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INTRODUCTION
Preparing and selling food in the street is an ancient activity, particularly widespread in developing countries. The food preparation, handling and servicing at large scale provides an ample opportunity for the spread of foodborne diseases, either sporadically or on epidemiological scale (Food and Agriculture Organization [FAO]/ World Health Organization [WHO], 1986). Streetvended foods are often the main source of cheap, convenient and significantly nutritious food for both urban and rural populations (Mensah, Yeboah-Manu, Owusu-Darko & Ablordey, 2002). The food and beverages defined as “readyto-eat” (RTE) are prepared for being sold especially in streets and similar public places (Samapundo, Climat, Xhaferi & Devlieghere, 2015). There is an increasing global interest in RTE items as part of a general concern for health and food safety (Canet & N'Diaye, 1996), particularly those which are composed of meat/poultry and salads, having been recognized as potential vehicles of bacterial foodborne pathogens (Campos, Gil, Mourão, Peixe & Antunes, 2015), generally associated with consumption of food that has been exposed to pathogenic microorganisms such as bacteria, viruses and parasites, and to their metabolic toxins, which results in health disorders, either chronic or acute (Ossai, 2012). Some of the microorganisms associated with these diseases are bacteria, such as Escherichia coli, Salmonella spp., Clostridium spp., Staphylococcus spp., Bacillus cereus, etc. (Food and Agriculture Organization [FAO], 1982, Campos et al., 2015). The aim of this study was to evaluate the microbiological quality of street-vended flavored water and fresh fruit by determining the presence of aerobic mesophilic microorganisms, fecal coliforms and pathogens such as Escherichia coli (E. coli), Salmonella spp., Shigella spp. and Staphylococcus aureus (S. aureus).
MATERIALS AND METHODS
Collection of Samples
This research was conducted in the city of Reynosa, state of Tamaulipas, Mexico. 48 samples of flavored water and 20 samples of fruit salad were collected from some ice cream stores and supermarkets, respectively, summing a total of 28 establishments. The samples were preserved in their original containers, transported to the laboratory in a cooler and immediately analyzed.
Sample Preparation
In the case of liquid samples, 60 ml of each sample were analyzed, and then homogenized by stirring for 1 min. In the case of solid samples, 60 g of each sample were analyzed, and then liquefied with diluent for not more than 2 min. After that, decimal and seeded broth dilutions were performed according to the required analysis.
Aerobic mesophiles
The presence of these microorganisms was determined according to the NOM-092-SSA1-1994 standard (DOF, 1994), using the technique of culture in depth; this method consists in counting colonies grown on agar after an incubation period of 48 h at 35 °C ± 2 °C. The results were expressed in CFU/ml or g of sample. The official NOM-093-SSA1-1994 standard (DOF, 1994) establishes the allowable limits for these microorganisms.
Fecal coliforms and Escherichia coli
The MPN (most probable number) technique, described by Camacho et al. (2009), was used; this method relies on the ability of some coliform bacteria to ferment lactose when incubated at 44 °C for 24 h. To determine fecal microorganisms, a presumptive test in sodium lauryl sulfate broth was conducted, including incubation for 48 h at 35 °C ± 2 °C, as well as a confirmatory test in EC-MUG broth, including also incubation but in water bath for 24 h at 44 °C. To determine the presence of Escherichia coli, positive confirmatory tests were irradiated with a UV lamp; to isolate and confirm the strain, Eosin Methylene Blue Agar (Levine) was used, incubating samples for 24 h at 35 °C ± 2 °C, and subsequent biochemical tests. The official NOM093-SSA1-1994 standard (DOF, 1994) establishes the allowable limits for these microorganisms.
Determination of Staphylococcus aureus
A total count of S. aureus was conducted by direct plating on agar Baird-Parker, with an incubation time of 48 h at 35 °C ± 2 °C. The presence of typical colonies was confirmed using coagulase and thermonuclease tests, as established by the NOM-115-SSA1-1994 standard (DOF, 1995).
Detection of Salmonella spp.
Following the NOM-114-SSA1-1994 standard (DOF, 1995), a pre-enrichment culture with a lactose broth was used, followed by an enrichment culture with tetrathionate broth, subsequent seeding on selective media, such as Hektoen Enteric Agar, Agar XLD, Salmonella Shigella Agar and Bismuth Sulfite Agar, and biochemical tests (glucose fermentation, urease, lysine descarboxylase, indole test, H2S production and dulcitol fermentation); all cultures were incubated for 24 h at 35 °C ± 2 °C.
Determination of Shigella spp.
An enrichment culture with GN broth was used, following Barrantes & Achí (2011); samples were then replated on selective media such as Hektoen Enteric Agar, XLD Agar, Salmonella Shigella Agar, and further biochemical tests were conducted. All cultures were incubated for 24 h at 35 °C ± 2 °C.
RESULTS
This study analyzed 28 street food establishments, including some ice cream stores and supermarkets. The water samples included 6 different flavors: “rice water” (horchata) (n = 8), pineapple (n = 9), lemon (n = 8), melon (n = 8), “roselle flowers” (jamaica) (n = 8) and tamarind (n = 7). As for fruit salads, 20 samples of fruits such as papaya, pineapple, strawberry, mango, peach, watermelon, jicama, orange, apple, melon and banana were collected. These samples were divided into two groups: salads with orange juice (n = 7) and salads without orange juice (n = 13).
Determination of aerobic mesophiles
Table 1 shows the overall results of aerobic mesophiles in fruit water and fruit salad samples. Among the total samples analyzed, mesophilic contamination was found in 82.3% (n = 56/68). Among the 48 fruit water samples analyzed, mesophilic contamination was found in 77% (n = 37/48), with a range of 20 CFU/ml - 940 000 CFU/ml, which exceeded 6.2 times the maximum value dictated by the standard (150 000 CFU/ml). Pineapple water showed a high degree of contamination, with an average of 225 190 CFU/ml; it was also the water flavor with more out-of-standard samples, a 44.4% (n = 4/9), while lemon water showed the lowest degree of contamination, with 501 CFU/ml. On the other hand, 95% of fruit salad samples were contaminated (n = 19/20), with counts of 95 CFU/g to 240 000 CFU/g, exceeding 1.6 times the maximum value, according to the standard. The group of fruit salads without orange juice showed 92.3% (n = 12/13) of positive samples, with a bacterial count average of 41 009 CFU/g, but only one sample was found to be out of standard (14.2%; n = 1/7). The group of fruit salads with orange juice showed 100% (n = 7/7) contamination, with a bacterial count average of 69 040 CFU/g and 16.6 % (n = 2/12) of out-of-standard samples.
Table 1 Results for aerobic mesophiles by type of water and group of fruits
| Positive samples | Samples outside the Norm | Average CFU/g or ml | Range | |
| Fruit Waters (n = 48) | 37 (77%) | 10 (27%) | 95 769 | 20 - 940 000 |
| Horchata (n = 8) | 8 (100%) | 3 (37.5%) | 102 802 | 640 - 340 000 |
| Pineapple (n = 9) | 8 (100%) | 4 (44.4%) | 225 190 | 5 100 - 710 000 |
| Lemon (n = 8) | 3 (37.5%) | 0 | 501 | 110 - 3 400 |
| Melon (n = 8) | 8 (100%) | 1 (12.5%) | 138 795 | 2100 - 940 000 |
| Jamaica (n = 8) | 6 (75%) | 2 (25%) | 101 896 | 20 - 430 000 |
| Tamarind (n = 7) | 3 (42.8%) | 0 | 5428 | 1000 - 220 000 |
| Fruit Salads (n = 20) | 19 (95%) | 3 (15.7%) | 433 209 | 5 - 240 000 |
| Fruit Salads with orange juice (n = 7) | 7 (100%) | 1 (14.2%) | 690 409 | 5 - 240 000 |
| Fruit Salads without Orange juice (n = 13) | 12 (92.3%) | 2 (16.6%) | 41 009 | 130 - 220 000 |
Source: Author´s own elaboration
Fecal coliforms and Escherichia coli
Table 2 shows the general data about fecal coliforms and Escherichia coli isolation. Among the total samples, 35.2% (n = 24/68) were found positive for bacterial contamination. Among the flavored water samples analyzed, 41.6% (n = 20/48) showed contamination by these microorganisms, with a count of < 3 MPN/ml - 654 MPN/ml. All positive samples were out of the official Mexican standard, which states that there must be no fecal contamination in these foods. Horchata water had the largest number of contaminated samples, with an 87.5% (n = 7/8) and a contamination average of 640 MPN/ml; however, melon water showed the highest average of fecal contamination with 654 MPN/ml, while jamaica water showed the lowest average with 5 MPN/ml. Fruit salads showed less contamination, with only 20% (n = 4/20) of the samples showing fecal contamination; again, any positive sample was considered as out of standard. The counts of these organisms were of 9 MPN/g - 23 MPN/g. In the group of fruit salads with orange juice, 28.5% (n = 2/7) of the samples tested positive for contamination, with an average of 2.46 MPN/g, while in the group of fruit salads without orange juice, only 15.3% (n = 2/13) were positive, with an average of 4.57 MPN/g. No presence of Escherichia coli was detected in any sample analyzed in this study.
Table 2 Results for fecal coliforms by type of water and group of fruits
| Positive samples | Samples outside the Norm | Average CFU/g or ml | Range | E coli | |
| Fruit Waters (n = 48) | 20 (41.6%) | 20 (100%) | 294 | < 3 - 2400 | 0 |
| Horchata (n = 8) | 7 (87.5%) | 7 (100%) | 640 | < 3 - 2400 | 0 |
| Pineapple (n = 9) | 6 (66.6%) | 6 (100%) | 323 | < 3 - 2400 | 0 |
| Lemon (n = 8) | 0 | 0 | 0 | 0 | 0 |
| Melon (n = 8) | 5 (62.5%) | 5 (100%) | 654 | < 3 - 2400 | 0 |
| Jamaica (n = 8) | 1 (12.5%) | 1 (100%) | 5 | < 3 - 43 | 0 |
| Tamarind (n = 7) | 1 (14.2%) | 1 (100%) | 100 | < 3 - 1000 | 0 |
| Fruit Salads (n = 20) | 4 (20%) | 4 (100%) | 3.5 | < 3 - 23 | 0 |
| Fruit Salads with orange juice (n = 7) | 2 (28.5%) | 2 (100%) | 2.5 | < 3 - 24 | 0 |
| Fruit Salads without Orange juice (n = 13) | 2 (15.3%) | 2 (100%) | 4.6 | < 3 - 25 | 0 |
Source: Author's own elaboration.
Detection of Staphylococcus aureus
Table 3 shows the overall results for S. aureus; 35.2% (n = 24/68) of the samples showed contamination by this microorganism. 37.5% (n = 18/48) of the flavored water samples showed contamination, with counts of 100 CFU/ml - 200 000 CFU/ml. Horchata water showed higher contamination than the rest of the samples, with 62.5% (n = 5/8) and a contamination index of 26 625 CFU/ml. Even so, tamarind water showed the highest contamination level, with an average of 41 428 CFU/ml. Lemon water showed the lowest contamination level, with 1375 CFU/ml. Fruit salads showed less contamination by this microorganism, with 30% (n = 6/20) of positive samples and a contamination range of 100 CFU/g to 10 000 CFU/g. Fruit salads with orange juice showed 42.8% (n = 3/7) of positive samples, with an average of 1471 CFU/g, while fruit salads without orange juice had 23% (n = 7/13) of contaminated samples, with an average of 69 CFU/g.
Table 3 Results for Staphylococcus aureus by type of water and group of fruits
| Positive samples | Average CFU/g or ml | Range | |
| Fruit Waters (n = 48) | 18 (37.5%) | 21 963 | 100 - 200 000 |
| Horchata (n = 8) | 5 (62.5%) | 26 625 | 1000 - 100 000 |
| Pineapple (n = 9) | 4 (44.4%) | 2230 | 100 - 20 000 |
| Lemon (n = 8) | 2 (25%) | 1375 | 1000 - 10 000 |
| Melon (n = 8) | 2 (25%) | 20 125 | 1000 - 160 000 |
| Jamaica (n = 8) | 2 (25%) | 40 000 | 120 000 - 200 000 |
| Tamarind (n = 7) | 3 (42.8%) | 41 428 | 90 000 - 100 000 |
| Fruit Salads (n = 20) | 6 (30%) | 560 | 100 - 10 000 |
| Fruit Salads with orange juice (n = 7) | 3 (42.8%) | 69.2 | 100 - 400 |
| Fruit Salads without Orange juice (n = 13) | 3 (23%) | 1471 | 100 - 10 000 |
Source: Author's own elaboration.
Detection of Salmonella and Shigella spp.
Table 4 shows the overall results. The presence of Salmonella spp. was detected in 16.2% (n = 11/68) of all samples analyzed. Among fruit water samples, 16.6% (n = 8/48) were positive for contamination with this microorganism; the most contaminated were horchata, melon and jamaica, with 25% (n = 2/8). In the case of fruit salads, this microorganism was present in 15% of all samples (n = 3/20); the group of fruit salads with orange juice was the most contaminated with 28.6% (n = 2/7) of them infected. In the case of Shigella spp., only 2.9% (n = 2/68) of all samples tested were contaminated; only flavored water samples showed total contamination by this organism (4.1%; n = 2/48), horchata and melon water having one positive sample each (12.5%; n = 1/8).
Table 4 Results for Salmonella ssp. and Shigella ssp. by type of water and group of fruits
| Salmonella ssp. Positive samples | Shigella ssp. Positive samples | |
| Fruit Waters (n = 48) | 8 (16.6%) | 2 (10%) |
| Horchata (n = 8) | 2 (25%) | 1 (12.5%) |
| Pineapple (n = 9) | 1 (11.1%) | 0 |
| Lemon (n = 8) | 0 | 0 |
| Melon (n = 8) | 2 (25%) | 1 (12.5%) |
| Jamaica (n = 8) | 2 (25%) | 0 |
| Tamarind (n = 7) | 1 (14.2%) | 0 |
| Fruit Salads (n = 20) | 3 (15%) | 0 |
| Fruit Salads with orange juice (n = 7) | 2 (28.6%) | 0 |
| Fruit Salads without Orange juice (n = 13) | 1 (7.7%) | 0 |
Source: Author's own elaboration.
Other isolated pathogens
Table 5 shows the general data. In addition to the microorganisms that were specifically sought in this study, the isolation of some other pathogens, such as Klebsiella pneumoniae (K. pnuemoniae), Klebsiella oxytoca (K. oxytoca), Enterobacter aerogenes (E. aerogenes) and Serratia marcescens (S. marcescens) was successfully achieved. These microorganisms were isolated in 36.8% (n = 25/68) of the samples. The most frequently isolated was K. pneumoniae, in 33.8% (n = 23/68) of the samples. Among the flavored water samples, 50% (n = 24/48) showed contamination; horchata was the most frequently contaminated with 87.5% (n = 7/8) of the samples. The presence of K. pneumoniae was detected in 41.6% (n = 20/48) of the flavored water samples; it was more prevalent in horchata water, as 100% of the samples (n = 7/7) were positive for its presence. These microorganisms were found in only 15% (n = 3/20) of the fruit salad samples; the group of fruit salads without orange juice showed greater contamination with 15.3% (n = 2/13). It is worth noting that K. pneumoniae was the only bacteria that prevailed in this type of sample.
Table 5 Results of K. pneumoniae, K oxytoca, E. aerogenes and S. marcescens by type of water and group of fruits
| Positive samples | K. pneumoniae | K. oxytoca | E. aerogenes | S. marcescens | |
| Fruit Waters (n = 48) | 22 (45.8%) | 20 (42%) | 3 (6%) | 4 (8%) | 0 |
| Horchata (n = 8) | 7 (87.5%) | 7 (15%) | 2 (4%) | 0 | 0 |
| Pineapple (n = 9) | 7 (77.7%) | 5 (10%) | 0 | 2 (4%) | 0 |
| Lemon (n = 8) | 0 | 0 | 0 | 0 | 0 |
| Melon (n = 8) | 6 (75%) | 6 (13%) | 0 | 1 (2%) | 0 |
| Jamaica (n = 8) | 2 (25%) | 1 (2%) | 0 | 1 (2%) | 0 |
| Tamarind (n = 7) | 2 (25%) | 1 (2%) | 0 | 0 | |
| Fruit Salads (n = 20) | 3 (15%) | 0 | 0 | 0 | 0 |
| Fruit Salads with orange juice (n = 7) | 1 (14.2%) | 1 | 0 | 0 | 0 |
| Fruit Salads without Orange juice (n = 13) | 2 (15.3%) | 2 | 0 | 0 | 0 |
Source: Author's own elaboration.
DISCUSSION
Food security has been defined as the ability of a society to address the food needs of its people (Barcelo, 1989). However, this definition should not be limited only to the field of food availability, because even if it is enough food available, the biological utility of food can be affected if its safety is not taken into account (Daley, David & Robertson, 1982). Today, it is well known that food-handling personnel play an important role in ensuring food safety, since mismanagement and the breach of hygiene regulations by those engaged in the selling of food can help pathogens get into contact with it, survive in it and multiply to a scale at which they are able to cause diseases in consumers. Thus, the results of this study reflect improper practices of food preparation and the lack of knowledge among employees of food selling establishments about proper food handling, processing and hygiene, which causes food to suffer contamination by the microorganisms mentioned above.
This study showed that flavored waters are more contaminated than fruit salads, which is attributable to the fact that most of the flavored water samples were collected in open-air exposed establishments; in addition, the preparation of flavored water requires a lot of handling. In contrast, fruit salad samples were collected from supermarkets and there was less handling of the fruits compared to fruits for making flavored water. There is a remarkable difference between these results and those reported by Félix, Campas & Meza (2005), who evaluated the presence of aerobic mesophiles, fecal coliforms and Salmonella spp. They found a range of 200 - 450 000 and 11 600 - 850 000 of aerobic mesophiles in salads and flavored water respectively. On the other hand, Salmonella was present in only 3% of salads. This can be explained because in their study the flavored waters were all collected from fixed establishments, while fruit salads were collected from street establishments; consequently, fruit salads showed higher contamination by mesophiles, fecal coliforms and Salmonella spp. than flavored waters, the opposite of what was found hereby.
In the present research work, S. aureus was isolated in 37.5% of flavored water samples, largely above the percentage reported by Tambekar, Jaiswal, Dhanorkar, Gulhane & Dudhane (2009), who isolated this microorganism in only 6% of the fruit juice samples. In this study, the presence of this bacteria was much higher in fruit water samples than in fruit salads, yet the amount of enterotoxigenic S. aureus was not enough to be toxic. The reason for the low counts of S. aureus is that this microorganism is not competitive, and in most cases, it was isolated together with other bacteria. The presence of this bacteria is attributable to the fact that it is part of the normal flora of the human body, where it is found in the nasopharyngeal tract and on the skin. When collecting the samples, it was observed that, during the preparation of flavored water, employees tend to use their bare hands when picking the ice, and they also speak during the preparation; it was also realized that, when serving water, it makes contact with their skin, and droplets of it fall into the main container of flavored water.
No E. coli was isolated in this study, despite being considered the most important fecal microorganism. However, the isolation of K. pneunomiae is not surprising, since this microorganism is considered part of fecal coliforms and its presence is consistent with other studies in which it was isolated in juice samples (Reddy, Chandrakanth, Indu, Venkata & Usha 2009; Tambekar et al., 2009).
Salmonella spp. was isolated in 16.6% of the total samples, a very similar result to that reported by Tambekar et al. (2009) and Ukwo, Ndaeyo & Udoh (2011), both of whom reported the presence of this microorganism in 17% of the juice samples analyzed. However, only 15% of fruit salad samples showed contamination by this microorganism, a low percentage compared with those reported by Bayona (2009), who presented that 25% of the fruit samples were contaminated by this organism.
In this study, Shigella spp. was isolated in 4.1% of fruit water samples, unlike the study by Lewis, Thompson, Rao, Kalavati & Rajanna (2006), which reported contamination by these bacteria in 16.6% of his juice samples. Regarding the absence of this microorganism in fruit salads, this coincides with the studies made by Utzinger, Arias, Monge & Antillón (1992).
In conclusion, street-vended foods have clearly a higher degree of contamination than those sold in closed establishments. Aerobic mesophilic microorganisms were detected in 82.3% of all samples, while contamination by fecal coliforms and Staphylococcus aureus was detected in 35.2% of them, Salmonella spp. was present in 16.1% of them, Klebsiella in 33.8% of the samples and Shigella spp. in only 2.9% of them. No presence of Escherichia coli was detected. In addition to the excessive handling and the lack of water for washing, the environmental pollution also contributes to contaminate food and drinks. For all these reasons, it is necessary to establish preventive measures, such as the training of the sales personnel, focusing on hygiene practices in handling and processing of food. These could prevent many foodborne diseases, since access to good quality food is a basic requirement for the preservation of human health.
