Introduction

Bacterial genus Vibrio comprises of 151 validly published species (https://lpsn.dsmz.de/genus/vibrio) occurring mainly in aquatic habitats worldwide (Murray et al. 2012). The most significant species is V. cholerae, causative agent of acute diarrheal disease called cholera (Baker-Austin et al. 2018). Cholera is manifested by watery diarrhea resulting in severe dehydration. Annually, 4 million cases and 21 000–143 000 deaths are estimated (https://www.who.int/news-room/fact-sheets/detail/cholera). Since 1817, seven cholera pandemics have been recorded. The last one, still ongoing, has begun in 1961 in Indonesia (Hu et al. 2016).

V. cholerae is divided into 2 serogroups based on the somatic antigen O. The non-O1 serogroup includes serotypes O2 to O200 (Oliver et al. 2013). Serotypes of serogroup O1 are Ogawa, Inaba and Hikojima, serogroup O1 includes also biotypes called El Tor and classical. Only serogroup O1 and serotype O139, belonging to the serogroup non-O1, can induce cholera, but only toxigenic strains producing cholera toxin (CT), toxin-co-regulated pilus (TCP) and the regulatory protein ToxR, which co-regulates CT and TCP expression (Cheasty 2006). Other serotypes of the non-O1 serogroup generally do not produce CT and do not cause diarrheal epidemics but may produce other toxins. They occasionally cause diarrhoea and infections of ears, wounds, or cerebrospinal fluid (Kaper et al. 1995; Weil and Ryan 2018). Other species as V. alginolyticus, V. anguillarum, V. cincinnatiensis, V. fluvialis, V. furnissii, V. harveyi, V. metschnikovii, V. mimicus, V. parahaemolyticus, V. vulnificus and recently described species V. cidicii, V. injenensis, V. metoecus, V. navarrensis are associated with human infections causing gastrointestinal and extraintestinal infections, including eyes, ears, wounds infections and septicemia. The source of infection is contaminated water or raw shellfish and fish (Janda 2020).

Epidemic of cholera has occurred in 1970 in Slovakia with an epicentre in the village of Vojany in Zemplín region (Seman 2005). The occurrence of vibrios in the Danube River was documented by Seman et al. (2012). In Slovakia, in 2017 V. cholerae non-O1/non-O139 non-CT producing strain caused gastroenteritis in an immunocompetent woman who became infected by ingesting contaminated water from a well (Lacková and Sojka 2018). Potential pathogenicity and antibiotic resistance of aquatic Vibrio isolates from freshwater in Slovakia documented Valáriková et al. (2020).

The longest river within today’s European Union– and second-longest on the continent (after the Volga in Russia) has length 2,872 km (1,785 miles). It flows into the territory of Slovakia through the Devín Gate in rkm 1880. It is already a mighty river in Bratislava. The average discharge of the Danube in Bratislava is 2,025 m³/s and the width of the riverbed is approximately 300 m (https://www.icpdr.org/sites/default/files/nodes/documents/icpdr_facts_figures.pdf).

From a hygienic and ecological point of view, water quality in the Danube is important. It has been improving over the years. To improve the water quality, an ambitious programme of measures for the whole Danube River Basin District has been agreed under the European Union Water Framework Directive (Kirschner et al. 2017).

To assess trends in water quality, the International Commission for the Protection of the Danube River (ICPDR) oversees the TransNational Monitoring Network (TNMN). The network carefully monitors physical, chemical, biological and microbiological conditions in the Danube and its tributaries and provides in TNMN Yearbooks an annual overview of pollution levels as well as long term trends for water quality in the basin (http://www.icpdr.org/main/issues/water-quality).

For Slovakia, the quality of the Danube water is of great importance. The Danube is the source of water for the irrigation of the most agriculturally important part of Slovakia– Žitný ostrov. Ecological status is based on the assessment of the status of all biological and microbiological quality elements, the supportive physico-chemical parameters as well as hydromorphological parameters (Fľaková et al. 2014). The Danube River in area of Bratislava City is also used by inhabitants for recreational activities and fishing.

In the hygienic assessment of water quality, in addition to the legislatively defined indicators, it would be appropriate to focus attention on other risk groups, such as emergent pathogens, for example.

Therefore, our work was focused on the occurrence, isolation and identification potentially dangerous emergent pathogens of the genus Vibrio in the Danube River when it enters the territory of Slovakia in the area of the city of Bratislava. The work follows on previous studies on the occurrence of vibrios in the Danube River.

Materials and methods

Sample collection and physicochemical analyses of water

Water samples were collected during the seasonal period from January to July of 2011 from two sampling points of the Danube River in Bratislava City, located on opposite banks of the river at rkm 1871,5: sampling point no. 1, left bank (48°8’38” N), approximately 150 meters above the Lanfranconi bridge in Bratislava and sampling point no. 2, right bank (17°4’26” E), about 150 m above the Lanfranconi bridge. The span between the banks is 510,97 m, distance from the Austrian-Slovak border about 1000 m (Fig. 1).

Fig. 1
figure 1

Sampling points at the entrance of the Danube to the territory of Slovakia (Map: Ivana Ondrejková)

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Samples of water were taken in sterile 0.5 L dark glass bottles with ground glass joints from both sampling points from depth of 20–30 cm and 2–3 m from the shore. Physicochemical parameters of water (water temperature, pH, electrical conductivity (EC), dissolved oxygen, oxygen saturation) were performed in situ at each sampling point. The EC measurements were performed using a WTW Multi 350i portable instrument with a TetraCon® 325 electrode. The WTW Multi 350i portable instrument with SenTix® 41 electrode was used for pH measurement and the WTW Oxi 340i/SET portable instrument with DurOx® 325-3 electrode for dissolved oxygen and oxygen saturation. Samples were transported in a portable refrigerator (5 °C) to the laboratory and processed immediately upon arrival in the laboratory.

Determination of total microbial profile

The total microbial profile of the culturable heterotrophic microbiota was performed by determining indicator microorganisms in the range: colony count at 22 °C (CC 22), colony count at 36 °C (CC 36), total coliform (TC) and enterococci (EC) in accordance with the Häusler reference method (Häusler 1994).

Isolation of vibrios

Cultivation in enrichment broth alkaline peptone water (APW) (1% peptone, 1% NaCl, 100 mL of distilled water, pH 8.6) was used for isolation of vibrios. Five hundred milliliters of water were added to one hundred milliliters of 5-times concentrated APW and incubated at 37 °C for 6 h. After that, broth was streaked on solid selective medium thiosulfate-citrate-bile-salts-sucrose (TCBS) agar (HiMedia Laboratories, India) and cultivation was performed at 37 °C for 24–48 h. Yellow colonies were selected and inoculated separately on Mueller-Hinton agar (MHA) (HiMedia Laboratories, India) to obtain pure cultures for species identification. Cultivation took place at 37 °C for 24–48 h. Isolates are maintained at -80 °C in Luria–Bertani Broth (pH 8.5) (Sigma-Aldrich, Germany) containing 20% glycerol.

Biochemical identification

Oxidase test was performed using reagent for OXI test (Erba-Lachema, Czech Republic). Strains Pseudomonas aeruginosa CCM 1960 and Escherichia coli CCM 3954 were used as a positive and negative control, respectively. Biochemical identification was performed using ENTEROtest 24 kit (Erba-Lachema, Czech Republic) according to manufacturer’s instructions. Tests were evaluated by program TNW Lite CZ 7.0 (Erba Lachema, Czech Republic). The identification result was characterized by two statistical values: the identification score (ID %, interval 0–100), expressing the probability of being the determined taxon, and the taxonomical index (T-index, interval 0–1), expressing the typicality of the strain (distance from the reference strain). The level of identification is characterized by 5 categories: A (ID = 99.01–100.00%), B (ID = 95.01–99.00%), C (ID = 90.01–95.00%), D (80.01–90.00%), E (˂ 80.00%).

Serological identification

Polyvalent O antiserum kits for O1 and O139 V. cholerae (Denka Seiken, Japan) were used to determine the serotype of positive isolates of V. cholerae by slide agglutination, according to the manufacturer’s instructions.

DNA isolation for 16S rRNA amplification

Isolates were incubated in Mueller-Hinton broth (MHB) (HiMedia Laboratories, India) at 37 °C overnight. DNA was isolated from the grown culture using SiMax™– Genomic DNA Extraction kit according to manufacturer’s instructions. The volume of 1.5 mL of the culture was centrifuged for 30 s at 13,000 rpm. The cell pellet was resuspended in 0.5 mL of TE buffer and 1 mL of GN binding buffer was added and mixed thoroughly. The mixture was transferred to a Miniprep spin column placed in a 2 mL Collection tube. The complex stood for 5 min and then was centrifuged for 30 s at 13,000 rpm. This step was repeated with the rest of the lysate. Subsequently, 0.5 mL of washing buffer was added to the Spin column and centrifuged for 30 s at 13,000 rpm. This step was repeated 2 times. The spin columns were placed in a new 1.5 mL microtube and placed with the lid open for 3 min. 100 µL of 60 °C TE buffer was added into center part of the SiMax™ membrane in the spin column and incubated for 5 min at room temperature. This was followed by centrifugation for 1 min at 13,000 rpm. Isolated DNA was used to amplify 16S rRNA by PCR.

  16S rRNA amplification

The PCR reaction mixture (20 µL) contained 2.5 µL of 10 × buffer with MgCl2 (BioThermStar); 0.25 µL of 200 µM deoxyribonucleotide triphosphates (dNTPs); 0,25 µL of 2.5 U DNA Polymerase (BioThermStar), 5 µL of 0.5 µM primers 27f (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1390r (5´-AGGCCCGGGAACGTATTCAC-3´); 10 µL of deionized sterile H2O and 2 µL of DNA. Amplification was performed in Peltier Thermal Cycler Biometra Tprofessional Gradient 96 (Analytic Jena, Germany). The reaction conditions were as follows: initial denaturation at 95 °C for 15 min followed by 30 cycles consisting of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 3 min. Amplified product (10 µL) mixed with 2 µL of Orange 6 × Loading Dye was analyzed using agarose gel electrophoresis (1%) in 1 × TAE buffer, 70 V, 1.5 h. DNA was stained with Gold View™ (Genetech, China) and visualized with UV-light at a wavelength of 254 nm.

Amplified ribosomal DNA restriction analysis (ARDRA)

The volume of 2 µL of BSA buffer, 5 µL of analyzed DNA, 0.5 µL of AluI enzyme (Promega) were pipetted into the microtube and the volume was made up to 20 µL with sterile deionized water. The restriction mixture was incubated for 3 h at 37 °C. Products were analyzed using agarose gel electrophoresis (1%) in 1 × TAE buffer at 70 V for 1.5 h. The size of the fragments was determined by comparison with the 100 bp and 2000 bp ladder (Genetech, China). Restriction profiles of isolates and reference strains were compared.

As a control, reference strains Vibrio fluvialis CCM 3689, Vibrio furnissii CCM 3696, Vibrio metschnikovii CCM 4065, Vibrio vulnificus CCM 2838 from the Czech Collection of Microorganisms (CCM; Brno, Czech Republic) and Vibrio cholerae CNCTC 71/89 from the Czech National Collection of Type Cultures (CNCTC; Prague, Czech Republic) were used.

PCR identification and determination of virulence factors

Isolates were cultivated on MHA (HiMedia Laboratories, India) at 37 °C for 24 h. From the grown culture, one inoculation loop (approximately 5 morphologically identical colonies) was resuspended in 300 µL of sterile deionized water in a microtube. The suspension was incubated at 100 °C for 10 min and then centrifuged at 4 °C and 10,500 rpm for 8 min. The obtained cell lysate contained in the supernatant was transferred to a new microtube and could be used immediately in the PCR reaction or stored at a temperature of -20 °C for a maximum of 2 weeks.

DNA polymerization mix (Genecraft, Germany) containing dNTP, Taq DNA polymerase, 10 × buffer with 15 mM MgCl2 was used for the PCR reaction. The instructions recommended by the manufacturer have been followed.

For species identification primers for gene ompW– (F) CAC CAA GAA GGT GAC TTT ATT GTG, (R) GAA CTT ATA ACC ACC CGC G (Nandi et al. 2000) (0.2 µL, 0.5 µM) were used. Virulence factors determination was performed using primers for genes toxR– (F) CCT TCG ATC CCC TAA GCA ATA C, (R) AGG GTT AGC AAC GAT GCG TAA G (Singh et al. 2002) (0.4 µL, 0.5 µM), ctxA– (F) CGG GCA GAT TCT AGA CCT CCT G, (R) CGA TGA TCT TGG AGC ATT CCC AC (Singh et al. 2002) (0.2 µL, 0.5 µM) and tcpA– (F) CAC GAT AAG AAA ACC GGT CAA GAG, (R) TTA CCA AAT GCA ACG CCG AAT G (Singh et al. 2002) (0.25 µL, 0.5 µM).

Amplification was performed in Peltier Thermal Cycler Biometra Tprofessional Gradient 96 (Analytic Jena, Germany). The reaction conditions were as follows: initial denaturation at 94 °C for 2 min followed by 30 cycles consisting of denaturation at 90 °C for 30 s, annealing at 62 °C for 30 s, and elongation at 72 °C for 60 s, and a final elongation at 72 °C for 8 min (Drahovská, personal communication).

After completion of the PCR reaction, the product was stored at a temperature of -20 °C.

The size of the amplified products of the PCR reaction was determined after electrophoretic separation in an agarose gel. A horizontal apparatus with 1.5% agarose gel was used for electrophoresis of nucleic acids. To confirm the presence of the PCR product, 8 µL of the sample mixed with 2 µL of STOP C application solution was applied to the gel. The molecular weight standard (100 bp DNA Ladder, Genetech, China) was applied to the first lane, the positive control containing the detected gene to the second lane, and the samples obtained to the other lanes from the tested isolates. Electrophoresis was carried out in a solution of 1 × TAE buffer (pH 8.0) at 90 V for 1.5 h. After running gel electrophoresis, the gel was stained with Gold View™ (Genetech, China) for approximately 30 min. DNA was visualized on a UV transilluminator at a wavelength of 254 nm. The approximate size of the amplified DNA fragments was determined by comparison with a molecular weight standard.

RESULTS

Sample collection and physico-chemical parameters of water

During the seasonal period, samples of the Danube waters were taken from two sampling points. In situ analysis of basic physico-chemical parameters was performed at sampling points. The samples were then transported to a laboratory for microbiological analysis.

We carried out 7 samplings. During all sampling, physico-chemical parameters of water were determined, data shown in Table 1.

Table 1 Physico-chemical parameters of analyzed water
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Determination of total microbial profile

Heterotrophic aerobic microorganisms cultivable at temperatures of 22 °C and 36 °C as well as total coliforms and enterococci were applied to determine the microbial profile and hygienic quality of Danube water samples. During all sampling the limit set by Slovak Government Regulation No 269/2010 was not exceeded for any microbiological indicator (Table 2).

Table 2 Total microbial profile of water samples
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Isolation of vibrios

APW (pH 8.8) was used for isolation of members of the genus Vibrio. Incubations took place at time intervals of 6, 24 and 48 h. at 37 °C. At each time interval, one bacteriological loop was inoculated onto two parallel Petri dishes containing TCBS agar and subsequently incubated at 37 °C for 24 h. Ten arbitrarily selected colonies with typical micromorphology on TCBS agar– yellow, flat, with diameter of 1–3 mm were obtained from all samplings and inoculated separately on MHA. A total of 70 (10 from each sampling) colonies were selected for presumptive identification by ENTEROtest 24.

Phenotypic identification

Using ENETEROtest 24, 48 isolates were identified as the genus Aeromonas and 22 isolates as the genus Vibrio. Of them, 14 isolates were identified as V. cholerae, 5 as V. fluvialis, 2 as V. metschnikovii and 1 isolate as V. vulnificus (Table 3).

All isolates of V. metschnikovii were oxidase-negative, other Vibrio spp. were oxidase-positive.

Acceptable identification at the species level (IL = B, C) had 9 isolates, 3 strains at the genus level (IL = D), and there were 10 intermediate isolates (IL = E).

All strains were stored in Luria–Bertani Broth (pH 8.5) (Sigma-Aldrich, Germany) supplemented with 20% glycerol and stored at -80 °C for further use.

Table 3 Phenotypic identification of isolates by ENTEROtest 24
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Molecular ARDRA identification

By comparison of restriction profiles of isolates (Fig. 2) and reference strains obtained with ARDRA, species identity of 19 isolates of total of 22 identified Vibrio spp. was confirmed. Genus’s identity has not changed (Table 4).

Fig. 2
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AluI restriction profiles of amplified region 16 S rRNA of isolates 14–22, 23 molecular size marker 2,000 bp

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Table 4 Comparison of results of identification by ENTEROtest 24 and ARDRA
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PCR genotyping

All isolates identified as V. cholerae by ARDRA possessed species-specific gene ompW.

Results of multiplex PCR assays (Table 5) indicated that two V. cholerae isolates were ctxA positive, three other different V. cholerae isolates were tcpA positive, of which two isolates were also toxR positive. Another isolate was also toxR positive. None of the isolates had all three virulence genes.

Table 5 Molecular identification of isolates and their virulence factors
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DISCUSSION

The goal of this present study was to assess the incidence of vibrios in Danube River near the Bratislava City agglomeration (half a million inhabitants) and to determine the presence of the main virulence factors of Vibrio cholerae.

Seven samplings were carried out during the year 2011 from January (winter season) to July (summer season). A total of 70 presumptive isolates were obtained (10 isolates from each sampling), of which 22 isolates were identified as Vibrio spp. The number of isolates from each month was as follows: January (1), February (0), March (2), April (3), May (4), June (5), July (7). We can conclude that vibrios are present in the Danube River already in its upper reaches, at the entrance to the territory of Slovakia. Species V. cholerae dominated (17/22), also V. fluvialis and V. metschnikovii have been detected.

Identification score (ID) (the percentage of probability whether certain isolate belongs to the given taxa) had acceptable rate of dissimilarity (ID ≥ 90%) only at 8 isolates, but 19 isolates were correctly identified, confirmed with molecular method ARDRA. Three misidentified isolates had ID lower than 57%. These isolates represent intermediary taxa.

None of the V. cholerae strains agglutinated with antisera against somatic antigen O1 and O139, which means all V. cholerae isolates are V. cholerae non-O1/non-O139.

In years 1974–1976 and subsequently in 1986–1988 Ďurkovský et al. (1989) isolated from the Danube River near the city Komárno (south Slovakia) up to 20 isolates of V. cholerae O1, 272 isolates of V. cholerae non-O1, 113 isolates of V. metschnikovii and 66 isolates of Vibrio spp.

An extensive study in Romania from 1977 to 1995 was conducted by Israil et al. (1998), 624 strains of Vibrio cholerae O1 were studied of which more than half were linked to the Delta Danube.

During our sampling we did not detect V. cholerae O1 nor V. cholerae O139, these strains are known to be difficult to isolate, even in areas where cholera is endemic. The reason may be that these strains are often in a dormant state when they are viable but nonculturable (VBNC) (Alam et al. 2006). Unfavorable environmental factors can turn vibrio cells into VBNC state (Xu et al. 1982; Amel et al. 2006), but their fitness and virulence factors can be remained (Colwell et al. 1985).

The presence of species V. cholerae non-O1/non-O139, V. metschnikovii is documented in several freshwaters of Slovakia. In 2009 Rosinský and his colleagues isolated from 306 isolates of V. cholerae non-O1 and 25 isolates of V. metschnikovii. Subsequently, in 2010, he isolated 150 isolates of V. cholerae non-O1, 21 isolates of V. metschnikovii and one isolate each of V. alginolyticus and V. parahaemolyticus (Rosinský, personal communication).

Valáriková et al. (2020) isolated 21 strains of V. cholerae non-O1/non-O139 and 10 strains of V. metschnikovii from surface waters of Slovakia: gravel pits Kava and Bohatá (the municipality Komárno), from the river arm of Váh (Apáli, Komárno) and water reservoir Zemplínska Šírava (east Slovakia).

In other countries, they have isolated V. cholerae from surface waters, too. Between 2000 and 2005, 13 human isolates of V. cholerae non-O1/non-O139 were documented in Austria, five of which were associated with Lake Neusiedl in Austria and resulted in three ear infections, one asymptomatic case, and one fatal septicemia (Huhulescu et al. 2007).

The permanent presence of V. cholerae in Lake Neusiedl, which can be considered a “hot spot” for microbial transport associated with migrating avifauna, has been confirmed by a study by Austrian authors (Kirschner et al. 2008). This alkaline lake has a relatively high temperature and organic matter content, which make it an ideal habitat for vibrios.

In our study physico-chemical parameters, pH, EC, O2, O2%, were relatively stable during the monitored period at the sampling sites. The pH value of all water samples was above 8.00, which is optimal pH value for occurrence of vibrios (Oliver et al. 2013). The water temperature gradually increased from 1.1 °C in January to 20.3 °C in July. With increasing temperature, we also noticed an increased number of isolated vibrios.

We also isolated V. fluvialis, which is together with V. furnissii, an important pathogen in coastal areas. In 2020, these vibrios were detected in samples of mineralized pool waters and in surface waters used for bathing in Slovakia. Their occurrence in our waters can probably be related to global warming. Also, one infection caused by V. fluvialis and two infections caused by V. furnissii were recorded in Slovakia, while another pathogen was not detected in clinical samples (Ministry of Agriculture of the Slovak Republic 2021).

Species identity of all V. cholerae isolates was also confirmed using PCR identification of species-specific gene ompW. Also, the main virulence factors of V. cholerae (ctxA, tcpA and toxR) were determined.

Our study revealed that freshwater V. cholerae non-O1/non-O139 isolates contain potential virulence genes. Six of 17 isolates had one virulence gene: ctxA (2 isolates), tcpA (1) isolate), toxR (3 isolates). None of the isolates possessed all three main virulence genes (ctxA, tcpA and toxR), however two isolates contained two genes tcpA and toxR. Cholera toxin can be gained through transduction via lysogenic bacteriophage (CTXφ), which encodes two genes of cholera toxin, ctxA and ctxB. Study of Choi et al. (2010) demonstrated gene transfer via vibriophage CTXφ-positive strains to environmental V. cholerae non-O1/non-O139 strains.

Isolates V. cholerae non-O1/non-O139 are not causative agents of cholera, however they can harbor virulence genes related to pathogenesis of cholera. The presence of ctxA gene in freshwater isolates of V. cholerae non-O1/non-O139 has also been documented by Daboul et al. (2020). During 2017–2018 13 strains of V. cholerae non-O1/non-O139 were collected from freshwaters in Toledo, Ohio. The gene ctxA (one of two genes encoding CT) was detected in two isolates, ctxB gene, as well as tcpA gene were absent. The gene toxR was not determined. Seman et al. (2012) isolated three strains of V. metschnikovii, 10 strains of V. cholerae from the Danube River and two strains of V. cholerae and two strains of V. metschnikovii from the Little Danube River, the longest left-hand tributary of the Danube. The presence of gene encoding CT at either V. cholerae strain was not confirmed, but 7 strains had genes encoding the main regulatory protein ToxR.

The present study confirms that the waters of the Danube are reservoirs of important aquatic bacteria V. cholerae, V. fluvialis and V. metschnikovii which are considered human pathogens. It is important to continue with monitoring of pathogenic Vibrio spp. in aquatic biotopes to identify potential sources of infection, as many surface waters are used for recreational activities and irrigation of the field. Future studies will investigate the possibility that O1/O139 strains are also present in Danube River in Slovakia.