**5. Interaction of** *Salmonella* **with foods**

*Salmonella* serotypes can grow and survive on a large number of foods (Harris et al., 2003). Their behavior in foods is controlled by a variety of environmental and ecological factors, including water activity, pH, Eh, chemical composition, the presence of natural or added antimicrobial compounds and storage temperature; as well as processing factors such as heat application and physical handling. For example, optimum pH for growth in *Salmonella* is approximately neutral, with values > 9.0 and < 4.0 being bactericidal. Minimum growth in some serotypes can occur at pH 4.05 (with HCl and citric acids), although this minimum can occur at pH as high as 5.5, depending on the acid used to lower pH (Harris, et al., 2003). Growth in *Salmonella* can continue at temperatures as low as 5.3 °C (*S.* Heidelberg) and 6.2 °C (*S.* Typhimurium), and temperatures near 45 °C (temperatures ≥ 45 °C are bactericidal). In addition, available moisture (aw) inhibits growth at values below 0.94 in neutral pH media, although higher aw values are required as pH declines to near the minimum growth values (Harris, et al., 2003).

Extensive data is available on the effects of individual environmental factors on *Salmonella*  strains, but the effects of their interactions are not as well understood. Parish et al. (1997) determined survival for several *Salmonella* serotypes in orange juice. To achieve a 6 log reduction in *Salmonella* serotypes, orange juice (pH 3.5) had to be stored at 4 °C for 15-24 days. A similar reduction took 43-57 days when the orange juice was at pH 4.1 and 4 °C. Using apple juice, Uljas & Ingham (1999) demonstrated that *S*. Typhimurium DT104 could be reduced by at least 5 log units at pH 3.3 after storage at 25 °C for 12 hours or at 35 °C for 2 hours. These treatments did not achieve a 5 log reduction in *E.* coli O157. At pH 4.1, a 5 log reduction in *S.* Typhimurium DT104 was produced by storage at 35 °C for 6 hours in the presence of 0.1% sorbic acid or by a combination of storage at elevated temperature (25 °C for 6 hours or 35 °C for 4 hours) followed by a freeze/thaw cycle without sorbic acid (Uljas & Ingham, 1999). In the field, the physical environment of vegetables surfaces is considered to be inhospitable for growth and survival of *Salmonella* (for example, temperature and humidity fluctuations, and ultraviolet light) (Dickinson, 1986). Environmental conditions, however, can greatly influence bacterial populations; the presence of free moisture on vegetable surfaces from precipitation, dew or irrigation can promote survival and growth of bacterial populations (Shaper et al., 2006). Certain conditions such as sunlight, particularly shorter ultraviolet wavelengths, can damage bacterial cells (Shaper et al., 2006); selection therefore occurs for bacteria with adaptations to stressful conditions. Microorganisms' ability to survive on plants depends on the environmental, physicochemical and genetic features of the plant and specific properties (Shaper et al., 2006*)*. Many microorganisms have developed mechanisms to attach to, survive and/or grow in microniches on different vegetables (Shaper et al., 2006). For instance, surface moisture on vegetables may provide a protective environment for *Salmonella* strains. On vegetable surfaces, microorganisms interact in aggregates and may compete for the limited nutrients available in microniches at the junction of epidermal cells, where water accumulates, cuticular waxes are less dense and nutrients are more available than in other sites (Shaper et al., 2006). Free water in the surface apertures of vegetables (e.g. stomata) constitutes a water channel connecting a plant's apoplast with its external environment. Microorganisms can enter vegetables through these water channels in various ways. Once internalized, the microorganisms are protected from environmental stress (Shaper et al., 2006). Survival of pathogenic microorganisms on or in raw produce is

*Salmonella* serotypes can grow and survive on a large number of foods (Harris et al., 2003). Their behavior in foods is controlled by a variety of environmental and ecological factors, including water activity, pH, Eh, chemical composition, the presence of natural or added antimicrobial compounds and storage temperature; as well as processing factors such as heat application and physical handling. For example, optimum pH for growth in *Salmonella* is approximately neutral, with values > 9.0 and < 4.0 being bactericidal. Minimum growth in some serotypes can occur at pH 4.05 (with HCl and citric acids), although this minimum can occur at pH as high as 5.5, depending on the acid used to lower pH (Harris, et al., 2003). Growth in *Salmonella* can continue at temperatures as low as 5.3 °C (*S.* Heidelberg) and 6.2 °C (*S.* Typhimurium), and temperatures near 45 °C (temperatures ≥ 45 °C are bactericidal). In addition, available moisture (aw) inhibits growth at values below 0.94 in neutral pH media, although higher aw values are required as pH declines to near the minimum growth

Extensive data is available on the effects of individual environmental factors on *Salmonella*  strains, but the effects of their interactions are not as well understood. Parish et al. (1997) determined survival for several *Salmonella* serotypes in orange juice. To achieve a 6 log reduction in *Salmonella* serotypes, orange juice (pH 3.5) had to be stored at 4 °C for 15-24 days. A similar reduction took 43-57 days when the orange juice was at pH 4.1 and 4 °C. Using apple juice, Uljas & Ingham (1999) demonstrated that *S*. Typhimurium DT104 could be reduced by at least 5 log units at pH 3.3 after storage at 25 °C for 12 hours or at 35 °C for 2 hours. These treatments did not achieve a 5 log reduction in *E.* coli O157. At pH 4.1, a 5 log reduction in *S.* Typhimurium DT104 was produced by storage at 35 °C for 6 hours in the presence of 0.1% sorbic acid or by a combination of storage at elevated temperature (25 °C for 6 hours or 35 °C for 4 hours) followed by a freeze/thaw cycle without sorbic acid (Uljas & Ingham, 1999). In the field, the physical environment of vegetables surfaces is considered to be inhospitable for growth and survival of *Salmonella* (for example, temperature and humidity fluctuations, and ultraviolet light) (Dickinson, 1986). Environmental conditions, however, can greatly influence bacterial populations; the presence of free moisture on vegetable surfaces from precipitation, dew or irrigation can promote survival and growth of bacterial populations (Shaper et al., 2006). Certain conditions such as sunlight, particularly shorter ultraviolet wavelengths, can damage bacterial cells (Shaper et al., 2006); selection therefore occurs for bacteria with adaptations to stressful conditions. Microorganisms' ability to survive on plants depends on the environmental, physicochemical and genetic features of the plant and specific properties (Shaper et al., 2006*)*. Many microorganisms have developed mechanisms to attach to, survive and/or grow in microniches on different vegetables (Shaper et al., 2006). For instance, surface moisture on vegetables may provide a protective environment for *Salmonella* strains. On vegetable surfaces, microorganisms interact in aggregates and may compete for the limited nutrients available in microniches at the junction of epidermal cells, where water accumulates, cuticular waxes are less dense and nutrients are more available than in other sites (Shaper et al., 2006). Free water in the surface apertures of vegetables (e.g. stomata) constitutes a water channel connecting a plant's apoplast with its external environment. Microorganisms can enter vegetables through these water channels in various ways. Once internalized, the microorganisms are protected from environmental stress (Shaper et al., 2006). Survival of pathogenic microorganisms on or in raw produce is

**5. Interaction of** *Salmonella* **with foods** 

values (Harris, et al., 2003).

also dictated by its metabolic capabilities. However, the manifestations of these capabilities can be greatly influenced by intrinsic (e.g. vegetable moisture surface) and extrinsic ecological factors naturally present in the raw produce or imposed at one or more points during production, processing and distribution (Harris et al., 2003)*. Salmonella* strains may be able to enter a viable but nonculturable state (VBNC) on the surface of fruit and vegetables, resulting in underestimation of viable population size by direct plating on culture medium. Brandl and Mandrell (2002), suggested that *S.* Thompson may enter into a VBNC state on *Cilantro phyllosphere* due to exposure to dry pre-harvest conditions on the plant surface. Improved understanding of microbial ecosystems on the surface of foods such as raw fruits and vegetables would be extremely useful in developing strategies to minimize contamination, prevent pathogen growth, and kill or remove pathogens at different stages in production, processing, marketing and preparation for consumption. Food ecosystems are extremely diverse and complex. *Salmonella* survival and/or growth on foods are influenced by the organism, produce item and environmental conditions in the field and post-harvest, including storage conditions.

For many years, the interaction of *Salmonella* with animal hosts and animal-origin foods has received intense attention. In contrast, little research has been done on the interaction between *Salmonella* spp. and fruits and vegetables, and more specifically on its frequency and behavior in fruits and vegetables which may pose a special risk to humans [e.g. radish root (*Raphanus sativus*), beetroot (*Beta vulgaris* var. *conditiva*), jicama (*Pachyrhizus erosus*), loroco (*Fernaldia pandurata*), prickly pear (*Opuntia* spp.), zucchini squash (*Cucurbita pepo*), chili peppers (Jalapeño and Serrano peppers) and others]. It is particularly urgent to study fruits and vegetables not previously considered health hazards and those with the potential to function as pathogen microorganism vehicles but are as yet unidentified.

In a recent *Salmonella* outbreak in the US, jalapeño and serrano peppers were the food vehicle and the isolated serovar was Saintpaul (CDC, 2008). It affected at least 1,442 persons in 43 states, the District of Columbia and Canada, and was traced back to distributors in the United States which had received produce grown and packed in Mexico. The outbreak strain was isolated from samples of jalapeño peppers collected from a US warehouse and a patient's home, as well as from samples of serrano peppers and water collected from a farm in Mexico. We have studied the behavior of *Salmonella* serotypes in zucchini squash and chili peppers. In zucchini, we tested the behavior of four *Salmonella* serotypes (Typhimurium, Typhi, Gaminara and Montevideo) and a cocktail of three *Escherichia coli* strains on whole and sliced zucchini squash at 25±2 and 3-5 °C. No growth was observed for any of the tested microorganisms or the cocktail on whole fruit stored at 25±2 or 3-5 °C. After 15 days at 25±2 °C, the tested *Salmonella* serotypes had decreased from an initial inoculum level of 7 log CFU to <1 log and at 3-5 °C they decreased to approximately 2 log (Figure 1). Among the *E. coli* strains, survival was significantly higher than for the *Salmonella* strains at the same times and temperatures: after 15 days at 25±2 °C, *E. coli* cocktail strains had decreased to 3.4 log CFU/fruit and at 3-5 °C they decreased to 3.6 log CFU/fruit (Figure 1). The observed differences in survival between the *Salmonella* and *E. coli* strains on zucchini squash fruit could be due to factors such as the area inoculated, fruit ripeness and physical

and chemical characteristics of the studied fruit and strains. Different strains of *E. coli* O157:H7, *Pseudomonas*, *Salmonella*, and *Listeria monocytogenes* attach to different regions of cut lettuce leaves, indicating different and specific attachment mechanisms among different species or strains (Takeuchi et al., 2000).

The Role of Foods in *Salmonella* Infections 35

the serrano peppers and to 1.2 log on the jalapeño peppers. In contrast, when inoculated onto slices of both peppers and into the blended sauce, the *Salmonella* serotypes and *E. coli* grew: after 24 h at 25±2 °C, both bacteria types had grown to approximately 4 log CFU on the slices and 5 log CFU in the sauce (Figures 4-5). Bacterial growth was inhibited at 3-5 °C. In summary, the four tested *Salmonella* serotypes can survive on whole or sliced zucchini squash, serrano and jalapeño peppers and in sauce made of raw chili peppers, indicating them to be effective

0 0.5 1 1.5 2 2.5 3

APC *E. coli* S. Typhimurium S. Gaminara S. Typhi S. Montevideo

> Typhimurium Typhi Gaminara Montevideo *E.coli*

**Days**

0123456

**Days**

Fig. 3. Behavior of 4 *Salmonella* serotypes and a cocktail of three *E. coli* strains on whole

Fig. 2. Behavior of 4 *Salmonella* serotypes, *E. coli* and Aerobic Plate Count on zucchini slices

transmission vehicles and potential public health threats.

0

jalapeño peppers at 25±2 ° C (Castro-Rosas, 2011).

1

2

3

**log CFU/chili**

4

5

6

7

at 25±2 °C (Castro-Rosas et al., 2010).

**log CFU/squash**

Fig. 1. Behavior of 4 *Salmonella* serotypes and *E. coli* on zucchini squash at 25±2 °C (Castro-Rosas et al., 2010).

When inoculated onto zucchini squash slices and incubated at 25±2 °C, the studied *Salmonella* and *E. coli* strains grew (Figure 2). After a short lag period (approx. 4 h), the *Salmonella* and *E. coli* populations increased from 2 log to 6 log CFU/slice at 24 h, and the *E. coli* strains increased a further 1 log CFU by 72 h. Initial *Salmonella* and *E. coli* inocula levels were close to that of Aerobic Plate Count bacteria (APC) in the studied zucchini squash fruit (approx. 2.5 log CFU/slice), and the APC growth rate (7.6 log CFU/slice by 24 h; 8.9 log CFU/slice by 72 h) was comparable to the studied strains (Figure 2). The behavior of *Salmonella* under these conditions does not differ greatly from that of *Salmonella* strains in other foods. For instance, *S*. Typhimurium inoculated in shredded cooked beef and stored at 20 ºC/8 h, increased from 2.3 to 3.4 log CFU/g *(16)*, while after 22 h incubation on sliced tomatoes *S.* Montevideo increased by ca. 1.5 log CFU/g at 20 °C and 2.5 log CFU/g at 30 °C (Zhuang et al. 1995).

Under refrigeration (3-5 °C), growth in the *Salmonella* serotypes and *E. coli* strains was inhibited (Figure 4): bacterial concentration at 5 days was essentially similar to initial inocula levels. Nonetheless, survival of even a small concentration of *E. coli* and/or *Salmonella* under refrigeration poses a serious health hazard to consumers since salmonellosis outbreaks have been reported as originating in different foods at low pathogen concentrations (Greenwood and Hopper, 1983).

In a separate study, we tested the growth behavior of the same four *Salmonella* serotypes and three *E. coli* strains at the same temperatures (25±2 and 3-5 °C) on whole and sliced jalapeño and serrano peppers, as well as in a blended chili pepper sauce (Castro-Rosas et al., 2011). The sauce was an aqueous suspension containing mixed peppers, tomatoes, coriander, onion and salt (NaCl) in specific proportions. Both types of microorganisms exhibited similar behavior on/in the serrano and jalapeño peppers. No growth was observed in rifampicin-resistant *Salmonella* and *E. coli* strains on the surface of whole serrano and jalapeño peppers stored at 25±2 or 3-5 °C. After 6 days at 25±2 °C, the tested *Salmonella* serotypes and *E. coli* had decreased from an initial inoculum level of 5 log CFU to 1 log on the serrano peppers and to 2.5 log on the jalapeño peppers (Figure 3). At 3-5 °C they decreased to approximately 1.8 log in

Fig. 1. Behavior of 4 *Salmonella* serotypes and *E. coli* on zucchini squash at 25±2 °C (Castro-

When inoculated onto zucchini squash slices and incubated at 25±2 °C, the studied *Salmonella* and *E. coli* strains grew (Figure 2). After a short lag period (approx. 4 h), the *Salmonella* and *E. coli* populations increased from 2 log to 6 log CFU/slice at 24 h, and the *E. coli* strains increased a further 1 log CFU by 72 h. Initial *Salmonella* and *E. coli* inocula levels were close to that of Aerobic Plate Count bacteria (APC) in the studied zucchini squash fruit (approx. 2.5 log CFU/slice), and the APC growth rate (7.6 log CFU/slice by 24 h; 8.9 log CFU/slice by 72 h) was comparable to the studied strains (Figure 2). The behavior of *Salmonella* under these conditions does not differ greatly from that of *Salmonella* strains in other foods. For instance, *S*. Typhimurium inoculated in shredded cooked beef and stored at 20 ºC/8 h, increased from 2.3 to 3.4 log CFU/g *(16)*, while after 22 h incubation on sliced tomatoes *S.* Montevideo increased

Under refrigeration (3-5 °C), growth in the *Salmonella* serotypes and *E. coli* strains was inhibited (Figure 4): bacterial concentration at 5 days was essentially similar to initial inocula levels. Nonetheless, survival of even a small concentration of *E. coli* and/or *Salmonella* under refrigeration poses a serious health hazard to consumers since salmonellosis outbreaks have been reported as originating in different foods at low pathogen concentrations (Greenwood

In a separate study, we tested the growth behavior of the same four *Salmonella* serotypes and three *E. coli* strains at the same temperatures (25±2 and 3-5 °C) on whole and sliced jalapeño and serrano peppers, as well as in a blended chili pepper sauce (Castro-Rosas et al., 2011). The sauce was an aqueous suspension containing mixed peppers, tomatoes, coriander, onion and salt (NaCl) in specific proportions. Both types of microorganisms exhibited similar behavior on/in the serrano and jalapeño peppers. No growth was observed in rifampicin-resistant *Salmonella* and *E. coli* strains on the surface of whole serrano and jalapeño peppers stored at 25±2 or 3-5 °C. After 6 days at 25±2 °C, the tested *Salmonella* serotypes and *E. coli* had decreased from an initial inoculum level of 5 log CFU to 1 log on the serrano peppers and to 2.5 log on the jalapeño peppers (Figure 3). At 3-5 °C they decreased to approximately 1.8 log in

by ca. 1.5 log CFU/g at 20 °C and 2.5 log CFU/g at 30 °C (Zhuang et al. 1995).

Rosas et al., 2010).

and Hopper, 1983).

the serrano peppers and to 1.2 log on the jalapeño peppers. In contrast, when inoculated onto slices of both peppers and into the blended sauce, the *Salmonella* serotypes and *E. coli* grew: after 24 h at 25±2 °C, both bacteria types had grown to approximately 4 log CFU on the slices and 5 log CFU in the sauce (Figures 4-5). Bacterial growth was inhibited at 3-5 °C. In summary, the four tested *Salmonella* serotypes can survive on whole or sliced zucchini squash, serrano and jalapeño peppers and in sauce made of raw chili peppers, indicating them to be effective transmission vehicles and potential public health threats.

Fig. 2. Behavior of 4 *Salmonella* serotypes, *E. coli* and Aerobic Plate Count on zucchini slices at 25±2 °C (Castro-Rosas et al., 2010).

Fig. 3. Behavior of 4 *Salmonella* serotypes and a cocktail of three *E. coli* strains on whole jalapeño peppers at 25±2 ° C (Castro-Rosas, 2011).

The Role of Foods in *Salmonella* Infections 37

Food is clearly a major *Salmonella* infection vehicle. This vital role in salmonellosis outbreaks calls for strict measures to minimize transmission, such as appropriate animal husbandry and agriculture practices, protection of feeds and water from contamination, adequate waste disposal methods and an overall effort to maintain a clean environment around food from farm to fork. Additionally, much of the risk posed by *Salmonella* can be mitigated through proper handling and correct food safety practices, including thorough washing and disinfection, prevention of pre-consumption, human-borne contamination during preparation and storage, leftovers disposal, cooking before consumption and refrigerated storage (3-5 ºC). Continuous monitoring and generation of data on *Salmonella* and salmonellosis outbreaks, and improved surveillance measures are also vital to controlling this public health hazard. A deeper understanding of *Salmonella* and its behavior in foods is

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**6. Conclusion** 

**7. References** 

0362-028X

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still needed to ensure food safety and quality.

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(February, 2009), pp. 35-40, ISSN 19722680

Fig. 4. Behavior of 4 *Salmonella* serotypes and a cocktail of three *E. coli* strains in jalapeño peppers slices at 25±2° C (Castro-Rosas, 2011).

Fig. 5. Behavior of 4 *Salmonella* serotypes and a cocktail of three *E. coli* strains in a chili pepper sauce at 25±2 °C (Castro-Rosas, 2011).
