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Microbial and Parasitic Contamination on Fresh Vegetables Sold in Traditional Markets in Hue City, Vietnam

Ho Le Quynh Chau*, Ho Trung Thong, Nguyen Van Chao, Pham Hoang Son Hung, Vu Van Hai, Le Van An, Ayako Fujieda, Tanaka Ueru, Miki Akamatsu

College of Agriculture and Forestry, Hue University, Vietnam

Abstract: This study was conducted to evaluate microbial and parasitic contamination in twelve types of popular vegetables in Hue city. A total of 108 vegetable samples, equal numbers of young mustard greens, celery, amaranth, cilantro, water spinach, rice paddy herb, Vietnamese cilantro, basil, centella, lettuce, watercress, and iceberg lettuce were collected from three traditional markets in Hue city. All of samples were tested for total aerobic bacteria counts and E. coli by traditional culture-based methods. The Salmonella and parasites on the vegetables were detected by PCR technique and microscopic methods, respectively. All samples were highly contaminated with aerobic bacteria and E. coli. The aerobic bacteria counts ranged from 6.84 to 8.40 log CFU/g. Escherichia coli levels ranged from 5.47 – 6.88 log CFU/g. Salmonella was detected in 19/108 of test samples. Water spinach was found to have the highest contamination prevalence with Salmonella (55.56%). Contamination by multiple parasites was detected in all vegetable samples. The contamination rates of Fasciola, Ascaris, Trichuris and Clonorchis sinensis eggs were 83.33%, 85.19%, 64.81% and 16.67%, respectively. The oocysts of Cryptosporidium, Isospora and Cyclospora were found on the samples at rates of 47.22%, 27.78% and 27.78%, respectively. These bacteria and parasites may become potential sources of cross contamination and pose a serious risk to human health.

Keywords: aerobic bacteria, E. coli, parasite, Salmonella, vegetable

Received October 29, 2014; Revised November 12, 2014; Accepted November 20, 2014

Citation: Ho Le Quynh Chau, Ho Trung Thong, Nguyen Van Chao, Pham Hoang Son Hung, Vu Van Hai, Le Van An, Ayako Fujieda, Tanaka Ueru, Miki Akamatsu, Microbial and Parasitic Contamination on Fresh Vegetables Sold in Traditional Markets in Hue City, Vietnam. Journal of Food and Nutrition Research, 2 (12). 959-964. January 2014. doi:10.12691/jfnr-2-12-16

1. Introduction

Vegetables are essential ingredients in Vietnamese diet. Many kinds of vegetables are consumed fresh (raw) and provide benefit nutrients as vitamins, fiber and ash for human body. However, these vegetables may be a potential source of infection if they are contaminated. Evaluation of hygienic-sanitary quality of vegetables is not only based on chemical safety (pesticide and hormone residues, heavy metals) but biological safety (bacteria and parasites).

Cases of food poisoning due to vegetable consumption have been reported. Contamination with bacteria (especially E. coli and Salmonella) and parasites on vegetables have been reported in many countries ([11,16,24]). Salmonella was detected in 5.76% of the examined vegetables in Mexico [16]. Meanwhile, E. Salmonella and E. coli were found on 7.5% and 86.1% of the vegetable samples collected from farms, a wholesale market, supermarkets and small shops in Granada, Spain [19]. Vegetables may be infected by bacteria and parasites during the planting (through contaminated fertilizer, water source and soil), transportation, post-harvest or unhygienic cooking ([5,8,12]).

Using fresh stool as fertilizer is still popular in Vietnam. A previous survey implemented by Uga et al (2009) has shown that 81% (121/149) farmers who lived in suburban Hanoi, Vietnam used animal and human feces as fertilizer frequently [23]. Therefore, the risk of microbiological infection related to vegetable consumption is still high whilst using raw vegetables in daily meals continues to play a key role in Vietnamese cuisine. The objective of this study was to evaluate the contamination of vegetables in traditional wet market in Hue city, Vietnam. Information obtained from this study will help vegetable consumers understand the current situation, and provide information to help local governments to develop policy to improve food safety and safeguard public health.

2. Materials and Methods

2.1. Sample Collection

A total of 108 samples of twelve vegetables types were randomly collected from three traditional markets (Tay Loc, Ben Ngu and An Cuu) in Hue city, Vietnam between March 2013 and January 2014. For each vegetable type, three samples from three different stalls were collected. The twelve types of vegetables analyzed in this study were young mustard greens (Brassica juncea), celery (Apium graveolens), amaranth (Amaranthus tricolor), cilantro (Coriandrum sativum), water spinach (Ipomoea aquatica), rice paddy herb (Limnophila aromatica), Vietnamese cilantro (Persicaria odorata), basil (Ocimum basilicum), centella (Centella asiatica), lettuce (Lactuca sativa var. longifolia), watercress (Nasturtium officinale), and iceberg lettuce (Lactuca sativa var. capitata). Five hundred grams of each vegetable were collected, identified and placed in separate sterilized bags and immediately transported to the laboratory in an ice box.

2.2. Evaluation of Aerobic Bacteria and E. coli Contamination

Twenty five grams of chopped vegetable sample were aseptically transferred into a stomacher bag. Samples were homogenized for 2 min at high speed (260 rpm) with 225 ml of sterile peptone water (0.1% peptone) using a Stomacher®400 Circulator (Seward, UK) [6]. Serial decimal dilutions of the supernatant were made in sterile peptone water and aliquots of 0.1 ml of the appropriate dilutions were surface spread in duplicate onto appropriate media. Total aerobic bacteria was enumerated on Plate Count Agar (PCA) (Oxoid CM 325, Unipath Ltd, Basingstoke, Hampshire, England) after incubation at 30°C for 48h [6]. Escherichia coli contamination on vegetables was evaluated by counting all colonies exhibiting typical dark to purple red colonies with metallic sheen grown on Eosin methylene blue (EMB) agar (Conda Laboratories, S.A., Spain) after incubation at 37°C for 24h. Confirmation tests were made on E. coli colonies by Gram stain, catalase, oxydase and biochemical tests (API 20E strips, Biomerieux, USA) [22].

2.3. Salmonella Detection

Buffered Peptone Water (10.0g/l peptone, 5.0g/l sodium chloride, 3.5g/l disodium phosphate, 1.5g/l monopotassium phosphate, pH 7.2) was used for preenrichment [1]. Twenty five grams of chopped vegetable sample were aseptically transferred into a stomacher bag containing 225 ml of Buffered Peptone Water, followed by homogenized for 2 min at high speed (260 rpm) using a Stomacher®400 Circulator (Seward, UK) [14]. The homogenized samples were incubated at 37°C for 24h.

Bacterial chromosomal DNA was isolated using DNA - boiling method. Bacteria suspension was centrifuged at 8,000 rpm for 8 min. The supernatant was removed and the pellet was resuspended in PBS (8g/l NaCl, 0.2g/l KCl; 1.44g/l Na₂HPO₄, 0.24 g/l KH₂PO₄, pH 7.2). The suspension was boiled in water bath at 100°C for 10 min and transferred into ice box immediately. After cooling down, it was centrifuged at 3,000 rpm for 10 min. The supernatant was collected for PCR [2]. DNA of Salmonella enterica subsp. diarizonae (ATCC 12325TM) was used as positive control.

The invA gene of Salmonella was amplified using PCR technique with primer set 139: 5’GTG AAA TTA TCG CCA CGT TCG GGC AA-3’ and 141: 5’TCA TCG CAC CGT CAA AGG AAC C-3’ [17]. PCR product size was 284bp. The PCR mixture contained 12.5 µl of GoTaq Green Master Mix, 1.0 pmol of each primer, 4.0 µl of DNA template and nuclease-free water for a final volume of 25 µl. Amplification was performed using a gradient thermal cycler (MaxyGene, Axygen). The cycling conditions were as follows: heat denaturation at 94°C for 5 min, and then 35 cycles with heat denaturation at 94°C for 1 min, primer annealing at 64°C for 1 min, DNA elongation at 72°C for 1 min and final extension for 7 min at 72°C. Amplified products were electrophoresed in 2% agarose gel, stained with ethidium bromide (0.5 µl/ml), visualized and photographed with Gel Doc™ XR+ system (Biorad).

2.4. Parasitic Contamination Detection

A 10 gram chopped vegetable sample was transferred into a stomacher bag containing 100ml of sterile distilled water and homogenized for 2 min at 260 rpm using a Stomacher®400 Circulator (Seward, UK). Homogenized samples were centrifuged at 1200 ×g for 10 min. The supernatant was discarded and 2 ml of the pellet was collected [24]. The next steps were done following the modified Bailenger method [3]. The pellet was suspended in an equal volume of acetoacetic buffer (1 liter of buffer contained 15g sodium acetate trihydrate and 3.6ml glacial acetic acid, pH 4.5) and two volumes of ethyl acetate or ether. The mixture was mixed throughly in a vortex mixer and centrifuged at 1000 ×g in 15 min. After centrifugation, the sample was separated into three phases. The bottom phase contained heavy debris, including helminth eggs, larvae and protozoa. The middle layer was buffer. The materials dissolved into ethyl ether or ether formed were in the top layer. The supernatant was discarded and 5 volumes of 33% zinc sulfate solution was used to resuspend the pellet. After, thorough mixing the sample was then tested for the presence of parasites.

Helminth eggs were detected in the sample under a light microscope using ×40 magnification and identified according to the description of Ayres and Mara (1996) [3]. Protozoa were detected by staining the specimens following modified Ziehl-Neelsen technique [4]. The sample was smeared directly onto a microscope slide and air dried. The slide was then prefixed in absolute methanol for 5-10 sec, stained in carbol fuchsine – DMSO (4g of basic fuchsin crystals, 25ml of 99% ethyl alcohol, 12g of phenol crystals, 25ml of glycerol, 25ml of dimethyl sulfoxide and 75ml of distilled water) for 5 min. The slide was rinsed under running tap water for 10-30 sec until excess solution no longer ran off. The slide was placed in decolorizer-counter-stain solution (220ml of 2% aqueous solution of malachite green, 30ml of glacial acid acetic and 50ml of glycerol) for 1 min or until a green background appeared. The slide was then rinsed under running tap water for 10 sec, drained and air dried. Observing the slide under a light microscope using ×100 magnification and identifying the parasites according to the description of Ribes et al (1996) [18].

2.6. Statistical data Analysis

The data were analyzed using general linear model (GLM) in SPSS software version 18.0. Results presented in tables are mean ± standard deviation. Significant differences among means were separated by Tukey test with a 5% level of probability.

3. Results and Discussion

3.1. Bacterial Contamination on Vegetables

The study results showed that all vegetable samples were contaminated with aerobic bacteria and E. coli. The average concentrations of aerobic bacteria and E. coli ranged from 6.84 to 8.40 log CFU/g and 5.47 – 6.88 log CFU/g, respectively (Table 1). The E. coli concentrations detected were higher than the acceptable limit (10² – 10³ CFU/g) for fresh vegetables according to Vietnamese technical regulation of microbiological contaminants in food QCVN 8-3: 2012/BYT. There was a significant difference in E. coli load detected in the same type of vegetable between the three markets, especially in celery, cilantro and Vietnamese cilantro (Table 1). Meanwhile, the highest variation of total aerobic bacteria concentration was detected on water spinach (7.52 – 9.45 log CFU/g), followed by Vietnamese cilantro (6.82 – 7.81 log CFU/g).

This result was higher than that in other previous reports [11,19]. The total aerobic bacteria found on leafy vegetables from small shops in Granada (Spain) was 10⁴ – 10⁶ CFU/g or 4 – 6 log CFU/g [19]. In the same study, the authors also indicated a high degree of fecal contamination with 86.1% vegetable samples examined contaminated with E. coli [19]. Meanwhile, the concentrations of aerobic bacteria (4.5 – 6.2 log CFU/g) and E. coli (0.7 – 1.0 log CFU/g) were found on leafy vegetables and herbs from the southern United States [11].

Table 1. Aerobic bacteria and E. coli concentrations on the examined vegetables

Table 2. Aerobic bacteria and E. coli contamination on the vegetables between markets

There was no significant difference in total aerobic bacteria count on vegetable samples between the three markets (Table 2, p > 0.05). However, the concentrations of E. coli detected on the samples differed significantly between Tay Loc, An Cuu and Ben Ngu markets. The lowest degree of E. coli contamination on vegetables was found at Tay Loc market (5.79 log CFU/g). Conversely, the highest contamination with E. coli was detected on vegetables collected from An Cuu market (6.88 log CFU/g).

Being able to get results quickly is one of the advantages of using PCR technique on Salmonella detection, compared to traditional culture method. Moreover, the dead Salmonella cells are can be detected using PCR. Amplification of invA gene sequence of Salmonella has been being used for detecting Salmonella in poultry, meats and dairy products, and in vegetables and fruits [9]. The result of invA gene amplification is presented in Figure 1.

Figure 1. Detection of Salmonella by PCR from the examined vegetables

Eighteen percent (19/108) of samples were contaminated with Salmonella (Table 3). The percentage of Salmonella-positive samples from each market were Tay Loc 7/36 (19.44%), Ben Ngu 5/36 (13.89%) and An Cuu 7/36 (19.44%). Most vegetable types were contaminated with Salmonella except cilantro, Vietnamese cilantro, basil and centella. The highest prevalence of Salmonella contamination was found on water spinach (55.56%). A previous study carried out by Ruiz et al (1987) has shown that Salmonella was detected in 7.5% of the examined vegetables in Granada (Spain). Furthermore, the study result of Quiroz-Santiago et al (2009) showed that 98/1700 vegetable samples collected in Mexico was contamniated with Salmonella [16]. The prevalence of Salmonella contamination in each type of vegetables ranged from 1% to 12% [16].

Table 3. Prevalence of Salmonella contamination on the examined vegetables

Bacteria can contaminate vegetables in different ways and at different points in time. The study result of Enabulele and Uraih (2009) indicated that the source of E. coli contamination on vegetables sold in Benin (Nigeria) was contaminated irrigated water [5]. This contaminated water had come from contact with grazing cattle and cross contamination from the vehicles that were used for animal transportation prior to carrying vegetables. In addition to this, Salmonella-contaminated wastewater used for irrigation in Marrakesh city (Morocco) caused contamination on vegetables was also reported [13]; and a study on the microbiological quality of carrots used for fresh juice preparation in India showed that there was a large increase in total aerobic bacteria and Staphylococcus on carrots as they travelled further through the distribution chain [8]. The total aerobic bacteria and Staphylococcus counts detected on carrots increased from 10⁴ to 3.6×107 CFU/g and from 1.9×104 to 7.7×10⁶ CFU/g, respectively during the transportation from the central distribution site to street vendors. It is posited that unhygienic handling, cross contamination and water quality caused the contamination of the carrots [8].

3.2. Parasitic Contamination on Vegetables

All vegetable samples were contaminated with parasites. The parasites most commonly detected were Fasciola (eggs, 83.33%), Ascaris (eggs, 85.19%) and Trichuris (eggs, 64.81%) (Table 4). Clonorchis sinensis eggs was also detected with the frequency of 16.67%. In addition, oocysts of some protozoa as Cryptosporidium, Isospora and Cyclospora were found on the test samples. The Cryptosporidium contamination ratio on 12 types of examined vegetables ranged from 22.22% to 66.67%. The number of samples contaminated with Isospora and Cyclospora were relatively low. Cilantro was Cyclospora-free. Of twelve types of examined vegetables, watercress was the vegetable most infected by parasites, followed by basil and lettuce.

The parasitic contamination on vegetables in Vietnam was also reported [23]. There were 26% vegetable samples collected from a suburban market in Hanoi (Vietnam) contaminated with helminth eggs. Leafy vegetables had the highest contamination ratio (31%; [23]). The five main types of parasite eggs were found in that study were Ascaris sp., Trichuris sp., Toxocara sp., Taenia sp. and Ascaridia galli [23]. Furthermore, Kłapeć and Borecka (2012) also reported the contamination of vegetables, fruits and soil on organic and conventional farms with parasite eggs (Ascaris, Trichuris and Toxocara) in south-eastern Poland [12]. The situation of contamination with parasites on vegetables in other countries was also reported, for example in Iran [7], Nigeria [10], Semnan [15], Egypt [20] and Philippines [21]. The reasons of parasitic contamination on vegetables were probably contaminated from fertilizer ([23], [12]), wastewater source [24], soil [12].

Table 4. Prevalence of parasitic contamination on vegetables

4. Conclusion

Contamination with bacteria and parasites was detected on a range of vegetables at the three big, traditional markets in Hue city. All of examined vegetables samples were contaminated with multiple bacteria and parasites. Contamination levels of E. coli and Salmonella on most of vegetables examined were higher than acceptable limits for fresh vegetables according to Vietnamese technical regulations on microbiological contamination in food. Vegetables contaminated in this way can become potential sources of cross contamination and pose a serious risk to human health. Therefore, with the aim of reducing the risk of cross contamination and food poisoning, washing vegetables after harvest and before using is very important. Studies aimed at reducing pathogen contamination in the field, during transportation, and to limit or remove pathogens on vegetables after harvest are necessary to supply safe vegetables to consumers.

Acknowledgement

We would like to acknowledge the financial support of the Graduate School of Global Environmental Studies, Kyoto University, Japan. We also thank Dr. Mary Kathryn Finlay-Doney for her support in English writing.

Statement of Competing Interests

The authors have no competing interests.

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