The Prevalence of Bacterial Pathogens in Vegetable Salads and Juices in Urban West Region of Zanzibar, Tanzania

Fruit juices and vegetable salads are the most common street foods vended in developing countries. Improperly processed juices and vegetables can cause food borne illnesses, which is a long-standing problem in Zanzibar. The study’s objective was to determine the bacterial quality in the vegetable salads and juices in Zanzibar. The cross-sectional design that involved field sampling of juices and vegetables was used. Bacteriological analysis was performed using standard techniques of culturing in designated media. The investigated samples were mango juice (n= 49), tamarind juice (n= 49), and vegetable salads (n= 98). One-way analysis of variance (ANOVA) was used to compare the mean bacterial counts between tamarind and mango juice as well as bacterial counts in salads. The result of this study revealed that 88/98 (89.7%) of salad samples had high counts of E. coli with colony-forming units between 2.7 × 10² to 7.8 × 10³ and E. coli contamination levels of 55% (27/49) in tamarind juice and 30/49 (61%) in mango juice respectively. Salmonella Typhi was found in two samples of mango juice but none in tamarind juice and salad. The overall E. coli contamination level in all juices was 58%. Average Most Probable Number (MPN) of coliforms in tamarind and mango juice were 450.48 MPN/m and 952.08 MPN/ml, respectively. Comparatively, mango juice is more contaminated than tamarind juice. The majority of the juice and salads in the study were found to be severely contaminated, which implies that there was inadequate handling and hygiene during the food processing process. Health authorities should keep a close eye on the quality of food being sold on the streets.

Introduction

Raw fruit juices and freshly prepared vegetable salads are among the vendor street foods that are primarily eaten in urban regions of developing countries [1]. These foods are known to be rich in Vitamins, minerals, carbohydrates such as sucrose, fructose, glucose, and sorbitol, as well as dietary fibers that are necessary for digestion [1]. The majority of the food vendors who sell juice and vegetable salad are low-income individuals operating within harsh environments that make it difficult to maintain the required hygiene standards. Their working environments are characterized by inadequate sanitary facilities without basic services like potable water [2]. Juice and vegetable salads are favored foods by the Zanzibar population, usually served at roadside kiosks.

Despite the health benefits derived from the consumption of fresh juice and vegetable salads, they can be a source of bacteria pathogens that may cause food poisoning, a public health problem affecting many people at a time [3]. The main sources of contamination in fruit and vegetables are linked to handling practices and the use of organic fertilizers, such as animal manure from cattle, goats, and chickens. These fertilizers can pose a potential risk if applied shortly before harvest. Contaminated water containing fecal material, disposed of in streams, rivers, and ponds, along with postharvest issues or improper handling during food processing, can lead to contamination of food when handled by unhygienic workers [4]. Additionally, pre-prepared fruits and vegetables can offer the right environment for human pathogens to grow and survive if properly stored. Cut surfaces release nutrients that can be used by microbes to grow, especially in foods such as fruits which are used to make juices as well as tomatoes and cabbage used to make vegetable salads. An estimated 2 million individuals die globally each year from food-borne illnesses caused by eating contaminated food [5].

Numerous microorganisms are implicated as the causative agents of food poisoning and diarrheal illnesses; among the most common species that contaminate juices and salad vegetables are Salmonella and Escherichia coli (E. coli) [6]. E. coli possess virulence factors that give them a selective advantage of surviving within unfavorable environmental conditions and evading the immune system of the infected hosts, leading to infection and inflammation after consuming contaminated food [7]. While most strains of E. coli are intestinal normal flora but once enters foods some strains can cause food poisoning leading to symptoms such as fever, vomiting, abdominal crampsb, and diarrhea. In rare cases, these symptoms can progress to systemic invasive infections, which can lead to respiratory disorders, urinary tract infections, and other potentially fatal infections [8]. There are six pathogenic categories of E. coli that cause pathogenic diarrhea illness: Enteropathogenic E. Coli (EPEC), Enteroaggregative E. Coli (EAEC), Entero-invasive E. Coli (EIEC), Diffusely Adherent E. Coli, Enterotoxigenic E. Coli (ETEC), and Shiga toxin-producing E. Coli (STEC). The most significant and prevalent group among these is STEC E. coli serotype O157: H7 While outbreaks of this pathogen are frequent, the diseases it produces are relatively new [9].

Vegetable and juice salads may also be contaminated by the intestinal bacterium Salmonella. These are Gram-negative, rod-shaped, facultative, anaerobic members of the Enterobacteriaceae family. In general, Salmonella is an extremely effective pathogen that can survive in the guts of warm and cold-blooded animals [1]. Salmonella enterica and S. bongori are the two species that make up the genus, S. enterica is further divided into six subspecies, that can be distinguished from one another both biochemically and by genetic relatedness. Salmonella bongori is the second species which is less pathogenic to humans and is rarely associated with human infections, but it primarily infects cold-blooded animals, such as reptiles [1]. Salmonella became one of the most significant and newly discovered zoonotic bacteria causing both typhoid fever and acute gastroenteritis [1]. Typhoid fever is brought on by bacterial invasion of the bloodstream, whereas gastroenteritis is a foodborne illness that can cause diarrhea. Salmonellosis can be harmful either because of the pathogen itself or because of a toxin generated from the lipopolysaccharide layer [1].

Food-borne illness cases, particularly Salmonellosis, have increased recently [3]. Humans with salmonellosis experience a wide range of clinical symptoms, including severe diarrhea and abdominal cramps. Every year, diarrheal illnesses cause 1.8 million deaths worldwide; 89% of these infections are caused by Salmonella, particularly in underdeveloped nations [10]. Eating infected and/or improperly prepared foods, such as seafood, eggs, chicken, milk, cattle, pig, vegetables, and fruits that haven’t been completely cleaned, can spread salmonellosis [11]. The salmonella infections can occur due to unhygienic meal handling procedures, that can lead to food contamination and make food unfit for consumption [12].Foods may also be contaminated by coliforms. Coliform bacteria are characterized as facultative or aerobes. Gram-negative, non-spore-forming, motile, or non-motile bacilli that have β-galactosidase, which allows them to create acids and gasses at their ideal growth temperature of 35°C–37°C [13]. They are widely utilized as a sign of bad hygienic quality in foods [12]. As they are known to reside in the gastrointestinal tract, coliforms are ubiquitously present in large numbers in the feces of warm-blooded animals [13]. Their presence is used to infer that other pathogenic organisms of fecal origin may be present in a sample or that the sample is unsafe for consumption. Coliforms can be found in the aquatic environment, in soil, and in vegetation [13]. Citrobacter is one common coliform genus that contaminates foods. These are 0.6 μm–6 μm long, facultative anaerobic, peritrichous bacilli [14]. While certain species of Citrobacter live in the gut as normal flora without harming humans, if given the chance, they can cause meningitis, urinary tract infections, bacteremia, brain abscesses, pneumonia, intra-abdominal sepsis, and joint infections.in human [14]. A Citrobacter infection can be fatal, with a 33%–48% death rate. Newborns and those with impaired immune systems are more vulnerable to Citrobacter infections [14]. Enterobacter is another variety of coliform. These are motile, flagellated bacteria that cause infections such as osteomyelitis, meningitis, sinusitis, and bacteremia, as well as infections of the surgical site, respiratory tract, urinary tract, and other tract infections [15]. Another kind of coliform is called Klebsiella, which are non-motile, Gram-negative bacilli with a length of 1 μm–2 μm [16]. They are facultative anaerobes that can endure drying for several months because of their complex acid polysaccharide capsule [16]. Klebsiella pneumoniae is the most prevalent species of Klebsiella that can be found in soil, sewage, animal gastrointestinal tracts, and human bodies [16]. Colonies of Klebsiella are greyish-white in color on media high in carbohydrates in particular. Agar containing ornithine, raffinose, and Koser citrate is the medium used to select for Klebsiella species in a mixed sample. Members of this genus will form yellow, wet-looking colonies on this medium [17]. Klebsiella can contaminate Juice and salads, like any other meal ingested by humans. Street vendors often operate in environments with limited access to clean water, proper sanitation, and preservation facilities like refrigeration [18]. This situation increases the risk of contamination and can make street food a potential vector for foodborne illnesses, especially for items that are consumed fresh and without further cooking. Contaminated food can lead to outbreaks of diseases such as diarrhea, food poisoning, and more severe infections. Assessing microbial contamination in street food helps to identify potential health risks and prevent disease outbreaks. Ensuring that these foods are safe to eat protects consumers from dangerous pathogens such as E. coli and Salmonella. With the rapid expansion of urban areas in Zanzibar, there is a growing demand for fast food, which is often served by street vendors. Unfortunately, the hygienic conditions at many street food serving points are substandard. Freshly consumed foods like salads and juices are popular components of street food. Given this context, determining microbial contamination in street vendors’ juices and salads is crucial for protecting public health, ensuring food safety, and supporting regulatory compliance.

This study assessed the prevalence of E. coli, Salmonella, and coliforms in freshly produced juice and vegetable salads sold in Zanzibar. It also measured the quantity of coliforms and E. coli bacterium using the MPN method and total plate count to assess contamination levels.

Materials and Methods

Study Site

The study was carried out in the Urban West region of Unguja, Tanzania (Fig. 1). The region hosts the capital city of Zanzibar and Stone Town and is characterized by both high population and high socio-economic activities. Food vending services are widely distributed within the city and surrounding urban areas. Like many other cities within developing countries, Zanzibar faces various challenges related to sanitation and hygiene. This makes the food vending service to be potentially vulnerable to microbial contamination.

Fig. 1. Study site Urban West region.

Study Design

The study used a cross-sectional design to examine the prevalence of bacteria pathogens in juices and vegetable salads at a given specific time.

Data Analysis

The data was analyzed using the Statistical Package for Social Studies (SPSS) software (version 20) and Pearson correlation was used to compare pH and bacterial load in MPN/ml between tamarind and mango juice.

Sample Size and Sampling

The sample size was calculated using the following formula:

$$\begin{array}{r}N=\frac{{(Z1-\alpha )}^2\times P{(1-P)}^2}{E^2}\end{array}$$

(1) $$\begin{array}{r}N=\frac{{\left(1.6\right)}^2\times P{\left(1.7\right)}^2}{{0.05}^2}\end{array}$$

where N is 98 samples, Z_1-X = Z_0.95 = 1.96 This value of 1.96 is standard for a confidence level of 95%. P is the prevalence of juice vegetable salad samples. E is the degree of cumulative 5% is 0.05, and 1–α = 95% is the confidence interval. In accordance with the formula above. Total number of juices collected was 98, of which 49 were tamarind and 49 were mango juices samples. The number of salad samples collected was 98.

Sample Collection

A total of 98 samples of juice and 98 salad samples were collected. The juice samples were composed of 49 tamarind juice and 49 mango juice. The juice samples (350 ml) were bought as parked in plastic bottles from vendors to reflect the real situation. For salad samples, an approximate mass of 30 g was collected in sterilized plastic containers from each vendor to avoid cross-contamination. The samples were labeled, placed in a sterile icebox, and transported to the Zanzibar Food and Drug Agency (ZFDA) laboratory for analysis within 24 hours of collection.

Microbial Analysis of E. coli in Salad

A mass of 25 g of each sample of salad was homogenized containing 225 ml, placed in sterile bottles, and serially diluted in Beffered Peptone Water (BPW). The serial dilutions of 1:10, 1:100, and 1:1000 was used. The total colony count of the bacteria was carried out using the pour plate count method on plate count agar (PCA) on triplicate plates (3 plates for each dilution) using ISO 4833-1:2013(E) [18]. About 15 ml–20 ml of Tryptone Bile Glucuronic (TBX) media was poured into Petri dishes containing 1 ml of sample dilutions and left for solidification, then the plates were incubated at 44 °C for 24 hours.Blue/green colors were observed in Petri dishes, indicating the presence of E. coli in TBX agar, Fig. 2.

Fig. 2. Blue-green colonies in E.coli endo TBX agar.

Bacterial counts in c.f.u per g were estimated using the formula: [19].

(2) $$\begin{array}{r}C.F.U=\frac{EC}{n1+0.1n2+0.01n3}\times d\end{array}$$

where C.F.U is colony forming unit, n1 is the total number of plates, EC is the sum of the colonies counted on all the plates, n2 is the number of plates counted in the first dilution, n3 is the number of plates counted in the second dilution, d is the dilution used to get the first count.

Detection of E. coli. Coli in Juices

The detection of Escherichia coli in juice samples was performed using the ISO 7251:2005 (E) protocol. For each juice sample, 25 ml was transferred into sterile bottles containing 225 ml of Buffered Peptone Water (BPW). A dilution series was then prepared by pipetting 1 ml of the BPW mixture into test tubes containing 9 ml of Lauryl Sulphate Tryptose Broth (LSTB). The second dilution was made by adding 1 ml of the first dilution to 9 ml of LSTB, resulting in a 1:100 dilution. Similarly, the third dilution (1:1000) was prepared by adding 1 ml of the second dilution to 9 ml of LSTB [18]. The test tubes were incubated at 37°C for 24 hours. Positive tubes, showing turbidity and gas formation, were transferred into Brilliant Green Broth for further confirmation. After incubation, the contents of the positive tubes were streaked onto Endo agar and incubated again at 37°C for 24 hours. Colonies exhibiting a green metallic sheen were identified as E. coli. These colonies were then sub-cultured onto Triple Sugar Iron (TSI) agar for final confirmation [20].

MPN Method for Coliform Estimation in Juices

For each juice sample, a volume of 25 ml was placed in sterile bottles containing 225 ml of BPW. Enumeration of coliforms by the most probable number (MPN) method was done by inoculation 1 ml from BPW into nine test tubes for each triplet with dilutions of 1 ml, 0.1 ml, and 0.01 ml were used. 1 ml of each of the three consecutive dilution tubes was inoculated into tubes containing lactose broth (LB) with Durham’s tubes and incubated at 35 °C for 48 hours. From positive cultures, a loop-full of suspension was transferred to tubes containing 2% Brilliant Green LB and incubated at 35°C for 48 hours. The number of positive tubes showing turbidity and gas production in Durham tubes were recorded for each dilution, and MPN index per ml was calculated using MPN tables.

Microbial Analysis of Salmonella in Juice and Salads

Twenty-five g of each sample of salad and juices were put separately into a conical flask containing 225 ml of BPW and then incubated at 37°C for 24 hours. Then, one ml of the solution was transferred into the test tube containing 10 ml of Rappaport Vassiliadis medium (RVS) broth and incubated at 41.5 °C for 24 hours. Then streaked from Rappaport Vassiliadis medium (RV) into a surface Xylose-lysine deoxycholate agar (XLD agar) plate and incubated at 37°C for 24 hours. Red colonies with black centers were observed and suspected as Salmonella colonies (Fig. 3). Suspected Salmonella colonies were sub-cultured into nutrient agar to obtain a pure culture of Salmonella under an incubation temperature of 37°C for 24 hours, and then colonies were inoculated to TSI for confirmation at an incubation temperature of 37°C for 24 hours. The yellowish coloration on the agar butt, bubbles, cracks, and blackening on the butt confirmed the presence of Salmonella Typhi (Fig. 4).

Fig. 3. Red colonies with black centers were observed and suspected as Salmonella colonies in Xylose-lysine deoxycholate agar (XLD agar).

Fig. 4. Yellowish coloration on the agar butt, bubbles, cracks, and blackening on the butt confirmed the presence of Salmonella Typhi from two samples [21].

Results and Discussion

Prevalence of E. coli and Salmonella in Vegetable Salads

The levels of contamination of vegetable salads were presented in colony-forming units per g (CFU/g) (Table I). The study revealed that the majority (89.7%, N = 88) of salad samples are contaminated with E. coli. The samples gave high counts of E. coli with colony-forming units ranging between 2.7 × 10² and 7.8 × 10^3. Contamination level showed no significant difference (P > 0.05) in the E. coli counts among selected sampled areas (Shehias). Vegetable salads were highly contaminated due to poor and unhygienic handling. On the other hand, salads were not contaminated with Salmonella Typhi.

Table I. Mean C.F.U/Ml for Vegetable Salads

Shehia/village	Number of samples	C.F.U/g	Salmonella	P-value
Uwandani	5	7.8 × 103	−ve	0.51889
Mbweni	5	0	−ve
Mombasa	5	4.6 × 101	−ve
Tomondo	5	5 × 102	−ve
Fuoni kipungani	5	4.7 × 103	−ve
Amani	5	3.7 × 105	−ve
Muembe makumbi	5	1.5 × 104	−ve
Nyerere	5	2.7 × 102	−ve
Shauri moyo	5	7.6 × 102	−ve
Bububu	5	4.5 × 104	−ve
Mkunazini	5	7.9 × 104	−ve
Kiponda	5	0	−ve
Shangani	5	6 × 101	−ve
Kwa ali nato	5	5.1 × 102	−ve
Gulioni	5	4.8 × 103	−ve
Kikwajuni bondeni	5	3.7 × 105	−ve
Jangombe	5	1.5 × 104	−ve
Kidongo chekundu	5	2.7 × 102	−ve
Kwahani	4	7.5 × 102	−ve
Kikwajuni juu	4	4.5 × 104	−ve
Total	98	88/98 (89%)	0%

These ranges are comparable to the findings reported by Hosein et al. [22] however, lower counts were reported by Ramesh and Thiyagarajan [23]. Furthermore, this study is in coincidence with the study done in Basra which demonstrated the isolation of E. coli, coliforms, and other bacteria from vegetables. In this study, E. coli was identified as a principal contaminant in different vegetables investigated. A similar study that investigated juices in Kampala, Uganda by Kaddumukasa et al. [24] demonstrated high counts of E. coli, coliforms, and S. aureus, the latter being the most prevalent bacteria, which may be due to processor’s hand isolations. Regarding the source of contamination, nearly all cited studies agree with Kaddumukasa et al. [24] stated that E. coli contamination in street vended salad and juice is likely due to contamination from unhygienic handling and processing practices. Consumption of E. coli-contaminated juice and salad predisposes the consumer to food borne diseases, leading to various health effects such as diarrhea and cholera.

Prevalence of E. coli and Salmonella in Juices

Results for analysis of E’ coli and salmonella bacteria in juice are presented in Table II. The prevalence of E. coli in juice from the study areas was 58%, while only 2% of the juice samples showed contamination of salmonella. The analysis revealed no significant difference (p > 0.05) in levels of contamination of E. coli among the sampling sites. The two samples with Salmonella contamination were collected from Uwandani and Nyerere Shehias. Since contamination of E. coli is associated with gastrointestinal disturbances or even diarrhea outbreaks, the presence of the bacteria in more than half of the juice samples is an indication of potential health risks associated with the consumption of juice. As both salad and juice were served in the same environment, the source of the bacteria contamination could also be associated with the same poor hygiene status of handlers.

Table II. Prevalence of E.coli and Salmonella Typhi for Juice

Shehia	Number of samples	E. Coli +Ve samples	Salmonella Typhi	P-value
Uwandani	5	3	+ve	0.549
Mbweni	5	5	−ve
Mambosasa	5	4	−ve
Tomondo	5	0	−ve
Fuoni kipungani	5	4	−ve
Amani	5	4	−ve
Mwembe makumbi	5	2	−ve
Nyerere	5	4	+ve
Shurimoyo	5	1	−ve
Bububu	5	0	−ve
Mkunazini	5	4	−ve
Kiponda	5	4	−ve
Shangani	5	2	−ve
Kwa ali natu	5	1	−ve
Gulioni	5	2	−ve
Kikwajuni bondeni	5	5	−ve
Jangombe	5	4	−ve
Kidongo chekundu	5	3	−ve
Kikwajuni chini	4	1	−ve
Kikwajuni juu	4	4	−ve
	98	57 (58%)	−ve

Similar to this study, the study conducted by Hosein et al. [20] on vented juices revealed high E. coli counts Similar findings were also reported by [23]. Unlike the study conducted by [18] the current study didn’t explore the molecular identity of the microbes isolated, this study showed that out of 98 samples, two (2) were contaminated with Salmonella., in contrast to a study conducted by Hosein et al. [19] in Dar Es Salaam Salmonella was not isolated from juice samples. A comparable investigation was conducted by [20] to look at the frequency of Salmonella spp., Salmonella Typhi, and Salmonella Typhimurium in fruit juices. Poor sanitation practices and cross-contamination were factors that led to the prevalence of Salmonella in these juices. Salmonella also grew as a result of carelessness with regard to storage conditions, including temperature.

Coliform Counts

Total coliform analysis in juice revealed the presence of the coliforms in all juice samples. The results showed that mango juice showed higher total coliform counts (952.08/ml) compared to tamarind juice (450.48 MPN/ml). The comparisons of coliform counts between the two types of juice showed a statistically significant difference of MPN/ml (P-value = 0 .000157) between mango and tamarind juices. The presence of coliforms indicates the probability of existing other pathogenic organisms of fecal origin in the samples. The f-ratio value is 16.8325 (Table III).

Table III. E. coli Counts in Tamarind and Mango Juice using MPN/ml

Mango Juice (MPN/ml)	Frequency	Tamarind juice (MPN/ml)	Frequency	P-Value
36	2	38	4	0.595
110	3	240	7
1000	2	290	5
1500	4	460	3
1100	14	1100	6
952.08	25	450.48	25

Comparison of pH of Tamarind Juice and Mango Juice

The mean pH of tamarind juice was 3.04, while that of mango juice was 3.53, making tamarind juice more acidic than mango juice. The difference in pH between tamarind and mango juice was statistically insignificant (P > 0.05). The determined pH values of tamarind juice and mango juice are given in Table IV. In this investigation, tamarind juice had a little lower microbial contamination than mango juice from the same vendor. This finding indicates the presence of other contamination sources other than poor hygienic handling. It is probable that the lower pH values of tamarind juices considerably reduced the microbial contamination of the juices when compared to the relatively higher pH values of mango juices, which appeared to favor the growth of microorganisms. The influence of the acidic nature of water or juice in reducing microbial contamination has also been reported.

Table IV. pH. Value in Mango Juice and Tamarind

pH of mango juice	pH of tamarind juice	P-Value
3.2	3.0	0.883
3.5	3.3
3.0	3.2
3.1	3.5
3.3	3.0
3.3	3.2
3.5	3.3
3.8	3.0
3.6	2,8
3.2	2.9
3.9	2.9
3.7	3.0
3.5	3.2
3.2	2.9
3.5	2.9
3.4	2.9
3.0	3.1
3.6	2.8
3.6	2.8
3.3	2.9
3.9	2.9
3.8	2.8
4.4	3.4
4.3	3.2
4.6	3.1
3.53	3.04

Pearson correlation revealed no significant positive correlation (P > 0.05 and r (23) = 0.014 between pH and levels of contamination in the tamarind juice. Similarly, there was no positive correlation (P > 0.05 and r (23) = 0.072) between pH and contamination levels of bacteria in mangoes. This implies that the higher the pH. the higher level of contamination because of lower pH values of tamarind juices, it was found that tamarind juices had lower E. coli counts compared to mango juices. The same findings were reported by El-Safey [21], demonstrating that using higher acid concentrations ought to lead to a greater overall decrease in the bacterial burden. Similar to this, bacterial reduction occurs best at greater acid concentrations, and higher acid temperature [22].

Conclusion and Recomendation

The study found that most of the juice and salad samples were highly contaminated with E. coli, while Salmonella was absent in the salad and tamarind juices but present in only two mango juice samples. The findings suggest that poor hygiene practices were likely the source of contamination. Interestingly, tamarind juice, which has a lower pH than mango juice, was less contaminated, indicating that the acidic environment may help reduce microbial contamination. Overall, while E. coli was the predominant pathogen found in most of the juice and salad samples, Salmonella was present in only a few. These results underscore the importance of regular monitoring by health authorities to ensure the safety and quality of street-vending foods.