**2.** *Salmonella* **sp. as a pontential contamination microorganism of minimally processed vegetables**

#### **2.1 Taxonomy**

*Salmonella* is a genus of Rod-shaped gram negative bacteria that belong to the family *Enterobacteriaceae*. Their species are motile, oxidase-negative, catalase positive and utilize glucose and other carbohydrates with the production of acid and gas.

Officially the genus is composed of a single species, *Salmonella choleraesuis,* divided into seven subspecies, which are also known by Roman numerals: I. *choleraesuis*, II. *salamae,* IIIa. *arizonae,* IIIb. *diarizonae*, IV. *houtenae*, V. *bongori* and VI. *indicates*. In 1987 a proposal was made to change the name *Salmonella choleraesuis* for *Salmonella enterica* and in 1989 the

<sup>\*</sup> Corresponding author

Occurrence of *Salmonella* in Minimally Processed Vegetables 111

for minimally processed vegetables, a portion of 25 g or 25 mL of the sample, is taken and placed in 225 ml of buffered peptone water (BPW). Incubate for 18 hours at 37°C (SILVA et

Selective enrichment of portion of pre-enriched culture in nine volumes of tetrathionate brilliant green (TBG), selenite cistine (SC) or Rappaport-Vassilads (RV) broth medium for 18-24h represses the growth of competitive microflora and makes easy the recovery in

Selective differential plating objectives to promote the preferential development of colonies with typical *Salmonella* sp. It is recommended to be done in one or more culture media. The most common are the Hectoen Enteric Agar (HE), Xylose Lysine Desicolato Agar (XLD) agar and Xylose Lysine tergitol4 (XLT4) (Silva et al., 2010). Each culture purview a streak SVR (depletion) in the differential media recommended. Repeat this procedure with the broth MKTTn. Incubate plates inverted XLD 37oC/24 hours. Follow the incubation plates of the

Confirmation is a step that aims to verify whether the colonies obtained in the typical differential plating are actually colonies of *Salmonella* sp. is carried out through biochemical

In the XLD medium, typical colonies are dark pink in color with black center and a reddish zone, slightly transparent around. In the second chosen medium, after plating, following the manufacturer's guidelines for evaluating features of typical colonies of *Salmonella* sp. Select

Confirmation checks the biochemical profile biochemical characteristics of strains of *Salmonella enterica* subsp. enterica. Miniaturized kits are also recommended for this aim. Series recommended for biochemical analysis method for *Salmonella* sp.: Incubation of all

 Growth test Agar Triple Sugar Iron-TSI: Initial color: orange. Positive test: ramp alkaline (red), background acid (yellow) with production of gas (bubbles) with or

Urease test: deep pink colour. Negative for *Salmonella* sp. The medium maintains its

 Test Lysine Carboxylase: Most lysine-positive strains. Serotype Parathypi are negative. Voges-Proskauer test: tubes with 3 mL of methyl red (VM)-VP, Voges-Proskauer.

 Beta-galactosidase test. Most strains of *Salmonella enterica* subsp. *enterica* are negative. A serological confirmation checks for the presence of antigens "O", "Vi" and "H" for tests agglutination polyvalent antisera. The results for confirmed positive *Salmonella* sp. by the ISO 6579 method (2007) are: typical for biochemical tests, no self-agglutination and antibody positive serological test for O, Vi, or H. The methods of analysis of food end up confirming this stage, since the full characterization of *Salmonella* sp. is usually done by reference

Over the past ten years there was a breakthrough in developing new methods, especially immunological methods and to a lesser extent, methods based on nucleic acids. These methods follow the current trend of development kits analytical trademarked defined by AOAC (Association of Official Analytical Chemists) as "a system containing all key components to the analysis of one or more microorganisms, one or more types of food,

others differential culture media, according to the manufacturer.

at least two colonies of each medium for further confirmation.

tests: 37C/24 hours (SILVA et al., 2010).

without H2S production.

*Salmonella* sp. are negative.

Indole: indole-negative: the most of them.

laboratories in each specific country (SILVA et al., 2010).

original color.

al., 2010).

different plating media.

and serological tests.

proposed elevation of the subspecies to the species category bongori. This proposal received unanimous support of the Subcommittee on *Enterobacteriaceae* of the International Committee on Systematic Bacteriology at the Fourteenth International Congress of Microbiology, but was not made official by the International Committee of Nomenclature of Bacteria. Still, it was adopted and used by the CDC (U.S. Center for Disease Control and Prevention), ASM (American Society for Microbiology) and WHO (World Health Organization). The strains most frequently involved in human disease are *S. enterica* subsp. *enterica*, which is the habitat for warm-blooded animals and are responsible for 99% of human salmonellosis. *S. enterica* subsp. *salamae* subsp. arizonae and subsp. diarizonae, are often isolated from the intestinal contents of cold-blooded animals and rarely humans or warm-blooded animals. *S. enterica* subsp. *houtenae* and *S. bongori* are predominantly isolated from the environment and are rarely pathogenic to humans (SILVA et al., 2010).

More than 50% of the serotypes of *Salmonella* belong to the *Salmonella enterica* subsp. *enterica*, and the most common somatic serogroups are; A, B, C1, C2, D, E1, and E4. Approximately, 99% of *Salmonella* infections in humans and warm-blooded animals, are due these serogroups, including widely known serotypes Parathyphi A (A group), Paratyphi B and Thyphimurium (B group), Paratyphi C and cholerasuis (C group), Typhi, Enteritidis and Gallinarum (D group) (SILVA et al., 2010).

#### **2.2 Growth and survival**

*Salmonella* sp. has the ability to growth in the temperature range of 2-45oC, with the optimum at 35-37oC. The psychotropic attribute of *Salmonellae* and ability to growth slowly at cold temperature raises concerns on cold-induced bacteriostasis as a food safety measure. *Salmonellae* can growth in the pH range with an optimum pH range of 6.5-7.5 for growth. The water activity for this genus is 0.93 or greater (SILVA et al., 2010).

The propensity of *Salmonella* sp. to survive bactericidal food process and to persist for years in frozen foods and in dry foods stores at ambient temperature is a food safety concern. The thermal process in food industry widely used to eliminated bacterial human pathogens is a challenge concerning to *Salmonella* sp*.*, because of its heat resistance in foods with low water activity. The classical study on solute dependent thermal resistance showed that heating of *Salmonella* sp. at 57.2oC in aqueous solutions of sucrose and glycerol adjust the AW = 0.90 yields D value of 40-55 minutes and 1.8-8.3 min. respectively (GOEPFERT et al., 1970).

#### **2.3 Detection methods**

The traditional technique for detecting *Salmonella* sp. in food is a classic culture method for presence/absence, developed in order to ensure detection even under extremely unfavorable conditions. This is the case of food microbiology with a competitor microbiota much larger than the population of *Salmonella* and / or food in which the cells of *Salmonella* sp. are very few in number and/or foods in which the cells are injured by the process of preservation (application of heat, freezing, drying) (SILVA et al., 2010).

The procedures recommended by different regulatory bodies, although they present some variations in the selection of culture media and method of sample preparation basically follow five steps: pre-enrichment, selective enrichment, plating, biochemical and serological confirmation (DÁOUST, 1994). All samples should be pre-enriched in a non-selective broth medium for 18-24h at 35-37oC. The aim of this step is to resuscitate the few injured or stressed cells of *Salmonella* sp. By the method ISO 6579 (2007), one of the most recommended

proposed elevation of the subspecies to the species category bongori. This proposal received unanimous support of the Subcommittee on *Enterobacteriaceae* of the International Committee on Systematic Bacteriology at the Fourteenth International Congress of Microbiology, but was not made official by the International Committee of Nomenclature of Bacteria. Still, it was adopted and used by the CDC (U.S. Center for Disease Control and Prevention), ASM (American Society for Microbiology) and WHO (World Health Organization). The strains most frequently involved in human disease are *S. enterica* subsp. *enterica*, which is the habitat for warm-blooded animals and are responsible for 99% of human salmonellosis. *S. enterica* subsp. *salamae* subsp. arizonae and subsp. diarizonae, are often isolated from the intestinal contents of cold-blooded animals and rarely humans or warm-blooded animals. *S. enterica* subsp. *houtenae* and *S. bongori* are predominantly isolated

from the environment and are rarely pathogenic to humans (SILVA et al., 2010).

The water activity for this genus is 0.93 or greater (SILVA et al., 2010).

preservation (application of heat, freezing, drying) (SILVA et al., 2010).

Gallinarum (D group) (SILVA et al., 2010).

**2.2 Growth and survival** 

**2.3 Detection methods** 

More than 50% of the serotypes of *Salmonella* belong to the *Salmonella enterica* subsp. *enterica*, and the most common somatic serogroups are; A, B, C1, C2, D, E1, and E4. Approximately, 99% of *Salmonella* infections in humans and warm-blooded animals, are due these serogroups, including widely known serotypes Parathyphi A (A group), Paratyphi B and Thyphimurium (B group), Paratyphi C and cholerasuis (C group), Typhi, Enteritidis and

*Salmonella* sp. has the ability to growth in the temperature range of 2-45oC, with the optimum at 35-37oC. The psychotropic attribute of *Salmonellae* and ability to growth slowly at cold temperature raises concerns on cold-induced bacteriostasis as a food safety measure. *Salmonellae* can growth in the pH range with an optimum pH range of 6.5-7.5 for growth.

The propensity of *Salmonella* sp. to survive bactericidal food process and to persist for years in frozen foods and in dry foods stores at ambient temperature is a food safety concern. The thermal process in food industry widely used to eliminated bacterial human pathogens is a challenge concerning to *Salmonella* sp*.*, because of its heat resistance in foods with low water activity. The classical study on solute dependent thermal resistance showed that heating of *Salmonella* sp. at 57.2oC in aqueous solutions of sucrose and glycerol adjust the AW = 0.90 yields D value of 40-55 minutes and 1.8-8.3 min. respectively (GOEPFERT et al., 1970).

The traditional technique for detecting *Salmonella* sp. in food is a classic culture method for presence/absence, developed in order to ensure detection even under extremely unfavorable conditions. This is the case of food microbiology with a competitor microbiota much larger than the population of *Salmonella* and / or food in which the cells of *Salmonella* sp. are very few in number and/or foods in which the cells are injured by the process of

The procedures recommended by different regulatory bodies, although they present some variations in the selection of culture media and method of sample preparation basically follow five steps: pre-enrichment, selective enrichment, plating, biochemical and serological confirmation (DÁOUST, 1994). All samples should be pre-enriched in a non-selective broth medium for 18-24h at 35-37oC. The aim of this step is to resuscitate the few injured or stressed cells of *Salmonella* sp. By the method ISO 6579 (2007), one of the most recommended for minimally processed vegetables, a portion of 25 g or 25 mL of the sample, is taken and placed in 225 ml of buffered peptone water (BPW). Incubate for 18 hours at 37°C (SILVA et al., 2010).

Selective enrichment of portion of pre-enriched culture in nine volumes of tetrathionate brilliant green (TBG), selenite cistine (SC) or Rappaport-Vassilads (RV) broth medium for 18-24h represses the growth of competitive microflora and makes easy the recovery in different plating media.

Selective differential plating objectives to promote the preferential development of colonies with typical *Salmonella* sp. It is recommended to be done in one or more culture media. The most common are the Hectoen Enteric Agar (HE), Xylose Lysine Desicolato Agar (XLD) agar and Xylose Lysine tergitol4 (XLT4) (Silva et al., 2010). Each culture purview a streak SVR (depletion) in the differential media recommended. Repeat this procedure with the broth MKTTn. Incubate plates inverted XLD 37oC/24 hours. Follow the incubation plates of the others differential culture media, according to the manufacturer.

Confirmation is a step that aims to verify whether the colonies obtained in the typical differential plating are actually colonies of *Salmonella* sp. is carried out through biochemical and serological tests.

In the XLD medium, typical colonies are dark pink in color with black center and a reddish zone, slightly transparent around. In the second chosen medium, after plating, following the manufacturer's guidelines for evaluating features of typical colonies of *Salmonella* sp. Select at least two colonies of each medium for further confirmation.

Confirmation checks the biochemical profile biochemical characteristics of strains of *Salmonella enterica* subsp. enterica. Miniaturized kits are also recommended for this aim.

Series recommended for biochemical analysis method for *Salmonella* sp.: Incubation of all tests: 37C/24 hours (SILVA et al., 2010).


A serological confirmation checks for the presence of antigens "O", "Vi" and "H" for tests agglutination polyvalent antisera. The results for confirmed positive *Salmonella* sp. by the ISO 6579 method (2007) are: typical for biochemical tests, no self-agglutination and antibody positive serological test for O, Vi, or H. The methods of analysis of food end up confirming this stage, since the full characterization of *Salmonella* sp. is usually done by reference laboratories in each specific country (SILVA et al., 2010).

Over the past ten years there was a breakthrough in developing new methods, especially immunological methods and to a lesser extent, methods based on nucleic acids. These methods follow the current trend of development kits analytical trademarked defined by AOAC (Association of Official Analytical Chemists) as "a system containing all key components to the analysis of one or more microorganisms, one or more types of food,

Occurrence of *Salmonella* in Minimally Processed Vegetables 113

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

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

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

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

 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

of Brazil verified that *Salmonella* sp. was present in 66% of the samples.

worldwide (MEAD et al., 1999).

detected in 12.7% of the samples.

during this period.

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

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. (DÁOUST, 1994).

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 biochemical bacteria identification.
