Simultaneous and individual quantitative estimation of Salmonella, Shigella and Listeria monocytogenes on inoculated Roma tomatoes (Lycopersicon esculentum var. Pyriforme) and Serrano peppers (Capsicum annuum) using an MPN technique

Highlights

• The MPN technique was effective at quantifying the 3 pathogens simultaneously.

• The MPN technique was effective in quantifying Salmonella on both commodities.

• The MPN technique was effective for individual recovery of Shigella on tomatoes.

• The MPN technique was more effective for Salmonella than for Shigella and L. monocytogenes.

• Recovery of the pathogens by MPN technique was influenced by food matrix.

Abstract

Simultaneous and individual enumeration of Salmonella, Shigella and Listeria monocytogenes was compared on inoculated Roma tomatoes and Serrano peppers using an Most Probable Number (MPN) technique. Samples consisting of tomatoes (4 units) or peppers (8 units) were individually inoculated with a cocktail of three strains of Salmonella, Shigella or L. monocytogenes, or by simultaneous inoculation of three strains of each pathogen, at low (1.2–1.7 log CFU/sample) and high (2.2–2.7 log CFU/sample) inocula. Samples were analyzed by an MPN technique using universal pre-enrichment (UP) broth at 35 °C for 24 ± 2 h. The UP tubes from each MPN series were transferred to enrichment and plating media following adequate conventional methods for isolating each pathogen. Data were analyzed using multifactorial analysis of variance (p < 0.05) and LSD multiple rang test. There were differences (p < 0.05) in recovery of simultaneous and individual bacteria inoculated (individual > simultaneous), type of bacteria (Salmonella > Shigella and L. monocytogenes), type of sample (UP broth > pepper and tomato), and inoculum level (high > low). The MPN technique was effective for Salmonella on both commodities. Shigella counts were higher on tomatoes compared to peppers, (p < 0.05), and for L. monocytogenes on peppers (p < 0.05).

Introduction

Tomatoes and peppers are popular produce commodities in homes and food service worldwide. Both products have been recognized as potential vehicles of foodborne pathogens (Klontz et al., 2010, Olaimat and Holley, 2012). In the United States (US), 186 outbreaks were related to consumption of tomatoes and peppers during the period 1998–2016. These outbreaks resulted in 8857 illnesses and most of them were caused by nontyphoidal Salmonella, Shigella, Clostridium perfringens, Bacillus cereus, Campylobacter jejuni, Staphylococcus aureus and Norovirus (CDC, 2017).

Although several studies have reported the presence of pathogens on fresh tomatoes and peppers (Orozco et al., 2008; Castro-Rosas et al., 2011, Cárdenas et al., 2013), the quantification of foodborne pathogens on these commodities has not been reported. Other studies have reported low numbers of pathogens on fresh produce and minimally processed vegetables (Sant'Ana et al., 2011, Sant'Ana et al., 2012, Althaus et al., 2012). Finding low concentrations of pathogens on fresh produce that will be consumed raw is a matter of concern, because of the potential for multiplication and survival of pathogens such as Salmonella and Listeria monocytogenes, which has been reported on tomatoes (Beuchat and Brackett, 1991, Wei et al., 1995) and peppers (Han et al., 2001, Castro-Rosas et al., 2011, Salazar et al., 2017). Effective methods for the quantification of low levels of Salmonella, Shigella and L. monocytogenes on fresh produce are necessary to conduct prevalence studies that provide quantitative data for microbial risk assessments (MRA) in the productive chains of produce commodities.

Molecular methods for quantification of foodborne pathogens, mainly PCR-based techniques, are widely used. These methods are faster and more specific than traditional methods. However, one disadvantage of using molecular methods in food matrices is the minimum limit of quantification, which is frequently higher (up to 1 CFU/g or ml) than the level at which the pathogen is usually found (Postollec et al., 2011). The concentration of a foodborne pathogen in the analyzed food may be increased using a pre-enrichment stage; however, by doing this the initial level of the pathogen is unknown. Another disadvantage is the presence of inhibitors in the analyzed food that may affect different steps of the PCR assay (Wilson, 1997). Some of these inhibitors include phenolic compounds which are present in both tomatoes and peppers (Materska and Perucka, 2005, Barros et al., 2012). These limitations may be overcome by the application of culture-based methods such as the Most Probable Number (MPN), a sensitive method in which the initial concentration of the target microorganism can be estimated and which can be designed to detect one single target cell in samples, but requires several days to isolate and enumerate foodborne pathogens (Oblinger and Koburger, 1975). Using the MPN, the proportion between the amount of food sample analyzed and the pre-enrichment broth can be modified to dilute the inhibitors present in the food matrix, thus reducing their negative effect on the pathogens detection.

Numerous studies have used different pre-enrichment broths to detect Salmonella, Shigella and L. monocytogenes in food samples (Nam et al., 2004, Warren et al., 2006, Hammack et al., 2005, Hammack et al., 2008, Jacobson et al., 2017). Universal Pre-enrichment (UP) broth is used for recovering sub-lethally injured Salmonella and Listeria from food products (Bailey and Cox, 1992). This medium allows bacteria to resuscitate and grow. The UP broth is highly buffered, mildly acidic (pH 6.3), low in carbohydrates, and prevents the pH of the medium from dropping rapidly in the presence of microorganisms found in foods. Nevertheless, no studies have included its use as diluent to revitalize cells of Shigella in foods.

The use of methods to simultaneously detect and quantify two or more pathogens in one single assay have been reported (Jofré et al., 2005, Kim et al., 2006, Kawasaki et al., 2009, Garrido et al., 2013a, Garrido et al., 2013b). This approach is economically favorable because it reduces the total space, the amount of reagents, and the labor needed for handling a large number of samples. The aim of this study was to evaluate an MPN technique for the simultaneous and individual enumeration of Salmonella, Shigella and L. monocytogenes on inoculated Roma tomatoes (Lycopersicon esculentum var. Pyriforme) and Serrano peppers (Capsicum annuum) using UP broth as a single pre-enrichment.