Detection of marine Vibrio species from environmental samples with a nested PCR technique.

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1 Detection of marine Vibrio species from environmental samples with a nested PCR technique. By Olof Peterson Supervisors Anna Godhe and Maria Asplund BIO311 Tillämpningskurs (7,5p), Marine ecology, University of Gothenburg Abstract Bacteria of the genus Vibrio are indigenous for the marine environment and temporarily in abundances in the ocean, causing infections to humans and commercially important species of crustaceans, bivalves and fish. These bacteria can therefore cause severe complications for human health and large economic losses to aquaculture. A polymerase chain reaction (PCR) technique was used to detect presence of four different Vibrio species from two different seasons in marine environmental water samples, collected in the south west coast of India. Two species, V. alginolyticus and V. harveyi, were detected in both post- and pre-monsoon seasons. However, no difference in presence between the seasons could be statistically determined. V. cholerae was not detected in any of the samples and the results for V. vulnificus were inconclusive due to failure to optimize the PCR technique for this species. The qualitative experiment set up was found to be too weak to detect difference in Vibrio species presence between the two seasons. However, using nested PCR to detect pathogenic bacteria from environmental samples is an important tool for risk assessments in the food industry.

2 Introduction Marine bacteria are naturally abundant inhabitants of the marine environment. Some of the bacterial species act as pathogens and constitute a threat to human economic or health interests. One group with many species of infectious bacteria is the genus Vibrio, which are common in both tropic and temperate waters. In temperate waters infections are rare, but the occurrence of Vibrio spp. in the ocean might be a potentially overseen health risk (Eiler et al., 2006). In tropical waters, on the other hand, the pathogenic strains of different Vibrio species cause severe outbreaks of diseases and have large economical consequences on aquaculture and other economical sectors (Liu et al., 2004; Saravanan et al., 2007). In this project, four different species of Vibrio were studied; V. alginolyticus, V. cholerae, V. harveyi and V. vulnificus. V alginolyticus is known to infect shrimp in tropical waters and can, when occurring together with different abiotic stress factors, cause severe losses in shrimp farms (Liu et al., 2004). V. cholerae, known for causing endemic or pandemic outbreaks of the diarrheal disease cholera, is regarded as a common component in some marine ecosystems (Islam et al., 1994). Even if only two out of approximately 200 serogroups of V. cholerae contain the genotype needed to induce such large spread diseases, the other subgroup can cause small outbreaks of disease and pose a potential threat to public health (Saravanan et al., 2007). V. harveyi is a common bacterium causing severe infections in marine fauna, especially marine invertebrates (Oakley et al., 2003). This bacteria has caused severe economical losses to aquaculture in several Asian countries (Liu et al., 2004), infecting fish (Conejero & Hedreyda, 2004) and invertebrates such as prawns, lobsters, oysters and seahorses (Oakley et al., 2003). V. vulnificus is a human pathogen, spread through raw or under-cooked seafood, and can cause severe and life threatening diseases (Kumar et al., 2006). In this project, a polymerase chain reaction (PCR) based technique has been used to detect four different species of Vibrio from environmental samples, taken outside Mangalore, India. PCR is a simple but effective method for detecting presence of marine bacteria from natural samples. However, the method requires species-specific primers and thus DNA-sequences that are unique for the respective species must be known in order for these primers to be designed and manufactured. For many pathogenic and well-studied bacteria there are several published primers to be found in the scientific literature, and for many of them there are not only species-specific primers but also primers that can detect strains that contain certain toxic genes only (e.g. Saravanan et al., 2007). Since many bacteria, when taken out of their natural environment, enter a viable but not culturable (VBNC) state, PCR may be a more accurate way of detecting marine bacteria, compared to specific plate growth (Campbell & Wright, 2003). However, the technique used in this project is mainly qualitative and the abundance of bacteria in the samples cannot be quantified. This could be further done by quantitative real time polymerase chain reaction (qpcr), which on the other hand is a more costly technique. The PCR technique is a method which amplifies a specific fragment of DNA. By adding short manufactured DNA sequences, primers, only DNA from the target species is copied. The reaction goes thorough a number of cycles, with three different temperatures. First at a high temperature (denaturation) the DNA strains are separated. After, at a lower temperature (annealing) the primers bind in to the target DNA sequence. After that the temperature is slightly raised and the target DNA sequence is copied with the use of an enzyme, Taq-polymerase. Each cycle copies the target DNA sequence, creating the amplification. i*2^c (where i is the start concentration of the target gene sequence, and c is the number of cycles). Nested PCR is a technique used for further amplification and purification of the PCR product. Here, the PCR product from a PCR is used as template in a second, nested, PCR. With the nested PCR, the same protocol can be used again, or some modifications can be used (e.g. raised annealing

3 temperature or new primers) to further increase specificity of the procedure. The aim of the conducted survey was to detect the presence of four different Vibrio species (V. alginolyticus, V. cholerae, V. harveyi and V. vulnificus), in marine environmental water samples collected during two different seasons, one post- and one pre-monsoon, from the southwest coast of India. Secondarily, the study aimed to evaluate the adaptations and reliability of the used nested PCR technique. Methods Field water samples were taken during post-monsoon (December 2007) and pre-monsoon (February-March, 2008) season, in the Arabic Sea offshore of Mangalore, India. At each sampling occasion (numbered for post-monsoon and for pre-monsoon), samples were taken at three different locations, labeled a, b & c (see appendix A for sample date and coordinates). Sea water was collected with a water sampler at the depth of the fluorescence maximum, where the highest chlorophyll levels were expected (this varied between 2-6 m), and was transferred to a 5L dark container. The water immediately was brought back to the laboratory where a known volume (between ml) was filtered through a 0.2 μm membrane filter at low vacuum (about 100 mm Hg). The cells were lysed and DNA was extracted according to the protocol used by Godhe et al. (2008). This was made within another study (Asplund et al., unpublished) and thus not by the author of this report. For species-specific PCR detection of Vibrio, eight samples from each season were randomly selected, to a total of 16 samples for each species survey. Different protocol was used for the different species, based on the optimal PCR protocols obtained from literature (eg. Saravanan et al., 2007; Conejero & Hedreyda, 2004; Liu et al., 2004; Kumar et al., 2006). In the first pilot tests, no positive results was detected for any species in any of the samples. Therefore some adjustments of the protocols were made, such as diluting of the samples and lowering of annealing temperature, in order to minimize inhibition of the PCR amplification from cellular and DNA extraction rest products and to decrease the number of longer unspecific products. The protocols and reaction mixtures described below are the ones optimized in these pilot tests and that gave the clearest results. At the end of this report, the applications and effects of different protocol adjustments will be discussed. The species with their respective primers, fraction length, references and adjustments made in this survey are listed in table 1. For all reaction mixtures and protocols, a positive control containing eukaryotic LSU primers was used, to be certain that the amplification reaction was effective. One negative control containing MilliQ filtered water instead of template was added to every PCR procedure. For V. cholerae, primers targeting a 883bp sequence of the toxr gene was used (Miller et al., 1987). The reaction mixture was made with a total reaction volume of 20 μl in PCR tubes and contained; 10 μl Promega GoTaq green Master Mix (2X), 5 µm Forward Primer, 5 µm Reverse Primer and 4 μl template. The thermal cycling protocol was made according to Saravanan et al. (2007) with a lowered annealing temperature to 45 C. The amplification product from this reaction was used in a second, nested, PCR with the same conditions except that the annealing temperature was slightly raised to 49 C. For V. harveyi two different sets of primers were used for initial PCR and nested PCR, respectively. In the initial PCR, primers VHF1 and VHR1 targeting a 1,3 kbp sequence was used, after which the product was submitted to the nested PCR, with primers VHF1 and VhhemoR targeting a 308 bp

4 sequence (Conejero & Hedreyda, 2004). The mixture was made with a total reaction volume of 25 μl with 1 μm of each primer, 200 μm dntp, 1U 5Prime Taq DNA polymerase, 1X Taq buffer, 1 μl template (diluted 1:10) and 19,9 μl MilliQ filtered water. The thermal cycling followed the protocol by Conejero & Hedreyda (2004) with a lowered annealing temperature to 40 C. In the nested PCR the annealing temperature was raised to 45 C. Also for V. alginolyticus, two sets of primers were used for the initial and the nested PCR, respectively. First primers VA16F1 and VA16R2 targeting a 1,5 kbp sequence and, second, primers VA16F2 and VA16R2 targeting a 1,0kbp sequence (Liu et al., 2004). The thermocycling protocol according to Liu et al. (2004) was used with a lowered annealing temperature to 45 C. For the nested PCR the annealing was raised to 47 C. The same GoTaq mixture and volumes as described for V. cholerae was used. For V. vulnificus the same reaction mixture and volumes as for V. harveyi was used, with the primers gyr-vv1 and gyr-vv2, targeting a 285 bp sequence of the gyrb gene. The thermocycling followed the protocol used by Kumar et al. (2006) with a lowered annealing temperature to 45 C. For the nested PCR the annealing temperature was 49 C. For all species, the nested PCR was done with the PCR product, diluted 1:10, as template. Table 1. The primers used, target fragment length, references for each species, with the adjustments made in this survey to optimize the technique. The PCR products were submitted to gelelectrophoresis in agarose gel and stained with ethidium bromide to visualize the results under UV illumination. Results Diluting the templates 1:10 with MilliQ filtered water increased the efficiency of the procedure significantly. The amount of disturbing fragments and primer dimers were decreased, enabling positive detection to be found. This can be seen in Fig 1, comparing results from the diluted and non diluted template of sample 19B.

Fig 1. Picture of gel electrophoresis of diluted and non diluted DNA template. Non-diluted samples are to the right and diluted to the left (x indicate diluted DNA template). 0 is negative control.

5 Fig 1. Picture of gel electrophoresis of diluted and non diluted DNA template. Non-diluted samples are to the right and diluted to the left (x indicate diluted DNA template). 0 is negative control. Note that the band at 308bp in the diluted sample 19Bx does not appear in the non-diluted 19B. Presence of the target Vibrio species in the randomly chosen samples, ranged between 0 for V. cholerae to 38% for V. alginolyticus. V. cholerae was not detected in any samples from the two seasons (Fig 2). V. alginolyticus was detected in three out of eight samples from both seasons. V. harveyi was detected in 20% of the samples with a slight decrease in presence from 29% in postmonsoon to 13% in pre-monsoon conditions (Fig 3)(see appendix B for complete raw results). Fig 2. Picture of one gel elctrophoresis of V. cholerae. No bands appeared at the target 883bp. L2 and L1 are both positive controls. 0 is negative control.

6 0,4 0,38 0,38 0,35 0,3 0,29 0,25 0,2 0,15 0,1 0,13 V. alginolyticus V. harveyi 0,05 0 Post monsoon Pre monsoon Fig 3. Proportion of samples were the target Vibrio species that was present (n=8, except for post monsoon V. harveyi n=7). The results from the PCR method detecting V. vulnificus was inconclusive with several bands appearing at 150 and 350 bp (Fig 4) and thus need further optimization. Therefore no results could be drawn from the experiment on this species. 285bp Fig 4. Photo of one electrophoresis of V. vulnificus. 0 is negative control. The arrow indicate where the target fragment band of 285bp should be. Note the unexpected bands appearing at approximately 150bp and 350bp. The presence of V. harveyi and V. alginolyticus on a time axis indicate occasionally simultaneous or nearly simultaneous presence of the two species, as seen in samples 16, 19 and 28/29 (Fig 5). V. harveyi and V. alginolyticus in sample 16 and 19 were found in the same DNA samples (16A and 19B, see appendix B), which indicate that the two species do not only occur at the same time, but co-occur in the same water sample. In samples 28/29, on the other hand, the species occur close in time, but were not found in the same water samples.

7 Post monsoon Post monsoon Pre monsoon Fig 5. The presence (y=1) or absence (y=0) of V. alginolyticus and V. harveyas as a function of time (sample number). Note the time gap between sample 20 and 21. See appendix A for the true dates. Discussion Occasional presence of Vibrio harveyi and V. alginolyticus could be detected in both seasons. While V. alginolyticus was present in equal number of samples in both seasons, V. harveyi showed 56% decrease in the number of positive samples in pre-monsoon conditions. However, comparison between the two seasons could not be done statistically due to the small amount of samples. This result for V. harveyi should therefore not be taken for a significant decrease. It is more plausible that the factor season is too weakly defined to explain any variation of Vibrios in the environment. Rather, there are a variety of other factors such as chlorophyll concentration, nutrients or phytoplankton community composition that may explain the temporal variation of Vibrios better (Islam at al. 1987). This is supported by the occasional co-occurrence of these two species within the two seasons, shown in Fig 5 and this indicates that some unknown factors during these periods (sample 16, 18 and possibly 28/29) have provided V. harveyi and V. alginolyticus with favorable conditions. The reason why the survey failed to prove any presence or absence of V. vulnificus is not clear. Since No bands appeared in the negative control, the problems are probably not due to contamination. A more likely explanation is that the primer and the protocol used was effective enough for these samples. The primer may have created hairpin loops or bound in to other DNA in the samples, leading to false bands. The low annealing temperatures used to increase the primer ability to bind DNA, further increase the risk of unspecific bands. V. cholerae was not detected. Since the LSU primers yielded a positive PCR product this indicates that the DNA extracts were of sufficient standard, and presumably that no V. cholerae was present in the tested samples (see Fig 2). However, usage of eukaryotic LSU primers as positive control is not optimal. Even though the LSU was positive in the V. cholerae samples, it is not certain that the protocol was efficient enough for the amplification by V. cholerae primers. Therefore, the throughout negative results in this test may be fallacious. The optimal positive control would be usage of DNA extracted from an isolated culture of the target species as template, but as these are costly they were not included in this survey. In such case the same primers could have been used in the treatment, as both positive and negative controls. Then, the results showing absence of V. cholerae would have been more certain. This is naturally an issue concerning all species, but since

8 no bands appeared it becomes an evident problem in the V. cholerae survey. On the other hand, V. cholerae is not as abundant as e.g. V. harveyi or V. alginolyticus in the investigated area; Saravanan et al. (2007) did only find V. cholerae in two out of 20 tested water samples from the same region. The use of PCR technique for detection of the presence of different Vibrio species in this study was carried out with some difficulties. The used protocols for the PCR reactions from the literature were optimized from isolated bacteria (eg. Conejero & Hedreya, 2004; Oakley et al., 2003) or from enriched environmental samples from bivalves (Kumar et al., 2006; Saravanan et al., 2007) or crustaceans (Liu et al., 2004; Saravanan et al., 2007) in contrast to our samples which were extracted from water samples. Unmodified, these protocols failed to detect the presence of free living Vibrio spp. in the water samples. This is probably due to the high concentration of extracted DNA from other co-occurring taxa, both prokaryote and eukaryote. Furthermore, in situ samples contain high concentrations of dissolved and particulate matter, which may inhibit the efficiency of the PCR technique. However, diluting the template 10X with MilliQ filtered water dilutes any obstructing matter, resulting in better efficiency (see Fig 1). Although lowering the annealing temperature may lead to less specificity of the primers (Kidd et al., 1995), it further increases the chance of primers annealing to the template DNA. These measures, along with the use of nested PCR, made the technique strong enough to detect the presence of at least two of the target bacterial species. The used PCR technique has its limitation in only showing presence or absence of the target species. Any variation in abundance will not be detected, which will prevent any quantitative conclusions to be drawn. To detect the quantitative differences between the two seasons, a qpcr that quantifies the abundance of the species would yield more dynamic data to make comparisons. Using PCR is however useful for knowing the presence of a target Vibrio species in a sample and to determine if further quantitative tests are required. Furthermore, if it is assumed that any detectable number of a pathogenic species is enough to alarm for elevated risk of infections, this technique is highly useful for preventive detection. As an example, in aquaculture or food analysis, which have zero tolerance for pathogenic bacteria, the detection of the species tested in this survey would imply the need of additional security measures, veterinary examinations and possibly discard of the complete yield. Thus, the PCR technique is an efficient and reliable tool for the food industry. References Campbell, M.S. & Wright, A.C. (2003) Real-time PCR analysis of Vibrio vulnificus from oysters. Applied Environmental Microbiology 69: Conejero, M.J.U. & Hedreyda, C.T. (2004) PCR detection of hemolysin (vhh) gene in Vibrio harveyi. Journal of General and Applied Microbiology 50: Eiler, A., Johansson, M., Bertilsson, S. (2006) Environmental Influences on Vibrio Populations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas). Applied and environmental microbiology, 72(9): Godhe, A., Asplund, M., Härnström, K., Saravanan, V., Tyagi, A., Karunasagar, I. (2008) Quantifying diatom and dinoflagellate biomass in coastal marine sea water samples by real-time PCR. Applied and Environmental Microbiology 74: Islam, M.S., Drasar, B.S., Sack, R.B. (1994) The aquatic flora and fauna as reservoirs of Vibrio Cholerae. Journal of Diarrheal Disease Research 12(2):87-96

9 Kidd, K.K., & Ruano, G., Optimizing PCR In: McPherson, J., Hames, B.D., Taylor, G.R., (ed) (1995) PCR2: A practical approach. Oxford University Press Inc, New York. Kumar, H.S., Parvathi, A., Karunasagar, I., Karunasagar, I. (2006) A gyrb-based PCR for the detection of Vibrio vulnificus and its application for direct detection of this pathogen in oyster enrichment broths. International Journal of Food Microbiology 111: Liu, C-H., Cheng, W., Hsu, J-P., Chen, J-C. (2004) Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rdna sequencing. Diseases of Aquatic Organisms 61: Miller, V.L., Taylor, R.K., Mekalanos, J.J. (1987) Cholera toxin transcriptional activator toxr is a trans-membrane DNA binding protein. Cell 48: Oakley, H.J., Levy. N., Bourne, D.G., Cullen, B., Thomas, A. (2003) The use of PCR to aid in the rapid identification of Vibrio harveyi isolates. Journal of Applied Microbiology 95: Saravanan, V., Sanath Kumar, H., Karunasagar, I., Karunasagar I. (2007) Putative virulence genes of Vibrio cholerae from seafoods and the coastal environment of Southwest India. International Journal of Food Microbiology 119(3):

10 Appendix A. Field sample code, the date and the coordinates they were taken. Post monsoon Pre monsoon

11 Appendix B. Results from the nested PCR.

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