Genomic characterization of non-O1, non-O139 Vibrio cholerae causing rare clinical manifestation

Genomic characterization of non-O1, non-O139 Vibrio cholerae causing rare clinical manifestation | 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 Case Report Genomic characterization of non-O1, non-O139 Vibrio cholerae causing rare clinical manifestation Judit Henczkó, Márta Vargha, András Kállai, Katalin Kristóf, Ákos Tóth, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5935550/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Dec, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted 9 You are reading this latest preprint version Abstract Background Non-O1, non-O139 Vibrio cholerae is an uncommon cause of pneumonia, particularly following freshwater exposure. Non-O1, non-O139 Vibrio cholerae was identified from bronchoalveolar lavage through culture and quantitative polymerase chain reaction (qPCR) in Hungary. Epidemiological investigation traced the source of infection in a designated bathing site in a lake in Central Hungary, where non-O1,non-O139 Vibrio cholerae was isolated from surface water. Methods We conducted whole-genome sequencing and comparative genomic analysis on a clinical isolate (N=1) and three phenotypically distinct environmental isolates (N=3). In addition, we reviewed the available literature on pulmonary infections associated with Vibrio cholerae . Results Core genome multilocus sequence typing (cgMLST) revealed that the clinical and environmental isolates clustered together with zero allelic differences. Multilocus sequence typing (MLST) identified a new sequence type (ST1605), representing a novel combination of known allele variants. In silico analysis of antibiotic resistance genes identified the presence of bla CARB-7. Both the clinical and environmental isolates exhibited identical virulence gene profiles, reinforcing the hypothesis that the infection was acquired from the local water source. Conclusions This study represents the first investigation of a primary pulmonary Vibrio cholerae infection in Europe following a near-drowning event. While Vibrio vulnificus and Vibrio metschnikovii have been implicated in similar pneumonia cases, the precise virulence mechanisms of these species remain poorly understood. Although non-O1, non-O139 Vibrio cholerae infections linked to recreational water exposure are rare in Hungary (1-2 cases annually), this study underscores the importance of ongoing surveillance to detect potential outbreaks and inform public health responses. Non-O1 non-O139 Vibrio cholerae pneumonia WGS cgMLST Figures Figure 1 Figure 2 Background Non-toxigenic Vibrio cholerae (NTVC) occurs naturally in both marine and freshwater environments (1). In the last decades NTVC has been detected in different lakes, rivers, and seawaters, including the North Sea, the Baltic Sea, and the Mediterranean Sea. Humans can be infected through the consumption of contaminated fish or seafood, or through direct water exposure. The incidence of human NTVC cases is uncertain, but the number of reports on their prevalence in water environments and their human infections have been increasing in the recent years across Europe (1–4). There is a strong association between NTVC incidence and elevated water temperature, increased salinity or dissolved oxygen, and it is historically well known that Vibrio species have the fastest growth rates among bacteria, responding almost immediately to preferred environmental conditions(5). NTVC can be also isolated from different fish, mollusks, shrimps, water birds, mammals etc. (6). NTVC are found often in viable but not culturable form (UVF) in the environment and are found frequently in biofilms (7,8). Despite that currently more than 200 Vibrio cholerae serogroups are known, the cholera toxin gene is usually carried by the O1 and O139. However, other serogroups, such as O75 or O141 are sometimes toxigenic and are responsible for smaller epidemics (9). Non-O1, non-O139 Vibrio cholerae , has also been implicated as etiologic agents of asymptomatic to severe human disease, including gastroenteritis and extraintestinal infections: sinusitis, otitis, sepsis, peritonitis, conjunctivitis, wound infections, soft tissue infections, etc. (2,10–20). The infections are usually self-limiting, but underlying conditions (e.g. immunosuppressed patients) may cause an increased risk of severe infection. The healthcare system recognizes only the laboratory confirmed cases, which are only the tip of the iceberg (1). Empiric antibiotic therapy is recommended as a first line treatment for the extraintestinal infections and during the therapy of the two most frequently identified types of infection(21)(22) (23,24). Non-toxigenic serogroups are invisible to the public health systems in the majority of the countries, and this poses a potential public health risk. In Hungary passive surveillance is available since the early 1950s, however, all Vibrio cholerae isolates should be sent to the reference laboratory for species confirmation and cholera toxin detection. In this case report, we present the details regarding this atypical infection and the genomic features of the isolates. According to our best knowledge this case is the first primary pneumonia case in Europe due to non-O1, and non-O139 Vibrio cholerae . Methods Clinical sample A previously healthy 21 year old male patient was admitted in critical condition to the Intensive Care Unit (ICU) of the Department of Anesthesiology and Intensive Therapy, Semmelweis University, Budapest after a near-drowning event and 2 minutes of resuscitation. The primary diagnosis was adult respiratory distress syndrome (ARDS) with bilateral infiltrates on chest x-ray and a low PaO2/FiO2 ratio (the lowest value recorded was 109 within the first 24 hours, despite the application of 10 cmH₂O positive end-expiratory pressure (PEEP)). No bacteria were cultured in significant colony counts from a bronchoalveolar lavage (BAL) sample taken shortly after admission. After an initial two-day period of transient improvement, the patient's condition deteriorated, marked by the onset of fever and impaired gas exchange. In response to the worsening of ventilatory status, prone positioning and airway pressure release ventilation (APRV) were implemented. The markedly elevated inflammatory markers (peak values on day 4 of treatment: procalcitonin 230 µg/L and CRP 368 mg/L), together with the development of purulent tracheal secretions, raised suspicion of aspiration pneumonia, supported by findings on chest X-ray and computed tomography. The CT scan also revealed a 2 cm pulmonary abscess. Vibrio species, identified with a low confidence score using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS, Biotyper®, database v.1.), was cultured in significant colony counts from a second BAL sample. In line with the national guidelines, the laboratory sent the clinical isolate for further confirmation to the National Center for Public Health and Pharmacy, National Biosafety Laboratory, where non-O1, non-O139 Vibrio cholerae was confirmed by classical and molecular methods (see below). Following a complex intensive care approach, including prone positioning with APRV ventilation, combined high-dose vasopressor and inotropic therapy, and six days of continuous renal replacement therapy), antibiotic therapy, guided by infectious disease consultation, was administered. Treatment began with empiric amoxicillin/clavulanic acid and was subsequently escalated to meropenem and cefazolin based on culture results, leading to stabilization of the patient. This antibiotic therapy was maintained until day 20 of treatment; the antibiotic therapy was discontinued due to decreasing inflammatory markers and the absence of infectious clinical symptoms. Given previous case reports that occasionally documented fungal infections following near-drowning in freshwater, empiric antifungal therapy with amphotericin B was initiated. This treatment was subsequently discontinued based on culture results and infectious disease consultation. After a prolonged critical condition complicated by a lung abscess, the length of stay was 27 days in the ICU and 34 days in the hospital. Standard epidemiological investigation was performed after the accident and identified the lake in Central Hungary and the exact location where the near-drowning event occurred. The place of exposure was a designated European Union bathing site. The patient fully recovered after the therapy. Bacterial isolates and antibiotic susceptibility Clinical isolate was plated to Thiosulfate Citrate Bile Sucrose agar plates, TCBS (Merck, Darmstadt, Germany) and Vibrio ChromoSelect Agar (Sigma, Germany) and incubated for 24 h at 37 °C at ambient air. Additionally, sheep blood agar (SBA) was inoculated and incubated for 24 hours at 37 ˚C at ambient air. For rapid identification of the main toxigenic serogroups, in-house slide- agglutination was performed by using rabbit anti- V. cholerae O1 and O139 sera (National Center for Public Health and Pharmacy, Budapest) from a 24-hour log-phase growth culture. Basic biochemical tests, including oxidase, catalase and indole test were performed. A water sample was collected at the exact location one week after the exposure in August 2023 by an accredited laboratory by a sampling rod in a sterile 500 ml glass bottle from a water depth of 30 cm. Water temperature, electrical conductivity, pH and oxygen content were measured directly during sampling by a portable device. Aliquots of the water sample from 1 mL to 100 mL were filtered through 0.45 μm pore-size mixed cellulose ester membrane filters (Ø 47 mm; Merck, Darmstadt, Germany), which was placed onto TCBS agar (Merck, Darmstadt, Germany), and Vibrio ChromoSelect Agar (Sigma, Germany) plates in parallel and incubated for 24 h at 37 °C at ambient air. Typical bacterial colonies on the membrane filters were enumerated by ISO 8199:2018 standard (25) in the colony forming unit (CFU) per 100 ml. The isolates were identified by using MALDI -TOF MS Biotyper Syrius One, (database v. 7.) Antimicrobial susceptibility testing was performed according to the EUCAST disk diffusion technical guideline(23) with cefotaxime (5 μg) ceftazidime (10 μg), doxycycline (30 μg), pefloxacin (5 μg), meropenem (10 μg), erythromycin (15 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg) and piperacillin-tazobactam (30-6 μg) disks.[18][21]. The selection of antibiotics was based on their frequent usage in clinical practices and according to the EUCAST Clinical Breakpoint Tables v. 13.1 guideline for Vibrio spp. Molecular identification, Whole genome sequencing and bioinformatics Total genomic DNA (gDNA) was extracted by using the Qiagen DNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Genesiq advanced real-time PCR kit (Genesiq, USA) was used for species identification targeting to the outer membrane protein W ( omp W). The Vibrio cholerae cholera enterotoxin subunit A gene ( ctx A) gene was detected by applying an in-house quantitative polymerase chain reaction (qPCR) method (26). In total, four Vibrio cholerae strains were subjected to WGS originating from the clinical (N=1, signed as Vibrio-33) and environmental (N=3, signed as Vibrio-34, Vibrio-35, Vibrio-36) samples. Genomic libraries were prepared by using the DNA prep Kit (Illumina, CA, United States) according to the manufacturer’s instructions. All isolates were paired-end sequenced by using MiSeq Reagent Kit v2 with a read length of 2 × 150 base pairs on a MiSeq instrument (Illumina, CA, United States). Raw reads were de-novo assembled by using the Ridom SeqSphere+ software pipeline (v. 9.0.8, Ridom GmbH, Münster, Germany) and SKESA (27).For Prediction of antimicrobial resistance (AMR) and virulence, genes draft genomes were analyzed by using The Comprehensive Antibiotic Resistance Database (CARD) and the ResFinder database. In silico target scan procedure settings in SeqSphere+ for reference sequences were defined with 90% required identity to reference sequence and 99% aligned to reference sequence. (28–30). Only resistance genes with a coverage of > 80% and > 75% identity were accepted. For virulence prediction and annotation Virulence Factor Database (VDFB) and rapid and standardized annotation of bacterial genomes via alignment-free sequence identification (Bakta) tool was used (29,31). For Phylogenetic analysis and genomic visualization, multi locus sequence typing (MLST) data was extracted in silico from the whole genome sequencing (WGS) data according to the dedicated non-O, non-O139 Vibrio cholerae MLST scheme. Sequence types (STs) were determined by using pubmlst database https://pubmlst.org/organisms/vibrio-cholerae (4). The possible clonal relationships were investigated by core genome multilocus sequence typing (cgMLST) performed on Ridom SeqSphere + by using the Vibrio cholerae scheme. To determine the cgMLST gene set, a genome-wide gene-by-gene comparison was performed by using Ridom SeqSphere+. For comparison 101 assembled NTVC genomes were downloaded from the National Center for Biotechnology Information (NCBI) genome database (supplementary material). Selection criteria were included from the neighboring country with Hungary, and from countries which share waterbody with Hungary. Literature review A systematic literature review was performed on July in 2024 using Pubmed and Google Scholar without a duration limit. Additional articles were identified by checking the references of relevant articles and duplicates were excluded. We included articles which described confirmed V. cholerae related infections in humans. Our search strategy included the following terms in Pubmed [ "non-O1 Vibrio cholerae "[MeSH Terms] OR non-O139 Vibrio cholerae "[MeSH Terms] OR "Non-toxigenic Vibrio" MeSH Terms] OR [ "non-O1 Vibrio cholerae "[TextWord] OR non-O139 Vibrio cholerae "[TextWord] OR "Non-toxigenic Vibrio" [TextWord] "non-O1, non-O139 Vibrio cholerae respiratory infection"[Text Word] OR "[TextWord] OR " Vibrio Cholerae pneumonia"[Text Word] AND "Vibrio Infections" [TextWord] OR “Vibrio Infections*"[Text Word] OR "Vibrio pneumonia*"[Text Word] OR "non-O1,non-O139 Vibrio cholerae pneumonia"[Text Word] in Google Scholar. In parallel, three authors screened the identified articles based on the defined parameters. We excluded non- V. cholerae , articles written in non-English, non- human infections, environmental. Clinical characteristics, risk factors, microbiological data, demographic data as well as treatment and clinical outcomes were collected and analyzed. Nucleotide Sequence Accession Numbers Sequences have been deposited at the GenBank under the accession of SUB13776351, BioProject PRJNA1007372. Results Microbiological results Culture and biochemical identification methods and in vitro susceptibility to antimicrobials After 24 hours medium-sized yellow colonies appeared on the TCBS agar, while BSA showed white medium-sized colonies with pronounced β-haemolytic zone. On Vibrio ChromoSelect Agar purple colonies were observed. These were identified as Vibrio cholerae with high scores (>2,3) by using MALDI-TOF MS. Basic biochemical tests, including oxidase, catalase and indole test were performed with positive results. The slide-agglutination test identified the isolates as non-O1, non-O139 serogroup. The colonies from the concentrated water samples were not totally identical in appearance, therefore three different colonies were selected for further analysis, including WGS, and all colonies were identified as V. cholerae by MALDI-TOF MS. The V. cholerae colony count of the surface water was 1300 CFU/100 ml. The clinical and all environmental isolates were susceptible to ceftazidime, cefotaxime, pefloxacin, doxycycline, meropenem, trimethoprim-sulfamethoxazole, piperacillin-tazobactam and erythromycin. Whole genome sequencing The average coverage per genome was 99% (84-fold depth in average), the N50 was 205412, 205410, 178740 and 178754 respectively, and the assembly size was 3,9 Mb with 47,5% GC content in average. A total of 3481 coding sequences (CDS) were predicted in average. No putative plasmids were determined from raw data. Detection of antimicrobial resistance genes (AMR), virulence genes and genomic comparison All four isolates carried the bla CARB–7 and the alm EFG. Main virulence features were determined by using VDFB. Common NTVC virulence genes were present, including VCA0109, clp B/ vas G, hly -A, icm F/ vas K, lux S, rtx B-D, vas A-L, vip A/ mgl A, vip B/ mgl B, vgr B-3 and tox R. Virulence profiles were identical between the sequenced clinical and environmental isolates. Some parts of the Vibrio 7 th pandemic islands VSP1 (VC0174 and VC0186) and VSP2 (VC0489, VC0495, VC0496, VC0497, VC0501a, VC0504, VC0505, VC0506, VC0507, VC0508, VC0509, VC0510, VC0517) were detectable, most of them without known function. The clinical and environmental isolates were negative for cholera toxin ( ctx A and ctx B), zonula occludens toxin ( zot ), accessory cholera enterotoxin (ace ), toxin coregulated pilus (TCP) and heat-labil enterotoxin gene ( stn ) and the Type 3 secretion system (T3SS). According to the cgMLST results, the allelic distance between the clinical and three environmental isolates was zero which indicates that the isolates are identical. The in silico analysis of MLST resulted a new combination of the existing allele variants that has been assigned as ST1605. Genomic comparison between the selected isolates from NCBI and our isolates showed high allelic distance (20<), but a possible epidemiological linkage was not identified. Review results The initial search yielded 1098 articles in Pubmed and 1038 articles in Google scholar. We excluded 117 non-English and 570 non-human articles. The remaining articles were screened based on title or abstract, 687 were excluded due to containing epidemiological studies, environmental or non- V. cholerae infection. After eliminating duplicate cases and including the present case, we identified a total of 4 adult (≥ 18 years of age) patients (3 males, 1 females) with non-O1, non-O139 V. cholerae related pulmonary infection (Table 1). The result was congruent with the three independent data collectors. The summary of the screening process is shown in the PRISMA flow diagram (32) (Fig.1). Phylogenetic analysis As of 2024 July, the pubMLST database consisted of 576 records from Europe and of which 209 records represented isolates from human infection caused by non-O1, non-O139 serogroup; none of them was associated with pneumonia. MLST profiles of all four isolates were identical and represented a new ST assigned as ST1605. The allele profile was the following: adh 1; gyr B 41; mdh 7; met E 273; pnt A 58; pur M 1; pyr C 176. According to the cgMSLT analysis the tested four isolates had identical cgMLST profiles and belonged to the same Cluster Type. In addition, we performed a core-genome-based phylogenetic analysis on the four tested isolates and on further 101 selected isolates downloaded from the NCBI database (Supplementary data). The relatedness of the isolates included in the analysis is shown in the phylogenetic trees generated from the CT analysis performed with Ridom Seqsphere+ (Fig.2), The clinical sample Vibrio-33 showed 0 allele difference from the environmental isolate Vibrio-35, Vibrio-36 and Vibrio-37. High allele distances were observed between the other selected isolates. Discussion Pneumonia is an extremely rare and atypical manifestation of non-O1/non-O139 Vibrio cholerae infections(19,20).Here we presented an autochthonous pulmonary infection case due to non-O1, non-O139 Vibrio cholerae in Hungary after a near-drowning accident. By using WGS the clinical and environmental isolates were identical. Literature draws attention to the emergence of antibiotic resistance, in particular beta-lactam resistance among NTVC isolates worldwide, which may significantly influence the strategies for controlling NTVC at the national level (33,34) . Ampicillin or amoxicillin-clavulanate are the first choose empiric antibiotics used in many countries, including in Hungary as well against otitis, sinusitis or pneumonia, etc., which are the most frequent types of NTVC infections also(21,22,24). However, EUCAST has not determined breakpoints for ampicillin and amoxicillin-clavulanate. Therefore, close partnership and continued consultation between the microbiological laboratory and the clinicians on the proper antibiotic therapy in all cases of NTVC diagnosis is necessary. According to previously described cases, pneumonia associated with near-drowning are commonly caused by Gram-negative bacteria, especially Aeromonas sp. etc, and if the clinician decided to use empirical antimicrobial therapy in particularly amoxicillin-clavulanate, they should always consider covering them due to the potential for antibiotic resistance (18,24,34–36). All four isolates carried the bla CARB–7 gene, which is one of the main determinants of beta-lactam resistance and frequent among Vibrio cholerae isolates(34). The isolates carried Both isolates possessed the alm EFG operon, which is a determinant antibiotic resistance of Cationic antimicrobial peptides, including polymyxins. Polymyxins are widely used as a last-line treatment against MDR Gram-negative pathogens, potentially in case of MDR NTVC infections. The major global regulator of catabolite-sensitive operon was also detected, which might cause significant increase the resistance to oxacillin, azithromycin, erythromycin and crystal violet according to the literature(37). Among the isolates NTVC associated virulence genes were detectable by using VDFB and Bakta. All isolates possessed RTX toxin, which is one of the most important virulence factors or in NTVC isolates and plays an important role in cellular rounding and depolymerization of the actin cytoskeleton in host cells and haemolysin A ( hly A), which is responsible for lysis of red blood cells and finally the type IV pilus, mannose-sensitive haemagglutinin ( msh A), which is responsible for biofilm formation. Both isolates carried the tox R transcriptional regulator, which is one of the main and ancestral key virulence factors of vibrios, and lux R, which is a part of the quorum sensing system. Proteins associated with Type 6 Secretion System (T6SS)– vas A-L, vip A, vib B, vgrG-3, VC0109 were present(34,38). The allelic distance among the clinical and three environmental isolates was zero which indicates that the isolates are identical. The in silico analysis of MLST resulted a new combination of the existing allele variants that has been assigned as ST1605. A genomic comparison of the selected isolates (N = 97) revealed no allelic differences between our isolates (N = 4), while a significant allelic distance (greater than 20) was observed when compared to the other isolates. No further genetic relationships were detected among the isolates we tested. Limited data are available on aspiration-associated pneumonia. However, acquisition of organisms directly from aspirated water may result in serious bacterial infection due to opportunistic pathogens, including Aeromonas sp., Staphylococcus aureus , Haemophilus influenzae or Pseudomonas aeruginosa , etc(39,40) but Vibrio cholerae is very rarely encountered. However, clarifying the aetiology of a severe pulmonary status is not simple, as drowning can directly cause aspiration pneumonia because the entry of water into the lungs triggers anti-inflammatory reaction with the release of different pro-inflammatory cytokines. (24). We found only three cases in the literature where non-O1, non-O139 Vibrio cholerae caused pneumonia connected to fresh water. Of these, two cases started as a bacteraemia and pneumonia where there was a later complication and only one case was a primary pulmonary infection connected to a near-drowning event in the US (19,20).To the best of our knowledge, this is the first case of primary pulmonary infection due to V. cholerae , associated with near -drowning event in Europe. Other vibrios, such as Vibrio vulnificus and Vibrio metschnikovii sometimes can cause pneumonia directly, but specific virulence features which can play a role in aetiology are unknown. All sequenced isolates showed the same virulence profile. None of the isolates carried significant virulence features, such as T3SS or VP-s, or other specific toxins were not found. Some parts of the VSP1 and VSP2 were present, however it is still unknown how they can contribute to virulence in humans(41). As of July 2024, the presented case is the only identified case of NTVC infection at this bathing site, and the overall number of reported NTVC in relation to recreational bathing is very low (1-2 cases/year) in Hungary. Mild or asymptomatic cases, however, might go unnoticed. NTVC counts observed in this study were similar to those detected in other temperate lakes used for bathing , with or without recognized human NTVC infections(42,43). Despite the low incidence, severe NTVC infections may be a real health risk especially with the observed increase in case numbers through Europe. Surface waters, especially shallow lakes are expected to grow warmer and contain more saline as a consequence of climate change, resulting in higher prevalence of non-cholerae vibrios. Subsequently, NTVC case numbers are also expected to rise in the temperate regions, potentially including such severe, life-threatening infections as reported in the present study(43). There are no known measures to limit the proliferation of NTVC in surface water, guidelines suggest increasing awareness of the public to the existing health risk and of physicians to potential water-related vibriosis cases(44). Abbreviations ace accessory cholera enterotoxin AMR antimicrobial resistance ARDS adult respiratory distress syndrome BAL bronchoalveolar lavage CARD The Comprehensive Antibiotic Resistance Database CFU colony forming unit CDS coding sequences ctx A cholera enterotoxin subunit A gene ctx B cholera enterotoxin subunit B gene CT Cluster Type cgMLST core genome multilocus sequence typing ECDC European Centre for Disease Prevention and Control EUCAST The European Committee on Antimicrobial Susceptibility Testing FSA bovine serum albumin ICU Intensive Care Unit MALDI TOF MS Matrix-assisted laser desorption ionization–time of flight mass spectrometry MDR multidrug resistant MLST multilocus sequence typing NTVC non toxigenic Vibrio cholerae omp W outer membrane protein W gene PCR polymerase chain reaction qPCR quantitative polymerase chain reaction PEEP positive end-expiratory pressure SBA sheep blood agar ST Sequence Type stn heat-labil enterotoxin TCBS thiosulfate-citrate-bile salts-sucrose agar TCP toxin gene toxin coregulated pilus T3SS Type 3 secretion system T6SS Type 3 secretion system UVF viable but not culturable form VDFB Virulence Factor Database VP Vibrio pathogenic island VSP1 Vibrio seventh pandemic island-1 VSP2 Vibrio seventh pandemic island- zot zonula occludens toxin WGS whole genome sequencing Declarations Ethics approval and consent to participate Ethics Committee approval was not required as the Hungarian legislation on handling of personal health information (Law no. 1997. XLVII.) empowers the National Center for Public Health and Pharmacy to analyse data and take necessary measures in the interest of public health. Personal data have been handled in accordance with legal regulations and the Center’s data protection rules. Consent for publication Written informed consent for publication was obtained from participants for publication of the data collected during the study, including any identifiable information. Ministerial Decree No. 18/1998. (VI. 3.) NM. on epidemiological measures for the prevention of communicable diseases and outbreaks authorizes the National Center for Public Health and Pharmacy to analyze clinical samples and bacterial strains, and to take measures necessary measures in the interest of public health, including the management of personal and health data. Availability of data and material Sequences have been deposited at the GenBank under the accession of SUB13776351, BioProject PRJNA1007372. Competing interests None declared Funding This research was entirely funded by the National Center for Public Health and Pharmacy without any specific grant from other agencies in the public, commercial or not-for-profit sectors. Authors’ contributions JH: Methodology, Software, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. ÁT: Writing-original draft, review & editing MV: Writing-original draft, review & editing BP: Revising the manuscript BK: Methology ER: Revising the manuscript KK, AK, and BN: Revising the manuscript, writing SzT, TM: Revising the manuscript, ZK: Revising the Manuscript, drafting ZsM, EM: Revising the manuscript Acknowledgements We thank Erika Ungváry for support and Zsuzsanna Richter for the technical assistance. We are thankful to Dr. Gábor Kardos for his constructive feedback on the manuscript. 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Pulmonary Cholera Due to Infection with a Non-O1 Vibrio cholerae Strain. J Clin Microbiol. 2006 Sep;44(9):3459–60. Marinello S, Marini G, Parisi G, Gottardello L, Rossi L, Besutti V, et al. Vibrio cholerae non-O1, non-O139 bacteraemia associated with pneumonia, Italy 2016. Infection. 2017 Apr 11;45(2):237–40. Shannon JD, Kimbrough RC. Pulmonary Cholera Due to Infection with a Non-O1 Vibrio cholerae Strain. J Clin Microbiol. 2006 Sep;44(9):3459–60. Castillo-Aguas G Del, García-Vera C, Urkin J, Moretto M, Spreitzer MV, Keronen P, et al. Acute otitis media management: A survey of European primary care pediatricians. Global Pediatrics. 2023 Jun;4:100057. Zara M Patel M. Uncomplicated acute sinusitis and rhinosinusitis in adults: Treatment. 2022. Matuschek E, Brown DFJ, Kahlmeter G. Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clinical Microbiology and Infection. 2014;20(4). Niederman MS, Cilloniz C. Aspiration pneumonia. Revista Española de Quimioterapia. 2022 Apr 22;35(Suppl1):73–7. International Standard ISO 8199:2018. Water quality. General requirements and guidance for microbiological examinations by culture [Internet]. 2018 [cited 2025 Apr 10]. Report No.: 3. Available from: https://www.iso.org/standard/65350.html Blackstone GM, Nordstrom JL, Bowen MD, Meyer RF, Imbro P, DePaola A. Use of a real time PCR assay for detection of the ctxA gene of Vibrio cholerae in an environmental survey of Mobile Bay. J Microbiol Methods. 2007;68(2). Souvorov A, Agarwala R, Lipman DJ. SKESA: Strategic k-mer extension for scrupulous assemblies. Genome Biol. 2018 Oct 4;19(1). McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, et al. The Comprehensive Antibiotic Resistance Database. Antimicrob Agents Chemother. 2013 Jul;57(7):3348–57. Chen L, Zheng D, Liu B, Yang J, Jin Q. VFDB 2016: hierarchical and refined dataset for big data analysis—10 years on. 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Schmidt K, Scholz HC, Appelt S, Michel J, Jacob D, Dupke S. Virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 acquired in Germany and other European countries. Front Microbiol. 2023 Nov 22;14. Cousin VL, Pittet LF. Microbiological features of drowning-associated pneumonia: a systematic review and meta-analysis. Ann Intensive Care. 2024 Apr 20;14(1):61. Mandell LA, Niederman MS. Aspiration Pneumonia. New England Journal of Medicine. 2019 Feb 14;380(7):651–63. Nishino K, Senda Y, Yamaguchi A. CRP Regulator Modulates Multidrug Resistance of Escherichia coli by Repressing the mdtEF Multidrug Efflux Genes. J Antibiot (Tokyo). 2008 Mar;61(3):120–7. Agyei FK, Scharf B, Duodu S. Vibrio cholerae Bacteremia: An Enigma in Cholera-Endemic African Countries. Trop Med Infect Dis. 2024 May 2;9(5):103. Reizine F, Delbove A, Tattevin P, Santos A Dos, Bodenes L, Bouju P, et al. Clinical and microbiological features of drowning-associated pneumonia: a retrospective multicentre cohort study. Clinical Microbiology and Infection. 2023 Jan;29(1):108.e7-108.e13. Cerland L, Mégarbane B, Kallel H, Brouste Y, Mehdaoui H, Resiere D. Incidence and Consequences of Near-Drowning–Related Pneumonia—A Descriptive Series from Martinique, French West Indies. Int J Environ Res Public Health. 2017 Nov 17;14(11):1402. Kumar A, Das B, Kumar N. Vibrio Pathogenicity Island-1: The Master Determinant of Cholera Pathogenesis. Front Cell Infect Microbiol. 2020 Oct 6;10. Bliem R, Reischer G, Linke R, Farnleitner A, Kirschner A. Spatiotemporal Dynamics of Vibrio cholerae in Turbid Alkaline Lakes as Determined by Quantitative PCR. Appl Environ Microbiol. 2018 Jun;84(11). Sterk A, Schets FM, de Roda Husman AM, de Nijs T, Schijven JF. Effect of Climate Change on the Concentration and Associated Risks of Vibrio Spp. in Dutch Recreational Waters. Risk Analysis. 2015 Sep;35(9):1717–29. World Health Organization. (‎2021)‎. WHO guidelines on recreational water quality: volume 1: coastal and fresh waters. World Health Organization. https://iris.who.int/handle/10665/342625. License: CC BY-NC-SA 3.0 IGO. Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. 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of Public Health Laboratory and Methodology, Department for Environmental Health Laboratory, National Center for Public Health and Pharmacy,Budapest","correspondingAuthor":false,"prefix":"","firstName":"Márta","middleName":"","lastName":"Vargha","suffix":""},{"id":449639362,"identity":"ffb04a05-f6f5-47e8-b708-3ab50127cdf6","order_by":2,"name":"András Kállai","email":"","orcid":"","institution":"Department of Anesthesiology and Intensive Therapy, Semmelweis University, Budapest","correspondingAuthor":false,"prefix":"","firstName":"András","middleName":"","lastName":"Kállai","suffix":""},{"id":449639363,"identity":"94f18994-7864-4333-b0ec-9ee00494320d","order_by":3,"name":"Katalin Kristóf","email":"","orcid":"","institution":"Department of Laboratory Medicine, Semmelweis University, 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Laboratory and Methodology, Department for Environmental Health Laboratory, National Center for Public Health and Pharmacy,Budapest","correspondingAuthor":false,"prefix":"","firstName":"Bernadett","middleName":"","lastName":"Khayer","suffix":""},{"id":449639367,"identity":"e48a1b0e-4c26-4a77-9621-042dfabb75c3","order_by":7,"name":"Eszter Róka","email":"","orcid":"","institution":"Division of Public Health Laboratory and Methodology, Department for Environmental Health Laboratory, National Center for Public Health and Pharmacy,Budapest","correspondingAuthor":false,"prefix":"","firstName":"Eszter","middleName":"","lastName":"Róka","suffix":""},{"id":449639368,"identity":"1a5b0ef9-3378-4dff-9774-9f8e6bd0d97f","order_by":8,"name":"Bereniké Novák","email":"","orcid":"","institution":"National Biosafety Laboratory, National Center for Public Health and Pharmacy, 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Kis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3OsUoDMRjA8a8E0iV663cE714hpSAOPswdhU4nrjdpQTiX9gHEp3DJfEcGl4prIB1aDjpf6XIOgvGGTiZdHfInISHkFwIQCv3DkAzLjZ2kHraXdtbD8BNEAJoNW3qWwIkwcSLgI/HzeDvqS3yIXpfH7lDIhOKsaXrY5AsH4cQ+jmtE3HzI+EWaKcV5phjsnSQhDGBS2Y/pO0kupMkrXggFoDxkvIXcklQX7fFbmseK33f2Y27CCQhoLBG6AD6SJqO8gJp5SPzERLNYY/ym59fxSppJle6FYkJNXQQ/33e7vryNEj1ruy9p0oip9tCX6spFfqv/OBOe+6FQKBQ62w9hN1mKUyvUMQAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Medical Microbiology, Faculty of Medicine, Semmelweis University, Budapest","correspondingAuthor":true,"prefix":"","firstName":"Zoltán","middleName":"","lastName":"Kis","suffix":""}],"badges":[],"createdAt":"2025-01-31 10:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5935550/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5935550/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-025-12191-9","type":"published","date":"2025-12-09T15:58:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82048000,"identity":"cbb5cccf-f6da-4d32-bdcb-3f360b1e06fb","added_by":"auto","created_at":"2025-05-06 09:44:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17312,"visible":true,"origin":"","legend":"\u003cp\u003ePRISMA flow diagram for selections of non-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e infections.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5935550/v1/fcc0b89be8a1135d2b0cf468.png"},{"id":82048003,"identity":"3cd9bd53-4332-4f58-9671-5ec02649bce5","added_by":"auto","created_at":"2025-05-06 09:45:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":306888,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-Joining Tree of the NTVC \u003cem\u003eVibrio cholerae\u003c/em\u003e based on 97 isolates. The data analysed by Ridom Seqsphere+ where missing pairwise values were ignored. The tree based on cgMLST NTVC \u003cem\u003eVibrio cholerae\u003c/em\u003e 2415 target with a cluster threshold ≤ 10. Different colors indicate sequence types. The isolate from this study is highlighted with red color. Each isolate is indicated in the supplementary.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5935550/v1/290e7b74a96e2247a91291af.png"},{"id":98243816,"identity":"4676852e-f87c-4f54-a4f1-f990963c2421","added_by":"auto","created_at":"2025-12-15 16:10:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1070715,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5935550/v1/178b28dc-3900-4143-a407-d1be95ef88d7.pdf"},{"id":82049396,"identity":"e41fbb55-6f8a-45f2-ab6f-d0d65036f98c","added_by":"auto","created_at":"2025-05-06 09:53:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1174859,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5935550/v1/4efdd896f175c8450f1711b1.xlsx"},{"id":82049393,"identity":"6e77dd65-1ae8-4893-8a9c-e0536a3dc9af","added_by":"auto","created_at":"2025-05-06 09:53:00","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":110155,"visible":true,"origin":"","legend":"","description":"","filename":"CAREchecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5935550/v1/8499a1d8baa77f789e8b3281.pdf"},{"id":82048002,"identity":"91b08344-92d2-42b7-8705-8b7777836180","added_by":"auto","created_at":"2025-05-06 09:45:00","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10401,"visible":true,"origin":"","legend":"","description":"","filename":"Table1SummaryofcasesdescribedinliteratureofnonOnonO139Vibriocholeraerelatedinfectionsincludingthepresentstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5935550/v1/7bd0a28f034454c881475e4d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eGenomic characterization of non-O1, non-O139 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVibrio cholerae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e causing rare clinical manifestation\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eNon-toxigenic \u003cem\u003eVibrio cholerae\u003c/em\u003e (NTVC) occurs naturally in both marine and freshwater environments (1). In the last decades NTVC has been detected in different lakes, rivers, and seawaters, including the North Sea, the Baltic Sea, and the Mediterranean Sea. Humans can be infected through the consumption of contaminated fish or seafood, or through direct water exposure. The incidence of human NTVC cases is uncertain, but the number of reports on their prevalence in water environments and their human infections have been increasing in the recent years across Europe (1\u0026ndash;4). There is a strong association between NTVC incidence and elevated water temperature, increased salinity or dissolved oxygen, and it is historically well known that \u003cem\u003eVibrio\u003c/em\u003e species have the fastest growth rates among bacteria, responding almost immediately to preferred environmental conditions(5). NTVC can be also isolated from different fish, mollusks, shrimps, water birds, mammals etc. (6). NTVC are found often in viable but not culturable form (UVF) in the environment and are found frequently in biofilms (7,8). Despite that currently more than 200 \u003cem\u003eVibrio cholerae\u003c/em\u003e serogroups are known, the cholera toxin gene is usually carried by the O1 and O139. However, other serogroups, such as O75 or O141 are sometimes toxigenic and are responsible for smaller epidemics (9). Non-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e, has also been implicated as etiologic agents of asymptomatic to severe human disease, including gastroenteritis and extraintestinal infections: sinusitis, otitis, sepsis, peritonitis, conjunctivitis, wound infections, soft tissue infections, etc. (2,10\u0026ndash;20). The infections are usually self-limiting, but underlying conditions (e.g. immunosuppressed patients) may cause an increased risk of severe infection. The healthcare system recognizes only the laboratory confirmed cases, which are only the tip of the iceberg (1). Empiric antibiotic therapy is recommended as a first line treatment for the extraintestinal infections and during the therapy of the two most frequently identified types of infection(21)(22) (23,24).\u003c/p\u003e \u003cp\u003eNon-toxigenic serogroups are invisible to the public health systems in the majority of the countries, and this poses a potential public health risk. In Hungary passive surveillance is available since the early 1950s, however, all \u003cem\u003eVibrio cholerae\u003c/em\u003e isolates should be sent to the reference laboratory for species confirmation and cholera toxin detection. In this case report, we present the details regarding this atypical infection and the genomic features of the isolates. According to our best knowledge this case is the first primary pneumonia case in Europe due to non-O1, and non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eClinical sample\u003c/h2\u003e\n\u003cp\u003eA previously healthy 21 year old male patient was admitted in critical condition to the Intensive Care Unit (ICU) of the Department of Anesthesiology and Intensive Therapy, Semmelweis University, Budapest after a near-drowning event and 2 minutes of resuscitation. The primary diagnosis was adult respiratory distress syndrome (ARDS) with bilateral infiltrates on chest x-ray and a low PaO2/FiO2 ratio (the lowest value recorded was 109 within the first 24 hours, despite the application of 10 cmH₂O positive end-expiratory pressure (PEEP)). No bacteria were cultured in significant colony counts from a bronchoalveolar lavage (BAL) sample taken shortly after admission.\u003c/p\u003e\n\u003cp\u003eAfter an initial two-day period of transient improvement, the patient\u0026apos;s condition deteriorated, marked by the onset of fever and impaired gas exchange. In response to the worsening of ventilatory status, prone positioning and airway pressure release ventilation (APRV) were implemented. The markedly elevated inflammatory markers (peak values on day 4 of treatment: procalcitonin 230 \u0026micro;g/L and CRP 368 mg/L), together with the development of purulent tracheal secretions, raised suspicion of aspiration pneumonia, supported by findings on chest X-ray and computed tomography. The CT scan also revealed a 2 cm pulmonary abscess.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVibrio\u003c/em\u003e species, identified with a low confidence score using matrix-assisted laser desorption ionization\u0026ndash;time of flight mass spectrometry (MALDI-TOF MS, Biotyper\u0026reg;, database v.1.), was cultured in significant colony counts from a second BAL sample. In line with the national guidelines, the laboratory sent the clinical isolate for further confirmation to the National Center for Public Health and Pharmacy, National Biosafety Laboratory, where non-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e was confirmed by classical and molecular methods (see below).\u003c/p\u003e\n\u003cp\u003eFollowing a complex intensive care approach, including prone positioning with APRV ventilation, combined high-dose vasopressor and inotropic therapy, and six days of continuous renal replacement therapy), antibiotic therapy, guided by infectious disease consultation, was administered. Treatment began with empiric amoxicillin/clavulanic acid and was subsequently escalated to meropenem and cefazolin based on culture results, leading to stabilization of the patient. This antibiotic therapy was maintained until day 20 of treatment; the antibiotic therapy was discontinued due to decreasing inflammatory markers and the absence of infectious clinical symptoms. Given previous case reports that occasionally documented fungal infections following near-drowning in freshwater, empiric antifungal therapy with amphotericin B was initiated. This treatment was subsequently discontinued based on culture results and infectious disease consultation.\u003c/p\u003e\n\u003cp\u003eAfter a prolonged critical condition complicated by a lung abscess, the length of stay was 27 days in the ICU and 34 days in the hospital. Standard epidemiological investigation was performed after the accident and identified the lake in Central Hungary and the exact location where the near-drowning event occurred. The place of exposure was a designated European Union bathing site. The patient fully recovered after the therapy.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eBacterial isolates and antibiotic susceptibility\u003c/h2\u003e\n\u003cp\u003eClinical isolate was plated to Thiosulfate Citrate Bile Sucrose agar plates, TCBS (Merck, Darmstadt, Germany) and Vibrio ChromoSelect Agar\u0026nbsp;(Sigma, Germany) and incubated for 24 h at 37 \u0026deg;C at ambient air. Additionally, sheep blood agar (SBA) was inoculated and incubated for 24 hours at 37 ˚C at ambient air. For rapid identification of the main toxigenic serogroups, in-house slide- agglutination was performed by using rabbit anti-\u003cem\u003eV. cholerae\u003c/em\u003e O1 and O139 sera (National Center for Public Health and Pharmacy, Budapest) from a 24-hour log-phase growth culture. Basic biochemical tests, including oxidase, catalase and indole test were performed.\u003c/p\u003e\n\u003cp\u003eA water sample was collected at the exact location one week after the exposure in August 2023 by an accredited laboratory by a sampling rod in a sterile 500\u0026thinsp;ml glass bottle from a water depth of 30\u0026thinsp;cm. Water temperature, electrical conductivity, pH and oxygen content were measured directly during sampling by a portable device. Aliquots of the water sample from 1 mL to 100 mL were filtered through 0.45 \u0026mu;m pore-size mixed cellulose ester membrane filters (\u0026Oslash; 47 mm; Merck, Darmstadt, Germany), which was placed onto TCBS agar (Merck, Darmstadt, Germany), and\u0026nbsp;Vibrio ChromoSelect Agar\u0026nbsp;(Sigma, Germany) plates in parallel and incubated for 24 h at 37 \u0026deg;C at ambient air. Typical bacterial colonies on the membrane filters were enumerated by ISO 8199:2018 standard (25) in the colony forming unit (CFU) per 100 ml.\u0026nbsp; The isolates were identified by using MALDI -TOF MS Biotyper Syrius One, (database v. 7.) Antimicrobial susceptibility testing was performed according to the EUCAST disk diffusion technical guideline(23) with cefotaxime (5 \u0026mu;g) ceftazidime (10 \u0026mu;g), doxycycline (30 \u0026mu;g), pefloxacin (5 \u0026mu;g), meropenem (10 \u0026mu;g), erythromycin (15 \u0026mu;g), trimethoprim-sulfamethoxazole (1.25/23.75 \u0026mu;g) and piperacillin-tazobactam (30-6 \u0026mu;g) disks.[18][21]. The selection of antibiotics was based on their frequent usage in clinical practices and according to the EUCAST Clinical Breakpoint Tables v. 13.1 guideline for \u003cem\u003eVibrio\u003c/em\u003e spp.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u0026nbsp;Molecular identification, Whole genome sequencing and bioinformatics\u003c/h2\u003e\n\u003cp\u003eTotal genomic DNA (gDNA) was extracted by using the Qiagen DNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer\u0026rsquo;s instructions. Genesiq advanced real-time PCR kit (Genesiq, USA) was used for species identification targeting to the outer membrane protein W (\u003cem\u003eomp\u003c/em\u003eW). The \u003cem\u003eVibrio cholerae\u003c/em\u003e cholera enterotoxin subunit A gene\u003cem\u003e\u0026nbsp;(\u003c/em\u003e\u003cem\u003ectx\u003c/em\u003eA) gene was detected by applying an in-house quantitative polymerase chain reaction (qPCR) method\u003cem\u003e\u0026nbsp;\u003c/em\u003e(26).\u0026nbsp; In total, four \u003cem\u003eVibrio cholerae\u003c/em\u003e strains were subjected to WGS originating from the clinical (N=1, signed as Vibrio-33) and environmental (N=3, signed as Vibrio-34, Vibrio-35, Vibrio-36) samples. Genomic libraries were prepared by using the DNA prep Kit (Illumina, CA, United States) according to the manufacturer\u0026rsquo;s instructions. All isolates were paired-end sequenced by using MiSeq Reagent Kit v2 with a read length of 2 \u0026times; 150 base pairs on a MiSeq instrument (Illumina, CA, United States). Raw reads were de-novo assembled by using the Ridom SeqSphere+ software pipeline (v. 9.0.8, Ridom GmbH, M\u0026uuml;nster, Germany) and SKESA (27).For Prediction of antimicrobial resistance (AMR) and virulence, genes draft genomes were analyzed by using The Comprehensive Antibiotic Resistance Database (CARD) and the ResFinder database. In silico target scan procedure settings in SeqSphere+ for reference sequences were defined with 90% required identity to reference sequence and 99% aligned to reference sequence. (28\u0026ndash;30). Only resistance genes with a coverage of \u0026gt;\u0026thinsp;80% and\u0026thinsp;\u0026gt;\u0026thinsp;75% identity were accepted. For virulence prediction and annotation Virulence Factor Database (VDFB) and rapid and standardized annotation of bacterial genomes via alignment-free sequence identification (Bakta) tool was used (29,31). \u0026nbsp;For Phylogenetic analysis and genomic visualization, multi locus sequence typing (MLST) data was extracted in silico from the whole genome sequencing (WGS) data according to the dedicated non-O, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e MLST scheme. Sequence types (STs) were determined by using pubmlst database https://pubmlst.org/organisms/vibrio-cholerae (4). \u0026nbsp;The possible clonal relationships were investigated by core genome multilocus sequence typing (cgMLST) performed on Ridom SeqSphere\u0026thinsp;+ by using the \u003cem\u003eVibrio cholerae\u003c/em\u003e scheme. To determine the cgMLST gene set, a genome-wide gene-by-gene comparison was performed by using Ridom SeqSphere+. For comparison 101 assembled NTVC genomes were downloaded from the\u0026nbsp;National Center for Biotechnology Information (NCBI) genome database (supplementary material). Selection criteria were included from the neighboring country with Hungary, and from countries which share waterbody with Hungary.\u003c/p\u003e\n\u003ch2\u003eLiterature review\u003c/h2\u003e\n\u003cp\u003eA systematic literature review was performed on July in 2024 using Pubmed and Google Scholar without a duration limit. Additional articles were identified by checking the references of relevant articles and duplicates were excluded. We included articles which described confirmed \u003cem\u003eV. cholerae\u003c/em\u003e related infections in humans. Our search strategy included the following terms in Pubmed [ \u0026quot;non-O1 \u003cem\u003eVibrio\u003c/em\u003e \u003cem\u003echolerae\u003c/em\u003e\u0026quot;[MeSH Terms] OR non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e\u0026quot;[MeSH Terms] OR \u0026quot;Non-toxigenic Vibrio\u0026quot; MeSH Terms] OR [ \u0026quot;non-O1 \u003cem\u003eVibrio cholerae\u003c/em\u003e\u0026quot;[TextWord] OR non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e\u0026quot;[TextWord] OR \u0026quot;Non-toxigenic Vibrio\u0026quot; [TextWord] \u0026quot;non-O1, non-O139 Vibrio cholerae respiratory infection\u0026quot;[Text Word] OR \u0026quot;[TextWord] OR \u0026quot;\u003cem\u003eVibrio Cholerae\u003c/em\u003e pneumonia\u0026quot;[Text Word] AND \u0026quot;Vibrio Infections\u0026quot; [TextWord] OR \u0026nbsp;\u0026ldquo;Vibrio Infections*\u0026quot;[Text Word] OR \u0026quot;Vibrio pneumonia*\u0026quot;[Text Word] OR \u0026quot;non-O1,non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e pneumonia\u0026quot;[Text Word] in Google Scholar. In parallel, three authors screened the identified articles based on the defined parameters. We excluded non- \u003cem\u003eV. cholerae\u003c/em\u003e, articles written in non-English, non- human infections, environmental. Clinical characteristics, risk factors, microbiological data, demographic data as well as treatment and clinical outcomes were collected and analyzed.\u003c/p\u003e\n\u003ch2\u003eNucleotide Sequence Accession Numbers\u003c/h2\u003e\n\u003cp\u003eSequences have been deposited at the GenBank under the accession of SUB13776351, BioProject PRJNA1007372.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cstrong\u003eMicrobiological results\u003c/strong\u003e\u003c/h2\u003e\n\u003ch4\u003e\u003cstrong\u003eCulture and biochemical identification methods and in vitro susceptibility to antimicrobials\u0026nbsp;\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eAfter 24 hours medium-sized yellow colonies appeared on the TCBS agar, while BSA showed white medium-sized colonies with pronounced \u0026beta;-haemolytic zone. On Vibrio ChromoSelect Agar purple colonies were observed. These were identified as \u003cem\u003eVibrio cholerae\u003c/em\u003e with high scores (\u0026gt;2,3) by using MALDI-TOF MS. Basic biochemical tests, including oxidase, catalase and indole test were performed with positive results. The slide-agglutination test identified the isolates as non-O1, non-O139 serogroup. The colonies from the concentrated water samples were not totally identical in appearance, therefore three different colonies were selected for further analysis, including WGS, and all colonies were identified as \u003cem\u003eV.\u0026nbsp;cholerae\u003c/em\u003e by MALDI-TOF MS. The \u003cem\u003eV. cholerae\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecolony count of the surface water was 1300 CFU/100 ml.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe clinical and all environmental isolates were \u0026nbsp; susceptible to ceftazidime, cefotaxime, pefloxacin, doxycycline, meropenem, trimethoprim-sulfamethoxazole, piperacillin-tazobactam and erythromycin. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe average coverage per genome was 99% (84-fold depth in average), the N50 was 205412, 205410, 178740 and 178754 respectively, and the assembly size was 3,9 Mb with 47,5% GC content in average. A total of 3481 coding sequences (CDS) were predicted in average. No putative plasmids were determined from raw data. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of antimicrobial resistance genes (AMR), virulence genes and genomic comparison\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll four isolates carried the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCARB\u0026ndash;7\u003c/sub\u003e and the \u0026nbsp;\u003cem\u003ealm\u003c/em\u003eEFG. \u0026nbsp;Main virulence features were determined by using VDFB. Common NTVC virulence genes were present, including VCA0109, \u003cem\u003eclp\u003c/em\u003eB/\u003cem\u003evas\u003c/em\u003eG, \u0026nbsp;\u003cem\u003ehly\u003c/em\u003e-A, \u003cem\u003eicm\u003c/em\u003eF/\u003cem\u003evas\u003c/em\u003eK, \u003cem\u003elux\u003c/em\u003eS, \u003cem\u003ertx\u003c/em\u003eB-D, \u003cem\u003evas\u003c/em\u003eA-L, \u003cem\u003evip\u003c/em\u003eA/\u003cem\u003emgl\u003c/em\u003eA, \u003cem\u003evip\u003c/em\u003eB/\u003cem\u003emgl\u003c/em\u003eB, \u003cem\u003evgr\u003c/em\u003eB-3 and \u003cem\u003etox\u003c/em\u003eR. Virulence profiles were identical between the sequenced clinical and environmental isolates. Some parts of the Vibrio 7\u003csup\u003eth\u003c/sup\u003e pandemic islands VSP1 (VC0174 and VC0186) and VSP2 (VC0489, VC0495, VC0496, VC0497, VC0501a, VC0504, VC0505, VC0506, VC0507, VC0508, VC0509, VC0510, VC0517) were detectable, most of them without known function. The clinical and environmental isolates were negative for cholera toxin (\u003cem\u003ectx\u003c/em\u003eA and \u003cem\u003ectx\u003c/em\u003eB), zonula occludens toxin (\u003cem\u003ezot\u003c/em\u003e), accessory cholera enterotoxin \u003cem\u003e(ace\u003c/em\u003e), toxin coregulated pilus (TCP) and heat-labil enterotoxin gene (\u003cem\u003estn\u003c/em\u003e) and the Type 3 secretion system (T3SS). According to the cgMLST results, the allelic distance between the clinical and three environmental isolates was zero which indicates that the isolates are identical. The in silico analysis of MLST resulted a new combination of the existing allele variants that has been assigned as ST1605. Genomic comparison between the selected isolates from NCBI and our isolates showed high allelic distance (20\u0026lt;), but a possible epidemiological linkage was not identified. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReview results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial search yielded 1098 articles in Pubmed and 1038 articles in Google scholar. We excluded 117 non-English and 570 non-human articles. The remaining articles were screened based on title or abstract, 687 were excluded due to containing epidemiological studies, environmental or non-\u003cem\u003eV. cholerae\u003c/em\u003e infection.\u0026nbsp;After eliminating duplicate cases and including the present case, we identified a total of 4 adult (\u0026ge;\u0026thinsp;18 years of age) patients (3 males, 1 females) with non-O1, non-O139 \u003cem\u003eV.\u0026nbsp;cholerae\u003c/em\u003e related pulmonary infection\u0026nbsp;\u0026nbsp; (Table\u0026nbsp;1).\u0026nbsp;\u0026nbsp;The result was congruent with the three independent data collectors. The summary of the screening process is shown in the PRISMA flow diagram (32) (Fig.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs of 2024 July, the pubMLST database consisted of 576 records from Europe and of which 209 records represented isolates from human infection caused by non-O1, non-O139 serogroup; none of them was associated with pneumonia. MLST profiles of all four isolates were identical and represented a new ST assigned as ST1605. The allele profile was the following: \u003cem\u003eadh\u003c/em\u003e 1; \u003cem\u003egyr\u003c/em\u003eB 41; \u003cem\u003emdh\u0026nbsp;\u003c/em\u003e7; \u003cem\u003emet\u003c/em\u003eE 273; \u003cem\u003epnt\u003c/em\u003eA 58; \u003cem\u003epur\u003c/em\u003eM 1; \u003cem\u003epyr\u003c/em\u003eC 176. According to the cgMSLT analysis the tested four isolates had identical cgMLST profiles and belonged to the same Cluster Type. In addition, we performed a core-genome-based phylogenetic analysis on the four tested isolates and on further 101 selected isolates downloaded from the NCBI database\u0026nbsp;(Supplementary data). The relatedness of the isolates included in the analysis is shown in the phylogenetic trees generated from the CT analysis performed with Ridom Seqsphere+ (Fig.2), The clinical sample Vibrio-33 showed 0 allele difference from the environmental isolate Vibrio-35, Vibrio-36 and Vibrio-37. High allele distances were observed between the other selected isolates.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePneumonia is an extremely rare and atypical manifestation of non-O1/non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e infections(19,20).Here we presented an autochthonous pulmonary infection case due to non-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e in Hungary after a near-drowning accident. By using WGS the clinical and environmental isolates were identical. Literature draws attention to the emergence of antibiotic resistance, in particular beta-lactam resistance among NTVC isolates worldwide, which may significantly influence the strategies for controlling NTVC at the national level (33,34)\u0026nbsp;. Ampicillin or amoxicillin-clavulanate are the first choose empiric antibiotics used in many countries, including in Hungary as well against otitis, sinusitis or pneumonia, etc., which are the most frequent types of NTVC infections also(21,22,24). However, EUCAST has not determined breakpoints for ampicillin and amoxicillin-clavulanate. Therefore, close partnership and continued consultation between the microbiological laboratory and the clinicians on the proper antibiotic therapy in all cases of NTVC diagnosis is necessary.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to previously described cases, pneumonia associated with near-drowning are commonly caused by Gram-negative bacteria, especially \u003cem\u003eAeromonas\u003c/em\u003e sp. etc, and if the clinician decided to use empirical antimicrobial therapy in particularly amoxicillin-clavulanate, they should always consider covering them due to the potential for antibiotic resistance (18,24,34\u0026ndash;36). All four isolates carried the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCARB\u0026ndash;7\u003c/sub\u003e gene, which is one of the main determinants of beta-lactam resistance and frequent among \u003cem\u003eVibrio cholerae\u003c/em\u003e isolates(34). The isolates carried Both isolates possessed the \u003cem\u003ealm\u003c/em\u003eEFG operon, which is a determinant antibiotic resistance of Cationic antimicrobial peptides, including\u0026nbsp;polymyxins. Polymyxins are widely used as a last-line treatment against \u0026nbsp;\u0026nbsp;MDR Gram-negative pathogens, potentially in case of MDR NTVC infections. \u0026nbsp;The major global regulator of catabolite-sensitive operon was also detected, which might cause significant increase the resistance to oxacillin, azithromycin, erythromycin and crystal violet according to the literature(37). Among the isolates NTVC associated virulence genes were detectable by using VDFB and Bakta. All isolates possessed RTX toxin, which is one of the most important virulence factors or in NTVC isolates and plays an important role in cellular rounding and depolymerization of the actin cytoskeleton in host cells and haemolysin A (\u003cem\u003ehly\u003c/em\u003eA), which is responsible for lysis of red blood cells and finally the type IV pilus, mannose-sensitive haemagglutinin (\u003cem\u003emsh\u003c/em\u003eA), which is responsible for biofilm formation. Both isolates carried the \u003cem\u003etox\u003c/em\u003eR transcriptional regulator, which is one of the main and ancestral key virulence factors of vibrios, and \u003cem\u003elux\u003c/em\u003eR, which is a part of the quorum sensing system. Proteins associated with Type 6 Secretion System (T6SS)\u0026ndash;\u003cem\u003evas\u003c/em\u003eA-L, \u003cem\u003evip\u003c/em\u003eA, \u003cem\u003evib\u003c/em\u003eB, vgrG-3, VC0109 were present(34,38). The allelic distance among the clinical and three environmental isolates was zero which indicates that the isolates are identical. The in silico analysis of MLST resulted a new combination of the existing allele variants that has been assigned as ST1605.\u0026nbsp;A genomic comparison of the selected isolates (N = 97) revealed no allelic differences between our isolates (N = 4), while a significant allelic distance (greater than 20) was observed when compared to the other isolates. No further genetic relationships were detected among the isolates we tested.\u003c/p\u003e\n\u003cp\u003eLimited data are available on aspiration-associated pneumonia. However, acquisition of organisms directly from aspirated water may result in serious bacterial infection due to opportunistic pathogens, including \u003cem\u003eAeromonas\u003c/em\u003e sp., \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eHaemophilus influenzae\u003c/em\u003e or \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, etc(39,40) but \u003cem\u003eVibrio cholerae\u003c/em\u003e is very rarely encountered. However, clarifying the aetiology of a severe pulmonary status is not simple, as drowning can directly cause aspiration pneumonia because the entry of water into the lungs triggers anti-inflammatory reaction with the release of different pro-inflammatory cytokines. (24). We found only three cases in the literature where non-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e caused pneumonia connected to fresh water. Of these, two cases started as a bacteraemia and pneumonia where there was a later complication and only one case was a primary pulmonary infection connected to a near-drowning event in the US (19,20).To the best of our knowledge, this is the first case of primary pulmonary infection due to \u003cem\u003eV. cholerae\u003c/em\u003e, associated with near -drowning event in Europe. Other vibrios, such as \u003cem\u003eVibrio vulnificus\u003c/em\u003e and \u003cem\u003eVibrio metschnikovii\u003c/em\u003e sometimes can cause pneumonia directly, but specific virulence features which can play a role in aetiology are unknown. All sequenced isolates showed the same virulence profile. None of the isolates carried significant virulence features, such as T3SS or VP-s, or other specific toxins were not found. Some parts of the VSP1 and VSP2 were present, however it is still unknown how they can contribute to virulence in humans(41).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs of July 2024, the presented case is the only identified case of NTVC infection at this bathing site, and the overall number of reported NTVC in relation to recreational bathing is very low (1-2 cases/year) in Hungary. Mild or asymptomatic cases, however, might go unnoticed. NTVC counts observed in this study were similar to those detected in other temperate lakes used for bathing\u003cem\u003e,\u0026nbsp;\u003c/em\u003ewith or without recognized human NTVC infections(42,43). Despite the low incidence, severe NTVC infections may be a real health risk especially with the observed increase in case numbers through Europe. Surface waters, especially shallow lakes are expected to grow warmer and contain more saline as a consequence of climate change, resulting in higher prevalence of non-cholerae vibrios. Subsequently, NTVC case numbers are also expected to rise in the temperate regions, potentially including such severe, life-threatening infections as reported in the present study(43). There are no known measures to limit the proliferation of NTVC in surface water, guidelines suggest increasing awareness of the public to the existing health risk and of physicians to potential water-related vibriosis cases(44). \u0026nbsp;\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eace\u003c/em\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;accessory cholera enterotoxin\u003c/p\u003e\n\u003cp\u003eAMR \u0026nbsp;antimicrobial resistance\u003c/p\u003e\n\u003cp\u003eARDS adult respiratory distress syndrome\u003c/p\u003e\n\u003cp\u003eBAL \u0026nbsp; bronchoalveolar lavage\u003c/p\u003e\n\u003cp\u003eCARD The Comprehensive Antibiotic Resistance Database\u003c/p\u003e\n\u003cp\u003eCFU\u003cem\u003e\u0026nbsp;\u003c/em\u003ecolony forming unit\u003c/p\u003e\n\u003cp\u003eCDS coding sequences\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ectx\u003c/em\u003eA cholera enterotoxin subunit A gene\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ectx\u003c/em\u003eB cholera enterotoxin subunit B gene\u003c/p\u003e\n\u003cp\u003eCT Cluster Type\u003c/p\u003e\n\u003cp\u003ecgMLST core genome multilocus sequence typing\u003c/p\u003e\n\u003cp\u003eECDC \u0026nbsp;European Centre for Disease Prevention and Control\u003c/p\u003e\n\u003cp\u003eEUCAST \u0026nbsp;The European Committee on Antimicrobial Susceptibility Testing\u003c/p\u003e\n\u003cp\u003eFSA bovine serum albumin\u003c/p\u003e\n\u003cp\u003eICU Intensive Care Unit\u003c/p\u003e\n\u003cp\u003eMALDI TOF MS Matrix-assisted laser desorption ionization\u0026ndash;time of flight mass spectrometry\u003c/p\u003e\n\u003cp\u003eMDR multidrug resistant\u003c/p\u003e\n\u003cp\u003eMLST multilocus sequence typing\u003c/p\u003e\n\u003cp\u003eNTVC non toxigenic Vibrio cholerae\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eomp\u003c/em\u003eW outer membrane protein W gene\u003c/p\u003e\n\u003cp\u003ePCR polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eqPCR quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003ePEEP positive end-expiratory pressure\u003c/p\u003e\n\u003cp\u003eSBA sheep blood agar\u003c/p\u003e\n\u003cp\u003eST Sequence Type\u003c/p\u003e\n\u003cp\u003e\u003cem\u003estn\u003c/em\u003e heat-labil enterotoxin\u003c/p\u003e\n\u003cp\u003eTCBS\u003cem\u003e\u0026nbsp;\u003c/em\u003ethiosulfate-citrate-bile salts-sucrose agar\u003c/p\u003e\n\u003cp\u003eTCP toxin gene toxin coregulated pilus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eT3SS Type 3 secretion system\u003c/p\u003e\n\u003cp\u003eT6SS Type 3 secretion system\u003c/p\u003e\n\u003cp\u003eUVF viable but not culturable form\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVDFB Virulence Factor Database\u003c/p\u003e\n\u003cp\u003eVP Vibrio pathogenic island\u003c/p\u003e\n\u003cp\u003eVSP1\u003cem\u003e\u0026nbsp;\u003c/em\u003eVibrio seventh pandemic island-1\u003c/p\u003e\n\u003cp\u003eVSP2\u003cem\u003e\u0026nbsp;\u003c/em\u003eVibrio seventh pandemic island-\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ezot\u003c/em\u003e zonula occludens toxin\u003c/p\u003e\n\u003cp\u003eWGS whole genome sequencing\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics Committee approval was not required as the Hungarian legislation on handling of personal health information (Law no. 1997. XLVII.) empowers the National Center for Public Health and Pharmacy to analyse data and take necessary measures in the interest of public health. Personal data have been handled in accordance with legal regulations and the Center\u0026rsquo;s data protection rules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from participants for publication of the data collected during the study, including any identifiable information.\u003c/p\u003e\n\u003cp\u003eMinisterial Decree No. 18/1998. (VI. 3.) NM. on epidemiological measures for the prevention of communicable diseases and outbreaks authorizes the National Center for Public Health and Pharmacy to analyze clinical samples and bacterial strains, and to take measures necessary measures in the interest of public health, including the management of personal and health data. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequences have been deposited at the GenBank under the accession of SUB13776351, BioProject PRJNA1007372.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was entirely funded by the National Center for Public Health and Pharmacy without any specific grant from other agencies in the public, commercial or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJH: Methodology, Software, Formal analysis, Investigation, Writing - original draft, Writing - review \u0026amp; editing, Visualization.\u003c/p\u003e\n\u003cp\u003e\u0026Aacute;T: Writing-original draft, review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eMV: Writing-original draft, review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eBP: Revising the manuscript\u003c/p\u003e\n\u003cp\u003eBK: Methology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eER: Revising the manuscript\u003c/p\u003e\n\u003cp\u003eKK, AK, and BN: Revising the manuscript, writing\u003c/p\u003e\n\u003cp\u003eSzT, TM: \u0026nbsp;Revising the manuscript,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZK: Revising the Manuscript, drafting\u003c/p\u003e\n\u003cp\u003eZsM, EM: Revising the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Erika Ungv\u0026aacute;ry for support and Zsuzsanna Richter for the technical assistance.\u003c/p\u003e\n\u003cp\u003eWe are thankful to Dr. G\u0026aacute;bor Kardos for his constructive feedback on the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also would like to thank the colleagues from the Governmental Office of the Capital City Budapest, Division of Public Health, Department of Laboratory for the environmental sampling.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVezzulli L, Baker‐Austin C, Kirschner A, Pruzzo C, Martinez‐Urtaza J. 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J Clin Microbiol. 2006 Sep;44(9):3459\u0026ndash;60. \u003c/li\u003e\n\u003cli\u003eMarinello S, Marini G, Parisi G, Gottardello L, Rossi L, Besutti V, et al. \u003cem\u003eVibrio cholerae\u003c/em\u003e non-O1, non-O139 bacteraemia associated with pneumonia, Italy 2016. Infection. 2017 Apr 11;45(2):237\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eShannon JD, Kimbrough RC. Pulmonary Cholera Due to Infection with a Non-O1 \u003cem\u003eVibrio cholerae\u003c/em\u003e Strain. J Clin Microbiol. 2006 Sep;44(9):3459\u0026ndash;60. \u003c/li\u003e\n\u003cli\u003eCastillo-Aguas G Del, Garc\u0026iacute;a-Vera C, Urkin J, Moretto M, Spreitzer MV, Keronen P, et al. Acute otitis media management: A survey of European primary care pediatricians. Global Pediatrics. 2023 Jun;4:100057. \u003c/li\u003e\n\u003cli\u003eZara M Patel M. Uncomplicated acute sinusitis and rhinosinusitis in adults: Treatment. 2022. \u003c/li\u003e\n\u003cli\u003eMatuschek E, Brown DFJ, Kahlmeter G. 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SKESA: Strategic k-mer extension for scrupulous assemblies. Genome Biol. 2018 Oct 4;19(1). \u003c/li\u003e\n\u003cli\u003eMcArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, et al. The Comprehensive Antibiotic Resistance Database. Antimicrob Agents Chemother. 2013 Jul;57(7):3348\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eChen L, Zheng D, Liu B, Yang J, Jin Q. VFDB 2016: hierarchical and refined dataset for big data analysis\u0026mdash;10 years on. Nucleic Acids Res. 2016 Jan 4;44(D1):D694\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eFlorensa AF, Kaas RS, Clausen PTLC, Aytan-Aktug D, Aarestrup FM. ResFinder \u0026ndash; an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes. Microb Genom. 2022 Jan 17;8(1). \u003c/li\u003e\n\u003cli\u003eSchwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J, Goesmann A. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb Genom. 2021 Nov 5;7(11). \u003c/li\u003e\n\u003cli\u003ePage MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021 Mar 29;n71. \u003c/li\u003e\n\u003cli\u003eBier N, Schwartz K, Guerra B, Strauch E. Survey on antimicrobial resistance patterns in \u003cem\u003eVibrio vulnificus\u003c/em\u003e and \u003cem\u003eVibrio cholerae\u003c/em\u003e non-O1/non-O139 in Germany reveals carbapenemase-producing Vibrio cholerae in coastal waters. Front Microbiol. 2015 Oct 28;6. \u003c/li\u003e\n\u003cli\u003eSchmidt K, Scholz HC, Appelt S, Michel J, Jacob D, Dupke S. Virulence and resistance patterns of \u003cem\u003eVibrio cholerae\u003c/em\u003e non-O1/non-O139 acquired in Germany and other European countries. Front Microbiol. 2023 Nov 22;14. \u003c/li\u003e\n\u003cli\u003eCousin VL, Pittet LF. Microbiological features of drowning-associated pneumonia: a systematic review and meta-analysis. Ann Intensive Care. 2024 Apr 20;14(1):61. \u003c/li\u003e\n\u003cli\u003eMandell LA, Niederman MS. Aspiration Pneumonia. New England Journal of Medicine. 2019 Feb 14;380(7):651\u0026ndash;63. \u003c/li\u003e\n\u003cli\u003eNishino K, Senda Y, Yamaguchi A. CRP Regulator Modulates Multidrug Resistance of Escherichia coli by Repressing the mdtEF Multidrug Efflux Genes. J Antibiot (Tokyo). 2008 Mar;61(3):120\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eAgyei FK, Scharf B, Duodu S. \u003cem\u003eVibrio cholerae\u003c/em\u003e Bacteremia: An Enigma in Cholera-Endemic African Countries. Trop Med Infect Dis. 2024 May 2;9(5):103. \u003c/li\u003e\n\u003cli\u003eReizine F, Delbove A, Tattevin P, Santos A Dos, Bodenes L, Bouju P, et al. Clinical and microbiological features of drowning-associated pneumonia: a retrospective multicentre cohort study. Clinical Microbiology and Infection. 2023 Jan;29(1):108.e7-108.e13. \u003c/li\u003e\n\u003cli\u003eCerland L, M\u0026eacute;garbane B, Kallel H, Brouste Y, Mehdaoui H, Resiere D. Incidence and Consequences of Near-Drowning\u0026ndash;Related Pneumonia\u0026mdash;A Descriptive Series from Martinique, French West Indies. Int J Environ Res Public Health. 2017 Nov 17;14(11):1402. \u003c/li\u003e\n\u003cli\u003eKumar A, Das B, Kumar N. \u003cem\u003eVibrio\u003c/em\u003e Pathogenicity Island-1: The Master Determinant of Cholera Pathogenesis. Front Cell Infect Microbiol. 2020 Oct 6;10. \u003c/li\u003e\n\u003cli\u003eBliem R, Reischer G, Linke R, Farnleitner A, Kirschner A. Spatiotemporal Dynamics of \u003cem\u003eVibrio cholerae\u003c/em\u003e in Turbid Alkaline Lakes as Determined by Quantitative PCR. Appl Environ Microbiol. 2018 Jun;84(11). \u003c/li\u003e\n\u003cli\u003eSterk A, Schets FM, de Roda Husman AM, de Nijs T, Schijven JF. Effect of Climate Change on the Concentration and Associated Risks of \u003cem\u003eVibrio\u003c/em\u003e Spp. in Dutch Recreational Waters. Risk Analysis. 2015 Sep;35(9):1717\u0026ndash;29. \u003c/li\u003e\n\u003cli\u003eWorld Health Organization. (\u0026lrm;2021)\u0026lrm;. WHO guidelines on recreational water quality: volume 1: coastal and fresh waters. World Health Organization. https://iris.who.int/handle/10665/342625. License: CC BY-NC-SA 3.0 IGO. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Non-O1, non-O139 Vibrio cholerae, pneumonia, WGS, cgMLST","lastPublishedDoi":"10.21203/rs.3.rs-5935550/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5935550/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNon-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e is an uncommon cause of pneumonia, particularly following freshwater exposure. Non-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e was identified from bronchoalveolar lavage through culture and quantitative polymerase chain reaction (qPCR) in Hungary. Epidemiological investigation traced the source of infection in a designated bathing site in a lake in Central Hungary, where non-O1,non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e was isolated from surface water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted whole-genome sequencing and comparative genomic analysis on a clinical isolate (N=1) and three phenotypically distinct environmental isolates (N=3). In addition, we reviewed the available literature on pulmonary infections associated with \u003cem\u003eVibrio cholerae\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCore genome multilocus sequence typing (cgMLST) revealed that the clinical and environmental isolates clustered together with zero allelic differences. Multilocus sequence typing (MLST) identified a new sequence type (ST1605), representing a novel combination of known allele variants. In silico analysis of antibiotic resistance genes identified the presence of \u003cem\u003ebla\u003c/em\u003eCARB-7. Both the clinical and environmental isolates exhibited identical virulence gene profiles, reinforcing the hypothesis that the infection was acquired from the local water source.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study represents the first investigation of a primary pulmonary \u003cem\u003eVibrio cholerae\u003c/em\u003e infection in Europe following a near-drowning event. While \u003cem\u003eVibrio vulnificus\u003c/em\u003e and \u003cem\u003eVibrio metschnikovii\u003c/em\u003e have been implicated in similar pneumonia cases, the precise virulence mechanisms of these species remain poorly understood. Although non-O1, non-O139 \u003cem\u003eVibrio cholerae\u003c/em\u003e infections linked to recreational water exposure are rare in Hungary (1-2 cases annually), this study underscores the importance of ongoing surveillance to detect potential outbreaks and inform public health responses.\u003c/p\u003e","manuscriptTitle":"Genomic characterization of non-O1, non-O139 Vibrio cholerae causing rare clinical manifestation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 09:44:55","doi":"10.21203/rs.3.rs-5935550/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T11:57:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-17T00:44:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-06T16:20:00+00:00","index":"hide","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-05T17:15:03+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"290229011044496320637609877893678580929","date":"2025-05-01T11:25:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52274173446430785207970959828123109313","date":"2025-05-01T11:22:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-29T10:48:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-28T23:21:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-04-23T14:43:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2428e9c1-fde0-40a7-b2f7-9be8a48c86b8","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:03:07+00:00","versionOfRecord":{"articleIdentity":"rs-5935550","link":"https://doi.org/10.1186/s12879-025-12191-9","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2025-12-09 15:58:14","publishedOnDateReadable":"December 9th, 2025"},"versionCreatedAt":"2025-05-06 09:44:55","video":"","vorDoi":"10.1186/s12879-025-12191-9","vorDoiUrl":"https://doi.org/10.1186/s12879-025-12191-9","workflowStages":[]},"version":"v1","identity":"rs-5935550","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5935550","identity":"rs-5935550","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}