Abstract: Background and Objective: Vibrio sp. are a crucial constituent of marine ecosystems worldwide. Vibrio alginolyticus is extremely significant and major pathogenic agent facing wild and farmed fishes. The main objective of this study was to record the molecular characterization (IGS-Typing pattern) by single PCR step and virulence genes in V. alginolyticus isolates. Materials and Methods: For this study 360 fishes were collected, including gilt head seabream (Sparus auratus), two bar seabream (Acanthopagrus bifasciatus), black bream (Acanthopagrus latus), red sea seabream (Diplodus noct), brown-spotted grouper (Epinephilus coioides) and rabbitfish (Siganus canaliculatus) between July, 2015-June, 2016. Primary identification of isolates was done conventionally using API 20NE systems (Biomerieux), while confirmation was done by sequencing of 16S rDNA and V. alginolyticus specific gene targets, construction of phylogenetic tree using Molecular Evolutionary Genetics Analysis (MEGA) 6.0 software. IGS-Typing and antibiogram test screening were also carried out for isolates. Results: Two hundred and eighty isolates of V. alginolyticus were recovered from farmed fishes. Clinical signs expressed as general septicaemia, ulcers and corneal opacity. Conventional methods identified isolates as V. alginolyticus (ID% 96-99.9), sequencing and phylogenetic tree confirmed this identification. The PCR results for IGS-Typing were expressed as four typical band patterns ≈ 420, 580, 650 and 820 bp, while those for virulence genes were positive amplification for collagenase, toxR and ompK, while tlh and VPI virulence genes were not recognized. Conclusion: Isolated V. alginolyticus were highly pathogenic to farmed fishes as determined by virulence genes identified. Results of IGS-Typing facilitate further identification of isolates by single PCR step as it records the pattern for V. alginolyticus.
INTRODUCTION
Fish aquaculture is a growing industry in Saudi Arabia. However, vibriosis outbreak caused by various pathogenic Vibrio sp. is considered a significant problem to the development of farmed fish and shellfish with severe economic losses1,2. Moreover, Many Vibrio sp. are serious pathogen in human causing septicemia, gastroenteritis as well as wound infection3,.
Vibrio alginolyticus is Gram-negative halophilic bacterium, ubiquitous in marine and estuarine ecosystems and has been associated with extensive body ulceration of gilthead sea bream (Sparus aurata), silver sea bream (Sparus sarba), sea bass (Dicentrarchus labrax), turbot (Scophthalmus maximus), crimson snapper (Lutjanus erythopterus), sea mullet (Mugilcephalus) and freshwater fish, Nile tilapia (Oreochromis niloticus)2-10. Recently, V. alginolyticus was recorded as a responsible pathogen for Porites andrewsi white syndrome in coral reefs and was associated with white spot disease in black tiger shrimp (Penaeus monodon)11,12. Hassan et al.1 isolated V. vulnificus and V. alginolyticus from cage-cultured marine fish Acanthopagrus bifasciatus and Sparus aurata in Arabian Gulf.
More than 74 distinct Vibrio spp. has been identified till now (http://www.vibriobiology.net/) and continuously increases annually. This gave rise to the extreme importance for identification of Vibrio sp. based on well-known IGS-regions located between the 16S and 23S rRNA genes on the bacterial chromosomes13. This rapid, reliable and efficient IGS-Fingerprinting was able to identify Vibrio sp. not only at species level, but also, in some cases, at subspecies level using one primer for one PCR reaction especially if combined with 16S-rRNA gene sequence13.
Previous studies reported V. alginolyticus as the dominant living species in water and sediments14. It can maintain virulence properties and pull out from several stressors including nutrient stress8. Presence of V. cholerae pathogenicity island (VPI) virulence gene and thermolabile haemolysin (tlh) virulence gene of V. parahemolyticus in V. alginolyticus reported by Zhang et al.15 and Snoussi et al.16 respectively. OmpK virulence gene of V. alginolyticus believed to play important role in infection and pathogenicity to host and the expression of this gene is controlled by toxR gene17. Collagenase has been widely utilized as a biomarker of V. alginolyticus as well as capable of causing severe pathology and distribution to blood stream18. Although V. alginolyticus has been reported in many cases from Arabian Gulf fish and shellfish19-21,1. No data available on the presence of virulence genes especially those derived from V. cholera as well as V. parahaemolyticus. Similarly, to the best of author’s knowledge, no studies till now clarified the most important virulence genes for pathogenicity of V. alginolyticus.
The aims of the present study were to clarify the molecular identification of V. alginolyticus isolated from Saudi coast in Arabian gulf, by sequencing of targets in the 16S rDNA and V. alginolyticus specific gene regions, phylogenetic tree, recording 16S-23S rRNA Intergenic Spacer Regions pattern (IGS-Fingerprinting) and detect presence of V. cholerae pathogenicity island (VPI), thermolabile haemolysin (tlh), collagenase, toxR and ompK virulence genes by means of PCR for better understanding how to control or eradicate the disease in an aquaculture setting.
MATERIALS AND METHODS
Fish sampling: About 360 fishes were collected and examined as a part of ongoing monitoring program related to Fisheries Research Center, Ministry of Environment, Water and Agriculture, Saudi Arabia from cage cultured fish owned by private farm in the eastern province. About 6 species of fishes (60 fish/Species) were examined include, gilthead seabream (Sparus auratus), two bar seabream (Acanthopagrus bifasciatus), sobaity seabream (Sparidentex hasta), red sea seabream (Diplodus noct), brown-spotted grouper (Epinephilus coioides) and rabbitfish (Siganus canaliculatus) exhibits septicemic signs such as dermal ulcers and/or external hemorrhage, in addition to 96 water samples were also collected aseptically in sterile bottles and examined, during the period from June, 2015 to May, 2016.
Fishes were transferred alive in a special vessels supplied with oxygen to the Lab. of Fisheries Research Center in Al-Qatif (17025:2005 ISO Certified) where clinical and postmortem examination were carried out according to Noga22. Salinity, DO and water temperature were analyzed for water samples.
Bacterial strains and culturing conditions: Samples were taken from each fish separately from hematogenous organs of each fish (liver, kidney, spleen) in addition to water samples by using sterile bacteriological loop and inoculated in TSB and TSA (Oxoid) supplemented with 2% NaCl, incubated at 28°C for 24 h and further purificationin TSA with 2% NaCl according to Austin and Austin23.
Phenotypic identification: Gram staining and oxidase test were done as a preliminary step of identification.
| Table 1: | Selected primer pairs sequence used in the study |
| Table 2: | Cycling conditions for different PCR reactions in the study |
Purification and further phenotypic identification were done with the mean of Thiosulphate Citrate Bile Salt Sucrose agar (TCBS) as a selective media, growth in presence of vibriostatic agent 0/129 and API 20NE identification system (Biomérieux) using ApiwebTM identification software according to manufacturer’s instructions. Pooling of strains were done and kept in TSB (15% glycerol) in -80°C.
DNA extraction: Pooled strains (phenotypically identified) were incubated at 28°C in TSB supplemented with 2% NaCl and DNA were extracted using a commercial kit (Qiagen, Germany), DNA quantification were done by mean of Nano Drop Technology (Thermo Scientific NanoDrop 2000 UV-Vis spectrophotometer) and stored in -20°C. Molecular identification, characterization and detection of virulence genes were carried out as follow:
| • | Sequencing of 16S rDNA gene target: Amplification of a specific target of the 16s rDNA using a pan-bacterial universal primer set (27F and 1492R, Table 1) following the method described by Polz and Cavanaugh24. The reaction was carried out in 25 μL reaction and the cyclic condition explained in (Table 2). PCR product directly sequenced and results were compared with those held in GenBank database (National Center for Biotechnology Information “NCBI”, Bethesda, MD, USA) by using Blast program |
| • | Sequencing of V. alginolyticus specific gene target: Using a Species-specific primer set (VA 1198230 F and VA 1198230 R, Table 1) following the method described by Kim et al.25. The PCR product directly sequenced and data BLASTed |
Phylogenetic tree: A phylogenetic analysis was conducted using the Molecular Evolutionary Genetics Analysis (MEGA) 6.0 software26.
PCR of V. alginolyticus targeting 16S-23S rRNA intergenic spacer regions (IGS-fingerprinting): PCR of V. alginolyticus targeting 16S-23S rRNA Intergenic Spacer Regions (IGS-Fingerprinting) was performed in a 50 μL reaction following the method described by Hoffmann et al.13. The cyclic condition includes a second round amplification step to eliminate artifacts as possible as explained in Table 2.
PCR for Detection of V. alginolyticus virulence genes were performed in 50 μL reaction following the procedures described by Xie et al.27 for tlh and toxR virulence genes, Sechi et al.28 for VPI virulence gene, Cai et al.29 for OmpK and Di Pinto et al.30 for Collagenase virulence genes. Primer sequence for all reactions were described in Table 1 and detailed cyclic condition in Table 2.
Gel electrophoresis: The PCR products in all reactions were run on 1.5% agarose gel stained with Ethidium bromide (10 mg mL–1) in Tris Acetate EDTA buffer (TAE) using Biorad PowerPac universal electrophoresis and visualized with UV Transilluminator using Molecular Imager Gel DOCTM with Image LabTM software (USA).
RESULTS
Clinical signs of examined fishes: Symptoms of septicemic vibriosis are clear with Sluggish movement, nervous manifestation represented by listlessness, loss of body reflexes and Fin erosions (Fig. 1a). Generalized erythematic hemorrhage distributed all over the body surface especially anal fins and operculum, marked ulcers with different sizes especially on grouper fish (Fig. 1b and d), corneal opacity (bilateral Fig. 1c) or (unilateral Fig. 1e), hemorrhagic swollen vent and sometimes dark pigmentation were also observed.
Postmortem examination: Severe congestion in gills, liver, kidney and intestine were observed. Distended gall bladder, the abdominal cavity filled with copious exudate (hemorrhagic or ascetic) (Fig. 1f). Inflammation and thickening of swim bladder wall were also recorded.
| Fig. 1 (a-f): | Gilthead seabream (Sparus auratus) showing fin erosion, (b) Black bream (Acanthopagrus latus) showing deep skin ulcer, (c) Brown-spotted grouper (Epinephilus coioides) showing bilateral corneal opacity, (d) Brown-Spotted Grouper (Epinephilus coioides) showing skin lesion and ulcers with different sizes. (E) Brown-spotted Grouper (Epinephilus coioides) showing unilateral corneal opacity and (f) Black bream (Acanthopagrus latus) showing bloody ascites |
| Fig. 2: | Phylogenetic tree based on 16S r DNA gene sequence showing the relationships of V. alginolyticus with related species. The tree was created using MEGA 6.0 software |
| Table 3: | Nucleotides sequence results for both 16S r DNA and V. alginolyticus specific gene targets |
Phenotypic results: About 280 isolates recovered from examined fishes were Gram-negative, straight or slightly curved (comma shaped) rods, motile, oxidase positive, sensitive to vibriostatic agents 0/129. Small, circular creamy colored colonies on TSA supplemented with 2% NaCl. Large yellow colonies on TCBS agar plates were also recorded.
Biochemical results using API 20NE system: Indicated that all isolates were phenotypically identified as V. alginolyticus (ID % 96-99.9) with reference No. 7474644, 7464644, 7456744 and 7456744.
Sequencing results: BLASTing of sequence results from 16S rDNA (1500 bp) and from sequencing of V. alginolyticus specific target (199 bp) (Table 3) against those in GenBank “NCBI” resulted in accurate identification of isolates with 99% similarities to reference strains V. alginolyticus strain NBRC 15630-16S-rRNA gene (accession No. NR_121709) and V. alginolyticus strain ANC5-1 (accession No. KP330587) respectively.
Phylogenetic tree: Construction of phylogenetic tree using sequences output from both 16S rDNA and V. alginolyticus specific target accurately put isolates of the present study in V. alginolyticus group and indicated its closest proximity to V. alginolyticus strain NBRC 15630-16S-rRNA (Fig. 2) and V. alginolyticus strains ANC5-1 (Fig. 3) respectively.
| Fig. 3: | Phylogenetic tree based on sequencing of targets of V. alginolyticus specific gene regions showing the relationships of V. alginolyticus with related species. The tree was created using MEGA 6.0 software |
| Fig. 4: | About 1.5% agarose electrophoretic gel picture showing the 16S-23S intergenic spacer pattern (IGS-Fingerprinting) of V. alginolyticus where Lane 1: 100 bp DNA ladder, Lane 2 and 3: 4 bands of V. alginolyticus near 420, 580, 650 and 820 bp |
Intergenic spacers (IGS) results: A polymerase chain reaction (PCR) targeting 16S-23S rRNA intergenic spacer (IGS) region was conducted with extracted DNA from pooled samples and resulted in amplicons. The latter were analyzed by gel electrophoresis and resulted in 4 typical bands ≈420, 580, 650 and 820 bp (Fig. 4).
Presence of virulence genes: The extracted DNA from pooled samples in the present study was subjected to virulence genes detection by mean of a conventional PCR and gave similar results. They were positive for the presence of collagenase, toxR and ompK genes and lacking tlh and VPI genes (Fig. 5).
The accurate molecular identification of V. alginolyticus isolates in present study revealed that the highest incidence of infection was in rabbitfish (Siganus canaliculatus) (29.64%) followed by brown-spotted grouper (Epinephilus coioides) (27.14%), two bar seabream (Acanthopagrus bifasciatus) (20.36%), red sea seabream (Diplodus noct) (8.93%), black bream (Acanthopagrus latus) (7.5%) and gilthead seabream (Sparus auratus) (6.43%) as shown in Table 4. As well as the highest incidence of infection was recorded from kidney tissues (46.79%) followed by liver (31.43%) and spleen (21.78%) as shown in Table 5.
| Table 4: | Total no. of V. alginolyticus isolates/fish species |
| Table 5: | Total No. of V. alginolyticus isolates/organs of various fish species |
| Table 6: | Seasonal prevalence of V. alginolyticus in the examined fishes |
| Fig. 5: | About 1.5% agarose electrophoretic gel picture showing amplification of V. alginolyticus virulence genes under study where Lane 1: 100 bp DNA ladder, Lane 2 and 3: Positive toxR-gene amplification, Lane 4 and 5: Positive collagenase-gene amplification, Lane 6 and 7: Positive ompK-gene amplification, Lane 8 and 9: Negative tlh-gene amplification and Lane 10 and 11: Negative VPI-gene amplification |
The seasonal prevalence of V. alginolyticus infection among the total number of examined fishes revealed that, the highest prevalence was in summer season (61.78%) followed by spring (18.93%), Autumn (11.43) and winter (7.86%) as shown in Table 6.
DISCUSSION
Arabian gulf area characterized by higher salt concentration and extreme higher temperature specially in summer season which favor Vibrio sp. Mainly V. alginolyticus2. Virulence of V. alginolyticus strain for fish epizootics outbreaks have been reported in gilthead seabream (Sparus auratus) by Paperna31 and in two bar seabream (Acanthopagrus bifasciatus), black bream (Acanthopagrus latus), red sea seabream (Diplodus noct), brown-spotted grouper (Epinephilus coioides) and rabbitfish (Siganus canaliculatus) by Hassan et al.1.
General septicemic picture were obvious on the examined fishes. In some cases, the symptoms were markedly prominent such as ulcers with different sizes and corneal opacity (uni-bilateral) especially in grouper fish. Internally, congestion of internal organs, distension of gall bladder and abdominal cavity with copious exudate (hemorrhagic or ascetic) were also recorded. Similar clinical signs and postmortem lesions stated by Labella et al.32 and Moustafa et al.33, which may came as a result of higher salinity, lack of dissolved oxygen by the rise of water temperature especially in summer season together with the presence of V. alginolyticus virulence factors. Gomathi et al.34 stated that natural disease caused by V. alginolyticus in sea bream includes, septicemia, hemorrhage and skin ulcers in some cases. Internally, fish accumulate fluid in the peritoneal cavity and in some cases have hemorrhagic livers.
Due to plastic nature and higher number of identified vibrios (73 spp.) till now, Phenotypic and conventional methods including culture-based, API 20E and 20NE identification system were unreliable for identification of Vibrio sp.13,35. Hence, molecular biological DNA-based methods have been well established for rapid and accurate identification of Vibrio spp.25. It provide privileges than/or sequel to conventional biochemical and culture-based methods36.
Sequencing of 16S rRNA and V. alginolyticus specific targets is of quiet importance for accurate identification and classification of isolates in the present study. The 16S rRNA gene sequence alone is less trust worthy in differentiation between species or even subspecies of G. vibrio37 due to continuous increases in the number of known Vibrio sp., moreover several Vibrio sp. Contain almost similar sequence of 16S rRNA gene with fewer differences. Hence, in the present study, 16S rRNA sequence combined with V. alginolyticus specific gene sequence for accurate identification. BLASTing of sequence results against those held in GenBank (NCBI, Bethesda, USA) identified these isolates as V. alginolyticus with 99% similarity to V. alginolyticus strain NBRC 15630-16SrRNA gene and V. alginolyticus strain ANC5-1 respectively.
In bacterial chromosome, Intergenic Spacer (IGS) regions situated among 16S and 23S rRNA genes and considered as a fingerprinting to discriminate vibrios not only at species level but also at subspecies level as well13. The IGS-Typing pattern of V. alginolyticus on agarose gel was recorded in the present study as illustrated in Fig. 3 and represented by 4 typical bands ≈420, 580, 650 and 820 bp. The differences in the length and sequence of the 16S-23S intergenic spacer regions (IGSs) of rRNA persons were used to develop IGS-Typing system for Vibrio species, hence, Vibrio species can be distinguished from each other by one PCR reaction13.
Concerning the results of virulence genes detection in the present study, V. alginolyticus possess positive results for collagenase, toxR and ompK genes and lacking tlh and VPI virulence genes. These results were supported by Effendy et al.5, who stated that 67% of tested V. alginolyticus strains carry ompK, toxR and collagenase genes, while, 19% of them carrying both ompK and toxR genes and supported as well by Xie et al.27, who detect the presence of tlh virulence gene which is homologous to those of V. parahaemolyticus and presence of VPI gene in 2 strains of V. alginolyticus. Hence, it proposed that V. alginolyticus act as substantial reservoir of several known virulence genes from other species of genus Vibrio.
The pathogenicity mechanism of V. alginolyticus might be different from other pathogenic Vibrio species or the virulence may be caused by multi virulent factors as reported by Kahla-Nakbi et al.8 and Xieet al.27 when they did not found any correlation between presence or absence or virulence genes in V. alginolyticus and its virulent strains.
In present study, the highest incidence of infection was in rabbitfish (Siganus canaliculatus) (29.64%) while, the lowest was in gilthead seabream (Sparus auratus) (6.43%). Similarly, Al-Sunaiher et al.2 stated that Vibrio species detected in 16.8% of the total examined fish with higher incidence in rabbitfish (50%). On the contrary, Balebona et al.38 reported that incidence of infection was (67.8%) intensive cultured gilt-head seabream (Sparus aurata L.). This variation in prevalence may be attributed to the differences salinity level, the sample sizes of studies, different climate.
The seasonal prevalence of V. alginolyticus infection among the total number of examined fishes revealed that, the highest prevalence was in summer season (61.78%) and these were supported by Giorgetti and Ceschia39, who revealed that the main predisposing factors in all Vibrios outbreaks were increased in water temperature and epizootics were always associated with temperature higher than 15°C.
CONCLUSION
The present study suggested that the isolated V. alginolyticus was virulent to fish, shellfish, shrimp and crustacean present in Arabian gulf as it carry virulence genes. Subsequently, it should receive enough attention, may be by vaccination to avoid the resulting mortality and hence economic losses in commercial farms. Accurate identification and characterization of the dominant strain of local bacteria greatly support the epidemiological studies and tracing of diseases outbreaks. Moreover knowing the type of dominant bacteria is very helpful in developing of protection programs against it as well as to discover suitable methods to control the infection transmission to consumers.
SIGNIFICANCE STATEMENT
This study discovered the molecular characterization and virulence properties of V. alginolyticus isolated from Saudi coast of Arabian gulf and recorded its IGS-Typing pattern. This study will help the researcher to definitely identify V. alginolyticus using one PCR reaction in references to data of this study.