Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Attachment and colonization of Salmonella on 'Rayada', 'Golden Delicious', and 'Red Delicious' apples.

BACKGROUND: Fruits and vegetables have been associated with outbreaks of disease in different countries. The apple (Malus domestica Borkh) and its products have been reported as vehicles for illness outbreaks. To create strategies to prevent pathogen survival it is necessary to understand how pathogens persist on fruit. This paper assessed the ability of Salmonella to attach to, and to colonize, the surface of three apple cultivars: 'Rayada', 'Golden Delicious' and 'Red Delicious'. RESULTS: Salmonella was able to colonize and generate biofilms on the surface of apples with a soil suspension as the only source of nutrients. Significant differences in Salmonella attachment were seen among the three cultivars of apple studied. Using SEM, attached cells and the formation of exopolysaccharides and biofilms on the three apple cultivars were demonstrated. In all cultivars, the development of Salmonella was only seen in apples stored at 15 and 22 °C, with average increases in the population of 1.4 and 2.3 Log CFU/apple, respectively. At 5 °C, Salmonella growth was inhibited. CONCLUSION: Salmonella can colonize apple surfaces under environmental conditions (relative humidity, temperature and nutrients) occurring in primary apple production. © 2018 Society of Chemical Industry.

Enological Qualities and Interactions Between Native Yeast and Lactic Acid Bacteria from Queretaro, Mexico.

Despite the importance of strain compatibility, most of the enological strain selection studies are carried out separately on yeasts and lactic acid bacteria (LAB). In this study, the enological traits and interactions between native yeasts and LAB were studied. The H2 S and acetic acid production, growth rates at 8 °C, killer phenotypes, flocculation, and tolerance to must and wine inhibitors were determined for 25 Saccharomyces yeasts. The ability to grow under two wine-like conditions was also determined in 37 LAB (Oenococcus oeni and Lactobacillus plantarum). The yeast-LAB compatibility of selected strains was tested in a sequential scheme. Finally, microvinification trials were performed using two strains from each group to determine the efficiencies and quality parameters. The phenotypic characterization by the K-means and hierarchical clusters indicated a correlation between flocculation and optical density increase in simulated must and wine medium (r = -0.415) and grouped the prominent yeasts SR19, SR26, and N05 as moderately flocculent, killer, acid producing, and highly tolerant strains. Among the LAB, L. plantarum FU39 grew 230% more than the rest. With regard to interactions, LAB growth stimulation (14-fold on average) due to the previous action of yeasts, particularly of SR19, was observed. The final quality of all wines was similar, but yeast SR19 performed a faster and more efficient fermentation than did N05, Also L. plantarum FU39 fermented faster than did O. oeni VC32. The use of quantitative data, and multivariate analyses allowed an integrative approach to the selection of a compatible and efficient pair of enological yeast-LAB strains. PRACTICAL APPLICATION: An alternative scheme is proposed for the joint selection of yeast and lactic acid bacteria strains, which allows us to foresee the interactions that may occur between them during winemaking. The kinetic parameters, turbidimetrically measured and analyzed by multivariate methods, simplify the detection of outstanding selectable microorganisms. This methodology can be implemented at any cellar or even any fermentative industry that aims to select compatible yeast and lactic acid bacteria.

Microbiological Quality and Incidence of Salmonella on Cherry Tomatoes at Retail in Querétaro, México.

Multiple outbreaks related to Salmonella in tomatoes require an evaluation of the risk associated with cherry tomatoes due to the increase in its production, consumption, and marketing in Mexico's central region. The purpose of this study was to determine the microbial quality of cherry tomatoes obtained from two retail sale points (supermarkets and local markets). Cherry tomato samples (333) were collected from four supermarkets and from four local markets, and the contents of aerobic plate count, molds and yeasts, total coliforms, and Escherichia coli were quantified; the presence of Salmonella was simultaneously determined. The median values of the microbial populations were obtained, and the data were analyzed per the sampling site by using the Wilcoxon and Kruskal-Wallis tests. The median of aerobic plate count content in tomatoes obtained from supermarkets ranged between 2.2 and 4.4 log CFU/g, and in markets from 2.9 to 4.8 log CFU/g. For molds and yeasts, the tomatoes from supermarkets (2.0 to 4.1 log CFU/g) and markets (1.5 to 4.5 log CFU/g) showed similar contents. Regardless of the sampling site, the values of total coliforms were very low, ranging from 1.0 to 1.8 log CFU/g. E. coli was detected in 5.4 and 20.1% of samples from supermarkets and markets, respectively; in both sites, the content was low (0.3 to 5.8 most probable number per g). The incidence of Salmonella was 14.1% in supermarkets and 7.8% in local markets. The results obtained from this investigation highlight the elevated risk for consumer health associated with the ingestion of cherry tomatoes.

An Improve Protocol for PCR Using LM1 and LM2 Primers for Listeria monocytogenes Detection in Food Matrices.

Several studies have observed that a conventional PCR protocol using primers LM1 and LM2 for the identification of gene hlyA Listeria monocytogenes generates non-specific PCR amplifications and false positives. For this reason in this study, we provide a modified PCR protocol that improves the specificity of the LM1 and LM2 primers.

Dynamic light scattering: A fast and reliable method to analyze bacterial growth during the lag phase.

A comparison between plate counting (PC) and dynamic light scattering (DLS) is reported. PC is the standard technique to determine bacterial population as a function of time; however, this method has drawbacks, such as the cumbersome preparation and handling of samples, as well as the long time required to obtain results. Alternative methods based on optical density are faster, but do not distinguish viable from non-viable cells. These inconveniences are overcome by using DLS. Two different bacteria strains were considered: Escherichia coli and Staphylococcus aureus. DLS was performed at two different illuminating conditions: continuous and intermittent. By the increment of particle size as a function of time, it was possible to observe cell division and the formation of aggregates containing very few bacteria. The scattered intensity profiles showed the lag phase and the transition to the exponential phase of growth, providing a quantity proportional to viable bacteria concentration. The results revealed a clear and linear correlation in both lag and exponential phase, between the Log10(colony-forming units/mL) from PC and the Log10 of the scattered intensity Is from DLS. These correlations provide a good support to use DLS as an alternative technique to determine bacterial population.

Distribution of Native Lactic Acid Bacteria in Wineries of Queretaro, Mexico and Their Resistance to Wine-Like Conditions.

Native lactic acid bacteria (LAB) are capable of growing during winemaking, thereby strongly affecting wine quality. The species of LAB present in musts, wines during malolactic fermentation (MLF), and barrels/filters were investigated in wineries from the emerging wine region of Queretaro, México using multiplex PCR and culture. The resistance to wine-like conditions (WLC): ethanol (10, 12, and 13%), SO2 (30 mg⋅l-1), and low pH (3.5) of native LAB strains was also studied. Five species were detected within 61 samples obtained: Oenococcus oeni, Lactobacillus plantarum, Pediococcus parvulus, Lactobacillus hilgardi, and Lactobacillus brevis. Four species (excepting L. brevis) were found in must; O. oeni and P. parvulus were ubiquitous in wine and L. plantarum and L. brevis were mainly present at the initial stage of MLF, while L. hilgardii was mostly detected at the advanced stage. Furthermore, some species detected in barrel/filter, prove them to be hazardous reservoirs. From 822 LAB isolates, only 119 resisted WLC with 10% ethanol; the number of strains able to grow in WLC with 13% ethanol decreased approximately by 50%, O. oeni being the most versatile species with 65% of resistant isolates, while Lactobacillus spp. and P. parvulus were the most strongly affected, especially those recovered from barrel/filter, with less than 10% of resistant isolates. This study evidences the presence of local strains able to be used as starter cultures, and also enabled the assessment of the risks derived from the presence of spoilage LAB strains resistant to WLC.

Inactivation of spoiling microorganisms in apple juice by a combination of essential oils' constituents and physical treatments.

A combination of different hurdles, such as mild heat (54 ℃ for 10 min) or pulsed electric field (25 pulses; 25 kV/cm; 3.35 kJ/cm per pulse) treatments and essential oils constituents (carvacrol, citral, and (+)-limonene), to reduce spoiling bacteria and yeasts in apple juice was evaluated. For this purpose, the heat and pulsed electric field resistances of five strains of Leuconostoc spp. and five Saccharomyces spp. strains were assayed, achieving different inactivation levels for each treatment and strain. For instance, Leuconostoc fallax 74, the most heat-resistant strain, was the second-most sensitive strain to pulsed electric field. The most resistant strains were exposed to combined processes of heat or pulsed electric field and 0.2 µl/ml essential oils constituents. The combination of heat and essential oils constituents proved to be synergistic against both microorganisms in apple juice. The most effective was the combination of mild heat and carvacrol, which caused the inactivation of 99% of L. fallax 74 and 99.99% of Saccharomyces cerevisiae CECT 1172 cells. Therefore, this study shows the great potential of carvacrol, citral, and (+)-limonene in combined treatments with mild heat to achieve a higher degree of inactivation of spoiling microorganisms in apple juice, and thus, to extend its shelf life.

Investigation on the Factors Affecting Cronobacter sakazakii Contamination Levels in Reconstituted Powdered Infant Formula.

INTRODUCTION: Certain strains of Cronobacter sakazakii can cause serious invasive infections in children, mainly those <2 months old and fed with powdered infant formula (PIF). The infectious dose of C. sakazakii is unknown but evidence suggests that it is approximately 1000 colony forming units (CFU). PIF is currently considered safe if its end-product C. sakazakii level is <1 CFU/g. In this study, we determined the lag time, generation time (GT), and growth rate of five pooled C. sakazakii isolates to evaluate the factors affecting contamination levels in reconstituted PIF. METHODS: 1.71 log CFU/ml of C. sakazakii were inoculated into 100 and 3000 ml of reconstituted PIF and incubated at 22 and 35°C. Growth was evaluated over a 24-h period. ComBase was used for modeling. RESULTS: In 3000 ml, the growth rate was 0.45 ± 0.02 log CFU/h with a lag phase of 3 ± 0.05 h and GT of 0.67 h at 22°C, while the growth rate was 0.73 ± 0.01 log CFU/h with a lag phase of 0.45 ± 0.03 h and GT of 0.41 h at 35° C. CONCLUSION: Cronobacter sakazakii grows rapidly in reconstituted PIF, especially at 35° C.

Microbiological profile and incidence of Salmonella and Listeria monocytogenes on hydroponic bell peppers and greenhouse cultivation environment.

The aim of this study was to generate information regarding the microbiological profile, including Salmonella and Listeria monocytogenes incidence, of hydroponically grown bell peppers and materials associated with their production in greenhouses located in Mexico. Samples of coconut fiber (24), knives (30), drippers (20), conveyor belts (161), pepper transportation wagons (30), air (178), water (16), nutrient solution for plant irrigation (78), and bell pepper fruits (528) were collected during one cycle of production (2009 to 2010) for the quantification of microbial indicators (aerobic plate counts [APC], molds, coliforms, and Escherichia coli) and the detection of Salmonella and L. monocytogenes. With regard to surfaces (conveyor belts and wagons) and utensils (knives and drippers), the APC, coliform, and mold counts ranged from 3.0 to 6.0, from 1.4 to 6.3, and from 3.6 to 5.2 log CFU/100 cm(2) or per utensil, respectively. The air in the greenhouse contained low median levels of APC (1.2 to 1.4 log CFU/100 liters) and molds (2.2 to 2.5 log CFU/100 liters). The median content of APC and coliforms in water were 0.5 log CFU/ml and 0.3 log MPN/100 ml, respectively. The median content of coliforms in nutrient solution ranged from 1.8 to 2.4 log MPN/100 ml, and E. coli was detected in 18 samples (range, <0.3 to 1.2 log MPN/100 ml). On bell pepper analyzed during the study, populations (median) of APC, coliforms, and molds were 5.4, 3.6, and 5.8 log CFU per fruit, respectively; E. coli was detected in 5.1% of the samples (range, 0.23 to 1.4 log MPN per fruit). Salmonella was isolated from only one sample (1.6%) of conveyor belt located at the packing area and in four bell pepper samples (3%). L. monocytogenes was not detected. This information could help producers to establish effective control measures to prevent the presence of foodborne pathogens in bell peppers based on a scientific approach.

Effect of acid shock with hydrochloric, citric, and lactic acids on the survival and growth of Salmonella typhi and Salmonella typhimurium in acidified media.

The effect of acid shock with hydrochloric, citric, or lactic acid on the survival and growth of Salmonella Typhi and Salmonella Typhimurium in acidified broth was evaluated. Salmonella serovars were acid shocked (1 h at 35 degrees C) in Trypticase soy broth acidified with hydrochloric, citric, or lactic acid at pH 5.5. Unshocked cells were exposed to the same media that had been neutralized before use to pH 7.0. Shocked and unshocked cells were inoculated into broth acidified with hydrochloric acid (pH 3.0), citric acid (pH 3.0), or lactic acid (pH 3.8), and growth and survival ability were evaluated. The acid shock conferred protection to Salmonella against the lethal effects of low pH and organic acids. The adaptive response was not specific to the anion used for adaptation. The biggest difference in reduction of survival between shocked and unshocked strains (approximately 2 log CFU/ml) was observed when the microorganisms were shocked with lactic acid and then challenged with citric acid. Salmonella Typhi was more tolerant of citric acid than was Salmonella Typhimurium, but Salmonella Typhimurium had higher acid tolerance in response to acid shock than did Salmonella Typhi. The acid shock decreased the extension of the lag phase and enhanced the physiological state values of Salmonella Typhi and Salmonella Typhimurium when the pH of growth was 4.5. This increased ability to tolerate acidity may have an important impact on food safety, especially in the case of Salmonella Typhi, given the very low infectious dose of this pathogen.

Behavior of Listeria monocytogenes in avocado pulp and processed guacamole.

The potential ability of Listeria monocytogenes to grow or survive in avocado pulp (AP) and processed guacamole (PG) stored at 22, 4 to 7, and -18 degrees C was studied. Both products were obtained from a factory in Michoacan, Mexico. PG consisted of AP mixed with dehydrated vegetables, antioxidants, and preservatives. Populations of L monocytogenes in AP stored at 22 degrees C increased from 2 to 6 and 9 log CFU/g after 24 and 48 h, respectively. At 4 to 7 degrees C, the growth rate of L monocytogenes in AP was greatly decreased; generation time was 8.2 h, in contrast with 1.35 h observed at 22 degrees C. L. monocytogenes populations did not increase in PG either at 22 degrees C for 48 h or at 4 to 7 degrees C for 15 days. The bacteriostatic effect in PG may have resulted from the presence of added substances, especially citric acid and disodium dihydrogen pyrophosphate. Aerobic plate counts and coliforms increased in AP and PG stored at ambient temperature and under refrigeration. However, these increments did not affect the growth of the pathogen. L. monocytogenes (50,000 most probable number [MPN]/g) survived at least 58 weeks in both products stored frozen at -18 degrees C; the final population was 335 MPN/g in AP and 23 MPN/g in PG. Although the composition of avocado fruit differs significantly (high content of lipids and scarcity of simple carbohydrates) from that typical of most fruits, these results underline AP as a potential vehicle of human listeriosis and indicate that freezing should not be used as the sole mechanism to control this pathogen.