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Microbial and Parasitic Contamination on Vegetables Collected From Retailers in Main Market, Akure, Nigeria

Dada E. O., Olusola-Makinde O. O.*

Department of Microbiology, The Federal University of Technology, Akure, Nigeria

Abstract: Laboratory investigations were carried out on six different vegetable samples; Amaranthus cruentus (Amaranth), Talinum triangulare (Waterleaf), Solanecio biafrae (Worowo), Brassica olerecea (Cabbage), Lactuca sativa (Lettuce) and Daucus carota (Carrot) purchased from retailers in main market, Akure, Nigeria to determine the microbial and parasitic contamination. One part of each sample was washed with distilled water while the other part was washed with physiological saline. For parasitological analysis, the solutions were centrifuged, decanted and viewed under the microscope while culturing was done for microbial analysis using the pour plate technique. Only L. sativa was contaminated with helminth; Ascaris lumbricoides. All samples were found to be populated with various species of microorganisms. Seven bacteria belonging to different genera and six fungi were isolated randomly and identified from the vegetables. Bacteria include Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Salmonella typii and Proteus vulgaris while fungi are Aspergillus niger, Saccharomyces sp, Penicillium sp, Rhizopus stolonifer, Fusarium sp and Mucor mucedo. The total bacterial count ranged from 3.2×10⁶ to 7.2 ×10⁶cfu/g for samples washed with distilled water and 1.6 x 10⁶ to 4.8×10⁶cfu/g for samples washed with physiological saline. S. aureus had the highest occurrence of 25% while Salmonella typhii had the least occurrence of 10%. The fungal count ranged from 3.0×10⁵ to 5.0×10⁵sfu/g for samples washed with distilled water and 1.0×10⁵ to 3.0×10⁵sfu/g for samples washed with physiological saline. R. stolonifer had the highest percentage of occurrence of 33.3%. Saccharomyces had the occurrence of 22.2% and Penicillium sp, A. niger, Mucor mucedo, Fusarium sp had the least occurrence of 11.1%. This study showed the presence of organisms of health significance on retail vegetables. Reduction of risk of human illness associated with raw produce can be achieved through controlling points of potential contamination.

Keywords: vegetables, bacteria, helminth, physiological saline, distilled water

Received March 09, 2015; Revised April 13, 2015; Accepted June 07, 2015

Citation: Dada E. O., Olusola-Makinde O. O., Microbial and Parasitic Contamination on Vegetables Collected From Retailers in Main Market, Akure, Nigeria. American Journal of Microbiological Research, 3 (3). 112-117. January 2015. doi:10.12691/ajmr-3-3-3

1. Introduction

Consumption of leafy vegetables has increased in recent years because of their nutritional value and beneficial health effects [1]. Vegetables harbour a wide range of microbial contaminants which undermine their nutritional and health benefit thus increasing outbreaks of human infections associated with the consumption of fresh or minimally processed fruits and vegetables [2]. The number of outbreaks of food borne illnesses associated with consumption of fresh vegetables has increased due to the increase in demand for fresh produce [3]. Surveillance of vegetables has indicated that these vegetables can be contaminated with various bacterial pathogens, including Salmonella sp, Shigella sp, E. coli O157:H7, Listeria monocytogenes and Campylobacter at any of several points from the field through to the time of consumption [4]. Several factors contributed to increase in occurrence and outbreaks of numerous food borne diseases associated with raw fruits and vegetables. Some of the factors are globalization of food supply, introduction of pathogens into new geographical areas through import, use of untreated waste water and water supplies contaminated with sewage, manure as fertilizers for crop production, irrigation and various agronomic practices, post-harvest handling, and hygienic conditions of preparation in food service or home settings [5]. High incidence of human intestinal parasites has been found in communities that consume raw vegetables, especially where the vegetables are cultivated on farm lands fertilized with untreated human and animal sewage [6]. Although most processors and consumers assume that washing fresh vegetables and fruits will reduce the microbial load on their surfaces, but studies have shown that water washing alone is not effective in reducing microbial population on the fresh vegetables [4]. This research is therefore aimed at determining the carriage rate and identification of human intestinal helminth ova and associated microorganisms on some selected leafy vegetables.

2. Materials & Methods

2.1. Sample Collection

A total of six (6) types of fresh vegetables (Daucus carota, Amaranthus cruentus, Lactuca sativa, Brassica oleracea, Solanecio biafrae and Talinum triangulare) were collected from Akure main market using a random sampling technique. Mature leaves of the selected vegetables were sampled at early maturity according to methods used by [7,8]. All samples were collected aseptically in a sterilized universal container and plastic bags and transported to The Federal University of Technology, Akure for laboratory processing [9]. Analysis was conducted within 24 hour of arrival at the Microbiology Laboratory of the University.

2.2. Parasitological analyses of vegetables

Different solvents were assayed for the washing of vegetable samples; these include sterile water and physiological saline solution (0.90% NaCl). In the laboratory, 100g of each fresh vegetable sample was chopped into small pieces and put into a clean beaker containing 500ml physiological saline solution (0.90% NaCl), to wash the sample. After removing fragments of the vegetable sample from the washing saline using clean forceps, it was kept for 24 hour to allow sedimentation to take place. The same experiment was carried out with the use of 500ml sterile water for each of the samples but was washed two times. After 24 hour sedimentation, the top layer of the washing solvent was carefully discarded leaving 5 ml of the sediment. This was finally centrifuged at 2000 revolution/minute for 20 minutes by HERMLE Z200A centrifuge.

After discarding the supernatant, the residue was mounted on slides, stained with Lugol’s iodine solution and examined under the compound light microscope for the presence of intestinal helminth parasites or their ova, cysts of E. histolytica and G. intestinalis in the vegetable samples [10,11]. These were identified as described by [12].

2.3. Microbiological Analysis of Vegetables

Vegetable samples were washed with 100ml of saline water and 100ml of distilled water respectively into different sterile containers. The microbiological assay of sampled vegetables was determined by the pour plate technique as described by [8]. 1ml of dilution 10⁻⁴ and 10-5 were assayed using nutrient, MacConkey and potato dextrose agar as growth media. The plates were observed for growth after 24 hours and 72 hours respectively and the numbers of colonies on the plates were counted [13].

2.4. Statistical analysis

All experiments were carried out in triplicate. Data obtained were analyzed by one way analysis of variance (ANOVA) and means were compared by duncan multiple range test (DMRT) using SPSS 18.0 version. Differences were considered significant at P ≤ 0.05.

3. Results

3.1. Helminth Parasite Ova and Microbial Load of Vegetable Samples

Ascaris lumbricoides ova were found in the distilled water solution and physiological saline solution of lettuce. Other samples were not contaminated with human intestinal helminth parasites as shown in Table 1. The total bacteria count on nutrient agar ranged from 3.2×106 to 7.2×106 cfu/g. In all the samples washed with distill water, D. carota was found to have the highest microbial load followed by B. oleracea, T. triangulare, L. sativa, A. cruentus and S. biafrae was the least contaminated on nutrient agar. The microbial count on MacConkey agar ranged between 1.0×10⁶ to 5.2×10⁶ cfu/g. Table 2 shows the highest microbial count on MacConkey agar to be found in D. carota, followed by B. oleracea, S. biafrae, T. triangulare, L. sativa and the least count was found in A. cruentus. The fungal count for distilled water ranged from 3.0×105 to 5.0×10⁵sfu/g. The highest fungal count was found in D. carota and L. sativa followed by A. cruentus and the least fungal count was found in B. oleracea, S. biafrae and T. triangulare as shown in Table 2. The total bacteria count on nutrient agar ranged from 1.6×10⁶ to 4.8×106 cfu/g. Of all the samples, B. oleracea was found to have the highest microbial load followed by L. sativa, D. carota, S. biafrae, A. cruentus and T. triangulare was the least contaminated. The bacteria count on MacConkey agar ranged from 5.0×10⁵ to 3.5×10⁶. D. carota has the highest microbial count on MacConkey agar, followed by L. sativa, S. biafrae, B. oleracea and the least count were found in A. cruentus and T. triangulare. The fungal count for physiological saline was between 1.0×10⁵ to 3.0×10⁵ (Table 3). The highest fungal count was found in D. carota and L. sativa followed by A. cruentus, B. oleracea and T. triangulare while the least count was found in S. biafrae as shown in Table 3.

Table 1. Ova of Intestinal Helminth Parasite Encountered In Vegetable Samples

3.2. Identification and Distribution of Isolated Organisms

The seven bacteria and six fungi species isolated and identified from vegetable samples are Staphylococcus aureus, Proteus vulgaris, Bacillus subtilis, Salmonella typii, Serratia marcescens, Pseudomonas aeruginosa, Escherichia coli., Penicillium sp, Aspergillus niger, Mucor mucedo, Rhizopus stolonifer, Fusarium sp and Saccharomyces sp were organism isolated and identified as contaminant of vegetable samplesas shown in Table 4. S. aureus have 25% occurrence rate, P. vulgaris, E. coli and B. subtilis have 15% occurrence rate and P. aeruginosa, S. marcescence and S. typii have the least occurrence rate of 10%. Among the fungi encountered, R. stolonifer have the highest percentage of occurrence of 33.3% followed by Saccharomyces sp. with 22.2% and Penicillium sp, Aspergillus sp, Mucormucedoand Fusariumsp having the lowest occurrence of 11.1% as shown in Table 5.

Table 2. Mean Microbial Load of Vegetable Samples Washed With Distilled Water

Table 3. Mean Microbial Load of Vegetable Samples Washed With Physiological Saline

Table 4. Morphological and Biochemical Characteristics of Bacteria Isolates

Table 5. Morphological characteristics of isolated fungi

4. Discussion

This study revealed the carriage rate of human intestinal helminth ova and associated microorganisms in some selected leafy vegetables. In line with [10], the presence of only the ova of A. lumbricoides was found in lettuce in this study whereas the presence of helminths and other parasites on vegetables were observed in the study of [9,14]. This present study did not observe any human intestinal helminths, their ova any other parasite in other vegetable samples. This is not unexpected as this could be due to prewashing of the samples in clean water by farmers or retailers before sales and high level of hygiene could have been maintained during pre-harvest or post-harvest handling.

The observed difference in prevalence rates of the different intestinal helminth parasite ova from fresh vegetables was also reported by [14]. Several factors may contribute to such differences; geographical location, type and number of samples examined, methods used for detection of the intestinal parasites, type of water used for irrigation, and post-harvest handling methods of such vegetables.

A. lumbricoides is the causative agent of ascariosis. The reservoir host of the parasite is man. The eggs contaminates the soil or vegetable though promiscuous defecation or through the use of manure as organic fertilizer on farmland. The eggs are ingested by humans with contaminated food, soil and less frequently drinking water [15]. The occurrence of A. lumbricoides in some stream, river and other sources of water has been reported by the work of [11,16] which is also a predisposing factor to the contamination of vegetables when such water is used for irrigation.

The high microbial load obtained from the isolation of microorganism from the vegetable samples could probably be directly linked to the recorded use of waste water in irrigation for watering the field, or the manure used for fertilization and the unhygienic condition of the area where the vegetables were being grown and sold. The result correspond to the findings of [17] who reported that the presence of many pathogens in the soil was thought to be from historical application or environmental presence of feaces or untreated sewage and pathogens existing in the soil or water can be the source of both pre- and post-harvest contamination respectively. The slight variation in the microbial load from other sources can be traced to prewashing of vegetables with portable water.

Among the organisms encountered during this study Staphylococcus aureus with the highest occurrence is in agreement with the studies of [7]. Poorna [18] also showed presence of Salmonella, Serratia, Enterobacter, Staphylococcus aureus, E. coli and P. aeruginosa in vegetables and fruits. S. aureus is a normal microbial flora of the mucus membrane and on the human skin. It is an opportunistic pathogen and enterotoxigenic strains which are known to cause serious food borne disease and has been reported that ingestion of the thermostable enterotoxins, rather than the bacterium itself is responsible for food borne illness [7]. They cause boils, abscesses, post-operative infections, toxic shock syndrome and food poisoning in man [13].

Other organisms encountered during the study and their respective health implications include Bacillus subtilis causing diarrhoeal. The occurrence of Bacillus subtilisin vegetables agrees with the findings of [19] who reported that estimated 27,000 cases of food borne illness are due to Bacillus species. Bacillus spp. are part of the natural flora and normally lives in the soil. They are among the most common vegetable spoilage bacteria [13], though some Bacillus species are capable of causing food borne illness.

Table 6. Microorganisms isolated from Vegetables

The occurrence of E. coli may be linked to animal dung and manure used during the cultivation of vegetables as fertilizers. Other organisms encountered include S. typhii which has been reported to be responsible for typhoid fever [20] and several cases of an outbreak of typhoid fever have been associated with consumption of contaminated vegetable grown in or fertilized with contaminated soil or sewage [21].

Pseudomonas aeruginosa is a prominent inhabitant of soil and organism is responsible for diseases of vegetables like angular leaf spot of many vegetables it has become an important cause of infection and It is a frequent cause of nosocomial infections such as pneumonia, urinary tract infections (UTIs), and bacteremia [22]. It invades burns area, causes septic shock and responsible for cystic fibrosis in human [23]. They are also associated with spoilage of vegetable.

Michael et al., [13] described Proteus vulgaris as an organism thatoccur in the intestine of human and in a wide variety of animals, in polluted water, soil and they are opportunistic pathogens. Michael et al., [13] also reported Serratia sp. that they were once thought to be harmless and it is now clear that they can be opportunistic human pathogen and are particularly prone to infect hospitalized patients.

The fungi isolate Rhizopus stolonifer and Mucor mucedo has been reported to cause food spoilage. They grow on fruits and vegetables [13]. These fungi grow on the surface of moist, carbohydrate-rich foods, such as breads, fruits, and vegetables [23]. The presence of Penicillium sp and Aspergillus niger agreed with the study of Akinyele et al., [7] and could be due to the fact that these organisms are spore formers and are known as common environmental contaminants [13,23] nevertheless, they have been implicated as food borne pathogens [7].

Table 7. Distribution Of Bacteria And Fungi Contaminant In Vegetable

Several fungi from different region has been isolated and identified to be associated with fruits and vegetables. The most common fungi found in a study by Akintobi et al., [24] were Aspergillus flavus, A. niger, Fusarium solani, Penicillium digitatum, Rhizopus stolonifer and yeasts. Baiyewu et al., [25] had also reported that Rhizopus nigricans, Aspergillus flavus, Aspergillusniger and Fusarium moniliforme among others, were responsible for post-harvest losses in pawpaw in South Western, Nigeria. The isolation of Rhizopus stolonifer from vegetables further confirmed the studies of Efiuvwerwere, [26], Chuku et al., [27], Akinmusire [28], and Akintobi et al., [24] who reported that Fusarium sp, and Rhizopus stolonifer were responsible for the soft rot of tomato. Onyia et al. [29] also reported that Fusarium monilifome, Aspergillus niger and Rhizopus.

In this study, the high prevalence of fungi, bacteria and parasites was further enhanced by unhygienic mode of transportation of these consumable products. Also local practice of using organic manure, such as human, animal and poultry dropping as fertilizer contributed immensely to the occurrence of the pathogen research. The contamination of vegetables by pathogenic bacteria, fungi and parasites could also be as a result of poor handling practices in food supply chain, storage conditions, distribution, marketing practices and transportation [26].

5. Conclusion and Recommendation

This is a preliminary study of an on-going research, it showed the presence of organisms that have serious public health significance while others fasten spoilage of the vegetables. High numbers of these microorganisms in raw consumed vegetables and salad produce would lead to the consumer’s illness with symptoms of the particular or combined microbial presence. Reduction of risk for human illness associated with raw product can be better achieved through controlling points of potential contamination in the field during harvesting, during processing or shipment, storage or distribution in the retail markets, food services facilities or home. The result of this study also shows that salt washing can reduce microbial loads on vegetables by lysing the cell wall.

It is therefore necessary and important for regulatory authorities in conjunction with the government to formulate a technique or safe way of producing, handling, processing, storing and retailing leafy vegetables especially in developing countries such as Nigeria. To control food borne infections during post-harvest handling, vegetables should be thoroughly washed with clean portable water before consuming raw and before cooking, addition of salt to the rinse water may also reduce microbial load as indicated in this study. It is therefore necessary and important that both the farmer who harvests the vegetables for transportation, the marketers and consumers take necessary and appropriate precautions in preventing contamination and eating of contaminated vegetables.

Seminars, random examinations and research can also be organized and carried out on sellers and vegetables to educate sellers on the implication and hazard of improper storage, handling and display of leafy vegetables.

Further studies on the enteroxigenicity tests for Escherichia coli, Salmonella spp and Staphylococcus aureus can be conducted also the effect of different salt concentrations on microorganisms isolated from leafy vegetables.

Acknowledgement

We appreciate the financial assistance and contribution of Miss Abiola Ojo to this study. We are indeed grateful to the laboratory technical staff in the Department of Microbiology, The Federal University of Technology, Akure for been there when we need their assistance.

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