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.

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.

Animal and environmental impact on the presence and distribution of Salmonella and Escherichia coli in hydroponic tomato greenhouses.

From 2003 to 2004, we studied the impact of environmental influences on the microbiological quality of a hydroponic tomato farm. The presence of Salmonella was investigated on 906 samples of tomatoes and 714 environmental samples. The farm comprised 14 greenhouses and a technologically advanced packinghouse, and operated under a sanitary agricultural practices plan. The objective of the present study was to determine the operating sources of contamination. During the course of the study, two independent natural events affected the farm. In 2003, water runoff entered some of the greenhouses. A year later, wild animals (opossums, mice, and sparrows) gained entry into several of the greenhouses. Salmonella and Escherichia coli were found in samples of tomatoes, water puddles, soil, shoes, and the feces of local wild and farm animals. Salmonella Montevideo, Salmonella Newport, and strains of the F serogroup were isolated from tomatoes. Almost all of the Salmonella Newport strains were isolated from samples collected during or immediately after the flood. Analysis by pulsed-field gel electrophoresis revealed that some Salmonella Montevideo isolates from tomatoes, opossums, and mice displayed identical XbaI or AvrII patterns, suggesting that these wild animals represented one source of contamination. F serogroup strains were found mostly on samples of goat feces and personnel shoes when standard working practices were in place. Shoes were found to be an important vehicle for dissemination of Salmonella into the greenhouses. The level of protection provided by hydroponic greenhouses does not exclude the eventuality that enteric pathogenic bacteria can gain access through various avenues.

Colonization of tomatoes by Salmonella montevideo is affected by relative humidity and storage temperature.

The influences of the relative humidity (RH) and storage temperature on the colonization of tomato surfaces by Salmonella Montevideo were studied. Red, ripe tomatoes (Lycopersicon esculentum) were spot inoculated in three separate trials with 100 pl (approximately 10(6) CFU) of Salmonella Montevideo and stored for 90 min at 22 degrees C under 97% RH to facilitate attachment of cells to the blossom end of tomato surfaces. Following this attachment step, tomatoes were washed to remove loosely adhered cells and then stored at 22 or 30 degrees C for up to 10 days under RH of 60, 75, 85, or 97%. At 0, 0.4, 1, 4, 7, and 10 days of storage, three tomatoes were individually hand massaged in 50 ml of 0.1% peptone water and the washes were separately analyzed to enumerate populations of Salmonella Montevideo. The number of Salmonella Montevideo cells attached after 90 min at 22 degrees C was 3.8 log CFU per tomato; this level was determined to be the initial colonizing population. After 10 days of storage at 30 degrees C, the Salmonella Montevideo population increased to 0.7, 1.0, 1.2, and 2.2 log CFU per tomato at 60, 75, 85, and 97% RH, respectively. A similar trend was observed at 22 degrees C, although populations were lower than at 30 degrees C. Scanning electron micrographs of tomato cuticles after storage revealed a well-defined biofilm containing bacteria. These findings reinforce the importance of maintaining stored tomatoes at temperatures that do not support growth of pathogenic bacteria and demonstrate the growth-promoting effects of high humidity.

Influence of variations in methodology on populations of Listeria monocytogenes recovered from lettuce treated with sanitizers.

The elimination of Listeria monocytogenes inoculated onto a piece of cut iceberg lettuce (3.8 by 3.8 cm) by treatment with chlorinated water (200 micrograms/ml free chlorine) and a 0.5% (wt/vol) solution of FIT Professional Line Antibacterial Cleaner (FIT) was investigated. The efficacy of the two sanitizers was not influenced by the composition of the medium used to culture the L. monocytogenes used in the inocula, the number of strains in the inoculum, or the recovery medium used to enumerate the pathogen on lettuce after treatment. Drying inoculum on lettuce for 45 min at 37 degrees C caused more cells to die or not be retrieved compared with drying inoculum for 30 min at 25 degrees C. However, the percentage of cells in the inoculum recovered from lettuce treated with chlorine or FIT was not significantly different, regardless of the drying method. Stomaching, homogenizing, or stomaching followed by homogenizing lettuce treated with sanitizers resulted in recovery of similar numbers of L. monocytogenes, indicating that stomaching and homogenizing are equivalent in extracting cells; the sequential use of both processing methods did not substantially increase the efficiency of recovery. Washing lettuce with water or treating lettuce with 200 micrograms/ml chlorine or FIT resulted in decreases in populations of 0.60, 1.76, and 1.51 log CFU per lettuce piece, respectively, regardless of variations in test parameters. Reductions caused by sanitizers were significantly greater (alpha = 0.05) than that observed for water but not significantly different from each other. It is concluded that evaluation of sanitizers for their efficacy in killing L. monocytogenes on lettuce can be determined by spot inoculating 50 microliters of a five-strain mixture of cells from 24-h cultures suspended in 5% horse serum albumen, followed by drying the inoculum for 45 min at 37 degrees C, treatment by submerging in 50 ml of sanitizer for 5 min, stomaching samples in 50 ml of Dey-Engley neutralizing broth for 2 min, and enumerating survivors on modified Oxford medium.

Effect of inoculum size, relative humidity, storage temperature, and ripening stage on the attachment of Salmonella Montevideo to tomatoes and tomatillos.

The influence of inoculum populations and environmental factors on attachment of Salmonella Montevideo to the surface of tomatoes and tomatillos was evaluated. To study the effect of inoculum size, red, ripe tomatoes were spot-inoculated with bacterial suspensions (10(5) and 10(8) CFU/fruit) and stored at 22 degrees C under 100% relative humidity. The effects of temperature (12, 22, and 30 degrees C) and relative humidity (75, 85, and 97%) on attachment of the pathogen (10(7) CFU/fruit) to tomatoes (red and green) and ripe tomatillos were also evaluated. Inoculated fruits were stored for 90 min at all combinations of temperature and relative humidity, and after rinsing with water, the number of cells attached to the surface was determined. Salmonella Montevideo attached to the surface of tomatoes within 90 min. A direct correlation between the number of attached cells and the population in the inoculum was observed. The percentage of cells that attached immediately after inoculation was approximately 0.3% for the three test products. After storage for 90 min at various temperature and relative humidity conditions, the number of adhering cells ranged from 4.0 to 5.4 log CFU/fruit (1.2% of inoculum). Both the type of product and the temperature/relative humidity combination had a significant (P < 0.05) effect on attachment of Salmonella Montevideo to the surfaces of tomatoes and tomatillos. Scanning electron micrographs of the cuticles of inoculated washed tomatoes and tomatillos revealed typical skin cell patterns, and only a few randomly dispersed Salmonella Montevideo were observed. Deposition of Salmonella Montevideo on the surface of tomatoes and tomatillos could result in attachment and subsequent colonization under suitable conditions.

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.