Detection and quantification of Vibrio parahaemolyticus in vegetables and environmental samples at farm level

The purpose of this study was to detect and quantify total and pathogenic Vibrio parahaemolyticus from vegetables and environmental samples at the farm level in Cameron Highlands, Pahang, Malaysia. Most Probable Number (MPN) – Polymerase Chain Reaction (PCR) method was used to detect toxR, tdh and trh genes and to quantify their concentration in samples. Samples obtained were cabbage (20), carrot (10), cucumber (10), lettuce (31), tomato (18), manure (10), soil (12), surface swab (21) and water (14), with a total of 146 samples. Sampling locations involved were three vegetable farms, two packing houses and one loading bay. Based on the results, overall, 13.7% of samples were present with V. parahaemolyticus toxR (maximum concentration 1100 MPN/g), with the highest detection in cabbage (6%). Vibrio parahaemolyticus tdh was detected in 1.4% samples (maximum concentration 7.3 MPN/g), and V. parahaemolyticus trh could not be detected in any samples. No tdh and trh genes could be detected from the recovered isolates. This finding highlighted that vegetables and environmental samples could potentially be contaminated with V. parahaemolyticus which poses risk to consumers. This study could be useful in future food safety risk communication and management programmes.


Introduction
Fruits and vegetables play a significant role in human nutrition by supplying nutrients such as vitamins, minerals and dietary fibre. The production and processing of fruits and vegetables involve a complex supply chain from the farm to the point of consumption (Pilizota, 2013). The number of produce-related outbreaks has increased in the past decade (Kalantar et al., 2018). These products could be contaminated with biological hazards at any point throughout the supply chain. Consumers eating fresh fruits and vegetables are at risk because this product may be grown on contaminated soil. These bacterial-tainted fruits and vegetables may have come from fields that used to contain animals. Their faeces, faeces-laced irrigation water, or raw manure may have been used as soil additives. Other contributing factors may include changes in agronomic and processing practices, increased international trade and distribution, and an increase in the number of immuno-compromised consumers. Contamination of produce is a concern in developing countries that lack sanitary basic conditions eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER (Western Farm Press, 2007;Pilizota, 2014).
Vibrio parahaemolyticus is a halophilic bacterium that has been known to cause foodborne disease through the consumption of contaminated raw or undercooked seafood (Prabhakaran et al., 2020;Martinez-Urtaza and Baker-Austin, 2020). However, there have been reports of vibrio gastroenteritis associated with the consumption of raw vegetables. V. parahaemolyticus could be found in freshwater or streams and is occasionally isolated from the infected skin of fish handlers. This shows that V. parahaemolyticus could cross-contaminate and survive on non-marine sources. Vibrio gastroenteritis has sometimes been associated with the consumption of raw vegetables which have been contaminated through kitchen utensils (Sakazaki et al., 2006). An outbreak in Kedah, Malaysia in 2003 was reported to be caused by V. parahaemolyticus linked to 'kerabu tauge', a local dish mixed with raw vegetables (Mohamad et al., 2006). In Wenzhou, China, Li (2007) reported a 6.12% (20/112) presence of V. parahaemolyticus in vegetable fruit salad and other food types. Besides that, Okafo et al. (2003) reported the presence of Vibrio spp. in raw vegetables harvested from soils irrigated with contaminated streams in Nigeria (Sakazaki et al., 2006;Mohamad et al., 2006;Li, 2007). This indicated that there is a risk for consumers when consuming raw vegetables and fruits, and can cause acute gastroenteritis in immunocompromised individuals. Therefore, it is important to investigate the presence of V. parahaemolyticus in raw vegetables and fruits, especially in Malaysia.
V. parahaemolyticus regulatory gene toxR is present in all strains, and PCR based on toxR reported to be specific for V. parahaemolyticus has been found useful for confirmation of this species. Pathogenic V. parahaemolyticus produce either thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) or both, and TDH and TRH encoded by tdh and trh genes are recognized as major virulence factors of V. parahaemolyticus (Zhang et al., 2018;Prabhakaran et al., 2020;Martinez-Urtaza and Baker-Austin, 2020). Most Probable Number (MPN) combined with PCR has been successfully applied in enumerating various pathogens in food samples including raw vegetables (Chai et al., 2009;Sandra et al., 2012;Wong et al., 2012).
In order to understand the risk of acquiring V. parahaemolyticus from the consumption of raw vegetables, it is necessary to assess the prevalence and quantity of total and pathogenic V. parahaemolyticus in raw vegetables. This study will assess V. parahaemolyticus in vegetables and environmental samples collected at the farm level in Cameron Highlands, Pahang, Malaysia. This will provide an insight into the scenario in Malaysia and will be useful for further risk assessment studies.

Sample collection
Samples were collected from three vegetable farms, two packing houses and one loading bay, in Cameron Highlands, Pahang. The location is one of the main sources and distributors of fresh produce throughout Peninsular Malaysia. The sampling locations were located at three of the eight sub-districts and were randomly chosen. The samples collected were freshly cut vegetables (n = 89), soil (n = 12), animal manure (n = 10), irrigation water from the reservoir and pipes (n = 14), and swabs (n = 21). Vegetable types collected from the three farms were conducted at random and were dependent on the vegetables that were available at the time of the visit. Soil and animal manure were only collected from Farms 2 and 3 which practised organic farming. The samples collected from the loading bay were freshly harvested vegetables and about to be transported. The samples collected from the packing houses were freshly harvested vegetables, surface swabs from vats, food sorting equipment and packaging tools. Vegetable samples were freshly harvested and placed in sterile plastic bags. Soil and animal manure samples (approximately 200 g each) were collected from different points of the planting site and placed in sterile plastic sampling tubes. Water samples were collected from different points at the main reservoir, distribution tanks, taps and irrigation pipes, and were placed into sterile tubes. Surface swabs were taken from baskets, vehicles, knives, food sorting equipment and vats using sterile cotton swabs, and were placed in sterile plastic tubes. All samples were transported to the laboratory immediately and analyzed within 24 hrs of sample collection.

Sample preparation
The sampling method performed in this study was based on methods by Tunung et al. (2010) and . A 10 g portion of each vegetable sample was placed in a stomacher bag added with 90 mL of Tryptic Soy Broth (TSB; Bacto TM , France) with 3% sodium chloride (NaCl; Merck, Germany) and pummeled in a stomacher (Interscience, France) for 60 s, followed with pre-enrichment by incubation at 37°C for 6 hrs.

MPN-PCR
A 100-fold and 1000-fold dilutions of the stomacher fluid were prepared with Salt Polymyxin Broth (SPB; Nissui, Japan). Portions of each dilution were transferred eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER into three tubes and incubated at 37°C for 18 to 24 hrs. After incubation, a loopful of culture from each tube was streaked onto CHROMagar TM Vibrio. The MPN tubes were first preceded by DNA extraction, which was carried out using the boil cell method  with slight modifications. A 1 mL portion of each MPN broth was centrifuged at 13400×g for 1 min and the pellet was resuspended in 500 μL of sterile distilled water. The mixture was boiled for 10 mins and then immediately cooled at -20°C for 10 mins before it was centrifuged at 13400×g for 3 mins. The supernatant was kept for use in PCR for detection of toxR. The reference V. parahaemolyticus strains (V. parahaemolyticus strain coded 1808, 1896, 2053) used for positive control in the PCR reaction was obtained from Kyoto University, Japan.

Direct PCR
The direct-PCR detection method was used only for environmental samples such as soil, animal manure, swab and water samples (Chai et al., 2009). For soil and animal manure samples, 10 g of the sample was weighed into a 50 mL Falcon tube, mixed with sterile distilled water up to a final volume of 40 mL and incubated at 37°C for 1 hr. The mixture was centrifuged at 600×g for 15 mins and the supernatant was filtered through a sterile cheesecloth. The supernatant was discarded and 400 µL of sterile distilled water was added. The mixture was then transferred to a sterile 1.5 mL microcentrifuge tube and was boiled for 10 mins. Then the sample was cooled at -20°C for 5 mins before it was centrifuged at 12000×g for 10 mins. The supernatant was transferred to a new 1.5 mL centrifuge tube and 800 µL of 95% ethanol was added. The tube was inverted several times and left on ice for 5 mins prior to centrifugation at 12000×g for 10 mins. The supernatant was discarded and the pellet was washed with 1 mL of 97% ethanol. Then it was centrifuged at 12000×g for 10 mins and the pellet was dried under laminar airflow. The DNA was resuspended with 200 µL of sterile distilled water and proceeded for PCR analysis.
For water samples, 50 mL of the sample was poured into a 50 mL Falcon tube and centrifuged at 600×g for 15 mins. The supernatant was transferred to a new tube and subjected to centrifugation at 12000×g for 30 mins. The supernatant was discarded and the pellet was resuspended with 400 µL of sterile distilled water. The mixture was transferred to a sterile 1.5 mL centrifuge tube and boiled for 10 mins. The sample was cooled at -20°C for 5 mins before centrifugation at 15000×g for 10 mins. The supernatant was transferred to a new 1.5 mL centrifuge tube and 800 µL of 95% ethanol was added. The tube was inverted several times and was left on ice for 5 mins prior to centrifugation at 12000×g for 10 mins. The supernatant was discarded and the pellet was washed with 1 mL of 97% ethanol. Centrifugation was carried out again at 12000×g for 10 mins and the pellet was dried under laminar airflow. The DNA was resuspended with 200 µL of sterile distilled water and proceeded for PCR analysis.

Isolates recovery
A loopful of culture from each MPN tubes was streaked onto CHROMagar TM Vibrio (CV). The plates were then incubated at 37°C for 18 to 24 hrs. Presumptive colonies of V. parahaemolyticus (mauve purple colour) was picked and subcultured onto Tryptic Soy Agar (TSA; Bacto TM , France) with 3% sodium chloride (NaCl; Merck, Germany). Isolates were confirmed by gram-strain, colonial and microscopic morphology, catalase test, oxidase test (data not shown), and specific PCR targeting toxR, tdh and trh genes of V. parahaemolyticus. Pure confirmed isolates were maintained on TSA 3% NaCl agar slants and stock cultred in 20% glycerol.

Statistical analysis
To determine if there was any significant difference eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER between the prevalence of total and pathogenic V. parahaemolyticus among the samples, sample types, sampling locations, temperatures during sampling, and hygiene levels of the sampling locations, SPSS software (version 16.0) was utilised to analyze the data. The level of significance was set at P<0.05. Whenever there was a significant difference, post-hoc comparison was done using Tukey's Honestly Significant Difference (HSD) to identify the groups that are significantly different (data not shown).

Results
The total frequency of V. parahaemolyticus detected in 146 samples collected at pre-harvest level was 13.7% (Table 1). Vibrio parahaemolyticus was found highest in cabbage (30%), followed by soil (25%), cucumber and animal manure (20% respectively), tomato (16.7%), carrot (10%), lettuce (6.5%), and a surface swab of the basket (4.8%). Vibrio parahaemolyticus in water samples was not detected (0%). For sampling locations, the prevalence of V. parahaemolyticus was highest in samples collected from Farm 3 (21.2%), followed by Farm 2 (20%), 18.75% from Packing House 1, 11.5% from Loading Bay, and 3.9% from Farm 1. No V. parahaemolyticus was detected in samples from Packing House 2. The concentrations of V. parahaemolyticus in the samples ranged from <3 MPN/g up to 1100 MPN/g. The highest maximum numbers of V. parahaemolyticus were in samples from Farm 2 and Farm 3 (1100 MPN/g respectively). Statistical analysis was carried out to determine any significant differences in MPN/g values of V. parahaemolyticus toxR between the samples, sample types, sampling locations and temperatures during sampling (data not shown). The results showed no significant differences between all the categories tested.
The prevalence of V. parahaemolyticus tdh was 1.4% (2/146) overall, as shown in Table 2. Vibrio parahaemolyticus tdh was found in one sample from Loading Bay (3.85%) and one sample from Farm 2 (3.33%), while none were detected in samples from the rest of the sampling locations. V. parahaemolyticus tdh was most predominant in sample type cabbage with 10% (2/20) prevalence; while no V. parahaemolyticus tdh could be detected in other types of samples. The MPN/g values of V. parahaemolyticus tdh detected in the samples ranged from <3 MPN/g up to 7.3 MPN/g. When the data were analyzed statistically, there was no significant difference in MPN/g values between the samples, sample types, sampling locations and temperatures during sampling. As for V. parahaemolyticus trh, it was not detected in all of the samples.
For the recovery of isolates from plating, only 1 isolate (0.7%) was confirmed positive V. parahaemolyticus toxR (Table 3). The sample was cabbage from Packing House 1. The confirmed V. parahaemolyticus isolate was further tested for the presence of virulent genes tdh and trh, however, none was detected (data not shown).

Discussion
This study revealed the presence of V. parahaemolyticus in vegetables and environmental samples at the pre-harvest stage in vegetable farms. In this study, V. parahaemolyticus was detected in nonmarine samples. The issue of how V. parahaemolyticus could survive and grow on these unnatural sources raises questions and requires explanation and further research. However, previous outbreaks and research have reported the presence of Vibrio spp. in vegetables and other nonmarine samples (Okafo et al., 2003;Mohamad et al., 2006;Sakazaki et al., 2006;Li, 2007;Noorlis et al., 2011). Changes in dietary habits, methods of fruit and vegetable production and processing, sources of produce, and the emergence of pathogens previously not recognized for their association with raw produce, could have enhanced the potential for outbreaks.
Overall, the percentage of samples with the presence of toxR and tdh were 13.7% and 1.4% respectively, while trh could not be detected in any of the samples. Higher prevalence and concentration of toxR were found in samples from Farm 2 and 3 compared to samples from Farm 1. This could be due to the fact that Farms 2 and 3 practised organic farming. It is tempting to speculate that the environmental samples were the most possible sources of contamination of V. parahaemolyticus to the vegetables since there was the presence of toxR in manure, soil and swab samples from Farm 2 and 3. Chai et al. (2009) also reported the possibility of Campylobacter spp. contamination in vegetables in their study was associated with the presence of Campylobacter spp. in soil and manure samples. However, vegetable samples from Loading Bay and Packing House 1 harboured V. parahaemolyticus toxR although none could be detected in environmental samples from the same location. The possible explanation is most probably due to cross-contamination introduced by other sources which were not sampled in this study, which include the handler's hands. Pickers, handlers, packers, and other individuals involved in the handling of produce may have the potential to contaminate the procedure along the harvesting chain.
In this study, the number of V. parahaemolyticus toxR and tdh in the samples were mostly <3 MPN/g, with the maximum number reaching only 1100 MPN/g and 7.3 MPN/g (respectively). Although the counts were (1/10) 20 (2/10) 6.5 (2/31) 16.7 (3/18) 20 (2/10) 25 (3/12) 4.8 (1/21) 0 (0/14) 13.7 (20/146) Table   1. Several researchers (Park and Sanders, 1992;Chai et al., 2009) have reported the prevalence of pathogens in leafy and roots vegetables, suggesting that vegetables in close contact with soil have a higher possibility of contamination by pathogens. Previous research by Tunung et. al. (2010) also reported a higher prevalence of V. parahaemolyticus in leafy vegetables, suggesting that the leafy structure, which allowed more surfaces for attachments, could contribute to the higher rate of survival of V. parahaemolyticus on the vegetables. However, in this study, there was no significant difference in the prevalence of V. parahaemolyticus toxR between the different types of vegetables. Both leafytype vegetable samples consisted of the highest (cabbage 30%) and lowest (lettuce 6.5%) prevalence of V. parahaemolyticus compared to root-type and smoothsurface vegetables samples. In addition to that, cucumber and tomato, which are smooth-surface vegetables, contained a higher prevalence of V. parahaemolyticus (20% and 16.7% respectively) compared to carrot (10%), which is a root-type vegetable. However, it may be worthy to note that in this study; only sample type cabbage contained virulent V. parahaemolyticus tdh (1.4%). Bruised and cut surface tissue discharge fluids containing nutrients and numerous phytoalexins and other antimicrobials that may improve or retard the growth of naturally occurring microflora and pathogens (Thompson, 2009). Soil or faecal material present on the surface of products that may permeate bruised tissues may alter the ecological environment and the behaviour of pathogens and microflora (Beuchat, 2002;Alegbeleye et al., 2018). Mould growths in these environments may result in increased pH and enhance the probability of growth of pathogenic bacteria. Colonization and biofilm development may occur, resulting in conditions that would protect against the death of pathogens or promote the growth of pathogenic microorganisms. Their viability as affected by extrinsic and intrinsic factors unique to fruits and vegetables is unknown (Beuchat, 2002). Natural and food processing environments often impose various stresses on foodborne pathogens and cause morphological changes and adaptation to stress. In an investigation done by (Chen et al., 2009), V. parahaemolyticus was incubated under starvation conditions for 3 days and revealed a characteristic morphological change called viable but nonculturable (VBNC) state. Yoon et al. (2020) also published a report on the VBNC characteristic of V. parahaemolyticus.
The MPN-PCR detection method used in this study was effective compared to the conventional plating method. The plating method was only able to detect viable cells which resulted in a very low prevalence (0.7%), while the MPN-PCR technique was able to detect non-viable cells in which the total prevalence detected was 13.7%.

Conclusion
This study provided data on the presence of V. parahaemolyticus at the farm level in Cameron Highlands, Pahang, Malaysia, and indicated a 13.7% overall prevalence in vegetables and environmental samples. The findings in this study highlighted the potential of vegetables and environmental samples to be contaminated with V. parahaemolyticus and may pose risk to consumers, especially through the consumption of  (positive/total) = no. of positive sample/total sample