Microbiological study of vinaigrette salad sold at pasty stalls in street markets in the City of São Paulo, Brazil

Foodborne diseases (FBD) present high prevalence worldwide and more than 250 types have been reported. The main agents are Staphylococcus aureus, Escherichia coli, Salmonella and certain parasites. The habit of consuming foods sold in street markets in the city of São Paulo exposes consumers to the risk of acquiring FBD. 25 samples of vinaigrette salad were collected in the central, northern, southern, eastern and western regions of the city of São Paulo, Brazil. The samples were inoculated in selective, enriched culture media and biochemical analyses were performed. Bacterial and fungal growth occurred in all samples. The microorganisms detected were E. coli (64%), Enterobacter (60%), S. aureus (52%), Klebsiella (40%), Proteus sp. (32%), Shigella sp. (28%), Citrobacter sp. (16%), Edwardsiella sp. (12%), Alcaligenes sp. (8%), Serratia sp. (8%), Salmonella sp. (4%), Pseudomonas sp. (4%) and; 72% were positive for aflatoxins. Twelve species were identified, 50% of which can cause FBD. S. aureus and fecal coliforms can cause FBD, while Alcaligenes sp. can cause nosocomial infections and Edwardsiella sp. can cause hepatic abscess, meningitis and septicemia. Aflatoxins may cause mycotoxicoses and liver cancer. Therefore, inspections, and prevention and awareness measures should be reinforced to minimize the risks of contracting FBD from foods sold in street markets in the city of São Paulo. Research, Society and Development, v. 10, n. 11, e504101119738, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19738 2


Introduction
Foodborne Diseases (FBD) is caused by the ingestion of water or food contaminated with microorganisms and/or their toxins. There are over 250 types of FBDs, and the main causative agents include Staphylococcus aureus, Escherichia coli, Salmonella sp. and Shigella sp. Vegetables, fruits, milk and dairy products, eggs and meat are the vehicles most involved in outbreaks (Secretaria de Vigilância em Saúde, 2018). The symptoms of gastrointestinal FBD are colic, diarrhea, vomiting and fever, which may vary in intensity and characteristics according to the etiological agent, microbial load and health status of the individual affected (Secretaria de Vigilância em Saúde, 2018; Herrera et al., 2006;Riddle et al., 2016;Silva et al., 2008). Is outbreak considered when two or more people show events, signs and symptoms similar after ingesting food or water from the same source within the same time frame, or when cases of serious diseases occur, such as cholera and botulism (Secretaria de Vigilância em Saúde, 2018). The investigation is conducted to identify the etiological agent through laboratory, bromatological and clinical exams and/or epidemiological norms (Secretaria de Vigilância em Saúde, 2018; Ganho et al., 2011).
The consumption of food purchased in street markets is a common practice in the city of São Paulo, especially a type of deep-fried pasty (a pastel) that is served with a salad made of onion, tomato, pepper and cabbage with a vinaigrette dressing, known as vinaigrette salad. It is packaged in a disposable plastic bag or cup (Silva et al., 2008;Ganho et al., 2011). It is a fresh food that remains exposed on stalls where it is handled with minimal criteria concerning the prevention of contamination. The Research, Society andDevelopment, v. 10, n. 11, e504101119738, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19738 3 presence of pathogenic microorganisms in this food may contribute to an increase in the number of outbreaks and the development of FBD. Thus, the purpose of this research was to investigate the presence of pathogenic microorganisms in vinaigrette salad offered as an accompaniment to the pasties sold in street markets in the city of São Paulo (Silva et al., 2008).

Samples
Twenty-five samples of vinaigrette salad sold in street markets in São Paulo were collected, six from the central region, five from the northern region, four from the southern region, six from the eastern region and four from the western region, Table 1. Source: Own authorship.

Culture mediums
The culture media used were blood agar, MacConkey agar, cystine lactose electrolyte-deficient (CLED) agar, Salmonella-Shigella (SS) agar and Sabouraud agar (Microbac®, Jacarepaguá, RJ, Brazil), each chosen for their selective and differential characteristics. Each culture medium was prepared according to the manufacturer's recommendations. Following inoculation of the medium, the plates were incubated at 35±2°C for 18-24h (Blood, MacConkey, CLED and SS agars) or at 25°C for seven days (Sabouraud agar).

Enzymatic and biochemical tests
The following biochemical tests were performed: the Rugai method (Cecon® Vila Sonia, SP, Brazil) and the mannitol, DNAse and TSI tests (Microbac®, Jacarepaguá, RJ, Brazil), each according to the manufacturer's recommendations.

Aflatoxin test
A fluorescence detector was used in qualitative assays to verify the presence of aflatoxins. Sabouraud culture medium plates containing aflatoxins were exposed to a 366 nm ultraviolet lamp, such that aflatoxin molecules absorb the photons and emit a blue light that indicates the presence of aflatoxins B and green light when detect aflatoxins G (Rasooly et al., 2016).

Results
All the samples inoculated on blood, MacConkey CLED, SS and Sabouraud media (n=25) showed colonies of bacterial growth, except one (1.5), which showed no bacterial growth on MacConkey and SS media.
The trade in street food has grown as a practical and economical alternative the population (Silva et al., 2016;Santos et al., 2016). In São Paulo, street markets form part of the city's culture, they are distributed throughout the five regions and operate Tuesday through Sunday. The consumption of deep-fried pasties accompanied by a vinaigrette salad is a common habit that could be associated with the acquisition of FBD (Silva et al., 2016). Food poisoning caused by S. aureus occurs due to the presence of toxins that lead to symptoms like vomiting, diarrhea, nausea and colic (Secretaria de Vigilância em Saúde, 2018).
These toxins can trigger other infections, such as pneumonia and septicemia, important diseases in the hospital environment that can contribute to the development and permanence of methicillin resistant strains (MRSA) (Sciezyńska at al., 2012). S. aureus is part of the human microbiota, as well as being found in numerous environments, so food handling must be performed rigorously, because the handler can be a vehicle of contamination. In the United States of America (USA), 27.8% of raw meat samples analyzed were positive for S. aureus (Sciezyńska at al., 2012). In this study, we observed that 60% of the samples were contaminated with S. aureus.
Thermotolerant coliforms (E. coli, Klebsiella and Enterobacter) can contaminate water and food through untreated sewage disposal. They are useful indicators for assessing water and food contamination (Martin et al., 2016). Some species of E. coli stand out as producers of Shiga toxin, which leads to severe conditions like hemorrhagic colitis and uremic hemolytic syndrome that can lead to renal failure. The most severe cases can be fatal among children and the elderly with poor health (Martin et al., 2016). In India, E. coli was detected in 66% of samples of salads, eggs, chicken, sheep meat and unpasteurized buffalo milk, which corroborates the findings of our study, where 64% of samples were contaminated with E. coli (Rasheed et al., 2014). Besides being associated with FBD outbreaks, Enterobacter and Klebsiella are also involved in other diseases, such as pneumonia, and wound and urinary infections (Martin et al., 2016). In Ghana, 65% of pasta and plantain samples were contaminated with Enterobacter (Saba & Gonzalez-Zorn, 2012). In Barcelona, microbiological analysis performed on hospital food detected 8.8% of Klebsiella in the samples analyzed (Calbo et al., 2011). Among our samples, 60% were positive for Enterobacter and 40% for Klebsiella.
Salmonella sp. and Shigella sp. can cause typhoid, salmonellosis and enteric fever. Salmonellosis is one of the leading causes of human bacterial gastroenteritis and the second most reported in the European Union (Mughini-Gras et al., 2014). In Salvador, 11.25% of fresh eggs, and in Recife 12.7% of curd cheeses presented Salmonella (Evêncio-Luz et al., 2012). In Mexico, the presence of Salmonella was detected in 2% of cooked vegetable salad samples (Bautista-De Leon et al., 2013). In Ethiopia, 7.4% of beef samples were positive for Shigella (Garedew et al., 2016). In this study, 4% of the samples presented Salmonella and 28% presented Shigella sp.
Citrobacter sp., Pseudomonas sp., Proteus sp., Alcaligenes sp. and Serratia sp. are microorganisms resident in the human gut, and contaminated water and soil. They can cause various numerous diseases, including urinary and respiratory infections, meningitis, bacteremia and septicemia, and more severe cases can result in death (Jarvis et al., 2010). In China, 35.7% of fresh vegetable samples were positive for Citrobacter freudi (Liu et al., 2018). Among our samples, 16% were contaminated with Citrobacter sp. In Switzerland, a study of fish and seafood detected P. aeruginosa in 27% of the samples (Boss et al., 2016). In France, an analysis of cheese detected the presence of Proteus in 68% of samples (Deetae et al., 2009).
Here, we detected Pseudomonas in 4% of the samples studied and Proteus in 32%. In Dhaka, Bangladesh, 17.5% of frozen animal foods were contaminated with Alcaligenes (Sultana et al., 2014). In Germany, 26% of Serratia contamination was detected in fresh pork meat (Schill et al., 2017). In our research, 8% of the samples showed the growth of both Alcaligenes sp.
and Serratia sp.
Edwardsiella sp. is found in the aquatic environments and is responsible for enteric infection, endocarditis, liver abscess, and more severe cases of meningitis and septicemia. Mortality can affect 61.1% in patients with soft tissue infection (Ota et al., 2011;Takeuchi et al., 2009). In Spain, the presence of Edwardsiella was detected in 8% of the grouper fillets studied (Herrera et al., 2006). In this study, we detected Edwardsiella sp. in 12% of the samples.
Aflatoxins are products of the secondary metabolism of Aspergillus spp. and other fungi. Isoform B1 causes acute toxicity in mammals, fish, birds and humans. They are found in agricultural products like corn and peanuts (Rasooly et al., 2016). Aflatoxin contamination leads to suppression of the immune system and to liver failure, which can progress to cancer and in more severe cases, to death. Yearly estimates suggest that in South Africa and the Sahara, 26,000 deaths are associated with chronic aflatoxin exposure. This contamination is a serious food security problem worldwide (Rasooly et al., 2016;Shekhar et al., 2017). In India, Aspergillus flavus was detected on 83.33% of corn kernels (Shekhar et al., 2017), while herein, 72% of the samples were positive for aflatoxins.
In São Paulo, SP, Brazil, 45% of the fish sold in street markets was unfit for consumption due to the levels of thermotolerant coliforms (E. coli) and non Vibrio-cholerae. This indicates that the inadequate food handling and hygiene observed in street markets in São Paulo could be endangering public health and facilitating FBD (Silva et al., 2008).
Food sold on the street is a known vehicle for microorganisms that cause FBD. The source of bacterial contamination can be present throughout the production chain, from the use of contaminated water, poor hygiene with regard to the food, the utensils and even cross contamination from prepping surfaces, when handling fresh meat and fish, for example, in the case of Edwardsiella sp. (Santos et al., 2016;Schill et al., 2017).
During sample collection, we observed that vinaigrette salad is almost always exposed, with minimal protection or refrigeration. In all the stalls, the trash cans were close to where the product was stored. The sellers frequently manipulated money concomitant with serving vinaigrette salad. The sum of these factors makes it impossible to determine at what point product contamination occurs during the transaction.

Final Considerations
This study detected the presence of pathogenic microorganisms in vinaigrette salad offered as an accompaniment to