# Microbiological Quality of Salads Served along with Street Foods of Hyderabad, India

Authors: Alekhya Sabbithi; R. Naveen Kumar; L. Kashinath; V. Bhaskar; V. Sudershan Rao
Journal: International Journal of Microbiology
 (2014)

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## Abstract

A study has been done to analyse the microbiological quality of salads served along with street foods of Hyderabad. A total of 163 salad samples, 53 of carrot and 110 of onion samples, were collected from four different zones of Hyderabad. About 74% and 56% hadStaphylococcus aureus in carrots and onions, respectively. Fifty-eight percent of carrots and forty-five percent of onions samples contained Salmonella, 68% of carrots and 24% of onions had Yersinia. HACCP study was carried out with 6 street food vendors to identify the source of Salmonella contamination in salads. Food handlers were found to be responsible for Salmonella contamination in salads. The present study revealed the potential hazards of street vended salad vegetables, considering the handling practice usually carried out by vendors. Ninety-eight percent of the vendors did not wash the vegetables before processing and serving while about 56.6% of the vendors did not peel the vegetables. Majority of street vendors’ nails were uncut. A significant difference (P<0.01) was observed in Yersinia spp. and Salmonella spp. in wet-dirty chopping board when compared to clean-dry chopping board. A significant difference (P<0.05) of Staphylococcus spp. was observed when the status of cleaning cloth was neat/untidy.


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## Body

## 1. Introduction

Street vended food (SVF) includes food and beverages that are prepared and sold outdoors or in public spaces by street merchants for consumption at the location or later without further preparation [1]. Quality and safety are two common concerns with regard to street foods. Poor hygiene and sanitation practices are one of the major bottlenecks in street food vending [2].Salad can be defined as a food made primarily of mixture of raw vegetables and/or fruits [3]. The possibility of foodborne diseases is more when salad vegetables are consumed without any heat treatment, sometimes without washing and peeling [4]. Water used for rinsing the vegetables and sprinkling to keep them fresh is also a source of contamination [5]. Fresh vegetables and fruits become contaminated with microorganisms during production, harvest, packing, and distribution [6].Ready-to-eat fruit and vegetables requiring minimal or no further processing prior to consumption have been implicated as vehicles for transmission of infectious microorganisms of foodborne outbreaks related to gastrointestinal illness associated with fruit and vegetables [7]. In this context the present study was undertaken to analyse the microbiological quality of salads served along with street foods of Hyderabad city.


## 2. Materials and Methods

### 2.1. Study Area

The Study was carried out in Hyderabad which is the capital of Andhra Pradesh, India. The twin cities of Hyderabad and Secunderabad come under ambit of a single municipal unit, the Greater Hyderabad Municipal Corporation. For administrative purpose Greater Hyderabad Municipal Corporation was divided into many zones. Random sampling procedure was adopted to select four zones (Alwal: east zone, Yousufguda: west zone, Old City: south zone, and Secunderabad: north zone). The sample required for the study was obtained using proportionate representation according to size.


### 2.2. Questionnaire

Personal interview of the street vendor was taken and flow chart was prepared from which critical control points of contamination were examined.Data on salad preparation, handling, and storage practices was collected using structured questionnaire that had both observational and responsive questions. Equipment used for the preparation of salad, source of water for both utensil cleaning and vegetables, utensil cleaning methods, and hand washing were considered. Salad preparation, handling practices, storage of leftover salad, storage length, and salad storage practices were observed and noted. Information on status of the premises, storage conditions for salad before cooking, cutting and chopping place, status of the serving plate, peeling and cleaning of vegetables, cleanliness of the cloth and clothing used, provision for waste disposal, presence of rodent droppings in the outlet, and exposure to insects was gathered.


### 2.3. Hazard Analysis Critical Control Point (HACCP)

HACCP was carried outin order to understand the critical points for the contamination; information on source of purchasing of salad vegetables, their transportation, storage, and further processing before consumption was obtained through personal interviews of the street food vendors (n=53). Based on this information the process flow diagram was constructed. HACCP is a system which identifies, evaluates, and controls hazards which are significant for food safety. The hazard analysis includes observing salad preparation and practices to identify the sources and modes of contamination. Preparation of process flow diagram is a prerequisite for carrying out HACCP.Samples of hand washings, knife swab, and chopping board swab were collected from six street food vendors (n=6) and examined for presence of food pathogens.


### 2.4. Sample Collection and Processing

A total of 163 samples were collected from four different zones of Hyderabad. Samples were transported to the laboratory in aseptic condition. The polythene zip lock bags with salad samples were kept in an ice box maintained at 6–10 degree centigrade and processed within 2–4 hrs. Twenty-five grams of each salad sample was weighed and transferred to 225 mL of sterile buffered peptone water. The diluents of buffered peptone water were then inoculated onto the respective media.


### 2.5. Identification and Enumeration

Identification and enumeration of foodborne pathogens (S. aureus, Salmonellaspp., andYersinia enterocolitica) were performed as described by standard methods of bacteriological analytical manual. After thoroughly mixing the food sample (25 g) in buffered peptone water (225 mL) by 1 : 10 dilution, the diluents were inoculated onto their respective selective media such as MSA (mannitol salt agar) forStaphylococcus aureuswith small round pale colored colonies, XLD (xylose lysine deoxycholate agar) forSalmonellaspp. with red colored colonies with black centre, and YSA (Yersinia selective agar) forYersiniaspp.with transparent colonies with pink centre. After inoculation the plates were incubated for 24 hrs at 37°C in incubator. Morphological tests such as grams staining and motility tests (coagulase test forS. aureus) were conducted followed by biochemical tests using Hi assorted biochemical test kit (A combination of 12 tests for identification of gram-negative rods. Kits contain sterile media for citrate utilization, lysine utilization and ornithine utilization, urease detection phenylalanine deamination (TDA), nitrate reduction, H2S production test, and 5 different carbohydrates for utilization test: glucose, adonitol, lactose, arabinose, and sorbitol) followed by latex agglutination test kit supplied by HiMedia.


### 2.6. Statistical Analysis

The analysis was carried out by descriptive analysis (mean, minimum, and maximum) for each category of the group. Difference between the groups was tested by nonparametric Kruskal-Wallis ANOVA considering the heterogeneity of variance. Individual pair difference was tested by Mann-WhitneyU test (SPSS-14.8 windows version was used).


## 2.1. Study Area

The Study was carried out in Hyderabad which is the capital of Andhra Pradesh, India. The twin cities of Hyderabad and Secunderabad come under ambit of a single municipal unit, the Greater Hyderabad Municipal Corporation. For administrative purpose Greater Hyderabad Municipal Corporation was divided into many zones. Random sampling procedure was adopted to select four zones (Alwal: east zone, Yousufguda: west zone, Old City: south zone, and Secunderabad: north zone). The sample required for the study was obtained using proportionate representation according to size.


## 2.2. Questionnaire

Personal interview of the street vendor was taken and flow chart was prepared from which critical control points of contamination were examined.Data on salad preparation, handling, and storage practices was collected using structured questionnaire that had both observational and responsive questions. Equipment used for the preparation of salad, source of water for both utensil cleaning and vegetables, utensil cleaning methods, and hand washing were considered. Salad preparation, handling practices, storage of leftover salad, storage length, and salad storage practices were observed and noted. Information on status of the premises, storage conditions for salad before cooking, cutting and chopping place, status of the serving plate, peeling and cleaning of vegetables, cleanliness of the cloth and clothing used, provision for waste disposal, presence of rodent droppings in the outlet, and exposure to insects was gathered.


## 2.3. Hazard Analysis Critical Control Point (HACCP)

HACCP was carried outin order to understand the critical points for the contamination; information on source of purchasing of salad vegetables, their transportation, storage, and further processing before consumption was obtained through personal interviews of the street food vendors (n=53). Based on this information the process flow diagram was constructed. HACCP is a system which identifies, evaluates, and controls hazards which are significant for food safety. The hazard analysis includes observing salad preparation and practices to identify the sources and modes of contamination. Preparation of process flow diagram is a prerequisite for carrying out HACCP.Samples of hand washings, knife swab, and chopping board swab were collected from six street food vendors (n=6) and examined for presence of food pathogens.


## 2.4. Sample Collection and Processing

A total of 163 samples were collected from four different zones of Hyderabad. Samples were transported to the laboratory in aseptic condition. The polythene zip lock bags with salad samples were kept in an ice box maintained at 6–10 degree centigrade and processed within 2–4 hrs. Twenty-five grams of each salad sample was weighed and transferred to 225 mL of sterile buffered peptone water. The diluents of buffered peptone water were then inoculated onto the respective media.


## 2.5. Identification and Enumeration

Identification and enumeration of foodborne pathogens (S. aureus, Salmonellaspp., andYersinia enterocolitica) were performed as described by standard methods of bacteriological analytical manual. After thoroughly mixing the food sample (25 g) in buffered peptone water (225 mL) by 1 : 10 dilution, the diluents were inoculated onto their respective selective media such as MSA (mannitol salt agar) forStaphylococcus aureuswith small round pale colored colonies, XLD (xylose lysine deoxycholate agar) forSalmonellaspp. with red colored colonies with black centre, and YSA (Yersinia selective agar) forYersiniaspp.with transparent colonies with pink centre. After inoculation the plates were incubated for 24 hrs at 37°C in incubator. Morphological tests such as grams staining and motility tests (coagulase test forS. aureus) were conducted followed by biochemical tests using Hi assorted biochemical test kit (A combination of 12 tests for identification of gram-negative rods. Kits contain sterile media for citrate utilization, lysine utilization and ornithine utilization, urease detection phenylalanine deamination (TDA), nitrate reduction, H2S production test, and 5 different carbohydrates for utilization test: glucose, adonitol, lactose, arabinose, and sorbitol) followed by latex agglutination test kit supplied by HiMedia.


## 2.6. Statistical Analysis

The analysis was carried out by descriptive analysis (mean, minimum, and maximum) for each category of the group. Difference between the groups was tested by nonparametric Kruskal-Wallis ANOVA considering the heterogeneity of variance. Individual pair difference was tested by Mann-WhitneyU test (SPSS-14.8 windows version was used).


## 3. Results

Microbiological analysis of salads indicated that both onion and carrot were contaminated with three pathogens.Salmonellawas present in 58% in carrots and 45% of onion samples. Compared to onions, carrots harboured higher percentage of contamination (Table 1). Among the carrot samples procured from four zones of Hyderabad, Old City, that is, south zone, has shown high levels of contamination compared to other zones. The mean concentration ranges ofStaphylococcus,Salmonella, andYersiniain carrot and onion are shown (Table 2).Table 1
Incidence of foodborne pathogens in salads given along with street foods in Hyderabad.
Pathogen
Carrot (n=53)
Onion (n=110)
Staphylococcus
39 (73.6%)
62 (56.4%)
Salmonella
31 (58.5%)
50 (45.5%)
Yersinia
36 (67.9%)
26 (23.6%)Table 2
Mean concentration ranges ofStaphylococcus, Salmonella, and Yersiniain salad samples.
Pathogens
Carrot (min–max)
Mean
Onion (min–max)
Mean
Staphylococcus
2.0–5.2 (102–105)
4
2.0–5.0 (102–105)
3.2
Salmonella
3.0–5.4 (103–105)
4.4
2.0–5.4 (102–105)
3.9
Yersinia
3.3–5.4 (103–105)
4.5
2.9–4.8 (102–105)
3.9
*Range of microbial counts (log⁡10 cfu/g).Seventy-six percent of onion samples from east zone hadStaphylococcus which is high when compared to other zones.Salmonellawas not detected in onion samples of east zone. In Old City, that is, south zone, 76% of onion samples hadSalmonellawhich is high when compared to other zones (Figure 2). Samples of carrots have high levels of contamination when compared to onions.Process flow diagram of onion and carrot indicated that, after peeling the outer skin of the onions, they were directly cut into small pieces and served without washing them. It was observed that the peeling which is very important step in processing of carrots was not done; they were directly grated and served (Figure1). HACCP study on contamination ofSalmonella in salads indicated that food handlers were responsible forSalmonella contamination (Table 4).Figure 1
Process flow diagram for onions.Figure 2
Prevalence of foodborne pathogens in onion samples of Hyderabad city.Most of the vendors got vegetables from whole sale market and 56% of the vendors stored them in their house and 44% of them stored it in the stall itself. About 98% of the vendors did not wash the vegetables before processing and 56% of the vendors did not peel carrots before cutting. Ninety-two percent of the vendors used knife to cut the vegetables out of which 77% of the knives were wet and dirty and 88% of the vendors did not wash knife frequently. About 54% of the vendors kept salads in open air without covering them properly. Ninety percent of them used wooden board for cutting and chopping vegetables. About 90% of the vendors’ cleaning cloths were untidy and none of them wore gloves before serving salads. There were about 60% of the vendors whose nails were uncut. About 64% of the stalls are adjacent to road (Table3).Table 3
Salad handling practices and preparation and storage practices of 53 street food venders in Hyderabad city.
Practices
Frequency of responses  (n=53) %
Vegetables from
(a) Market
49 (92.5)
(b) Shop
4 (7.5)
Vegetables brought in
(a) Polythene bag
23 (43.4)
(b) Gunny bags
30 (56.6)
Frequency
(a) Once in a day
8 (15.1)
(b) Once in 2 days
15 (28.3)
(c) Once in 5 days
16 (30.2)
(d) Once in a week
14 (26.4)
Storage
(a) House
30 (56.6)
(b) Stall
23 (43.4)
Storage before cutting
(a) Table
10 (18.9)
(b) Bowl
11 (20.8)
(c) Bucket
12 (22.6)
(d) Refrigerator
10 (18.9)
(e) Others
10 (18.9)
Wash vegetables
(a) Yes
1 (1.9)*
(b) No
52 (98.1)
Peeling vegetables
(a) Yes
23 (43.4)
(b) No
30 (56.6)
Peeled with
(a) Peeler
1 (1.9)
(b) Knife
22 (41.5)
(c) Not peeled
30 (56.6)
Cutting vegetable
(a) Knife
49 (92.5)
(b) Vegetable cutter
3 (5.7)
(c) Grater
1 (1.9)
Status of knife
(a) Clean-dry
15 (28.3)
(b) Wet-dirty
38 (71.7)
Storage after cutting
(a) Open place
29 (54.7)
(b) Container
24 (45.3)
Selling of non-veg. food
(a) Yes
6 (11.3)~
(b) No
47 (88.6)
Frequent washing of knife
(a) Yes
6 (11.3)
(b) No
47 (88.7)
Cutting and chopping place
(a) Wooden
48 (90.6)
(b) Stainless table
5 (9.4)
Status of cleaning cloth
(a) Neat
5 (9.4)
(b) Dirty
48 (90.6)
Does vendor wear gloves?
(a) No
53 (100)
Frequent washing of hands
(a) Yes
3 (5.7)
(b) No
50 (94.3)
Status of his nails
(a) Neat
21 (39.6)
(b) Dirty
32 (60.4)
Wounds
(a) Cut
6  (11.3)
(b) Wound
1 (1.9)
(c) No wound
46 (86.8)
Status of nails
(a) Cut
21 (39.7)
(b) Uncut
32 (60.3)
Neatly combed
(a) Yes
16 (30.2)
(b) No
37 (69.8)
Uniform
(a) Neat
12 (22.6)
(b) Dirty
41 (77.4)
Status of premises
(a) Near to the water drain
9 (17)
(b) Adjacent to road
34 (64.2)
(c) Near public toilet
3 (5.7)
(d) Near municipal garbage
4 (7.5)
(e) Others
3 (5.7)
Waste disposal
(a) Garbage bin
40 (75.5)
(b) Plastic bag
12 (22.6)
(c) Outside
1 (1.9)
Stray dogs and cats in vicinity
(a) Yes
53 (100)
Rodent droppings
(a) Yes
31 (58.5)
(b) No
22 (41.5)
Insect exposure
(a) Yes
51 (96.2)
(b) No
2 (3.8)
*Washing is done with still water. ~Use same knife to cut vegetables and meat.Table 4
HACCP analysis ofSalmonella (cfu/g) contamination in salads.
Salad sample
Hand wash
Knife swab
Chopping board swab
Vendor 1
ND
1
×
10
3
ND
ND
Vendor 2
7
×
10
4
12
×
10
4
ND
ND
Vendor 3
3
×
10
5
6
×
10
3
18
×
10
3
5
×
10
4
Vendor 4
ND
6
×
10
3
ND
ND
Vendor 5
3
×
10
5
ND
4
×
10
3
3
×
10
5
Vendor 6
ND
1
×
10
4
ND
1
×
10
3
ND: not detected.


## 4. Discussion

The study demonstrated that salads served along with popularly sold street foods are contaminated with one or many pathogens. A recent foodborne disease outbreak during 2011 in Europe and Germany was mainly due to contamination of salads such as cucumber, tomatoes, and lettuces by harmful strain ofE. coli O104:H4 [8].SalmonellaandStaphylococcus aureuswere the other major pathogens found in salads. A study conducted in Dehradun, India, has shown that different types of salads such as carrot, radish, tomato, coriander leaves, turnip, onion, cucumber, and beetroot were contaminated withSalmonella (32.28%),E. coli (33.14%),S. aureus (50.57%), andP. aeruginosa (60.85%) [9].In this present study carrot and onion were chosen because they are the most commonly served salads in street foods of Hyderabad. About 74% and 56% tested positive forStaphylococcusin carrots and onions, respectively. About 58% of carrots and 45% of onions containedSalmonella and 68% of carrots and 24% of onions hadYersinia. Type of pathogens reported in salads, specifically with carrot, wasE. coliO157:H7,Salmonellaspp., andL. monocytogenesand for onions wasE. coliandSalmonella typhimurium[10]. The presence of microbes in salads can be linked to a number of factors such as improper handling and processing, use of contaminated water during washing and dilution, cross-contamination from rotten vegetables, or the use of dirty processing utensils like knife and trays [11, 12]. The present study indicated that the bacterial load of carrots was high when compared to onions because carrots were not peeled. Peeling as expected reduces the bacterial load; a study done by Ankitha et al. indicated that a reduction of about 40% of bacterial load was observed in peeled carrot [3].The contamination byStaphylococcus aureusmay be due to its carriage in nasal passages of food handlers or infected workers [13]. The contamination ofSalmonellaspp. in vegetables was due to washing of vegetables with contaminated water and handling of vegetables by infected workers, vendors, and consumers in the market place which helps spread pathogenic microorganisms. HACCP study revealed that raw vegetables (carrot and onion) themselves carried pathogens and since they were not washed they continued to be present at the time of consumption. Hand washings also containedSalmonella which indicate that even food handlers contaminate the salads.Previous studies have demonstrated thatSalmonellacross-contamination occurs frequently through the use of contaminated vegetables that are improperly cleaned and undisinfected [14–16]. The serving stage is a critical point in the street food vending. Poor personal hygiene often facilitates transmission of 60 pathogens via food to humans [17]. Most of the vendors were not using hand gloves while preparing and serving street food. Vendors were carriers of a variety of bacterial enteropathogens, includingS. typhimurium[18]. SimilarlyEscherichia coliwas detected in hand washings of high income and low income mothers in India at levels of 7.0±4.2 log 10 cfu/mL and 9.0±5.7 log 10 cfu/mL, respectively [18]. A significant difference (P<0.01) was observed in load ofSalmonellaspp. in wet, dirty chopping board when compared to the load ofSalmonellain clean and dry chopping board. Similarly the load ofYersinia spp. was high in wet, dirty chopping board when compared to the load ofYersiniaspp. in clean and dry chopping board. A significant difference (P<0.05) was observed in the load ofStaphylococcusspp. in cleaning cloth which was dirty used by the vendor when compared to the load ofStaphylococcusin neat cloth used by the vendor. The pathogenic bacteria population was significantly higher in south circle because demographically this circle belongs to the old part of the city wherein the density of the population is high and most of the population was inhabited by lower income group which may be one of the factors contributing to poor hygienic practices.


## 5. Conclusions

The present study revealed that the potential hazard of street vended salad vegetables may be due to handling practices of vendors and poor hygienic conditions in which they are sold. Salads can be a potential source ofSalmonellainfection among the consumers. A simple thorough washing of vegetables with safe running water before further processing reduces the risk of microbiological hazards. There is a need to provide basic training on food hygiene to food vendors to ensure food safety and also create consumer awareness of consuming improperly processed salads as one of the possible sources of microbial hazards.



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*Source: 932191-2014-05-14.xml*