Genomic characteristics of Vibrio vulnificus isolated from clinical and environmental sources

Genomic characteristics of Vibrio vulnificus isolated from clinical and environmental sources | 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 characteristics of Vibrio vulnificus isolated from clinical and environmental sources Jinkyeong Lee, Jeong-Ih Shin, Woo Young Cho, Kun Take Park, Yeun-Jun Chung, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5229211/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 Vibrio vulnificus is a gram-negative pathogenic bacterium that is transmitted through undercooked seafood or contaminated seawater, causing septicemia and wound infections. In this study, 15 clinical isolates and 11 environmental isolates were analyzed, revealing a total of 20 sequence types (STs), 8 of which were newly identified. Antibiotic resistance gene analysis revealed that the CRP gene was frequently present in both clinical and environmental isolates. Interestingly, the clinical and environmental isolates showed notable not-susceptible to third-generation cephalosporins, such as ceftazidime and cefotaxime, which may complicate treatment strategies for V. vulnificus infections. The Multiple Antibiotic Resistance (MAR) index ranged from 0.1 to 0.5, with clinical isolates having a higher mean MAR index compared to environmental isolates, indicating a broader spectrum of resistance in clinical strains. No quantitative (124.3 versus 126.5) and qualitative (adherence, antiphagocytosis, and chemotaxis/motility) differences in virulence factors were observed between environmental and clinical strains. The molecular characteristics identified in this study may provide a basis for understanding the virulence of Vibrio vulnificus strains in South Korea and highlight the need for continuous surveillance of antibiotic resistance in emerging V. vulnificus strains. Vibrio vulnificus Whole-Genome Sequencing Antimicrobial Resistance Virulence Factor Multi-Locus Sequence Typing Figures Figure 1 Figure 2 Introduction Vibrio vulnificus is a gram-negative aquatic bacterium that is naturally found in warm coastal waters, especially in regions with higher salinity [ 1 ]. It belongs to the Vibrionaceae family and is closely related to V. cholerae , the causative agent of cholera. V. vulnificus is known as an opportunistic pathogen commonly transmitted by consumption of seafood, particularly raw or undercooked shellfish like oysters [ 2 ]. It can also rarely enter the body through open wounds when exposed to contaminated water, leading to invasive infections [ 2 ]. V. vulnificus infections can range from gastroenteritis to life-threatening septicemia, which has the highest fatality rate among the many foodborne pathogens [ 3 ]. While healthy individuals may experience mild symptoms, the bacterium poses a significant risk to those with weakened immune systems, particularly those with liver disease or diabetes [ 4 ]. In South Korea, 69 cases of V. vulnificus sepsis were reported in 2023, resulting in 27 deaths and a fatality rate of 39.1% [ 5 ]. Virulence factors contribute to the infection capability of V. vulnificus , and therefore play an important role in its pathogenicity [ 6 ]. The vvhA gene is related to the virulence of V. vulnificus and is used by public health authorities to assess human pathogenicity [ 7 ]. Additionally, the vcgC and vcgE genes have been reported as useful markers for distinguishing strains that originate from human or environmental cases [ 8 ]. Recent studies indicate that vcgC is more frequently found in strains associated with human infections, while vcgE is widely used to identify strains originating from the environment [ 7 , 9 ]. Indeed, vcgC produces proteins involved in invading the host, whereas vcgE produces proteins that respond to environmental stress and evade immune responses [ 7 ]. However, there is no definitive consensus on whether a specific virulence factor can distinguish between pathogenic and non-pathogenic strains of V. vulnificus [ 10 ]. In addition to virulence factors, antibiotic resistance is another important factor associated with V. vulnificus infections [ 11 , 12 ]. It can spread by both clonal transmission and horizontal gene transfer, which makes infections difficult to treat and control [ 13 , 14 ]. Therefore, it is necessary to analyze the virulence profile of infected isolates to determine their pathogenic potential and monitor antibiotic resistance. In this study, 26 V. vulnificus strains collected from coastal waters or infected patients were subjected to whole-genome sequencing (WGS) to investigate (i) their molecular subtypes and diversity, and (ii) their genomic characteristics, including antimicrobial resistance genes and virulence factors. Materials and Methods Bacterial collection and culture A total of 11 V. vulnificus strains were collected in South Korea between August 2022 and September 2022 as part of the national foodborne pathogen surveillance research. The V. vulnificus strains were isolated from coastal waters in five provinces of South Korea (3 isolated from the Namhae-gun, Gyeongsangnam-do, 2 isolated from Geoje-si, Gyeongsangnam-do, 3 isolated from Mokpo-si, Jeollanam-do, 2 isolated from Busan, and 1 isolated from Ulsan). Additionally, 15 clinical strains of V. vulnificus isolated during the period of 2010–2020 were obtained from nationwide bacterial biobanks in Korea (GNUH-NCCP). Of the 15 clinical strains, 11 were isolated from blood, while the remaining four were isolated from pus. The details of each isolate are provided in Supplementary Table S1 . The V. vulnificus strains were cultured on Blood Agar Plates at 37°C under ambient air conditions for 18 to 24 hours and stored at -80°C until further use analysis. This study was approved by the Institutional Review Board of the Catholic University of Korea College of Medicine (approval number: MC24SASI0019), and the requirement for informed consent was waived. Drug susceptibility testing Antimicrobial susceptibility testing for 15 drugs (gentamicin [GEN], meropenem [MEM], chloramphenicol [CHL], cefoxitin [FOX], cefotaxime [CTX], ceftazidime [CAZ], cefepime [FEP], ampicillin [AMP], colistin [COL], tetracycline [TET], amoxicillin/clavulanic acid [AUG2], nalidixic acid [NAL], ciprofloxacin [CIP], sulfisoxazole [FIS], and trimethoprim/sulfamethoxazole [SXT]) was performed using the Sensititre TM KRNV6F Kit (Thermo Fisher Scientific, Waltham, MA, USA) with the microbroth dilution method. Antimicrobial breakpoints were determined according to the Clinical and Laboratory Standards Institute guideline [ 15 ], with the minimum inhibitory concentration (MIC) range not in the ‘S’ being resistance. Escherichia coli ATCC 25922 was used as a standard strain. For antibiotics without established resistance determination criteria (COL, NAL, and FIS), MIC50 and MIC90 values were used to assess susceptibility. Three strains (two environmental and one clinical) were not tested for drug susceptibility due to the lack of regrowth activity. The Multiple Antibiotics Resistance (MAR) method, as described by Osundiya et al. [ 16 ], was employed to generate the MAR index [ 17 ]. The MAR index is calculated as the ratio of the number of antibiotics to which the organism is resistant divided by the total number of antibiotics to which the organism is exposed. A MAR index of ≥ 0.2 was defined as a high risk of the used antibiotics [ 13 ]. Whole-genome sequencing analysis Genomic DNA was extracted using iDetect gDNA Prep Kit for Microbes (ConnectaGen, Hanam, South Korea). The extracted DNA was evaluated by NanoDrop (Thermo Fisher Scientific) and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). NGS sequencing libraries were prepared using a TruSeq Nano DNA sample preparation kit (Illumina, San Diego, CA, USA). Sequencing libraries were then pooled and subjected to sequencing on the Illumina NovaSeq 6000 system. Sequencing adapters and low-quality bases were trimmed using Trimmomatic. The trimmed reads were assembled using SPAdes [ 18 ], and assembled contigs were annotated and evaluated using Prokka [ 19 ] and Quast [ 20 ], respectively. Kraken2 was used to confirm the species of each strain [ 21 ]. Multi-locus sequence types (STs) were determined using the MLST tools ( https://github.com/tseemann/mlst ) with the allelic profiles of the 10 housekeeping genes ( glp , gyrB , mdh , metG , purM , dtdS , lysA , pntA , pyrC , and tnaA ) from the PubMLST database [ 22 ]. The newly discovered STs were submitted to PubMLST database. Acquisition of antimicrobial resistance genes and virulence factors were determined using Abricate employing the Comprehensive Antibiotic Resistance Database [ 23 ] and Virulence Factor Database [ 24 ]. The virulence correlated gene (vcg) typing was performed using in silico PCR ( https://www.bioinformatics.org/sms2/pcr_products.html ) with previously reported primer sets [ 25 , 26 ]. A core single nucleotide polymorphism (SNP) alignment for 26 V. vulnificus genomes was performed using snippy ( https://github.com/tseemann/snippy ). A maximum likelihood phylogenetic tree based on core SNPs was constructed using RAxML with a generalized time-reversible gamma model [ 27 ]. The resulting phylogenetic tree was visualized in Microreact [ 28 ]. Statistical test Categorical variables were compared using the Fisher’s exact test. All tests were two-tailed, and P -value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software (IBM Corp., Armonk, NY, USA). Results Sample distributions Of the 26 V. vulnificus strains isolated from 2010 to 2022, 11 environmental strains (42.3%) were collected from the coastal waters of Gyeongsanam-do, Jeollanam-do, Busan, and Ulsan, and 15 clinical strains (57.7%) were collected from Gyeongsangnam-do, South Korea. The phylogenetic tree showed relatively clear differences between clinical and environmental strains (Fig. 1 ). Clinical strains exhibited a high degree of phylogenetic coherence, with 70% (11/15) forming a monophyletic clade, suggesting a conserved genetic lineage and potential clonal expansion. In contrast, environmental strains were phylogenetically dispersed across multiple distinct branches, reflecting a greater degree of genetic heterogeneity and suggesting a broader evolutionary divergence. Sequence types Through WGS analysis, we obtained high-quality sequence reads for 26 V. vulnificus strains. The average N50 value was 375.8Kb (range 149.0Kb-770.0Kb), and the average genome size was 5.17Mb (range 4.83Mb-7.53Mb). A total of 20 STs were identified, of which eight STs were newly discovered based on housekeeping genes: ST705 (n = 1), ST711 (n = 1), ST714 (n = 1), ST715 (n = 1), ST717 (n = 1), ST718 (n = 1), ST712 (n = 3), and ST713 (n = 3) (Supplementary Table S1 ). The new alleles and STs were submitted to the PubMLST database for assignment. Four STs were recurrently detected (ST449, n = 2; ST648, n = 2; ST712, n = 3; and ST713, n = 3), while the remaining STs were singletons (Fig. 1 ). These results suggest that the genetic background of V. vulnificus is largely heterogenous and that various STs have yet to be discovered. Antimicrobial resistance Phenotypic drug susceptibility testing was performed on 15 antibiotics, revealing prominent colistin resistance in both environmental (8/9, 88.9%) and clinical (11/14, 78.6%) strains. (Fig. 1 and Table 1 ). Notably, clinical strains exhibited higher resistance to cefepime, ceftazidime, and meropenem than environmental strains. Five clinical strains (35.7%, 5/14) were resistant to cefepime, whereas only one environmental strain (11.1%, 1/9) showed resistance. Similarly, 28.6% (4/14) of clinical strains were resistant to each of ceftazidime and meropenem, whereas only one environmental strain (11.1%, 1/9) was resistant to ceftazidime and meropenem, respectively. In contrast, resistance to trimethoprim/sulfamethoxazole (33.3%, 3/9) and cefotaxime (33.3%, 3/9) was relatively higher in environmental strains than in clinical strains (21.4% for trimethoprim/sulfamethoxazole and 14.3% for cefotaxime) (Table 1 ). Table 1 Phenotypic drug susceptibility test for V. vulnificus strains Antibiotic class Drugs Environment (n = 8) Clinical (n = 14) S I R S I R Fluoroquinolones CIP 8 (100%) 0 0 14 (100%) 0 0 Cephalosporins FOX 4 (50)% 2 (25%) 2 (25%) 9 (64.3%) 3 (21.4%) 2 (14.3%) FEP 7 (87.5%) 0 1 (12.5%) 9 (64.3%) 0 5 (35.7%) Amphenicols CHL 8 (100%) 0 0 13 (92.9%) 0 1 (7.1%) Aminoglycosides GEN 8 (100%) 0 0 14 (100%) 0 0 Tetracyclines TET 8 (100%) 0 0 14 (100%) 0 0 Quinolones NAL 8 (100%) 0 0 14 (100%) 0 0 Cephems CAZ 7 (87.5%) 0 1 (12.5%) 10 (71.4%) 0 4 (28.6%) CTX 5 (62.5%) 0 3 (37.5%) 12 (85.7%) 0 2 (14.3%) Carbapenem MEM 7 (87.5%) 0 1 (12.5%) 10 (71.4%) 0 4 (28.6%) Penicillin AMP 7 (87.5%) 0 1 (12.5%) 13 (92.9%) 0 1 (7.1%) AUG2 7 (87.5%) 0 1 (12.5%) 14 (100%) 0 0 Polymyxin COL 0 1 (12.5%) 7 (87.5%) 0 3 (21.4%) 11 (78.6%) Sulfonamide FIS 8 (100%) 0 0 14 (100%) 0 0 SXT 5 (62.5%) 0 3 (37.5%) 11 (78.6%) 0 3 (21.4%) CIP: ciprofloxacin, FOX: cefoxitin, FEP: cefepime, CHL: chloramphenicol, GEN: gentamicin, TET: tetracycline, NAL: nalidixic acid, CAZ: ceftazidime, CTX: cefotaxime, MEM: meropenem, AMP: ampicillin, AMC: amoxicillin/clavulanic acid, COL: colistin, FIS: sulfisoxazole, SXT: trimethoprim/sulfamethoxazole, S: susceptible, I: intermediate, R: resistant All strains harbored efflux pump related gene CRP. In addition, norA , ant(4’)-Ia , msrA , fusB , blaZ , and dfrC were found in CMCVV2817 (Supplementary Table S1 ). However, we did not observe any association between the detected resistance genes and phenotypic resistance. Multiple Antibiotics Resistance index The MAR index was calculated only for strains with phenotypic resistance to at least 2 antibiotics. The most common resistance pattern found in 11 strains included cefoxitin, cefotaxime, colistin, cefepime, ceftazidime, meropenem, and trimethoprim/sulfamethoxazole, with a MAR index of 0.5 (Table 2 ). This pattern was observed more frequently in clinical strains (46.7%, 7/15) than in environmental strains (36.4%, 4/11). However, as the number of antibiotics decreased in the resistance pattern, the gaps between clinical and environmental strains also decreased. Of note, for cefoxitin and cefotaxime (MAR index of 0.1), the resistance rate in environmental strains (18.2%) was higher than in clinical strains (13.3%) (Table 2 ). These results suggest that clinical strains tend to develop resistance to a broader range of antibiotics, whereas environmental strains may show similar or higher resistance rates when fewer antibiotics are involved. Table 2 Multiple Antibiotic Resistance (MAR) index of V. vulnificus strains Resistance pattern Frequency of occurrence Source No. (%) MAR index Cefoxitin + Cefotaxime + Colistin + Cefepime + Ceftazidime + Meropenem + Trimethoprim/sulfamethoxazole 11 Environment 4/11 (36.4%) 0.5 Human 7/15 (46.7%) Cefoxitin + Cefotaxime + Colistin + Cefepime + Ceftazidime + Meropenem 9 Environment 3/11 (27.3%) 0.4 Human 6/15 (40.0%) Cefoxitin + Cefotaxime + Colistin + Cefepime + Ceftazidime 8 Environment 3/11 (27.3%) 0.3 Human 5/15 (33.3%) Cefoxitin + Cefotaxime + Colistin + Cefepime 7 Environment 3/11 (27.3%) 0.3 Human 4/15 (26.7%) Cefoxitin + Cefotaxime + Colistin 6 Environment 3/11 (27.3%) 0.2 Human 3/15 (20.0%) Cefoxitin + Cefotaxime 4 Environment 2/11 (18.2%) 0.1 Human 2/15 (13.3%) Virulence factors To distinguish between clinical (C-type) and environmental (E-type) V. vulnificus , we analyzed the virulence correlated genes (vcg) and found 19 vcg-C and 7 vcg-E types (Supplementary Table S1 ). Six vcg-C and five vcg-E types were identified in environmental strains, while 13 vcg-C and two vcg-E types were identified in clinical strains. The high proportion of vcg-C types in environmental strains (54.5%, 6/11) suggests the presence of potentially virulent strains in the environment. Notably, the presence of vcg-E types in human isolates (13.3%, 2/15) indicates that vcg type alone may not be a definitive predictor of pathogenicity. We identified 201 virulence genes in 26 V. vulnificus strains, with an average of 125.6 (110–161) detected per strain (Supplementary Table S1 ). Among these, 85 genes were ubiquitous, while the remaining 116 genes were detected at variable rates: 36 genes were found in 80–99% of the isolates, two in 60–80%, one in 40–60%, four in 20–40%, and 73 in 0–20% (Fig. 2 ). Overall, the virulence gene counts per strain for each virulence functional class were similar between the two groups (124.3 versus 126.5), indicating comparable virulence potential. Furthermore, clinical strains showed a similar number of virulence genes in several key categories compared to environmental strains, including adherence (18.5 versus 18.4), antiphagocytosis (15.5 versus 14.4), and chemotaxis/motility (54.5 vs. 53.9), suggesting that their host interaction and immune evasion abilities may not differ significantly (Table 3 ). A clinical strain, CMCVV2817 (novel ST type), contained virulence genes not found in environmental strains, including those for acid resistance, anaerobic respiration, copper uptake, intracellular survival, invasion, and surface protein anchoring (Supplementary Table S1 ). Table 3 Differences in the number of Virulence Factors between clinical and environmental isolates Virulence class (number of related genes) Environmental isolates (n = 11) Clinical isolates (n = 15) Virulence genes per strain(%)* Virulence genes per strain(%)* Acid resistance(n = 2) 0 0 2 0.13 Adherence(n = 51) 202 18.36 277 18.47 Anaerobic respiration(n = 1) 0 0 1 0.07 Antiphagocytosis (n = 32) 158 14.36 233 15.53 Chemotaxis and motility(n = 56) 593 53.91 818 54.53 Copper uptake(n = 1) 0 0 1 0.07 Endotoxin(n = 2) 1 0.09 1 0.07 Enzyme(n = 7) 11 1 20 1.33 Immune evasion(n = 6) 11 1 19 1.27 Intracellular survival(n = 2) 0 0 1 0.07 Invasion(n = 1) 0 0 1 0.07 Iron uptake(n = 25) 166 15.09 227 15.13 Quorum sensing(n = 2) 10 0.91 15 1 Regulation(n = 2) 0 0 2 0.13 Secretion system(n = 86) 145 13.18 182 12.13 Serum resistance(n = 1) 1 0.09 0 0 Stress adaptation(n = 2) 1 0.09 1 0.07 Surface protein anchoring(n = 1) 0 0 1 0.07 Toxin(n = 14) 65 5.91 90 6 Others(n = 4) 3 0.27 5 0.33 *Total VF genes divided by the number of isolates Discussion Although V. vulnificus inhabits coastal environments worldwide, research on this pathogen remains relatively limited [ 29 ]. This study provides comprehensive insights into the genetic diversity, virulence factors, and antibiotic resistance profiles of V. vulnificus strains isolated from environmental and clinical sources. The wide diversity of STs and the presence of both vcg-C and vcg-E types in both environmental and clinical strains of this study indicate that the vcg typing system alone cannot definitively predict pathogenicity. No quantitative and qualitative differences in virulence factors were found between environmental and clinical strains, and the high ST diversity, especially the identification of novel STs, further complicates the understanding of the pathogenicity potential of V. vulnificus . Environmental and clinical strains of Vibrio spp. have similar virulence gene profiles, making it difficult to distinguish them based solely on the presence of virulence genes [ 30 , 31 ], which is consistent with this study. However, we identified unique virulence factors related to acid resistance, anaerobic respiration, intracellular survival, and invasion in one clinical strain with a novel ST (CMCVV2817), indicating potential adaptations for survival in the human host. These results suggest that it may have acquired specific genetic adaptations that enhance its pathogenicity. The accumulation of multiple virulence factors in a single strain could potentially indicate an increased risk of severe infections, making it a critical target for future monitoring and research. While polymyxin class resistance has been reported in previous a study of V. vulnificus [ 32 ], both clinical and environmental strains in this study were remarkably not-susceptible to third-generation cephalosporins, including ceftazidime and cefotaxime. Considering that the CDC recommends the use of a third-generation cephalosporin for the treatment of V. vulnificus wound infections [ 33 ], this finding indicates potential therapeutic challenges in the management of V. vulnificus infections. In this study, 11.1% (1/9) of environmental strains and 28.6% (4/14) of clinical strains were resistant to ceftazidime. For cefotaxime, 33.3% (3/9) of environmental strains and 14.3% (2/14) of clinical strains were classified as resistant. In addition, the MAR index ranged from 0.1 to 0.5, indicating varying levels of MAR. When the MAR index exceeds 0.2, contamination from high-risk sources may pose a health risk to humans [ 32 ]. Taken together, these findings support the need for stringent surveillance programs to track the emergence and spread of resistant V. vulnificus strains. The study is limited by a small sample size that may not fully represent the broader distribution of V. vulnificus strains. Future studies with larger sample sizes and more diverse geographic locations are needed. Nonetheless, our results provide valuable insight into the broad spectrum of sequence types with novel STs, virulence factors, and antibiotic resistance in V. vulnificus , contributing to a better understanding of its pathogenic potential in clinical and environmental settings and informing treatment and surveillance strategies. Declarations Ethics approval and consent to participate This study was approved by the Institutional Review Board of the Catholic University of Korea College of Medicine (approval number: MC24SASI0019), and the requirement for informed consent was waived. Consent for publication Not applicable Availability of data and material The datasets generated and analyzed during the study are available in Supplementary Tables. The raw FASTQ files are available from the corresponding authors upon reasonable request. Competing interests The authors declare no competing interests. Funding This research was supported by grants from the Ministry of Food and Drug Safety (22192MFDS021). Authors' contributions Conceptualization: YJC and SHJ; Data collection and processing: JL, JIS, and KTP; Formal analysis and interpretation: JL and JIS; Writing – Original Draft: JL, YJC, and SHJ. All authors read and approved the final version of the manuscript. Acknowledgments The pathogen resources for this study were provided by Gyeongsang National University Hospital Branch of the National Culture Collection for Pathogens (GNUH-NCCP). We also appreciate the support by Basic Medical Science Facilitation Program through the Catholic Medical Center of the Catholic University of Korea funded by the Catholic Education Foundation. References Leng F, Lin S, Wu W, Zhang J, Song J, Zhong M. Epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection: a case report and literature review. Eur J Clin Microbiol Infect Dis (2019) 38:1999–2004. 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Supplementary Files SupplementaryTableS1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Oct, 2024 Reviews received at journal 15 Oct, 2024 Reviews received at journal 14 Oct, 2024 Reviewers agreed at journal 13 Oct, 2024 Reviewers agreed at journal 12 Oct, 2024 Reviewers agreed at journal 10 Oct, 2024 Reviewers invited by journal 10 Oct, 2024 Editor assigned by journal 10 Oct, 2024 Submission checks completed at journal 10 Oct, 2024 First submitted to journal 09 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5229211","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370986059,"identity":"8832478d-3805-40e8-b83a-ba83a4c2981b","order_by":0,"name":"Jinkyeong Lee","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Jinkyeong","middleName":"","lastName":"Lee","suffix":""},{"id":370986060,"identity":"d14f8cd7-a7c7-4259-ae6e-b954e7947018","order_by":1,"name":"Jeong-Ih Shin","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Jeong-Ih","middleName":"","lastName":"Shin","suffix":""},{"id":370986061,"identity":"c1a1f964-1403-4c85-b97b-e3149a17c9a7","order_by":2,"name":"Woo Young Cho","email":"","orcid":"","institution":"ConnectaGen","correspondingAuthor":false,"prefix":"","firstName":"Woo","middleName":"Young","lastName":"Cho","suffix":""},{"id":370986062,"identity":"3f95b56f-1e93-47f1-9b3c-fc928f9c582e","order_by":3,"name":"Kun Take Park","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"Take","lastName":"Park","suffix":""},{"id":370986063,"identity":"6e35c167-5f1b-464f-a66a-7878243f4fee","order_by":4,"name":"Yeun-Jun Chung","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Yeun-Jun","middleName":"","lastName":"Chung","suffix":""},{"id":370986064,"identity":"a936a839-a509-4f86-83f5-3587b7e33459","order_by":5,"name":"Seung-Hyun Jung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACAxDxAcxgbjgAFuIhQgvjDAYGCQMGRhK0MPNAtTAQpcVcIsfssU2NXZ05e2PjgR8MdvIMPGcf4NViOSPH3DjnWLKEZc/BhoM9DMmGDbztBvgddiPHTDq3gVnC4EZiw2GgIxMY+NkI+AWkxbKhHqalnkgtjA2HYVoOJzDwthHQcuZZmWTPseOSG86A/GJw3LCN5xgBLceTt0n8qKnmNzjefPjDj4pqeX6eNPxaGAQSUExgYCDgEyDgP0BQySgYBaNgFIx0AADRiUGQX3+iLgAAAABJRU5ErkJggg==","orcid":"","institution":"Graduate School of The Catholic University of Korea","correspondingAuthor":true,"prefix":"","firstName":"Seung-Hyun","middleName":"","lastName":"Jung","suffix":""}],"badges":[],"createdAt":"2024-10-09 04:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5229211/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5229211/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68318650,"identity":"b4da7f15-bca7-4400-9121-9c4e70baa20a","added_by":"auto","created_at":"2024-11-06 04:00:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":522267,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood tree of 26 \u003cem\u003eV. vulnificus\u003c/em\u003e strains collected from South Korea. Sample sources are represented by colored circles, while other phenotypic and genotypic characteristics are represented by colored squares. ARG: antimicrobial resistance gene, DST: drug susceptibility test.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229211/v1/bdf7c98a3673ca0411e175f6.jpg"},{"id":68318651,"identity":"0aa72d99-6783-425d-b5ea-e579558169be","added_by":"auto","created_at":"2024-11-06 04:00:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230734,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of 201 virulence genes present in 26 \u003cem\u003eV. vulnificus\u003c/em\u003e isolates. The X-axis and Y-axis represent the virulence functional categories and sample sources, respectively.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229211/v1/362026539c7959a610b15142.jpg"},{"id":68319076,"identity":"4e0a5f74-1978-45c3-a8dd-d9abe506d872","added_by":"auto","created_at":"2024-11-06 04:08:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1386639,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5229211/v1/54f6e848-aa59-4089-940e-8ff6506c3ce9.pdf"},{"id":68318648,"identity":"5167f470-3c6a-4aff-a329-dad20763222c","added_by":"auto","created_at":"2024-11-06 04:00:43","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":42187,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5229211/v1/2cbcec2711fc0bc630662954.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic characteristics of Vibrio vulnificus isolated from clinical and environmental sources","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eVibrio vulnificus\u003c/em\u003e is a gram-negative aquatic bacterium that is naturally found in warm coastal waters, especially in regions with higher salinity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It belongs to the \u003cem\u003eVibrionaceae\u003c/em\u003e family and is closely related to \u003cem\u003eV. cholerae\u003c/em\u003e, the causative agent of cholera. \u003cem\u003eV. vulnificus\u003c/em\u003e is known as an opportunistic pathogen commonly transmitted by consumption of seafood, particularly raw or undercooked shellfish like oysters [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It can also rarely enter the body through open wounds when exposed to contaminated water, leading to invasive infections [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eV. vulnificus\u003c/em\u003e infections can range from gastroenteritis to life-threatening septicemia, which has the highest fatality rate among the many foodborne pathogens [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While healthy individuals may experience mild symptoms, the bacterium poses a significant risk to those with weakened immune systems, particularly those with liver disease or diabetes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In South Korea, 69 cases of \u003cem\u003eV. vulnificus\u003c/em\u003e sepsis were reported in 2023, resulting in 27 deaths and a fatality rate of 39.1% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVirulence factors contribute to the infection capability of \u003cem\u003eV. vulnificus\u003c/em\u003e, and therefore play an important role in its pathogenicity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The \u003cem\u003evvhA\u003c/em\u003e gene is related to the virulence of \u003cem\u003eV. vulnificus\u003c/em\u003e and is used by public health authorities to assess human pathogenicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, the \u003cem\u003evcgC\u003c/em\u003e and \u003cem\u003evcgE\u003c/em\u003e genes have been reported as useful markers for distinguishing strains that originate from human or environmental cases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recent studies indicate that \u003cem\u003evcgC\u003c/em\u003e is more frequently found in strains associated with human infections, while \u003cem\u003evcgE\u003c/em\u003e is widely used to identify strains originating from the environment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Indeed, \u003cem\u003evcgC\u003c/em\u003e produces proteins involved in invading the host, whereas \u003cem\u003evcgE\u003c/em\u003e produces proteins that respond to environmental stress and evade immune responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, there is no definitive consensus on whether a specific virulence factor can distinguish between pathogenic and non-pathogenic strains of \u003cem\u003eV. vulnificus\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition to virulence factors, antibiotic resistance is another important factor associated with \u003cem\u003eV. vulnificus\u003c/em\u003e infections [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It can spread by both clonal transmission and horizontal gene transfer, which makes infections difficult to treat and control [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, it is necessary to analyze the virulence profile of infected isolates to determine their pathogenic potential and monitor antibiotic resistance.\u003c/p\u003e \u003cp\u003eIn this study, 26 \u003cem\u003eV. vulnificus\u003c/em\u003e strains collected from coastal waters or infected patients were subjected to whole-genome sequencing (WGS) to investigate (i) their molecular subtypes and diversity, and (ii) their genomic characteristics, including antimicrobial resistance genes and virulence factors.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial collection and culture\u003c/h2\u003e \u003cp\u003eA total of 11 \u003cem\u003eV. vulnificus\u003c/em\u003e strains were collected in South Korea between August 2022 and September 2022 as part of the national foodborne pathogen surveillance research. The \u003cem\u003eV. vulnificus\u003c/em\u003e strains were isolated from coastal waters in five provinces of South Korea (3 isolated from the Namhae-gun, Gyeongsangnam-do, 2 isolated from Geoje-si, Gyeongsangnam-do, 3 isolated from Mokpo-si, Jeollanam-do, 2 isolated from Busan, and 1 isolated from Ulsan). Additionally, 15 clinical strains of \u003cem\u003eV. vulnificus\u003c/em\u003e isolated during the period of 2010\u0026ndash;2020 were obtained from nationwide bacterial biobanks in Korea (GNUH-NCCP). Of the 15 clinical strains, 11 were isolated from blood, while the remaining four were isolated from pus. The details of each isolate are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The \u003cem\u003eV. vulnificus\u003c/em\u003e strains were cultured on Blood Agar Plates at 37\u0026deg;C under ambient air conditions for 18 to 24 hours and stored at -80\u0026deg;C until further use analysis. This study was approved by the Institutional Review Board of the Catholic University of Korea College of Medicine (approval number: MC24SASI0019), and the requirement for informed consent was waived.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDrug susceptibility testing\u003c/h3\u003e\n\u003cp\u003eAntimicrobial susceptibility testing for 15 drugs (gentamicin [GEN], meropenem [MEM], chloramphenicol [CHL], cefoxitin [FOX], cefotaxime [CTX], ceftazidime [CAZ], cefepime [FEP], ampicillin [AMP], colistin [COL], tetracycline [TET], amoxicillin/clavulanic acid [AUG2], nalidixic acid [NAL], ciprofloxacin [CIP], sulfisoxazole [FIS], and trimethoprim/sulfamethoxazole [SXT]) was performed using the Sensititre TM KRNV6F Kit (Thermo Fisher Scientific, Waltham, MA, USA) with the microbroth dilution method. Antimicrobial breakpoints were determined according to the Clinical and Laboratory Standards Institute guideline [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], with the minimum inhibitory concentration (MIC) range not in the \u0026lsquo;S\u0026rsquo; being resistance. \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 was used as a standard strain. For antibiotics without established resistance determination criteria (COL, NAL, and FIS), MIC50 and MIC90 values were used to assess susceptibility. Three strains (two environmental and one clinical) were not tested for drug susceptibility due to the lack of regrowth activity.\u003c/p\u003e \u003cp\u003eThe Multiple Antibiotics Resistance (MAR) method, as described by Osundiya et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], was employed to generate the MAR index [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The MAR index is calculated as the ratio of the number of antibiotics to which the organism is resistant divided by the total number of antibiotics to which the organism is exposed. A MAR index of \u0026ge;\u0026thinsp;0.2 was defined as a high risk of the used antibiotics [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eWhole-genome sequencing analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted using iDetect gDNA Prep Kit for Microbes (ConnectaGen, Hanam, South Korea). The extracted DNA was evaluated by NanoDrop (Thermo Fisher Scientific) and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). NGS sequencing libraries were prepared using a TruSeq Nano DNA sample preparation kit (Illumina, San Diego, CA, USA). Sequencing libraries were then pooled and subjected to sequencing on the Illumina NovaSeq 6000 system. Sequencing adapters and low-quality bases were trimmed using Trimmomatic. The trimmed reads were assembled using SPAdes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and assembled contigs were annotated and evaluated using Prokka [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and Quast [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], respectively.\u003c/p\u003e \u003cp\u003eKraken2 was used to confirm the species of each strain [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Multi-locus sequence types (STs) were determined using the MLST tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/mlst\u003c/span\u003e\u003cspan address=\"https://github.com/tseemann/mlst\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the allelic profiles of the 10 housekeeping genes (\u003cem\u003eglp\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003emetG\u003c/em\u003e, \u003cem\u003epurM\u003c/em\u003e, \u003cem\u003edtdS\u003c/em\u003e, \u003cem\u003elysA\u003c/em\u003e, \u003cem\u003epntA\u003c/em\u003e, \u003cem\u003epyrC\u003c/em\u003e, and \u003cem\u003etnaA\u003c/em\u003e) from the PubMLST database [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The newly discovered STs were submitted to PubMLST database. Acquisition of antimicrobial resistance genes and virulence factors were determined using Abricate employing the Comprehensive Antibiotic Resistance Database [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Virulence Factor Database [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The virulence correlated gene (vcg) typing was performed using in silico PCR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.org/sms2/pcr_products.html\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.org/sms2/pcr_products.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with previously reported primer sets [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA core single nucleotide polymorphism (SNP) alignment for 26 \u003cem\u003eV. vulnificus\u003c/em\u003e genomes was performed using snippy (\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). A maximum likelihood phylogenetic tree based on core SNPs was constructed using RAxML with a generalized time-reversible gamma model [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The resulting phylogenetic tree was visualized in Microreact [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStatistical test\u003c/h3\u003e\n\u003cp\u003eCategorical variables were compared using the Fisher\u0026rsquo;s exact test. All tests were two-tailed, and \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were performed using SPSS software (IBM Corp., Armonk, NY, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample distributions\u003c/h2\u003e \u003cp\u003eOf the 26 \u003cem\u003eV. vulnificus\u003c/em\u003e strains isolated from 2010 to 2022, 11 environmental strains (42.3%) were collected from the coastal waters of Gyeongsanam-do, Jeollanam-do, Busan, and Ulsan, and 15 clinical strains (57.7%) were collected from Gyeongsangnam-do, South Korea. The phylogenetic tree showed relatively clear differences between clinical and environmental strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Clinical strains exhibited a high degree of phylogenetic coherence, with 70% (11/15) forming a monophyletic clade, suggesting a conserved genetic lineage and potential clonal expansion. In contrast, environmental strains were phylogenetically dispersed across multiple distinct branches, reflecting a greater degree of genetic heterogeneity and suggesting a broader evolutionary divergence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSequence types\u003c/h3\u003e\n\u003cp\u003eThrough WGS analysis, we obtained high-quality sequence reads for 26 \u003cem\u003eV. vulnificus\u003c/em\u003e strains. The average N50 value was 375.8Kb (range 149.0Kb-770.0Kb), and the average genome size was 5.17Mb (range 4.83Mb-7.53Mb). A total of 20 STs were identified, of which eight STs were newly discovered based on housekeeping genes: ST705 (n\u0026thinsp;=\u0026thinsp;1), ST711 (n\u0026thinsp;=\u0026thinsp;1), ST714 (n\u0026thinsp;=\u0026thinsp;1), ST715 (n\u0026thinsp;=\u0026thinsp;1), ST717 (n\u0026thinsp;=\u0026thinsp;1), ST718 (n\u0026thinsp;=\u0026thinsp;1), ST712 (n\u0026thinsp;=\u0026thinsp;3), and ST713 (n\u0026thinsp;=\u0026thinsp;3) (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The new alleles and STs were submitted to the PubMLST database for assignment. Four STs were recurrently detected (ST449, n\u0026thinsp;=\u0026thinsp;2; ST648, n\u0026thinsp;=\u0026thinsp;2; ST712, n\u0026thinsp;=\u0026thinsp;3; and ST713, n\u0026thinsp;=\u0026thinsp;3), while the remaining STs were singletons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results suggest that the genetic background of \u003cem\u003eV. vulnificus\u003c/em\u003e is largely heterogenous and that various STs have yet to be discovered.\u003c/p\u003e\n\u003ch3\u003eAntimicrobial resistance\u003c/h3\u003e\n\u003cp\u003ePhenotypic drug susceptibility testing was performed on 15 antibiotics, revealing prominent colistin resistance in both environmental (8/9, 88.9%) and clinical (11/14, 78.6%) strains. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, clinical strains exhibited higher resistance to cefepime, ceftazidime, and meropenem than environmental strains. Five clinical strains (35.7%, 5/14) were resistant to cefepime, whereas only one environmental strain (11.1%, 1/9) showed resistance. Similarly, 28.6% (4/14) of clinical strains were resistant to each of ceftazidime and meropenem, whereas only one environmental strain (11.1%, 1/9) was resistant to ceftazidime and meropenem, respectively. In contrast, resistance to trimethoprim/sulfamethoxazole (33.3%, 3/9) and cefotaxime (33.3%, 3/9) was relatively higher in environmental strains than in clinical strains (21.4% for trimethoprim/sulfamethoxazole and 14.3% for cefotaxime) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhenotypic drug susceptibility test for \u003cem\u003eV. vulnificus\u003c/em\u003e strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibiotic class\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDrugs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eEnvironment (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eClinical (n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluoroquinolones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCephalosporins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (50)%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (64.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3 (21.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2 (14.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFEP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (64.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (35.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmphenicols\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCHL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13 (92.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 (7.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminoglycosides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGEN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetracyclines\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTET\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuinolones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCephems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10 (71.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (28.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12 (85.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2 (14.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbapenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10 (71.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (28.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePenicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13 (92.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 (7.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAUG2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymyxin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCOL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3 (21.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11 (78.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSulfonamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSXT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11 (78.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3 (21.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eCIP: ciprofloxacin, FOX: cefoxitin, FEP: cefepime, CHL: chloramphenicol, GEN: gentamicin, TET: tetracycline, NAL: nalidixic acid, CAZ: ceftazidime, CTX: cefotaxime, MEM: meropenem, AMP: ampicillin, AMC: amoxicillin/clavulanic acid, COL: colistin, FIS: sulfisoxazole, SXT: trimethoprim/sulfamethoxazole, S: susceptible, I: intermediate, R: resistant\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll strains harbored efflux pump related gene CRP. In addition, \u003cem\u003enorA\u003c/em\u003e, \u003cem\u003eant(4\u0026rsquo;)-Ia\u003c/em\u003e, \u003cem\u003emsrA\u003c/em\u003e, \u003cem\u003efusB\u003c/em\u003e, \u003cem\u003eblaZ\u003c/em\u003e, and \u003cem\u003edfrC\u003c/em\u003e were found in CMCVV2817 (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, we did not observe any association between the detected resistance genes and phenotypic resistance.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMultiple Antibiotics Resistance index\u003c/h2\u003e \u003cp\u003eThe MAR index was calculated only for strains with phenotypic resistance to at least 2 antibiotics. The most common resistance pattern found in 11 strains included cefoxitin, cefotaxime, colistin, cefepime, ceftazidime, meropenem, and trimethoprim/sulfamethoxazole, with a MAR index of 0.5 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This pattern was observed more frequently in clinical strains (46.7%, 7/15) than in environmental strains (36.4%, 4/11). However, as the number of antibiotics decreased in the resistance pattern, the gaps between clinical and environmental strains also decreased. Of note, for cefoxitin and cefotaxime (MAR index of 0.1), the resistance rate in environmental strains (18.2%) was higher than in clinical strains (13.3%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results suggest that clinical strains tend to develop resistance to a broader range of antibiotics, whereas environmental strains may show similar or higher resistance rates when fewer antibiotics are involved.\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\u003eMultiple Antibiotic Resistance (MAR) index of \u003cem\u003eV. vulnificus\u003c/em\u003e strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResistance pattern\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrequency of \u003c/p\u003e \u003cp\u003eoccurrence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMAR index\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCefoxitin + Cefotaxime\u0026thinsp;+\u0026thinsp;Colistin\u0026thinsp;+\u0026thinsp;Cefepime\u0026thinsp;+\u0026thinsp;Ceftazidime\u0026thinsp;+\u0026thinsp;Meropenem\u0026thinsp;+\u0026thinsp;Trimethoprim/sulfamethoxazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnvironment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4/11 (36.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7/15 (46.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCefoxitin + Cefotaxime\u0026thinsp;+\u0026thinsp;Colistin\u0026thinsp;+\u0026thinsp;Cefepime\u0026thinsp;+\u0026thinsp;Ceftazidime\u0026thinsp;+\u0026thinsp;Meropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnvironment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3/11 (27.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6/15 (40.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCefoxitin + Cefotaxime\u0026thinsp;+\u0026thinsp;Colistin\u0026thinsp;+\u0026thinsp;Cefepime\u0026thinsp;+\u0026thinsp;Ceftazidime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnvironment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3/11 (27.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5/15 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCefoxitin + Cefotaxime\u0026thinsp;+\u0026thinsp;Colistin\u0026thinsp;+\u0026thinsp;Cefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnvironment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3/11 (27.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4/15 (26.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCefoxitin + Cefotaxime\u0026thinsp;+\u0026thinsp;Colistin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnvironment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3/11 (27.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3/15 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCefoxitin + Cefotaxime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnvironment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2/11 (18.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2/15 (13.3%)\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\u003eVirulence factors\u003c/h2\u003e \u003cp\u003eTo distinguish between clinical (C-type) and environmental (E-type) \u003cem\u003eV. vulnificus\u003c/em\u003e, we analyzed the virulence correlated genes (vcg) and found 19 vcg-C and 7 vcg-E types (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Six vcg-C and five vcg-E types were identified in environmental strains, while 13 vcg-C and two vcg-E types were identified in clinical strains. The high proportion of vcg-C types in environmental strains (54.5%, 6/11) suggests the presence of potentially virulent strains in the environment. Notably, the presence of vcg-E types in human isolates (13.3%, 2/15) indicates that vcg type alone may not be a definitive predictor of pathogenicity.\u003c/p\u003e \u003cp\u003eWe identified 201 virulence genes in 26 \u003cem\u003eV. vulnificus\u003c/em\u003e strains, with an average of 125.6 (110\u0026ndash;161) detected per strain (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among these, 85 genes were ubiquitous, while the remaining 116 genes were detected at variable rates: 36 genes were found in 80\u0026ndash;99% of the isolates, two in 60\u0026ndash;80%, one in 40\u0026ndash;60%, four in 20\u0026ndash;40%, and 73 in 0\u0026ndash;20% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Overall, the virulence gene counts per strain for each virulence functional class were similar between the two groups (124.3 versus 126.5), indicating comparable virulence potential. Furthermore, clinical strains showed a similar number of virulence genes in several key categories compared to environmental strains, including adherence (18.5 versus 18.4), antiphagocytosis (15.5 versus 14.4), and chemotaxis/motility (54.5 vs. 53.9), suggesting that their host interaction and immune evasion abilities may not differ significantly (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A clinical strain, CMCVV2817 (novel ST type), contained virulence genes not found in environmental strains, including those for acid resistance, anaerobic respiration, copper uptake, intracellular survival, invasion, and surface protein anchoring (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\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\u003eDifferences in the number of Virulence Factors between clinical and environmental isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVirulence class\u003c/p\u003e \u003cp\u003e(number of related genes)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eEnvironmental isolates (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eClinical isolates (n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVirulence genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eper strain(%)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVirulence genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eper strain(%)*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid resistance(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdherence(n\u0026thinsp;=\u0026thinsp;51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnaerobic respiration(n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntiphagocytosis (n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemotaxis and motility(n\u0026thinsp;=\u0026thinsp;56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCopper uptake(n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndotoxin(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnzyme(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImmune evasion(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntracellular survival(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInvasion(n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIron uptake(n\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuorum sensing(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegulation(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecretion system(n\u0026thinsp;=\u0026thinsp;86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum resistance(n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStress adaptation(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurface protein anchoring(n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eToxin(n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e*Total VF genes divided by the number of isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough \u003cem\u003eV. vulnificus\u003c/em\u003e inhabits coastal environments worldwide, research on this pathogen remains relatively limited [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This study provides comprehensive insights into the genetic diversity, virulence factors, and antibiotic resistance profiles of \u003cem\u003eV. vulnificus\u003c/em\u003e strains isolated from environmental and clinical sources. The wide diversity of STs and the presence of both vcg-C and vcg-E types in both environmental and clinical strains of this study indicate that the vcg typing system alone cannot definitively predict pathogenicity. No quantitative and qualitative differences in virulence factors were found between environmental and clinical strains, and the high ST diversity, especially the identification of novel STs, further complicates the understanding of the pathogenicity potential of \u003cem\u003eV. vulnificus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eEnvironmental and clinical strains of \u003cem\u003eVibrio\u003c/em\u003e spp. have similar virulence gene profiles, making it difficult to distinguish them based solely on the presence of virulence genes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which is consistent with this study. However, we identified unique virulence factors related to acid resistance, anaerobic respiration, intracellular survival, and invasion in one clinical strain with a novel ST (CMCVV2817), indicating potential adaptations for survival in the human host. These results suggest that it may have acquired specific genetic adaptations that enhance its pathogenicity. The accumulation of multiple virulence factors in a single strain could potentially indicate an increased risk of severe infections, making it a critical target for future monitoring and research.\u003c/p\u003e \u003cp\u003eWhile polymyxin class resistance has been reported in previous a study of \u003cem\u003eV. vulnificus\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], both clinical and environmental strains in this study were remarkably not-susceptible to third-generation cephalosporins, including ceftazidime and cefotaxime. Considering that the CDC recommends the use of a third-generation cephalosporin for the treatment of \u003cem\u003eV. vulnificus\u003c/em\u003e wound infections [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], this finding indicates potential therapeutic challenges in the management of \u003cem\u003eV. vulnificus\u003c/em\u003e infections. In this study, 11.1% (1/9) of environmental strains and 28.6% (4/14) of clinical strains were resistant to ceftazidime. For cefotaxime, 33.3% (3/9) of environmental strains and 14.3% (2/14) of clinical strains were classified as resistant. In addition, the MAR index ranged from 0.1 to 0.5, indicating varying levels of MAR. When the MAR index exceeds 0.2, contamination from high-risk sources may pose a health risk to humans [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Taken together, these findings support the need for stringent surveillance programs to track the emergence and spread of resistant \u003cem\u003eV. vulnificus\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003eThe study is limited by a small sample size that may not fully represent the broader distribution of \u003cem\u003eV. vulnificus\u003c/em\u003e strains. Future studies with larger sample sizes and more diverse geographic locations are needed. Nonetheless, our results provide valuable insight into the broad spectrum of sequence types with novel STs, virulence factors, and antibiotic resistance in \u003cem\u003eV. vulnificus\u003c/em\u003e, contributing to a better understanding of its pathogenic potential in clinical and environmental settings and informing treatment and surveillance strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of the Catholic University of Korea College of Medicine (approval number: MC24SASI0019), and the requirement for informed consent was waived.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the study are available in Supplementary Tables. The raw FASTQ files are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the Ministry of Food and Drug Safety (22192MFDS021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: YJC and SHJ; Data collection and processing: JL, JIS, and KTP; Formal analysis and interpretation: JL and JIS; Writing \u0026ndash; Original Draft: JL, YJC, and SHJ. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pathogen resources for this study were provided by Gyeongsang National University Hospital Branch of the National Culture Collection for Pathogens (GNUH-NCCP). We also appreciate the support by Basic Medical Science Facilitation Program through the Catholic Medical Center of the Catholic University of Korea funded by the Catholic Education Foundation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeng F, Lin S, Wu W, Zhang J, Song J, Zhong M. Epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection: a case report and literature review. Eur J Clin Microbiol Infect Dis (2019) 38:1999\u0026ndash;2004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuehn S, Eichhorn C, Urmersbach S, Breidenbach J, Bechlars S, Bier N, et al. Pathogenic vibrios in environmental, seafood and clinical sources in Germany. Int J Med Microbiol (2014) 304:843\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaker-Austin C, Oliver JD. Vibrio vulnificus. Trends Microbiol (2020) 28:81\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMenon MP, Yu PA, Iwamoto M, Painter J. Pre-existing medical conditions associated with Vibrio vulnificus septicaemia. Epidemiol Infect (2014) 142:878\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark S, Park S, Won J, Kim H, Yang S, Yang J. Epidemiological Characteristics of Cases and Deaths of Vibrio vulnificus Sepsis, 2023. 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Vibrio vulnificus genome suggests two distinct ecotypes. Environ Microbiol Rep (2010) 2:128\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrom MS, Paranjpye RN. Epidemiology and pathogenesis of Vibrio vulnificus. Microbes Infect (2000) 2:177\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaker-Austin C, McArthur JV, Lindell AH, Wright MS, Tuckfield RC, Gooch J, et al. Multi-site analysis reveals widespread antibiotic resistance in the marine pathogen Vibrio vulnificus. Microb Ecol (2009) 57:151\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElmahdi S, DaSilva LV, Parveen S. Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: A review. Food Microbiol (2016) 57:128\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM MKA-D, Abd Mutalib S, Abd Ghani M, Mohd Zaini NA, Ariffin AA. 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J Food Prot (2005) 68:2533\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGxalo O, Digban TO, Igere BE, Olapade OA, Okoh AI, Nwodo UU. Virulence and Antibiotic Resistance Characteristics of Vibrio Isolates From Rustic Environmental Freshwaters. Front Cell Infect Microbiol (2021) 11:732001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SE, Shin SU, Oh TH, Kim UJ, Darboe KS, Kang SJ, et al. Outcomes of Third-Generation Cephalosporin Plus Ciprofloxacin or Doxycycline Therapy in Patients with Vibrio vulnificus Septicemia: A Propensity Score-Matched Analysis. PLoS Negl Trop Dis (2019) 13:e0007478.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genomics-and-informatics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Genomics \u0026 Informatics](https://genomicsinform.biomedcentral.com/)","snPcode":"44342","submissionUrl":"https://submission.springernature.com/new-submission/44342/3","title":"Genomics \u0026 Informatics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vibrio vulnificus, Whole-Genome Sequencing, Antimicrobial Resistance, Virulence Factor, Multi-Locus Sequence Typing","lastPublishedDoi":"10.21203/rs.3.rs-5229211/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5229211/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eVibrio vulnificus\u003c/em\u003e is a gram-negative pathogenic bacterium that is transmitted through undercooked seafood or contaminated seawater, causing septicemia and wound infections. In this study, 15 clinical isolates and 11 environmental isolates were analyzed, revealing a total of 20 sequence types (STs), 8 of which were newly identified. Antibiotic resistance gene analysis revealed that the CRP gene was frequently present in both clinical and environmental isolates. Interestingly, the clinical and environmental isolates showed notable not-susceptible to third-generation cephalosporins, such as ceftazidime and cefotaxime, which may complicate treatment strategies for \u003cem\u003eV. vulnificus\u003c/em\u003e infections. The Multiple Antibiotic Resistance (MAR) index ranged from 0.1 to 0.5, with clinical isolates having a higher mean MAR index compared to environmental isolates, indicating a broader spectrum of resistance in clinical strains. No quantitative (124.3 versus 126.5) and qualitative (adherence, antiphagocytosis, and chemotaxis/motility) differences in virulence factors were observed between environmental and clinical strains. The molecular characteristics identified in this study may provide a basis for understanding the virulence of \u003cem\u003eVibrio vulnificus\u003c/em\u003e strains in South Korea and highlight the need for continuous surveillance of antibiotic resistance in emerging \u003cem\u003eV. vulnificus\u003c/em\u003e strains.\u003c/p\u003e","manuscriptTitle":"Genomic characteristics of Vibrio vulnificus isolated from clinical and environmental sources","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 04:00:39","doi":"10.21203/rs.3.rs-5229211/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-28T02:35:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-16T01:06:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-15T00:09:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163494235790179471204555146037298637371","date":"2024-10-13T07:27:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53777408410676330446188631449813266190","date":"2024-10-13T00:16:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37862729287339889450740958042254918293","date":"2024-10-11T00:22:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-11T00:17:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-10T22:16:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-10T22:15:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genomics \u0026 Informatics","date":"2024-10-09T04:33:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genomics-and-informatics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Genomics \u0026 Informatics](https://genomicsinform.biomedcentral.com/)","snPcode":"44342","submissionUrl":"https://submission.springernature.com/new-submission/44342/3","title":"Genomics \u0026 Informatics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df7f8fac-38ce-47bb-976e-717167328041","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-11-06T04:00:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-06 04:00:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5229211","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5229211","identity":"rs-5229211","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}