| Abstract|| |
Context: A comparatively small number of species, e.g., Vibrio parahaemolyticus and V. vulnificus, cause disease in both aquatic animals and humans. V. harveyi is marine animal pathogen and rarely causes infections in humans; however, it might become a reservoir of antibiotic-resistant bacteria forms and virulence genes. Aims: 1) to develop rapid multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of V. harveyi, V. parahaemolyticus, and V. vulnificus by using vhhP2, tl, and rpoS genes as the respective target genes and 2) to evaluate specificity and determined detection of multiplex PCR technique. Materials and Methods: The multiplex PCR assay was developed and evaluated for specificity on 36 isolates of V. harveyi, 30 isolates of V. parahaemolyticus, and 14 isolates of V. vulnificus, along with other species of Vibrio and non-Vibrio bacterial isolates. Sensitivity of test was described as detection limit of pathogens in lowest amount of sample (CFU/mL or CFU/g) was determined by diluted DNA extracts of the pure cultures and spiked pacific white shrimp (Litopenaeus vannamei) samples Results: This developed multiplex PCR was proved as an accurate method, which was specific for three Vibrio species. The detection limits of V. harveyi, V. parahaemolyticus, and V. vulnificus in pure cultures and spiked shrimp samples ranged 1.05-4.8 × 103 CFU/mL and 1.9-7 × 104 CFU/g, respectively. Conclusions: This rapid multiplex PCR assay can decrease amount and process of sample preparation, which was time-consuming, and had preferable accuracy. This developed technique will be suitable and useful for food-borne pathogen detection in shrimp and horizontal gene transfer study among different Vibrio species in aquatic animals.
Keywords: Vibrio species, Litopenaeus vannamei, multiplex PCR, rpoS gene, vhhP2 gene, tl gene
|How to cite this article:|
Thongkao K, Sudjaroen Y, Chaivisuthangkura P. Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei). Ann Trop Med Public Health 2016;9:255-62
|How to cite this URL:|
Thongkao K, Sudjaroen Y, Chaivisuthangkura P. Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei). Ann Trop Med Public Health [serial online] 2016 [cited 2019 Aug 19];9:255-62. Available from: http://www.atmph.org/text.asp?2016/9/4/255/184792
| Introduction|| |
Bacteria of the genus Vibrio are widely distributed in aquatic environments and include both nonpathogenic and pathogenic species. The organisms are generally widespread in the coastal and estuarine environments; some species, e.g., V. parahaemolyticus, are common in aquatic animals, notably invertebrates. A comparatively small number of species, e.g., V. parahaemolyticus and V. vulnificus, cause disease in both aquatic animals and humans, which are of concern as they are responsible for diseases in humans ranging from seafood-borne gastroenteritis to cholera, wound infections, and septicemia.,,, Nakaguchi (2013) reported the prevalence of three genetic markers of V. parahaemolyticus in seafoods such as fish, shrimp, squid, crab, and molluscan shellfish that were purchased from provinces in Thailand and three Southeast Asian countries and data suggest that the molluscan shellfish sold in the Southeast Asian markets are highly contaminated. Uncooked seafood samples were collected from open markets and supermarkets in Bangkok, Thailand, which had contamination from 27% of Vibrio species and their resistance to antibiotics was relatively high.
V. harveyi has been reported to be a factor in loose shell syndrome and white gut disease in Penaeus monodon in India and also in Southeast Asia including Thailand. V. harveyi is found in the aquatic environment and defined as nonpathogenic for humans; however, it is interesting that they are pathogens of marine animals and they have been isolated, albeit rarely, in association with infections in humans.,, In terms of human disease, V. harveyi has been recovered from wound infections, specifically from a leg wound resulting from a shark bite in South Carolina, USA. A pediatric oncology patient presented with central line sepsis caused by V. harveyi after swimming in the sea.V. harveyi is also the one of the autochthonous marine microbial communities, and in specific ecological niches such as fish farms where antibiotics are frequently used, they might become a reservoir of antibiotic-resistant bacteria forms and virulence genes or their homologs; it could also be present in strains from environmental sources and the acquisition of such genes might take place in the aquatic environment.,, Horizontal gene transfer (HGT) in the genus Vibrio is process by genetic islands (GEIs), are classified according to the different functions, which include antibiotic resistances and virulence factors [pathogenicity islands (PAIs)] and V. harveyi may related to flrA gene involved in the regulation of V. cholerae flagella synthesis, nanH gene encoded for V. cholerae neuraminidase, tdh gene involved in V. parahaemolyticus virulence, and luxA gene included in the lux operon involved in bioluminescent expression and quorum sensing. Development this present method for simultaneous detections of V. harveyi, V. parahaemolyticus, and V. vulnificus by mulitiplex PCR may be useful in clinical and epidemiological studies on the relation of HGT between pathogenic and nonpathogenic Vibrio species in clinical specimens and seafood such as shrimps and shellfish, which as reservoirs.
Conventional methods for Vibrio identification are including selective enrichment, colony morphology, biochemical tests, and serological identifications, which are very laborious and time-consuming. Also, Vibrio species may display similar biochemical characteristics, which difficult to identify especially within related species.,, Immunoassay techniques for Vibrio Identification might be low sensitivity, which may affected by low affinity of antibodies and antigen interaction. The detectability can improve sensitivity of the original bacterial content after preincubating samples in enrichment media. Expanded cultures may more detectable by various immunoassays such as latex agglutination, enzyme-linked immunosorbent assay (ELISA), and immunochromatographic strip test. The polymerase chain reaction (PCR) method is more rapid, sensitive, and specific than conventional methods for the detection of viable but nonculturable (VBNC) pathogens or low microbial concentrations. Moreover, multiplex PCR assays have been used more than one primer to detect or identify more than one species or multiple species by amplification of more than one target gene. Each organism can be detected simultaneously by targeting unique sequences of oligonucleotide primer. The reactions of target genes are provided in the same tube. The separation of PCR amplicons by agarose gel electrophoresis are based on the size and different molecular weights, which are the visualized by fluorescence dye-stained gel. The advantages of multiplex PCR in microbiology laboratories included rapid, sensitive, turnaround time reduction, and reagent cost effectiveness. Furthermore, the identification of Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, and V. mimicus on rpoA and rpoB genes by multiplex primer extension method has been reported., Previously developed tetraplex PCR for the detectionof V. parahaemolyticus, V. vulnificus, V. cholerae, and V. mimicus in cockle using tl, hsp60, OmpW, and sodB genes had been reported. Pentaplex PCR for the detection of V. parahaemolyticus, V. vulnificus, V. cholerae, and V. mimicus by using dnaJ gene has also been reported. The species-specific markers were successfully developed for V. harveyi, V. parahaemolyticus, and V. vulnificus.,, The vhhP2 gene was originally identified encoding a putative outer membrane protein from a pathogenic V. harveyi strain T4., Later, the new set primer was designed to be a more appropriate species marker for V. harveyi. The hemolysin genes of V. parahaemolyticus including thermostable direct hemolysin (tdh), thermostable direct-related hemolysin (trh), and thermolabile hemolysin (tlh) were chosen in early studies to be the target genes for the detection of these bacteria by PCR methods. PCR targeting of tdh and/or trh gene has been used to detect the clinical strain of V. parahaemolyticus. The tlh gene has been used to detect the total V. parahaemolyticus in the sample.V. vulnificus has been developed by targeting an alternative gene called RNA polymerase subunit sigma factor S (rpoS) gene. This gene has been shown to be specific to V. vulnificus since in some cases mutation or rearrangements within the vvhA gene, which were can be detectable., The present study optimized appropriated conditions of multiplex PCR method for the detection of V. harveyi, V. parahaemolyticus, and V. vulnificus on various types of isolated sources and evaluated the specificity and sensitivity of this test for Vibrio detection in both pure culture (CFU/mL) and spiked shrimp samples (CFU/g), which have been reported as detection limit.
| Materials and Methods|| |
Bacterial isolation and DNA extraction
A total of 177 bacterial isolates were obtained from clinical samples, food sources, or environmental sources including V. harveyi (36 isolates), V. parahaemolyticus (30 isolates), V. vulnificus (14 isolates), and other Vibrio species (59 isolates). The 38 isolates of non-Vibrio bacteria were as follows: three isolates of Aeromonas hydrophila, three isolates of Aeromonas caviae, three isolates of Aeromonas sobria, four isolates of Pseudomonas aeruginosa, two isolates of Plesiomonas shigelloides, one isolate each of Aeromonas veronii, Aeromonas jandei, Escherichia coli, Edwardsiella tarda, Enterobacter cloacae, Enterococcus faecalis ATCC 25922, Klebsiella Pneumoniae, Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida, Proteus vulgaris, Pseudomonas stutzeri, Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas boreopolis, Pseudomonas oleovorans, Pseudomonas syringae, Pseudomonas japonica, Pseudomonas fluorescens, Salmonella enteric serotype Enteritidis, S. enteric serotype Typhimurium, Shigella flexneri, Staphylococcus aureus, and Yersinia ruckeri.
Conventional biochemical tests were performed to identify the bacterial isolates. The 16S rRNA gene analysis was also employed to verify the bacterial identification as previously described. The isolate of VH 14126, VP22092, and VV497001 was utilized for the assay of optimization and sensitivity testing. To extract bacterial DNA, a single loopful of culture on thiosulfate citrate Bile salts sucrose (TCBS) agar or tryptic soy agar (TSA) was used with QIAamp DNA mini-kit (Qiagen, Hilden, Germany) according to the manufacturer's specification. The extracted DNA was then stored at −70°C until use.
Optimization and specificity of multiplex polymerase chain reaction identification
The 177 of bacterial isolates were used to test for specificity and DNA templates were prepared from bacterial cultures for amplification. The oligonucleotide primers were specific for their respective targeted gene of three Vibrio species as shown in [Table 1]. The annealing temperature were performed at 55-59°C in 2XKAPA2G Fast Hot start ReadyMix PCR kit (Kapa Biosystems, Boston, Massachusetts, USA). The PCR master mix (25 µL) was contained 0.625 µL of 20 µM forward and reverse primers (for each), 0.5 µL of 50 mM MgCl2, 2.5 µL of 50 mM KCl, 12.5 µL of 2XKAPA2G Fast Hot start ReadyMix (1X), 1 µL of DNA template and distilled water (the remaining volume). The PCR amplifying condition was included, 1) initial denaturation of DNA template (95°C for 3 min), 2) DNA denature (95°C for 15 s), 3) primer annealing (59°C for 15 s) 4) primer extension (72°C for 1 s) and 5) final extension (72°C for 10 min). Process 2) to 4) were run repeat in 35 cycles, The successfully multiplexed three targeted genes, vhhP2 gene (157 bp) for V. harveyi, tl gene (450 bp) for V. parahaemolyticus and rpoS (273bp) for V. vulnificus. Multiplex PCR targeting the simultaneous amplification of the three targeted gene segments from the three Vibrio species in a single reaction tube produced consistent results, when detected on one, two or three Vibrio species [Figure 1].
|Figure 1: Electrophoresis of multiplex PCR products from three Vibrio species. Lane M: 100 bp DNA ladder (Bioline), Lane 1: V. harveyi, Lane 2: V. vulnificus, Lane 3: V. parahaemolyticus, Lane 4: V. harveyi and V. vulnificus, Lane 5: V. harveyi and V. parahaemolyticus, Lane 6: V. harveyi, V. vulnificus and V. parahaemolyticus, Lane N: Negative control (no DNA template)|
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Determinations of sensitivities of multiplex polymerase chain reaction assay with pure culture
The sensitivity of the multiplex PCR assay for the detection V. harveyi, V. parahaemolyticus, and V. vulnificus in pure cultures was determined as previously described with some modifications using the known amounts of V. harveyi, V. parahaemolyticus, and V. vulnificus. In brief, a single colony on TCBS agar was inoculated in 4 mL of tryptic soy broth (TSB) (Difco) supplemented with 2% NaCl and incubated overnight at 37°C. Subsequently, 40 µl of TSB culture was transferred to a new 4 mL of TSB and incubated at 37°C with shaking at 225 rev/min - at 37°C to obtain mid-log phase cells (OD600nm= 0.5). Serial tenfold dilutions of the cultures were prepared in phosphate buffered saline (PBS). For preparation of DNAs from pure cultures, 100 µl of each dilution was transferred to a 1.5 mL microcentrifuge tube (Eppendorf AG, Germany), and was centrifuged at 18,000 × g for 5 min, then the pellet was resuspended in 50 µl of 25 mM NaOH, and the mixture was heated at 95°C for 5 min. After neutralization with 4 µl of 1 M Tris-HCl buffer (pH 7.5), the suspension was centrifuged; the supernatant was used as a template for multiplex PCR assay. The sensitivity tests were performed in triplicate, the last dilution with all three samples tested positive and was considered as the detection limit. In parallel, to enumerate the bacteria, 100 µl of each bacterial dilution was spread on TCBS in duplicate and incubated at 37°C overnight. The colonies were counted at the dilution yielding 30-300 colony forming units (CFUs), and the CFU/mL of bacterial suspension was calculated.
Determinations of sensitivities of multiplex polymerase chain reaction assay with spiked shrimp sample
The pacific white shrimp (Litopenaeus vannamei) samples tested negative for V. harveyi, V. parahaemolyticus, and V. vulnificus according to the microbiological examination by enrichment in alkaline peptone water (APW) overnight. The shrimp homogenate was cultured onto TCBS agar. The DNA samples were extracted from bacterial colonies and shrimp homogenate as the template for testing the presence of V. harveyi, V. parahaemolyticus, and V. vulnificus by PCR targeted vhhP2, tl, and rpoS genes. Only shrimp homogenates that were negative for V. harveyi were used in the following spiked shrimp experiments. The detection limit of multiplex PCR assay for V. harveyi, V. parahaemolyticus, and V. vulnificus in spiked shrimp samples was evaluated as previously described, with some modifications using known amounts of V. harveyi, V. parahaemolyticus, and V. vulnificus. Nine milliliters of APW were added to 1 g of the shrimp sample and homogenized thoroughly. Serial ten-fold dilutions of three bacteria during mid-log phase were prepared as described in above. One hundred microliters of each dilution of each bacterium with known amounts was spiked into 900 µl of each of the shrimp homogenates and mixed well. The shrimp homogenate was centrifuged at 200 g for 5 min to remove shrimp tissues. The supernatant was transferred to a new tube and centrifuged at 18,000 g for 5 min. After removal of the supernatant, the pellet was resuspended in 100 µl of 25 mM NaOH, and the mixture was heated at 95°C, for 5 min. After neutralization with 8 µl of 1 M Tris-HCl buffer (pH 7.5), the debris was pelleted by centrifugation at 20,000 g (at 4°C) for 5 min. For multiplex PCR assay, 1 µl of each supernatant was used as a template. The sensitivity tests were conducted in triplicate. The last dilution with all three samples tested positive and was considered as the detection limit dilution of sample test.
| Results|| |
The separation of PCR products by using electrophoresis were presented as banding on lane 1, 2, 3, 4, 5, 6 and N, which were represented to V. harveyi (VH), V. vulnificus (VV), V. parahaemolyticus (VP), mixture of VH and VV, mixture of VH and VP, mixture of three Vibrio species and negative control, respectively. Specificity of multiplex PCR assay for three targets and nontargets of Vibrio species in various isolates from different sources of samples, including clinical samples, aquatic animals and environmental was presented in [Table 2]. This assay was regarded for specificity on V. harveyi, V. parahaemolyticus, and V. vulnificus detection, which no cross reaction to other Vibrio species and also other bacterial isolates in different sample types. The sensitivity of multiplex PCR for V. parahaemolyticus detection was evaluated in pure culture and spiked shrimp samples [Figure 2], which can be detected on beginning concentration at 7.5 × 106 CFU/mL and 1.0 × 108 CFU/g and limited to detect at 7.5 × 103 CFU/mL and 1.0 × 104 CFU/g, respectively. The sensitivity of multiplex PCR for V. vulnificus detection was evaluated in pure culture and spiked shrimp samples, which was detected on beginning concentration at 4.8 × 107 CFU/mL and 7.0 × 108 CFU/g and limited to detect at 4.8 × 103 CFU/mL and 7.0 × 104 CFU/g, respectively. The successfully multiplexed amplification of three targeted genes, including vhhP2 gene (157 bp) for V. harveyi, tl gene (450 bp) for V. parahaemolyticus and rpoS (273bp) for V. vulnificus.
|Table 2: Specificity of multiplex PCR assay for three targets and nontargets of Vibrio species in various types of samples|
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|Figure 2: The comparison of sensitivity among V. harveyi, V. parahaemolyticus, V. vulnificus detections by multiplex PCR (a) PCR products from pure culture at various dilutions (CFU/mL) included V. harveyi (157 bp); V. vulnificus (273 bp) and V. parahaemolyticus (450 bp) (b) PCR products from spiked shrimp samples at various dilutions (CFU/g). Lane M and Lane N were molecular markers and negative control (none for DNA template)|
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| Discussion|| |
The development of this new method was for improvement in efficacy, which can be applied in many related areas of microbiology including medicine, food science, environmental science, and aquatic animal study. For example, this method can be suitable for rapid interpretation in clinical samples such as in septicemia cases and can be applied to screen shrimp diseases and frozen marine foods for microbial contaminant detection that should meet international standards of marine foods and products. Several previous studies were designed primer sets to detect V. parahaemolyticus in shellfish by using tdh, trh, and tl genes  and found that tdh and trh genes were appropriated to detect pandemic strains O3:K6, O4:K68, and O1:KUT. The presence of tdh gene and trh genes were presented by a ß-type hemolysis on Wagatsuma agar and positive on urease test, respectively. Both of which appearances served as markers for pathogenic strains. The tlh gene encoding of thermolabile hemolysin (TLH) was first cloned and characterized by Taniguchi, et al., This gene was detected in all strains of V. parahaemolyticus, which were isolates from both clinical and environmental sources and were absent in other Vibrio species; consequently, this gene is considered to be a species-specific gene in V. parahaemolyticus. Several studies have suggested using this gene as a target gene for the identification of V. parahaemolyticus. McCarthy et al. developed multiplex real-time PCR method on internal amplification control to screen V. parahaemolyticus contamination in oyster by using of specific tlh gene probe (combined with tdh and trh gene probes) and comparing with API-20E method. The detection of rpoA, rpoB, and atpA genes were targeted genes to identify V. parahaemolyticus and V. vulnificus separated from other Vibrio species. However, these target genes were conserving as housekeeping genes, which could not be classified on the basis of phenotypic test or the multiplex PCR and may caused by similar sequences to other bacteria species. The sequence analysis of the rpoB gene has been used to identify bacterial isolates in a number of taxa including Pseudomonas, Corynebacterium, and Staphylococcus.
Probe was targeted on rpoS gene, which gave positive results for detection of all V. vulnificus and did not show any cross-reactivity with the other bacteria. In addition, the commonly used vvhA PCR amplification for V. vulnificus identification yielded negative results with two isolates of V. vulnificus. It has been shown that rpoS gene was suitable for the detection of V. vulnificus. These results showed rpoS gene detection on V. vulnificus, which was specific. The hly gene was conducted with multiplex PCR of V. harveyi, V. campbellii, and V. parahaemolyticus. Later, in preliminary study this gene was found to be nonspecific to V. harveyi and could not be detected for the all isolates. However, Thongkao et al. had improved the specificity of vhhP2 gene for V. harveyi detection by a new primer design that had coverage of all the strains of V. harveyi detection, which had various phenotypic expression and cannot be distinguished the color and/or characteristics of colonies on TCBS agar (sucrose-positive or sucrose-negative species).
The sensitivity of multiplex PCR for V. parahaemolyticus detection was evaluated in pure culture and spiked shrimp samples [Figure 2]. Several studies reported that sensitivity of multiplex PCR for the detection of V. vulnificus was 105 CFU/mL and for V. parahaemolyticus 106 CFU/mL. After enrichment for 6 h, the optimized tetraplex PCR condition achieved a detection limit of 1 CFU/10 g of spiked cockle tissue for both V. parahaemolyticus and V. vulnificus.
Nhung et al. reported the sensitivity of multiplex PCR for V. vulnificus and V. parahaemolyticus detections and detection limits of the test were 105 CFU/mL and 106 CFU/mL, respectively. When compared to our finding, detection limits of this multiplex PCR were better than a previous study by 4.8 CFU/mL and 7.5 × 103 CFU/mL for V. vulnificus and V. parahaemolyticus, respectively. Many relating factors may affect the detection limits of the test, including different periods of bacterial storage on mid-log phase (OD = 0.4-0.6), target gene characteristics, DNA template concentration, amount and activity of taq DNA polymerase, primer concentration, MgCl2 concentration, and dNTP concentration. The sensitivity of multiplex PCR for Vibrio detection in aquatic animals was increased to 1 CFU/mL after using enrichment process [Figure 3]. Detection limits of the test for the three Vibrio species in pure culture were higher than in spiked shrimp samples, approximately 10 CFU/mL; however, they were still better than a previous study. The enrichment of media in pure culture was more appropriate rather than spiked shrimp samples. The sensitivity improvement was enhancing from the empirical choice of oligonucleotide primers and using of hot start-based PCR method to facilitate automation. This method might be affecting from the containing of DNA from dead cells or viable but non-culturable cells (VBNC) state, which were not performing colonies on high-nutrient containing solid media, however, there are considered as alive, because of metabolic activity can still be detected.,
|Figure 3: The comparison of processes and time spending between (a) conventional method (b) multiplex PCR method for V. harveyi, V. vulnificus, and V. parahaemolyticus detection|
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This method was not only for the detection of three pathogens simultaneously; some steps of the process were also reduced, which resulted from the decrease of the turnaround time of the method from 90 min to 22 min and the PCR process was done by preparation of mixture containing with primer set, DNA template, and HotStart ReadyMix PCR kit (Kapa Biosystems, Boston, Massachusetts, USA) to running the PCR reaction. Overall, the processes of conventional and multiplex PCR are shown in [Figure 3]. The time consumed by the conventional method and multiplex PCR was approximately 60-78 h and 9 h, respectively. This implied that time consumption of the conventional method was about six to nine times of multiplex PCR. In addition, the amount of biochemical tests spending for the conventional method might be higher volume when comparing to multiplex PCR method and conventional method had lower cost effectiveness by lower sensitivity and specificity (accuracy), and difficult to interpretation, such as, similarly results on colony characteristics and biochemical tests.
| Conclusion|| |
In conclusion, the development multiplex PCR assay may turn-around time by reduced the amount and process of sample preparation, and had high accurate results. In addition, reagent spending for multiplex PCR was also cost-effective. This method can be applied in epidemiological studies, especially food-borne pathogen detection when compared to conventional methods because it can be operate on many samples and report in a short time. Moreover, the export of marine frozen products, especially shrimps is important in Thailand. Our results may be useful for Vibrio-contaminated detection in aquatic products, which is one process of quality control for exporting food products. Nonpathogenic Vibrio strains such as, V. harveyi might become a significant reservoir of virulence and fitness genes, which will emerge to be new virulence traits of environmental bacteria and might concern for public health and a risk for human health. Thus, this present method may be useful for the simultaneous detection of pathogenic and nonpathogenic Vibrio species, which might be appropriated for further study on horizontal gene transfer.
The authors express their sincere appreciation to Suan Sunandha Rajabhat University, Bangkok, Thailand for granting support for this work. The authors are indebted to all institutes in [Table 2] for providing various bacterial isolates. We would like to sincerely thank the Research Division of Srinakharinwirot University, Thailand for use of its research facility and helping with this research.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Faculty of Science and Technology, Suan Sunandha Rajabhat University, 1 U-Thong-Nok Road, Dusit, Bangkok - 10300
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]