Potential pathogenicity and antibiotic resistance of aquatic Vibrio isolates from freshwater in Slovakia

  • Jana ValárikováEmail author
  • Jana Korcová
  • Jana Ziburová
  • Jozef Rosinský
  • Alžbeta Čížová
  • Sandra Bieliková
  • Martin Sojka
  • Pavol Farkaš
Original Article


This study aimed to evaluate the potential pathogenicity and antibiotic resistance of 31 environmental Vibrio isolates obtained from surface water in southern and eastern Slovakia. Isolates were identified as Vibrio cholerae non-O1/non-O139 and Vibrio metschnikovii by biochemical tests, MALDI biotyping, and 16S RNA gene sequencing. Analysis of the susceptibility to 13 antibacterial agents showed susceptibility of all isolates to ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, gentamicin, imipenem, tetracyclin, and doxycycline. We recorded high rates of resistance to β-lactams and streptomycin. Investigation of antibiotic resistance showed five different antibiotic profiles with resistance to antibacterials from three classes, but no multidrug resistance was observed. The investigation of the pathogenic potential of V. cholerae isolates showed that neither the cholera toxin coding gene ctxA nor the genes zot (zonula occludens toxin), ace (accessory cholera toxin), and tcpA (toxin-coregulated pilus) were present in any of 31 isolated samples. Gene ompU (outer membrane protein) was confirmed in 80% and central regulatory protein-coding gene toxR in 71% of V. cholerae isolates, respectively. A high prevalence of the hemolysin coding gene hlyA in all V. cholerae was observed. The data point toward the importance of systematic monitoring and comparative studies of potentially pathogenic vibrios in European countries.


Vibrio cholerae non-O1/non-O139 Vibrio metschnikovii Antibiotic resistance Virulence MALDI biotyping 



We thank Dr. Kovac (National Institutes of Health, Maryland) for providing the strain V. cholerae O139 CIRS 245. We also thank Dr. Slavomír Bystrický for valuable suggestions to improve the paper.

Funding information

This study was funded by Centre of Excellence for Glycomics, supported by the Research & Development Operational Programme funded by the ERDF (ITMS 26240120031) and by the grant VEGA (2/0093/17) supported by the Ministry of Education, Science, Research and Sport of the Slovak Republic.

Compliance with ethical standards

Conflict of interest

The autors declare that they have no conflict of interest.

Supplementary material

12223_2019_760_MOESM1_ESM.doc (120 kb)
ESM 1 (DOC 120 kb)


  1. Andersson Y, Ekdahl K (2006) Wound infections due to Vibrio cholerae in Sweden after swimming in the Baltic Sea, summer 2006. Euro Surveill 11(8):E060803.2. CrossRefPubMedGoogle Scholar
  2. Baker-Austin C, Stockley L, Rangdale R, Martinez-Urtaza J (2010) Environmental occurrence and clinical impact of Vibrio vulnificus and Vibrio parahaemolyticus: a European perspective. Environ Microbiol Rep 2:7–18. CrossRefPubMedGoogle Scholar
  3. Baker-Austin C, Trinanes J, Gonzalez-Escalona N, Martinez-Urtaza J (2017) Non-cholera vibrios: the microbial barometer of climate change. Trends Microbiol 25:76–84. CrossRefPubMedGoogle Scholar
  4. Baker-Austin C, Trinanes J, Salmenlinna S, Löfdahl M, Siitonen A, Taylor NG, Martinez-Urtaza J (2016) Heat wave-associated vibriosis, Sweden and Finland, 2014. Emerg Infect Dis 22:1216–1220. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Baron S, Larvor E, Chevalier S, Jouy E, Kempf I, Granier SA, Lesne J (2017) Antimicrobial susceptibility among urban wastewater and wild shellfish isolates of non-O1/non-O139 Vibrio cholerae from La Rance estuary (Brittany, France). Front Microbiol 8:1637. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Baron S, Lesne J, Jouy E, Larvor E, Kempf I, Boncy J, Rebadet S, Piarroux R (2016) Antimicrobial susceptibility of autochthonous aquatic Vibrio cholerae in Haiti. Front Microbiol 7:1671. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bhuyan SK, Vairale MG, Arya N, Yadev P, Veer V, Singh L, Yadava PK, Kumar P (2016) Molecular epidemiology of Vibrio cholerae associated with flood in Brahamputra river valley, Assam, India. Infect Genet Evol 40:352–356. CrossRefPubMedGoogle Scholar
  8. Bier N, Schwartz K, Guerra B, Strauch E (2015) Survey on antimicrobial resistance patterns in Vibrio vulnificus and Vibrio cholerae non-O1/non-O139 in Germany reveals carbapenemase-producing Vibrio cholerae in coastal waters. Front Microbiol 6:1179. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cabral JP (2010) Water microbiology. Bacterial pathogens and water. Int J Environ Res Public Health 7:3657–3703. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Cheng WC, Jan IS, Chen JM, Teng SH, Sheng WH, Ko WC, Hsueh PR (2015) Evaluation of the Bruker Biotyper matrix-assisted laser desorption ionization–time of flight mass spectrometry system for identification of blood isolates of Vibrio species. J Clin Microbiol 53:1741–1744. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Clinical and Laboratory Standards Institute (2012). Performance standards for antimicrobial disk susceptibility tests; approved standard—eleventh edition. CLSI document M02-A11. Clinical and Laboratory Standards Institute, Wayne, PA, USAGoogle Scholar
  12. Clinical and Laboratory Standards Institute (2015). Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria—3rd edition. CLSI guideline M45. Clinical and Laboratory Standards Institute, Wayne, PA, USAGoogle Scholar
  13. Clinical and Laboratory Standards Institute (2018). Performance standards for antimicrobial susceptibility testing—28th edition. CLSI supplement M100. Clinical and Laboratory Standards Institute, Wayne, PA, USAGoogle Scholar
  14. Farmer JJ III, Hickman-Brenner FW (2006) The genera Vibrio and Photobacterium. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E (eds) The prokaryotes: a handbook on the biology of bacteria, 3rd edn. Springer, New York, pp 508–563CrossRefGoogle Scholar
  15. Faruque SM, Biswas K, Udden SM, Ahmad QS, Sack DA, Nair GB, Mekalanos JJ (2006) Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proc Natl Acad Sci U S A 103:6350–6355. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Faruque SM, Chowdhury N, Kamruzzaman M, Dzjejman M, Rahman MH, Sack DA, Nair GB, Mekalanos JJ (2004) Genetic diversity and virulence potential of environmental Vibrio cholerae population in a cholera-endemic area. Proc Natl Acad Sci U S A 101:2123–2128. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Fasano A, Baudry B, Pumplin DW, Wasserman SS, Tall BD, Ketley JM, Kaper JB (1991) Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions. Proc Natl Acad Sci U S A 88:5242–5246. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fong JC, Syed KA, Klose KE, Yildiz FH (2010) Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis. Microbiology 156:2757–2769. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Frank C, Littman M, Alpers K, Hallauer J (2006) Vibrio vulnificus wound infections after contact with the Baltic Sea. Germany Weekly Releases (1997–2007) 11:3024. CrossRefGoogle Scholar
  20. Gupta P, Mankere B, Chekkoora Keloth S, Tuteja U, Pandey P, Thava Chelvam K (2018) Increased antibiotic resistance exhibited by the biofilm of Vibrio cholerae O139. J Antimicrob Chemother 73:1841–1847. CrossRefPubMedGoogle Scholar
  21. Heiberg B (1936) The biochemical reaction of vibrios. J Hyg (Lond) 36:114–117. CrossRefGoogle Scholar
  22. Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Taylor RK, Levine MM (1988) Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med 168:1487–1492. CrossRefPubMedGoogle Scholar
  23. Hirk S, Huhulescu S, Allerberger F, Lepuschitz S, Rehak S, Weil S, Gschwandtner E, Hermann M, Neuhold S, Zoufaly A, Indra A (2016) Necrotizing fasciitis due to Vibrio cholerae non-O1/non-O139 after exposure to Austrian bathing sites. Wien Klin Wochenschr 128:141–145. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hsueh PR, Kuo LC, Chang TC, Lee TF, Teng SH, Chuang YCH, Teng LJ, Sheng WH (2014) Evaluation of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of blood isolates of Acinetobacter species. J Clin Microbiol 52:3095–3100. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Huhulescu S, Indra A, Feierl G, Stoeger A, Ruppitsch W, Sarkar B, Allerberger F (2007) Occurrence of Vibrio cholerae serogroups other than O1 and O139 in Austria. Wien Klin Wochenschr 119:235–241. CrossRefPubMedGoogle Scholar
  26. Kirschner AKT, Schlesinger J, Farnleitner AH, Hornek R, Süß B, Golda B, Herzig A, Reitner B (2008) Rapid growth of planktonic Vibrio cholerae non-O1/non-O139 strains in a large alkaline lake in Austria: dependence on temperature and dissolved organic carbon quality. Appl Environ Microbiol 74:2004–2015. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kokashvili T, Whitehouse CA, Tskhvediani A, Grim CJ, Elbakidze T, Mitaishvili N, Janelidze N, Jaiani E, Haley BJ, Lashkhi N, Huq A, Colwell RR, Tediashvili M (2015) Occurrence and diversity of clinically important Vibrio species in the aquatic environment of Georgia. Front Public Health 3:232. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kumar PA, Patterson J, Karpagam P (2009) Multiple antibiotic resistance profiles of Vibrio cholerae non-O1 and non-O139. Jpn J Infect Dis 62:230–232PubMedGoogle Scholar
  29. Lackova D, Sojka M (2018) A rare case of infection caused by Vibrio cholerae non-O1, non-O139 in clinical practice in Slovakia [Ojedinelý prípad infekcie spôsobenej Vibrio cholerae non-O1, non-O139 v klinickej praxi na Slovensku]. Newslab. 1:50–53Google Scholar
  30. Le Roux F, Wegner KM, Baker-Austin C et al (2015) The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis. Front Microbiol 6:830. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Leibovici-Weissman Y, Neuberger A, Bitterman R, Sinclair D, Salam MA, Paul M (2014) Antimicrobial drugs for treating cholera. Cochrane Database Syst Rev 6:1–182. CrossRefGoogle Scholar
  32. Lukinmaa S, Mattila K, Lehtinen V, Hakkinen M, Koskela M, Siitonen A (2006) Territorial waters of the Baltic Sea as a source of infections caused by Vibrio cholerae non-O1, non-O139: report of 3 hospitalized cases. Diagn Microbiol Infect Dis 54:1–6. CrossRefPubMedGoogle Scholar
  33. Machado A, Bordalo AA (2016) Detection and quantification of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in coastal waters of Guinea-Bissau (West Africa). Ecohealth 13:339–349. CrossRefPubMedGoogle Scholar
  34. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268–281. CrossRefGoogle Scholar
  35. Marek A, Inkster T, Anderson E, Jenkins C, Boyd J, Kerr S, Cowden J (2013) Non-toxigenic Vibrio cholerae bacteraemia: case report and review of the literature. J Med Microbiol 62:1357–1359. CrossRefPubMedGoogle Scholar
  36. Mathur J, Waldor MK (2004) The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides. Infect Immun 72:3577–3583. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Mohammed Y, Aboderin AO, Okeke IN, Olayinka AT (2018) Antimicrobial resistance of Vibrio cholerae from sub-Saharan Africa: a systematic review. Afr J Lab Med 7:778. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Nair GB, Oku Y, Takeda Y, Ghosh A, Ghosh RK, Chattopadhyay S, Pal SC, Kaper JB (1988) Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India. Appl Environ Microbiol 54:3180–3182PubMedPubMedCentralGoogle Scholar
  39. Nandi B, Nandy RK, Mukhopadhyay S, Nair GB, Shimada T, Ghose AC (2000) Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. J Clin Microbiol 38:4145–4151PubMedPubMedCentralGoogle Scholar
  40. Nandi B, Nandy RK, Sarkar A, Ghose AC (2005) Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae. Microbiology 151:2975–2986. CrossRefPubMedGoogle Scholar
  41. Ottaviani D, Leoni F, Rocchegiani E, Santarelli S, Masini L, di Trani V, Canonico C, Pianetti A, Tega L, Carraturo A (2009) Prevalence and virulence properties of non-O1 non-O139 Vibrio cholerae strains from seafood and clinical samples collected in Italy. Int J Food Microbiol 132:47–53. CrossRefPubMedGoogle Scholar
  42. Ottemann KM, Mekalanos JJ (1996) The ToxR protein of Vibrio cholerae forms homodimers and heterodimers. J Bacteriol 178:156–162. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Parte AC (2018) LPSN—list of prokaryotic names with standing in nomenclature (, 20 years on. Int J Syst Evol Microbiol 68:1825–1829. CrossRefPubMedGoogle Scholar
  44. Paz S, Bisharat N, Paz E, Kidar O, Cohen D (2007) Climate change and the emergence of Vibrio vulnificus disease in Israel. Environ Res 103:390–396. CrossRefPubMedGoogle Scholar
  45. Rahman MH, Biswas K, Hossain MA, Sack RB, Mekalanos JJ, Faruque SM (2008) Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains. DNA Cell Biol 27:347–355. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Raissy M, Moumeni M, Ansari M, Rahimi E (2012) Occurrence of Vibrio spp. in lobster and crab from the Persian gulf. J Food Saf 32:198–203. CrossRefGoogle Scholar
  47. Rashed SM, Hasan NA, Alam M, Sadique A, Sultana M, Hoq MM, Sack RB, Colwell RR, Huq A (2017) Vibrio cholerae O1 with reduced susceptibility to ciprofloxacin and azithromycin isolated from a rural coastal area of Bangladesh. Front Microbiol 8:252. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Rivera ING, Chun J, Huq A, Sack B, Colwell RR (2001) Genotypes associated with virulence in environmental isolates of Vibrio cholerae. Appl Environ Microbiol 67:2421–2429. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy MC, Michael I, Fatta-Kassinos D (2013) Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. Sci Total Environ 447:345–360. CrossRefPubMedGoogle Scholar
  50. Rosinsky J (1996) The unwritten history of microbiological diagnostics. The causative agent of cholera isolated in Slovakia 25 years ago. [Z nepísaných dejín mikrobiologickej diagnostiky. Pred 25 rokmi Bol na Slovensku izolovaný pôvodca cholery]. Lekárske listy, príloha Zdravotníckych novín 18:7Google Scholar
  51. Schirmeister F, Dieckmann R, Bechlars S, Bier N, Faruque SM, Strauch E (2014) Genetic and phenotypic analysis of Vibrio cholerae non-O1, non-O139 isolated from German and Austrian patients. Eur J Clin Microbiol Infect Dis 33:767–778. CrossRefPubMedGoogle Scholar
  52. Seman M, Prokšová M, Rosinský J, Ferianc P (2012) Isolation, identification, and characterization of Vibrio cholerae from the Danube river in Slovakia. Folia Microbiol 57:191–197. CrossRefGoogle Scholar
  53. Shin OS, Tam VC, Suzuki M, Ritchie JM, Bronson RT, Waldor MK, Mekalanos JJ (2011) Type III secretion is essential for the rapidly fatal diarrheal disease caused by non-O1, non-O139 Vibrio cholerae. mBio. 2:e00106-11.
  54. Silva AJ, Benitez JA (2016) Vibrio cholerae biofilms and cholera pathogenesis. PLoS Negl Trop Dis 10:e0004330. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Singh DV, Isac SR, Colwell RR (2002) Development of a hexaplex PCR assay for rapid detection of virulence and regulatory genes in Vibrio cholerae and Vibrio mimicus. J Clin Microbiol 40:4321–4324. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Smith HL, Goodner K (1965) On the classification of vibrios. In: Proceedings of the cholera research symposium (pp. 4–8), January 24–29, 1965, Honolulu, Hawaii. Public Health Service Publication No. 1328. Government Printing Office: Washington, USAGoogle Scholar
  57. Spangler BD (1992) Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 56:622–647PubMedPubMedCentralGoogle Scholar
  58. Spira WM, Fedorka-Cray PJ (1984) Purification of enterotoxins from Vibrio mimicus that appear to be identical to cholera toxin. Infect Immun 45:679–684PubMedPubMedCentralGoogle Scholar
  59. Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Meth 40:175–179. CrossRefGoogle Scholar
  60. Stypulkowska-Misiurewicz H, Pancer K, Roszkowiak A (2006) Two unrelated cases of septicaemia due to Vibrio cholerae non-O1, non-O139 in Poland, July and August 2006. Euro Surveill 11:3088. CrossRefGoogle Scholar
  61. Tan KK, Sin KS, Ng AJ, Yahya H, Kaur P (1994) Non-O1 Vibrio cholerae septicaemia: a case report. Singap Med J 35:648–649Google Scholar
  62. Trucksis M, Galen JE, Michalski J, Fasano A, Kaper JB (1993) Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette. Proc Natl Acad Sci U S A 90:5267–5271. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Turner S, Pryer KM, Miao VPW, Palmer JD (1999) Investigating deep phylogenetic relationships among Cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryot Microbiol 46:327–338. CrossRefPubMedGoogle Scholar
  64. Watts J, Schreier H, Lanska L, Hale MS (2017) The rising tide of antimicrobial resistance in aquaculture: sources, sinks and solutions. Mar Drugs 15:158. CrossRefPubMedCentralGoogle Scholar
  65. Yildiz FH, Visick KL (2009) Vibrio biofilms: so much the same yet so different. Trends Microbiol 17:109–118. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019

Authors and Affiliations

  1. 1.Institute of ChemistrySlovak Academy of SciencesBratislavaSlovakia
  2. 2.Institute of Epidemiology, Faculty of MedicineComenius UniversityBratislavaSlovakia
  3. 3.National Reference Centre for Vibrionaceae, Regional Public Health AuthorityKomárnoSlovakia

Personalised recommendations