Next-Generation Sequencing of Klebsiella Pneumoniae Isolated from Exudates: A Comprehensive Analysis of Antimicrobial and Virulence Genes, Serotypes, and Sequence Types

Next-Generation Sequencing of Klebsiella Pneumoniae Isolated from Exudates: A Comprehensive Analysis of Antimicrobial and Virulence Genes, Serotypes, and Sequence Types | 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 Next-Generation Sequencing of Klebsiella Pneumoniae Isolated from Exudates: A Comprehensive Analysis of Antimicrobial and Virulence Genes, Serotypes, and Sequence Types Chitra Rajalakshmi P, Vallab Ganesh Bharadwaj Balasubramanian, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9016172/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The global expansion of antimicrobial resistance (AMR) in Klebsiella pneumoniae represents a significant threat to clinical treatment success, often termed a "silent epidemic". This study utilized next-generation sequencing (NGS) and whole-genome sequencing (WGS) to characterize nine multidrug-resistant (MDR) isolates recovered from patients with pyogenic infections. Results indicated that the blaCTX-M-15 gene was the most predominant resistance marker (88.88%), followed by blaOXA-1 and blaTEM-18 (77.77%), while the carbapenemase blaNDM-5 was present in 33.33% of isolates.Genomic analysis via multilocus sequence typing (MLST) identified ST-2096 and ST-985 as common sequence types, with KL-64 and KL-39 being the most prevalent capsular polysaccharide types. The investigation further detailed an extensive array of virulence genes, including the ybt and ent complexes, and identified key plasmid replicons such as IncFIB and IncFII. These findings highlight the emergence of hypervirulent (hvKp) global sequence types that also exhibit MDR. Ultimately, the molecular characterization provided by WGS is essential for understanding the epidemiology of bacterial strains, enabling the development of targeted antibiotic prescribing procedures and enhanced patient management strategies Health sciences/Diseases Biological sciences/Microbiology Klebsiella pneumoniae Antimicrobial Resistance (AMR) Whole-Genome Sequencing (WGS) Next-Generation Sequencing (NGS) Multi Drug Resistant(MDR) Figures Figure 1 Introduction According to World Health Organization reports from 2025, one in six illnesses are now drug-resistant, showing that rising antimicrobial resistance (AMR) among common bacterial isolates has reached catastrophic proportions. The accurate identification of AMR genes, plasmids, and virulence factors is made possible by next-generation sequencing (NGS), particularly whole-genome sequencing (WGS), which is quickly displacing phenotypic approaches in order to counteract this and enable molecular mapping of resistance propagation [ 1 , 2 ]. Due to their remarkable capacity to "escape" the effects of traditional antibiotics, the ESKAPE pathogens—which include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii, Pseudomonas aeruginosa , and Enterobacter species —are acknowledged worldwide as serious, high-priority threats to human health. These pathogens are major causes of the AMR crisis, which raises global healthcare expenses, morbidity, and mortality [ 3 – 6 ]. Over the past ten years, there has been a sharp rise in the prevalence of multi-drug resistance (MDR) K. pneumoniae strains, particularly the carbapenem-resistant Klebsiella pneumoniae (CRKP). The rise of hypervirulent K. pneumoniae (hvKP), which is resistant to the majority of therapeutically available β-lactams, and carbapenem-resistant K. pneumoniae has been particularly concerning recently, severely restricting treatment options. There are two ways that this type of bacteria can arise: either hypervirulent strains acquire carbapenem-resistant plasmid (CR-hvKP) or carbapenem-resistant strains acquire hypervirulence determining genes/plasmid (hv-CRKP) [ 7 , 8 ]. To understand the development of AMR, epidemiological study is required, to facilitate the identification of specific high-risk bacterial lineages known as "global clones" or "clonal complexes." As a result, tracking these epidemic clones and elucidating their genetic features have emerged as important scientific priorities. The purpose of this study was to identify the potential virulence genes, plasmids, AMR genes, and sequence types (STs) and serotypes of K. pneumoniae that were isolated from pyogenic infections. Methods This observational, analytical, and cross-sectional investigation examined nine MDR K. pneumoniae isolates obtained from individuals with pyogenic illnesses. All methods were carried out in accordance with relevant guidelines and regulations. This study was approved by the Institutional Ethics Committee (1136/TSRMMCH&RC/ME-1/2023).The study utilized anonymised and de-identified clinical samples collected during routine clinical care, and the requirement for informed consent was waived by the ethics committee.. All isolates were identified, and antimicrobial susceptibility profiles were obtained using traditional microbiological procedures and validated using automated systems [ 9 – 12 ]. Furthermore, all of the isolates were examined using NGS or WGS to discover resistance genes such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs), and virulence factors. Multilocus sequence typing (MLST) was carried out to determine the sequence types, and serotyping was carried out using Kaptive Web ( https://kaptive-web.erc.monash.edu/ ), a bioinformatics tool for identifying capsular (K) and lipopolysaccharide (O) antigen loci., In silico serotyping of Klebsiella isolates was carried out. After uploading "FASTA"-formatted genome assemblies to the Kaptive Web interface, they were compared to the K. pneumoniae reference K- and O-locus databases. Based on sequence similarity, gene content, and locus completeness, Kaptive predicts serotypes. For every forecast, a confidence level (such as "good," "very good," or "perfect") was noted. For further study, the K- and O-locus types that were produced were utilized. Genomic characterisation and whole-genome sequencing to identify virulence and resistance genes and sequence types The Qiagen QIAamp DNA Mini kit (Qiagen, Hilden, Germany) was used to extract the deoxyribonucleic acid (DNA) from K. pneumoniae isolates in accordance with the manufacturer's instructions. Using 150 bp paired-end chemistry, double-stranded DNA libraries with a 450 bp insert size were created and sequenced on the Illumina platform. Prokka v1.5 [ 14 ] was used to annotate the genomes that passed sequence quality control after they were assembled using Spades v3.14 [ 13 ] to create contigs. A bactinspector was used to identify species, and Confindr was used to evaluate contamination. Web-based reports were created by combining all of the quality measures using Qualifyr and MultiQC. The ARIBA program v2.14.4 [ 15 ] was used to identify MLST, AMR, and virulence factors using the BIGSdb-Pasteur MLST database, the National Center for Biotechnological Information (NCBI) AMR-acquired gene, the PointFinder databases, and the VFDB, respectively [ 16 – 18 ]. Nextflow pipelines, which were created as part of the Global Health Research Unit (GHRU), United Kingdom, for AMR surveillance, were used for all bioinformatic analyses. Results Among the K . pneumoniae isolates studied, 5 (55.55%) were isolated from females, and 4 (44.44%) were isolated from male patients. The mean age of the patients was 50.50±9.25 years. The WGS analysis revealed blaCTX-M-15 (8, 88.88%) was the most predominant gene, followed by blaOXA-1, and blaTEM-18 in 7 (77.77%) isolates, and blaNDM-5, blaSHV-187, and blaSHV-106 in 3 (33.33%) isolates. Other AMR genes observed included, aac3IIa, aac6Ibcr, aadA2, aph3Ia, aph3Ib, aph6Id, blaDHA-1, blaOXA-9, blaOXA-232, blaSHV-110, catA1, dfrA1, dfrA5, dfrA12, dfrA14, dfrA17, fosA, fosA6, mphA, mphE, msrE, oqxA, oqxB, qnrB1, qnrB4, qnrS1, rmtB, sul1, sul2, and tetA. entA, The virulence genes that were identified were entB, fepC, fyuA, irp1, irp2, iucA, iucB, iucC, iutA, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR . (Fig 1 & Table 1) Table 1: The antimicrobial and virulence genes identified among the isolates Strain Age (years) Sex AMR genes Virulence genes G20250305 53 Female aac6Ib, aph3Ia, aph3VI, armA, bla CTX-M-15 , bla NDM-5 , bla OXA-1 , bla OXA-232 , bla OXA-9 , bla SHV-106 , bla TEM-1B , dfrA1, dfrA5, fosA6, mphA, mphE, msrE, oqxA, oqxB, qnrS1, sul1, sul2 entA, entB, fepC, fyuA, irp1, irp2, iucA, iucB, iucC, iutA, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR G20250306 53 Female aac6Ib, aph3Ia, aph3VI, bla CTX-M-15 , bla NDM-5 , bla OXA-1 , bla OXA-232 , bla OXA-9 , bla SHV-106 , bla TEM-1B , dfrA1, fosA6, oqxA, oqxB, qnrS1 entA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR G20250307 60 Male aac3IIa, aac6Ibcr, aph3Ib, aph6Id, bla CTX-M-15 , bla OXA-1 , bla SHV-187 , bla TEM-1B , fosA6, oqxA, oqxB, qnrB1, sul2, tetA entA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR G20250308 45 Female aac3IIa, aac6Ibcr, aph3Ib, aph6Id, bla CTX-M-15 , bla OXA-1 , bla SHV-187 , bla TEM-1B , fosA6, oqxA, oqxB, qnrB1, sul2, tetA entA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR G20250309 53 Female aac3IIa, aac6Ibcr, aadA2, aph3Ia, aph3Ib, aph6Id, bla CTX-M-15 , bla OXA-1 , catA1, dfrA12, fosA, mphA, oqxA, oqxB, sul1, sul2, tetA entA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR G20250310 30 Male fosA, oqxA, oqxB entB, fepC, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR G20250311 55 Female aac6Ibcr, aph3Ib, aph6Id, bla CTX-M-15 , bla OXA-1 , bla SHV-110 , bla TEM-1B , dfrA14, fosA, oqxA, oqxB, qnrB1, sul2, tetA entA, entB, fepC, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR G20252895 55 Male aac3IIa, aac6Ibcr, aadA2, aph3Ib, aph6Id, bla CTX-M-15 , bla NDM-5 , bla OXA-1 , bla SHV-106 , bla TEM-1B , dfrA12, dfrA14, ermB, fosA6, mphA, oqxA, oqxB, qnrB1, rmtB, sul1, sul2, tetA entA, net, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR G20252896 59 Male aph3Ib, aph6Id, bla CTX-M-15 , bla DHA-1 , bla SHV-187 , bla TEM-1B , dfrA17, fosA6, mphA, oqxA, oqxB, qnrB4, qnrS1, sul1, sul2 entA, net, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR Kleborate analysis found that KL-64 and KL-39 were the most prevalent, followed by KL-62, KL-63, KL-16, KL-23, and KL-112 capsular polysaccharide types. The cell wall lipopolysaccharide antigen and its variations detected were O1v2 (44.44%), O1v1 (44.44%), and O3 (11.11%). (Table 2) Table 2: Kleborate analysis revealing serotypes of K. pneumoniae isolates Strain Serotype K (capsular polysaccharide antigen) O/v (cell wall lipopolysaccharide antigen and its variant) G20250305 KL64 O1v1 G20250306 KL64 O1v1 G20250307 KL39 O1v2 G20250308 KL39 O1v2 G20250309 KL62 O1v1 G20250310 KL63 O3 G20250311 KL16 O1v1 G20252895 KL23 O1v2 G20252896 KL112 O1v2 MLST analysis revelaed ST-2096 and ST-985 were regularly observed, while other STs detected included ST-147, ST-198, ST-15, and ST-17. The most prevalent plasmid replicons were IncFIB (K), IncFIB (pNDM-Mar), IncFII, and ColRNAIThe most prevalent plasmid replicons were IncFIB (K), IncFIB (pNDM-Mar), IncFII, and ColRNAI. (Table 3) Table 3: Sequence typing results and the plasmid replicons identified in the isolates Strain MLST type Housekeeping genes Plasmid replicons G20250305 ST-2096 gapA , infB , mdh , pgi , phoE , rpoB , tonB IncFIB (K), IncFIB(pNDM-Mar), IncHI1B(pNDM-MAR) , ColRNAI, Col440I, ColKP3 G20250306 ST-2096 gapA , infB , mdh , pgi , phoE , rpoB , tonB IncFIB (K), IncFIB(pNDM-Mar), IncHI1B(pNDM-MAR) , ColRNAI, Col440I, ColKP3 G20250307 ST-985 gapA , infB , mdh , pgi , phoE , rpoB , tonB IncFIB (K), IncFII, ColRNAI G20250308 ST-985 gapA , infB , mdh , pgi , phoE , rpoB , tonB IncFIB (K), IncFII, ColRNAI G20250309 ST-48 gapA , infB , mdh , pgi , phoE , rpoB , tonB IncFIB (K), IncFIB(pNDM-Mar), IncHI1B(pNDM-MAR) , IncFII, ColRNAI, Col440I, ColpVC G20250310 ST-147 gapA , infB , mdh , pgi , phoE , rpoB , tonB None G20250311 ST-198 gapA , infB , mdh , pgi , phoE , rpoB , tonB None G20252895 ST-15 gapA , infB , mdh , pgi , phoE , rpoB , tonB IncX4, IncFIB (K), IncFII, Col(MG828) G20252896 ST-17 gapA , infB , mdh , pgi , phoE , rpoB , tonB IncFII (pKP91) , IncFIB (K) , IncFII , Col440II Discussion The classical K. pneumoniae , which has a strong propensity to acquire antibiotic resistance, and the recently discovered hvKP are the two primary K. pneumoniae pathogenic variants of public health significance. Hypervirulence (hvkp), which is connected to invasive illness, abscess formation, and metastatic infection, has been linked to a number of virulence factors for K. pneumoniae . Moreover, infections with strains that produce MDR, carbapenemase and ESBL have been linked to higher fatality rates in critically ill patients [ 19 , 20 ]. The WGS of K. pneumoniae strains aids researchers in identifying epidemic clones of drug-resistant infections. Furthermore, the MDR can be converted into a hypervirulent phenotype by acquiring virulence-carrying plasmids. Understanding the diversity of genomes and resistance phenotypes is critical for determining transmission patterns [ 21 ]. Antimicrobial resistance genes The most common AMR gene found in 88.88% (8/9) of the strains in the current investigation was blaCTX-M-15. In K. pneumoniae, blaCTX-M-15 is a common, widely distributed ESBL gene that confers high-level resistance to third-generation cephalosporins (such as ceftazidime and ceftriaxone). It is mainly carried on mobile genetic elements, such as IncFIIk plasmids, and is linked to the insertion sequence (ISEcp1), which allows for frequent outbreaks and quick dissemination, particularly in hospital critical care units (ICUs) [ 22 ]. The production of ESBL contributing blaOXA-1 results in resistance to ampicillin, ticarcillin, piperacillin and cephalosporins. Furthermore, it was observed that most blaOXA-1 carrying strains simultaneously acquire blaCTX-M-15, thereby contributing to widespread resistance to broad spectrum antibiotics including cephalosporins [ 23 , 24 ]. This study revealed seven out of nine (88.88%) strains carried both genes. Three strains (33.33%) with the blaNDM-5 gene were found in the current investigation. Penicillin, cephalosporins, and carbapenems are among the β-lactam antibiotics that NDM can hydrolyze. There are variations of the blaNDM gene. The most common ones in the world are blaNDM-1, blaNDM-5, and blaNDM-7. According to earlier studies, blaNDM-5 has greater carbapenemase activity than NDM-1, which leads to higher carbapenem minimum inhibitory concentrations. Furthermore, it was established that NDM genes have been shown to be highly transmissible and often associated with travel [ 25 ]. In the current investigation, the aac gene and its variants, including the aac6Ib, were found in seven (77.77%) strains. Through enzymatic modification of the amino or hydroxyl groups of the aminoglycoside group of antibiotics, such as gentamicin and amikacin, these genes may confer resistance to aminoglycosides [ 26 ]. The aph gene and its variations, including aph3Ia, aph3Ib, aph6Id, and aph3VI, were detected in eight (88.88%) strains in this investigation. Aminoglycoside aminotransferase, which is encoded by the aph gene, alters and deactivates aminoglycoside antibiotics, frequently resulting in resistance to gentamicin, tobramycin, and amikacin [ 27 ]. In MDR clinical isolates, these gene variations are often detected on plasmids or transposons. The armA gene, which codes for a 16S rRNA methyltransferase that alters the ribosomal target site and makes aminoglycoside antibiotics ineffective, was found in only one strain (11.11%) in our investigation. It facilitates quick horizontal transmission among Klebsiella species and is frequently carried on plasmids or transposons. Additionally, it was discovered that the expression of the armA gene restricts the available treatments for serious infections, including sepsis, bloodstream infections, and pneumonia, requiring robust molecular surveillance [ 28 ]. The aadA2 gene, which codes for an aminoglycoside nucleotidyl transferase—a crucial enzyme conferring resistance to aminoglycoside antibiotics, specifically streptomycin and spectinomycin—was found in one strain (11.11%). It usually co-occurs with ESBL genes (such as blaCTX-M and blaSHV) and other resistance markers. It is often found on class 1 integrons and plasmid-transmitted, contributing to MDR in clinical isolates [ 29 ]. The blaSHV gene and its variations were detected in six (66.66%) of the isolates in this investigation. Intrinsic ampicillin resistance in K. pneumoniae is caused by the blaSHV gene, a common beta-lactamase that is frequently expressed on chromosomes. It has a wide range of variations, from ESBL, which hydrolyzes third-generation cephalosporins, to narrow-spectrum. Plasmids frequently contain these genes, which facilitate their propagation [ 30 ]. In this study, blaTEM-IB was found in seven (77.77%) isolates. This gene is a common beta-lactamase found in K. pneumoniae . It belongs to the TEM-1 family of penicillinases, which are enzymes encoded by plasmids that provide resistance to primary-generation cephalosporins and penicillins [ 31 ]. One strain (11.11%) was found to have the class C beta-lactamase blaDHA-1, which hydrolyzes cephalosporins. BlaDHA-1 can be acquired by K. pneumniae on big plasmids. Plasmid-borne blaDHA-1 isolates have been reported from various geographical places, and it has been discovered that blaDHA-1 can be induced when linked to the ampR gene. This gene may not have a major impact on β-lactam susceptibility, but when paired with other mechanisms such as porin abnormalities, it may become more prominent in some isolates, underestimating their prevalence [ 32 ]. Virulence genes All the isolates tested in this study revealed the presence of Outer membrane protein A (OmpA), an essential virulence and pathogenic factor of K. pneumoniae . A recent study demonstrated that OmpA promotes K. pneumoniae penetration of the blood-brain barrier and mediates meningitis via multiple pathways [ 33 ]. In this investigation, fosA and its variation fosA6 were present in eight (88.88%) strains. One important mechanism of resistance to the antibiotic fosfomycin in K. pneumoniae is the fosA gene, which is typically found as a chromosomally encoded glutathione transferase that clears the drug. High-level resistance in CRKP isolates is largely caused via plasmid-mediated transmission and fosA variations (such as fosA3 and fosA6), even though these are frequently innate [ 34 ]. In this investigation, the mphA was detected in four (44.44%) K. pneumoniae strains. The enzyme that confers resistance to 14-membered macrolide antibiotics, such as roxithromycin and erythromycin, is encoded by the mphA gene. Along with other resistance genes (such as sul1 and qnrB4), it is frequently found on plasmids and transposons, which contribute to MDR and spread in clinical settings [ 35 ]. In this investigation, one strain (11.11%) had the ermB gene. By altering the (50S) ribosomal subunit, the erm gene codes for erythromycin ribosome methylase and methyltransferase enzymes that provide resistance to macrolide antibiotics like azithromycin. Important erm genes found in Klebsiella include erm(B), erm(T), and erm(42), which are frequently carried on plasmids and linked to high-level resistance and dissemination [ 36 ]. The msrE gene was found in one (11.11%) strain of K. pneumoniae in this investigation. High-level resistance to macrolides and streptogramin B is provided by the ABC-F subfamily protein that is encoded by the msrE gene and functions as a ribosomal protection protein. It is frequently detected in MDR clinical isolates and is frequently linked to the msr(E)-mph(E) operon on plasmids. It frequently co-occurs with other resistance mechanisms [ 37 ]. In this investigation, one (11.11%) K. pneumoniae strain had the rmtB gene. K. pneumoniae's rmtB gene produces a 16S rRNA methyltransferase, which results in high-level resistance to aminoglycosides (gentamicin, amikacin, and tobramycin). It typically co-occurs with armA on mobile plasmids with ESBL (such as blaCTX-M or carbapenemase genes blaKPC-2, blaNDM) [ 38 ]. Serotypes Among the numerous K antigens identified in this investigation, KL64 and KL39 were most commonly (22.22%) noticed. KL63, which was found in one strain (11.11%), is known to be a common or emerging variety that is quite pathogenic. Additionally, KL62 confers greater levels of AMR, and KL16 and KL64 have been identified as hypervirulent strains [ 39 , 40 ]. Multilocus sequence types K. pneumoniae ST2096, a recently reported highly pathogenic, MDR clone that is frequently associated with bloodstream infections, was found in two (22.22%) strains in this investigation. It is sometimes referred to as a "hybrid" strain because it possesses both high-level resistance genes (like blaOXA) and hypervirulence factors (like iucA, iutA, and rmpA2). It is considered a serious clinical risk in regions such as Saudi Arabia and India [ 41 ]. K. pneumoniae ST985, which was found in two (22.22%) isolates, is frequently linked to AMR and has been documented in clinical, environmental, and animal contexts. It has been found to possess several AMR genes that could lead to infections in hospital settings and to be a component of the polyclonal distribution of strains in various locales [ 42 ]. The hybrid resistant virulence plasmids IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) are known to be carried by ST147, which was found in one (11.11%) strain in our study. Additionally, ST147 may carry plasmids that combine virulence and resistance factors, raising patient risk. Recently discovered in the United Kingdom, ST147 is widely acknowledged as a high-risk clone [ 43 ]. K. pneumoniae ST-48 strains found in this investigation is one of the MDR strains that are widely distributed around the world and are frequently linked to the synthesis of carbapenemases (such as OXA-48 and NDM-1). This ST was classified as an opportunistic pathogen due to its high genetic plasticity, rich virulome, and capacity to cause serious, challenging hospital-acquired infections (HAIs), such as bloodstream infections, pneumonia, and urinary tract infections, especially in patients with compromised immune systems. Additionally, ST-48 has been found in environmental sources, such as chicken meat, indicating a possibility of food chain transmission [ 44 ]. K. pneumoniae ST198, a developing MDR ST linked to CTX-M-15 ESBL production and, occasionally, hypervirulence, was discovered in one (11.11%) strain in this investigation. This strain poses a danger to food safety since it has been found in both clinical settings and environmental sources like lettuce [ 45 ]. One strain (11.11%) of K. pneumoniae ST15 is a new, high-risk CRKP clone that is generating HAI outbreaks worldwide, especially in intensive care units. It is typified by MDR, which includes the generation of carbapenemases (such as KPC-2, NDM-1, and OXA-232) and ESBLs [ 46 ]. K. pneumoniae sequence type 17 (ST17) found in one (11.11%) strain in this study is a globally disseminated, MDR clone frequently responsible for opportunistic, HAIs, particularly in neonatal intensive care units. It is highly diverse, ST that can carry ESBL-encoding plasmids and virulence genes [ 47 ]. Conclusions The WGS analysis of K. pneumoniae isolates from pyogenic infections revealed a concerning convergence of MDR and virulence traits. The predominance of ESBL genes such as blaCTX-M-15 , alongside carbapenemase genes like blaNDM-5 , underscores the growing challenge of treating infections caused by these strains. The simultaneous presence of siderophore clusters and virulence-associated genes suggests that these isolates are not only resistant to frontline antibiotics but also highly capable of establishing severe infections. The identification of high-risk global clones (e.g., ST-2096, ST-147) and diverse capsular and O-antigen types highlights the genetic adaptability of K. pneumoniae , enabling it to persist and spread across different patient populations. The plasmid replicons detected further emphasize the role of mobile genetic elements in disseminating resistance and virulence determinants, raising the risk of rapid outbreaks. From a clinical perspective, these findings reinforce the urgent need for enhanced molecular surveillance using WGS to track the evolution and spread of epidemic clones, strict antibiotic stewardship programs to minimize the misuse of broad-spectrum antibiotics and slow resistance development, improved infection control measures in healthcare settings to prevent transmission of MDR strains, and Integration of genomic data into patient management, allowing clinicians to tailor therapies based on resistance and virulence profiles. Ultimately, the study demonstrates that K. pneumoniae is evolving into a formidable pathogen with both resistance and virulence advantages. Without coordinated global efforts in surveillance, stewardship, and clinical management, these strains could significantly undermine current treatment options and pose a major public health threat. Limitations of this study The limited number of isolates and their specific origin from exudates may restrict the generalizability of the results. Additionally, the study did not correlate serotype or ST data with clinical outcomes or bacterial virulence. Declarations Funding Declaration The authors declare that no funds,grants or other support were received during the preparation of this manuscript. Author Contribution C.P Writing original draft ,Software,Table 1,2&3; V.G.B.B Writing Original draft , Formal analysis, Software , Data Collection,Coordination,Table 1,2&3; V.K Writing original draft ,Table 1,2&3,Concept and Design of the paper; A.D Review and Editing,Supervision, F.A Review and Editing,Supervision; H.P.K.S Supervision Editing, Fig 1 ;D.S Formal analysis,Software Table 1,2&3 Acknowledgement Kempegowda Institute of Medical Sciences has made a memorandum of understanding (MOU) with Dr. P. Chitra Rajalakshmi, Trichy SRM Medical College Hospital and Research Centre, to collaborate under the project supported by the Global Health Research Unit (GHRU), United Kingdom Data Availability The sequence data for isolates G20250305, G20250306, G20250307, G20250308, G20250309, G20250310, G20250311, G20252895, and G20252896 have been deposited in GenBank under the BioProject number PRJNA1435588. The corresponding BioSample accession numbers are SAMN56449246, SAMN56449247, SAMN56449248, SAMN56449249, SAMN56449250, SAMN56449251, SAMN56449252, SAMN56449253, and SAMN56449254. The raw sequencing reads are available in the NCBI Sequence Read Archive (SRA). References Global antibiotic resistance surveillance report 2025. (2025). https://iris.who.int/server/api/core/bitstreams/8831cd08-44cc-4741-9e30-5b1293150bda/content Last Accessed: January 27, 2026. van Belkum, A. Next generation sequencing as a panacea for antibiotic susceptibility testing: yea or nay? Front. Public. 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Agents Chemother. 62 (10), e01202–e01218. 10.1128/AAC.01202-18 (2018). PMID: 30061296; PMCID: PMC6153798. Zeng, W. et al. Outer membrane protein A mediates Klebsiella pneumoniae penetration of the blood-brain barrier and induces bacterial meningitis. Microbiol. Res. 299 , 128262 (2025). Epub 2025 Jun 23. PMID: 40578143. Wang, Y. P. et al. Transporter Genes and fosA Associated With Fosfomycin Resistance in Carbapenem-Resistant Klebsiella pneumoniae . Front. Microbiol. 13 , 816806. 10.3389/fmicb.2022.816806 (2022). PMID: 35173700; PMCID: PMC8841775. Pustam, A., Jayaraman, J. & Ramsubhag, A. Whole genome sequencing reveals complex resistome features of Klebsiella pneumoniae isolated from patients at major hospitals in Trinidad, West Indies. J. Glob Antimicrob. Resist. 37 , 141–149. 10.1016/j.jgar.2024.03.019 (2024). Epub 2024 Apr 10. PMID: 38608934. Yang, X., Zhang, H., Chan, E. W., Zhang, R. & Chen, S. Transmission of azithromycin-resistant gene, erm(T), of Gram-positive bacteria origin to Klebsiella pneumoniae. Microbiol Res. ;282:127636. (2024). 10.1016/j.micres.2024.127636 . Epub 2024 Feb 3. PMID: 38359498. Duan, Y. et al. Macrolides mediate transcriptional activation of the msr(E)-mph(E) operon through histone-like nucleoid-structuring protein (HNS) and cAMP receptor protein (CRP). J Antimicrob Chemother. ;77(2):391–399. (2022). 10.1093/jac/dkab395 . PMID: 34747464. Zhou, Y. et al. Co-Occurrence of Rare ArmA-, RmtB-, and KPC-2-Encoding Multidrug-Resistant Plasmids and Hypervirulence iuc Operon in ST11-KL47 Klebsiella pneumoniae. Microbiol. Spectr. 10 (2), e0237121. 10.1128/spectrum.02371-21 (2022). Epub 2022 Mar 24. PMID: 35323034; PMCID: PMC9045180. Kinney, E. L. et al. Connections between Klebsiella pneumoniae bloodstream dynamics and serotype-independent capsule properties. Infect. Immun. 2026 Jan 29 :e0064125. 10.1128/iai.00641-25 . Epub ahead of print. PMID: 41609496. Huang, X. et al. Capsule type defines the capability of Klebsiella pneumoniae in evading Kupffer cell capture in the liver. PLoS Pathog . 18 (8), e1010693. 10.1371/journal.ppat.1010693 (2022). PMID: 35914009; PMCID: PMC9342791. Hala, S. et al. The emergence of highly resistant and hypervirulent Klebsiella pneumoniae CC14 clone in a tertiary hospital over 8 years. Genome Med. 16 (1), 58. 10.1186/s13073-024-01332-5 (2024). PMID: 38637822; PMCID: PMC11025284. Habib, S. et al. The Diversity, Resistance Profiles and Plasmid Content of Klebsiella spp. Recovered from Dairy Farms Located around Three Cities in Pakistan. Antibiot. (Basel) . 12 (3), 539. 10.3390/antibiotics12030539 (2023). PMID: 36978406; PMCID: PMC10043998. Turton, J. F., Perry, C., McGowan, K., Turton, J. A. & Hope, R. Klebsiella pneumoniae sequence type 147: a high-risk clone increasingly associated with plasmids carrying both resistance and virulence elements. J. Med. Microbiol. 73 (4), 001823. 10.1099/jmm.0.001823 (2024). 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The accurate identification of AMR genes, plasmids, and virulence factors is made possible by next-generation sequencing (NGS), particularly whole-genome sequencing (WGS), which is quickly displacing phenotypic approaches in order to counteract this and enable molecular mapping of resistance propagation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to their remarkable capacity to \"escape\" the effects of traditional antibiotics, the ESKAPE pathogens\u0026mdash;which include \u003cem\u003eEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii, Pseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eEnterobacter species\u003c/em\u003e\u0026mdash;are acknowledged worldwide as serious, high-priority threats to human health. These pathogens are major causes of the AMR crisis, which raises global healthcare expenses, morbidity, and mortality [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the past ten years, there has been a sharp rise in the prevalence of multi-drug resistance (MDR) K. pneumoniae strains, particularly the carbapenem-resistant Klebsiella pneumoniae (CRKP). The rise of hypervirulent K. pneumoniae (hvKP), which is resistant to the majority of therapeutically available β-lactams, and carbapenem-resistant K. pneumoniae has been particularly concerning recently, severely restricting treatment options. There are two ways that this type of bacteria can arise: either hypervirulent strains acquire carbapenem-resistant plasmid (CR-hvKP) or carbapenem-resistant strains acquire hypervirulence determining genes/plasmid (hv-CRKP) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo understand the development of AMR, epidemiological study is required, to facilitate the identification of specific high-risk bacterial lineages known as \"global clones\" or \"clonal complexes.\" As a result, tracking these epidemic clones and elucidating their genetic features have emerged as important scientific priorities. The purpose of this study was to identify the potential virulence genes, plasmids, AMR genes, and sequence types (STs) and serotypes of \u003cem\u003eK. pneumoniae\u003c/em\u003e that were isolated from pyogenic infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis observational, analytical, and cross-sectional investigation examined nine MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates obtained from individuals with pyogenic illnesses. All methods were carried out in accordance with relevant guidelines and regulations. This study was approved by the Institutional Ethics Committee (1136/TSRMMCH\u0026amp;RC/ME-1/2023).The study utilized anonymised and de-identified clinical samples collected during routine clinical care, and the requirement for informed consent was waived by the ethics committee.. All isolates were identified, and antimicrobial susceptibility profiles were obtained using traditional microbiological procedures and validated using automated systems [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, all of the isolates were examined using NGS or WGS to discover resistance genes such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs), and virulence factors.\u003c/p\u003e \u003cp\u003eMultilocus sequence typing (MLST) was carried out to determine the sequence types, and serotyping was carried out using Kaptive Web (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kaptive-web.erc.monash.edu/\u003c/span\u003e\u003cspan address=\"https://kaptive-web.erc.monash.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a bioinformatics tool for identifying capsular (K) and lipopolysaccharide (O) antigen loci., In silico serotyping of \u003cem\u003eKlebsiella\u003c/em\u003e isolates was carried out. After uploading \"FASTA\"-formatted genome assemblies to the Kaptive Web interface, they were compared to the \u003cem\u003eK. pneumoniae\u003c/em\u003e reference K- and O-locus databases. Based on sequence similarity, gene content, and locus completeness, Kaptive predicts serotypes. For every forecast, a confidence level (such as \"good,\" \"very good,\" or \"perfect\") was noted. For further study, the K- and O-locus types that were produced were utilized.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenomic characterisation and whole-genome sequencing to identify virulence and resistance genes and sequence types\u003c/h2\u003e \u003cp\u003eThe Qiagen QIAamp DNA Mini kit (Qiagen, Hilden, Germany) was used to extract the deoxyribonucleic acid (DNA) from \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates in accordance with the manufacturer's instructions. Using 150 bp paired-end chemistry, double-stranded DNA libraries with a 450 bp insert size were created and sequenced on the Illumina platform. Prokka v1.5 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] was used to annotate the genomes that passed sequence quality control after they were assembled using Spades v3.14 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] to create contigs. A bactinspector was used to identify species, and Confindr was used to evaluate contamination. Web-based reports were created by combining all of the quality measures using Qualifyr and MultiQC. The ARIBA program v2.14.4 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] was used to identify MLST, AMR, and virulence factors using the BIGSdb-Pasteur MLST database, the National Center for Biotechnological Information (NCBI) AMR-acquired gene, the PointFinder databases, and the VFDB, respectively [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nextflow pipelines, which were created as part of the Global Health Research Unit (GHRU), United Kingdom, for AMR surveillance, were used for all bioinformatic analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the \u003cem\u003eK\u003c/em\u003e. \u003cem\u003epneumoniae\u003c/em\u003e isolates studied, 5 (55.55%) were isolated from females, and 4 (44.44%) were isolated from male patients. The mean age of the patients was 50.50\u0026plusmn;9.25 years. The WGS analysis revealed blaCTX-M-15 (8, 88.88%) was the most predominant gene, followed by blaOXA-1, and blaTEM-18 in 7 (77.77%) isolates, and blaNDM-5, blaSHV-187, and blaSHV-106 in 3 (33.33%) isolates. \u0026nbsp;Other AMR genes observed included, aac3IIa, aac6Ibcr, aadA2, aph3Ia, aph3Ib, aph6Id, blaDHA-1, blaOXA-9, blaOXA-232, blaSHV-110, catA1, dfrA1, dfrA5, dfrA12, dfrA14, dfrA17, fosA, fosA6, mphA, mphE, msrE, oqxA, oqxB, qnrB1, qnrB4, qnrS1, rmtB, sul1, sul2, and tetA. entA, The virulence genes that were identified were entB, fepC, fyuA, irp1, irp2, iucA, iucB, iucC, iutA, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR . \u003cstrong\u003e(Fig 1 \u0026amp; Table 1)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: The antimicrobial and virulence genes identified among the isolates\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.8e+2pt\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAMR genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVirulence genes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20250305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaac6Ib, aph3Ia, aph3VI, armA, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eNDM-5\u003c/sub\u003e, bla\u003csub\u003eOXA-1\u003c/sub\u003e, bla\u003csub\u003eOXA-232\u003c/sub\u003e, bla\u003csub\u003eOXA-9\u003c/sub\u003e, bla\u003csub\u003eSHV-106\u003c/sub\u003e, bla\u003csub\u003eTEM-1B\u003c/sub\u003e, dfrA1, dfrA5, fosA6, mphA, mphE, msrE, oqxA, oqxB, qnrS1, sul1, sul2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, entB, fepC, fyuA, irp1, irp2, iucA, iucB, iucC, iutA, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20250306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaac6Ib, aph3Ia, aph3VI, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eNDM-5\u003c/sub\u003e, bla\u003csub\u003eOXA-1\u003c/sub\u003e, bla\u003csub\u003eOXA-232\u003c/sub\u003e, bla\u003csub\u003eOXA-9\u003c/sub\u003e, bla\u003csub\u003eSHV-106\u003c/sub\u003e, bla\u003csub\u003eTEM-1B\u003c/sub\u003e, dfrA1, fosA6, oqxA, oqxB, qnrS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20250307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaac3IIa, aac6Ibcr, aph3Ib, aph6Id, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eOXA-1\u003c/sub\u003e, bla\u003csub\u003eSHV-187\u003c/sub\u003e, bla\u003csub\u003eTEM-1B\u003c/sub\u003e, fosA6, oqxA, oqxB, qnrB1, sul2, tetA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20250308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaac3IIa, aac6Ibcr, aph3Ib, aph6Id, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eOXA-1\u003c/sub\u003e, bla\u003csub\u003eSHV-187\u003c/sub\u003e, bla\u003csub\u003eTEM-1B\u003c/sub\u003e, fosA6, oqxA, oqxB, qnrB1, sul2, tetA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20250309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaac3IIa, aac6Ibcr, aadA2, aph3Ia, aph3Ib, aph6Id, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eOXA-1\u003c/sub\u003e, catA1, dfrA12, fosA, mphA, oqxA, oqxB, sul1, sul2, tetA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, entB, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20250310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003efosA, oqxA, oqxB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentB, fepC, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20250311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaac6Ibcr, aph3Ib, aph6Id, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eOXA-1\u003c/sub\u003e, bla\u003csub\u003eSHV-110\u003c/sub\u003e, bla\u003csub\u003eTEM-1B\u003c/sub\u003e, dfrA14, fosA, oqxA, oqxB, qnrB1, sul2, tetA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, entB, fepC, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20252895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaac3IIa, aac6Ibcr, aadA2, aph3Ib, aph6Id, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eNDM-5\u003c/sub\u003e, bla\u003csub\u003eOXA-1\u003c/sub\u003e,\u0026nbsp;bla\u003csub\u003eSHV-106\u003c/sub\u003e,\u0026nbsp;bla\u003csub\u003eTEM-1B\u003c/sub\u003e, dfrA12, dfrA14, ermB,\u0026nbsp;fosA6,\u0026nbsp;mphA,\u0026nbsp;oqxA, oqxB, qnrB1, rmtB, sul1, sul2, tetA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, net, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eG20252896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eaph3Ib, aph6Id, bla\u003csub\u003eCTX-M-15\u003c/sub\u003e, bla\u003csub\u003eDHA-1\u003c/sub\u003e, bla\u003csub\u003eSHV-187\u003c/sub\u003e, bla\u003csub\u003eTEM-1B\u003c/sub\u003e, dfrA17, fosA6,\u0026nbsp;mphA,\u0026nbsp;oqxA, oqxB, qnrB4, qnrS1, sul1, sul2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eentA, net, fepC, fyuA, irp1, irp2, ompA, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX, ykgK/ecpR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eKleborate analysis found that KL-64 and KL-39 were the most prevalent, followed by KL-62, KL-63, KL-16, KL-23, and KL-112 capsular polysaccharide types. The cell wall lipopolysaccharide antigen and its variations detected were O1v2 (44.44%), O1v1 (44.44%), and O3 (11.11%). \u003cstrong\u003e(Table 2)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eTable 2: Kleborate analysis revealing serotypes of K. pneumoniae isolates\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.8e+2pt\" cellspacing=\"3\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003eSerotype\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eK (capsular polysaccharide antigen)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO/v (cell wall lipopolysaccharide antigen and its variant)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20252895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20252896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKL112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eO1v2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eMLST analysis revelaed ST-2096 and ST-985 were regularly observed, while other STs detected included ST-147, ST-198, ST-15, and ST-17. The most prevalent plasmid replicons were IncFIB (K), IncFIB (pNDM-Mar), IncFII, and ColRNAIThe most prevalent plasmid replicons were IncFIB (K), IncFIB (pNDM-Mar), IncFII, and ColRNAI. \u003cstrong\u003e(Table 3)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Sequence typing results and the plasmid replicons identified in the isolates\u003c/strong\u003e\u003c/p\u003e\n\u003ctable cellspacing=\"3\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMLST type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHousekeeping genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmid replicons\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-2096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eIncFIB (K), IncFIB(pNDM-Mar),\u003c/em\u003e \u003cem\u003eIncHI1B(pNDM-MAR)\u003c/em\u003e, \u003cem\u003eColRNAI, Col440I, ColKP3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-2096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eIncFIB (K), IncFIB(pNDM-Mar),\u003c/em\u003e \u003cem\u003eIncHI1B(pNDM-MAR)\u003c/em\u003e, \u003cem\u003eColRNAI, Col440I, ColKP3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eIncFIB (K), IncFII,\u003c/em\u003e \u003cem\u003eColRNAI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eIncFIB (K), IncFII,\u003c/em\u003e \u003cem\u003eColRNAI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eIncFIB (K), IncFIB(pNDM-Mar),\u0026nbsp;\u003c/em\u003e\u003cem\u003eIncHI1B(pNDM-MAR)\u003c/em\u003e, \u003cem\u003eIncFII,\u003c/em\u003e \u003cem\u003eColRNAI, Col440I, ColpVC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eNone\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20250311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eNone\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20252895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eIncX4, IncFIB (K), IncFII, Col(MG828)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eG20252896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eST-17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003egapA\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ephoE\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003etonB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eIncFII (pKP91)\u003c/em\u003e, \u003cem\u003eIncFIB (K)\u003c/em\u003e, \u003cem\u003eIncFII\u003c/em\u003e, \u003cem\u003eCol440II\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe classical \u003cem\u003eK. pneumoniae\u003c/em\u003e, which has a strong propensity to acquire antibiotic resistance, and the recently discovered hvKP are the two primary \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenic variants of public health significance. Hypervirulence (hvkp), which is connected to invasive illness, abscess formation, and metastatic infection, has been linked to a number of virulence factors for \u003cem\u003eK. pneumoniae\u003c/em\u003e. Moreover, infections with strains that produce MDR, carbapenemase and ESBL have been linked to higher fatality rates in critically ill patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The WGS of \u003cem\u003eK. pneumoniae\u003c/em\u003e strains aids researchers in identifying epidemic clones of drug-resistant infections. Furthermore, the MDR can be converted into a hypervirulent phenotype by acquiring virulence-carrying plasmids. Understanding the diversity of genomes and resistance phenotypes is critical for determining transmission patterns [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAntimicrobial resistance genes\u003c/h3\u003e\n\u003cp\u003eThe most common AMR gene found in 88.88% (8/9) of the strains in the current investigation was blaCTX-M-15. In K. pneumoniae, blaCTX-M-15 is a common, widely distributed ESBL gene that confers high-level resistance to third-generation cephalosporins (such as ceftazidime and ceftriaxone). It is mainly carried on mobile genetic elements, such as IncFIIk plasmids, and is linked to the insertion sequence (ISEcp1), which allows for frequent outbreaks and quick dissemination, particularly in hospital critical care units (ICUs) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The production of ESBL contributing blaOXA-1 results in resistance to ampicillin, ticarcillin, piperacillin and cephalosporins. Furthermore, it was observed that most blaOXA-1 carrying strains simultaneously acquire blaCTX-M-15, thereby contributing to widespread resistance to broad spectrum antibiotics including cephalosporins [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This study revealed seven out of nine (88.88%) strains carried both genes. Three strains (33.33%) with the blaNDM-5 gene were found in the current investigation. Penicillin, cephalosporins, and carbapenems are among the β-lactam antibiotics that NDM can hydrolyze. There are variations of the blaNDM gene. The most common ones in the world are blaNDM-1, blaNDM-5, and blaNDM-7. According to earlier studies, blaNDM-5 has greater carbapenemase activity than NDM-1, which leads to higher carbapenem minimum inhibitory concentrations. Furthermore, it was established that NDM genes have been shown to be highly transmissible and often associated with travel [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the current investigation, the aac gene and its variants, including the aac6Ib, were found in seven (77.77%) strains. Through enzymatic modification of the amino or hydroxyl groups of the aminoglycoside group of antibiotics, such as gentamicin and amikacin, these genes may confer resistance to aminoglycosides [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The aph gene and its variations, including aph3Ia, aph3Ib, aph6Id, and aph3VI, were detected in eight (88.88%) strains in this investigation. Aminoglycoside aminotransferase, which is encoded by the aph gene, alters and deactivates aminoglycoside antibiotics, frequently resulting in resistance to gentamicin, tobramycin, and amikacin [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In MDR clinical isolates, these gene variations are often detected on plasmids or transposons. The armA gene, which codes for a 16S rRNA methyltransferase that alters the ribosomal target site and makes aminoglycoside antibiotics ineffective, was found in only one strain (11.11%) in our investigation. It facilitates quick horizontal transmission among Klebsiella species and is frequently carried on plasmids or transposons. Additionally, it was discovered that the expression of the armA gene restricts the available treatments for serious infections, including sepsis, bloodstream infections, and pneumonia, requiring robust molecular surveillance [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The aadA2 gene, which codes for an aminoglycoside nucleotidyl transferase\u0026mdash;a crucial enzyme conferring resistance to aminoglycoside antibiotics, specifically streptomycin and spectinomycin\u0026mdash;was found in one strain (11.11%). It usually co-occurs with ESBL genes (such as blaCTX-M and blaSHV) and other resistance markers. It is often found on class 1 integrons and plasmid-transmitted, contributing to MDR in clinical isolates [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The blaSHV gene and its variations were detected in six (66.66%) of the isolates in this investigation. Intrinsic ampicillin resistance in \u003cem\u003eK. pneumoniae\u003c/em\u003e is caused by the blaSHV gene, a common beta-lactamase that is frequently expressed on chromosomes. It has a wide range of variations, from ESBL, which hydrolyzes third-generation cephalosporins, to narrow-spectrum. Plasmids frequently contain these genes, which facilitate their propagation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, blaTEM-IB was found in seven (77.77%) isolates. This gene is a common beta-lactamase found in \u003cem\u003eK. pneumoniae\u003c/em\u003e. It belongs to the TEM-1 family of penicillinases, which are enzymes encoded by plasmids that provide resistance to primary-generation cephalosporins and penicillins [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. One strain (11.11%) was found to have the class C beta-lactamase blaDHA-1, which hydrolyzes cephalosporins. BlaDHA-1 can be acquired by \u003cem\u003eK. pneumniae\u003c/em\u003e on big plasmids. Plasmid-borne blaDHA-1 isolates have been reported from various geographical places, and it has been discovered that blaDHA-1 can be induced when linked to the ampR gene. This gene may not have a major impact on β-lactam susceptibility, but when paired with other mechanisms such as porin abnormalities, it may become more prominent in some isolates, underestimating their prevalence [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eVirulence genes\u003c/h3\u003e\n\u003cp\u003eAll the isolates tested in this study revealed the presence of Outer membrane protein A (OmpA), an essential virulence and pathogenic factor of \u003cem\u003eK. pneumoniae\u003c/em\u003e. A recent study demonstrated that OmpA promotes \u003cem\u003eK. pneumoniae\u003c/em\u003e penetration of the blood-brain barrier and mediates meningitis via multiple pathways [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this investigation, fosA and its variation fosA6 were present in eight (88.88%) strains. One important mechanism of resistance to the antibiotic fosfomycin in \u003cem\u003eK. pneumoniae\u003c/em\u003e is the fosA gene, which is typically found as a chromosomally encoded glutathione transferase that clears the drug. High-level resistance in CRKP isolates is largely caused via plasmid-mediated transmission and fosA variations (such as fosA3 and fosA6), even though these are frequently innate [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this investigation, the mphA was detected in four (44.44%) K. pneumoniae strains. The enzyme that confers resistance to 14-membered macrolide antibiotics, such as roxithromycin and erythromycin, is encoded by the mphA gene. Along with other resistance genes (such as sul1 and qnrB4), it is frequently found on plasmids and transposons, which contribute to MDR and spread in clinical settings [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this investigation, one strain (11.11%) had the ermB gene. By altering the (50S) ribosomal subunit, the erm gene codes for erythromycin ribosome methylase and methyltransferase enzymes that provide resistance to macrolide antibiotics like azithromycin. Important erm genes found in Klebsiella include erm(B), erm(T), and erm(42), which are frequently carried on plasmids and linked to high-level resistance and dissemination [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The msrE gene was found in one (11.11%) strain of K. pneumoniae in this investigation. High-level resistance to macrolides and streptogramin B is provided by the ABC-F subfamily protein that is encoded by the msrE gene and functions as a ribosomal protection protein. It is frequently detected in MDR clinical isolates and is frequently linked to the msr(E)-mph(E) operon on plasmids. It frequently co-occurs with other resistance mechanisms [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this investigation, one (11.11%) K. pneumoniae strain had the rmtB gene. K. pneumoniae's rmtB gene produces a 16S rRNA methyltransferase, which results in high-level resistance to aminoglycosides (gentamicin, amikacin, and tobramycin). It typically co-occurs with armA on mobile plasmids with ESBL (such as blaCTX-M or carbapenemase genes blaKPC-2, blaNDM) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerotypes\u003c/h2\u003e \u003cp\u003eAmong the numerous K antigens identified in this investigation, KL64 and KL39 were most commonly (22.22%) noticed. KL63, which was found in one strain (11.11%), is known to be a common or emerging variety that is quite pathogenic. Additionally, KL62 confers greater levels of AMR, and KL16 and KL64 have been identified as hypervirulent strains [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMultilocus sequence types\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e ST2096, a recently reported highly pathogenic, MDR clone that is frequently associated with bloodstream infections, was found in two (22.22%) strains in this investigation. It is sometimes referred to as a \"hybrid\" strain because it possesses both high-level resistance genes (like blaOXA) and hypervirulence factors (like iucA, iutA, and rmpA2). It is considered a serious clinical risk in regions such as Saudi Arabia and India [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eK. pneumoniae\u003c/em\u003e ST985, which was found in two (22.22%) isolates, is frequently linked to AMR and has been documented in clinical, environmental, and animal contexts. It has been found to possess several AMR genes that could lead to infections in hospital settings and to be a component of the polyclonal distribution of strains in various locales [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The hybrid resistant virulence plasmids IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) are known to be carried by ST147, which was found in one (11.11%) strain in our study. Additionally, ST147 may carry plasmids that combine virulence and resistance factors, raising patient risk. Recently discovered in the United Kingdom, ST147 is widely acknowledged as a high-risk clone [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. \u003cem\u003eK. pneumoniae\u003c/em\u003e ST-48 strains found in this investigation is one of the MDR strains that are widely distributed around the world and are frequently linked to the synthesis of carbapenemases (such as OXA-48 and NDM-1). This ST was classified as an opportunistic pathogen due to its high genetic plasticity, rich virulome, and capacity to cause serious, challenging hospital-acquired infections (HAIs), such as bloodstream infections, pneumonia, and urinary tract infections, especially in patients with compromised immune systems. Additionally, ST-48 has been found in environmental sources, such as chicken meat, indicating a possibility of food chain transmission [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. \u003cem\u003eK. pneumoniae\u003c/em\u003e ST198, a developing MDR ST linked to CTX-M-15 ESBL production and, occasionally, hypervirulence, was discovered in one (11.11%) strain in this investigation. This strain poses a danger to food safety since it has been found in both clinical settings and environmental sources like lettuce [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. One strain (11.11%) of \u003cem\u003eK. pneumoniae\u003c/em\u003e ST15 is a new, high-risk CRKP clone that is generating HAI outbreaks worldwide, especially in intensive care units. It is typified by MDR, which includes the generation of carbapenemases (such as KPC-2, NDM-1, and OXA-232) and ESBLs [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. K. pneumoniae sequence type 17 (ST17) found in one (11.11%) strain in this study is a globally disseminated, MDR clone frequently responsible for opportunistic, HAIs, particularly in neonatal intensive care units. It is highly diverse, ST that can carry ESBL-encoding plasmids and virulence genes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe WGS analysis of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from pyogenic infections revealed a concerning convergence of MDR and virulence traits. The predominance of ESBL genes such as \u003cem\u003eblaCTX-M-15\u003c/em\u003e, alongside carbapenemase genes like \u003cem\u003eblaNDM-5\u003c/em\u003e, underscores the growing challenge of treating infections caused by these strains. The simultaneous presence of siderophore clusters and virulence-associated genes suggests that these isolates are not only resistant to frontline antibiotics but also highly capable of establishing severe infections. The identification of high-risk global clones (e.g., ST-2096, ST-147) and diverse capsular and O-antigen types highlights the genetic adaptability of \u003cem\u003eK. pneumoniae\u003c/em\u003e, enabling it to persist and spread across different patient populations. The plasmid replicons detected further emphasize the role of mobile genetic elements in disseminating resistance and virulence determinants, raising the risk of rapid outbreaks.\u003c/p\u003e \u003cp\u003eFrom a clinical perspective, these findings reinforce the urgent need for enhanced molecular surveillance using WGS to track the evolution and spread of epidemic clones, strict antibiotic stewardship programs to minimize the misuse of broad-spectrum antibiotics and slow resistance development, improved infection control measures in healthcare settings to prevent transmission of MDR strains, and Integration of genomic data into patient management, allowing clinicians to tailor therapies based on resistance and virulence profiles. Ultimately, the study demonstrates that \u003cem\u003eK. pneumoniae\u003c/em\u003e is evolving into a formidable pathogen with both resistance and virulence advantages. Without coordinated global efforts in surveillance, stewardship, and clinical management, these strains could significantly undermine current treatment options and pose a major public health threat.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLimitations of this study\u003c/h2\u003e \u003cp\u003eThe limited number of isolates and their specific origin from exudates may restrict the generalizability of the results. Additionally, the study did not correlate serotype or ST data with clinical outcomes or bacterial virulence.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThe authors declare that no funds,grants or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eC.P Writing original draft ,Software,Table 1,2\u0026amp;3; V.G.B.B Writing Original draft , Formal analysis, Software , Data Collection,Coordination,Table 1,2\u0026amp;3; V.K Writing original draft ,Table 1,2\u0026amp;3,Concept and Design of the paper; A.D Review and Editing,Supervision, F.A Review and Editing,Supervision; H.P.K.S Supervision Editing, Fig 1 ;D.S Formal analysis,Software Table 1,2\u0026amp;3\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eKempegowda Institute of Medical Sciences has made a memorandum of understanding (MOU) with Dr. P. Chitra Rajalakshmi, Trichy SRM Medical College Hospital and Research Centre, to collaborate under the project supported by the Global Health Research Unit (GHRU), United Kingdom\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe sequence data for isolates G20250305, G20250306, G20250307, G20250308, G20250309, G20250310, G20250311, G20252895, and G20252896 have been deposited in GenBank under the BioProject number PRJNA1435588. The corresponding BioSample accession numbers are SAMN56449246, SAMN56449247, SAMN56449248, SAMN56449249, SAMN56449250, SAMN56449251, SAMN56449252, SAMN56449253, and SAMN56449254. The raw sequencing reads are available in the NCBI Sequence Read Archive (SRA).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlobal antibiotic resistance surveillance report 2025. 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Genom\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e (5), mgen001005. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/mgen.0.001005\u003c/span\u003e\u003cspan address=\"10.1099/mgen.0.001005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023). PMID: 37200066; PMCID: PMC10272876.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella pneumoniae, Antimicrobial Resistance (AMR), Whole-Genome Sequencing (WGS), Next-Generation Sequencing (NGS), Multi Drug Resistant(MDR)","lastPublishedDoi":"10.21203/rs.3.rs-9016172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9016172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe global expansion of antimicrobial resistance (AMR) in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e represents a significant threat to clinical treatment success, often termed a \"silent epidemic\". This study utilized next-generation sequencing (NGS) and whole-genome sequencing (WGS) to characterize nine multidrug-resistant (MDR) isolates recovered from patients with pyogenic infections. Results indicated that the \u003cem\u003eblaCTX-M-15\u003c/em\u003e gene was the most predominant resistance marker (88.88%), followed by \u003cem\u003eblaOXA-1\u003c/em\u003e and \u003cem\u003eblaTEM-18\u003c/em\u003e (77.77%), while the carbapenemase \u003cem\u003eblaNDM-5\u003c/em\u003e was present in 33.33% of isolates.Genomic analysis via multilocus sequence typing (MLST) identified ST-2096 and ST-985 as common sequence types, with KL-64 and KL-39 being the most prevalent capsular polysaccharide types. The investigation further detailed an extensive array of virulence genes, including the \u003cem\u003eybt\u003c/em\u003e and \u003cem\u003eent\u003c/em\u003e complexes, and identified key plasmid replicons such as IncFIB and IncFII. These findings highlight the emergence of hypervirulent (hvKp) global sequence types that also exhibit MDR. Ultimately, the molecular characterization provided by WGS is essential for understanding the epidemiology of bacterial strains, enabling the development of targeted antibiotic prescribing procedures and enhanced patient management strategies\u003c/p\u003e","manuscriptTitle":"Next-Generation Sequencing of Klebsiella Pneumoniae Isolated from Exudates: A Comprehensive Analysis of Antimicrobial and Virulence Genes, Serotypes, and Sequence Types","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 17:16:02","doi":"10.21203/rs.3.rs-9016172/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96048ad1-4002-446b-9a5e-289cc4d4349b","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64945456,"name":"Health sciences/Diseases"},{"id":64945457,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-14T04:39:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 17:16:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9016172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9016172","identity":"rs-9016172","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}