Genomic, virulent and phenotypic characterization of a cerebrospinal fluid- derived ST86-KL2 hypervirulent Klebsiella pneumoniae isolate from a patient with meningitis and diabetes mellitus

Genomic, virulent and phenotypic characterization of a cerebrospinal fluid- derived ST86-KL2 hypervirulent Klebsiella pneumoniae isolate from a patient with meningitis and diabetes mellitus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genomic, virulent and phenotypic characterization of a cerebrospinal fluid- derived ST86-KL2 hypervirulent Klebsiella pneumoniae isolate from a patient with meningitis and diabetes mellitus Xiyun Lai, Xinyu Zhu, Jie Yan, Tingjian Zou, Mengyuan Chen, Heng Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9398214/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Hypervirulent Klebsiella pneumoniae (hvKP) is an important cause of invasive community-acquired infection, particularly in individuals with diabetes mellitus. However, cerebrospinal fluid (CSF)-derived hvKP isolates, especially those belonging to the ST86-KL2 lineage, remain poorly characterized at the integrated clinical, genomic, and phenotypic levels. Methods A K. pneumoniae isolate, designated BP9811, was recovered from the CSF of a patient with meningitis and diabetes mellitus and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA sequencing. Antimicrobial susceptibility testing and whole-genome sequencing were performed to define its resistance, virulence, sequence type, capsular type, and plasmid content. Virulence was evaluated using the Galleria mellonella infection model. In addition, interaction with human brain microvascular endothelial cells was preliminarily assessed using adhesion, gentamicin protection, and transmission electron microscopy assays, together with measurement of relative ompA transcription by reverse transcription-quantitative polymerase chain reaction. Comparative phylogenetic analyses were performed using publicly available CSF-derived and KL2 K. pneumoniae genomes. Results BP9811 was identified as a hypermucoviscous ST86-KL2 hvKP isolate that remained susceptible to all tested antimicrobial agents. Whole-genome sequencing revealed an IncHI1B virulence plasmid carrying canonical hvKP-associated determinants, including rmpA/rmpA2 , peg-344 , iucABCD , and iroBCDE . In the Galleria mellonella model, BP9811 showed high virulence comparable to that of the hypervirulent reference strain NTUH-2044. In HCMEC/D3 cells, BP9811 exhibited increased adhesion and intracellular recovery under the tested conditions, and transmission electron microscopy confirmed bacterial internalization. BP9811 also showed higher ompA transcript levels than the control strain. Phylogenetic analysis indicated that BP9811 was genetically distinct from currently available CSF-derived isolates and occupied a related branch within the KL2 population. Conclusions This study provides an integrated clinical, genomic, and phenotypic characterization of BP9811, a CSF-derived ST86-KL2 hvKP isolate recovered from a patient with meningitis and diabetes mellitus. BP9811 carried a canonical hvKP virulence plasmid, displayed marked virulence-associated phenotypes, and showed enhanced interaction with human brain microvascular endothelial cells in vitro under the tested conditions. These findings expand the limited isolate-level evidence on central nervous system-associated hvKP and provide a basis for future comparative and mechanistic studies. Hypervirulent Klebsiella pneumoniae Meningitis Cerebrospinal fluid ST86 KL2 Whole-genome sequencing OmpA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Klebsiella pneumoniae is an important opportunistic pathogen capable of causing a wide spectrum of healthcare-associated and community-acquired infections, including pneumonia, urinary tract infection, bloodstream infection, and central nervous system (CNS) infection [1, 2] . Among CNS infections, K. pneumoniae meningitis is clinically significant because of its rapid progression, high mortality, and potential for severe neurological sequelae [3] . Clinically and microbiologically, K. pneumoniae meningitis can be broadly divided into trauma/postoperative-associated meningitis and spontaneous meningitis [4] . Trauma/postoperative-associated infection usually follows craniotomy, head trauma, or cerebrospinal fluid drainage and arises in the context of iatrogenic exposure or disruption of anatomical barriers [4] . Correspondingly, these infections are caused by strains spanning a broader range of sequence types (STs) and capsular locus (KL) types and more frequently include extended-spectrum β-lactamase-producing isolates [4, 5, 6, 7] . In contrast, spontaneous meningitis typically occurs in patients without neurosurgical intervention or traumatic brain injury and is often associated with diabetes mellitus or other forms of impaired immunity [8] . Compared with trauma/postoperative-associated disease, spontaneous meningitis is more commonly linked to hypervirulent strains enriched in the K1/K2 capsular background and carrying canonical hypervirulence-associated determinants, including rmpA , rmpA2 , and iuc /aerobactin, often together with a hypermucoviscous phenotype [4, 9] . In addition to canonical hypervirulent K. pneumoniae (hvKP) virulence determinants, recent evidence suggests that outer membrane protein A (OmpA) may contribute to interactions between K. pneumoniae and the blood-brain barrier [10] . This raises the possibility that, beyond classical hypervirulence markers, specific bacterial factors involved in host cell interaction may also be relevant to meningitis pathogenesis. However, such features have been insufficiently explored in CSF-derived hvKP isolates. Although K. pneumoniae has been well recognized in liver abscess, bacteremia, and metastatic infections, its role in CNS infection remains less well defined at the isolate level. Whole-genome studies on meningitis-associated K. pneumoniae , particularly CSF-derived hypervirulent isolates of the ST86-KL2 lineage, remain limited [3, 11, 12] . Most currently available data are derived from case reports or small retrospective studies. Therefore, we aimed to describe the clinical presentation and perform an integrated clinical, genomic, and phenotypic characterization of a CSF-derived ST86-KL2 isolate recovered from a patient with community-acquired spontaneous meningitis and diabetes mellitus. In addition, given the emerging evidence implicating OmpA in blood-brain barrier interaction, we performed preliminary analyses of bacterial interaction with human brain microvascular endothelial cells and relative ompA transcription. These analyses were intended to provide isolate-level evidence on central nervous system-associated hvKP and a basis for future mechanistic investigation. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang Chinese Medical University (ethical approval no. Y2026-038-01). Methods Bacterial isolation and antimicrobial susceptibility testing (AST) K. pneumoniae BP9811 was isolated from CSF at the Second Affiliated Hospital of Zhejiang Chinese Medical University (Hangzhou, China). Species identification was performed via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Autof ms1500; Auto-bio) and confirmed via 16S rRNA sequencing. AST was performed in the clinical laboratory. Susceptibilities to 12 antibiotics were determined by the broth microdilution method. Fosfomycin susceptibility was determined by Kirby-Bauer disk diffusion using 200 µg fosfomycin disks supplemented with 50 µg glucose-6-phosphate. MIC results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, except for tigecycline and colistin [13] . Tigecycline MIC breakpoints (susceptible, ≤ 2 mg/L; resistant, ≥ 8 mg/L) were interpreted according to the Food and Drug Administration (FDA) criteria. The susceptibility of the isolate to colistin was extrapolated from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) colistin breakpoints (susceptible, ≤ 2 mg/L; resistant, > 2 mg/L) [14] . Fosfomycin zone diameter breakpoints (susceptible, ≥ 18 mm; resistant, ≤ 14 mm) were interpreted according to FDA criteria. The strains used for quality control were Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27853, and K. pneumoniae ATCC700603 (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). String test String tests were conducted as previously described [15] . Briefly, the isolates were cultured on Columbia Blood Agar plates (BIO-KONT, Wenzhou, China) and incubated overnight at 37°C. The colonies were gently touched and lifted using an inoculation loop. A positive result was defined as the formation of a viscous string typically exceeding 5 mm in length. Whole-genome sequencing (WGS) and data analysis Genomic DNA was extracted and purified using the OMEGA Bacterial DNA Kit (OMEGA Bio-tek, Norcross, GA, USA), followed by DNA sequencing on Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) and PacBio Sequel IIe. Whole-genome assembly was performed using Unicycler v0.4.8 ( https://github.com/rrwick/Unicycler ). The Canu program was used for self-correction. GATK ( https://www.broadinstitute.org/gatk/ ) was used for single-base corrections. The assembled genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The multilocus sequence typing (MLST) profiles were determined using the MLST database ( https://bigsdb.pasteur.fr/klebsiella/ ). The capsular serotype (KL type) was determined in silico using Kaptive v2.0 ( https://github.com/klebgenomics/Kaptive ). Antibiotic resistance and virulence genes in the assembled genome sequences were identified using ResFinder 4.1 ( https://cge.cbs.dtu.dk/services/ResFinder/ ) and the Virulence Factor Database ( http://www.mgc.ac.cn/VFs/ ). PlasmidFinder2.1 ( https://cge.cbs.dtu.dk/services/PlasmidFinder/ ) was used to predict the plasmid types. Snippy was applied to run core single nucleotide polymorphism (SNP) calling ( https://github.com/tseemann/snippy ), and maximum-likelihood phylogenetic trees were inferred using FastTree with K. pneumoniae NUHL30457 (GenBank no. CP026586.1) as the reference. ITOL ( https://itol.embl.de/ ) was used to visualize the phylogenetic trees. Plasmid sequences were compared using BRIG v0.95. The genome sequence of K. pneumoniae BP9811 has been submitted to NCBI GenBank under BioSample accession number SAMN56319679. Transmission electron microscopy (TEM) HCMEC/D3 cells (Jinyuan Biotechnology, Shanghai, China) were seeded on Thermonox coverslips in 24-well plates (5 × 10 5 cells/well). The cells were infected at a multiplicity of infection of 10 (5 × 10 6 bacteria/well) for 8 h at 37°C. Following infection, the samples were gently washed with phosphate-buffered saline (PBS) to remove non-adherent bacteria and subjected to TEM by a commercial service provider (Sevier, Wuhan, China). The samples were fixed with 2.5% glutaraldehyde at 4°C, post-fixed with 1% osmium tetroxide for 1 h, dehydrated through graded ethanol, infiltrated with Spurr resin, and polymerized at 70°C for 12 h. Finally, ultrathin sections were prepared using an ultramicrotome and examined using a transmission electron microscope at an accelerating voltage of 120 kV. Adhesion and invasion assays HCMEC/D3 cells (Jinyuan Biotechnology, Shanghai, China) were cultured in the Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in 5% CO₂ until reaching confluence. To induce bacterial infection, the cells were cultured in 24-well tissue culture plates containing DMEM without FBS or penicillin/streptomycin at 37°C in 5% CO₂ for 12 h. Log-phase bacteria were prepared from overnight cultures, harvested via centrifugation (10,000 × g , 5 min), washed thrice with PBS, and resuspended in antibiotic-free FBS-free DMEM to 1 × 10 7 colony-forming units (CFU)/mL. The cells were infected at a multiplicity of infection of 10 at 37°C in 5% CO₂ for 2 h. Uninfected wells received an equal volume of medium and served as controls. Adhesion and invasion assays were performed as previously described [16,17] , with minor modifications. For the adhesion assay, wells were washed thrice with PBS and lysed with 200 µL of PBS containing 1% Triton X-100. After 10 min, the lysates were serially diluted and plated on Luria–Bertani agar for CFU enumeration after incubation at 37°C for 18–24 h. For the invasion assay, extracellular bacteria were killed using gentamicin (100 µg/mL) for 1 h, followed by washing and CFU enumeration as described above. Negative control wells containing only bacteria were used to confirm that all extracellular bacteria were killed. All assays were performed in three independent experiments, each with three technical replicate wells. RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Bacteria were grown to the logarithmic phase, and total RNA was extracted using the SteadyPure Quick RNA Extraction Kit (Accurate Biotechnology, Hunan, China). The total RNA from each isolate was reverse-transcribed into cDNA using the Evo M-MLV RT Mix Kit (with gDNase; Accurate Biotechnology). Finally, gene expression data were obtained via RT-qPCR using the CFX96 Real-Time System (Bio-Rad, USA) and SYBR Green Premix Pro Taq HS qPCR Kit (Rox Plus; Accurate Biotechnology). The 16S rRNA served as an internal reference gene. Each experiment was repeated thrice, and the relative expression of each gene in different strains was calculated using the 2 −ΔΔCt method. Comparative sequence analysis of ompA and selected candidate regulators Given the elevated ompA transcript level detected in BP9811, we compared the nucleotide sequences of ompA and selected candidate regulatory genes ( rpoE , hfq , ompR , and envZ ) between BP9811 and the phylogenetically closest comparator strain, CG43. For each locus, the complete coding sequence and the 500-bp upstream region relative to the annotated start codon were retrieved from the assembled genomes and aligned using MAFFT v7.526 ( https://mafft.cbrc.jp/alignment/software/ ). The deduced amino acid sequences were further compared. Galleria mellonella infection model Virulence was evaluated using the G. mellonella infection model, as previously described [18] . NTUH-2044 was used as the hypervirulent control strain, and ATCC 700603 served as the low-virulence control strain. Overnight K. pneumoniae cultures were diluted in sterile PBS to achieve a concentration of 1 × 10 8 CFU/mL. G. mellonella larvae weighing 250–300 mg (Henan Jiyuan Baiyun Industry Co., Ltd., Henan, China) were injected with 10 µL bacterial suspension and incubated for five days at 35°C. Survival was recorded daily from day 1 to day 5. Ten larvae per strain were used for each experiment, and the experiments were performed in triplicate. Kaplan–Meier curves were generated using GraphPad Prism (10.1.2). The results were presented as the mean of three independent experiments. Statistical analyses Differences among groups were statistically analyzed via one-way analysis of variance or Student’s t -test in GraphPad Prism 10.1.2, as appropriate. Survival rates of G. mellonella larvae were analyzed using the log-rank test. Statistical significance was set at P < 0.05. Results Clinical characteristics of the CSF isolate BP9811 K. pneumoniae BP9811 was isolated from the CSF of a 50-year-old male patient with diabetes mellitus who presented with high fever and confusion. The patient had not been hospitalized within the preceding 3 months. Blood culture revealed no bacterial growth. Chest and abdominal computed tomography scans on admission showed no abnormalities. Brain magnetic resonance imaging on admission revealed ventricular enlargement suggestive of hydrocephalus (Fig. 1 ). The isolate was identified as K. pneumoniae via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Autof ms1500; Auto-bio) and confirmed via 16S rRNA sequencing (Fig. 2 A). A hypermucoviscous phenotype was observed using string tests, with a viscous string exceeding 5 mm in length (Fig. 2 B). Laboratory analysis of the CSF showed significant abnormalities, including Pandy’s test (+), elevated mononuclear cell count (56200/µL), decreased glucose level (0.01 mmol/L), and markedly increased total trace protein level (6382.2 mg/L), consistent with bacterial meningitis (Table 1 ). Empirical antimicrobial therapy with meropenem (2 g IV q8h) and levofloxacin (500 mg IV qd) was initiated upon admission. After seven days of combination therapy, the infection-related symptoms subsided, the patient became afebrile, and no neurological sequelae were observed. The patient was discharged after 21 days of hospitalization following systemic treatment. Table 1 Clinical and laboratory indicators of the patient Laboratory Indicators Measurements Normal Interval Cerebrospinal fluid (CSF) tests Appearance Light yellow Colorless, transparent Pandy's test Positive Negative RBC 1600/µL 0/µL Mononuclear cell count 56200/µL 0/µL Microprotein 6382.20 mg/L 80–430 mg/L CSF glucose 0.01 mmol/L 2.22–3.88 mmol/L Chloride 120.20 mmol/L 120–130 mmol/L Cryptococcal antigen Negative Negative Blood tests WBC 4.50×10 9 /L 3.50–9.50×10 9 /L Neutrophil 78.70% 40–75% Lymphocyte 14.30% 20–50% Eosinophil 0.30% 0.40–0.80% Blood glucose 19.46 mmol/L 3.89–6.11 mmol/L HbA1c 10.70% 4.0–6.0% C-reactive protein 13.47 mg/L 0–10 mg/L Procalcitonin 5.05 ng/mL 0-0.05 ng/mL Immunological tests IgG 12.40 g/L 7.51–15.60 g/L IgA 2.90 g/L 0.7-4.0 g/L IgM 0.44 g/L 0.46–3.04 g/L C3 1.31 g/L 0.79–1.52 g/L C4 0.32 g/L 0.16–0.38 g/L Antibiotic susceptibility test results Comprehensive AST results for the 13 agents are presented in Table 2 . Isolate BP9811 was susceptible to all tested antibiotics, namely piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, colistin, ceftazidime-avibactam, tigecycline, amikacin and fosfomycin. This finding indicated susceptibility to multiple drug classes, including aminoglycosides, cephalosporins, carbapenems, and fluoroquinolones, as well as to colistin. Notably, despite carrying fosA , BP9811 remained susceptible to fosfomycin. Table 2 Antimicrobial susceptibility of the Klebsiella pneumoniae isolate by broth microdilution and Kirby-Bauer methods Antibiotics Method Result Breakpoint Unit Interpretation ≤S ≥ R Piperacillin/Tazobactam MIC ≤ 4 8 32 mg/L S Ceftazidime MIC ≤ 0.25 4 16 mg/L S Cefepime MIC ≤ 0.12 2 16 mg/L S Imipenem MIC ≤ 0.25 1 4 mg/L S Meropenem MIC ≤ 0.25 1 4 mg/L S Ertapenem MIC ≤ 0.12 0.5 2 mg/L S Ciprofloxacin MIC ≤ 0.25 0.25 1 mg/L S Levofloxacin MIC ≤ 0.12 0.5 2 mg/L S Colistin MIC ≤ 0.5 0.5 mg/L S Ceftazidime/Avibactam MIC 2 8 16 mg/L S Tigecycline MIC ≤ 0.5 2 8 mg/L S Amikacin MIC ≤ 2 4 16 mg/L S Fosfomycin KB 22 18 14 mm S Antimicrobial resistance and virulence determinants The resistance genes bla SHV−28 , oqxA, oqxB , and fosA were located on the chromosome via WGS. Virulence Factor Database analysis showed that BP9811 carried the core hypervirulence-associated determinant rmpA/rmpA2 , peg-344 , and siderophore genes iucABCD and iroBCDE , supporting its classification as an hvKP isolate. BP9811 also harbored other colonization and fitness determinants, including the enterobactin biosynthesis gene entABCD and genes involved in envelope homeostasis and efflux, such as rcsAB , acrAB , and fepABCDG . Furthermore, BP9811 encoded virulence factors potentially associated with host cell interactions, including types 1 and 3 fimbrial gene clusters ( fimABCDEFGH and mrkABCDFH ; Table 3 ). Table 3 Characteristics of K. pneumoniae BP9811 Strain ST KL Source Virulence factors Resistance genes BP9811 ST86 KL2 CSF entABCD, iroBCDE, iucABCD, fimABCDEFGH, mrkABCDFH, acrAB, fepABCDG, peg-344, rcsAB, rmpA / rmpA2 bla SHV−28 oqxA oqxB fosA Characterization of the BP9811 genome BP9811 was assigned to sequence type 86 by MLST and to capsular locus KL2 by Kaptive analysis. The complete genome of BP9811 comprised a 5,170,607 bp chromosome and a 224,627 bp plasmid (Table 4 ), yielding a total genome size of 5,395,234 bp with an overall G + C content of 57.3%. Genome annotation identified 4,965 protein-coding sequences, 84 tRNA genes, and 25 rRNA genes. Four putative secondary metabolite biosynthetic gene clusters, including siderophore-, lankacidin-, and O-antigen-associated clusters, were also identified. Table 4 Genomic features of the strain BP9811 Features BP9811 Chromosome Plasmid pIM057_vir Total number of bases (bp) 5,170,607 224,627 G + C content (%) 57.62% 50.33% No. of tRNA genes 84 0 No. of protein-coding sequences 4,735 230 Plasmid pIM057_vir was identified as an IncHI1B virulence plasmid. As shown in Fig. 3 , a set of virulence genes (including the previously reported hvKP-associated iucABCD and iroBCD ) was located on the plasmid pIM057_vir, whereas no antibiotic resistance genes were detected. Basic Local Alignment Search Tool analysis further indicated that pIM057_vir shared 99% sequence identity with pE16KP0290-1 (GenBank accession no. CP052258.1), pGDFK0476-unnamed (CP167435.1), and pKP48326-1 (CP167190.1), with a query coverage of 99–100%. Virulence analysis using the G. mellonella infection model The G. mellonella infection assay was conducted to investigate the virulence of BP9811. BP9811 caused a significantly accelerated killing trajectory compared to the low-virulence control strain ATCC 700603 and showed virulence comparable to that of the hypervirulent reference strain NTUH-2044 (Fig. 4 ). These data support the high pathogenicity of BP9811 in vivo. Enhanced interaction of BP9811 with human cerebral microvascular endothelial cells To evaluate the interaction of BP9811 with human cerebral microvascular endothelial cells, we conducted adhesion and gentamicin protection assays using HCMEC/D3 cells. TEM revealed that BP9811 was internalized by HCMEC/D3 cells, with membrane-bound bacteria observed within the host cell cytoplasm (Fig. 5 A). Adhesion experiments revealed that BP9811 showed greater adhesion to HCMEC/D3 cells than NTUH-2044 (Fig. 5 B). Gentamicin protection experiments showed a higher intracellular recovery of BP9811 than of NTUH-2044 under the same assay conditions (Fig. 5 C). Next, we examined ompA transcript levels in BP9811. RT-qPCR revealed that the ompA mRNA levels were higher in BP9811 than in the control strain (Fig. 5 D). Taken together, BP9811 exhibited increased interaction with HCMEC/D3 cells in the in vitro assays, along with high ompA transcript levels. Sequence conservation of ompA and related regulatory genes To determine whether increased ompA transcript levels in BP9811 were accompanied by obvious sequence variation in ompA or selected regulatory determinants, we performed a comparative sequence analysis using the phylogenetically closely related strain CG43 as the reference comparator. Notably, the ompA coding sequence and analyzed upstream region were highly conserved between BP9811 and CG43. Similarly, the coding sequences of rpoE, hfq, ompR , and envZ , including their upstream regions, showed a high degree of conservation, with no amino acid-altering substitutions identified in the analyzed loci. These findings indicate that higher ompA transcript levels in BP9811 were not accompanied by obvious sequence differences in ompA or coding regions of the selected candidate regulators examined in this study. Phylogenetic analyses of CSF isolates To further clarify the phylogenetic relationships between the CSF-derived isolate BP9811 and previously reported CSF isolates, 26 CSF-derived K. pneumoniae genomes were retrieved from the National Center for Biotechnology Information database and analyzed together with BP9811 using a core SNP-based phylogenetic framework (Fig. 6 ). Core-genome SNP analysis revealed that the CSF isolates were genetically diverse, comprising multiple STs and KL types. Notably, BP9811 did not cluster with the other 26 isolates, and the substantial core-SNP distances between them argue against a recent common origin or transmission cluster. Collectively, these findings suggest that BP9811 is a sporadic ST86-KL2 isolate. Phylogenetic analysis of KL2 To further define the phylogenetic position of BP9811 within the KL2 K. pneumoniae population, we constructed a core-genome SNP-based phylogeny (Fig. 7 ). BP9811 showed the smallest core-SNP distance to CG43 (318 SNPs), whereas the distances to Kpn154, HK787, 18CP0060, Kpn2166, and KPN55602 ranged from 344 to 373 SNPs. This suggests that BP9811 occupies a relatively closely related phylogenetic branch among the tested KL2 isolates, although the observed divergence is insufficient to support recent clonal transmission. Notably, although BP9811 was most closely related to CG43 at the core-genome level, their virulence gene profiles differed markedly. This pattern suggests that the hypervirulent phenotype of BP9811 is attributable, at least in part, to the acquisition of virulence determinants mediated by mobile genetic elements rather than vertical diversification of the core genome alone. Discussion In this study, we characterized a CSF-derived ST86-KL2 hvKP isolate, BP9811, from a patient with diabetes and community-acquired meningitis. The presentation was consistent with the established clinical spectrum of invasive hvKP infection, in which diabetes is a frequent host factor and community-onset severe disease is well recognized [19] . However, published studies on CSF-derived hvKP isolates remain limited, particularly for the ST86-KL2 lineage, for which the available evidence is largely restricted to sporadic case reports and small series [3, 11, 12] . This study provided an integrated strain-level clinical, genomic, and phenotypic dataset for a CSF-derived ST86-KL2 hvKP isolate, broadening the currently available evidence on hvKP-associated meningitis. The virulence profile of BP9811 was consistent with that of canonical hvKP. The isolate carried rmpA , rmpA2 , iuc, iro , and peg-344 , along with an IncHI1B virulence plasmid. These features were widely accepted markers of hvKP [20, 21] . BP9811 also showed high lethality in the G. mellonella model and increased adhesion to and invasion of HCMEC/D3 cells. These results suggest preserved functional virulence. Additionally, ompA transcription was increased in this strain. This finding is of interest because a recent study implicated ompA in blood-brain barrier penetration and meningitis pathogenesis [10, 22] . Notably, BP9811 did not closely cluster with currently available CSF-derived genomes, suggesting that CNS invasion was not restricted to a single meningitis-associated clone. Instead, it possibly emerged in different genetic backgrounds when key virulence determinants act in a susceptible host [23] . The conservation of ompA coding sequences and increased transcription indicated that regulatory differences, rather than structural variation alone, contributed to the observed phenotype; however, this remained speculative in the absence of direct functional validation. The resistance profile also merits attention. Although BP9811 carried resistance-associated genes, including bla SHV−28 , oqxA/oqxB , and fosA , it remained susceptible to most clinically relevant agents. This pattern was consistent with the traditional view that canonical hvKP typically combines high virulence with a relatively limited resistance burden [24] . However, the genotype did not fully predict the phenotype. The presence of fosA did not translate to fosfomycin resistance in the tested isolate, highlighting the need to interpret genomic resistance data together with phenotypic susceptibility testing, especially for CSF-derived isolates with direct therapeutic implications [25, 26] . Recent literature had also shown that hypervirulent lineages acquire mobile resistance determinants and evolve toward a convergent, hypervirulent, and multidrug-resistant phenotype [23, 27] . Therefore, BP9811 might represent a susceptible hvKP background, but not the only evolutionary trajectory of ST86-KL2 or other K2 lineages. This study had several limitations that should be considered when interpreting the results. First, this single case couldn’t define the epidemiology or prognosis of ST86-KL2 meningitis. Second, the contribution of OmpA from the strains in this study to CNS invasion required further validation using gene knockout and complementation or blood-brain barrier models. Third, plasmid transferability and stability were not assessed in this study. These factors limited inferences regarding virulence plasmid dissemination and the risk of recombination with multidrug-resistant lineages. Conclusions In summary, we found that a CSF-derived ST86-KL2 hvKP isolate harbored a canonical virulence plasmid and exhibited increased ompA transcription and prominent virulence-associated phenotypes. Overall, our findings provided an integrated clinical, genomic, and phenotypic dataset for a CSF-derived hvKP isolate, supporting systematic surveillance of invasive hvKP lineages in high-risk populations, including patients with diabetes. Abbreviations AST antimicrobial susceptibility testing CSF cerebrospinal fluid DMEM Dulbecco’s modified Eagle’s medium FBS fetal bovine serum hvKP hypervirulent Klebsiella pneumoniae MLST multilocus sequence typing PBS phosphate-buffered saline RT-qPCR reverse transcription-quantitative polymerase chain reaction SNP single nucleotide polymorphism TEM transmission electron microscopy WGS whole-genome sequencing Declarations Ethics approval and consent to participate Informed consent was obtained from the patient in the study. The protocol was reviewed by the Ethics Committee (IRB) of the Second Affiliated Hospital of Zhejiang Chinese Medical University. As this was an observational study and strains cultured from residual samples were used in clinical diagnosis, the confidentiality of patient data is preserved and compliance with the Declaration of Helsinki is ensured. After consulting the IRB of the Second Affiliated Hospital of Zhejiang Chinese Medical University, a formal ethical review approval was obtained (ethical approval no.: Y2026-038-01). Consent for publication Written informed consent for publication of patient clinical details and clinical images was obtained from the patient. Clinical trial number: not applicable. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the Chinese Medicine Scientific Research Foundation of Zhejiang Province, China (Grant No. 2026ZL0059); The funder had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Authors’ contributions QW conceived and designed the experiments. XL, XZ, MC, TZ and XT performed the experiments. XL, JY and HC analyzed the data. XL wrote the manuscript. All authors read and approved the final manuscript. All authors contributed toward data analysis, drafting and revising the manuscript, and agreed to be accountable for all aspects of the work. Acknowledgements The author thanks Editage (www.editage.cn) for English language editing. Availability of data and materials The data used and/or analyzed in this study are available from the corresponding author on reasonable request. The genome sequence of K. pneumoniae BP9811 has been submitted to NCBI GenBank under BioSample accession number SAMN56319679 (https://www.ncbi.nlm.nih. gov/biosample/SAMN56319679/). References Kubicskó AS, Juhász JN, Kamotsay K, Szabo D, Kocsis BL. Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant Klebsiella pneumoniae . Antibiotics. 2025; 14(1):1-14. doi: 10.3390/antibiotics14010062 Zhao C, Liu P, Lin X, Wan C, Liao K, Guo P, et al. The type VI secretion system as a potential predictor of subsequent bloodstream infection of carbapenem-resistant Klebsiella pneumoniae strains on intestinal colonization. Infection. 2025; 53(2):667-678. doi: 10.1007/s15010-024-02456-x Huang N, Jia H, Zhou B, Zhou C, Cao J, Liao W, et al. Hypervirulent carbapenem-resistant Klebsiella pneumoniae causing highly fatal meningitis in southeastern China. Frontiers in Public Health. 2022; 10:1-11. doi: 10.3389/fpubh.2022.991306 Rollin G, Rossi B, Brisse S, Decré D, Leflon-Guibout V, Bert F, et al. Spontaneous and postsurgical/traumatic Klebsiella pneumoniae meningitis: two distinct clinico-microbiological entities. International Journal of Infectious Diseases. 2022; 114:185-191. doi: 10.1016/j.ijid.2021.11.013 Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae . Clinical Microbiology Reviews. 2019; 32(3):e00001-19. doi: 10.1128/CMR.00001-19 Hu H, Wang H, Yu M, Feng H, Zhang S, Zhang Y, et al. Clinical and microbiological characteristics of carbapenem-resistant Enterobacteriaceae causing post-operative central nervous system infections in China. Journal of Global Antimicrobial Resistance. 2023; 35:35-43. doi: 10.1016/j.jgar.2023.08.006 Li Y, Hu D, Ma X, Li D, Tian D, Gong Y, et al. Convergence of carbapenem resistance and hypervirulence leads to high mortality in patients with postoperative Klebsiella pneumoniae meningitis. Journal of Global Antimicrobial Resistance. 2021; 27:95-100. doi: 10.1016/j.jgar.2021.02.035 Huang CR, Lu CH, Chang HW, Lee PY, Lin MW, Chang WN. Community-acquired spontaneous bacterial meningitis in adult diabetic patients: an analysis of clinical characteristics and prognostic factors. Infection. 2002; 30(6):346-350. doi: 10.1007/s15010-002-3010-4 Marr CM, Russo TA. Hypervirulent Klebsiella pneumoniae : a new public health threat. Expert Review of Anti-infective Therapy. 2019; 17(2):71-73. doi: 10.1080/14787210.2019.1555470 Zeng W, Sun Y, Zhang J, Yao Z, Cao J, Hu P, et al. Outer membrane protein A mediates Klebsiella pneumoniae penetration of the blood-brain barrier and induces bacterial meningitis. Microbiological Research. 2025; 299:1-15. doi: 10.1016/ j.micres.2025.128262 Zhang Z, Wen H, Wang H, Zhang P, Li J, Liang Y, et al. A Case of Meningitis in an Infant Due to Hypervirulent Klebsiella pneumoniae Transmission Within a Family. Infection and Drug Resistance. 2022; 15:4927-4933. doi: 10.2147/IDR. S376055 Ku YH, Chuang YC, Chen CC, Lee MF, Yang YC, Tang HJ, et al. Klebsiella pneumoniae Isolates from Meningitis: Epidemiology, Virulence and Antibiotic Resistance. Scientific Reports. 2017; 7(1):1-10. doi: 10.1038/s41598-017-06878-6 CLSI (2025). Performance standards for antimicrobial susceptibility testing. 35th informational supplement (CLSI M100-S35). Wayne, PA: Clinical and Laboratory Standards Institute. European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. version 16.0; 2026. Available at https:// www.eucast.org/fileadmin/eucast/pdf/breakpoints/ v_16.0_Breakpoint_Tables.pdf Hagiya H, Watanabe N, Maki M, Murase T, Otsuka F. Clinical utility of string test as a screening method for hypermucoviscosity‐phenotype Klebsiella pneumoniae . Acute Medicine & Surgery. 2014; 1(4):245-246. doi: 10.1002/ams2.40 Xiao S, Suo W, Zhang J, Zhang X, Yin Y, Guo X, et al. MgaSpn is a negative regulator of capsule and phosphorylcholine biosynthesis and influences the virulence of Streptococcus pneumoniae D39. Virulence. 2021; 12(1):2366-2381. doi: 10.1080/21505594.2021.1972539 Deng L, Spencer BL, Holmes JA, Mu R, Rego S, Weston TA, et al. The Group B Streptococcal surface antigen I/II protein, BspC, interacts with host vimentin to promote adherence to brain endothelium and inflammation during the pathogenesis of meningitis. PLoS Pathogens. 2019; 15(6):1-30. doi: 10.1371/journal.ppat. 1007848 Chen J, Zeng Y, Zhang R, Cai J. In vivo Emergence of Colistin and Tigecycline Resistance in Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae During Antibiotics Treatment. Frontiers in Microbiology. 2021; 12:1-8. doi: 10.3389/fmicb.2021.702956 Namikawa H, Oinuma KI, Yamada K, Kaneko Y, Kakeya H, Shuto T. Predictors of hypervirulent Klebsiella pneumoniae infections: a systematic review and meta-analysis. The Journal of Hospital Infection. 2023; 134:153-160. doi: 10.1016/j.jhin. 2023.02.005 Russo TA, Lebreton F, McGann PT. A Step Forward in Hypervirulent Klebsiella pneumoniae Diagnostics. Emerging Infectious Diseases. 2025; 31(1):1-3. doi: 10.3201/eid3101.241516 Russo TA, Olson R, Fang CT, Stoesser N, Miller M, MacDonald U, et al. Identification of Biomarkers for Differentiation of Hypervirulent Klebsiella pneumoniae from Classical K. pneumoniae . Journal of Clinical Microbiology. 2018; 56(9):1-12. doi: 10.1128/JCM.00776-18 Shin S, Lu G, Cai M, Kim KS. Escherichia coli outer membrane protein A adheres to human brain microvascular endothelial cells. Biochemical and Biophysical Research Communications. 2005; 330(4):1199-1204. doi: 10.1016/j.bbrc.2005. 03.097 Chen T, Ying L, Xiong L, Wang X, Lu P, Wang Y, et al. Understanding carbapenem-resistant hypervirulent Klebsiella pneumoniae : Key virulence factors and evolutionary convergence. hLife. 2024; 2(12):611-624. doi: 10.1016/j.hlife. 2024.06.005 Lee CR, Lee JH, Park KS, Jeon JH, Kim YB, Cha CJ, et al. Antimicrobial Resistance of Hypervirulent Klebsiella pneumoniae : Epidemiology, Hypervirulence-Associated Determinants, and Resistance Mechanisms. Frontiers in Cellular and Infection Microbiology. 2017; 7:1-13. doi: 10.3389/fcimb. 2017.00483 Zhou X, Yang M, Chen F, Wang L, Han P, Jiang Z, et al. Prediction of antimicrobial resistance in Klebsiella pneumoniae using genomic and metagenomic next-generation sequencing data. Journal of Antimicrobial Chemotherapy. 2024; 79(10):2509-2517. doi: 10.1093/jac/dkae248 Bixby ML, Collins LB, Daley EC, Salay JM, Oliver S, Bryson AL, et al. Heteroresistance associated with the production of fosfomycin-resistant inner colonies during disk diffusion testing among a geographically diverse collection of Klebsiella pneumoniae clinical isolates. JAC-Antimicrobial Resistance. 2025; 7(1):1-8. doi: 10.1093/jacamr/dlaf013 Arcari G, Carattoli A. Global spread and evolutionary convergence of multidrug-resistant and hypervirulent Klebsiella pneumoniae high-risk clones. Pathogens and Global Health. 2023; 117(4):328-341. doi: 10.1080/20477724.2022.2121362 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 29 Apr, 2026 Reviews received at journal 27 Apr, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 21 Apr, 2026 Editor invited by journal 21 Apr, 2026 Submission checks completed at journal 20 Apr, 2026 First submitted to journal 20 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9398214","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631482531,"identity":"e52ef514-89a8-4087-8ded-fb25006bd0ef","order_by":0,"name":"Xiyun Lai","email":"","orcid":"","institution":"The Second Clinical Medical College of Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiyun","middleName":"","lastName":"Lai","suffix":""},{"id":631482532,"identity":"628c33e6-c683-4455-b303-3ba22b98d434","order_by":1,"name":"Xinyu Zhu","email":"","orcid":"","institution":"The Second Clinical Medical College of Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Zhu","suffix":""},{"id":631482534,"identity":"0501a239-e8ef-4700-a9fd-f49515fb22a2","order_by":2,"name":"Jie Yan","email":"","orcid":"","institution":"The Second Clinical Medical College of Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yan","suffix":""},{"id":631482540,"identity":"1795a73e-567e-4806-8549-5164eb0b9fbe","order_by":3,"name":"Tingjian Zou","email":"","orcid":"","institution":"The Second Clinical Medical College of Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingjian","middleName":"","lastName":"Zou","suffix":""},{"id":631482541,"identity":"367ae031-b041-4f32-b011-39ab6bc8583d","order_by":4,"name":"Mengyuan Chen","email":"","orcid":"","institution":"Department of Clinical Laboratory, The Second Affiliated Hospital of Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengyuan","middleName":"","lastName":"Chen","suffix":""},{"id":631482542,"identity":"66d8f085-5feb-4bfc-82d3-218c62534b48","order_by":5,"name":"Heng Chen","email":"","orcid":"","institution":"Department of Clinical Laboratory, The Second Affiliated Hospital of Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Heng","middleName":"","lastName":"Chen","suffix":""},{"id":631482543,"identity":"4a25b702-f1fb-4919-a5d1-cca760f380a3","order_by":6,"name":"Xiaofang Tang","email":"","orcid":"","institution":"Department of General Medicine, Zhejiang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaofang","middleName":"","lastName":"Tang","suffix":""},{"id":631482544,"identity":"7ac253fe-8ba5-4f65-b7fe-bbed0046cd36","order_by":7,"name":"Qiang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYNACNgY5Nvb2A6RpMebjOZNAmpbEeRIOBsQpNjieYybxoexwepsEQwLDj4ptRGg588bYcMa5w7lt0o0HGHvO3CasxexGjuFj3jagFpkDCcyMbcRpMTj8t+1wOptEggHRWgwfM7YdTiBei/2ZZ8WGPefSDduAgXyQKL9Itidvk/hRZi0v395+8MGPCiK0MDBkIKLjADHqgSD9AZEKR8EoGAWjYMQCADq4PlzFEfcvAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Clinical Laboratory, The Second Affiliated Hospital of Zhejiang Chinese Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-04-13 03:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9398214/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9398214/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108398737,"identity":"0eef0441-df4b-48c1-ac72-dd8050a16bc6","added_by":"auto","created_at":"2026-05-04 08:35:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2128753,"visible":true,"origin":"","legend":"\u003cp\u003eBrain magnetic resonance imaging (MRI) on admission. (A) Axial T1-weighted image showing marked enlargement of the lateral ventricles. (B) Axial T2-weighted/FLAIR image confirming ventriculomegaly, suggestive of hydrocephalus.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/a6276687079e6dcb0d3dfbb6.png"},{"id":108493691,"identity":"33115d28-b2d0-424f-9e97-6978bf8da40a","added_by":"auto","created_at":"2026-05-05 10:01:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2783139,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobiological identification of a cerebrospinal fluid (CSF) isolate. (A) \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e was identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Autof ms1500; Auto-bio). (B) Bacterial string test showed the formation of a viscous string exceeding 5 mm in length, indicating a positiveresult.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/601069237b63a724dc4ed1cb.png"},{"id":108804381,"identity":"51ccaf6c-2d26-4536-bdc4-fe8ba831e4ad","added_by":"auto","created_at":"2026-05-08 15:20:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":765725,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genomic sequence analysis of plasmid pIM057_vir and three reported virulence plasmids of hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e (hvKP) strains. Circles from inside to outside indicate the GC content of pIM057_vir, GC skew of pIM057_vir, and genomic sequences of the plasmids \u003cem\u003epE16KP0290-1\u003c/em\u003e, \u003cem\u003epGDFK0476-unnamed1\u003c/em\u003e, \u003cem\u003epKP48326-1\u003c/em\u003e, and pIM057_vir. White and colored regions of the rings indicate absence and presence, respectively. Locations of the virulence genes \u003cem\u003eiucABCD\u003c/em\u003e and \u003cem\u003eiroBCD\u003c/em\u003e are indicated.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/93b1fa15877736f36cab8ad9.png"},{"id":108398739,"identity":"4cc5b1a7-e087-4054-945c-3ae8c2bc15e0","added_by":"auto","created_at":"2026-05-04 08:35:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41315,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae over a five-day observation periodafter infection with \u003cem\u003eK. pneumoniae\u003c/em\u003estrains. NTUH-2044 and ATCC 700603 were included as high- and low-virulence reference strains, respectively. Curves represent the mean of three independent experiments.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/a9b7864d545bd054e33373ce.png"},{"id":108803941,"identity":"064f1d93-3a37-488c-81dd-464bff880744","added_by":"auto","created_at":"2026-05-08 15:12:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10422635,"visible":true,"origin":"","legend":"\u003cp\u003eBP9811 increased interaction with HCMEC/D3 cells, and elevated \u003cem\u003eompA\u003c/em\u003etranscription. (A) Representative transmission electron microscopy images demonstrate bacterial internalization by HCMEC/D3 cells (arrowheads indicate internalized bacteria in HCMEC/D3 cells). Scale bar, 2 µm. (B) Adhesion of BP9811 to HCMEC/D3 cells at a multiplicity of infection (MOI) of 10 measured 2 h after infection by enumerating cell-associated colony-forming units (CFU) after washing and host cell lysis. (C) Intracellular recovery of BP9811 assessed via gentamicin protection assay (100 µg/mL for 1 h), followed by host cell lysis and CFU counting. (D) Relative \u003cem\u003eompA\u003c/em\u003e transcript levels, normalized to 16S rRNA levels, in ATCC 700603 and BP9811 determined via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Data are represented as the mean ± standard deviation (SD) of three independent experiments. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, determined via Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/0a8330f6ae09954af8fd2370.png"},{"id":108398741,"identity":"5d45b8d7-4f33-4b1a-8c47-313ae214d18b","added_by":"auto","created_at":"2026-05-04 08:35:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8070307,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships, resistance genes, and virulence genes of BP9811 analyzed in this study and 26 CSF isolates retrieved from the National Center for Biotechnology Information (NCBI) database. The darker blue box indicates the presenceof genes. \u003cem\u003eK. pneumoniae\u003c/em\u003e NUHL30457 was used as the reference for core-single nucleotide polymorphism (SNP) calling.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/c7918a6038050e7389a59b5d.png"},{"id":108493419,"identity":"a253b1a2-15a4-455f-8a53-1d5457aed82d","added_by":"auto","created_at":"2026-05-05 10:00:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3290260,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolates from KL2 strains. The outer ring of the phylogenetic tree was categorized based on genetic distance (branch length). The inner ring shows the strain names, with red indicating the strains in this study, and the outer ring shows the source, sequence type, and country. The darker purple box indicates the presence of genes. \u003cem\u003eK. pneumoniae\u003c/em\u003e NUHL30457 was used as the reference for core-SNP calling.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/214b4bd56564ebf9bba25a41.png"},{"id":108816618,"identity":"9002a484-c892-4013-8d40-aa8e7659d93e","added_by":"auto","created_at":"2026-05-08 16:25:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2862416,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9398214/v1/5820bcf9-4a92-4787-9d7c-c6cfc36da865.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic, virulent and phenotypic characterization of a cerebrospinal fluid- derived ST86-KL2 hypervirulent Klebsiella pneumoniae isolate from a patient with meningitis and diabetes mellitus","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is an important opportunistic pathogen capable of causing a wide spectrum of healthcare-associated and community-acquired infections, including pneumonia, urinary tract infection, bloodstream infection, and central nervous system (CNS) infection \u003csup\u003e[1, 2]\u003c/sup\u003e. Among CNS infections, \u003cem\u003eK. pneumoniae\u003c/em\u003e meningitis is clinically significant because of its rapid progression, high mortality, and potential for severe neurological sequelae \u003csup\u003e[3]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eClinically and microbiologically, \u003cem\u003eK. pneumoniae\u003c/em\u003e meningitis can be broadly divided into trauma/postoperative-associated meningitis and spontaneous meningitis \u003csup\u003e[4]\u003c/sup\u003e. Trauma/postoperative-associated infection usually follows craniotomy, head trauma, or cerebrospinal fluid drainage and arises in the context of iatrogenic exposure or disruption of anatomical barriers \u003csup\u003e[4]\u003c/sup\u003e. Correspondingly, these infections are caused by strains spanning a broader range of sequence types (STs) and capsular locus (KL) types and more frequently include extended-spectrum β-lactamase-producing isolates \u003csup\u003e[4, 5, 6, 7]\u003c/sup\u003e. In contrast, spontaneous meningitis typically occurs in patients without neurosurgical intervention or traumatic brain injury and is often associated with diabetes mellitus or other forms of impaired immunity \u003csup\u003e[8]\u003c/sup\u003e. Compared with trauma/postoperative-associated disease, spontaneous meningitis is more commonly linked to hypervirulent strains enriched in the K1/K2 capsular background and carrying canonical hypervirulence-associated determinants, including \u003cem\u003ermpA\u003c/em\u003e, \u003cem\u003ermpA2\u003c/em\u003e, and \u003cem\u003eiuc\u003c/em\u003e/aerobactin, often together with a hypermucoviscous phenotype \u003csup\u003e[4, 9]\u003c/sup\u003e. In addition to canonical hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e (hvKP) virulence determinants, recent evidence suggests that outer membrane protein A (OmpA) may contribute to interactions between \u003cem\u003eK. pneumoniae\u003c/em\u003e and the blood-brain barrier \u003csup\u003e[10]\u003c/sup\u003e. This raises the possibility that, beyond classical hypervirulence markers, specific bacterial factors involved in host cell interaction may also be relevant to meningitis pathogenesis. However, such features have been insufficiently explored in CSF-derived hvKP isolates.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eK. pneumoniae\u003c/em\u003e has been well recognized in liver abscess, bacteremia, and metastatic infections, its role in CNS infection remains less well defined at the isolate level. Whole-genome studies on meningitis-associated \u003cem\u003eK. pneumoniae\u003c/em\u003e, particularly CSF-derived hypervirulent isolates of the ST86-KL2 lineage, remain limited \u003csup\u003e[3, 11, 12]\u003c/sup\u003e. Most currently available data are derived from case reports or small retrospective studies. Therefore, we aimed to describe the clinical presentation and perform an integrated clinical, genomic, and phenotypic characterization of a CSF-derived ST86-KL2 isolate recovered from a patient with community-acquired spontaneous meningitis and diabetes mellitus. In addition, given the emerging evidence implicating OmpA in blood-brain barrier interaction, we performed preliminary analyses of bacterial interaction with human brain microvascular endothelial cells and relative \u003cem\u003eompA\u003c/em\u003e transcription. These analyses were intended to provide isolate-level evidence on central nervous system-associated hvKP and a basis for future mechanistic investigation. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang Chinese Medical University (ethical approval no. Y2026-038-01).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolation and antimicrobial susceptibility testing (AST)\u003c/h2\u003e \u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e BP9811 was isolated from CSF at the Second Affiliated Hospital of Zhejiang Chinese Medical University (Hangzhou, China). Species identification was performed via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Autof ms1500; Auto-bio) and confirmed via 16S rRNA sequencing. AST was performed in the clinical laboratory. Susceptibilities to 12 antibiotics were determined by the broth microdilution method. Fosfomycin susceptibility was determined by Kirby-Bauer disk diffusion using 200 \u0026micro;g fosfomycin disks supplemented with 50 \u0026micro;g glucose-6-phosphate. MIC results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, except for tigecycline and colistin \u003csup\u003e[13]\u003c/sup\u003e. Tigecycline MIC breakpoints (susceptible, \u0026le;\u0026thinsp;2 mg/L; resistant, \u0026ge;\u0026thinsp;8 mg/L) were interpreted according to the Food and Drug Administration (FDA) criteria. The susceptibility of the isolate to colistin was extrapolated from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) colistin breakpoints (susceptible, \u0026le;\u0026thinsp;2 mg/L; resistant, \u0026gt;\u0026thinsp;2 mg/L) \u003csup\u003e[14]\u003c/sup\u003e. Fosfomycin zone diameter breakpoints (susceptible, \u0026ge;\u0026thinsp;18 mm; resistant, \u0026le;\u0026thinsp;14 mm) were interpreted according to FDA criteria. The strains used for quality control were \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC25922, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853, and \u003cem\u003eK. pneumoniae\u003c/em\u003e ATCC700603 (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eString test\u003c/h3\u003e\n\u003cp\u003eString tests were conducted as previously described \u003csup\u003e[15]\u003c/sup\u003e. Briefly, the isolates were cultured on Columbia Blood Agar plates (BIO-KONT, Wenzhou, China) and incubated overnight at 37\u0026deg;C. The colonies were gently touched and lifted using an inoculation loop. A positive result was defined as the formation of a viscous string typically exceeding 5 mm in length.\u003c/p\u003e\n\u003ch3\u003eWhole-genome sequencing (WGS) and data analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted and purified using the OMEGA Bacterial DNA Kit (OMEGA Bio-tek, Norcross, GA, USA), followed by DNA sequencing on Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) and PacBio Sequel IIe. Whole-genome assembly was performed using Unicycler v0.4.8 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rrwick/Unicycler\u003c/span\u003e\u003cspan address=\"https://github.com/rrwick/Unicycler\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The Canu program was used for self-correction. GATK (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.broadinstitute.org/gatk/\u003c/span\u003e\u003cspan address=\"https://www.broadinstitute.org/gatk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for single-base corrections. The assembled genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The multilocus sequence typing (MLST) profiles were determined using the MLST database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bigsdb.pasteur.fr/klebsiella/\u003c/span\u003e\u003cspan address=\"https://bigsdb.pasteur.fr/klebsiella/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The capsular serotype (KL type) was determined in silico using Kaptive v2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/klebgenomics/Kaptive\u003c/span\u003e\u003cspan address=\"https://github.com/klebgenomics/Kaptive\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Antibiotic resistance and virulence genes in the assembled genome sequences were identified using ResFinder 4.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.cbs.dtu.dk/services/ResFinder/\u003c/span\u003e\u003cspan address=\"https://cge.cbs.dtu.dk/services/ResFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the Virulence Factor Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.mgc.ac.cn/VFs/\u003c/span\u003e\u003cspan address=\"http://www.mgc.ac.cn/VFs/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlasmidFinder2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.cbs.dtu.dk/services/PlasmidFinder/\u003c/span\u003e\u003cspan address=\"https://cge.cbs.dtu.dk/services/PlasmidFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the plasmid types. Snippy was applied to run core single nucleotide polymorphism (SNP) calling (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/snippy\u003c/span\u003e\u003cspan address=\"https://github.com/tseemann/snippy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and maximum-likelihood phylogenetic trees were inferred using FastTree with \u003cem\u003eK. pneumoniae\u003c/em\u003e NUHL30457 (GenBank no. CP026586.1) as the reference. ITOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to visualize the phylogenetic trees. Plasmid sequences were compared using BRIG v0.95. The genome sequence of \u003cem\u003eK. pneumoniae\u003c/em\u003e BP9811 has been submitted to NCBI GenBank under BioSample accession number SAMN56319679.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eHCMEC/D3 cells (Jinyuan Biotechnology, Shanghai, China) were seeded on Thermonox coverslips in 24-well plates (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well). The cells were infected at a multiplicity of infection of 10 (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e bacteria/well) for 8 h at 37\u0026deg;C. Following infection, the samples were gently washed with phosphate-buffered saline (PBS) to remove non-adherent bacteria and subjected to TEM by a commercial service provider (Sevier, Wuhan, China). The samples were fixed with 2.5% glutaraldehyde at 4\u0026deg;C, post-fixed with 1% osmium tetroxide for 1 h, dehydrated through graded ethanol, infiltrated with Spurr resin, and polymerized at 70\u0026deg;C for 12 h. Finally, ultrathin sections were prepared using an ultramicrotome and examined using a transmission electron microscope at an accelerating voltage of 120 kV.\u003c/p\u003e\n\u003ch3\u003eAdhesion and invasion assays\u003c/h3\u003e\n\u003cp\u003eHCMEC/D3 cells (Jinyuan Biotechnology, Shanghai, China) were cultured in the Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37\u0026deg;C in 5% CO₂ until reaching confluence. To induce bacterial infection, the cells were cultured in 24-well tissue culture plates containing DMEM without FBS or penicillin/streptomycin at 37\u0026deg;C in 5% CO₂ for 12 h. Log-phase bacteria were prepared from overnight cultures, harvested via centrifugation (10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 5 min), washed thrice with PBS, and resuspended in antibiotic-free FBS-free DMEM to 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e colony-forming units (CFU)/mL. The cells were infected at a multiplicity of infection of 10 at 37\u0026deg;C in 5% CO₂ for 2 h. Uninfected wells received an equal volume of medium and served as controls. Adhesion and invasion assays were performed as previously described \u003csup\u003e[16,17]\u003c/sup\u003e, with minor modifications. For the adhesion assay, wells were washed thrice with PBS and lysed with 200 \u0026micro;L of PBS containing 1% Triton X-100. After 10 min, the lysates were serially diluted and plated on Luria\u0026ndash;Bertani agar for CFU enumeration after incubation at 37\u0026deg;C for 18\u0026ndash;24 h. For the invasion assay, extracellular bacteria were killed using gentamicin (100 \u0026micro;g/mL) for 1 h, followed by washing and CFU enumeration as described above. Negative control wells containing only bacteria were used to confirm that all extracellular bacteria were killed. All assays were performed in three independent experiments, each with three technical replicate wells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eBacteria were grown to the logarithmic phase, and total RNA was extracted using the SteadyPure Quick RNA Extraction Kit (Accurate Biotechnology, Hunan, China). The total RNA from each isolate was reverse-transcribed into cDNA using the Evo M-MLV RT Mix Kit (with gDNase; Accurate Biotechnology). Finally, gene expression data were obtained via RT-qPCR using the CFX96 Real-Time System (Bio-Rad, USA) and SYBR Green Premix Pro Taq HS qPCR Kit (Rox Plus; Accurate Biotechnology). The 16S rRNA served as an internal reference gene. Each experiment was repeated thrice, and the relative expression of each gene in different strains was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative sequence analysis of\u003c/b\u003e \u003cb\u003eompA\u003c/b\u003e \u003cb\u003eand selected candidate regulators\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the elevated \u003cem\u003eompA\u003c/em\u003e transcript level detected in BP9811, we compared the nucleotide sequences of \u003cem\u003eompA\u003c/em\u003e and selected candidate regulatory genes (\u003cem\u003erpoE\u003c/em\u003e, \u003cem\u003ehfq\u003c/em\u003e, \u003cem\u003eompR\u003c/em\u003e, and \u003cem\u003eenvZ\u003c/em\u003e) between BP9811 and the phylogenetically closest comparator strain, CG43. For each locus, the complete coding sequence and the 500-bp upstream region relative to the annotated start codon were retrieved from the assembled genomes and aligned using MAFFT v7.526 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mafft.cbrc.jp/alignment/software/\u003c/span\u003e\u003cspan address=\"https://mafft.cbrc.jp/alignment/software/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The deduced amino acid sequences were further compared.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGalleria mellonella\u003c/b\u003e \u003cb\u003einfection model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVirulence was evaluated using the \u003cem\u003eG. mellonella\u003c/em\u003e infection model, as previously described \u003csup\u003e[18]\u003c/sup\u003e. NTUH-2044 was used as the hypervirulent control strain, and ATCC 700603 served as the low-virulence control strain. Overnight \u003cem\u003eK. pneumoniae\u003c/em\u003e cultures were diluted in sterile PBS to achieve a concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. \u003cem\u003eG. mellonella\u003c/em\u003e larvae weighing 250\u0026ndash;300 mg (Henan Jiyuan Baiyun Industry Co., Ltd., Henan, China) were injected with 10 \u0026micro;L bacterial suspension and incubated for five days at 35\u0026deg;C. Survival was recorded daily from day 1 to day 5. Ten larvae per strain were used for each experiment, and the experiments were performed in triplicate. Kaplan\u0026ndash;Meier curves were generated using GraphPad Prism (10.1.2). The results were presented as the mean of three independent experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eDifferences among groups were statistically analyzed via one-way analysis of variance or Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test in GraphPad Prism 10.1.2, as appropriate. Survival rates of \u003cem\u003eG. mellonella\u003c/em\u003e larvae were analyzed using the log-rank test. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClinical characteristics of the CSF isolate BP9811\u003c/h2\u003e \u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e BP9811 was isolated from the CSF of a 50-year-old male patient with diabetes mellitus who presented with high fever and confusion. The patient had not been hospitalized within the preceding 3 months. Blood culture revealed no bacterial growth. Chest and abdominal computed tomography scans on admission showed no abnormalities. Brain magnetic resonance imaging on admission revealed ventricular enlargement suggestive of hydrocephalus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The isolate was identified as \u003cem\u003eK. pneumoniae\u003c/em\u003e via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Autof ms1500; Auto-bio) and confirmed via 16S rRNA sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A hypermucoviscous phenotype was observed using string tests, with a viscous string exceeding 5 mm in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Laboratory analysis of the CSF showed significant abnormalities, including Pandy\u0026rsquo;s test (+), elevated mononuclear cell count (56200/\u0026micro;L), decreased glucose level (0.01 mmol/L), and markedly increased total trace protein level (6382.2 mg/L), consistent with bacterial meningitis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Empirical antimicrobial therapy with meropenem (2 g IV q8h) and levofloxacin (500 mg IV qd) was initiated upon admission. After seven days of combination therapy, the infection-related symptoms subsided, the patient became afebrile, and no neurological sequelae were observed. The patient was discharged after 21 days of hospitalization following systemic treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical and laboratory indicators of the patient\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaboratory Indicators\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeasurements\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal Interval\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCerebrospinal fluid (CSF) tests\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAppearance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLight yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eColorless, transparent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePandy's test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1600/\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMononuclear cell count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56200/\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicroprotein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6382.20 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u0026ndash;430 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCSF glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.01 mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.22\u0026ndash;3.88 mmol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120.20 mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e120\u0026ndash;130 mmol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCryptococcal antigen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBlood tests\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.50\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.50\u0026ndash;9.50\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026ndash;75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocyte\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEosinophil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.40\u0026ndash;0.80%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.46 mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.89\u0026ndash;6.11 mmol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbA1c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.0\u0026ndash;6.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC-reactive protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.47 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026ndash;10 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProcalcitonin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.05 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0-0.05 ng/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eImmunological tests\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.40 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.51\u0026ndash;15.60 g/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.90 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.7-4.0 g/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.44 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.46\u0026ndash;3.04 g/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.31 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.79\u0026ndash;1.52 g/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.16\u0026ndash;0.38 g/L\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\u003eAntibiotic susceptibility test results\u003c/h2\u003e \u003cp\u003eComprehensive AST results for the 13 agents are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Isolate BP9811 was susceptible to all tested antibiotics, namely piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, colistin, ceftazidime-avibactam, tigecycline, amikacin and fosfomycin. This finding indicated susceptibility to multiple drug classes, including aminoglycosides, cephalosporins, carbapenems, and fluoroquinolones, as well as to colistin. Notably, despite carrying \u003cem\u003efosA\u003c/em\u003e, BP9811 remained susceptible to fosfomycin.\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 susceptibility of the \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e isolate by broth microdilution and Kirby-Bauer methods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibiotics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBreakpoint\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInterpretation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026le;S\u0026thinsp;\u0026ge;\u0026thinsp;R\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePiperacillin/Tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftazidime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErtapenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColistin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftazidime/Avibactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTigecycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFosfomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\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\u003eAntimicrobial resistance and virulence determinants\u003c/h2\u003e \u003cp\u003eThe resistance genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;28\u003c/sub\u003e, \u003cem\u003eoqxA, oqxB\u003c/em\u003e, and \u003cem\u003efosA\u003c/em\u003e were located on the chromosome via WGS. Virulence Factor Database analysis showed that BP9811 carried the core hypervirulence-associated determinant \u003cem\u003ermpA/rmpA2\u003c/em\u003e, \u003cem\u003epeg-344\u003c/em\u003e, and siderophore genes \u003cem\u003eiucABCD\u003c/em\u003e and \u003cem\u003eiroBCDE\u003c/em\u003e, supporting its classification as an hvKP isolate. BP9811 also harbored other colonization and fitness determinants, including the enterobactin biosynthesis gene \u003cem\u003eentABCD\u003c/em\u003e and genes involved in envelope homeostasis and efflux, such as \u003cem\u003ercsAB\u003c/em\u003e, \u003cem\u003eacrAB\u003c/em\u003e, and \u003cem\u003efepABCDG\u003c/em\u003e. Furthermore, BP9811 encoded virulence factors potentially associated with host cell interactions, including types 1 and 3 fimbrial gene clusters (\u003cem\u003efimABCDEFGH\u003c/em\u003e and \u003cem\u003emrkABCDFH\u003c/em\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of \u003cem\u003eK. pneumoniae\u003c/em\u003e BP9811\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eST\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVirulence factors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eResistance genes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP9811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eST86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eentABCD, iroBCDE, iucABCD, fimABCDEFGH, mrkABCDFH, acrAB, fepABCDG, peg-344, rcsAB, rmpA\u003c/em\u003e/\u003cem\u003ermpA2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;28\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eoqxA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eoqxB\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003efosA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of the BP9811 genome\u003c/h2\u003e \u003cp\u003eBP9811 was assigned to sequence type 86 by MLST and to capsular locus KL2 by Kaptive analysis. The complete genome of BP9811 comprised a 5,170,607 bp chromosome and a 224,627 bp plasmid (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), yielding a total genome size of 5,395,234 bp with an overall G\u0026thinsp;+\u0026thinsp;C content of 57.3%. Genome annotation identified 4,965 protein-coding sequences, 84 tRNA genes, and 25 rRNA genes. Four putative secondary metabolite biosynthetic gene clusters, including siderophore-, lankacidin-, and O-antigen-associated clusters, were also identified.\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\u003eGenomic features of the strain BP9811\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFeatures\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBP9811\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid pIM057_vir\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal number of bases (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,170,607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e224,627\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG\u0026thinsp;+\u0026thinsp;C content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.62%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.33%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of tRNA genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of protein-coding sequences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e230\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\u003ePlasmid pIM057_vir was identified as an IncHI1B virulence plasmid. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, a set of virulence genes (including the previously reported hvKP-associated \u003cem\u003eiucABCD\u003c/em\u003e and \u003cem\u003eiroBCD\u003c/em\u003e) was located on the plasmid pIM057_vir, whereas no antibiotic resistance genes were detected. Basic Local Alignment Search Tool analysis further indicated that pIM057_vir shared 99% sequence identity with pE16KP0290-1 (GenBank accession no. CP052258.1), pGDFK0476-unnamed (CP167435.1), and pKP48326-1 (CP167190.1), with a query coverage of 99\u0026ndash;100%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVirulence analysis using the\u003c/b\u003e \u003cb\u003eG. mellonella\u003c/b\u003e \u003cb\u003einfection model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eG. mellonella\u003c/em\u003e infection assay was conducted to investigate the virulence of BP9811. BP9811 caused a significantly accelerated killing trajectory compared to the low-virulence control strain ATCC 700603 and showed virulence comparable to that of the hypervirulent reference strain NTUH-2044 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These data support the high pathogenicity of BP9811 in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced interaction of BP9811 with human cerebral microvascular endothelial cells\u003c/h2\u003e \u003cp\u003eTo evaluate the interaction of BP9811 with human cerebral microvascular endothelial cells, we conducted adhesion and gentamicin protection assays using HCMEC/D3 cells. TEM revealed that BP9811 was internalized by HCMEC/D3 cells, with membrane-bound bacteria observed within the host cell cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Adhesion experiments revealed that BP9811 showed greater adhesion to HCMEC/D3 cells than NTUH-2044 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Gentamicin protection experiments showed a higher intracellular recovery of BP9811 than of NTUH-2044 under the same assay conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Next, we examined \u003cem\u003eompA\u003c/em\u003e transcript levels in BP9811. RT-qPCR revealed that the \u003cem\u003eompA\u003c/em\u003e mRNA levels were higher in BP9811 than in the control strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Taken together, BP9811 exhibited increased interaction with HCMEC/D3 cells in the in vitro assays, along with high \u003cem\u003eompA\u003c/em\u003e transcript levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSequence conservation of\u003c/b\u003e \u003cb\u003eompA\u003c/b\u003e \u003cb\u003eand related regulatory genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether increased \u003cem\u003eompA\u003c/em\u003e transcript levels in BP9811 were accompanied by obvious sequence variation in \u003cem\u003eompA\u003c/em\u003e or selected regulatory determinants, we performed a comparative sequence analysis using the phylogenetically closely related strain CG43 as the reference comparator. Notably, the \u003cem\u003eompA\u003c/em\u003e coding sequence and analyzed upstream region were highly conserved between BP9811 and CG43. Similarly, the coding sequences of \u003cem\u003erpoE, hfq, ompR\u003c/em\u003e, and \u003cem\u003eenvZ\u003c/em\u003e, including their upstream regions, showed a high degree of conservation, with no amino acid-altering substitutions identified in the analyzed loci. These findings indicate that higher \u003cem\u003eompA\u003c/em\u003e transcript levels in BP9811 were not accompanied by obvious sequence differences in \u003cem\u003eompA\u003c/em\u003e or coding regions of the selected candidate regulators examined in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analyses of CSF isolates\u003c/h2\u003e \u003cp\u003eTo further clarify the phylogenetic relationships between the CSF-derived isolate BP9811 and previously reported CSF isolates, 26 CSF-derived \u003cem\u003eK. pneumoniae\u003c/em\u003e genomes were retrieved from the National Center for Biotechnology Information database and analyzed together with BP9811 using a core SNP-based phylogenetic framework (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Core-genome SNP analysis revealed that the CSF isolates were genetically diverse, comprising multiple STs and KL types. Notably, BP9811 did not cluster with the other 26 isolates, and the substantial core-SNP distances between them argue against a recent common origin or transmission cluster. Collectively, these findings suggest that BP9811 is a sporadic ST86-KL2 isolate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of KL2\u003c/h2\u003e \u003cp\u003eTo further define the phylogenetic position of BP9811 within the KL2 \u003cem\u003eK. pneumoniae\u003c/em\u003e population, we constructed a core-genome SNP-based phylogeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). BP9811 showed the smallest core-SNP distance to CG43 (318 SNPs), whereas the distances to Kpn154, HK787, 18CP0060, Kpn2166, and KPN55602 ranged from 344 to 373 SNPs. This suggests that BP9811 occupies a relatively closely related phylogenetic branch among the tested KL2 isolates, although the observed divergence is insufficient to support recent clonal transmission. Notably, although BP9811 was most closely related to CG43 at the core-genome level, their virulence gene profiles differed markedly. This pattern suggests that the hypervirulent phenotype of BP9811 is attributable, at least in part, to the acquisition of virulence determinants mediated by mobile genetic elements rather than vertical diversification of the core genome alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we characterized a CSF-derived ST86-KL2 hvKP isolate, BP9811, from a patient with diabetes and community-acquired meningitis. The presentation was consistent with the established clinical spectrum of invasive hvKP infection, in which diabetes is a frequent host factor and community-onset severe disease is well recognized \u003csup\u003e[19]\u003c/sup\u003e. However, published studies on CSF-derived hvKP isolates remain limited, particularly for the ST86-KL2 lineage, for which the available evidence is largely restricted to sporadic case reports and small series \u003csup\u003e[3, 11, 12]\u003c/sup\u003e. This study provided an integrated strain-level clinical, genomic, and phenotypic dataset for a CSF-derived ST86-KL2 hvKP isolate, broadening the currently available evidence on hvKP-associated meningitis.\u003c/p\u003e \u003cp\u003eThe virulence profile of BP9811 was consistent with that of canonical hvKP. The isolate carried \u003cem\u003ermpA\u003c/em\u003e, \u003cem\u003ermpA2\u003c/em\u003e, \u003cem\u003eiuc, iro\u003c/em\u003e, and \u003cem\u003epeg-344\u003c/em\u003e, along with an IncHI1B virulence plasmid. These features were widely accepted markers of hvKP \u003csup\u003e[20, 21]\u003c/sup\u003e. BP9811 also showed high lethality in the \u003cem\u003eG. mellonella\u003c/em\u003e model and increased adhesion to and invasion of HCMEC/D3 cells. These results suggest preserved functional virulence. Additionally, \u003cem\u003eompA\u003c/em\u003e transcription was increased in this strain. This finding is of interest because a recent study implicated \u003cem\u003eompA\u003c/em\u003e in blood-brain barrier penetration and meningitis pathogenesis \u003csup\u003e[10, 22]\u003c/sup\u003e. Notably, BP9811 did not closely cluster with currently available CSF-derived genomes, suggesting that CNS invasion was not restricted to a single meningitis-associated clone. Instead, it possibly emerged in different genetic backgrounds when key virulence determinants act in a susceptible host \u003csup\u003e[23]\u003c/sup\u003e. The conservation of \u003cem\u003eompA\u003c/em\u003e coding sequences and increased transcription indicated that regulatory differences, rather than structural variation alone, contributed to the observed phenotype; however, this remained speculative in the absence of direct functional validation.\u003c/p\u003e \u003cp\u003eThe resistance profile also merits attention. Although BP9811 carried resistance-associated genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;28\u003c/sub\u003e, \u003cem\u003eoqxA/oqxB\u003c/em\u003e, and \u003cem\u003efosA\u003c/em\u003e, it remained susceptible to most clinically relevant agents. This pattern was consistent with the traditional view that canonical hvKP typically combines high virulence with a relatively limited resistance burden \u003csup\u003e[24]\u003c/sup\u003e. However, the genotype did not fully predict the phenotype. The presence of \u003cem\u003efosA\u003c/em\u003e did not translate to fosfomycin resistance in the tested isolate, highlighting the need to interpret genomic resistance data together with phenotypic susceptibility testing, especially for CSF-derived isolates with direct therapeutic implications \u003csup\u003e[25, 26]\u003c/sup\u003e. Recent literature had also shown that hypervirulent lineages acquire mobile resistance determinants and evolve toward a convergent, hypervirulent, and multidrug-resistant phenotype \u003csup\u003e[23, 27]\u003c/sup\u003e. Therefore, BP9811 might represent a susceptible hvKP background, but not the only evolutionary trajectory of ST86-KL2 or other K2 lineages.\u003c/p\u003e \u003cp\u003eThis study had several limitations that should be considered when interpreting the results. First, this single case couldn\u0026rsquo;t define the epidemiology or prognosis of ST86-KL2 meningitis. Second, the contribution of OmpA from the strains in this study to CNS invasion required further validation using gene knockout and complementation or blood-brain barrier models. Third, plasmid transferability and stability were not assessed in this study. These factors limited inferences regarding virulence plasmid dissemination and the risk of recombination with multidrug-resistant lineages.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we found that a CSF-derived ST86-KL2 hvKP isolate harbored a canonical virulence plasmid and exhibited increased \u003cem\u003eompA\u003c/em\u003e transcription and prominent virulence-associated phenotypes. Overall, our findings provided an integrated clinical, genomic, and phenotypic dataset for a CSF-derived hvKP isolate, supporting systematic surveillance of invasive hvKP lineages in high-risk populations, including patients with diabetes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eantimicrobial susceptibility testing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecerebrospinal fluid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehvKP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emultilocus sequence typing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereverse transcription-quantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSNP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingle nucleotide polymorphism\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewhole-genome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from the patient in the study. The protocol was reviewed by the Ethics Committee (IRB) of the Second Affiliated Hospital of Zhejiang Chinese Medical University. As this was an observational study and strains cultured from residual samples were used in clinical diagnosis, the confidentiality of patient data is preserved and compliance with the Declaration of Helsinki is ensured. After consulting the IRB of the Second Affiliated Hospital of Zhejiang Chinese Medical University, a formal ethical review approval was obtained (ethical approval no.: Y2026-038-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication of patient clinical details and clinical images was obtained from the patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number: not applicable.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Chinese Medicine Scientific Research Foundation of Zhejiang Province, China (Grant No. 2026ZL0059); The funder had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQW conceived and designed the experiments. XL, XZ, MC, TZ and XT performed the experiments. XL, JY and HC analyzed the data. XL wrote the manuscript. All authors read and approved the final manuscript. All authors contributed toward data analysis, drafting and revising the manuscript, and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks Editage (www.editage.cn) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and/or analyzed in this study are available from the corresponding author on reasonable request. The genome sequence of K. pneumoniae BP9811 has been submitted to NCBI GenBank under BioSample accession number SAMN56319679 (https://www.ncbi.nlm.nih. gov/biosample/SAMN56319679/).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKubicsk\u0026oacute; AS, Juh\u0026aacute;sz JN, Kamotsay K, Szabo D, Kocsis BL. Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Antibiotics. 2025; 14(1):1-14. doi: 10.3390/antibiotics14010062\u003c/li\u003e\n \u003cli\u003eZhao C, Liu P, Lin X, Wan C, Liao K, Guo P, et al. The type VI secretion system as a potential predictor of subsequent bloodstream infection of carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains on intestinal colonization. Infection. 2025; 53(2):667-678. doi: 10.1007/s15010-024-02456-x\u003c/li\u003e\n \u003cli\u003eHuang N, Jia H, Zhou B, Zhou C, Cao J, Liao W, et al. Hypervirulent carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e causing highly fatal meningitis in southeastern China. Frontiers in Public Health. 2022; 10:1-11. doi: 10.3389/fpubh.2022.991306\u003c/li\u003e\n \u003cli\u003eRollin G, Rossi B, Brisse S, Decr\u0026eacute; D, Leflon-Guibout V, Bert F, et al. Spontaneous and postsurgical/traumatic \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e meningitis: two distinct clinico-microbiological entities. International Journal of Infectious Diseases. 2022; 114:185-191. doi: 10.1016/j.ijid.2021.11.013\u003c/li\u003e\n \u003cli\u003eRusso TA, Marr CM. Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Clinical Microbiology Reviews. 2019; 32(3):e00001-19. doi: 10.1128/CMR.00001-19\u003c/li\u003e\n \u003cli\u003eHu H, Wang H, Yu M, Feng H, Zhang S, Zhang Y, et al. Clinical and microbiological characteristics of carbapenem-resistant Enterobacteriaceae causing post-operative central nervous system infections in China. Journal of Global Antimicrobial Resistance. 2023; 35:35-43. doi: 10.1016/j.jgar.2023.08.006\u003c/li\u003e\n \u003cli\u003eLi Y, Hu D, Ma X, Li D, Tian D, Gong Y, et al. Convergence of carbapenem resistance and hypervirulence leads to high mortality in patients with postoperative \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e meningitis. Journal of Global Antimicrobial Resistance. 2021; 27:95-100. doi: 10.1016/j.jgar.2021.02.035\u003c/li\u003e\n \u003cli\u003eHuang CR, Lu CH, Chang HW, Lee PY, Lin MW, Chang WN. Community-acquired spontaneous bacterial meningitis in adult diabetic patients: an analysis of clinical characteristics and prognostic factors. Infection. 2002; 30(6):346-350. doi: 10.1007/s15010-002-3010-4\u003c/li\u003e\n \u003cli\u003eMarr CM, Russo TA. Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e: a new public health threat. Expert Review of Anti-infective Therapy. 2019; 17(2):71-73. doi: 10.1080/14787210.2019.1555470\u003c/li\u003e\n \u003cli\u003eZeng W, Sun Y, Zhang J, Yao Z, Cao J, Hu P, et al. Outer membrane protein A mediates \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e penetration of the blood-brain barrier and induces bacterial meningitis. Microbiological Research. 2025; 299:1-15. doi: 10.1016/ j.micres.2025.128262\u003c/li\u003e\n \u003cli\u003eZhang Z, Wen H, Wang H, Zhang P, Li J, Liang Y, et al. A Case of Meningitis in an Infant Due to Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Transmission Within a Family. Infection and Drug Resistance. 2022; 15:4927-4933. doi: 10.2147/IDR. S376055\u003c/li\u003e\n \u003cli\u003eKu YH, Chuang YC, Chen CC, Lee MF, Yang YC, Tang HJ, et al. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Isolates from Meningitis: Epidemiology, Virulence and Antibiotic Resistance. Scientific Reports. 2017; 7(1):1-10. doi: 10.1038/s41598-017-06878-6\u003c/li\u003e\n \u003cli\u003eCLSI (2025). Performance standards for antimicrobial susceptibility testing. 35th informational supplement (CLSI M100-S35). Wayne, PA: Clinical and Laboratory Standards Institute.\u003c/li\u003e\n \u003cli\u003eEuropean Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. version 16.0; 2026. Available at https:// www.eucast.org/fileadmin/eucast/pdf/breakpoints/ v_16.0_Breakpoint_Tables.pdf\u003c/li\u003e\n \u003cli\u003eHagiya H, Watanabe N, Maki M, Murase T, Otsuka F. Clinical utility of string test as a screening method for hypermucoviscosity‐phenotype \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Acute Medicine \u0026amp; Surgery. 2014; 1(4):245-246. doi: 10.1002/ams2.40\u003c/li\u003e\n \u003cli\u003eXiao S, Suo W, Zhang J, Zhang X, Yin Y, Guo X, et al. MgaSpn is a negative regulator of capsule and phosphorylcholine biosynthesis and influences the virulence of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e D39. Virulence. 2021; 12(1):2366-2381. doi: 10.1080/21505594.2021.1972539\u003c/li\u003e\n \u003cli\u003eDeng L, Spencer BL, Holmes JA, Mu R, Rego S, Weston TA, et al. The Group B \u003cem\u003eStreptococcal\u003c/em\u003e surface antigen I/II protein, BspC, interacts with host vimentin to promote adherence to brain endothelium and inflammation during the pathogenesis of meningitis. PLoS Pathogens. 2019; 15(6):1-30. doi: 10.1371/journal.ppat. 1007848\u003c/li\u003e\n \u003cli\u003eChen J, Zeng Y, Zhang R, Cai J. In vivo Emergence of Colistin and Tigecycline Resistance in Carbapenem-Resistant Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e During Antibiotics Treatment. Frontiers in Microbiology. 2021; 12:1-8. doi: 10.3389/fmicb.2021.702956\u003c/li\u003e\n \u003cli\u003eNamikawa H, Oinuma KI, Yamada K, Kaneko Y, Kakeya H, Shuto T. Predictors of hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infections: a systematic review and meta-analysis. The Journal of Hospital Infection. 2023; 134:153-160. doi: 10.1016/j.jhin. 2023.02.005\u003c/li\u003e\n \u003cli\u003eRusso TA, Lebreton F, McGann PT. A Step Forward in Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Diagnostics. Emerging Infectious Diseases. 2025; 31(1):1-3. doi: 10.3201/eid3101.241516\u003c/li\u003e\n \u003cli\u003eRusso TA, Olson R, Fang CT, Stoesser N, Miller M, MacDonald U, et al. Identification of Biomarkers for Differentiation of Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e from Classical \u003cem\u003eK. pneumoniae\u003c/em\u003e. Journal of Clinical Microbiology. 2018; 56(9):1-12. doi: 10.1128/JCM.00776-18\u003c/li\u003e\n \u003cli\u003eShin S, Lu G, Cai M, Kim KS. \u003cem\u003eEscherichia coli\u003c/em\u003e outer membrane protein A adheres to human brain microvascular endothelial cells. Biochemical and Biophysical Research Communications. 2005; 330(4):1199-1204. doi: 10.1016/j.bbrc.2005. 03.097\u003c/li\u003e\n \u003cli\u003eChen T, Ying L, Xiong L, Wang X, Lu P, Wang Y, et al. Understanding carbapenem-resistant hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e: Key virulence factors and evolutionary convergence. hLife. 2024; 2(12):611-624. doi: 10.1016/j.hlife. 2024.06.005\u003c/li\u003e\n \u003cli\u003eLee CR, Lee JH, Park KS, Jeon JH, Kim YB, Cha CJ, et al. Antimicrobial Resistance of Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e: Epidemiology, Hypervirulence-Associated Determinants, and Resistance Mechanisms. Frontiers in Cellular and Infection Microbiology. 2017; 7:1-13. doi: 10.3389/fcimb. 2017.00483\u003c/li\u003e\n \u003cli\u003eZhou X, Yang M, Chen F, Wang L, Han P, Jiang Z, et al. Prediction of antimicrobial resistance in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e using genomic and metagenomic next-generation sequencing data. Journal of Antimicrobial Chemotherapy. 2024; 79(10):2509-2517. doi: 10.1093/jac/dkae248\u003c/li\u003e\n \u003cli\u003eBixby ML, Collins LB, Daley EC, Salay JM, Oliver S, Bryson AL, et al. Heteroresistance associated with the production of fosfomycin-resistant inner colonies during disk diffusion testing among a geographically diverse collection of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e clinical isolates. JAC-Antimicrobial Resistance. 2025; 7(1):1-8. doi: 10.1093/jacamr/dlaf013\u003c/li\u003e\n \u003cli\u003eArcari G, Carattoli A. Global spread and evolutionary convergence of multidrug-resistant and hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e high-risk clones. Pathogens and Global Health. 2023; 117(4):328-341. doi: 10.1080/20477724.2022.2121362\u003c/li\u003e\n\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":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hypervirulent Klebsiella pneumoniae, Meningitis, Cerebrospinal fluid, ST86, KL2, Whole-genome sequencing, OmpA","lastPublishedDoi":"10.21203/rs.3.rs-9398214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9398214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (hvKP) is an important cause of invasive community-acquired infection, particularly in individuals with diabetes mellitus. However, cerebrospinal fluid (CSF)-derived hvKP isolates, especially those belonging to the ST86-KL2 lineage, remain poorly characterized at the integrated clinical, genomic, and phenotypic levels.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA \u003cem\u003eK. pneumoniae\u003c/em\u003e isolate, designated BP9811, was recovered from the CSF of a patient with meningitis and diabetes mellitus and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA sequencing. Antimicrobial susceptibility testing and whole-genome sequencing were performed to define its resistance, virulence, sequence type, capsular type, and plasmid content. Virulence was evaluated using the \u003cem\u003eGalleria mellonella\u003c/em\u003e infection model. In addition, interaction with human brain microvascular endothelial cells was preliminarily assessed using adhesion, gentamicin protection, and transmission electron microscopy assays, together with measurement of relative \u003cem\u003eompA\u003c/em\u003e transcription by reverse transcription-quantitative polymerase chain reaction. Comparative phylogenetic analyses were performed using publicly available CSF-derived and KL2 \u003cem\u003eK. pneumoniae\u003c/em\u003e genomes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBP9811 was identified as a hypermucoviscous ST86-KL2 hvKP isolate that remained susceptible to all tested antimicrobial agents. Whole-genome sequencing revealed an IncHI1B virulence plasmid carrying canonical hvKP-associated determinants, including \u003cem\u003ermpA/rmpA2\u003c/em\u003e, \u003cem\u003epeg-344\u003c/em\u003e, \u003cem\u003eiucABCD\u003c/em\u003e, and \u003cem\u003eiroBCDE\u003c/em\u003e. In the \u003cem\u003eGalleria mellonella\u003c/em\u003e model, BP9811 showed high virulence comparable to that of the hypervirulent reference strain NTUH-2044. In HCMEC/D3 cells, BP9811 exhibited increased adhesion and intracellular recovery under the tested conditions, and transmission electron microscopy confirmed bacterial internalization. BP9811 also showed higher \u003cem\u003eompA\u003c/em\u003e transcript levels than the control strain. Phylogenetic analysis indicated that BP9811 was genetically distinct from currently available CSF-derived isolates and occupied a related branch within the KL2 population.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study provides an integrated clinical, genomic, and phenotypic characterization of BP9811, a CSF-derived ST86-KL2 hvKP isolate recovered from a patient with meningitis and diabetes mellitus. BP9811 carried a canonical hvKP virulence plasmid, displayed marked virulence-associated phenotypes, and showed enhanced interaction with human brain microvascular endothelial cells in vitro under the tested conditions. These findings expand the limited isolate-level evidence on central nervous system-associated hvKP and provide a basis for future comparative and mechanistic studies.\u003c/p\u003e","manuscriptTitle":"Genomic, virulent and phenotypic characterization of a cerebrospinal fluid- derived ST86-KL2 hypervirulent Klebsiella pneumoniae isolate from a patient with meningitis and diabetes mellitus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 08:35:18","doi":"10.21203/rs.3.rs-9398214/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-29T05:27:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T13:42:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211021896594203630224359215534644143461","date":"2026-04-27T08:16:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140266340209611928378977167543994036644","date":"2026-04-24T04:16:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50835376340561168572252447150077072030","date":"2026-04-22T06:56:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T01:36:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T01:35:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-21T11:18:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-20T10:35:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-04-20T08:57:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57dd272a-3c7e-46b2-bd9e-c93c92e39773","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-04-29T05:27:15+00:00","index":35,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T08:35:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 08:35:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9398214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9398214","identity":"rs-9398214","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}