Isolation and Whole-Genome Sequencing of a Less Prevalent Indian A. baumannii Strain Reveals Unique Uncharacterized Hypothetical Proteins and AMR-Linked ncRNAs.

Isolation and Whole-Genome Sequencing of a Less Prevalent Indian A. baumannii Strain Reveals Unique Uncharacterized Hypothetical Proteins and AMR-Linked ncRNAs. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Isolation and Whole-Genome Sequencing of a Less Prevalent Indian A. baumannii Strain Reveals Unique Uncharacterized Hypothetical Proteins and AMR-Linked ncRNAs. Sovon Acharya, Parmanand Kushwaha, Shailesh Desai, Langamba Longjam Angom, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8142534/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Acinetobacter baumannii is a high-priority pathogen due to its extensive antimicrobial resistance and persistence in clinical environments. This study describes the genomic features of AB_Varanasi, a carbapenem-resistant clinical isolate from India belonging to ST149 (Pasteur)/ST1506 (Oxford), a lineage not linked to major international clones. The isolate was resistant to almost all tested antibiotics, retaining susceptibility only to tigecycline and showing intermediate susceptibility to colistin. Whole-genome sequencing produced a 4.19 Mb circular chromosome encoding 4,039 predicted genes, with metabolism-associated functions forming the largest subsystem category. The genome carried multiple resistance determinants, including carbapenem-hydrolysing β-lactamases (OXA-23, OXA-104), extended-spectrum β-lactamases (ADC-26, PER-7), aminoglycoside-modifying enzymes, fluoroquinolone resistance–associated mutations, and several multidrug efflux systems (AdeABC, AdeIJK, AcrAB-TolC, and MFS transporters). Several of these genes were embedded within mobile genetic element–rich regions, suggesting a high potential for horizontal gene transfer. Virulence profiling identified the complete acinetobactin iron acquisition system, a functional Type VI secretion system, Type IV pilus components, and biofilm-associated operons. Variant analysis detected 30,385 high-confidence mutations with a transition-to-transversion ratio of 3.05. High-impact variants affected ribosomal protein L22 and the RNA polymerase β-subunit, while moderate-impact mutations involved metabolic and recombination-related genes. The regulatory landscape comprised 113 non-coding RNAs, including riboswitches and antisense RNAs. A total of 294 hypothetical proteins were identified, representing 192 DUF families, many predicted to encode membrane-associated or transport-related functions. Several of these uncharacterized proteins may serve as promising therapeutic or vaccine targets, given their potential surface exposure and species-specific conservation. Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Horizontal Gene Transfer (HGT) Antimicrobial Resistance (AMR) Whole-genome Sequencing (WGS) Hypothetical proteins (HP) Domain of Unknown Family (DUF) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Acinetobacter baumannii is a Gram-negative, opportunistic pathogen that has become one of the most challenging bacterial species in contemporary clinical practice. It is increasingly recognized for its capacity to cause a wide range of hospital-acquired infections (HAIs), especially among critically ill and immunocompromised patients. The pathogen is frequently isolated from intensive care units (ICUs), where it contributes substantially to ventilator-associated pneumonia (VAP), bloodstream infections, catheter-associated urinary tract infections (CAUTIs), wound infections, and surgical site infections. These infections often result in prolonged hospitalization, increased healthcare costs, and high morbidity and mortality rates. According to the World Health Organization’s (WHO) 2024 priority pathogen list, carbapenem-resistant A. baumannii (CRAB) ranks among the most critical threats to global health security, underscoring the urgent need for genomic and epidemiological surveillance ( 1 , 2 , 4 , 5 , 6 , 29 ) . Over the last few decades, A. baumannii has evolved into a paradigm of bacterial adaptability. Its success as a nosocomial pathogen is closely tied to its exceptional genomic plasticity and ability to acquire foreign genetic material. This species has accumulated a wide repertoire of antimicrobial resistance (AMR) and virulence genes, many of which are acquired through horizontal gene transfer (HGT). Such transfers are facilitated by mobile genetic elements (MGEs) including plasmids, transposons, insertion sequences, and integrons ( 30 , 31 ) . The interplay between these MGEs and selective pressure from antimicrobial use in hospital settings has driven the rapid evolution of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Early studies identified three predominant clonal groups circulating in Europe, designated as European clones I, II, and III. These lineages were later standardized as International Clones (ICs) 1, 2, and 3, respectively (37). Among them, IC1 and IC2 have achieved remarkable global dissemination and are responsible for many large-scale outbreaks involving carbapenem-resistant and MDR A. baumannii . Advances in whole-genome sequencing (WGS) and multilocus sequence typing (MLST) have subsequently expanded the classification of A. baumannii to at least eight recognized international clones, each characterized by distinct resistance mechanisms, virulence determinants, and geographical patterns of distribution ( 32 , 33 ) . The global spread of these clones has been largely driven by the carriage of carbapenemase-encoding genes such as bla OXA-23 , bla OXA-24/40 , and bla OXA-58 , frequently embedded within integrative conjugative elements and transposons ( 34 , 36 , 37 ) . The mobility of these genetic structures facilitates the horizontal dissemination of carbapenem resistance both within and between bacterial populations. Furthermore, A. baumannii possesses an intrinsic bla OXA-51-like gene that can become overexpressed through the insertion of an ISAba1 element upstream, further enhancing resistance to β-lactams. Together, these mechanisms have contributed to the establishment of globally dominant CRAB lineages that are highly adaptive in hospital environments. Genomic plasticity plays a central role in the ecological and clinical success of A. baumannii . The incorporation of type 1 integrons and pathogenicity islands enhances the bacterium’s ability to persist under environmental and antimicrobial stress. Integrons serve as efficient platforms for capturing and expressing resistance gene cassettes, while pathogenicity islands often encode virulence determinants associated with biofilm formation, iron acquisition, and adherence to abiotic surfaces. These genomic features collectively enable A. baumannii to survive in desiccated hospital environments, colonize medical equipment, and persist despite extensive disinfection efforts. While international clones dominate the global population structure of A. baumannii , several non-IC sequence types (STs) have been increasingly detected in clinical isolates. Sequence types such as ST25, ST78, and ST149 have emerged in different regions, each harboring distinct sets of resistance and virulence genes ( 35 , 36 , 37 ) . Although currently less prevalent, these STs represent potentially high-risk lineages that could play a more significant role in future outbreaks. The genetic diversity within these non-IC clones highlights the ongoing evolution of A. baumannii and its capacity to exploit new ecological niches under selective pressure. Carbapenem resistance in A. baumannii is primarily mediated by class D β-lactamases, commonly referred to as oxacillinases (OXAs), which hydrolyze carbapenems and other β-lactam antibiotics ( 28 ) . Other resistance mechanisms, including efflux pumps, porin loss, and target site mutations, also contribute to its MDR and XDR phenotypes. A recent study by Vijaykumar et al. (2022) reported that most A. baumannii isolates in India belong to the IC2 lineage, while clones IC7 and IC8 occur less frequently ( 32 ) . Despite the rising clinical significance of CRAB in India, comprehensive genomic data on Indian isolates remain scarce. In particular, the genomic architecture and evolutionary trajectory of A. baumannii ST149, a non-IC lineage, are not yet well characterized. Due to the widespread presence of carbapenemases and other antibiotic resistance determinants, treating A. baumannii infections in hospital settings has become increasingly challenging. This highlights an urgent need to identify novel therapeutic targets. Still a substantial proportion of the A. baumannii proteome remains uncharacterized, suggesting that unexplored proteins—particularly hypothetical proteins—may hold untapped potential for drug discovery ( 46 ) . In this study, we report the whole-genome sequencing of a carbapenem-resistant Acinetobacter baumannii isolate from India belonging to sequence type 149 (ST149). By systematically characterizing its resistome, virulome, and mobilome, we further delineate the repertoire of hypothetical proteins, with the goal of identifying novel candidates that may serve as therapeutic targets. 2. Materials and Methods 2.1. Bacterial isolation and identification The Acinetobacter baumannii isolate used in this study was obtained from a blood culture sample processed in the diagnostic microbiology laboratory of IQ City Medical College & NH Hospital, Durgapur, India. Ethical approval for this work was granted by the Institutional Ethics Committee of IQ City Medical College & NH Hospital (Approval No. IQMC/IEC/Project/17/28). The isolate was recovered from anonymized clinical culture plates with no access to identifiable patient information. According to the Council for International Organizations of Medical Sciences (CIOMS)–World Health Organization (WHO) International Ethical Guidelines for Health-related Research Involving Humans (2016) and the Indian Council of Medical Research (ICMR) guidelines, the use of de-identified bacterial isolates does not constitute human subject research and therefore does not require individual patient consent. Primary isolation was performed on MacConkey agar, and colony morphology was documented following 24 h incubation at 37°C. Gram staining was conducted to confirm cellular morphology. Species identification was performed using PCR amplification targeting the gyrB gene, a species-specific region of A. baumannii . The isolate was further validated through biochemical profiling and genomic analysis. 2.2. Antimicrobial susceptibility testing (AST) Antimicrobial susceptibility testing was performed using the VITEK® 2 Compact system (bioMérieux, France) based on the automated broth microdilution method. The antimicrobial agents tested included β-lactams, aminoglycosides, fluoroquinolones, and carbapenems commonly used against Acinetobacter infections. The results were validated by the Kirby–Bauer disk diffusion method to ensure concordance. Interpretation of susceptibility profiles was carried out according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2023). 2.3. Transmission electron microscopy (TEM) For ultrastructural visualization, bacterial cells were concentrated by gentle centrifugation and resuspended in sterile phosphate-buffered saline (PBS). A 5 µL aliquot of the suspension was placed on a formvar-coated copper grid and allowed to adsorb for 5 min. The sample was then negatively stained with 1% (w/v) uranyl acetate for 1 min, air-dried, and examined under a transmission electron microscope operated at 80 kV. Images were captured at various magnifications to assess cell morphology and surface architecture. 2.4 Genomic DNA extraction, library preparation, and whole-genome sequencing (WGS) Genomic DNA was extracted from an overnight culture of A. baumannii grown in Luria–Bertani broth using the cetyltrimethylammonium bromide (CTAB)–phenol–chloroform method. A 1.5 mL aliquot of culture was centrifuged at 4000 rpm for 5 min, and the resulting pellet was subjected to CTAB-mediated lysis followed by phenol–chloroform–isoamyl alcohol purification. DNA integrity was assessed on a 0.8% agarose gel, and concentration was quantified using a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, USA). Sequencing libraries were prepared with the QIAseq FX DNA Library Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Paired-end sequencing (2 × 150 bp) was performed on the Illumina NovaSeq 6000 platform using v1.5 chemistry (300-cycle kit). Raw reads were subsequently processed through a standard quality control workflow before assembly and annotation. 2.5 Genome assembly, annotation, and bioinformatic analysis Raw read quality was evaluated using FastQC (accessed 15 May 2025) to assess per-base sequence quality, GC distribution, and duplication levels. All downstream analyses were conducted on the Galaxy Europe platform ( https://usegalaxy.eu/ ; accessed 15 May 2025). Adapter sequences and low-quality bases were removed with Fastp (11) using default parameters, ensuring retention of high-quality reads for assembly. De novo assembly was generated using Unicycler v0.8.4.0, which employs short-read assembly with conservative bridging to resolve repeat-rich regions and improve contiguity. Assembly statistics, including N50, L50, GC content, and total genome size, were assessed using QUAST to confirm completeness and structural accuracy. Gene prediction and functional annotation were conducted using Prokka (12), which identified coding sequences, rRNAs, tRNAs, and additional genomic features using curated bacterial databases. Annotation reliability was further supported by cross-validation with the Rapid Annotations using Subsystems Technology (RAST) server, enabling subsystem-level functional categorisation of metabolic, stress-response, and virulence pathways. Sequence type assignment followed the A. baumannii MLST scheme hosted on PubMLST. Antimicrobial resistance (AMR) determinants were identified using ResFinder and the Comprehensive Antibiotic Resistance Database (CARD), with high-confidence hits defined by ≥ 90% identity and ≥ 80% coverage thresholds. Mobile genetic elements (MGEs) were detected using ISEScan for insertion sequences, Integron Finder 2.0 (17) for integron structures, and IslandViewer 4 (18) for genomic island identification based on SIGI-HMM, IslandPath-DIMOB, and IslandPick prediction algorithms. Genome visualization and mapping of annotated features were performed using Proksee. Virulence-associated genes were identified using the Virulence Factor Database (VFDB) (15) and independently verified with BacWGSTdb (16). Annotation and AMR predictions were cross-checked against outputs from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) to ensure concordance across platforms. Key resistance and virulence loci were further validated by pairwise nucleotide and protein alignments using BLASTn and BLASTp. Structural predictions for hypothetical proteins were generated using AlphaFold (38), enabling preliminary functional inference based on protein fold and domain architecture. 2.6. Variant detection using DeepVariant High-confidence SNPs and small indels were detected using DeepVariant v1.6.0 (Poplin et al., 2018), a deep-learning–based variant caller that improves base-level accuracy by modeling sequencing errors. The DeepVariant pipeline was run with the model type set to WGS and the reference genome CP000521. Resulting variant calls were exported in VCF format. Quality filters were applied using bcftools to retain variants with a minimum quality score (QUAL) ≥ 30 and read depth (DP) ≥ 10. Multi-allelic and low-confidence variants were excluded. 3. Results 3.1. Isolation, Identification, and Genomic Characterisation led to annotation yielding 4039 predicted proteins The combined Gram staining, TEM morphology, and gyrB-based PCR profiling consistently supported the identity of the isolate as a Gram-negative Acinetobacter strain. These results provided the foundational confirmation required for downstream genomic and phenotypic analyses (Fig. 1 ). The antimicrobial sensitivity test (AST) indicated that this isolate is only sensitive to Tigecycline and exhibited intermediate-level sensitivity to colistin (Table 1 ). The genome sequencing was performed using the WGS method. Using a standard quality control and adapter trimming tool, FastP on the Galaxy platform trimming was performed. Approximately 16.389052 million Illumina high-quality reads were trimmed to 16.099834 million. We obtained a genome size of the isolate approximately 4.1 Mb comprising a single circular chromosome (4,191,980 bp), which is circular. The whole genome contains 39% Guanine-Cytosine (GC) content. Annotation of the genome showed that the genome encodes 4039 predicted genes. Among them, 3970 CDS, 4 rRNAs, 64 tRNAs, and 1 tmRNA (Fig. 2 ). One CRISPR-related sequence was found. The sequence type of this isolate was ST1506 (31-33-67-40-1-95-7) under the Oxford MLST scheme. Under the Pasture MLST scheme, this genome belongs to ST149 (3-12-11-2-14-9-14). The strain is not part of the well-known highly pathogenic ICs, and yet it possesses all the AMR genes (Table 2 ) possibly exhibiting a hypervirulence phenotype. To evaluate genomic attributes, predicted genes were functionally categorised using subsystems (Fig. 3 ). The isolate's broad metabolic adaptability was highlighted by the fact that the majority of its genes (782 genes across 97 subsystems) were linked to metabolism. Genes related to energy production (230 genes, 30 subsystems), stress response, defence, and virulence (133 genes, 38 subsystems), and protein processing (220 genes across 40 subsystems) came next (Fig. 3 ). The bacterium's ability to adapt to its environment and possible resistance mechanisms is highlighted by the significant representation of genes linked to stress and virulence. Table 1 AST result by Kirby-Bauer disk diffusion test & MICs of antibiotics. S. No. Antibiotic Zone of Inhibition (mm) CLSI Breakpoint (Susceptible ≥ mm) Interpretation 1 Ceftazidime (CAZ 30) 0 ≥ 14 Resistant 2 Co-trimoxazole (COT 25) 0 ≥ 10 Resistant 3 Imipenem (IMP 10) 0 ≥ 18 Resistant 4 Colistin (CL 10) 12 ≥ 13 Intermediate 5 Ciprofloxacin (CIP 5) 0 ≥ 15 Resistant 6 Gentamicin (GEN 10) 0 ≥ 12 Resistant 7 Cefepime (FEP 30) 8 ≥ 14 Resistant 8 Meropenem (MEM 10) 0 ≥ 14 Resistant Table 2 Antimicrobial Resistance (AMR) Genes. Serial No. Resistance Gene Identity (%) Coverage (%) Phenotype Accession No. 1 armA 100.00 100.0 Amikacin, Gentamicin, Tobramycin, Isepamicin, Netilmicin AY220558 2 aph(3')-VI 99.23 100.0 Amikacin KC170992 3 aph(6)-Id 100.00 100.0 Streptomycin M28829 4 aph(3'')-Ib 100.00 99.88 Streptomycin AF024602 5 aph(3'')-Ib 99.88 100.0 Streptomycin AF321551 6 aph(3'')-Ib 99.88 100.0 Streptomycin AF313472 7 aph(3'')-Ib 99.88 100.0 Streptomycin AF321550 8 blaADC-25 96.70 99.91 Unknown Beta-lactam EF016355 9 blaOXA-203 99.88 100.0 Unknown Beta-lactam HQ998857 10 blaPER-7 100.00 100.0 Amoxicillin, Amoxicillin + Clavulanic acid, Ampicillin, Ampicillin + Clavulanic acid, Cefotaxime, Cefoxitin, Cefepime, Ceftazidime, Piperacillin, Piperacillin + Tazobactam, Ticarcillin, Ticarcillin + Clavulanic acid, Aztreonam HQ713678 11 blaOXA-23 100.00 100.0 Imipenem, Meropenem AY795964 12 msr(E) 100.00 100.0 Erythromycin, Azithromycin, Quinupristin, Pristinamycin IA, Virginiamycin S FR751518 13 mph(E) 100.00 100.0 Erythromycin DQ839391 14 cmlA1 99.68 100.0 Chloramphenicol M64556 15 ARR-2 100.00 100.0 Rifampicin HQ141279 16 sul1 100.00 100.0 Sulfamethoxazole U12338 17 sul1 100.00 100.0 Sulfamethoxazole U12338 18 sul2 100.00 85.42 Sulfamethoxazole AJ830710 19 sul2 100.00 85.42 Sulfamethoxazole AY034138 20 tet(B) 100.00 100.0 Doxycycline, Tetracycline, Minocycline AP000342 3.2. AMR Gene Profiling Reveals the presence of genes associated with the Active Efflux of the Chemotherapeutic Agents. The resistome of this isolate encompasses 11 functionally distinct categories, with macrolide-associated genes (205 genes), phenicol resistance determinants (79 genes), and multidrug efflux systems (79 genes) representing the predominant resistance mechanisms. Beta-lactam resistance is mediated by multiple clinically significant enzymes, including the carbapenem-hydrolyzing class D beta-lactamases OXA-23 and OXA-104, the extended-spectrum class C beta-lactamase ADC-26, the inhibitor-resistant extended-spectrum class A beta-lactamase PER-7, and the chromosomally-encoded AmpC cephalosporinase, collectively conferring resistance to penicillins, cephalosporins, and carbapenems. Aminoglycoside resistance mechanisms of this isolate are encoded by four distinct resistance genes: aminoglycoside O-phosphotransferases APH(3′)-VIa, APH(6)-Id, and APH(3″)-Ib, alongside the aminoglycoside nucleotidyltransferase ANT(3″)-IIa, providing enzymatic inactivation of gentamicin, tobramycin, amikacin, and kanamycin. Fluoroquinolone resistance is supported by mutations or upregulation of topoisomerase genes, specifically DNA gyrase subunits A and B ( gyrA , gyrB ) and DNA topoisomerase IV subunits A and B ( parC , parE ), conferring resistance to ciprofloxacin and levofloxacin. Tetracycline resistance is conferred by the major facilitator superfamily (MFS) efflux transporter Tet(B), regulated by the tetracycline resistance transcriptional repressor TetR(B), enabling active efflux-mediated resistance. The genome harbors a sophisticated multidrug efflux architecture comprising the tripartite resistance-nodulation-division (RND) systems AdeABC, AdeIJK, and AcrAB-TolC, alongside the efflux pumps EmrAB and additional MFS transporters, collectively mediating resistance to aminoglycosides, fluoroquinolones, macrolides, tetracyclines, tigecycline, and disinfectants. Sulfonamide resistance is mediated by sulfonamide-resistant dihydropteroate synthase variants Sul1 and Sul2, while fosfomycin resistance is conferred by the glutathione transferase FosLL and the MFS transporter AbaF. This multifaceted AMR profile, encompassing enzymatic inactivation, target modification, active efflux, and metabolic bypass, represents a clinically significant multi drug-resistant phenotype with substantial implications for therapeutic management. Additionally, the presence of the gene qacEΔ1, associated with the reduced susceptibility to quaternary ammonium compounds and disinfectants such as chlorhexidine, was identified, suggesting potential tolerance to antiseptics and disinfectants used in clinical settings (Table 2 & Fig. 4 ). 3.3. Virulence and Biofilm-Associated Genes Show Diverse Repertoire of Virulence-associated Genes The AB_Varanasi isolate encodes approximately 378 virulence-associated genes, of which 92 are identified in VFDB, reflecting a multifaceted pathogenic repertoire. Central to its virulence strategy is iron acquisition (52 genes), encompassing the complete acinetobactin biosynthesis system (BasA–BasJ) and associated transport machinery (BauA–BauF), ensuring efficient scavenging under host-imposed nutritional immunity. The isolate also possesses an extensive suite of adhesion and invasion determinants (171 genes), including outer membrane protein variants (OmpA and specialized receptors) and a Type IV pilus system (33 genes), facilitating host cell recognition, attachment, and surface colonization. For interbacterial competition and host manipulation, AB_Varanasi harbors a complete Type VI secretion system (14 genes) capable of effector protein delivery, alongside 13 phospholipase genes producing membrane-disrupting enzymes. Its biofilm persistence machinery (10 genes), including the PGA synthesis cluster, supports enhanced survival on both abiotic and biotic surfaces. Finally, regulatory networks (8 genes), particularly quorum-sensing systems, fine-tune virulence expression in response to population density, collectively underpinning the isolate’s adaptive pathogenic potential ( Table 3 ). AB_Varanasi's virulence genes may enhance tissue invasion, immune modulation, and persistence in clinical environments. Table 3 Virulence Genes Present in this isolate. S. No. Gene Contig Identity (%) Position Description 1 basJ 1 97.78 56573..57742 acinetobactin biosynthesis protein BasJ 2 basI 1 96.83 57867..58622 phosphopantetheinyl transferase component of acinetobactin biosynthesis protein BasI 3 basH 1 98.23 58633..59367 non-ribosomal peptide biosynthesis thioesterase BasH 4 barB 1 97.49 59439..61034 siderophore efflux system of the ABC superfamily 5 barA 1 96.52 61031..62641 siderophore efflux system of the ABC superfamily 6 basG 1 98.87 62887..64038 acinetobactin biosynthesis protein BasF 7 basF 1 97.7 64155..65024 aryl carrier protein BasF 8 entE 1 96.56 65042..66670 non-ribosomal peptide synthetase adenylate-forming enzyme of acinetobactin synthesis 9 basD 1 96.67 66925..69774 acinetobactin biosynthesis protein BasD 10 bauA 1 99.3 71199..73481 TonB-dependent siderophore receptor BauA 11 bauB 1 98.45 73567..74535 ferric siderophore ABC transporter, periplasmic siderophore-binding protein 12 bauC 1 97.15 75309..76256 ferric siderophore ABC transporter, permease protein BauC 13 bauD 1 96.44 76270..77197 ferric siderophore ABC transporter, permease protein BauD 14 bfmS 10 98.18 106102..107751 signal transduction histidine kinase 15 bfmR 10 98.88 107784..108500 biofilm-controlling response regulator 16 abaI 11 99.46 61431..61982 N-acyl-L-homoserine lactone synthetase 17 abaR 11 98.19 59461..60177 DNA-binding HTH domain-containing protein 18 plc 11 97.83 137693..139861 phospholipase C 19 ompA 16 99.25 95503..96573 outer membrane protein OmpA 20 adeG 2 97.04 19093..22272 cation/multidrug efflux pump 21 csuE 2 97.16 132548..133567 Csu pilus tip adhesin CsuE 22 pgaD 2 99.57 205626..206090 poly-beta-1,6-N-acetyl-D-glucosamine biosynthesis protein PgaD 23 plcD 25 98.89 24110..25735 phosphatidylserine/phosphatidylglycerophosphate/cardiolipin synthase 3.5. MGE Indicates Genomic Architecture Associated with Multiple Resistant Determinants Analysis of antimicrobial resistance gene-MGE co-localization demonstrated that 131 AMR genes (76.61% of total resistance determinants) are situated on MGE-rich contigs (≥ 10 MGE per contig), establishing a direct association between mobile genetic elements and resistance gene dissemination. High-density AMR-MGE co-occurrence was observed on contig_3 (16 AMR genes, 44 MGE), contig_7 (15 AMR genes, 29 MGE), contig_4 (14 AMR genes, 21 MGE), and contig_1 (10 AMR genes, 28 MGE), indicating genomic hotspots for resistance gene acquisition and mobilization. Clinically significant AMR genes identified on MGE-rich contigs include the carbapenem-hydrolyzing beta-lactamase blaOXA-104 (contig_4), the extended-spectrum beta-lactamase blaPER-7 (contig_27), the macrolide phosphotransferase mph(E) (contig_27), sulfonamide resistance genes sul1 (contig_27), the aminoglycoside nucleotidyltransferase ant(3″)-IIa (contig_12), and the fosfomycin resistance transferase fosLL (contig_6). The extensive mobilome, high frequency of IS elements, presence of functional integrons, multiple plasmid replicons, and prophage integration sites, combined with the substantial co-localization of resistance determinants on MGE-rich genomic regions, collectively indicate the significant horizontal gene transfer potential of this isolate and its capacity for rapid dissemination of antimicrobial resistance and virulence traits within microbial communities. ( Table 4 & Supplementary File 2). Analysis of the 18.9–22.4 kb integron region showed a class 1 integron with a well-defined integrase, attI/attC recombination sites, and several adjacent mobile-element–associated genes. Multiple hypothetical proteins were also present within this locus, indicating additional uncharacterized components potentially linked to cassette mobility or resistance acquisition ( Fig. 6 ). Table 4 Variant types present in this isolate Sl. No. Variant Type Count Example Genes Functional Category Predicted Impact 1 Synonymous 25 Lipid A phosphoethanolamine transferase, hypothetical proteins Neutral mutations Low 2 Missense 4 Glycine dehydrogenase, MerR recombinase Metabolic and regulatory adaptation Moderate 3 Frameshift 2 rpoB, L22p ribosomal protein Transcription and translation machinery High 3.6 Elevated Ti/Tv Ratio and Genomic Plasticity indicates Variant Landscape of A. baumannii Varanasi Analysis of AB_Varanasi with DeepVariant identified 30,385 high-confidence variants, comprising 29,926 SNPs and 458 indels, with an unusually high transition-to-transversion (Ti/Tv) ratio of 3.05, markedly above the typical bacterial range of 2.0–2.3. This elevated ratio likely reflects non-random mutational processes driven by multiple factors: oxidative stress from reactive oxygen species during persistent infection inducing characteristic G→A transitions; spontaneous cytosine deamination resulting in C→T changes; selective pressures favoring transitions over transversions at functionally important sites; and DNA polymerase error patterns arising from replication fidelity alterations during adaptation. Notably, the most frequent substitutions—G→A (5,836) and T→C (5,636)—account for 38.3% of all SNPs, reinforcing the notion of directed mutational biases rather than purely stochastic events. This distinctive mutational signature likely represents adaptation-driven evolution under hospital-associated selection pressures and is clinically significant, potentially revealing vulnerabilities in DNA repair or replication fidelity that could be therapeutically exploited ( Fig. 5 ). 3.7 Identification and distribution of SNPs in A. baumannii isolates Whole-genome variant analysis identified multiple synonymous and nonsynonymous substitutions distributed across key functional genes of Acinetobacter baumannii isolates. A total of 31 high-confidence single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) were detected, with variant frequencies ranging from 0.45 to 1.00, indicating the presence of both fixed and subclonal mutations within the populations. Approximately 80% of the substitutions were synonymous and classified as low-impact changes according to SnpEff annotations. In contrast, five missense variants and two frameshift mutations were categorized as moderate- to high-impact, potentially influencing protein function and fitness ( Table 4 ). 3.8 SNP-Based Functional Insights and Adaptive Significance of A. baumannii isolate Whole-genome SNP analysis revealed multiple genetic alterations with potential functional and adaptive implications in A. baumannii populations. Two high-impact frameshift mutations were identified among the analyzed isolates, indicating possible disruptions in essential cellular processes. The first was a frameshift deletion in the LSU ribosomal protein L22p (L17e) gene (470.25780.con.0005:191123), which resulted in a loss of the start codon (Met1fs) and predicted truncation of the encoded protein. Given that ribosomal protein L22p plays a crucial role in the assembly of the 50S ribosomal subunit and serves as part of the macrolide antibiotic-binding pocket, such a mutation is likely to impair ribosomal biogenesis and modulate antibiotic susceptibility, particularly towards macrolides and ketolides. The second high-impact event was a frameshift insertion in the DNA-directed RNA polymerase β subunit gene (470.25780.con.0009:5908), producing an Ala629 frameshift that may alter RNA polymerase structure and compromise transcriptional fidelity. Mutations in this subunit are known to influence global transcriptional regulation and may facilitate adaptive responses under antibiotic or oxidative stress, enhancing bacterial persistence in hostile environments. Beyond these severe disruptions, several moderate-impact missense mutations were detected in genes linked to metabolic and regulatory functions, reflecting ongoing microevolutionary diversification. An AlaLeuThr440ThrLeuAla substitution in glycine dehydrogenase (decarboxylating) (470.25780.con.0004:253773) could influence glycine cleavage efficiency and redox balance, critical for energy metabolism under nutrient limitation. Similarly, a Thr45Ser mutation in a resolvase family recombinase (470.25780.con.0019:47635) may enhance site-specific recombination, potentially increasing genome plasticity and facilitating horizontal gene transfer. Additionally, a Lys119Asn substitution in glycine dehydrogenase (P-protein) (470.25780.con.0064:357) might alter cofactor binding, improving metabolic adaptability during antibiotic stress. These high- and moderate-impact SNPs signify adaptive genomic fine-tuning that could reshape fundamental processes such as transcriptional regulation, ribosomal function, and metabolic plasticity, thereby promoting the survival of A. baumannii under diverse environmental and therapeutic pressures. Such mutations may underpin the evolutionary transition of emerging clones (e.g., ST149) towards enhanced persistence and resistance, reflecting a dynamic genomic response to continuous antibiotic selection (Supplementary file 4). 3.9 Non-Coding RNAs and Regulatory Control of Antimicrobial Resistance in AB Varanasi The regulatory landscape supporting extensive antimicrobial resistance in this isolate includes a sophisticated non-coding RNA (ncRNA) network comprising 113 RNA features (2.79% of total genomic elements) with direct implications for resistance gene expression, metabolic adaptation, and stress response. While the essential translational machinery—65 tRNA genes providing complete codon coverage with variable isoacceptor redundancy (1–7 per amino acid), a complete rRNA operon (5S-16S-23S), and a single tmRNA (SsrA) for ribosome quality control—ensures efficient synthesis of the 800 identified antimicrobial resistance proteins, the regulatory ncRNA complement establishes post-transcriptional control mechanisms that modulate resistance phenotypes. Critically, the genome encodes a putative aminoglycoside riboswitch/attI site that likely functions as a cis-acting metabolite-sensor capable of direct sensing of aminoglycoside antibiotics and potentially modulating expression of aminoglycoside resistance genes in response to antibiotic presence, representing a novel and previously uncharacterized direct link between antibiotic presence and resistance gene regulation. The ncRNA-mediated regulatory architecture includes 22 copies of C4 antisense RNA, representing the predominant regulatory class and suggesting extensive post-transcriptional suppression of mRNA targets, with potential roles in silencing susceptibility factors or modulating expression of multidrug efflux pump genes to optimize resistance phenotypes under varying environmental conditions. An additional 8 metabolite-sensing riboswitches were identified beyond the aminoglycoside-sensing element, including FMN riboswitch (modulating flavin-dependent oxidoreductases and energy metabolism supporting resistance), TPP riboswitch (controlling vitamin B12-independent metabolic pathways essential for nucleotide synthesis and DNA repair under antibiotic stress), cobalamin riboswitch (regulating B12-dependent pathways essential for stress responses), glycine riboswitch (governing amino acid metabolism affecting protein synthesis capacity for the synthesis of proteins associated with resistance mechanism), guanidine-I riboswitch (sensing nitrogen availability critical for growth under nutrient limitation encountered in biofilms), yybP-ykoY manganese riboswitch (modulating metal homeostasis and oxidative stress resistance mechanisms), and fluoride riboswitch (crcB) (enabling response to fluoride-containing disinfectants and antiseptics used in infection control). These metabolite-responsive elements establish direct links between nutrient and metabolite availability, stress conditions, and expression of metabolic genes that support growth, stress tolerance, and sustained antimicrobial resistance under nutrient-limiting or stress conditions. Four Acinetobacter -specific small regulatory RNAs (sRNAs)—including two copies of Acinetobacter sRNA 25, and single copies of Acinetobacter sRNA 11, Acinetobacter sRNA 28, and Aar sRNA—likely mediate species-specific regulatory programs governing virulence, stress adaptation, and metabolic capabilities unique to Acinetobacter baumannii , potentially including direct or indirect regulation of resistance gene expression, biofilm formation supporting antimicrobial tolerance, and stress-responsive adaptations to host immune pressures and antibiotic challenge. Structural RNAs essential for resistance gene expression include bacterial RNase P class A (mediating tRNA maturation critical for translation of resistance proteins), signal recognition particle RNA (directing membrane insertion of efflux pump components), and 6S/SsrS RNA (orchestrating stationary phase transcription including potential upregulation of resistance genes during nutrient-limited conditions encountered in biofilms or host tissues). The integration of cis-acting riboswitches (particularly aminoglycoside-sensing and metal-responsive elements), trans-acting antisense RNAs (C4; 22 copies), and species-specific regulatory sRNAs establishes a multi-layered post-transcriptional regulatory network that complements the transcriptional control of resistance genes, enabling rapid and reversible modulation of antimicrobial resistance phenotypes in response to nutrient availability, metabolite concentration, stress signals, and antibiotic challenge, thereby enhancing survival in the face of antibiotic pressure and environmental heterogeneity (Supplementary File 1). 3.10 Hypothetical and Uncharacterized Protein Repertoire of AB Varanasi Comprehensive genome annotation identified 294 hypothetical and uncharacterized proteins representing 7.48% of the total coding sequences, indicating a moderate proportion of the genome with undefined or poorly characterized functions. The hypothetical protein repertoire is predominantly composed of proteins containing Domains of Unknown Function (DUF), which constitute 222 proteins (75.5% of hypothetical proteins), representing 192 unique DUF families distributed across the genome. The most abundant DUF families include DUF559 (putative adhesion protein, 3 proteins), DUF1833 (uncharacterized conserved protein, 3 proteins), DUF3298 (predicted membrane protein, 3 proteins), DUF442 (putative metal-binding protein, 3 proteins), DUF2147 (predicted membrane protein, 3 proteins), and DUF606 (predicted lipoprotein, 3 proteins), suggesting potential roles in bacterial adherence, membrane structure and function, metal homeostasis, and cellular interactions (Supplementary File 3). Beyond DUF-containing proteins, 41 sequences (13.9%) are annotated as hypothetical proteins without assigned domain architecture, while 20 proteins (6.8%) belong to uncharacterized protein families (UPF) with conserved sequence motifs but undefined biochemical functions. An additional 10 proteins (3.4%) are designated as putative proteins with predicted but experimentally unvalidated functions. Analysis of product annotations revealed potential functional associations for a subset of hypothetical proteins, including membrane-associated proteins (6 proteins), transport and permease functions (6 proteins), signal transduction components (4 proteins), regulatory elements (2 proteins), DNA/RNA-binding proteins (1 protein), and stress response factors (1 protein), providing preliminary functional hypotheses for future experimental validation. The distribution of hypothetical proteins across genomic scaffolds demonstrates variable density, with contig_2 harboring the highest concentration (27 proteins), followed by contig_1 and contig_4 (21 proteins each), and an average distribution of 19.4 hypothetical proteins across the top 10 contigs. The substantial proportion of DUF-containing proteins and the diversity of represented DUF families (192 unique families) reflects the evolutionary plasticity and functional adaptability of this bacterial isolate, with uncharacterized genomic elements potentially contributing to niche-specific adaptations, environmental stress tolerance, host-pathogen interactions, or novel metabolic capabilities. The identification of membrane-associated and transport-related hypothetical proteins suggests potential involvement in nutrient acquisition, efflux-mediated resistance mechanisms, or host cell interactions, warranting targeted experimental characterization to elucidate their functional roles in bacterial physiology and pathogenesis. The moderate proportion (7.48%) of hypothetical proteins relative to total coding sequences indicates a well-characterized genome with established functional annotations for the majority of predicted genes, while the presence of 294 uncharacterized sequences may provide opportunities for discovery of novel bacterial functions and adaptive mechanisms unique to this extensively drug-resistant isolate. 4. Discussion This study characterises the genomic features of an Acinetobacter baumannii ST149 isolate recovered in India and demonstrates that this non-international clone lineage exhibits many of the hallmarks associated with high-risk, extensively drug-resistant strains. The increasing burden of A. baumannii infections among hospitalised and immunocompromised patients in India underscores the need for detailed genomic analyses of underrepresented lineages. Although ST149 is not part of the dominant international clone groups, its genomic configuration indicates substantial adaptive capacity and clinical relevance. The isolate displayed an extensively drug-resistant phenotype, with susceptibility restricted to tigecycline and intermediate susceptibility to colistin. This pattern is consistent with the widespread emergence of carbapenem-resistant A. baumannii driven largely by class D β-lactamases. The coexistence of bla OXA-23, bla OXA-104, bla ADC-26, and bla PER-7 within the same genome reflects the accumulation of diverse β-lactamase families, a feature commonly observed in global epidemic clones. The combined presence of aminoglycoside-modifying enzymes, sulfonamide resistance genes, and multiple efflux systems suggests a multidimensional resistance strategy integrating enzymatic inactivation, target modification, and active efflux. The detection of qacEΔ1 further indicates a potential for tolerance to disinfectants, supporting environmental persistence within healthcare settings. The strong association between antimicrobial resistance genes and mobile genetic elements highlights the dynamic nature of this genome. The localisation of most AMR determinants on MGE-rich contigs, including those encoding clinically relevant β-lactamases and macrolide resistance genes, suggests the presence of genomic regions analogous to resistance islands. This facilitates horizontal gene transfer events and may accelerate the spread of resistance traits within and between bacterial species. In addition to its extensive resistome, the isolate possesses a broad virulence genes repertoire. Complete acinetobactin clusters, multiple adhesion-associated genes, phospholipases, and a functional Type VI secretion system provide a foundation for host colonisation, nutrient acquisition, and interbacterial competition. The presence of biofilm-associated operons, including pgaABCD, supports the potential for long-term persistence on abiotic surfaces. These features collectively indicate a strain capable of maintaining fitness despite the metabolic costs associated with multidrug resistance. The variant analysis revealed an elevated transition-to-transversion ratio, suggesting a mutational bias consistent with oxidative and antibiotic stress. High-impact mutations in rplV (L22) and rpoB may modulate ribosomal and transcriptional processes, while moderate-impact substitutions affecting metabolic and recombination-related genes point toward ongoing microevolution. These findings support the view that selective pressures in clinical settings contribute to the emergence of distinctive mutational patterns in A. baumannii . The non-coding RNA landscape adds an additional regulatory dimension. The presence of multiple riboswitches, including a putative aminoglycoside-responsive element, indicates an ability to couple environmental cues with resistance gene expression. The abundance of C4 antisense RNAs and Acinetobacter-specific sRNAs suggests active post-transcriptional regulation influencing stress adaptation, virulence, and antimicrobial tolerance. This regulatory complexity likely enhances the strain’s ability to respond to fluctuating environmental and therapeutic conditions. Hypothetical proteins constituted a substantial portion of the AB_Varanasi genome, reflecting an unexplored genetic reservoir with potential functional relevance. Growing evidence suggests that hypothetical proteins can contribute to virulence, stress tolerance, and host interaction in several bacterial pathogens. Many of these proteins contained Domains of Unknown Function (DUFs), which are increasingly recognized as contributors to bacterial physiology, virulence and adaptation (46). The identification of 294 hypothetical or uncharacterized proteins, many belonging to diverse DUF families, points to a substantial reservoir of unexplored functional potential. Several predicted membrane-associated and transport-related proteins may contribute to adaptive processes that remain to be fully understood. Their characterisation could reveal novel mechanisms relevant to virulence, stress tolerance, or drug resistance. Overall, the genomic architecture of the ST149 isolate indicates a lineage with the potential to evolve into a clinically significant clone. Although ST149 remains less prevalent than major international clones, its extensive resistome, mobilome, virulence repertoire, and regulatory network suggest that it possesses the attributes required for successful adaptation and persistence. Broader genomic surveillance, particularly within South Asia, will be important for monitoring the emergence and spread of such lineages. The genomic insights presented here also provide a reference framework for future functional studies and may support the development of targeted interventions, including vaccine design and molecular diagnostics. 5. Conclusion This genomic analysis of the Acinetobacter baumannii ST149 isolate from India reveals a lineage with considerable adaptive potential despite its limited representation among globally dominant international clones unlike ST2. The genome harbours an extensive repertoire of antimicrobial resistance genes, a diverse set of virulence-associated genes, and a broad array of mobile genetic elements that collectively support its capacity for persistence, transmission, and therapeutic evasion. The unusually high transition-to-transversion ratio, together with several high-impact mutations in essential genes, indicates an active evolutionary trajectory shaped by strong selective pressures in clinical environments. The regulatory landscape, enriched with non-coding RNAs and metabolite-sensing riboswitches, further underscores the dynamic transcriptional and post-transcriptional control mechanisms contributing to its resilience. Finally, the presence of a substantial number of hypothetical and uncharacterized proteins highlights functional domains that remain unexplored and may represent novel contributors to host interaction, environmental survival, or antimicrobial resistance. Taken together, these findings broaden current understanding of emerging non-IC A. baumannii lineages and provide a foundation for future functional studies aimed at identifying diagnostic markers and potential therapeutic targets against this evolving pathogen strain. Declarations Data availability: The whole-genome sequencing (WGS) data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1201564. The assembled genome has been submitted to GenBank under the accession number JBRYYY000000000. All relevant data supporting the findings of this study are available within the article and its Supplementary Information files, or from the corresponding author upon reasonable request. Conflict of interest statement: The authors have stated explicitly that there are no conflicts of interest in connection with this article. Ethics statement: This study was approved by the Institutional Ethics Committee of IQ City Medical College & NH Hospital, Durgapur, India (Approval No. IQMC/IEC/Project/17/28). The Acinetobacter baumannii isolate in this study was recovered from discarded clinical culture plates without access to any patient-identifiable information. According to the International Ethical Guidelines for Health-related Research Involving Humans (CIOMS-WHO, 2016) and the Indian Council of Medical Research (ICMR) guidelines, such use of anonymized bacterial isolates without any associated personal data does not require individual patient consent, as it does not constitute human subject research. Authors’ contribution: SA, PK, PS & GC contributed to the study design. LAL, BC, PK, and SA collected bacterial samples. Genomic data was analysed and interpreted by SA, PK, AG, AV, SD1, SD2, PS, and GC. TEM was done by SA, PK & SG. SA and PK designed the figures. The manuscript was written by SA and PK. GC, RSPR, and PS co-edited the manuscript with SD1, MR, LK, SD1, SD2, AV. PS & GC supervised the study. All authors had full access to all the data in the study and accepted the responsibility for the decision to submit for publication. Authors’ Contributions (CRediT taxonomy) Conceptualization : SA, PK, PS, GC Sample collection : LAL, BC, PK, SA Data analysis : SA, PK, AG, AV, SD1, SD2, PS, GC Investigation & TEM : SA, PK, SG Visualization : SA, PK Writing – original draft : SA, PK Writing – review & editing : SD1, MR, LK, SD2, AV, GC, RSPR, PS Supervision : PS, GC All authors reviewed and approved the final manuscript. Ethics statement: This study was approved by the Institutional Ethics Committee of IQ City Medical College & NH Hospital, Durgapur, India (Approval No. IQMC/IEC/Project/17/28). The Acinetobacter baumannii isolate used in this work was obtained from discarded clinical culture plates, with no access to patient-identifiable information. In accordance with the International Ethical Guidelines for Health-related Research Involving Humans (CIOMS-WHO, 2016) and the Indian Council of Medical Research (ICMR) guidelines, the use of anonymized bacterial isolates without linked personal data does not constitute human subject research and therefore does not require individual informed consent. We have carefully followed the CIOMS-WHO (2016) International Ethical Guidelines for Health-related Research Involving Humans https://cioms.ch/publications/ ➤ Guideline 3: Use of de-identified, minimal-risk samples may not require consent. ICMR National Ethical Guidelines (2017) https://ethics.ncdirindia.org/ ➤ Section: “Biological Materials and Data” permits use of unlinked, anonymized clinical samples without consent under specified conditions. U.S. DHHS – Common Rule (45 CFR 46) (For international reviewers) https://www.ecfr.gov/current/ ➤ Research on non-identifiable biospecimens is not considered human subject research. 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Supplementary Files SupplementaryFile1.NonCodingRNAABVaranasi.csv SupplementaryFile2.ISAMRColocalizationAnalysis.csv SupplimentaryFile3CompleteHypotheticalProteinsInventory.csv SupplementaryFile4HighImpactVariantswithSlNo.csv Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 17 Apr, 2026 Reviews received at journal 13 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviews received at journal 25 Jan, 2026 Reviewers agreed at journal 27 Dec, 2025 Reviewers invited by journal 07 Dec, 2025 Editor invited by journal 24 Nov, 2025 Editor assigned by journal 18 Nov, 2025 Submission checks completed at journal 18 Nov, 2025 First submitted to journal 18 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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University","correspondingAuthor":false,"prefix":"","firstName":"Gyaneshwer","middleName":"","lastName":"Chaubey","suffix":""}],"badges":[],"createdAt":"2025-11-18 07:53:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8142534/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8142534/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97952236,"identity":"4c5ef9d8-acfb-40a9-a8d4-d4ab8fafc86a","added_by":"auto","created_at":"2025-12-11 07:21:54","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3275671,"visible":true,"origin":"","legend":"","description":"","filename":"AbPaper.docx","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/639f464fcbc8a88c6d37c2aa.docx"},{"id":98423639,"identity":"181f4cd7-fb42-44b6-b027-baa11954688f","added_by":"auto","created_at":"2025-12-17 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07:21:54","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":179858,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/fbb2ba2786faa09247a1042d.html"},{"id":98422587,"identity":"b6dc6c7a-fb57-4675-a1b3-4514101145c6","added_by":"auto","created_at":"2025-12-17 16:31:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":569502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the clinical isolate using Gram staining, transmission electron microscopy (TEM), and PCR analysis targeting the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egyrB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene.\u003cbr\u003e\n \u003c/strong\u003e\u0026nbsp;Gram staining revealed pink-stained cells, confirming that the isolate is Gram-negative. TEM imaging shows a typical coccobacillary morphology. PCR amplification of the \u003cem\u003egyrB\u003c/em\u003e gene produced a specific band for the clinical isolate (AB124), comparable to the positive control and distinct from the \u003cem\u003eE. coli\u003c/em\u003e control, confirming species identity.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/b448cc59ea9c9d5dc22206f1.jpeg"},{"id":97952230,"identity":"fffc1a7e-84ae-482c-b20e-715ffc952ca5","added_by":"auto","created_at":"2025-12-11 07:21:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":591372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA circular graphical display of the distribution of the genome annotations is prepared.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe outermost ring represents the complete circular genome of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. From outer to inner rings, the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known antimicrobial resistance genes, CDS with homology to know virulence factors, GC content and GC skew. The colors of the CDS on the forward and reverse strand are according to the subsystem . Image created using BV-BRC Server.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/de3eee6d4921367b87b65a93.png"},{"id":97952235,"identity":"1d9720d9-3025-4f9b-a49e-6b36f3083377","added_by":"auto","created_at":"2025-12-11 07:21:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubsystem analysis of this isolate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA subsystem is a set of proteins that together implement a specific biological process or structural complex\u003csup\u003e \u003c/sup\u003eand PATRIC annotation includes an analysis of the subsystems unique to each genome. An overview of the subsystems for this genome is provided in Figure 2. BV-BRC’s subsystem analysis identifies genes based on specific biological processes that they are hypothesized to be active in. The full genome report includes a pie chart showing the subsystems super classes[2], and an indication of the number of subsystems within that superclass (first number) and the number of annotated genes that are part of the superclass (second number).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/fd0767fd92331ed9c4a49f53.png"},{"id":97952233,"identity":"e10de183-33ef-454e-a936-52ed3b9f4207","added_by":"auto","created_at":"2025-12-11 07:21:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":433273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChord diagram illustrating the relationships between antibiotic resistance genes and associated antibiotic classes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAB Varanasi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diagram visualizes the connectivity between detected resistance genes (colored segments) and the antibiotics or antibiotic classes they confer resistance to (grey segments). Ribbons linking each gene to one or more antibiotics represent the functional associations inferred from genomic annotation. Segment size corresponds to the relative frequency or copy number of each resistance determinant. Distinct color groups highlight major resistance categories, including aminoglycosides, β-lactams, macrolides, tetracyclines, chloramphenicol, and sulfonamides. This integrative view emphasizes the multidrug-resistant profile of the isolate and demonstrates the co-occurrence of several clinically relevant resistance determinants, offering insights into potential selection pressures and the genomic architecture underlying extensive drug resistance.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/d5b256a2c83ddb1e554669c6.png"},{"id":98423333,"identity":"7a9aefe5-ae2b-4fa1-8d84-52bc92c89059","added_by":"auto","created_at":"2025-12-17 16:32:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":214906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole-genome variant quality and substitution metrics.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Nucleotide substitution spectrum illustrating the frequency of transition (cyan) versus transversion (red) events. \u003cstrong\u003e(B)\u003c/strong\u003eDistribution of variant classifications, highlighting the predominance of biallelic SNPs compared to insertions, deletions, and complex variants. \u003cstrong\u003e(C)\u003c/strong\u003eDetailed breakdown of biallelic base changes grouped by reference nucleotide (A, G, T, C), showing the specific alternate allele counts. \u003cstrong\u003e(D)\u003c/strong\u003eComparison of total transition and transversion counts, yielding a transition-to-transversion (Ti/Tv) ratio of 3.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/6b1be3ed17f76ddd5644ae35.png"},{"id":97952247,"identity":"e1a6a5e4-9930-49b0-9e52-7cffd76dcb3e","added_by":"auto","created_at":"2025-12-11 07:21:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGenetic organization of the integron region showing the arrangement of attC sites, integrase, promoters, functional annotations, and hypothetical proteins.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe schematic illustrates a genomic region spanning approximately 18.9–22.4 kb, highlighting a class 1 integron structure. The integrase gene (red) is positioned downstream of multiple promoter/attI elements (green, orange, yellow), enabling gene cassette recombination and transcription. attC recombination sites (blue) flank inserted gene cassettes. Several adjacent coding sequences are annotated as hypothetical proteins (grey), suggesting unexplored genetic content. Functional annotations (gold) indicate putative protein-coding genes associated with mobile element activity and resistance acquisition. The organization supports the role of integrons in capturing and mobilizing gene cassettes while harboring uncharacterized genes with potential clinical relevance.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/206e1164b6283f4e52927f14.png"},{"id":97952240,"identity":"bfa78c25-c919-4271-a02b-743ff29cef8e","added_by":"auto","created_at":"2025-12-11 07:21:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1300421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustrating the importance of investigating hypothetical proteins in bacterial genomes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diagram highlights four major research domains influenced by the functional characterization of hypothetical proteins. \u003cem\u003ePotential new functions\u003c/em\u003erepresent undiscovered biochemical activities that may expand current knowledge of bacterial physiology. \u003cem\u003eBacterial virulence\u003c/em\u003e indicates the possibility that uncharacterized proteins participate in adhesion, immune evasion, or host manipulation. \u003cem\u003eEvolutionary insights\u003c/em\u003e reflect the value of hypothetical proteins as markers of lineage-specific innovation, horizontal gene transfer, or adaptive diversification. \u003cem\u003eDrug-target discovery\u003c/em\u003e emphasizes their potential relevance in identifying novel vulnerabilities in drug-resistant pathogens. Collectively, these avenues demonstrate why systematic investigation of hypothetical proteins is essential for understanding microbial biology and developing future therapeutic strategies.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/e32f47d2a4ffc09c5b7957d7.png"},{"id":98622046,"identity":"c3b5a48f-d86a-4f5d-8894-98f27966de9e","added_by":"auto","created_at":"2025-12-19 16:43:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4972019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/d5124a08-3ab0-45b3-b85b-ed608247c616.pdf"},{"id":97952226,"identity":"5cc3b278-4bd2-4fd1-be31-c5e26c63d77c","added_by":"auto","created_at":"2025-12-11 07:21:54","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9324,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile1.NonCodingRNAABVaranasi.csv","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/8256275692917a0c64c3a6d9.csv"},{"id":98422782,"identity":"95e158e6-f227-4930-9f8a-06cf825f1deb","added_by":"auto","created_at":"2025-12-17 16:31:29","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8383,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile2.ISAMRColocalizationAnalysis.csv","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/7e14cd8b9fbcbafd0c5cc991.csv"},{"id":98422065,"identity":"3e4a25d0-4992-45b1-bf35-f3f33f1eb58a","added_by":"auto","created_at":"2025-12-17 16:30:24","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":32726,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryFile3CompleteHypotheticalProteinsInventory.csv","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/14ee9ac54e7bef77e91f2b22.csv"},{"id":98423670,"identity":"e8f58459-8e72-4f1e-a2d1-612de2eb2784","added_by":"auto","created_at":"2025-12-17 16:32:30","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2687,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile4HighImpactVariantswithSlNo.csv","url":"https://assets-eu.researchsquare.com/files/rs-8142534/v1/9b58a3a32feab24be54ab269.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation and Whole-Genome Sequencing of a Less Prevalent Indian A. baumannii Strain Reveals Unique Uncharacterized Hypothetical Proteins and AMR-Linked ncRNAs.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a Gram-negative, opportunistic pathogen that has become one of the most challenging bacterial species in contemporary clinical practice. It is increasingly recognized for its capacity to cause a wide range of hospital-acquired infections (HAIs), especially among critically ill and immunocompromised patients. The pathogen is frequently isolated from intensive care units (ICUs), where it contributes substantially to ventilator-associated pneumonia (VAP), bloodstream infections, catheter-associated urinary tract infections (CAUTIs), wound infections, and surgical site infections. These infections often result in prolonged hospitalization, increased healthcare costs, and high morbidity and mortality rates. According to the World Health Organization\u0026rsquo;s (WHO) 2024 priority pathogen list, carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e (CRAB) ranks among the most critical threats to global health security, underscoring the urgent need for genomic and epidemiological surveillance \u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOver the last few decades, \u003cem\u003eA. baumannii\u003c/em\u003e has evolved into a paradigm of bacterial adaptability. Its success as a nosocomial pathogen is closely tied to its exceptional genomic plasticity and ability to acquire foreign genetic material. This species has accumulated a wide repertoire of antimicrobial resistance (AMR) and virulence genes, many of which are acquired through horizontal gene transfer (HGT). Such transfers are facilitated by mobile genetic elements (MGEs) including plasmids, transposons, insertion sequences, and integrons \u003csup\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/sup\u003e. The interplay between these MGEs and selective pressure from antimicrobial use in hospital settings has driven the rapid evolution of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains.\u003c/p\u003e\u003cp\u003eEarly studies identified three predominant clonal groups circulating in Europe, designated as European clones I, II, and III. These lineages were later standardized as International Clones (ICs) 1, 2, and 3, respectively (37). Among them, IC1 and IC2 have achieved remarkable global dissemination and are responsible for many large-scale outbreaks involving carbapenem-resistant and MDR \u003cem\u003eA. baumannii\u003c/em\u003e. Advances in whole-genome sequencing (WGS) and multilocus sequence typing (MLST) have subsequently expanded the classification of \u003cem\u003eA. baumannii\u003c/em\u003e to at least eight recognized international clones, each characterized by distinct resistance mechanisms, virulence determinants, and geographical patterns of distribution \u003csup\u003e(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe global spread of these clones has been largely driven by the carriage of carbapenemase-encoding genes such as \u003cem\u003ebla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;OXA-23\u0026lt;/sub\u0026gt;\u003c/em\u003e, \u003cem\u003ebla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;OXA-24/40\u0026lt;/sub\u0026gt;\u003c/em\u003e, and \u003cem\u003ebla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;OXA-58\u0026lt;/sub\u0026gt;\u003c/em\u003e, frequently embedded within integrative conjugative elements and transposons \u003csup\u003e(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/sup\u003e. The mobility of these genetic structures facilitates the horizontal dissemination of carbapenem resistance both within and between bacterial populations. Furthermore, \u003cem\u003eA. baumannii\u003c/em\u003e possesses an intrinsic \u003cem\u003ebla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;OXA-51-like\u0026lt;/sub\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;gene that can become overexpressed through the insertion of an ISAba1 element upstream, further enhancing resistance to β-lactams. Together, these mechanisms have contributed to the establishment of globally dominant CRAB lineages that are highly adaptive in hospital environments. Genomic plasticity plays a central role in the ecological and clinical success of \u003cem\u003eA. baumannii\u003c/em\u003e. The incorporation of type 1 integrons and pathogenicity islands enhances the bacterium\u0026rsquo;s ability to persist under environmental and antimicrobial stress. Integrons serve as efficient platforms for capturing and expressing resistance gene cassettes, while pathogenicity islands often encode virulence determinants associated with biofilm formation, iron acquisition, and adherence to abiotic surfaces. These genomic features collectively enable \u003cem\u003eA. baumannii\u003c/em\u003e to survive in desiccated hospital environments, colonize medical equipment, and persist despite extensive disinfection efforts.\u003c/p\u003e\u003cp\u003eWhile international clones dominate the global population structure of \u003cem\u003eA. baumannii\u003c/em\u003e, several non-IC sequence types (STs) have been increasingly detected in clinical isolates. Sequence types such as ST25, ST78, and ST149 have emerged in different regions, each harboring distinct sets of resistance and virulence genes \u003csup\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/sup\u003e. Although currently less prevalent, these STs represent potentially high-risk lineages that could play a more significant role in future outbreaks. The genetic diversity within these non-IC clones highlights the ongoing evolution of \u003cem\u003eA. baumannii\u003c/em\u003e and its capacity to exploit new ecological niches under selective pressure.\u003c/p\u003e\u003cp\u003eCarbapenem resistance in \u003cem\u003eA. baumannii\u003c/em\u003e is primarily mediated by class D β-lactamases, commonly referred to as oxacillinases (OXAs), which hydrolyze carbapenems and other β-lactam antibiotics \u003csup\u003e(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/sup\u003e. Other resistance mechanisms, including efflux pumps, porin loss, and target site mutations, also contribute to its MDR and XDR phenotypes. A recent study by Vijaykumar \u003cem\u003eet al.\u003c/em\u003e (2022) reported that most \u003cem\u003eA. baumannii\u003c/em\u003e isolates in India belong to the IC2 lineage, while clones IC7 and IC8 occur less frequently \u003csup\u003e(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u003c/sup\u003e. Despite the rising clinical significance of CRAB in India, comprehensive genomic data on Indian isolates remain scarce. In particular, the genomic architecture and evolutionary trajectory of \u003cem\u003eA. baumannii\u003c/em\u003e ST149, a non-IC lineage, are not yet well characterized.\u003c/p\u003e\u003cp\u003eDue to the widespread presence of carbapenemases and other antibiotic resistance determinants, treating \u003cem\u003eA. baumannii\u003c/em\u003e infections in hospital settings has become increasingly challenging. This highlights an urgent need to identify novel therapeutic targets. Still a substantial proportion of the \u003cem\u003eA. baumannii\u003c/em\u003e proteome remains uncharacterized, suggesting that unexplored proteins\u0026mdash;particularly hypothetical proteins\u0026mdash;may hold untapped potential for drug discovery \u003csup\u003e(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we report the whole-genome sequencing of a carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolate from India belonging to sequence type 149 (ST149). By systematically characterizing its resistome, virulome, and mobilome, we further delineate the repertoire of hypothetical proteins, with the goal of identifying novel candidates that may serve as therapeutic targets.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Bacterial isolation and identification\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolate used in this study was obtained from a blood culture sample processed in the diagnostic microbiology laboratory of IQ City Medical College \u0026amp; NH Hospital, Durgapur, India. Ethical approval for this work was granted by the Institutional Ethics Committee of IQ City Medical College \u0026amp; NH Hospital (Approval No. IQMC/IEC/Project/17/28). The isolate was recovered from anonymized clinical culture plates with no access to identifiable patient information. According to the Council for International Organizations of Medical Sciences (CIOMS)\u0026ndash;World Health Organization (WHO) International Ethical Guidelines for Health-related Research Involving Humans (2016) and the Indian Council of Medical Research (ICMR) guidelines, the use of de-identified bacterial isolates does not constitute human subject research and therefore does not require individual patient consent. Primary isolation was performed on MacConkey agar, and colony morphology was documented following 24 h incubation at 37\u0026deg;C. Gram staining was conducted to confirm cellular morphology. Species identification was performed using PCR amplification targeting the \u003cem\u003egyrB\u003c/em\u003e gene, a species-specific region of \u003cem\u003eA. baumannii\u003c/em\u003e. The isolate was further validated through biochemical profiling and genomic analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Antimicrobial susceptibility testing (AST)\u003c/h2\u003e\u003cp\u003eAntimicrobial susceptibility testing was performed using the VITEK\u0026reg; 2 Compact system (bioM\u0026eacute;rieux, France) based on the automated broth microdilution method. The antimicrobial agents tested included β-lactams, aminoglycosides, fluoroquinolones, and carbapenems commonly used against \u003cem\u003eAcinetobacter\u003c/em\u003e infections. The results were validated by the Kirby\u0026ndash;Bauer disk diffusion method to ensure concordance. Interpretation of susceptibility profiles was carried out according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2023).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Transmission electron microscopy (TEM)\u003c/h2\u003e\u003cp\u003eFor ultrastructural visualization, bacterial cells were concentrated by gentle centrifugation and resuspended in sterile phosphate-buffered saline (PBS). A 5 \u0026micro;L aliquot of the suspension was placed on a formvar-coated copper grid and allowed to adsorb for 5 min. The sample was then negatively stained with 1% (w/v) uranyl acetate for 1 min, air-dried, and examined under a transmission electron microscope operated at 80 kV. Images were captured at various magnifications to assess cell morphology and surface architecture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Genomic DNA extraction, library preparation, and whole-genome sequencing (WGS)\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from an overnight culture of \u003cem\u003eA. baumannii\u003c/em\u003e grown in Luria\u0026ndash;Bertani broth using the cetyltrimethylammonium bromide (CTAB)\u0026ndash;phenol\u0026ndash;chloroform method. A 1.5 mL aliquot of culture was centrifuged at 4000 rpm for 5 min, and the resulting pellet was subjected to CTAB-mediated lysis followed by phenol\u0026ndash;chloroform\u0026ndash;isoamyl alcohol purification. DNA integrity was assessed on a 0.8% agarose gel, and concentration was quantified using a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, USA). Sequencing libraries were prepared with the QIAseq FX DNA Library Kit (QIAGEN, Germany) according to the manufacturer\u0026rsquo;s instructions. Paired-end sequencing (2 \u0026times; 150 bp) was performed on the Illumina NovaSeq 6000 platform using v1.5 chemistry (300-cycle kit). Raw reads were subsequently processed through a standard quality control workflow before assembly and annotation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Genome assembly, annotation, and bioinformatic analysis\u003c/h2\u003e\u003cp\u003eRaw read quality was evaluated using FastQC (accessed 15 May 2025) to assess per-base sequence quality, GC distribution, and duplication levels. All downstream analyses were conducted on the Galaxy Europe platform (\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; accessed 15 May 2025). Adapter sequences and low-quality bases were removed with Fastp (11) using default parameters, ensuring retention of high-quality reads for assembly.\u003c/p\u003e\u003cp\u003eDe novo assembly was generated using Unicycler v0.8.4.0, which employs short-read assembly with conservative bridging to resolve repeat-rich regions and improve contiguity. Assembly statistics, including N50, L50, GC content, and total genome size, were assessed using QUAST to confirm completeness and structural accuracy. Gene prediction and functional annotation were conducted using Prokka (12), which identified coding sequences, rRNAs, tRNAs, and additional genomic features using curated bacterial databases. Annotation reliability was further supported by cross-validation with the Rapid Annotations using Subsystems Technology (RAST) server, enabling subsystem-level functional categorisation of metabolic, stress-response, and virulence pathways.\u003c/p\u003e\u003cp\u003eSequence type assignment followed the \u003cem\u003eA. baumannii\u003c/em\u003e MLST scheme hosted on PubMLST. Antimicrobial resistance (AMR) determinants were identified using ResFinder and the Comprehensive Antibiotic Resistance Database (CARD), with high-confidence hits defined by \u0026ge;\u0026thinsp;90% identity and \u0026ge;\u0026thinsp;80% coverage thresholds. Mobile genetic elements (MGEs) were detected using ISEScan for insertion sequences, Integron Finder 2.0 (17) for integron structures, and IslandViewer 4 (18) for genomic island identification based on SIGI-HMM, IslandPath-DIMOB, and IslandPick prediction algorithms. Genome visualization and mapping of annotated features were performed using Proksee.\u003c/p\u003e\u003cp\u003eVirulence-associated genes were identified using the Virulence Factor Database (VFDB) (15) and independently verified with BacWGSTdb (16). Annotation and AMR predictions were cross-checked against outputs from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) to ensure concordance across platforms. Key resistance and virulence loci were further validated by pairwise nucleotide and protein alignments using BLASTn and BLASTp. Structural predictions for hypothetical proteins were generated using AlphaFold (38), enabling preliminary functional inference based on protein fold and domain architecture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Variant detection using DeepVariant\u003c/h2\u003e\u003cp\u003eHigh-confidence SNPs and small indels were detected using DeepVariant v1.6.0 (Poplin et al., 2018), a deep-learning\u0026ndash;based variant caller that improves base-level accuracy by modeling sequencing errors. The DeepVariant pipeline was run with the model type set to \u003cem\u003eWGS\u003c/em\u003e and the reference genome CP000521. Resulting variant calls were exported in VCF format. Quality filters were applied using bcftools to retain variants with a minimum quality score (QUAL)\u0026thinsp;\u0026ge;\u0026thinsp;30 and read depth (DP)\u0026thinsp;\u0026ge;\u0026thinsp;10. Multi-allelic and low-confidence variants were excluded.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Isolation, Identification, and Genomic Characterisation led to annotation yielding 4039 predicted proteins\u003c/h2\u003e\u003cp\u003eThe combined Gram staining, TEM morphology, and gyrB-based PCR profiling consistently supported the identity of the isolate as a Gram-negative \u003cem\u003eAcinetobacter\u003c/em\u003e strain. These results provided the foundational confirmation required for downstream genomic and phenotypic analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe antimicrobial sensitivity test (AST) indicated that this isolate is only sensitive to Tigecycline and exhibited intermediate-level sensitivity to colistin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The genome sequencing was performed using the WGS method. Using a standard quality control and adapter trimming tool, FastP on the Galaxy platform trimming was performed. Approximately 16.389052\u0026nbsp;million Illumina high-quality reads were trimmed to 16.099834\u0026nbsp;million. We obtained a genome size of the isolate approximately 4.1 Mb comprising a single circular chromosome (4,191,980 bp), which is circular. The whole genome contains 39% Guanine-Cytosine (GC) content. Annotation of the genome showed that the genome encodes 4039 predicted genes. Among them, 3970 CDS, 4 rRNAs, 64 tRNAs, and 1 tmRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e). One CRISPR-related sequence was found. The sequence type of this isolate was ST1506 (31-33-67-40-1-95-7) under the Oxford MLST scheme. Under the Pasture MLST scheme, this genome belongs to ST149 (3-12-11-2-14-9-14). The strain is not part of the well-known highly pathogenic ICs, and yet it possesses all the AMR genes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) possibly exhibiting a hypervirulence phenotype. To evaluate genomic attributes, predicted genes were functionally categorised using subsystems (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The isolate's broad metabolic adaptability was highlighted by the fact that the majority of its genes (782 genes across 97 subsystems) were linked to metabolism. Genes related to energy production (230 genes, 30 subsystems), stress response, defence, and virulence (133 genes, 38 subsystems), and protein processing (220 genes across 40 subsystems) came next (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The bacterium's ability to adapt to its environment and possible resistance mechanisms is highlighted by the significant representation of genes linked to stress and virulence.\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\u003eAST result by Kirby-Bauer disk diffusion test \u0026amp; MICs of antibiotics.\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=\"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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAntibiotic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eZone of Inhibition (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCLSI Breakpoint (Susceptible\u0026thinsp;\u0026ge;\u0026thinsp;mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInterpretation\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\u003eCeftazidime (CAZ 30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResistant\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\u003eCo-trimoxazole (COT 25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResistant\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\u003eImipenem (IMP 10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResistant\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\u003eColistin (CL 10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIntermediate\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\u003eCiprofloxacin (CIP 5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResistant\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\u003eGentamicin (GEN 10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResistant\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\u003eCefepime (FEP 30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResistant\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\u003eMeropenem (MEM 10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntimicrobial Resistance (AMR) Genes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"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\u003eSerial No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistance Gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIdentity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCoverage (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePhenotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAccession No.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003earmA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAmikacin, Gentamicin, Tobramycin, Isepamicin, Netilmicin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAY220558\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\u003eaph(3')-VI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAmikacin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKC170992\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\u003eaph(6)-Id\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eM28829\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\u003eaph(3'')-Ib\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e99.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAF024602\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\u003eaph(3'')-Ib\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAF321551\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\u003eaph(3'')-Ib\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAF313472\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\u003eaph(3'')-Ib\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAF321550\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\u003eblaADC-25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e96.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e99.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUnknown Beta-lactam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEF016355\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\u003eblaOXA-203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUnknown Beta-lactam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHQ998857\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\u003eblaPER-7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAmoxicillin, Amoxicillin\u0026thinsp;+\u0026thinsp;Clavulanic acid, Ampicillin, Ampicillin\u0026thinsp;+\u0026thinsp;Clavulanic acid, Cefotaxime, Cefoxitin, Cefepime, Ceftazidime, Piperacillin, Piperacillin\u0026thinsp;+\u0026thinsp;Tazobactam, Ticarcillin, Ticarcillin\u0026thinsp;+\u0026thinsp;Clavulanic acid, Aztreonam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHQ713678\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\u003eblaOXA-23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eImipenem, Meropenem\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAY795964\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\u003emsr(E)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eErythromycin, Azithromycin, Quinupristin, Pristinamycin IA, Virginiamycin S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFR751518\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\u003emph(E)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eErythromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDQ839391\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\u003ecmlA1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eChloramphenicol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eM64556\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\u003eARR-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRifampicin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHQ141279\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\u003esul1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSulfamethoxazole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eU12338\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\u003esul1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSulfamethoxazole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eU12338\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\u003esul2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e85.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSulfamethoxazole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAJ830710\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esul2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e85.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSulfamethoxazole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAY034138\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003etet(B)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDoxycycline, Tetracycline, Minocycline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAP000342\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2. AMR Gene Profiling Reveals the presence of genes associated with the Active Efflux of the Chemotherapeutic Agents.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe resistome of this isolate encompasses 11 functionally distinct categories, with macrolide-associated genes (205 genes), phenicol resistance determinants (79 genes), and multidrug efflux systems (79 genes) representing the predominant resistance mechanisms. Beta-lactam resistance is mediated by multiple clinically significant enzymes, including the carbapenem-hydrolyzing class D beta-lactamases OXA-23 and OXA-104, the extended-spectrum class C beta-lactamase ADC-26, the inhibitor-resistant extended-spectrum class A beta-lactamase PER-7, and the chromosomally-encoded AmpC cephalosporinase, collectively conferring resistance to penicillins, cephalosporins, and carbapenems.\u003c/p\u003e\u003cp\u003eAminoglycoside resistance mechanisms of this isolate are encoded by four distinct resistance genes: aminoglycoside O-phosphotransferases APH(3\u0026prime;)-VIa, APH(6)-Id, and APH(3\u0026Prime;)-Ib, alongside the aminoglycoside nucleotidyltransferase ANT(3\u0026Prime;)-IIa, providing enzymatic inactivation of gentamicin, tobramycin, amikacin, and kanamycin. Fluoroquinolone resistance is supported by mutations or upregulation of topoisomerase genes, specifically DNA gyrase subunits A and B (\u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e) and DNA topoisomerase IV subunits A and B (\u003cem\u003eparC\u003c/em\u003e, \u003cem\u003eparE\u003c/em\u003e), conferring resistance to ciprofloxacin and levofloxacin. Tetracycline resistance is conferred by the major facilitator superfamily (MFS) efflux transporter Tet(B), regulated by the tetracycline resistance transcriptional repressor TetR(B), enabling active efflux-mediated resistance. The genome harbors a sophisticated multidrug efflux architecture comprising the tripartite resistance-nodulation-division (RND) systems AdeABC, AdeIJK, and AcrAB-TolC, alongside the efflux pumps EmrAB and additional MFS transporters, collectively mediating resistance to aminoglycosides, fluoroquinolones, macrolides, tetracyclines, tigecycline, and disinfectants. Sulfonamide resistance is mediated by sulfonamide-resistant dihydropteroate synthase variants Sul1 and Sul2, while fosfomycin resistance is conferred by the glutathione transferase FosLL and the MFS transporter AbaF. This multifaceted AMR profile, encompassing enzymatic inactivation, target modification, active efflux, and metabolic bypass, represents a clinically significant multi drug-resistant phenotype with substantial implications for therapeutic management. Additionally, the presence of the gene qacEΔ1, associated with the reduced susceptibility to quaternary ammonium compounds and disinfectants such as chlorhexidine, was identified, suggesting potential tolerance to antiseptics and disinfectants used in clinical settings (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Virulence and Biofilm-Associated Genes Show Diverse Repertoire of Virulence-associated Genes\u003c/h2\u003e\u003cp\u003eThe AB_Varanasi isolate encodes approximately 378 virulence-associated genes, of which 92 are identified in VFDB, reflecting a multifaceted pathogenic repertoire. Central to its virulence strategy is iron acquisition (52 genes), encompassing the complete acinetobactin biosynthesis system (BasA\u0026ndash;BasJ) and associated transport machinery (BauA\u0026ndash;BauF), ensuring efficient scavenging under host-imposed nutritional immunity. The isolate also possesses an extensive suite of adhesion and invasion determinants (171 genes), including outer membrane protein variants (OmpA and specialized receptors) and a Type IV pilus system (33 genes), facilitating host cell recognition, attachment, and surface colonization. For interbacterial competition and host manipulation, AB_Varanasi harbors a complete Type VI secretion system (14 genes) capable of effector protein delivery, alongside 13 phospholipase genes producing membrane-disrupting enzymes. Its biofilm persistence machinery (10 genes), including the PGA synthesis cluster, supports enhanced survival on both abiotic and biotic surfaces. Finally, regulatory networks (8 genes), particularly quorum-sensing systems, fine-tune virulence expression in response to population density, collectively underpinning the isolate\u0026rsquo;s adaptive pathogenic potential \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e AB_Varanasi's virulence genes may enhance tissue invasion, immune modulation, and persistence in clinical environments.\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\u003eVirulence Genes Present in this isolate.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eContig\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIdentity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePosition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDescription\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\u003ebasJ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e56573..57742\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eacinetobactin biosynthesis protein BasJ\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\u003ebasI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e96.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e57867..58622\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ephosphopantetheinyl transferase component of acinetobactin biosynthesis protein BasI\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\u003ebasH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e58633..59367\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003enon-ribosomal peptide biosynthesis thioesterase BasH\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\u003ebarB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e59439..61034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003esiderophore efflux system of the ABC superfamily\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\u003ebarA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e96.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61031..62641\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003esiderophore efflux system of the ABC superfamily\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\u003ebasG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e62887..64038\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eacinetobactin biosynthesis protein BasF\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\u003ebasF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e64155..65024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003earyl carrier protein BasF\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\u003eentE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e96.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e65042..66670\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003enon-ribosomal peptide synthetase adenylate-forming enzyme of acinetobactin synthesis\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\u003ebasD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e96.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e66925..69774\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eacinetobactin biosynthesis protein BasD\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\u003ebauA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e99.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e71199..73481\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTonB-dependent siderophore receptor BauA\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\u003ebauB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e73567..74535\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eferric siderophore ABC transporter, periplasmic siderophore-binding protein\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\u003ebauC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e75309..76256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eferric siderophore ABC transporter, permease protein BauC\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\u003ebauD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e96.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e76270..77197\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eferric siderophore ABC transporter, permease protein BauD\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\u003ebfmS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e106102..107751\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003esignal transduction histidine kinase\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\u003ebfmR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e107784..108500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ebiofilm-controlling response regulator\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\u003eabaI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e99.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61431..61982\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eN-acyl-L-homoserine lactone synthetase\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\u003eabaR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e59461..60177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDNA-binding HTH domain-containing protein\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\u003eplc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e137693..139861\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ephospholipase C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eompA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e99.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e95503..96573\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eouter membrane protein OmpA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eadeG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19093..22272\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecation/multidrug efflux pump\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecsuE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e132548..133567\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCsu pilus tip adhesin CsuE\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epgaD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e99.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e205626..206090\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003epoly-beta-1,6-N-acetyl-D-glucosamine biosynthesis protein PgaD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eplcD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e24110..25735\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ephosphatidylserine/phosphatidylglycerophosphate/cardiolipin synthase\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\u003e3.5. MGE Indicates Genomic Architecture Associated with Multiple Resistant Determinants\u003c/h2\u003e\u003cp\u003eAnalysis of antimicrobial resistance gene-MGE co-localization demonstrated that 131 AMR genes (76.61% of total resistance determinants) are situated on MGE-rich contigs (\u0026ge;\u0026thinsp;10 MGE per contig), establishing a direct association between mobile genetic elements and resistance gene dissemination. High-density AMR-MGE co-occurrence was observed on contig_3 (16 AMR genes, 44 MGE), contig_7 (15 AMR genes, 29 MGE), contig_4 (14 AMR genes, 21 MGE), and contig_1 (10 AMR genes, 28 MGE), indicating genomic hotspots for resistance gene acquisition and mobilization. Clinically significant AMR genes identified on MGE-rich contigs include the carbapenem-hydrolyzing beta-lactamase \u003cem\u003eblaOXA-104\u003c/em\u003e (contig_4), the extended-spectrum beta-lactamase \u003cem\u003eblaPER-7\u003c/em\u003e (contig_27), the macrolide phosphotransferase \u003cem\u003emph(E)\u003c/em\u003e (contig_27), sulfonamide resistance genes \u003cem\u003esul1\u003c/em\u003e (contig_27), the aminoglycoside nucleotidyltransferase \u003cem\u003eant(3\u0026Prime;)-IIa\u003c/em\u003e (contig_12), and the fosfomycin resistance transferase \u003cem\u003efosLL\u003c/em\u003e (contig_6). The extensive mobilome, high frequency of IS elements, presence of functional integrons, multiple plasmid replicons, and prophage integration sites, combined with the substantial co-localization of resistance determinants on MGE-rich genomic regions, collectively indicate the significant horizontal gene transfer potential of this isolate and its capacity for rapid dissemination of antimicrobial resistance and virulence traits within microbial communities. \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003e\u0026amp; Supplementary File 2).\u003c/b\u003e Analysis of the 18.9\u0026ndash;22.4 kb integron region showed a class 1 integron with a well-defined integrase, attI/attC recombination sites, and several adjacent mobile-element\u0026ndash;associated genes. Multiple hypothetical proteins were also present within this locus, indicating additional uncharacterized components potentially linked to cassette mobility or resistance acquisition \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eVariant types present in this isolate\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"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\u003eSl. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVariant Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCount\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExample Genes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFunctional Category\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePredicted Impact\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\u003eSynonymous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLipid A phosphoethanolamine transferase, hypothetical proteins\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNeutral mutations\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLow\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\u003eMissense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlycine dehydrogenase, MerR recombinase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMetabolic and regulatory adaptation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eModerate\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\u003eFrameshift\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003erpoB, L22p ribosomal protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTranscription and translation machinery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHigh\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\u003e3.6 Elevated Ti/Tv Ratio and Genomic Plasticity indicates Variant Landscape of A. baumannii Varanasi\u003c/h2\u003e\u003cp\u003eAnalysis of AB_Varanasi with DeepVariant identified 30,385 high-confidence variants, comprising 29,926 SNPs and 458 indels, with an unusually high transition-to-transversion (Ti/Tv) ratio of 3.05, markedly above the typical bacterial range of 2.0\u0026ndash;2.3. This elevated ratio likely reflects non-random mutational processes driven by multiple factors: oxidative stress from reactive oxygen species during persistent infection inducing characteristic G\u0026rarr;A transitions; spontaneous cytosine deamination resulting in C\u0026rarr;T changes; selective pressures favoring transitions over transversions at functionally important sites; and DNA polymerase error patterns arising from replication fidelity alterations during adaptation. Notably, the most frequent substitutions\u0026mdash;G\u0026rarr;A (5,836) and T\u0026rarr;C (5,636)\u0026mdash;account for 38.3% of all SNPs, reinforcing the notion of directed mutational biases rather than purely stochastic events. This distinctive mutational signature likely represents adaptation-driven evolution under hospital-associated selection pressures and is clinically significant, potentially revealing vulnerabilities in DNA repair or replication fidelity that could be therapeutically exploited \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Identification and distribution of SNPs in A. baumannii isolates\u003c/h2\u003e\u003cp\u003eWhole-genome variant analysis identified multiple synonymous and nonsynonymous substitutions distributed across key functional genes of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolates. A total of 31 high-confidence single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) were detected, with variant frequencies ranging from 0.45 to 1.00, indicating the presence of both fixed and subclonal mutations within the populations. Approximately 80% of the substitutions were synonymous and classified as low-impact changes according to SnpEff annotations. In contrast, five missense variants and two frameshift mutations were categorized as moderate- to high-impact, potentially influencing protein function and fitness \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.8 SNP-Based Functional Insights and Adaptive Significance of A. baumannii isolate\u003c/h2\u003e\u003cp\u003eWhole-genome SNP analysis revealed multiple genetic alterations with potential functional and adaptive implications in \u003cem\u003eA. baumannii\u003c/em\u003e populations. Two high-impact frameshift mutations were identified among the analyzed isolates, indicating possible disruptions in essential cellular processes. The first was a frameshift deletion in the LSU ribosomal protein L22p (L17e) gene (470.25780.con.0005:191123), which resulted in a loss of the start codon (Met1fs) and predicted truncation of the encoded protein. Given that ribosomal protein L22p plays a crucial role in the assembly of the 50S ribosomal subunit and serves as part of the macrolide antibiotic-binding pocket, such a mutation is likely to impair ribosomal biogenesis and modulate antibiotic susceptibility, particularly towards macrolides and ketolides. The second high-impact event was a frameshift insertion in the DNA-directed RNA polymerase β subunit gene (470.25780.con.0009:5908), producing an Ala629 frameshift that may alter RNA polymerase structure and compromise transcriptional fidelity. Mutations in this subunit are known to influence global transcriptional regulation and may facilitate adaptive responses under antibiotic or oxidative stress, enhancing bacterial persistence in hostile environments. Beyond these severe disruptions, several moderate-impact missense mutations were detected in genes linked to metabolic and regulatory functions, reflecting ongoing microevolutionary diversification. An AlaLeuThr440ThrLeuAla substitution in \u003cem\u003eglycine dehydrogenase (decarboxylating)\u003c/em\u003e (470.25780.con.0004:253773) could influence glycine cleavage efficiency and redox balance, critical for energy metabolism under nutrient limitation. Similarly, a Thr45Ser mutation in a resolvase family recombinase (470.25780.con.0019:47635) may enhance site-specific recombination, potentially increasing genome plasticity and facilitating horizontal gene transfer. Additionally, a Lys119Asn substitution in glycine dehydrogenase (P-protein) (470.25780.con.0064:357) might alter cofactor binding, improving metabolic adaptability during antibiotic stress. These high- and moderate-impact SNPs signify adaptive genomic fine-tuning that could reshape fundamental processes such as transcriptional regulation, ribosomal function, and metabolic plasticity, thereby promoting the survival of \u003cem\u003eA. baumannii\u003c/em\u003e under diverse environmental and therapeutic pressures. Such mutations may underpin the evolutionary transition of emerging clones (e.g., ST149) towards enhanced persistence and resistance, reflecting a dynamic genomic response to continuous antibiotic selection \u003cb\u003e(Supplementary file 4).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Non-Coding RNAs and Regulatory Control of Antimicrobial Resistance in AB Varanasi\u003c/h2\u003e\u003cp\u003eThe regulatory landscape supporting extensive antimicrobial resistance in this isolate includes a sophisticated non-coding RNA (ncRNA) network comprising 113 RNA features (2.79% of total genomic elements) with direct implications for resistance gene expression, metabolic adaptation, and stress response. While the essential translational machinery\u0026mdash;65 tRNA genes providing complete codon coverage with variable isoacceptor redundancy (1\u0026ndash;7 per amino acid), a complete rRNA operon (5S-16S-23S), and a single tmRNA (SsrA) for ribosome quality control\u0026mdash;ensures efficient synthesis of the 800 identified antimicrobial resistance proteins, the regulatory ncRNA complement establishes post-transcriptional control mechanisms that modulate resistance phenotypes. Critically, the genome encodes a putative aminoglycoside riboswitch/attI site that likely functions as a cis-acting metabolite-sensor capable of direct sensing of aminoglycoside antibiotics and potentially modulating expression of aminoglycoside resistance genes in response to antibiotic presence, representing a novel and previously uncharacterized direct link between antibiotic presence and resistance gene regulation. The ncRNA-mediated regulatory architecture includes 22 copies of C4 antisense RNA, representing the predominant regulatory class and suggesting extensive post-transcriptional suppression of mRNA targets, with potential roles in silencing susceptibility factors or modulating expression of multidrug efflux pump genes to optimize resistance phenotypes under varying environmental conditions. An additional 8 metabolite-sensing riboswitches were identified beyond the aminoglycoside-sensing element, including FMN riboswitch (modulating flavin-dependent oxidoreductases and energy metabolism supporting resistance), TPP riboswitch (controlling vitamin B12-independent metabolic pathways essential for nucleotide synthesis and DNA repair under antibiotic stress), cobalamin riboswitch (regulating B12-dependent pathways essential for stress responses), glycine riboswitch (governing amino acid metabolism affecting protein synthesis capacity for the synthesis of proteins associated with resistance mechanism), guanidine-I riboswitch (sensing nitrogen availability critical for growth under nutrient limitation encountered in biofilms), yybP-ykoY manganese riboswitch (modulating metal homeostasis and oxidative stress resistance mechanisms), and fluoride riboswitch (crcB) (enabling response to fluoride-containing disinfectants and antiseptics used in infection control). These metabolite-responsive elements establish direct links between nutrient and metabolite availability, stress conditions, and expression of metabolic genes that support growth, stress tolerance, and sustained antimicrobial resistance under nutrient-limiting or stress conditions.\u003c/p\u003e\u003cp\u003eFour \u003cem\u003eAcinetobacter\u003c/em\u003e-specific small regulatory RNAs (sRNAs)\u0026mdash;including two copies of Acinetobacter sRNA 25, and single copies of Acinetobacter sRNA 11, Acinetobacter sRNA 28, and Aar sRNA\u0026mdash;likely mediate species-specific regulatory programs governing virulence, stress adaptation, and metabolic capabilities unique to \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, potentially including direct or indirect regulation of resistance gene expression, biofilm formation supporting antimicrobial tolerance, and stress-responsive adaptations to host immune pressures and antibiotic challenge. Structural RNAs essential for resistance gene expression include bacterial RNase P class A (mediating tRNA maturation critical for translation of resistance proteins), signal recognition particle RNA (directing membrane insertion of efflux pump components), and 6S/SsrS RNA (orchestrating stationary phase transcription including potential upregulation of resistance genes during nutrient-limited conditions encountered in biofilms or host tissues). The integration of cis-acting riboswitches (particularly aminoglycoside-sensing and metal-responsive elements), trans-acting antisense RNAs (C4; 22 copies), and species-specific regulatory sRNAs establishes a multi-layered post-transcriptional regulatory network that complements the transcriptional control of resistance genes, enabling rapid and reversible modulation of antimicrobial resistance phenotypes in response to nutrient availability, metabolite concentration, stress signals, and antibiotic challenge, thereby enhancing survival in the face of antibiotic pressure and environmental heterogeneity \u003cb\u003e(Supplementary File 1).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Hypothetical and Uncharacterized Protein Repertoire of AB Varanasi\u003c/h2\u003e\u003cp\u003eComprehensive genome annotation identified 294 hypothetical and uncharacterized proteins representing 7.48% of the total coding sequences, indicating a moderate proportion of the genome with undefined or poorly characterized functions. The hypothetical protein repertoire is predominantly composed of proteins containing Domains of Unknown Function (DUF), which constitute 222 proteins (75.5% of hypothetical proteins), representing 192 unique DUF families distributed across the genome. The most abundant DUF families include DUF559 (putative adhesion protein, 3 proteins), DUF1833 (uncharacterized conserved protein, 3 proteins), DUF3298 (predicted membrane protein, 3 proteins), DUF442 (putative metal-binding protein, 3 proteins), DUF2147 (predicted membrane protein, 3 proteins), and DUF606 (predicted lipoprotein, 3 proteins), suggesting potential roles in bacterial adherence, membrane structure and function, metal homeostasis, and cellular interactions \u003cb\u003e(Supplementary File 3).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBeyond DUF-containing proteins, 41 sequences (13.9%) are annotated as hypothetical proteins without assigned domain architecture, while 20 proteins (6.8%) belong to uncharacterized protein families (UPF) with conserved sequence motifs but undefined biochemical functions. An additional 10 proteins (3.4%) are designated as putative proteins with predicted but experimentally unvalidated functions. Analysis of product annotations revealed potential functional associations for a subset of hypothetical proteins, including membrane-associated proteins (6 proteins), transport and permease functions (6 proteins), signal transduction components (4 proteins), regulatory elements (2 proteins), DNA/RNA-binding proteins (1 protein), and stress response factors (1 protein), providing preliminary functional hypotheses for future experimental validation.\u003c/p\u003e\u003cp\u003eThe distribution of hypothetical proteins across genomic scaffolds demonstrates variable density, with contig_2 harboring the highest concentration (27 proteins), followed by contig_1 and contig_4 (21 proteins each), and an average distribution of 19.4 hypothetical proteins across the top 10 contigs. The substantial proportion of DUF-containing proteins and the diversity of represented DUF families (192 unique families) reflects the evolutionary plasticity and functional adaptability of this bacterial isolate, with uncharacterized genomic elements potentially contributing to niche-specific adaptations, environmental stress tolerance, host-pathogen interactions, or novel metabolic capabilities. The identification of membrane-associated and transport-related hypothetical proteins suggests potential involvement in nutrient acquisition, efflux-mediated resistance mechanisms, or host cell interactions, warranting targeted experimental characterization to elucidate their functional roles in bacterial physiology and pathogenesis. The moderate proportion (7.48%) of hypothetical proteins relative to total coding sequences indicates a well-characterized genome with established functional annotations for the majority of predicted genes, while the presence of 294 uncharacterized sequences may provide opportunities for discovery of novel bacterial functions and adaptive mechanisms unique to this extensively drug-resistant isolate.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study characterises the genomic features of an \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e ST149 isolate recovered in India and demonstrates that this non-international clone lineage exhibits many of the hallmarks associated with high-risk, extensively drug-resistant strains. The increasing burden of \u003cem\u003eA. baumannii\u003c/em\u003e infections among hospitalised and immunocompromised patients in India underscores the need for detailed genomic analyses of underrepresented lineages. Although ST149 is not part of the dominant international clone groups, its genomic configuration indicates substantial adaptive capacity and clinical relevance. The isolate displayed an extensively drug-resistant phenotype, with susceptibility restricted to tigecycline and intermediate susceptibility to colistin. This pattern is consistent with the widespread emergence of carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e driven largely by class D β-lactamases. The coexistence of bla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;OXA-23\u0026lt;/sub\u0026gt;, bla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;OXA-104\u0026lt;/sub\u0026gt;, bla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;ADC-26\u0026lt;/sub\u0026gt;, and bla\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026thinsp;\u0026gt;\u0026thinsp;PER-7\u0026lt;/sub\u0026thinsp;\u0026gt;\u0026thinsp;within the same genome reflects the accumulation of diverse β-lactamase families, a feature commonly observed in global epidemic clones. The combined presence of aminoglycoside-modifying enzymes, sulfonamide resistance genes, and multiple efflux systems suggests a multidimensional resistance strategy integrating enzymatic inactivation, target modification, and active efflux. The detection of qacEΔ1 further indicates a potential for tolerance to disinfectants, supporting environmental persistence within healthcare settings. The strong association between antimicrobial resistance genes and mobile genetic elements highlights the dynamic nature of this genome. The localisation of most AMR determinants on MGE-rich contigs, including those encoding clinically relevant β-lactamases and macrolide resistance genes, suggests the presence of genomic regions analogous to resistance islands. This facilitates horizontal gene transfer events and may accelerate the spread of resistance traits within and between bacterial species. In addition to its extensive resistome, the isolate possesses a broad virulence genes repertoire. Complete acinetobactin clusters, multiple adhesion-associated genes, phospholipases, and a functional Type VI secretion system provide a foundation for host colonisation, nutrient acquisition, and interbacterial competition. The presence of biofilm-associated operons, including pgaABCD, supports the potential for long-term persistence on abiotic surfaces. These features collectively indicate a strain capable of maintaining fitness despite the metabolic costs associated with multidrug resistance. The variant analysis revealed an elevated transition-to-transversion ratio, suggesting a mutational bias consistent with oxidative and antibiotic stress. High-impact mutations in rplV (L22) and rpoB may modulate ribosomal and transcriptional processes, while moderate-impact substitutions affecting metabolic and recombination-related genes point toward ongoing microevolution. These findings support the view that selective pressures in clinical settings contribute to the emergence of distinctive mutational patterns in \u003cem\u003eA. baumannii\u003c/em\u003e. The non-coding RNA landscape adds an additional regulatory dimension. The presence of multiple riboswitches, including a putative aminoglycoside-responsive element, indicates an ability to couple environmental cues with resistance gene expression. The abundance of C4 antisense RNAs and Acinetobacter-specific sRNAs suggests active post-transcriptional regulation influencing stress adaptation, virulence, and antimicrobial tolerance. This regulatory complexity likely enhances the strain\u0026rsquo;s ability to respond to fluctuating environmental and therapeutic conditions.\u003c/p\u003e\u003cp\u003eHypothetical proteins constituted a substantial portion of the AB_Varanasi genome, reflecting an unexplored genetic reservoir with potential functional relevance. Growing evidence suggests that hypothetical proteins can contribute to virulence, stress tolerance, and host interaction in several bacterial pathogens. Many of these proteins contained Domains of Unknown Function (DUFs), which are increasingly recognized as contributors to bacterial physiology, virulence and adaptation (46).\u003c/p\u003e\u003cp\u003eThe identification of 294 hypothetical or uncharacterized proteins, many belonging to diverse DUF families, points to a substantial reservoir of unexplored functional potential. Several predicted membrane-associated and transport-related proteins may contribute to adaptive processes that remain to be fully understood. Their characterisation could reveal novel mechanisms relevant to virulence, stress tolerance, or drug resistance.\u003c/p\u003e\u003cp\u003eOverall, the genomic architecture of the ST149 isolate indicates a lineage with the potential to evolve into a clinically significant clone. Although ST149 remains less prevalent than major international clones, its extensive resistome, mobilome, virulence repertoire, and regulatory network suggest that it possesses the attributes required for successful adaptation and persistence. Broader genomic surveillance, particularly within South Asia, will be important for monitoring the emergence and spread of such lineages. The genomic insights presented here also provide a reference framework for future functional studies and may support the development of targeted interventions, including vaccine design and molecular diagnostics.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis genomic analysis of the \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e ST149 isolate from India reveals a lineage with considerable adaptive potential despite its limited representation among globally dominant international clones unlike ST2. The genome harbours an extensive repertoire of antimicrobial resistance genes, a diverse set of virulence-associated genes, and a broad array of mobile genetic elements that collectively support its capacity for persistence, transmission, and therapeutic evasion. The unusually high transition-to-transversion ratio, together with several high-impact mutations in essential genes, indicates an active evolutionary trajectory shaped by strong selective pressures in clinical environments. The regulatory landscape, enriched with non-coding RNAs and metabolite-sensing riboswitches, further underscores the dynamic transcriptional and post-transcriptional control mechanisms contributing to its resilience. Finally, the presence of a substantial number of hypothetical and uncharacterized proteins highlights functional domains that remain unexplored and may represent novel contributors to host interaction, environmental survival, or antimicrobial resistance. Taken together, these findings broaden current understanding of emerging non-IC \u003cem\u003eA. baumannii\u003c/em\u003e lineages and provide a foundation for future functional studies aimed at identifying diagnostic markers and potential therapeutic targets against this evolving pathogen strain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e The whole-genome sequencing (WGS) data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1201564. The assembled genome has been submitted to GenBank under the accession number JBRYYY000000000. All relevant data supporting the findings of this study are available within the article and its Supplementary Information files, or from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement:\u003c/strong\u003e The authors have stated explicitly that there are no conflicts of interest in connection with this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u0026nbsp;\u003c/strong\u003eThis study was approved by the Institutional Ethics Committee of IQ City Medical College \u0026amp; NH Hospital, Durgapur, India (Approval No. IQMC/IEC/Project/17/28). The \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolate in this study was recovered from discarded clinical culture plates without access to any patient-identifiable information. According to the International Ethical Guidelines for Health-related Research Involving Humans (CIOMS-WHO, 2016) and the Indian Council of Medical Research (ICMR) guidelines, such use of anonymized bacterial isolates without any associated personal data does not require individual patient consent, as it does not constitute human subject research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution:\u003c/strong\u003e SA, PK, PS \u0026amp; GC contributed to the study design. LAL, BC, PK, and SA collected bacterial samples. Genomic data was analysed and interpreted by SA, PK, AG, AV, SD1, SD2, PS, and GC. TEM was done by SA, PK \u0026amp; SG. SA and PK designed the figures. The manuscript was written by SA and PK. \u0026nbsp;GC, RSPR, and PS co-edited the manuscript with SD1, MR, LK, SD1, SD2, AV. PS \u0026amp; GC supervised the study. All authors had full access to all the data in the study and accepted the responsibility for the decision to submit for publication.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; Contributions (CRediT taxonomy)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization\u003c/strong\u003e: SA, PK, PS, GC\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eSample collection\u003c/strong\u003e: LAL, BC, PK, SA\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eData analysis\u003c/strong\u003e: SA, PK, AG, AV, SD1, SD2, PS, GC\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eInvestigation \u0026amp; TEM\u003c/strong\u003e: SA, PK, SG\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eVisualization\u003c/strong\u003e: SA, PK\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eWriting \u0026ndash; original draft\u003c/strong\u003e: SA, PK\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eWriting \u0026ndash; review \u0026amp; editing\u003c/strong\u003e: SD1, MR, LK, SD2, AV, GC, RSPR, PS\u003cbr\u003e\u003cstrong\u003eSupervision\u003c/strong\u003e: PS, GC\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Ethics Committee of IQ City Medical College \u0026amp; NH Hospital, Durgapur, India (Approval No. IQMC/IEC/Project/17/28). The \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolate used in this work was obtained from discarded clinical culture plates, with no access to patient-identifiable information. In accordance with the International Ethical Guidelines for Health-related Research Involving Humans (CIOMS-WHO, 2016) and the Indian Council of Medical Research (ICMR) guidelines, the use of anonymized bacterial isolates without linked personal data does not constitute human subject research and therefore does not require individual informed consent. We have carefully followed the\u0026nbsp;\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003eCIOMS-WHO (2016)\u003cbr\u003e\u0026nbsp;International Ethical Guidelines for Health-related Research Involving Humans\u003cbr\u003e\u0026nbsp;https://cioms.ch/publications/\u003cbr\u003e\u0026nbsp;➤ Guideline 3: Use of de-identified, minimal-risk samples may not require consent.\u003c/li\u003e\n \u003cli\u003eICMR National Ethical Guidelines (2017)\u003cbr\u003e\u0026nbsp;https://ethics.ncdirindia.org/\u003cbr\u003e\u0026nbsp;➤ Section: \u0026ldquo;Biological Materials and Data\u0026rdquo; permits use of unlinked, anonymized clinical samples without consent under specified conditions.\u003c/li\u003e\n \u003cli\u003eU.S. DHHS \u0026ndash; Common Rule (45 CFR 46) (For international reviewers)\u003cbr\u003e\u0026nbsp;https://www.ecfr.gov/current/\u003cbr\u003e\u0026nbsp;➤ Research on non-identifiable biospecimens is not considered human subject research.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNemec, A., Dijkshoorn, L. \u0026amp; Van Der Reijden, T. 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Genome sequences of two multidrug-resistant Acinetobacter baumannii clinical strains isolated from southern India. \u003cem\u003eGenome Announcements\u003c/em\u003e. \u003cb\u003e3\u003c/b\u003e (5), 10\u0026ndash;128 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrasad, A., Sundararajan, V. S., JayaramanValadi, Nigam, V. K. \u0026amp; Suravajhala, P. Prediction of virulence factors in bacterial hypothetical proteins. \u003cem\u003eArch. Microbiol.\u003c/em\u003e \u003cb\u003e207\u003c/b\u003e (10), 244 (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Horizontal Gene Transfer (HGT), Antimicrobial Resistance (AMR), Whole-genome Sequencing (WGS), Hypothetical proteins (HP), Domain of Unknown Family (DUF)","lastPublishedDoi":"10.21203/rs.3.rs-8142534/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8142534/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a high-priority pathogen due to its extensive antimicrobial resistance and persistence in clinical environments. This study describes the genomic features of AB_Varanasi, a carbapenem-resistant clinical isolate from India belonging to ST149 (Pasteur)/ST1506 (Oxford), a lineage not linked to major international clones. The isolate was resistant to almost all tested antibiotics, retaining susceptibility only to tigecycline and showing intermediate susceptibility to colistin. Whole-genome sequencing produced a 4.19 Mb circular chromosome encoding 4,039 predicted genes, with metabolism-associated functions forming the largest subsystem category.\u003c/p\u003e\u003cp\u003eThe genome carried multiple resistance determinants, including carbapenem-hydrolysing β-lactamases (OXA-23, OXA-104), extended-spectrum β-lactamases (ADC-26, PER-7), aminoglycoside-modifying enzymes, fluoroquinolone resistance\u0026ndash;associated mutations, and several multidrug efflux systems (AdeABC, AdeIJK, AcrAB-TolC, and MFS transporters). Several of these genes were embedded within mobile genetic element\u0026ndash;rich regions, suggesting a high potential for horizontal gene transfer. Virulence profiling identified the complete acinetobactin iron acquisition system, a functional Type VI secretion system, Type IV pilus components, and biofilm-associated operons.\u003c/p\u003e\u003cp\u003eVariant analysis detected 30,385 high-confidence mutations with a transition-to-transversion ratio of 3.05. High-impact variants affected ribosomal protein L22 and the RNA polymerase β-subunit, while moderate-impact mutations involved metabolic and recombination-related genes. The regulatory landscape comprised 113 non-coding RNAs, including riboswitches and antisense RNAs. A total of 294 hypothetical proteins were identified, representing 192 DUF families, many predicted to encode membrane-associated or transport-related functions. Several of these uncharacterized proteins may serve as promising therapeutic or vaccine targets, given their potential surface exposure and species-specific conservation.\u003c/p\u003e","manuscriptTitle":"Isolation and Whole-Genome Sequencing of a Less Prevalent Indian A. baumannii Strain Reveals Unique Uncharacterized Hypothetical Proteins and AMR-Linked ncRNAs.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 07:21:45","doi":"10.21203/rs.3.rs-8142534/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-17T06:42:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T08:17:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210988826485720112775470035832572245723","date":"2026-04-06T17:13:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15702842616765453741458329409085004154","date":"2026-02-18T17:57:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-25T07:40:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223291265895842422416691611871646174135","date":"2025-12-28T01:16:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-07T15:57:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-24T13:11:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-18T13:14:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-18T13:14:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-18T07:38:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a0721d3-be7b-4edd-9ba3-5bfd89ad170f","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59231770,"name":"Biological sciences/Genetics"},{"id":59231771,"name":"Biological sciences/Microbiology"},{"id":59231772,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-05-07T13:53:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 07:21:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8142534","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8142534","identity":"rs-8142534","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}