Research on Antimicrobial Resistance (GRAM), a collaborative project between University of Oxford and Institute for Health Metrics and Evaluation, provides the first comprehensive analysis of global antimicrobial resistance (AMR) trends, reported one million people died each year due to AMR between 1990\u0026ndash;2021. It approximates 1.91\u0026nbsp;million potential deaths by 2050 as result of AMR, an increase of 75% over 2022 (Naghavi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn response to the antimicrobial resistance (AMR) challenges, the WHO Country Office for India, in partnership with the Department of Biotechnology, Government of India, announced a list of Indian Priority Pathogens, categorizing them into three distinct priority tiers depending on resistance: critical, high, and medium (WHO (INDIA) \u0026amp; DBT, GOI, 2021).\u003c/p\u003e \u003cp\u003eCritical-priority pathogens are antibiotic-resistant (ABR) bacteria posing the greatest public health threat due to limited treatment options, high morbidity and mortality, increasing resistance, and a lack of effective drugs in development. High-priority pathogens are ABR bacteria that are difficult to treat, with significant disease burden, show rising resistance, and may be highly transmissible; while not globally critical, they can be severe in specific populations or regions (WHO (INDIA) \u0026amp; DBT, GOI, 2021).\u003c/p\u003e \u003cp\u003eThe Indian high-priority bacterial pathogens encompass gram-negative \u003cem\u003eSalmonella\u003c/em\u003e species as well as the gram-positive pathogens \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e species. \u003cem\u003eSalmonella\u003c/em\u003e is a rod-shaped microorganism that is part of the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family. These pathogens are non-sporulating facultative anaerobes with significant zoonotic potential. They are the primary etiological agents of numerous gastrointestinal and systemic infectious diseases in both mammals and avians. \u003cem\u003eS. aureus\u003c/em\u003e is a clinically significant species, flexible, facultative aero-anaerobic coccus, serves as both a common commensal and a significant opportunistic pathogen (Tigabu \u0026amp; Getaneh, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eS. aureus\u003c/em\u003e causes a wide array of diseases, from localized skin and soft tissue infections (SSTIs) to severe systemic illnesses, such as pneumonia, septic arthritis, osteomyelitis, endocarditis, and bacteremia (Taylor et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). \u003cem\u003eEnterococcus faecium\u003c/em\u003e and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e are facultative anaerobic cocci and the principal etiological agents of human enterococcal diseases. Major enterococcal infections are caused by \u003cem\u003eE. faecalis\u003c/em\u003e, however, in clinical setting \u003cem\u003eE. faecium\u003c/em\u003e cases are increasing (Sangiorgio et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherapeutic management is increasingly compromised by the emergence of resistance phenotypes against last resort antibiotics. This includes alarming increase in azithromycin and third generation cephalosporins resistance in \u003cem\u003eSalmonella\u003c/em\u003e, vancomycin-resistance in \u003cem\u003eEnterococci\u003c/em\u003e (VRE), and methicillin-resistance in \u003cem\u003eS. aureus\u003c/em\u003e (MRSA). The clinical challenge is further compounded by delayed diagnostic turnaround times, which prevent the timely initiation of targeted antimicrobial therapy.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003emph(A)\u003c/em\u003e and \u003cem\u003eerm\u003c/em\u003e genes together with \u003cem\u003eAcrB\u003c/em\u003e efflux pump protein mutations and ribosomal protein 23S (rRNA) mutations function as essential factors that contribute to azithromycin resistance and extended spectrum beta-lactamases (ESBLs) genes such as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;12\u003c/sub\u003e in \u003cem\u003eSalmonella\u003c/em\u003e (Argim\u0026oacute;n et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chavan \u0026amp; Angadi, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). MRSA is characterized by the incorporation of the SCCmec genetic element, which harbors the \u003cem\u003emecA\u003c/em\u003e or \u003cem\u003emecC\u003c/em\u003e genes that provide resistance to methicillin. Although medicines such as Linezolid are often successful against these infections, the introduction of the \u003cem\u003ecfr\u003c/em\u003e gene is progressively enabling bacteria to circumvent this treatment (Suleiman et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Vancomycin-Resistant \u003cem\u003eEnterococcus\u003c/em\u003e (VRE) evades antibiotics by modifying its cell wall precursors through the \u003cem\u003evanA\u003c/em\u003e and \u003cem\u003evanB\u003c/em\u003e genes. Collectively, these genetic adaptations pose considerable obstacles in the treatment of contemporary gram-positive bacterial illnesses (Pidgeon \u0026amp; Pires, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDetecting AMR genes with high accuracy is crucial for managing infections, typically through Whole-genome sequencing (WGS) followed by bioinformatics analysis using tools like the Comprehensive Antibiotic Resistance Database (CARD) and Resistance Gene Identifier (RGI). These tools classify detected genes as perfect (100% match), strict (high sequence similarity to curated reference model, but not 100% match) and loose (variants with low similarity and below the threshold) hits to distinguish known resistance markers from distant homologs, new or unknown variants (Alcock et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Whole-genome sequencing augments antimicrobial resistance surveillance by offering extensive insights into AMR mechanisms, gene profiling, transmission dynamics, facilitating prompt investigation and implementation of infection control measures (Oniciuc et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Matsumura et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA combined data for high-priority pathogens \u003cem\u003eSalmonella species, Staphylococcus aureus\u003c/em\u003e and \u003cem\u003eEnterococcus species\u003c/em\u003e from India is limited. Further, to our knowledge, this is first such attempt from India utilizing publicly accessible whole genome sequencing data and clinical isolates for all three high-priority pathogens to elucidate the circulating antimicrobial resistance genes in the country. This study will aid us to understand the current AMR patterns in high-priority pathogens across India, rather than relying on global data, and address the challenges associated with locating fragmented data. This will further assist in diagnostics and healthcare management to combat antimicrobial resistance.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1 Sample collection \u0026amp; Identification\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eClinical specimens of \u003cem\u003eSalmonella enterica\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2), and \u003cem\u003eEnterococcus faecium\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1) were obtained from Toprani Advanced Lab Systems, Vadodara. All isolates were subcultured and reidentified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) prior to sending for whole genome sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Antibiotic susceptibility testing:\u003c/h2\u003e \u003cp\u003eAntibiotic susceptibility testing was conducted with the VITEK\u0026reg; system (bioM\u0026eacute;rieux, Marcy-l'\u0026Eacute;toile, France) and the disc diffusion method in accordance with M02, Clinical and Laboratory Standards Institute (CLSI) M100 standards. Antibiotics were selected for various species as delineated by (Magiorakos et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Isolates were further classified as sensitive, multidrug-resistant (MDR) and extensively drug-resistant (XDR), based on the AST profile for respective isolates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 DNA extraction and Whole genome sequencing:\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from 18 clinical isolates by using the XpressDNA Bacterial DNA extraction kit (MagGenome, Chennai, India) in accordance with the manufacturer's protocol. All isolates subjected to sequencing by Illumina MiSeq platform (Illumina, CA, USA) using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) to prepare NGS libraries according to the directions provided in the manual. All 18 sequences submitted to NCBI (BioProject no.: PRJNA1431137), with each sequence's accession number provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\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\u003eGenomic accession number of clinical isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenome accession no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSpecies\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\u003ePSMSU_001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVM000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\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\u003ePSMSU_002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVL000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\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\u003ePSMSU_003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVK000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\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\u003ePSMSU_004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVJ000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\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\u003ePSMSU_005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVI000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\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\u003ePSMSU_006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVH000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\u003c/em\u003e\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\u003ePSMSU_007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH242\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVG000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVF000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVE000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVD000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPS71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVC000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPS72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVB000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPS73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVVA000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\u003c/em\u003e\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\u003ePSMSU_014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPS74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVUZ000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPS75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVUY000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\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\u003ePSMSU_016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVUX000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEnterococcus faecium\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\u003ePSMSU_017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVUW000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEnterococcus faecalis\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\u003ePSMSU_018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSUH333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJBVVUV000000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEnterococcus faecalis\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 \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 NGS Data analysis:\u003c/h2\u003e \u003cp\u003eThe raw data were assessed in the Galaxy web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://usegalaxy.eu/\u003c/span\u003e\u003cspan address=\"https://usegalaxy.eu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). FastQC tool v0.74\u0026thinsp;+\u0026thinsp;galaxy1 was utilized for quality assessment of raw reads, Trimmomatic v0.39\u0026thinsp;+\u0026thinsp;galaxy2 was employed for filtering and trimming adaptors of low-quality reads, followed by De novo assembly preparation using the Unicycler tool v0.5.1\u0026thinsp;+\u0026thinsp;galaxy0. Assembly quality was assessed using the QUAST tool v5.3.0\u0026thinsp;+\u0026thinsp;galaxy1. The genome annotation was conducted using the Prokka tool v1.14.6\u0026thinsp;+\u0026thinsp;galaxy1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Genome retrieval:\u003c/h2\u003e \u003cp\u003eSince we had limited numbers of clinical isolates for genomic analysis, to define the clear picture of AMR gene pattern in India, we obtained the extensive genomes of these bacterial species from the bacterial and viral bioinformatics resource center (BV-BRC) tool v3.42.3, which included \u003cem\u003eSalmonella\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;344), \u003cem\u003eS. aureus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;142), and \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;49) which included \u003cem\u003eE. faecium\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;36), \u003cem\u003eE. faecalis\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), \u003cem\u003eE. lactis\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2), and \u003cem\u003eE. casseliflavus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1) till December 2024. We employed the filters \"Genome Quality: Good, Genome Status: Complete and/or WGS, Isolation Country: India, Host Group: Human\" for genome retrieval.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Bioinformatics analysis:\u003c/h2\u003e \u003cp\u003eTo identify the antimicrobial resistance genes, the Resistance Gene Identifier (RGI) v6.0.5 from the Comprehensive Antibiotic Resistance Database (CARD) v4.0.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://card.mcmaster.ca/\u003c/span\u003e\u003cspan address=\"https://card.mcmaster.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the ResFinder tool v4.7.2 from the Centre for Genomic Epidemiology (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genomicepidemiology.org/\u003c/span\u003e\u003cspan address=\"https://www.genomicepidemiology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were utilized, while their genomic locations (either on chromosomes or plasmids) were determined using PlasFlow Galaxy v1.1.0\u0026thinsp;+\u0026thinsp;galaxy0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/smaegol/PlasFlow\u003c/span\u003e\u003cspan address=\"https://github.com/smaegol/PlasFlow\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multi-locus sequence typing (MLST) was ascertained using organism-specific MLST from BIGSdb (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org/software/bigsdb\u003c/span\u003e\u003cspan address=\"https://pubmlst.org/software/bigsdb\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Plasmid replicons were identified using the Plasmidfinder tool v2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/PlasmidFinder/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/PlasmidFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A phylogenetic tree based on single nucleotide polymorphisms (SNPs) was constructed using CSI Phylogeny v1.4 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/CSIPhylogeny/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/CSIPhylogeny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). SNP calling and filtering were performed using the default CSI Phylogeny pipeline with the following thresholds: minimum depth at SNP positions of 10\u0026times;, minimum relative depth of 10%, minimum SNP quality score of 30, minimum read mapping quality of 25, and a minimum Z-score of 1.96. These filtering criteria were applied to ensure high-confidence SNP detection across the analyzed genomes. The reference strain LT2 (GenBank no.: GCA_000006945.2) for \u003cem\u003eS. enterica\u003c/em\u003e, strain NCTC 8325 (GenBank no.: GCA_000013425.1) for \u003cem\u003eS. aureus\u003c/em\u003e, and strain T5 (GenBank no.: GCA_000393015.1) for \u003cem\u003eEnterococcus\u003c/em\u003e spp. were selected due to their well-annotated and widely used complete genome for comparative analyses in respective species. Subsequently, the obtained data was annotated and compiled using the iTOL tool v7.5.1, accessible at (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/itol.cgi\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/itol.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 MALDI- TOF identification\u003c/h2\u003e \u003cp\u003eAll 18 clinical isolates grown overnight on Luria-Bertani (LB) and subjected to identification by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) showed a score greater than 2 with a confidence range (2.08 to 2.42), except 4 TPS (TPS71, TPS72, TPS73, and TPS74) isolates showed a range between 1.6 and 1.98 as listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand supplementary sheet 1.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Antibiotic susceptibility testing (AST)\u003c/h2\u003e \u003cp\u003eIn drug susceptibility testing for \u003cem\u003eSalmonella\u003c/em\u003e, all five MSUH isolates exhibited sensitivity to antibiotics, except for ciprofloxacin. On the other hand, the five TPS isolates were resistant to multiple antibiotics, demonstrating non-susceptibility to extended-spectrum cephalosporins (third and fourth generation: cefepime, cefotaxime, ceftazidime), fluoroquinolones (ciprofloxacin), folate pathway inhibitors (trimethoprim-sulfamethoxazole), monobactams (aztreonam), non-extended spectrum cephalosporins (first and second generation: cefazolin, cefuroxime, ampicillin), and tetracyclines (doxycycline, tetracycline). In case of \u003cem\u003eS. aureus\u003c/em\u003e, all five isolates exhibited lower multidrug resistance and demonstrated non-susceptibility to anti-staphylococcal β-lactams (oxacillin), fluoroquinolones (ciprofloxacin, moxifloxacin), lincosamides (clindamycin), macrolides (erythromycin), and streptogramins (quinupristin-dalfopristin). All five isolates were classified as MRSA, as they exhibited an intermediate level of resistance to Methicillin, except for isolate MSUH256, which showed full resistance. However, they exhibited susceptibility to major antibiotics such as oxazolidinone (linezolid), cyclic lipopeptide (daptomycin), and glycylcyclines (tigecycline). In \u003cem\u003eEnterococcus\u003c/em\u003e spp., the \u003cem\u003eE. faecium\u003c/em\u003e isolate MSUH265 exhibited non-susceptibility to carbapenems (doripenem and imipenem), fluoroquinolones (ciprofloxacin, levofloxacin), streptogramins (quinupristin-dalfopristin), tetracycline (doxycycline), and penicillins (ampicillin); it demonstrated intermediate susceptibility to tetracycline (minocycline) and aminoglycoside (gentamicin). Although both \u003cem\u003eE. faecalis\u003c/em\u003e isolates (MSUH331 and MSUH333) were classified as sensitive, they exhibited non-susceptibility to gentamicin, ciprofloxacin, levofloxacin and quinupristin-dalfopristin, as well as intermediate susceptibility to imipenem. However, all three isolates exhibited susceptibility to glycopeptide (vancomycin), tigecycline, daptomycin, and linezolid.\u003c/p\u003e \u003cp\u003eThe phenotypic analysis of 18 clinical isolates revealed (provided in supplementary file 1) a notable occurrence of multidrug resistance (MDR) across different bacterial species, with 11 isolates (61.1%) classified as MDR and 7 (38.9%) as susceptible listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cem\u003eSalmonella enterica\u003c/em\u003e isolates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), there was a split in the resistance profile: 50% were multidrug-resistant (TPS71, TPS72, TPS73, TPS74, and TPS75), and the other 50% were susceptible (MSUH241, MSUH242, MSUH245, MSUH246, and MSUH247). All five \u003cem\u003eS. aureus\u003c/em\u003e isolates (MSUH228, MSUH231, MSUH256, MSUH257, and MSUH258) were found to be MDR. Among the \u003cem\u003eEnterococcus\u003c/em\u003e species, the single \u003cem\u003eE. faecium\u003c/em\u003e isolate (MSUH265) displayed MDR, while both \u003cem\u003eE. faecalis\u003c/em\u003e isolates (MSUH331 and MSUH333) showed susceptibility. This data highlights a significant burden of multidrug resistance, particularly in \u003cem\u003eS. aureus\u003c/em\u003e and specific strains of \u003cem\u003eS. enterica\u003c/em\u003e, however the overall number of isolates was less.\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\u003eIdentification and resistance categorization of clinical isolates\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMALDI Identification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResistance Category\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMALDI Identification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eResistance Category\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTPS75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH242\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. faecium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSUH333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSusceptible\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\u003e3.3 Whole genome sequence data statistics\u003c/h2\u003e \u003cp\u003eThe whole-genome sequencing and de novo assembly of the 18 clinical isolates produced high-quality genomic blueprints, revealing species-specific characteristics and assembly inconsistencies as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The \u003cem\u003eSalmonella enterica\u003c/em\u003e isolates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) had the largest genomes, which ranged from 4.55 to 4.98 Mb, and their GC content was consistent at around 52%. They also had the most coding sequences (average 4,630 CDSs) and tRNA ranged from 67 to 75 in number. The TPS series has bigger genomes (4.96 Mb on an average) than the MSUH series (4.63 Mb on an average) in this species. On the other hand, the \u003cem\u003eS. aureus\u003c/em\u003e isolates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) had smaller genomes (2.75 to 2.83 Mb) and a much lower GC content (average 32.73%). The number of CDSs ranged from 2,537 to 2,638. Except MSUH228 with 54 tRNA, all four isolates had 58 tRNA in number. The three \u003cem\u003eEnterococcus\u003c/em\u003e species had genomic sizes between 2.72 and 2.81 Mb and GC contents of about 37.6%. \u003cem\u003eE. faecium\u003c/em\u003e had 2656 CDSs along with 54 tRNA, whereas in both the \u003cem\u003eE. faecalis\u003c/em\u003e isolates the number of CDSs were 2517 and 2634 respectively, with 48 and 44 tRNA. The \u003cem\u003eS. enterica\u003c/em\u003e TPS series demonstrated more fragmentation, encompassing up to 289 contigs. S. aureus showed moderate level of assembly contiguity with number of contigs in range of 51 to 65; whereas the assembly contiguity for \u003cem\u003eE. faecalis\u003c/em\u003e MSUH331 was high with 12 contigs and N50 of 1,410,860 bp. Across the 18 isolates tmRNA was confined to 1 in number, while the rRNA counts ranged from 2 to 10, which highlights the structural diversity among the clinical isolates.\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\u003eWhole genome sequencing data statistics of clinical isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"left\" 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=\"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=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of contigs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLargest contig\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGenome Size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCDS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003erRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003etRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003etmRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eRepeat\u003c/p\u003e \u003cp\u003eregion\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e466235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e171823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4669728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH242\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e436308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e241014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4559165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e261464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e131909\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4551637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4373\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e339749\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e143220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4706362\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e392766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e110055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4694109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e217626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e101842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4948661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4801\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\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e163791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4960659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e123415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4967293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4844\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\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e143581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33697\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4977480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4848\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\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPS75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e163354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e57652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4955158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e188020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e120466\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2762224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e188019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e109426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2762699\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e321337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e156267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2833727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e188019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e135952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2799556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e310602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e156282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2749925\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e173501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e55833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2813165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2656\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1410860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1410860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2721729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSUH333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e569421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e292188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2796204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2634\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\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=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Genome analysis for MLST, AMR genes \u0026amp; their location, Plasmids, and their association.\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e3.4.1 MLST\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eIn the clinical isolates shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e, six isolates of \u003cem\u003eSalmonella enterica\u003c/em\u003e were categorized as ST1, two were previously undetected ST6142, the remaining two isolates one categorized as ST2 and the other one ST129. All \u003cem\u003eS. aureus\u003c/em\u003e isolates were confined to a unique ST22 shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e. Whereas the three \u003cem\u003eEnterococcus\u003c/em\u003e isolates exhibited distinct STs; \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e was defined as ST117, and \u003cem\u003eE. faecalis\u003c/em\u003e isolates were designated as ST394 and ST134 shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMLST analysis of retrieved genomes as shown in Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e, 23 distinct STs identified within \u003cem\u003eSalmonella\u003c/em\u003e genomes (\u003cem\u003en\u003c/em\u003e = 344), with ST1 (\u003cem\u003en\u003c/em\u003e = 196, 56.98%) being the predominant type, succeeded by a varied array of other STs: ST2 \u0026amp; ST19 (\u003cem\u003en\u003c/em\u003e = 50, 14.53% each), ST198 (\u003cem\u003en\u003c/em\u003e = 7, 2.03%), ST13 (\u003cem\u003en\u003c/em\u003e = 5, 1.45%), and both ST36 and ST2344 at \u003cem\u003en\u003c/em\u003e = 4, 1.16% each, while the remaining 16 different STs accounted for less than 1% each. The analysis of 142 \u003cem\u003eS. aureus\u003c/em\u003e genomes, 31 different STs were identified shown in Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e. The predominant ST was ST772, accounting for 34.51% (\u003cem\u003en\u003c/em\u003e = 49), followed by ST239 at 9.86% (\u003cem\u003en\u003c/em\u003e = 14), ST22 at 8.45% (\u003cem\u003en\u003c/em\u003e = 12), ST672 at 7.75% (\u003cem\u003en\u003c/em\u003e = 11), ST1 at 4.93% (\u003cem\u003en\u003c/em\u003e = 7), ST1482 at 3.52% (\u003cem\u003en\u003c/em\u003e = 5), ST6 at 2.82% (\u003cem\u003en\u003c/em\u003e = 4), and ST121 at 2.11% (\u003cem\u003en\u003c/em\u003e = 3). The remaining 27 STs each exhibited a frequency of less than 2%. Whereas in \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e = 49), among four identified species, \u003cem\u003eE. faecium\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e = 36) represented across seven different STs as shown in Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e. Notably, ST80 accounted for 80.55% (\u003cem\u003en\u003c/em\u003e = 29), followed by ST17 at 5.55% (\u003cem\u003en\u003c/em\u003e = 2), while the remaining isolates confined to individual ST. Remarkably, none of the STs identified in the \u003cem\u003eE. faecalis\u003c/em\u003e genomes (\u003cem\u003en\u003c/em\u003e = 10) were repetitive, each genome exhibited distinct STs. In contrast, both genomes of \u003cem\u003eE. lactis\u003c/em\u003e were classified as ST798, while \u003cem\u003eE. casseliflavus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e = 1) could not be assigned any ST.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Association of AMR genes with plasmids and STs\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section4\"\u003e \u003ch2\u003e3.4.2.1 Perfect Hits:\u003c/h2\u003e \u003cp\u003eIn clinical isolates of \u003cem\u003eSalmonella\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) collected in the present work as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e, three distinct AMR genes, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e (ESBLs), \u003cem\u003esul2\u003c/em\u003e (sulfonamide resistance), and \u003cem\u003eqnrS1\u003c/em\u003e (fluoroquinolone resistance), were identified exclusively on plasmids, surprisingly all AMR genes were found only in ST1 except for the MSUH247 isolate that does not carry AMR genes. Genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e were identified in 66.66% isolates, whereas \u003cem\u003esul2\u003c/em\u003e, and \u003cem\u003eqnrS1\u003c/em\u003e were detected in 83.33% isolates of ST1. Interestingly both plasmids IncFIB (pHCM2) and IncFIB(K) were confined to ST1 only.\u003c/p\u003e \u003cp\u003eThe analysis of retrieved \u003cem\u003eSalmonella\u003c/em\u003e genomes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;344) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e for AMR genes revealed that the beta-lactamase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTem\u0026minus;1\u003c/sub\u003e had the highest prevalence at 13.95%, followed by \u003cem\u003esul1\u003c/em\u003e (sulfonamide resistance) at 12.50%, \u003cem\u003ecatA1\u003c/em\u003e (phenicol resistance) at 10.76%, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCMY\u003c/sub\u003e (cephalosporin resistance) and \u003cem\u003eAAC(6')-type\u003c/em\u003e (aminoglycoside resistance), each at 3.20%. Other resistance genes exhibited frequencies below 3% each. Fortunately, in Indian isolates, the occurrence of carbapenem resistance genes remains rare, with the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e (carbapenem resistance) gene identified solely in isolate P5558. Additionally, the prevalence of the ESBL gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e was also below 3%. All the perfect hits genes among the isolates were located on plasmids, with a few exceptions like \u003cem\u003eAAC(6')-type\u003c/em\u003e which was found primarily on chromosomes. Further, efflux genes were associated with resistance to specific antibiotic classes, such as \u003cem\u003earmA\u003c/em\u003e and \u003cem\u003ermtC\u003c/em\u003e (aminoglycoside resistance) and \u003cem\u003eqacEdelta1\u003c/em\u003e (disinfectant and antiseptic resistance), as well as broader antibiotic classes, \u003cem\u003egolS\u003c/em\u003e, \u003cem\u003emdsA\u003c/em\u003e, \u003cem\u003emdsB\u003c/em\u003e, \u003cem\u003emsrE\u003c/em\u003e, and \u003cem\u003esdiA\u003c/em\u003e were identified across the genomes of \u003cem\u003eSalmonella\u003c/em\u003e, with \u003cem\u003egolS, mdsA, mdsB\u003c/em\u003e, and \u003cem\u003esdiA\u003c/em\u003e exhibiting the highest frequencies at 22.0%, 15.40%, 15.40%, and 16.86%, respectively. The number of efflux genes ranged from 0 to 5 across genomes, however, the instance of efflux genes of 5 and 3 were detected only in Bareilly_strain_FC745 and AMRIR00166 strains. Further, 15.40% of isolates possessed four efflux genes, 1.74% had two efflux genes, and 15.98% exhibited just one efflux gene. Interestingly, the bulk of isolates, 66.27%, exhibited no efflux genes.\u003c/p\u003e \u003cp\u003eMost of the AMR genes were confined to a limited number of ST groups, such as ST1, ST13, ST198, and ST36. Genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1\u003c/sub\u003e, \u003cem\u003esul1\u003c/em\u003e, and \u003cem\u003ecatA1\u003c/em\u003e were predominantly associated with ST1 and ST13. Particularly, ST13 had several additional genes, namely \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e and \u003cem\u003eqnrB1\u003c/em\u003e (fluoroquinolone resistance), which were present in all instances except one, whereas \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;1\u003c/sub\u003e (beta-lactam resistance) was found in approximately half of the ST13. ST198 and ST36 possessed distinct gene sets: ST198 included \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1\u003c/sub\u003e, \u003cem\u003eAAC(3)-Id\u003c/em\u003e (aminoglycoside resistance), and \u003cem\u003esul1\u003c/em\u003e, whereas ST36 contained \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCMY\u003c/sub\u003e and \u003cem\u003eAAC(6')-type\u003c/em\u003e. Interestingly, neither of the second and third most prevalent sequence types, ST2 and ST19, exhibited any perfect gene hits, except two genomes of ST2 which contained a limited number of AMR genes, and each ST19 genome contained four efflux genes. Nonetheless, the quantity of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;12\u003c/sub\u003e (beta-lactam resistance) gene was confined to only five genomes, yet all \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;12\u003c/sub\u003e genes coexisted in five instances of the IncX3 plasmid. The majority of ST36 isolates possessed the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCMY\u003c/sub\u003e gene and harbored the IncI1-I(Alpha) plasmid. A significant association was observed, where all ST19 isolates had four efflux genes, and most genomes exhibited from the highly prevalent IncFIB(S) and IncFII(S) plasmids.\u003c/p\u003e \u003cp\u003eIn the clinical isolates of \u003cem\u003eS. aureus (n\u0026thinsp;=\u0026thinsp;5)\u003c/em\u003e collected in the present work as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e, only \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e (aminoglycoside resistance) gene was found across all five genomes and harbored four efflux genes. A plasmid, rep10 was identified across all genomes, rep22 along with \u003cem\u003eANT(4')-la\u003c/em\u003e (aminoglycoside resistance) gene was also observed in 60% of isolates.\u003c/p\u003e \u003cp\u003eThe AMR investigation of retrieved \u003cem\u003eS. aureus\u003c/em\u003e genomes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;142), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e, indicated the presence of 10 distinct AMR genes, including aminoglycoside resistance genes \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e (56.33%) and \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e (45.77%), the fosfomycin resistance gene \u003cem\u003efosB\u003c/em\u003e (48.59%), the diaminopyrimidine resistance gene \u003cem\u003edfrG\u003c/em\u003e (45.07%), the penicillin beta-lactam resistance gene \u003cem\u003eblaPC1\u003c/em\u003e (10.56%), and the macrolide resistance gene \u003cem\u003eermA\u003c/em\u003e (9.15%). Based on the genomic analysis, the large pool of genotypic resistance was noted with the predominant ST772, with 91.83% of genomes containing the \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e gene, followed by 89.79% each for \u003cem\u003efosB\u003c/em\u003e and \u003cem\u003edfrG\u003c/em\u003e genes, and 67.34% for \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e (aminoglycoside resistance) gene. Out of which 65.30% of ST772 isolates co-harbored \u003cem\u003eAPH(3')-IIIa and AAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e genes. Furthermore, ST772 also included efflux genes in a range of four to six. Except for a few genomes, all \u003cem\u003edfrG\u003c/em\u003e genes were located on plasmids, while all \u003cem\u003efosB\u003c/em\u003e genes (100%) were identified on chromosomes. Unfortunately, the locations of \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e and the bifunctional protein \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e could not be discovered by the employed tool.\u003c/p\u003e \u003cp\u003eThe second most prevalent ST239 exhibited the highest degree of resistance. Some of the isolates within this group possessed almost all varieties of AMR genes, except the \u003cem\u003efusC\u003c/em\u003e gene. 78.57% of isolates contained both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003ePC1\u003c/sub\u003e and \u003cem\u003eermA\u003c/em\u003e genes, \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e and \u003cem\u003efosB\u003c/em\u003e genes in 64.28%. The methicillin resistance gene \u003cem\u003emecR1\u003c/em\u003e was detected with frequency of 57.1%. ST239 also contained the highest number of efflux genes, varying from 6 to 8. Only in five instances \u003cem\u003etet(K)\u003c/em\u003e (tetracycline resistance) genes were recorded and located on plasmids only. In a single incidence it was detected in ST789 and ST2371. Additionally, it was associated with rep7a plasmids in 80% of occurrences.\u003c/p\u003e \u003cp\u003eThe third most prevalent ST22 possessed only \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e gene in 83.33% of this cohort, in conjunction with 3\u0026ndash;4 efflux genes. Unfortunately, the gene's location was not detected by tool. All ST672 (100%) isolates possessed the \u003cem\u003efosB\u003c/em\u003e gene, which were located on the chromosome; whereas 81.81% of isolates in this group also contained the \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e gene, which were located on plasmids, additionally 3 to 4 efflux genes also detected. Across genomes, ST1 exclusively harbored \u003cem\u003efusc\u003c/em\u003e gene except in one isolate 331100. The ST1 additionally possessed the \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e gene in 71.42%, followed by the \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e gene in 57.14% of the group, apart from that efflux genes were also detected in a range of 4\u0026ndash;5 in number. Certain ST groups, including ST6, ST291, ST1004, and ST1079 were devoid of all AMR genes, except for ST6, which had only efflux genes within a range of 5\u0026ndash;6.\u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003eS. aureus\u003c/em\u003e genomes exclusively contained only rep type plasmids, which includes \u003cem\u003erep5a\u003c/em\u003e (28.87%), succeeded by \u003cem\u003erep16\u003c/em\u003e (27.46%), \u003cem\u003erep21\u003c/em\u003e (17.61%), \u003cem\u003erep10\u003c/em\u003e (14.79%), \u003cem\u003erep7c\u003c/em\u003e (7.75%), \u003cem\u003erep20\u003c/em\u003e (4.93%), and \u003cem\u003erep7a\u003c/em\u003e (4.23%).\u003c/p\u003e \u003cp\u003eAmong clinical isolates of \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) collected in the present work as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e, only the \u003cem\u003eE. faecium\u003c/em\u003e isolate (MSUH265) exhibited the perfect hits of AMR genes, such as \u003cem\u003edfrG\u003c/em\u003e and efflux pump \u003cem\u003eefmA\u003c/em\u003e (macrolide resistance), both located on the chromosome. In contrast, neither of the \u003cem\u003eE. faecalis\u003c/em\u003e isolates (MSUH331 and MSUH333) possessed any AMR genes. Moreover, none of the isolates carried any plasmids.\u003c/p\u003e \u003cp\u003eWhereas retrieved data from \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;49) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e, \u003cem\u003eE. faecium\u003c/em\u003e exhibited the more genotypic resistance than other species in the group. All AMR genes identified within \u003cem\u003eEnterococcus\u003c/em\u003e spp. were confined to \u003cem\u003eE. faecium\u003c/em\u003e, except for two genomes of \u003cem\u003eE. faecalis\u003c/em\u003e. Both the \u003cem\u003eE. lactis\u003c/em\u003e and \u003cem\u003eE. casseliflavus\u003c/em\u003e, exhibited no genetic presence for AMR genes; nevertheless, two genomes of \u003cem\u003eE. faecalis\u003c/em\u003e, specifically LREF-1, harbored the \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia, ANT(9)-Ia\u003c/em\u003e (aminoglycoside resistance), and \u003cem\u003eermA\u003c/em\u003e genes, while the SVJ-EF01 isolate included the \u003cem\u003elnuG\u003c/em\u003e (lincosamide resistance) gene. Among the \u003cem\u003eE. faecium\u003c/em\u003e isolates, the majority of AMR genes were identified in ST80, gene \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e was predominant with 93.10%, followed by the vancomycin resistance gene cluster, including the \u003cem\u003evanR\u003c/em\u003e gene in the \u003cem\u003evanA\u003c/em\u003e cluster (86.20%), the \u003cem\u003evanZ\u003c/em\u003e gene in the \u003cem\u003evanA\u003c/em\u003e cluster (82.75%), and the \u003cem\u003evanH\u003c/em\u003e gene in the \u003cem\u003evanA\u003c/em\u003e cluster (79.31%); notably, all genes were located exclusively on plasmids. Genes such as \u003cem\u003ecfr(D\u003c/em\u003e) (multiclass resistance) and \u003cem\u003edfrG\u003c/em\u003e are occasionally detected only in ST80. However, few exceptions were observed in four isolates: BA3972, which exhibited a complete absence of all genes, while BA17063, BA7523, and A710, which were deficient in the \u003cem\u003evanH, vanR\u003c/em\u003e, and \u003cem\u003evanZ\u003c/em\u003e clusters of the \u003cem\u003evanA\u003c/em\u003e gene, they possessed the \u003cem\u003eAPH(3')-IIIa\u003c/em\u003e gene along with efflux genes.\u003c/p\u003e \u003cp\u003eThe plasmid analysis indicated that a col type plasmid ColKP3 was only detected in BA7523 of \u003cem\u003eE. faecium\u003c/em\u003e, which was linked to the carbapenem resistance gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;232\u003c/sub\u003e. The remaining plasmids were of rep type, which included repUS15 (69.39%) present in \u003cem\u003eE. faecium\u003c/em\u003e and \u003cem\u003eE. lactis\u003c/em\u003e, whereas it was absent in both \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eE. casseliflavus\u003c/em\u003e. Followed by rep2 (59.18%), rep17 (38.78%), rep18a (36.73%), rep11a (32.65%), rep14a (28.57%), repUS43 (24.49%), rep1 (14.29%), repUS57 (14.29%), and rep11C (10.20%). Except for repUS43 which was present in 30% of \u003cem\u003eE. faecalis\u003c/em\u003e, the other plasmids were not identified. Across \u003cem\u003eEnterococcus\u003c/em\u003e spp., only in \u003cem\u003eE. faecalis\u003c/em\u003e strain LREF-1, genes \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia, ANT(9)-Ia\u003c/em\u003e and \u003cem\u003eermA\u003c/em\u003e, and plasmid rep9b were found. The \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e gene was detected on plasmid, while both \u003cem\u003eANT(9)-Ia\u003c/em\u003e and \u003cem\u003eermA\u003c/em\u003e genes were on chromosome.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section4\"\u003e \u003ch2\u003e3.4.2.2 Strict Hits:\u003c/h2\u003e \u003cp\u003eIn clinical isolates, the analysis of strict hits to AMR genes in \u003cem\u003eSalmonella\u003c/em\u003e, genes conferring resistance to aminoglycosides such as \u003cem\u003eAAC(6')-Iy;\u003c/em\u003e to peptide resistance \u003cem\u003earnT, bacA, pmrF;\u003c/em\u003e to β-lactam resistance \u003cem\u003ePBP3\u003c/em\u003e seem to be intrinsic as detected throughout the genomes. Other genes, \u003cem\u003evanG\u003c/em\u003e (90%), and fosfomycin gene \u003cem\u003euhpT\u003c/em\u003e (80%) were also distributed across isolates. Majority of \u003cem\u003egyrA\u003c/em\u003e with mutations, and all five instances of both aminoglycoside resistance gene \u003cem\u003eAPH(6)-Id\u003c/em\u003e, and tetracycline resistance gene \u003cem\u003etet(A)\u003c/em\u003e were detected in ST1 isolates. Most of the \u003cem\u003eAPH(6)-Id\u003c/em\u003e gene and some of \u003cem\u003etet(A)\u003c/em\u003e genes were on plasmids, rest all other genes were detected on chromosomes. All isolates had large pool of efflux genes ranged between 17 to 19.\u003c/p\u003e \u003cp\u003eIn retrieved genomes of \u003cem\u003eSalmonella\u003c/em\u003e, a total of 33 distinct types of AMR genes were identified. Genes such as \u003cem\u003ePBP3\u003c/em\u003e, \u003cem\u003earnT\u003c/em\u003e, \u003cem\u003ebacA\u003c/em\u003e, \u003cem\u003epmrF\u003c/em\u003e; \u003cem\u003eglpT\u003c/em\u003e and \u003cem\u003euhpT\u003c/em\u003e (fosfomycin resistance); and \u003cem\u003evanG\u003c/em\u003e were predominant and detected in at least 95% of isolates, irrespective of their STs. Further, genes conferring resistance to aminoglycosides, such as \u003cem\u003eAAC(6')-Iy\u003c/em\u003e (80.23%), \u003cem\u003eAAC(6')-Iaa\u003c/em\u003e (17.73%), and \u003cem\u003eAPH(6)-Id\u003c/em\u003e (10.75%); fluoroquinolone resistance genes, including \u003cem\u003egyrA\u003c/em\u003e (48.54%) and \u003cem\u003eparC\u003c/em\u003e (17.44%); and the pulvomycin resistance gene \u003cem\u003eEf-Tu\u003c/em\u003e (14.82%) were detected across isolates irrespective of STs. While the remaining genes were observed in less than 10% of isolates. Nearly all genes, except for \u003cem\u003evanG\u003c/em\u003e, were situated on chromosomes. The \u003cem\u003eAAC(6')-Iaa\u003c/em\u003e gene had a strong association with the ST19 isolates. A significant association was seen with the \u003cem\u003esul2\u003c/em\u003e gene, it exhibited a 100% occurrence with IncQ1 plasmid, regardless of STs.\u003c/p\u003e \u003cp\u003eIn clinical isolates, all five \u003cem\u003eS. aureus\u003c/em\u003e genomes were identified as MRSA and were found to harbor the methicillin resistance gene \u003cem\u003emecA\u003c/em\u003e. Other genes \u003cem\u003eermC\u003c/em\u003e (multiclass resistance), \u003cem\u003eparC, gyrA\u003c/em\u003e, and \u003cem\u003evanT\u003c/em\u003e gene in \u003cem\u003evanG\u003c/em\u003e cluster were also detected across the isolates. Genes PC1 beta-lactamase (\u003cem\u003eblaZ\u003c/em\u003e) and \u003cem\u003edfrC\u003c/em\u003e (diaminopyrimidine resistance) were detected in all isolates except in MSUH231 and MSUH257 respectively. However, another gene \u003cem\u003eANT(4')-Ia\u003c/em\u003e was only detected in isolates that carried rep22 plasmid. All five genomes carried seven efflux genes.\u003c/p\u003e \u003cp\u003eIn retrieved genome of \u003cem\u003eS. aureus\u003c/em\u003e, the \u003cem\u003evanT\u003c/em\u003e gene of \u003cem\u003evanG\u003c/em\u003e cluster was most prevalent, which was found in 97.18% of isolates regardless of STs, and exclusively located on chromosomes. This was followed by \u003cem\u003emecA\u003c/em\u003e, present in 64.08% of isolates, predominantly associated with major STs such as ST772, ST239, ST22, and ST1, also located solely on chromosomes. The \u003cem\u003emphC\u003c/em\u003e (macrolide resistance) gene was identified in 49.30% of isolates, distributed among ST772, ST672, and ST1482, and located on plasmids. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003ePC1\u003c/sub\u003e gene was found in 47.89% isolates, primarily in ST772, ST671, ST1, and ST1482, mostly on plasmids except four isolates which were on chromosomes. The PC1 beta-lactamase (\u003cem\u003eblaZ\u003c/em\u003e) gene was present in 42.96% of isolates, specifically in ST772, ST22, and ST121, all were located on chromosomes.\u003c/p\u003e \u003cp\u003eGene conferring resistance to lincosamide \u003cem\u003elnuA\u003c/em\u003e (42.25%) was predominantly identified in ST239, whereas all ST1 isolates possessing the \u003cem\u003elnuA\u003c/em\u003e gene, the gene was also detected in few isolates of ST672 and ST1482. ST772, despite being the most widespread, \u003cem\u003elnuA\u003c/em\u003e genes were accounted only in 16.33% of the genomes and primarily situated on the plasmids. The \u003cem\u003emsrA\u003c/em\u003e (streptogramin resistance) gene (35.92%) was exclusively confined to ST772, ST672, and ST1482, and situated on plasmids. Nucleoside resistance gene \u003cem\u003eSAT-4\u003c/em\u003e (25.35%) was distributed among ST239, ST672, ST1482, and ST1. The genes were situated on plasmids in all STs, except for ST239, which had on chromosome. It is noteworthy that the most prevalent ST772 and ST22 completely lacked the \u003cem\u003eSAT-4\u003c/em\u003e gene. Gene \u003cem\u003efosB\u003c/em\u003e (21.83%) was identified in less prevalent STs, such as ST1482, ST121, occasionally in ST239 \u0026amp; ST772, all were located exclusively on the chromosome. Gene \u003cem\u003eermA/C\u003c/em\u003e (14.08%) identified in ST22, ST121, and ST6 exclusively on plasmids, and associated with the rep10 plasmid. Gene \u003cem\u003edfrC/G\u003c/em\u003e (12.68%) is confined to ST239 and ST22, whereas \u003cem\u003eAAC(6')-Ie-APH(2'')-Ia\u003c/em\u003e (9.15%) was restricted to ST239; unfortunately, the location of genes could not be discovered by the employed tool.\u003c/p\u003e \u003cp\u003eIn clinical isolates of \u003cem\u003eEnterococcus\u003c/em\u003e genomes, different sets of genes were identified in different species. Both the genomes of \u003cem\u003eE. faecalis\u003c/em\u003e (MSUH331 and MSUH33) carried \u003cem\u003edfrE\u003c/em\u003e (diaminopyrimidine resistance), \u003cem\u003evanT\u003c/em\u003e gene in \u003cem\u003evanG\u003c/em\u003e cluster, and multiclass resistance gene \u003cem\u003eefrA\u003c/em\u003e, whereas \u003cem\u003eE. faecium\u003c/em\u003e (MSUH265) carried \u003cem\u003eAAC(6')-Ii, ermT\u003c/em\u003e (multiclass resistance), and \u003cem\u003etet(45)\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eIn retrieved genome of \u003cem\u003eEnterococcus spp.\u003c/em\u003e, a distinctive pattern was identified, wherein the \u003cem\u003evanT\u003c/em\u003e gene in \u003cem\u003evanG\u003c/em\u003e cluster and \u003cem\u003edfrG\u003c/em\u003e were present in 100% of all \u003cem\u003eE. faecalis\u003c/em\u003e isolates situated on the chromosome. Efflux genes were identified in 90% of \u003cem\u003eE. faecalis\u003c/em\u003e genomes, they were also infrequently identified in two less prevalent ST17. The \u003cem\u003eAAC(6')-Ii\u003c/em\u003e gene was consistently seen throughout the genomes of \u003cem\u003eE. faecium\u003c/em\u003e and \u003cem\u003eE. lactis\u003c/em\u003e, regardless of the sequence types. The \u003cem\u003evanY\u003c/em\u003e gene in \u003cem\u003evanB\u003c/em\u003e cluster is predominantly found in \u003cem\u003eE. faecium\u003c/em\u003e (89.65%) and less frequently (20%) in \u003cem\u003eE. faecalis\u003c/em\u003e isolates, with both residing only on the chromosome. Additional genes, \u003cem\u003eermB\u003c/em\u003e (89.65%), \u003cem\u003evanY\u003c/em\u003e gene in \u003cem\u003evanA\u003c/em\u003e cluster (82.75%), both genes \u003cem\u003evanA, and vanX\u003c/em\u003e gene in \u003cem\u003evanA\u003c/em\u003e cluster (75.86% each), were confined to ST80 in \u003cem\u003eE. faecium\u003c/em\u003e isolates, and only found on plasmids. Additionally, few genes, including \u003cem\u003eaad(6), SAT-4\u003c/em\u003e, and \u003cem\u003etet(S)\u003c/em\u003e, were also identified, which were classified inside cluster 1 of the phylogenetic tree.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study provides the inaugural comprehensive analysis of high-priority Indian bacterial pathogens, encompassing a collection of 18 clinical samples of \u003cem\u003eSalmonella\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), \u003cem\u003eS. aureus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5), and \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3), alongside 535 retrieved genomes, which include \u003cem\u003eSalmonella\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;344), \u003cem\u003eS. aureus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;142), and \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;49), with respect to whole genome sequencing-based antimicrobial resistance profiling.\u003c/p\u003e \u003cp\u003eThe comprehensive genomic data from the clinical isolates of \u003cem\u003eSalmonella\u003c/em\u003e revealed two distinct sets of genomic statistics between susceptible (MSUH) and MDR (TPS), wherein all MSUH isolates exhibited a reduced number of contigs, an elevated N50 value, fewer coding sequences, and lower rRNA counts compared to the TPS isolates. This discrepancy is attributed to the fact that all TPS isolates were MDR and likely acquired antimicrobial resistance genes on plasmids. A comparable pattern was also seen in \u003cem\u003eEnterococcus\u003c/em\u003e spp. between susceptible strains (MSUH331 and MSUH333) and a MDR strain (MSUH265), despite the absence of plasmids among the isolates. This implies that the resistance may be associated with the acquisition of more rRNA. This observation contradicts previous findings, indicating that resistance is linked to a reduction in rRNA operon copies rather than an increase in \u003cem\u003eS. aureus\u003c/em\u003e isolates (Fluit et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Nonetheless, the uniformity observed across all \u003cem\u003eS. aureus\u003c/em\u003e genomes, characterized by a lower number of contigs and greater N50 values, substantiates the quality standards of the assembly.\u003c/p\u003e \u003cp\u003eIn the present study, the MLST results for both clinical and retrieved genomes of \u003cem\u003eSalmonella\u003c/em\u003e were anchored to ST1 with more than 50% frequency. This is due to specific evolutionary advantages such as enhanced virulence, augmented antibiotic resistance, or greater adaptation to human hosts. This study from retrieved genomes reveals that only a limited number of ST1 isolates contained AMR genes and plasmids, whereas the majority of ST2 isolates completely lacked both AMR genes and plasmids except for few isolates. These findings aligns with the currently available Indian as well as global data (Sharma et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, one study (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;122) on extensive drug-resistant \u003cem\u003eSalmonella Typhi\u003c/em\u003e during 2022\u0026ndash;23 in Gujarat indicated a prevalence of ST1 at 98.36%, which was higher than the percentage of clinical isolates from this study (Akshay et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Studies from India further reveals that \u003cem\u003eSalmonella\u003c/em\u003e resistance to carbapenem and azithromycin was infrequently identified at the genetic level across genomes. It is significant that approximately 15% of ST1 isolates exhibited positivity for the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1\u003c/sub\u003e, \u003cem\u003esul1\u003c/em\u003e, and \u003cem\u003ecatA1\u003c/em\u003e genes, like our findings. The heterogeneous distribution of antimicrobial resistance genes within identical \u003cem\u003eSalmonella\u003c/em\u003e ST1 is driven by the interplay of strain-specific genetic backgrounds, the presence of integrons or plasmid replicons, and localized evolutionary pressures (Liao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hern\u0026aacute;ndez-D\u0026iacute;az et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, it was observed that there were no perfect hits AMR genes, other than efflux in ST19, and a significant association was observed with IncFIB(S) \u0026amp; IncFII(S) plasmids, which aligns with earlier global reports (Elnekave et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Harmer \u0026amp; Hall, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite the less number of genomes, ST13 appears to be challenging, since large numbers of perfect hits genes particularly \u003cem\u003esul2\u003c/em\u003e, and the ESBL gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e a well-known genetic marker for 3rd generation cephalosporin resistance, along with strict hits genes especially \u003cem\u003eAPH(3')-IIIa, APH(6')-ld, fosA7\u003c/em\u003e, and \u003cem\u003edfrA14\u003c/em\u003e, with most types detected on plasmids, hence potential to transmit to other strains/species.\u003c/p\u003e \u003cp\u003eSimilar to perfect hits gene pattern, some of the ST1 also harbored exclusive strict hits genes \u003cem\u003eAPH(6)-Id, sul2\u003c/em\u003e, and \u003cem\u003edfrA7\u003c/em\u003e, out of which \u003cem\u003esul2\u003c/em\u003e showed a significant association with IncQ1. Nevertheless, certain strict hits genes, including \u003cem\u003ePBP3, arnT, bacA, pmrF, vanG, glpT\u003c/em\u003e, and \u003cem\u003euhpT\u003c/em\u003e, appear to be intrinsic across the genomes of \u003cem\u003eSalmonella\u003c/em\u003e. The phenotypic expression of these genes can be challenging, as it may confer resistance to specific antibiotics in bacteria.\u003c/p\u003e \u003cp\u003eA comparable trend was noted in clinical isolates of ST1, the genetic pattern for perfect hits AMR genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e, \u003cem\u003esul1\u003c/em\u003e, and \u003cem\u003ecatA1\u003c/em\u003e, differs from the retrieved Indian data. Despite the number of clinical isolates being less, we cannot ignore the possible threat of emergence of 3rd generation cephalosporin resistant \u003cem\u003eSalmonella\u003c/em\u003e, which warrants a large-scale genomic analysis to confirm or deny the facts. Almost similar patterns of strict hits genes, as seen in retrieved genomes, were also noted in clinical isolates except for additional gene \u003cem\u003etet(A)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn retrieved genomes of \u003cem\u003eS. aureus\u003c/em\u003e, the findings from present study showed ST772 as predominant strain, which diverge from prior reports from India, that indicated ST22 was the most prevalent, surpassing ST772 (Abrudan et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In worldwide statistics, ST5 serves as a significant international representative of the epidemic MRSA clone (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, across retrieved genomes ST772 identified as the predominant MRSA lineage, followed by ST239 and ST22. The second most prevalent ST239 carried the large pool of AMR genes both from perfect and strict hits, which could emerge as future challenge. This conclusion aligns with other Indian studies that reported MRSA prevalence of 27% in ST772 (Bakthavatchalam et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), 15.48% in ST239 and 29.7% in ST22 (Abrudan et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe AMR pattern of \u003cem\u003eS. aureus\u003c/em\u003e differed from \u003cem\u003eSalmonella\u003c/em\u003e. The predominant STs in \u003cem\u003eS. aureus\u003c/em\u003e, such as ST772 and ST239, appear to be more detrimental as they possessed many AMR genes from both the perfect and strict hits, on the other hand the top three STs in \u003cem\u003eSalmonella\u003c/em\u003e showed less genotypic resistance.\u003c/p\u003e \u003cp\u003eSurprisingly, there was no association identified between AMR genes and plasmids in \u003cem\u003eS. aureus\u003c/em\u003e. This could be likely arises from the limitations of short-read sequencing technologies like Illumina, which frequently encounter difficulties in accurately assembling plasmids because of their repetitive structures (Berbers et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which may lead to partial or complete loss of plasmid in assembly.\u003c/p\u003e \u003cp\u003eIn ST22, we found similar patterns of perfect and strict hits of AMR genes in both retrieved and clinical isolates. However, a distinct plasmid profile was observed. All clinical isolates possessed rep10, whereas it was absent in some of the isolates from retrieved genomes.\u003c/p\u003e \u003cp\u003eIn retrieved genomes of \u003cem\u003eEnterococcus\u003c/em\u003e, the importance of examining \u003cem\u003eE. faecium\u003c/em\u003e is more than other species in this genus. This work's genomic analysis reveals that \u003cem\u003eE. faecium\u003c/em\u003e, primarily in dominant ST80, identified nearly all perfect hits of AMR genes. Prevalence of sequence types observed in this investigation exhibited a consistent trend with earlier Indian studies (Rao et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), wherein ST80 was dominant strain followed by ST17. Another Indian publication which showed ST1643 was a predominant strain exceeded ST80, that contradicted with our findings (Bakthavatchalam et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, in global data, similar to this study ST80 was dominant, followed by ST117, whereas the ST17 was the 3rd most prevalent ST which differs from our findings (Tedim et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bezdicek et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Unfortunately, majority of ST80 genomes harbored the \u003cem\u003evanA\u003c/em\u003e gene cluster linked to vancomycin resistance, a similar trend was documented in other regions of the world, including China (Deng et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), Ireland (Egan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and Russia (Fedorova et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). In \u003cem\u003eE. faecalis\u003c/em\u003e, the resistance was minimal, as there were rarely perfect hits and the strict hits genes were remote. Despite the limited number of clinical samples, there was similar pattern observed against the retrieved genomes.\u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003eSalmonella\u003c/em\u003e genomes exhibited Col type and Inc type plasmids, while the \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e harbored Rep type plasmids. Overall, all high-priority pathogens possess distinct plasmid types, however they confer the antibiotic resistance. The existence of various plasmid types among these bacterial species highlights the intricacy of AMR gene transfer, which warrants the necessity for thorough monitoring of these mobile genetic elements. Comprehending these distinctions can facilitate the formulation of targeted methods to mitigate the proliferation of resistance in diverse bacterial species.\u003c/p\u003e \u003cp\u003eAn advantage of this study, which included \"Strict hits\" eliminates the likelihood of excluding AMR genes with high-confidence results of \u0026ge;\u0026thinsp;95% similarity. Moreover, there are limited number of publications that incorporate the strict hits genes in the research (Katiyar et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rose et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, a multispecies study from India also included strict hits in the surveillance of AMR genes; however, the study primarily concentrated on beta-lactamase genes (Gheewalla et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2026\u003c/span\u003e).The importance of including strict hits genes has highlighted in a study, the presence of the \u003cem\u003etet(B)\u003c/em\u003e gene can augment the minimum inhibitory concentration (MIC) of minocycline in carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e (Yang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A similar pattern was observed in clinical isolates of \u003cem\u003eSalmonella\u003c/em\u003e (TPS) for \u003cem\u003etet(A)\u003c/em\u003e gene; however, the ineffective agents were doxycycline and tetracycline. It was also observed in \u003cem\u003eS. aureus\u003c/em\u003e as well; MRSA was identified in clinical isolates, when the detection criteria for AMR genes broadened to strict hits.\u003c/p\u003e \u003cp\u003eWe analyzed \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;553 genomes of Indian high priority pathogens including retrieved (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;535) and clinical isolates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;18), for AMR profiling and their association with STs, and plasmids. Further the genomic data was correlated with phenotypic data with 18 isolates. The only limitation of the study is that we could not derive more samples to validate correlation of genomic data with phenotypic characteristics. Although the number of clinical isolates are less, it paves way for future studies with larger sample size to observe latest trends and predict emergence of new resistance variants.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo best of our knowledge, this is the first attempt from India to comprehensively analyze three high-priority AMR pathogens using both perfect (100% match) and strict hits (with mutations) criteria for gene identification. The findings indicate that \u003cem\u003eSalmonella\u003c/em\u003e sequence types ST1, ST2, and ST19 are predominant but exhibit relatively low levels of genotypic resistance. In contrast, ST13, despite its lower prevalence, harbors a greater number of AMR genes, underscoring the need for continued genomic surveillance. Among \u003cem\u003eS. aureus\u003c/em\u003e, ST772 and ST239 are the dominant lineages in retrieved genomes, whereas ST22 in clinical isolates, accounting for most MRSA. In \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eE. faecium\u003c/em\u003e ST80 emerged as the most prevalent lineage, and it was associated with key resistance determinants, including \u003cem\u003evan\u003c/em\u003e gene clusters. The study further emphasizes the importance of applying strict hits thresholds for AMR gene detection, particularly for \u003cem\u003etet\u003c/em\u003e and \u003cem\u003emec\u003c/em\u003e genes, to ensure accurate resistance profiling. Fortunately, the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u003c/sub\u003e gene conferring resistance to third-generation cephalosporins was detected only in a limited number of \u003cem\u003eSalmonella\u003c/em\u003e isolates, which keeps these stains manageable with available antibiotics.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e \u003cb\u003eEthics declarations\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by the Gujarat State Biotechnology Mission (GSBTM), Department of Science and Technology (DST), Government of Gujarat, under grant ID GSBTM/0018/09/2025.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eWe acknowledge Ms. Heli Upadhyaya and Mr. Jigresh Gohil for their support in sample collection. We would like to thank Mr. Raj Topagi for his assistance in experiments. We thank Dr. Tushar Toprani, Toprani Advanced Lab Systems, Vadodara for providing samples. We also thank Gujarat Biotechnology Research Centre, Gandhinagar for whole genome sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eContributions\u003c/b\u003e: P.S. and D.G. designed the study and contributed to the revision of the manuscript. P.S., and S.S. performed wet lab experiments, bioinformatical analysis and data management. G.M and D.G. helped to collect clinical bacterial samples. P.S. and S.S. drafted the first version of the manuscript, while P.S., S.S., S.P., and D.G participated in the discussion and manuscript revision. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbrudan MI, Shamanna V, Prasanna A, Underwood A, Argim\u0026oacute;n S, Nagaraj G, Di Gregorio S, Govindan V, Vasanth A, Dharmavaram S, Kekre M, Aanensen DM, Ravikumar KL (2023) Novel multidrug-resistant sublineages of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e clonal complex 22 discovered in India. mSphere 8(5):e00185\u0026ndash;e00123. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/msphere.00185-23\u003c/span\u003e\u003cspan address=\"10.1128/msphere.00185-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkshay SD, Upadhyaya H, Shukla N, Bhattacharjee R, Das S, Vyas U, Chavda P, Patel N, Jamkhandi D, Sabara P, Khandelwal N, Soni S, Upadhyaya K, Katira J, Maheshwari G, Joshi M, Gajjar D, Joshi C (2025) Comprehensive analysis of extensive drug-resistant \u003cem\u003eSalmonella\u003c/em\u003e Typhi in Gujarat region, India: Genomic findings and prospective alternative therapy. 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Front Microbiol 13:1073057. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.1073057\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.1073057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J-L, Yang C-J, Chuang Y-C, Sheng W-H, Chen Y-C, Chang S-C (2022) Minocycline Susceptibility and tetB Gene in Carbapenem-Resistant Acinetobacter baumannii in Taiwan. Infect Drug Resist 15:2401\u0026ndash;2408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IDR.S357344\u003c/span\u003e\u003cspan address=\"10.2147/IDR.S357344\" 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":true,"hideJournal":true,"highlight":"","institution":"Maharaja Sayajirao University of Baroda","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial resistance, Salmonella spp., Staphylococcus aureus, Enterococcus spp., High priority pathogens, Whole genome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-9677975/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9677975/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistance (AMR) has emerged as a key focus of research in infectious diseases, owing to the severity of infections and elevated worldwide death rates. Pathogens such as \u003cem\u003eSalmonella\u003c/em\u003e spp., \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eEnterococcus\u003c/em\u003e spp. have recently attracted attention because of their escalating resistance; therefore, they have been classified as high-priority pathogens by the WHO. This study aimed to obtain a comprehensive understanding of the AMR patterns among these pathogens\u003c/p\u003e \u003cp\u003eEighteen clinical isolates, comprising \u003cem\u003eSalmonella\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), \u003cem\u003eS. aureus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5), and \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3), were collected and tested for antibiotic susceptibility, subsequently subjected to whole-genome sequencing. Additionally, 535 genome submissions including \u003cem\u003eSalmonella\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;344), \u003cem\u003eS. aureus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;142), and \u003cem\u003eEnterococcus\u003c/em\u003e spp. (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;49) from India were retrieved from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) for bioinformatics analysis and were annotated for sequence types (STs), AMR genes, their locations, and plasmids.\u003c/p\u003e \u003cp\u003eMultidrug resistance (MDR) was identified in 50% of the \u003cem\u003eSalmonella\u003c/em\u003e isolates, all \u003cem\u003eS. aureus\u003c/em\u003e, and a single \u003cem\u003eE. faecium\u003c/em\u003e isolate; conversely, the remaining \u003cem\u003eSalmonella\u003c/em\u003e and both \u003cem\u003eE. faecalis\u003c/em\u003e isolates were susceptible. Genomic analysis identified the dominant STs: ST1 in both clinical isolates and retrieved genomes of \u003cem\u003eSalmonella\u003c/em\u003e; ST22 in clinical isolates, and ST772 in retrieved \u003cem\u003eS. aureus\u003c/em\u003e, while ST80 was observed in retrieved \u003cem\u003eE. faecium\u003c/em\u003e. All dominant STs were highly resistant, however, ST1 in \u003cem\u003eSalmonella\u003c/em\u003e retrieved genomes exhibited reduced resistance.\u003c/p\u003e \u003cp\u003eOverall, among high-priority pathogens, \u003cem\u003eSalmonella\u003c/em\u003e remains manageable due to lower resistance; however, \u003cem\u003eS. aureus\u003c/em\u003e, particularly ST772 and ST239, as well as \u003cem\u003eE. faecium\u003c/em\u003e, mainly ST80, exhibits a significant risk for severe infections due to their extensive gene reservoirs and high prevalence.\u003c/p\u003e","manuscriptTitle":"Genome based Investigation of AMR Determinants in High-Priority Bacterial Pathogens \nCirculating in India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 08:46:18","doi":"10.21203/rs.3.rs-9677975/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08dd76d0-2439-43b9-9cc3-225d6c073116","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":67921110,"name":"Molecular Epidemiology"},{"id":67921111,"name":"Infectious Diseases"},{"id":67921112,"name":"Bioinformatics"}],"tags":[],"updatedAt":"2026-05-13T08:46:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 08:46:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9677975","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9677975","identity":"rs-9677975","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}