Bacterial pathogens recovered from vegetables irrigated by wastewater in Morocco.

Introduction

An increase in consumption of fresh fruits and vegetables worldwide has been paralleled by an increase in the number of foodborne illnesses attributed to fresh products. Numerous reports have indicated that raw vegetables may harbor potential foodborne pathogens (Beuchat, 1996). In particular, tomatoes, cantaloupes, and sprouts have been linked to outbreaks of salmonellosis (Guo, Chen, Brackett, & Beuchat, 2001), and outbreaks of illnesses caused by Escherichia coli O157:H7 have been associated with melon, apple cider, lettuce, and radish sprouts (Breuer et al., 2001). Moreover, coleslaw, cabbage, potatoes, radishes, bean sprouts, and cucumbers contaminated with Listeria monocytogenes have been linked to disease outbreaks (Shearer, Strapp, & Joerger, 2001), and salad vegetables also may be contaminated with Campylobacter (Evans, Ribeiro, & Salmon, 2003).

Vegetables can become contaminated with pathogenic organisms during growth, harvest, postharvest handling, or distribution (McMahon & Wilson, 2001). Use of untreated wastewater in irrigation represents an important route for transmission of these pathogenic organisms. Raw vegetables are considered by some to represent an increased risk to public health when irrigation methods use untreated wastewater and no chemical treatments are employed to reduce the microbiological load on the raw product (Takeuchi, Hassan, & Frank, 2001).

In Morocco, vegetable products have been in great demand in recent years. Since the rate of precipitation has been very low during these last decades, wastewater is increasingly being used in agriculture. Little information is available on the number of human foodborne-illness outbreaks that have occurred from consumption of raw vegetables. The use of raw sewage to irrigate crops is an important mechanism that helps to propagate conditions conducive to cholera and typhoid fever (Castro-Rosas & Escartin, 2000). Increases in foodborne illnesses during the summer are not fully understood, although fresh produce likely plays a role since it is consumed in higher quantities during the summer.

The study reported here investigated the occurrence of pathogenic bacteria in vegetables irrigated by untreated wastewater in Morocco. Irrigated vegetables do not undergo any control before being exposed in the markets, after which they may be eaten cooked or raw. The purpose of the study was 1) to determine the bacterial quality of vegetables irrigated with untreated wastewater, 2) to sensitize farmers to the dangers from use of untreated wastewater for irrigation, and 3) to elucidate the risk to Moroccan public health.

Materials and Methods

Samples

A total of 50 vegetable samples were procured for bacteriological examination. Vegetables of various types were obtained from several wastewater-irrigated agricultural regions in Morocco. Sampling was conducted from August 2002 to July 2004. The vegetable samples were collected in sterile polyethylene bags, and steps were taken to avoid contamination of the vegetables by soil or other contamination sources. Each sample was collected in triplicate to prevent sampling error. The vegetables were tomato, radish, cucumber, eggplant, potato, pepper, garden pea, gourd, zucchini, artichoke, broad bean, turnip, onion, French bean, and lettuce. All the samples were transported to the laboratory under low temperature (<7[degrees]C) and stored at 4[degrees]C until testing. They were analyzed within 20 hours of sampling. Each sample was rinsed several times with sterile distilled water to eliminate the soil. Before analysis, 25 g of each sample was homogenized for two minutes with 225 mL of 0.1 percent sterile peptone water with a Model 400 Stomacher (Seward Medical, London) and serially diluted.

Bacteriological Analysis

Using the spread-plate technique and 100 [micro]L from the serial dilution, the authors prepared duplicate plates for the determination of aerobic plate counts (APC), Enterobactericiceae, fecal coliforms, total colilorms, Staphylococcus, and Streptococcus.

Aerobic plate counts were made with plate count agar (Merck), and plates were incubated at 30[degrees]C for 48 hours. Then all colonies on plates were counted. Enterococci counts were made with Slanetz and Bartley agar (Biokar). The plates were incubated at 37[degrees]C for 48 hours, and all typical colonies (pink or dark red with a narrow whitish border) were counted. For the coliform counts, violet red bile agar (from Merck) was used for direct plating, and plates were incubated at 37[degrees]C for 24 hours and 42[degrees]C for total coliforms and fecal coliforms, respectively. Typical colonies were round, red to pink, 0.5 to 2 mm in diameter, and surrounded with a red-to-pink halo. Staphylococcus aureus counts were determined with Baird-Parker Agar (Difco) with egg yolk--tellurite emulsion, and plates were incubated at 37[degrees]C for 24 hours to 48 hours. Colonies selected from the agar surface were examined under microscope for Gram stain and were tested for catalase reaction and then for coagulase activity with plasma rabbit (Biokar).

To isolate Salmonella spp., we pummeled a 25-g sample in a stomacher with 225 mL of buffered peptone water and pre-enriched the homogenate 37[degrees]C for 18 hours. A 100-[micro]L sample was subcultured into 10 mL of Rappaport Vassiliadis Broth (Difco) and enriched at 41.5[degrees]C for 24 hours and 48 hours. One mL of the pre-enrichment broth was simultaneously inoculated into 10 mL of selenite cysteine broth and enriched at 37[degrees]C for 22 hours and 48 hours. Both enrichment broths were streaked onto xylose lysine deoxycholate agar (Merck) and Salmonella-Shigella agar, and incubated at 37[degrees]C for 22 hours. For selective plating, presumptive Salmonella colonies from selective plates were confirmed with the API 20E identification system (BioMerieux). The Enterobacteriaceae strain was isolated with Levine-EMB agar (Merck). The plates were incubated at 37[degrees]C for 18 hours, and colonies growing on the plates were examined under a microscope for Gram stains and tested for catalase and oxydase reactions. For identification of all strains, the API 20E identification system (BioMerieux) was used.

Results

These analyses showed high aerobic-plate, total-coliform, fecal-coliform, and enterococci counts. Coagulase-positive Staphylococcus aureus was not detected in any samples (Table 1).

The frequencies with which the bacteria were recovered from samples are given in Table 2. Citrobacter freundii and Enterobacter cloacae were recovered most frequently (from 28 percent of samples). Other Gramnegative bacteria that were frequently isolated were Escherichia coli (16 percent), Enterobacter sakazakii (12 percent), Klebsiella pneumoniae (17 percent), and Serratia liquejaciens (11 percent).

Discussion

Foodborne diseases remain an important public health threat worldwide, and one of the most important food safety hazards is associated with raw vegetables. The large number of total microorganisms and fecal-contamination indicators (E. coli, coliform, and enterococci) detected in the vegetable samples we surveyed indicates a potential health hazard to consumers. Madden has discussed potential sources of microbial contamination of fresh fruit and vegetables during growth, harvest, distribution, and processing (1992). The bacteria that the authors found on samples belonged most frequently to the Citrobacter-Enterobacter-Serratia group of Enterobacteriaceae. Although usually regarded as human pathogens, these members of Enterobacteriaceae family have also been recognized as inhabitants of soil and plants (Wright, Kominos, & Yee, 1976). Thus, vegetables may serve as a reservoir from which the bacteria named above can colonize and infect a susceptible host.

In many countries, urban wastewater is used to irrigate agricultural land. This way of disposing of urban sewage water has several advantages. Wastewater contains a lot of nutrients, which increase crop yields without use of fertilizer. Furthermore, sewage water is an alternative water source in arid and semi-arid areas where water is scarce. Some disadvantages are that wastewater can contain heavy metals, organic compounds, and a wide spectrum of enteric pathogens that may have a negative impact on the environment and human health.

The study reported here demonstrates that a potential for disease transmission exists when wastewater is used for irrigation. Pathogens that have been transported by wastewater can survive in soil or on crops. The actual risk of disease transmission, however, is related to whether this survival time is long enough to allow transmission to a susceptible host. The crop and the field are the link between the pathogen in the wastewater and the potential for infection. The factors controlling transmission of disease are agronomic; examples of such factors are the crop grown, the irrigation method used to apply wastewater, and cultural and harvesting practices. Consumption of salad irrigated by wastewater has been found to be responsible for shigellosis in England (Frost, McEvoy, Bentley, Andersson, & Rowe, 1995). Numerous opportunities exist for attachment and penetration of pathogenic bacteria into lettuce in the field, as well as during harvesting, processing, and marketing, especially when a contaminated product is exposed to water or is damaged (Takeuchi et al., 2001). Guo and co-authors have demonstrated that soil and water are potential reservoirs from which Salmonella can contaminate tomatoes (Guo, Chen, Brackett, & Beuchat, 2002). The pathogen can survive in most soils in high numbers for at least 45 days and can infiltrate the tissues of tomatoes during contact with inoculated soil. This mechanism may explain the discovery of Salmonella arizonae on tomatoes in the investigation reported here. The hydroponics system used in another study provided a controlled environment in which to study the possible association of Salmonellae with aerial tissues, with minimal concerns about environmental contamination or temperature fluctuation (Guo, Iersel, Chen, Brackett, & Beuchat, 2002). That study provided evidence that Salmonellae can be transported from an inoculated nutrient solution to the hypocotyls, cotyledons, stems, and leaves of young tomato plants. In addition, the work of Guo and co-authors (2001) revealed the ability of Salmonella to survive on or in tomato fruits throughout the course of plant growth, flowering, fruit development, and fruit maturation (2001). Another study showed that Escherichia coli O157:H7 may be present not only on outer surfaces, but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the bacterium (Itoh et al., 1998). Indeed, this mechanism can be inferred from the fact that enteric pathogens were isolated from inside vegetables during our investigation.

Numerous foodborne diseases caused by Salmonella enterica and Escherichia coli O157:H7 have been associated with contaminated alfalfa, clover, and bean sprouts (Puohiniemi, Heiskanen, & Siitonen, 1997). Constant moisture, nutrients released by the sprouting seeds, and warm temperatures are conducive to the growth of human bacterial pathogens such as S. enterica and E. coli O157:H7 (Charkowski, Barak, Sarreal, & Mandrell, 2002). The fact that some bacteria and not others are present can be explained by a difference in their capacity to attach to plants. For example, Salmonella enterica attaches as well as plant-associated bacteria and significantly better than E. coli to alfalfa sprouts (Barak, Whitehand, & Charkowski, 2002).

Conclusion

The authors' study demonstrated that the vegetables analyzed may be an important source of foodborne bacterial-illness outbreaks in humans, especially when the vegetables are consumed raw. In addition, vegetables of these types can also be a vehicle for Giardia cysts and Ascaris eggs (Amahmid, Asmama, & Bouhoum, 1999). To protect public health, the use of raw sewage in the irrigation of the vegetable culture must be prohibited.

So it is necessary to consider the generalized program of wastewater treatment in Morocco. Moreover, the requirements for treated wastewater must respect the sanitary standards for agricultural reuse. There is an urgent need for development and validation of standard methods of eliminating the pathogenic microorganisms from raw vegetables.

Acknowledgements: This work was financially supported by the PARS and PROTARS programs of the Moroccan National Research Council. The authors acknowledge Hassan Tajnari (Service for Protection of Plants, Ministry for Agriculture, Marrakech-Morocco) and Meryem Aakill (Division of Plant Health Control, Ministry for Agriculture, Rabat-Morocco) for their assistance during sampling and for their technical help.

Corresponding Author: M.M. Ennaji, Professor, Laboratory of Virology and Hygiene & Microbiology, Department of Biology, Faculty of Science and Technology, University Hassan II-Mohammedia, Mohammedia, Morocco 20650. E-mail: [e-mail].

REFERENCES

Amahmid, O., Asmama, S., & Bouhoum, K. (1999). The effect of waste water reuse in irrigation on the contamination level of food crops by Giardia cysts and Ascaris eggs. International Journal of Food Microbiology, 49(1-2), 19-26.

Barak, J.D., Whitehand, L.C., & Charkowski, A.O. (2002). Differences in attachment of Salmonella enterica serovars and Escherichia coli O157:H7 to alfalfa sprouts. Applied and Environmental Microbiology, 68(10), 4758-4763.

Beuchat, L.R. (1996). Pathogenic microorganisms associated with fresh produce. Journal of Food Protection, 59(2), 204-216.

Breuer, T., Benkel, D.H., Shapiro, R.L., Hall, W.N., Winnett, M.M., Linn, M.J., Timothy, J.N., Barrett, J., Dietrich, S., Downes, F.P., Toney, D.M., Pearson, J.L., Rolka, H., Slutsker. L., & Griffin, P.M. (2001). A multi-state outbreak of Escherichia coli O157:H7 infections linked to alfalfa sprouts grown from contaminated seeds. Emerging Infectious Diseases, 7(6), 977-982.

Castro-Rosas, J., & Escartin, E.F. (2000). Survival and growth of Vibrio cholerae O1, Salmonella typhi, and Escherichia coli O157:H7 in alfalfa sprouts. Journal of Food Science, 65(1), 162-165.

Charkowski, A.O., Barak, J.D., Sarreal, C.Z., & Mandrell, R.E. (2002). Growth and colonization patterns of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts and the effects of sprouting temperature, inoculum dose, and frequency of irrigation on bacterial levels. Applied and Environmental Microbiology 68(6), 3114-3120.

Evans, M.R., Ribeiro, C.D., & Salmon, R.L. (2003). Hazards of healthy living: Bottled water and salad vegetables as risk factors for Campylobacter infection. Emerging Infectious Disease, 9(10), 1219-1225.

Frost, J.A., McEvoy, M.B., Bentley, C.A., Andersson, Y., & Rowe, B. (1995). An outbreak of Shigella sonnei infection associated with consumption of iceberg. Emerging Infectious Disease, 1(1), 26-28.

Guo, X., Chen, J., Brackett, R.E., & Beuchat, L.R. (2001). Survival of Salmonellae on and in tomato plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening. Applied and Environmental Microbiology, 67(10), 4760-4764.

Guo, X., Chen, J., Brackett, R.E., & Beuchat, L.R. (2002). Survival of Salmonellae on tomatoes stored at high relative humidity, in soil, and on tomatoes in contact with soil. Journal of Food Protection, 65(2), 274-279.

Guo, X., Iersel, M.W.V., Chen, J., Brackett, R.E., & Beuchat, L.R. (2002). Evidence of association of salmonellae with tomato plants grown hydroponically in inoculated nutrient solution. Applied and Environmental Microbiology, 68(7), 3639-3643.

Itoh, Y., Sugita-Konishi, Y., Kasuga, E, Iwaki, M., Hara-Kudo, Y., Saito, N., Noguchi, Y, Konuma, H., & Kumagai, S. (1998) Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts. Applied and Environmental Microbiology, 64(4), 1532-1535.

Madden, J.M. (1992). Microbial pathogens in fresh produce--The regulatory perspective. Journal of Food Protection, 55, 821-823.

McMahon, M.A.S., & Wilson, I.G. (2001). The occurrence of enteric pathogens and Aeromonas species in organic vegetables. International Journal of Food Microbiology, 70(1-2),155-162.

Puohiniemi, R., Heiskanen, T., & Siitonen, A. (1997). Molecular epidemiology of two international sprout-borne Salmonella outbreaks. Journal of Clinical Microbiology. 35(10), 2487-2491.

Shearer, A.E., Strapp, C.M., & Joerger, R.D. (2001). Evaluation of polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruit and vegetables. Journal of Food Protection, 64(6), 788-795.

Takeuchi, K., Hassan, A.N., & Frank, J.F. (2001). Penetration of Escherichia coli O157:H7 into lettuce as influenced by modified atmosphere and temperature. Journal of Food Protection, 64(11), 1820-1823.

Wright, C., Kominos, S.D., & Yee, R.B. (1976). Enterobacteriaceae and Pseudomonas aeruginosa recovered from vegetable salads. Applied and Environmental Microbiology, 31(3), 453-454.

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TABLE 1 Microbiological Profiles of Raw Vegetables Analyzed

                                    Number of
                      Range         Samples          Vegetable
Assay                 (CFUs/g)      in Range   Tomato  Radish  Cucumber

Aerobic plate counts
                      [10.sup.1-4]   3          -      -       -
                      [10.sup.5-8]  32          6      2       -
                      >[10.sup.8]   15          4      2       3
Total coliform
                      <10            2          -      -       -
                      [10.sup.1-4]  10          4      2       -
                      [10.sup.5-8]  38          6      2       3
Fecal coliform
                      <10            2          -      -       -
                      [10.sup.1-4]  20          5      4       -
                      [10.sup.5-8]  28          5      -       3
Enterococci
                      <10           16          4      2       -
                      [10.sup.1-4]  15          2      2       -
                      [10.sup.5-8]  19          4      -       3
                      Total                    10      4       3

                      Range                Vegetable          Gardenpa
Assay                 (CFUs/g)      Eggplant  Potato  Pepper  Pea

Aerobic plate counts
                      [10.sup.1-4]  2         -       1       -
                      [10.sup.5-8]  3         1       1       1
                      >[10.sup.8]   -         1       2       -
Total coliform
                      <10           1         -       1       -
                      [10.sup.1-4]  2         -       -       -
                      [10.sup.5-8]  2         2       3       1
Fecal coliform
                      <10           1         -       1       -
                      [10.sup.1-4]  3         1       -       -
                      [10.sup.5-8]  1         1       3       1
Enterococci
                      <10           3         -       1       -
                      [10.sup.1-4]  1         1       1       1
                      [10.sup.5-8]  1         1       2       -
                      Total         5         2       4       1

                      Range                 Vegetable           Broad
Assay                 (CFUs/g)      Gourd  Zucchini  Artichoke  Bean

Aerobic plate counts
                      [10.sup.1-4]  -      -         -          -
                      [10.sup.5-8]  2      4         2          2
                      >[10.sup.8]   1      1         -          -
Total coliform
                      <10           -      -         -          -
                      [10.sup.1-4]  -      1         1          -
                      [10.sup.5-8]  3      4         1          2
Fecal coliform
                      <10           -      -         -          -
                      [10.sup.1-4]  1      2         1          2
                      [10.sup.5-8]  2      3         1          -
Enterococci
                      <10           -      1         1          1
                      [10.sup.1-4]  2      3         -          1
                      [10.sup.5-8]  1      1         1          -
                      Total         3      5         2          2

                      Range                      Vegetable
Assay                 (CFUs/g)      Turnip  Onion  French Bean  Lettuce

Aerobic plate counts
                      [10.sup.1-4]  -       -      -            -
                      [10.sup.5-8]  -       3      3            2
                      >[10.sup.8]   1       -      -            -
Total coliform
                      <10           -       -      -            -
                      [10.sup.1-4]  -       -      -            -
                      [10.sup.5-8]  1       3      3            2
Fecal coliform
                      <10           -       -      -            -
                      [10.sup.1-4]          1      -            -
                      [10.sup.5-8]  1       2      3            2
Enterococci
                      <10           -       2      1            -
                      [10.sup.1-4]  -       1      -            -
                      [10.sup.5-8]  1       -      2            2
                      Total         1       3      3            2

TABLE 2 Enterobacteriaceae Recovered from Raw-Vegetable Analysis

Organism                       Frequency*  Percentage (%)**

Citrobacter freundii           28          18.7
Citrobacter yaungae             6           4.0
Enterobacter aerogenes          1           0.7
Enterobacter amnigenus          3           2.0
Enterobacter cloacae           27          18.0
Enterobacter cancerogens        1           0.7
Enterobacter intermedius        2           1.3
Enterobacter sakazakii         12           8.0
Escherichia coli 1             16          10.7
Escherichia coli 2              1           0.7
Klebsiella pneumoniae ozaenae   1           0.7
Klebsiella oxytoca              2           1.3
Klebsiella pneumoniae          17          11.3
Proteus mirabilis               8           5.3
Providencia rettgeri            3           2.0
Salmonella arizonae             1           0.7
Serratia ficolia                1           0.7
Serratia liquefaciens          11           7.3
Serratia odorifera              1           0.7
Serratia marcescens             1           0.7
Serratia phynuthica             2           1.3
Serratia rubidaea               5           3.3

* Frequency of strains in the 50 analyzed samples.
** Percentage of strains isolated.