The desire for a healthy lifestyle and faster mode of preparation has supported the consumption of ready to eat fresh salad. Street vended salads are recognized as a source of pathogenic transamination in different parts of the world. The present study was designed to evaluate the safety status of fresh vegetable and Russian salads being sold at various food outlets of Faisalabad. Samples of freshly prepared salads were collected from representative selected different areas of Faisalabad city divided into four different zones (zone 1, zone 2, zone 3 and zone 4). Prevalence and enumeration of Listeria was done through microbial testing via the spread plate method. Among samples of vegetable salad, the highest prevalence of Listeria was found in the zone 2 (75%) whereas Russian salad samples from zones 1 and 3 exhibited 62% prevalence, the highest among all 4 zones of study. On the whole, the lowest prevalence of Listeria was found in zone 4 (50% vegetable salad and 58% Russian salad). Biochemical conformation of Listeria done through different tests for the identification of various Listeria species, exhibited that Listeria monocytogenes and Listeria innocua were highly prevalent in samples from zones 1 and 3 respectively. The results will help to improve safety concerns associated with street vended foods.
Listeria, ready-to-eat, vended salads
Pathogenic microorganisms significantly exist in street vended foods and cause foodborne illnesses in all parts of world. Food borne illnesses not only effects consumer health also results in negative economic impacts, loss of customers, product recall and potential compensation costs1. Contaminated foodstuff is the main cause oftwenty to thirty per cent spreading of Listeria infection that causes ninety-five percent cases of food poisoning2. Every year, almost one third population is distressed with foodborne infections in industrialized countries whereas the problem is worse in developing countries due to poverty, overcrowding, inadequate sanitary conditions and poor general hygiene3.
Listeria is a Gram-positive, bacillus, non-spore forming, ubiquitous, motile, facultative anaerobe and one of the most environmental pathogenic microflora of food4. Listeria can easily deteriorate the raw ready-to-eat foods because of its high salt tolerance and ability to thrive at low preservation temperatures. Listeria increases the rate of death up to 25-30% just because of hygienically poor environment and inadequate management of food processing5. Due to consumption of contaminated and unhygienic foods nearly 76 million infections, 325,000 hospitalizations and 5000 deaths are caused annually in United States of America24.
Rapid change in the socioeconomic status and changing life style have greatly increased the consumption of street vended foods6. Control of safety of ready to eat street vended foods is difficult and much important because they have significant socioeconomic role in meeting food and nutritional requirements of consumers at affordable prices particularly to the lower and middle income people. As these food products are prepared by small street vendors, cheaper rates of these foods attract the low earners. Ready to eat street vended foods constitutes the primary source of meals for many low and middle income people7.Salad, a mixture of vegetables and fruits, is an excellent source of dietary fiber, phytochemicals, antioxidants, minerals, vitamins and phenolic that help to cure many diseases and reduce the risk of developing type 2 diabetes10.
Salads are economical, convenient in preparation and have delicious taste but the hygiene and safety levels are big challenges for the food administrators. Freshly consumed produce got contaminated on farm or point of sale in market. Reported sources of contamination include soil, handling during pre and post-harvest handling, water and dust11. Manures used to promote the growth and cultivation of these produce harbor the large number of pathogenic microorganisms such as Salmonella, Mycobacterium, Listeria monocytogenes, Klebsiella and Clostridium perfringens9.Globally, about 325,000 people are hospitalized along with death of 5,000 children each year due to food borne illnesses14. Therefore, despite the freshness and nutritional benefits of fruits and vegetables, there have been an increase in incidence of food borne illnesses thus bacteriologically safe fruit and vegetable products are required to maximize the health benefits promised by their consumption8. Ingredients of salads are contaminated with soil, dust, irrigation water which are proved to be associated with pathogenicity of Listeria. Findings have reported the highest prevalence of Listeria monocytogenes in ready to eat salad samples followed by chicken products, beverages, egg based products, beef and seafood products13. In salads, Listeria has been reported to be isolated from fresh produce such as parsley, cucumber, cabbage, sprouts and water cress and resulted in morbidity and mortality in various parts of world12. The average shelf life of ready to eats ranges from 5-7 days and after opening of package product can be stored for two days at refrigeration conditions. Highly perishable, favorable pH (6.0-7.0), poor abidance to decontamination procedures and temperature abuse can increase the risk associated with these products27. In Pakistan street vendors to large restaurants are responsible for sale of ready to eat salads. Most common salads on retail sale include vegetable salad and Russian salad. Common vegetables used in vegetable salads include onion, cucumber, tomatoes, lettuce green pepper, radish, salt and spices commonly stored at ambient temperature during retail conditions.. While ingredients in Russian salads include fresh or boiled vegetables, mayonnaise, cream and spices stored at refrigeration conditions. This study provides the prevalence data of Listeria monocytogenes for Vegetable salad and Russian salad. The objectives of this study were to assess microbiological quality of freshly prepared street vended salads and to get information regarding the major sources of contamination.
Sample of Vegetable and Russian salads were collected from four different zones of the Faisalabad city (Table 1). A total of 192 samples were picked during five weeks. Samples were kept in strict hygienic conditions in sterile zipper polythene bags, stored in an icebox under controlled temperature and brought to the Food Microbiology and Microbiology Laboratory, National Institute of Food Science and Technology, University of Agriculture, Faisalabad within an hour of its collection. Listeria selective agar was used for microbial analysis. Samples were homogenized in a peristaltic blender (Seward Europe) at 200 rpm for 2 minutes. For this purpose, 25g salad along with 25 mL of peptone water was taken in specialized stomacher bags15. Six serial dilutions were made by transferring 1 mL liquid sample from stomacher bag to the test tubes serial wise and transferred to the prepared Petri plates containing solidified Listeria Selective Agar Base (Oxoid). Spreading of the poured volume (0.1mL) was done using a sterile spreader and plates were allowed to dry for 15 minutes (Fig. 1)16. After drying, incubation was done for 24 hours at 37°C. After incubation, dark brown or blackish colored colonies grew over Listeria selective agar as depicted in Fig. 2. Colonies of Listeria were counted using the colony counter (galaxy 230-USA) and documented. Colony forming units per gram (CFU/g) were calculated using the following formula.
CFU/g = (Average No.of Colonies x Dilution Number) / (Dilution Factor x Volume plated)
DF = (Volume of sample) / (Sample Volume + Diluent Volume)
Sampling plan for vegetable and Russian salads collection.
|Zone 1 locations for collection of samples||Zone 2 locations for collection of samples|
|Sr. no.||Vegetable salad||Russian Salad||Sr. no.||Vegetable salad||Russian Salad|
|1||Yong Wala||Ghulam Muhammadabad road||1||Canal road||Sargodha road|
|2||Kotwali road||Tata bazar||2||Abdullahpur overpass||Millat road|
|3||Narwala road||Sadar bazar||3||Tahir road||Fsd.-Sanghla hill road|
|4||Jail road||Tariq road||4||Khaleeq road||Kashmir road|
|5||Ganda nala road||Gulberg road||5||Noorpur road||Iqbal stadium|
|6||Bakar mandi road||Makki road||6||Chibban road||Lhr.-Fsd. Shk. road|
|7||Chiniot bazar||Kachari bazar||7||Shadman road||College road|
|8||Oil depot road||Imam bargha road||8||Ittehad road||Wapda colony road|
|Zone 3 locations for collection of samples||Zone 4 locations for collection of samples|
|Sr no.||Vegetable salad||Russian Salad||Sr no.||Vegetable salad||Russian Salad|
|1||Rasool park||Susan road||1||Liaqat abad||Samundari road|
|2||Tufail shaheed road||Tezab mill road||2||Mulyanwala road||D-type colony road|
|3||Younas road||Jaranwala road||3||Naimatabad road||Dijkot road|
|4||Satyana road||D-ground||4||Sanni road||Samanabad road|
|5||Maqbool road||Lower canal road||5||Main road||Railway road|
|6||Usman road||Khawaja islam road||6||Saifabad road||Jhang road|
|7||Allama Iqbal road||Illahi abad road||7||Talianwala road||AARI colony road|
|8||Risalewala road||Warispura road||8||Bilal road||Fazal elahi road|
Samples were collected in triplicate from each location.
Two species of Listeria (Listeria monocytogenes and Listeria innocua) were identified and biochemically confirmed by through Gram staining, catalase, citrate, hydrogen sulfide, indole, methyl red and oxidase tests as described by the Laboratory Manual of Food Microbiology16. Statistical analysis was performed for to determine the significance of test results17 by using the completely randomized design with the help of Statistix 8.1 software.
Prevalence of Listeria in street vended vegetable salad samples gathered from zone 1, zone 2, zone 3 and zone 4 are shown in Table 2. Total of 24 samples of vegetable salad were collected separately from all four zones and the occurrence of Listeria was documented in percentage. Among 24 samples of vegetable salad 15 samples of vegetable salad illustrated positive results for the pathogen and the total prevalence of Listeria was recorded 62%. The highest count of 1.59×108 CFU/g was recorded from Ganda Nala Road and the lowest count of 4.47×106 CFU/g was recorded from oil depot road (Table 3). Highest prevalence was found in the area of Yong wala and Narwala road whereas Listeria was not detected from the samples of Chiniot bazar. The following results are in accordance with the findings of Christson et al18 who reported the presence of Listeria monocytogenes in street vended salad which may cause health hazards.
Prevalence of Listeria in salad samples from different zones.
Total prevalence in vegetable salad
Total prevalence in Russian salad
In zone 2, prevalence of the pathogen was recorded as 75% in vegetable salad. Total 18 samples were positive for Listeria as shown in Table 3. Tahir road, Noorpur road and Shadman road have shown highest prevalence while no pathogen was detected in samples taken from Canal road with the non-significant difference (P> 0.05). Enumeration results indicated highest count of 1.40×108 in samples from Shadman road while the lowest count of 3.86×106 CFU/g was recorded in samples collected from Noorpur road. The following results are in agreement with the study reported by El-Shenawy et al19 according to which Listeria is present everywhere in our environment and its various species like Listeria monocytogenes is responsible for many outbursts.
Prevalence and enumeration of Listeria in vegetable salad.
|Colony-forming unit (CFU/g)|
|Ganda nala road||33||66||1.59×108|
|Bakar mandi road||33||66||9.4×105|
|Oil depot road||66||33||4.47×106|
|Abdullah pur overpass||33||66||5.61×106|
|Tufail shaheed road||66||33||2.80×107|
|Allama iqbal road||33||66||3.56×106|
In zone 3, overall 55 % prevalence was observed in vegetable salads. None of the sample was found acceptable for the consumption. Highest count of 1.06×108 CFU/g was found in the area of Usman road whereas lowest count was shown in the area of Satyana road (4.86×105 CFU/g) with no detection in the area of Risalewala road as presented in the Table 3. The following results are in accordance with the study of Lianou, & Sofos20 who stated that inefficient methods of handling of street vended salads can support the growth of pathogenic micro flora in street vended foods. Most of the vendors openly store leftover food in an open place for the next day use21. Some of them also use stagnant water for washing which in result can cause serious health hazards.
In zone 4, vegetable salads have shown the total prevalence of 50%. Highest count was found in the area of Saifabad road that is 2.99×107 CFU/g whereas lowest count was observed in the samples taken from Naimatabad road (3.73×105 CFU/g). No pathogen was detected in the Main road samples. The results are in agreement with the work of Gelfand and colleague22 who describes the sources for the high prevalence of Listeria which may include the dusty unygenic premises and poor sanitary conditions.
Result of the study (Table 2) presented the significant difference in samples of Russian salad in zone 1 and zone 3 with 62% unsatisfactory results. None of the samples was found to be in the acceptable limits. In zone 1, highest count of 1.30×108 CFU/g from Makki roadand the least count of 3.86×105 CFU/g were observed in the area of Ghulam Muhammadabad road whereas no Listeria was detected in the samples taken from Tata bazar with the overall count ranging from 105-108 CFU/g. These results are closely related to the previous work done23 which reports that fresh and mixed salads sold by road merchants harbor numerous pathogenic micro flora.
In zone 2, the percentage of total prevalence in Russian salad samples was 45% as documented in the Table 2. Enumeration of Listeria in Russian salad samples collected from zone 2 shown the significant results and the highest count was found in the area of College road with 2.99×108 CFU/g while the lowest count of Listeria was recorded in samples taken from Faisalabad-Sanghla hill road (Fsd.-Sanghla hill road) as 7.66×105 CFU/g. No Listeria was detected in the samples gathered from the areas of Sargodha road and Iqbal Stadium as described in Table 4.These results are in accordance with the results of research in which shows that by adopting good hygiene practices like use of hygienic tops, aprons, utensils and good sanitary conditions can reduce the risk of Listeria by half.
Prevalence and enumeration of Listeria in Russian salad.
|Colony forming unit (CFU/g)|
|1||G.M Abad road||66||33||3.86×105|
|Imam bargha road||66||33||4.13×106|
|Fsd.-Sanghla hill road||33||66||7.66×105|
|Lhr Fsd Shk road||66||33||5.13×107|
|Wapda colony road||66||33||4.33×106|
|Tezab mill road||–||100||2.63×106|
|Lower canal road||33||66||4.44×107|
|Khawaja islam road||33||66||2×106|
|Illahi abad road||33||66||5.75×107|
|D-type colony road||100||–||ND*|
|AARI colony road||66||33||3.33×105|
|Fazal elahi road||33||66||1.06×108|
Similar to zone 1, percentage of total prevalence of Listeria in Russian salad samples was 62 % with the highest count in the area of D-groundas 2.33×108 CFU/g while the least count was recorded in the samples taken from Khawaja Islam road (2×106 CFU/g) and no Listeria was detected in the area of Susan road (Table 4).These results are in accordance with the work reported by Razzaq and colleagues21 that declared that most of the vendors openly store their leftover food in an open place for the next day use which is the significant reason of prevalence of Listeria. Results for the Russian salad samples collected from zone 4 depicted 58% total prevalence of Listeria with the highest count in the area of Fazal Elahi roadwith 1.06×108 CFU/g count while the least count was recorded in the samples taken from AARI colony road (3.3×105 CFU/g). No Listeria was detected in the area of D-type colony road (Table 4). The results are in agreement with the work of Gelfand and coworkers23 that describes the sources for the high prevalence of Listeria. Similarly in another study conducted in Malaysia commercially available minimally processed vegetable salads were analyzed microbiologically and Listeria monocytogens and Listeria spp. were detected in 22.5% and 33.3% samples respectively26 .
Biochemical conformation for the identification of various Listeria species (resulted shown in Table 5) indicated that the percentage of Listeria monocytogenes in vegetable salad and Russian salad was 62% and 45% respectively in the samples collected from zone 1, while the occurrence of Listeria innocua in the samples of vegetable salad and Russian salad was 16% and 8%. In the zone 2, the prevalence of Listeria monocytogenes in vegetable and Russian salad samples was 58% and 41% while that of Listeria innocua was 16% and 8% in vegetable and Russian salad samples respectively. In zone 3, the total percentage of Listeria monocytogenes in vegetable and Russian salad was 37% and 45%, whereas the percentage of Listeria innocua was 16% and 12% respectively. Similarly, in the zone 4, the occurrence of Listeria monocytogenes 41% and 45% while in case of Listeria innocua the prevalence was recorded as 16% and 8% in vegetable and Russian salad samples respectively. A study conducted in Brazil on 162 minimally processed vegetable salad samples sold locally also showed similar results containing Listeria monocytogenes, Salmonella and Escherichia coli at levels above (World Health Organization) WHO recommended limits25. In present study high Listeria count indicates increased consumer’s exposure to microorganisms as it is assumed that the pathogen level were not reduced from post salad production to the utilization point because salads were served as ready to eat form and were stored at room temperature for sale. Possibility is that rework salad from last day was also mixed in new lot to utilize it risking the overall quality of product. Therefore holding temperature and Thermal processing have strong impact on microbiological safety. The quality and safety of vegetables used for ready to eat salad preparation also depend upon use of clean irrigation water and appropriate washing to remove residues of soil and organic fertilizers. Thus for the prevention of increased bacterial load application of good hygiene practices, effective control on storage condition, decontamination and selection of appropriate packaging material is necessary.
Biochemical testing of Listeria spp.
|Zones||Types of Salad||Listeria monocytogenes||Listeria innocua|
|Zone 1||Vegetable Salad||+(15/24)||+ (3/24)|
|Zone 2||Vegetable Salad||+(14/24)||+(4/24)|
|Zone 3||Vegetable Salad||+(9/24)||+(5/24)|
|Zone 4||Vegetable Salad||+(10/24)||+(4/24)|
The study revealed that the microbiological quality of salads sold at various markets of Faisalabad were unwholesome for human consumption. On overall basis, Listeria monocytogenes was the most prevalent specie of Listeria that was found in most of the samples of salads collected from all zones. On the other hand, the prevalence of Listeria innocua was very low as compared to Listeria monocytogenes.
The contamination could be attributed to the productuion source of vegetables, use of contaminated water for washing, improper handling, use of dirty processing utensils, knives and trays, poor personal hygiene and improper storage conditions generally along environmental conditions and cross contamination from rotten vegetables in value chain Simple washing of vegetables before use can possibly reduce the risk of microbiological hazards. The need is to provide basic food hygiene training to food handlers and to create consumer awareness to consume properly processed food products. Further it is recommended that research should be carried out on salads to isolate bacterial pathogenic strains identified in present study and antimicobial susceptibility of identified bacteria.
Authors gratefully acknowledge the Food Microbiology and Dairy Technology Laboratory, National Institute of Food Science and Technology, University of Agriculture, Faisalabad and Department of Food Science and Technology, Khwaja Fareed University of Engineering and Information Technology, Rahim Yar Khan, Pakistan for the provision of technical and scientific expertise during this study.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
This research does not contain any studies with human participants or animals performed by any of the authors.
AVAILABILITY OF DATA
All data set generated or analyzed during the study are included in the manuscript.
- Carrasco E, Perez-Rodriguez F, Valero A, Garcia-Gimeno RM, Zurera G. Risk assessment and management of Listeria monocytogenes in ready-to-eat lettuce salads. Compr Rev Food Sci Food Saf. 2010;9(5):498-512.
- Thevenot D, Dernburg A, Vernozy-Rozand C. An updated review of Listeria monocytogenes in the pork meat industry and its products. J Appl Microbiol. 2006;101(1):7-17.
- Hu X, Swiecicka I, Timmery S, Mahillon J. Sympatric soil communities of Bacillus cereus sensu lato: population structure and potential plasmid dynamics of pXO1-and pXO2-like elements. FEMS Microbiol Ecol. 2009;70(3):344-355.
- Abebe E, Gugsa G, Ahmed M. Review on Major Food-Borne Zoonotic Bacterial Pathogens. J Trop Med. 2020;29;2020:4674235.
- Garode A, Waghode S. Bacteriological status of street-vended foods and public health significance: A Case Study of Buldana District, MS, India. ISCA J Biol Sci. 2012;1:69-71.
- Aptrakhimov D, Rebezov M, Slavyanskiy A, et al. Composite flour pasta processing technology and its nutritive value. Journal of Natural Remedies. 2021;21(9):67-71.
- FAO. Good hygienic practices in the preparation and sale of street food in Africa. FAO Corporate Document Repository. 2009.
- Eni AO, Oluwawemitan IA, Solomon OU. Microbial quality of fruits and vegetables sold in Sango Ota, Nigeria. African J Food Sci. 2010;4:291-296.
- Rahman F, Noor R. Prevalence of pathogenic bacteria in common salad vegetables of Dhaka Metropolis. Bangladesh J Botany. 2013;41(2):159-162.
- Campos J, Gil J, Mourao J, Peixe L, Antunes P. Ready-to-eat street-vended food as a potential vehicle of bacterial pathogens and antimicrobial resistance: an exploratory study in Porto region, Portugal. Int J Food Microbiol. 2015;206:1-6.
- Jeddi MZ, Yunesian M, Gorji MEH, Noori N, Pourmand MR, Khaniki GRJ. Microbial evaluation of fresh, minimally-processed vegetables and bagged sprouts from chain supermarkets. J Health Popul Nutr. 2014;32(3):391-399.
- Tonder IV, Lues JFR, Theron MM. The personal and general hygiene practices of food handlers in the delicatessen sections of retail outlets in South Africa. J Environ Health. 2007;70(4):33-38.
- Jamali H, Chai LC, Thong KL. Detection and isolation of Listeria spp. and Listeria monocytogenes in ready-to-eat foods with various selective culture media. Food Control. 2013;32(1):19-24.
- Yousef AE, Carlstrom C. Food microbiology: a laboratory manual, John Wiley & Sons. 2003.
- Uzeh RE, Alade FA, Bankole M. The microbial quality of pre-packed mixed vegetable salad in some retail outlets in Lagos, Nigeria. African Journal of Food Science. 2009;3(9):270-272.
- Kiiyukia C. Laboratory manual of food microbiology for Ethiopian health and nutrition research institute. UNIDO project. 2003. (YA/ETH/03/436/11-52).1-197.
- Montgomery DC. Design and Analysis of Experiments. 7th Ed. John Wiley and Sons. Inc. Hoboken, NJ, USA. 2008;1-656.
- Christison CA, Lindsay D, Von Holy A. Microbiological survey of ready-to-eat foods and associated preparation surfaces in retail delicatessens, Johannesburg, South Africa. Food Control. 2008;19(7):727-733.
- El-Shenawy M, El-Shenawy M, Manes J, Soriano JM. Listeria spp. in street-vended ready-to-eat foods. Interdiscip Perspect Infect Dis. 2011;2011:968031.
- Lianou A, Sofos JN. A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. J Food Prot. 2007;70:2172-2198.
- Razzaq R, Farzana K, Mahmood S, Murtaza G. Microbiological analysis of street vended vegetables in Multan City, Pakistan: A public health concern. Pakistan J Zool. 2014;46:1133-1138.
- Gelfand MS. Treatment, prognosis, and prevention of Listeria monocytogenes infection. UpToDate. 2013;25:1-9.
- Edelson BT, Unanue ER. Immunity to Listeria infection. Curr Opinion Immunol. 2000;12(4):425-431.
- Mustafa M, Yoshida T, Waheed Z. Seafood poisoning, symptom, treatment, and prevention. BBJoMSA. 2018;2:64-69.
- De Oliveira MA, De Souza VM, Bergamini AMM, De Martinis ECP. Microbiological quality of ready-to-eat minimally processed vegetables consumed in Brazil. Food Control. 2011;22(8):1400-1403.
- Ponniah J, Robin T, Paie MS, et al. Listeria monocytogenes in raw salad vegetables sold at retail level in Malaysia. Food Control. 2010;21(5):774-778.
- Arienzo A, Murgia L, Fraudentali I, Gallo V, Angelini R, Antonini G. Microbiological quality of ready-to-eat leafy green salads during shelf-life and home-refrigeration. Foods. 2020;9(10):1421.
Share This Article
© The Author(s) 2021. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.