**3. Foods involved in** *Salmonella* **sp. outbreaks**

*Salmonella* is a bacteria with wide occurrence in animals and in environment, and the main sources are water, soil, animal feces, insects and surfaces of factory's equipment and kitchen utensils. A disease is generally contracted mainly through consumption of contaminated food of animal origin. It is commonly accepted that at between 1 million to 1 billion bacteria are needed to cause infection although some investigators suggest some people may be infected by far fewer bacteria. Other authors mention that the infectious dose is 15 to 20 cells and can reach any age range, with the elderly and children under seven years the more susceptible to get ill (SILVA et al., 2010). Nevertheless, most data suggest food, water, or other sources of contamination contain large amounts of bacteria. Although human stomach acid can reduce and sometimes eliminate *Salmonella* spp., occasionally some bacteria get through to the intestine and then attach and penetrate the cells. Symptoms may include headache, muscle aches, diarrhea, vomiting, abdominal cramping, chills, fever, nausea and dehydration. According to the Illinois Department of Public Health, most persons infected with *Salmonella* bacteria develop diarrhea, fever and abdominal cramps 12 to 72 hours after infection. The illness usually lasts 4 to 7 days, and most persons recover without treatment. However, in some persons, the diarrhea may be so severe that the patient needs to be hospitalized. *Salmonella* sp. infection may spread from the intestines to the bloodstream, and then to other body sites and can cause death unless the person is treated promptly with antibiotics. On the other hand, persons can be infected with the bacteria without having symptoms. Persons with and without symptoms shed the bacteria in their stool, which is why proper handwashing after toileting and before handling food is so important. Children younger than 1 year old, people who have had ulcer surgery or take antacids, the elderly, infants and those with impaired immune systems are more likely to have a severe illness from *Salmonella* sp. which can contaminate a wide variety of foods. These include raw foods derived from animals like eggs and egg products, meat and meat products, unpasteurized milk and other dairy products, and raw poultry. Shell eggs and eggs products figured as prominently in recent years as a human salmonellosis. More recently, *Salmonella* sp. outbreaks have been tied to a variety of fresh produce like lettuce, salad mixes, sprouts, melons, tomatoes and even peanut butter. Minimally processed vegetables have risen, since

according to a particular method" (ANDREWS, 1997). The great advantage of the kits is that the material required for tests (all or part of it) is sold together, eliminating the preparation

The polymerase chain reaction (PCR) detection of *Salmonella* spp is based on the amplification of bacterial DNA sequence that is unique to salmonellae. The PCR assay consists of three different steps: denaturation of duplex bacterial DNA into single strands (94oC), annealing of synthetic oligonucleotide primers (45-65oC) that are highly- specific to *Salmonella* sp. DNA sequences that flank the *Salmonella*-specific DNA targed, and a polymerase-dependent extension (72oC) of the single-stranded DNA starting at the primer site where elongation progress from 3min to 5min end of template DNA strand. The commonly targeted sequence for amplification lies within the inVA gene of *Salmonella* sp.

Related to the minimally processed vegetables, the main method used is the traditional technique described in this chapter together with the use of miniaturized kits for

*Salmonella* is a bacteria with wide occurrence in animals and in environment, and the main sources are water, soil, animal feces, insects and surfaces of factory's equipment and kitchen utensils. A disease is generally contracted mainly through consumption of contaminated food of animal origin. It is commonly accepted that at between 1 million to 1 billion bacteria are needed to cause infection although some investigators suggest some people may be infected by far fewer bacteria. Other authors mention that the infectious dose is 15 to 20 cells and can reach any age range, with the elderly and children under seven years the more susceptible to get ill (SILVA et al., 2010). Nevertheless, most data suggest food, water, or other sources of contamination contain large amounts of bacteria. Although human stomach acid can reduce and sometimes eliminate *Salmonella* spp., occasionally some bacteria get through to the intestine and then attach and penetrate the cells. Symptoms may include headache, muscle aches, diarrhea, vomiting, abdominal cramping, chills, fever, nausea and dehydration. According to the Illinois Department of Public Health, most persons infected with *Salmonella* bacteria develop diarrhea, fever and abdominal cramps 12 to 72 hours after infection. The illness usually lasts 4 to 7 days, and most persons recover without treatment. However, in some persons, the diarrhea may be so severe that the patient needs to be hospitalized. *Salmonella* sp. infection may spread from the intestines to the bloodstream, and then to other body sites and can cause death unless the person is treated promptly with antibiotics. On the other hand, persons can be infected with the bacteria without having symptoms. Persons with and without symptoms shed the bacteria in their stool, which is why proper handwashing after toileting and before handling food is so important. Children younger than 1 year old, people who have had ulcer surgery or take antacids, the elderly, infants and those with impaired immune systems are more likely to have a severe illness from *Salmonella* sp. which can contaminate a wide variety of foods. These include raw foods derived from animals like eggs and egg products, meat and meat products, unpasteurized milk and other dairy products, and raw poultry. Shell eggs and eggs products figured as prominently in recent years as a human salmonellosis. More recently, *Salmonella* sp. outbreaks have been tied to a variety of fresh produce like lettuce, salad mixes, sprouts, melons, tomatoes and even peanut butter. Minimally processed vegetables have risen, since

in the laboratory (SILVA et al., 2010).

biochemical bacteria identification.

**3. Foods involved in** *Salmonella* **sp. outbreaks** 

(DÁOUST, 1994).

the 90"s, as a new source of *Salmonella* sp. in food industry. Fresh cut vegetables are by definition, perishables. The process of cutting, slicing, chopping, breaks the protective skin of fresh vegetables and increases their vulnerability to biological contamination. A poor hygiene in minimally processed vegetables, especially in developing countries are the main cause of food borne disease associated to this product. The minimally processed vegetables are products that have suffered some manipulation, thus, the useful life, compared to fresh produce is much lower (BOONER et al., 2003). Microorganisms that cause disease in humans as bacteria, protozoa, virus, has been the focus of many studies of minimally processed vegetables. *Salmonella* sp. serotypes however are estimated to be responsible for most cases of food poisoning due the consumption of this kind of product worldwide (MEAD et al., 1999).

According to Francys et al. (1999) *Salmonella* is the organism that are relevant to public health more commonly associated with food poisoning outbreaks involving vegetables ready for consumption. An outbreak of salmonellosis occurred in the UK in 1988, involved the consumption of green beans. Epidemiological studies in England and Wales between 1992 and 1996 linked the consumption of coleslaw with *Salmonella* outbreaks that occurred during this period.

Machado et al. (2009), in a research for microbiological evaluation of some minimally processed vegetables in Brazil, evaluated samples of watercress, lettuce, grated carrot, spinach, green cabbage and rocket minimally processed for some pathogens, including *Salmonella* sp. The vegetables were stored at a temperature of 5°C. *Salmonella* sp. was detected in 12.7% of the samples.

Bruno et al. (2005), evaluating the microbiological quality of 15 samples of vegetables including carrots, cabbage, chayote, all minimally processed and marketed in the north part of Brazil verified that *Salmonella* sp. was present in 66% of the samples.

 Santana et al. (2011) tested 512 samples of minimally processed vegetables in São Paulo, Brazil, and obtained that *Salmonella* sp. was detected in four samples. The serovars were *Salmonella* Typhimurium (three samples) and *Salmonella enterica* subsp. *enterica* (one sample). A small outbreak of *Salmonella* sp happened in five states of United States of America, in June, 2011. A total of 21 persons with the outbreak strain of *Salmonella enteritidis* have been reported from 5 states: Idaho (3), Montana (7), North Dakota (1), New Jersey (1) and Washington (9). Among persons for whom information is available, ill persons range in age from 12 years to 77 years old, with a median age of 35 years old. Seventy-one percent are female. Among the 10 ill persons with available information, 3 (30%) persons have been hospitalized. No deaths have been reported. It was announced by Los Angeles Times. The outbreak was linked with the consumption of alfafa sprouts (http:// articles.latimes.com/ 2011). In USA, a total of 99 individuals infected with the outbreak strain of *Salmonella* Agona have been reported from 23 states, late July, 2011. Epidemiologic, traceback, and laboratory investigations have linked this outbreak to eating fresh, whole papayas imported from Mexico (CDC, 2011). According to the Center for Disease Control (CDC), in the United States, food poisoning causes nearly 76 million illness cases with about 325,000 hospitalizations, and approximately 5,000 deaths yearly. The *Salmonellae* organisms are reportedly responsible for as much as \$1 billion in medical costs and lost time from work.

Concerning to salmonellosis preventions, it is important to say that *Salmonella* bacteria are killed when food is thoroughly cooked properly. Once cooked, any food held in a buffet should be kept hotter than 55oC. Cross contamination, may be avoided using different utensils, plates, cutting boards and count tops before and after cooking. Cooking food

Occurrence of *Salmonella* in Minimally Processed Vegetables 115

allowed a shelf-life of 6 days when stored at a temperature of 5°C ± 1°C. Nascimento (2002) showed that vegetables washed with a solution containing 50 ppm of free chlorine showed a significant reduction in the total count of aerobic and that fecal coliforms were even more sensitive to chlorine, not being more detected in vegetables after washing. Therefore, chlorine and its salts, especially hypochlorite, are effective and of low cost, and widely applied as a spray for bacteriological control in industries working with vegetables (KIM et al., 1999). However, in recent years there has been some concern in the use of chlorine due to the inconvenience of toxic compounds that can be formed and leave residual taste in food (OLIVEIRA & VALLE, 2000). Among these compounds, there are the trihalomethanes (THM), aldehydes, halocetonas and chloramines, which when hydrolyzed proved related to some types of cancer according to epidemiological studies of Meyer (1994). Depending on the toxicity of these compounds, there is a recognized need to find alternative sanitizers for hygiene and sanitization procedures for vegetables. Thus, chlorine dioxide (ClO2) has received special attention (ARENSTEIN, 2003) for, although it is a derivative of chlorine, generates negligible amount of by-products (trihalomethanes), characterized as a product of low carcinogenic potential (ANDRADE & MACEDO, 1999). In addition, chlorine dioxide is a strong oxidizing agent that reacts mostly through a mechanism of electron transfer by attacking the cell membrane, penetrating, dehydrating, and lastly, oxidizing the internal components of the microbial cell without however causing toxic effects, as most of the chlorine compounds do. It also has the advantage of being effective against gram negative and positive. Still, by the fact that hydrolyzes the phenolic compounds it reduces the

Another important aspect of chlorine dioxide is its sharp and sporicidal disinfectant action in lower concentrations than that of chlorine. The explanation of its high bactericidal action is due to the fact that it is soluble in oils, greases and substances of mixed composition, such as cells of virus and bacteria, whose membranes easily penetrates in, as opposed to other

Chlorine dioxide is stable under a wide pH range (6-10) and its decomposition are first

2 NaClO2 + Cl2 2 ClO2 + 2 NaCl

+ 2 ClO-

However, the major disadvantages of chlorine dioxide are its cost and its sensitivity to high

Currently, several studies are being conducted with chlorine dioxide in different countries. Felkey et al. (2003) and Rash (2003) showed in their studies the efficiency of chlorine dioxide in reducing *Salmonella* on the surface of tomato and melon, respectively. Another sanitizing agent that has been used quite successfully is peracetic acid, also known as peroxide of acetic acid or peroxyacetic acid. It is obtained by the reaction of acetic acid or acetic anhydride with hydrogen peroxide in the presence of sulfuric acid, which has the function of catalyst. The

The peroxyacetic acid has currently one of the largest application as disinfectants in the food industry and its efficiency is similar or superior to sodium hypochlorite (NASCIMENTO, 2002), but more potent than hydrogen peroxide. It is an excellent sanitizer for the great

2 + ClO-

2 + ClO-

3 + 2 H+

<sup>3</sup> + 2 H2O

formed chlorite (ClO3) and then chlorate (ClO2) which can be seen in the equations:

2 ClO2 + H2O ClO-

+ H2

decomposition products are acetic acid, hydrogen peroxide and water.

4 ClO2-

possibility of formation of tastes and odors.

disinfectants of polar nature.

temperatures.

stands at room temperature for a long time, such as two hours, is also at risk. It is important to assent that vegetables, now identified as a source of *Salmonella* sp., must be thoroughly washed in treated or healthy running water before they are eaten, as basic operations of food borne disease. In food industry, internal systems of quality control are essential to prevent occurrence of foodborne illness to consumer. As an example, the HACCP (Hazards Analysis and Control of Critical Points) system, adopted by major international markets, basically ensures that the manufactured products are developed without risk to public health, and also have uniform standards of identity and quality (SILVA, 1999).
