Occurrence of Escherichia coli, Campylobcter, Salmonella and Shiga-Toxin Producing E. coli in Norwegian Primary Strawberry Production.

The aim of this study was to investigate the bacteriological quality of strawberries at harvest and to study risk factors such as irrigation water, soil and picker's hand cleanliness. Four farms were visited during the harvest season in 2012. Samples of strawberries, irrigation water, soil and hand swabs were collected and analyzed for E. coli, Campylobacter, Salmonella and STEC Although fecal indicators and pathogens were found in environmental samples, only one of 80 samples of strawberries was positive for E. coli (1.0 log10 cfu/g) and pathogens were not detected in any of the strawberry samples. The water samples from all irrigation sources were contaminated with E. coli in numbers ranging from 0 to 3.3 log10 cfu/g. Campylobacter (8/16 samples) and Salmonella (1/16 samples) were isolated from samples with high numbers of E. coli. The water samples collected from a lake had lower numbers of E. coli than the samples from rivers and a stream. The present study indicated continuous background contamination in the primary production environment. Although the background contamination was not reflected on the strawberries tested here, the results must be interpreted with caution due to the limited number of samples.

Survival of Salmonella on basil plants and in pesto.

Leafy greens, including fresh herbs, have repeatedly been involved in outbreaks of foodborne disease. Although much effort has been put into studying leafy greens and products such as head lettuce and baby leaves, less is known about fresh leafy herbs, such as basil. The goal of this study was to investigate the survival of Salmonella on basil plants and in pesto. A mix of three Salmonella strains (Reading, Newport, and Typhimurium) was inoculated onto basil leaves and pesto and survived during the experimental period. Whereas the mix of Salmonella survived in pesto stored at 4°C for 4 days, Salmonella was recovered from inoculated leaves for up to 18 days at 20 to 22°C. Although the steady decline of Salmonella on leaves and in pesto suggests a lack of growth, it appears that pesto is a hostile environment for Salmonella because the rate of decline in pesto was faster (0.29 log CFU/g/day) than on leaves (0.11 log CFU/g/day). These findings suggest that the dilution of contaminated ingredients and the bactericidal effect of the pesto environment helped to further reduce the level of enteric organisms during storage, which may have applications for food safety.

Development of performance objectives for Listeria monocytogenes contaminated salmon (Salmo salar) intended used as sushi and sashimi based on analyses of naturally contaminated samples.

Raw salmon is commonly used in sushi and sashimi, indicating that fresh salmon may be considered as a ready-to-eat product. Listeria monocytogenes is occasionally present in fresh salmon, but studies of prevalence and growth of the bacterium in this matrix have been few. In the present study, salmon from a company where L. monocytogenes is present in low levels has been investigated in order to develop performance objectives for L. monocytogenes in fresh salmon intended for use in ready-to-eat products as sushi and sashimi. According to the European Food Law, the maximum level of L. monocytogenes on the last day of shelf life is 100 cfu/g. The variations between and within eight batches have been determined, and the results were used to estimate limit values for L. monocytogenes in salmon and develop a tentative sampling plan for the processing day. Various time-temperature scenarios for storage until the fish is consumed as sushi, sashimi or native fillets have been taken into account. The results indicate that limit values in the range from 0.5 to 10 cfu/g are sufficient to ensure that the regulatory limit of <100 cfu/g of L. monocytogenes on the last day of shelf life is not exceeded, provided that the recommended time-temperature conditions are respected. For instance, if the fish is intended for processing into sushi within one week of storage at 4°C after filleting, no samples should have higher levels of L. monocytogenes than 2 cfu/g at the day of filleting.

The effect of blast chilling on occurrence of human pathogenic Yersinia enterocolitica compared to Campylobacter spp. and numbers of hygienic indicators on pig carcasses.

In this study, the occurrence of Yersinia enterocolitica on pig carcasses was compared to the occurrence of Campylobacter spp., and to the numbers of aerobic micro-organisms, coliform bacteria, thermotolerant coliform bacteria, and Escherichia coli before and after blast chilling. Y. enterocolitica O:3/biovar 4 was isolated from five (8.3%) of 60 carcasses before blast chilling, and also from five of them 1 h after blast chilling. Therefore this procedure does not seem to have a significant effect on the occurrence of pathogenic Y. enterocolitica on pig carcasses. Y. enterocolitica O:9/biovar 2 was isolated from a pig source in Norway for the first time when this sero/biovariant was isolated from one of the carcasses before blast chilling. Campylobacter spp. was isolated from 34 (56.7%) of 60 carcass samples before blast chilling. After blast chilling Campylobacter spp. was isolated from only one (1.7%) of the 60 carcasses. There was a significant decrease of the numbers of coliform bacteria, thermotolerant coliform bacteria and E. coli after blast chilling. The number of aerobic micro-organisms did not decrease after this step. In contrast to the drastic decrease in the occurrence of campylobacter-positive carcasses and the significant decrease of the numbers of coliform bacteria, thermotolerant coliform bacteria and E. coli, blast chilling does not seem to have a significant effect on the occurrence of human pathogenic Y. enterocolitica on pig carcasses.

Testing of pathogenic Yersinia enterocolitica in pig herds based on the natural dynamic of infection.

This study was performed to evaluate testing methods of pathogenic Yersinia enterocolitica in pigs at different ages. Relevant tools and procedures are crucial if pig herds should be declared free from pathogenic Y. enterocolitica. Historical data based on serology showed that the two farms investigated in this study (herds A and B) were contaminated with Y. enterocolitica O:3 since at least 1995. Laboratory investigations of 60 pigs were sampled one to four times (herd A) and 20 pigs were sampled one to three times (herd B) at different ages were the basis for this report. The following testing procedures could be used to conclude that a herd is free from pathogenic Y. enterocolitica:--serological testing of pigs could be performed as a basis for categorisation for all ages from about 100 days including at slaughter when the pigs are 150-180 days old, --bacteriological examination of faeces could be used as a basis for categorisation at all ages from 85 days until about 135 days, --bacteriological examination of tonsils could be used as a basis for categorisation at all ages from 85 days including at slaughter when the pigs are 150-180 days old. However, due to animal welfare aspects, one should avoid sampling of tonsils. Accordingly, the serological method or bacteriological examination of faeces at relevant ages should be preferred. One aspect related to slaughter hygiene is that in pigs slaughtered at the age of 135 days or more, the tonsils may be a more significant source of human pathogenic Y. enterocolitica than faeces.

Occurrence of Yersinia enterocolitica and Campylobacter spp. in slaughter pigs and consequences for meat inspection, slaughtering, and dressing procedures.

The purpose of the present investigation was to assess the occurrence of Yersinia enterocolitica and Campylobacter spp. in the lymphoid tissues and intestinal tract in pigs and the risk for contamination during the compulsory meat inspection procedures and the procedures during slaughtering and dressing. Another objective of the investigation was to compare traditional isolation methods, the use of a polymerase chain reaction (PCR) method (BUGS'n BEADS bacterial DNA isolation kit) and an ELISA method (VIDAS CAM) as tools in risk management in the slaughterhouse. The results indicate that the compulsory procedure for the incision of the submaxillary lymph nodes represents a cross-contamination risk for virulent Yersinia. In the screening of 97 animals in 1999, 5.2% of the samples were positive, and by the sampling of 24 samples in 2000-2001, 12.5% of the samples were positive. In the last case, Y. enterocolitica O:3 was found in the kidney region in one of the subsequent carcasses that was only touched by the meat inspection personnel before sampling. In addition, incision of the mesenteric lymph nodes might represent a cross-contamination risk since 8.3% of the samples were positive. The association between antibody titres and the occurrence of virulent yersiniae in the tonsils (21-18) was striking, with virulent yersiniae found in the tonsils in most pigs with high titres. The contents of the stomach, ileum, caecum, and colon also represent contamination risks for Y. enterocolitica O:3 if the slaughterhouse personnel cuts into the viscera with their knives by accident; the frequency of virulent Yersinia varied from 4.2% to 16.7% within these sections. Campylobacter was detected in the gastrointestinal tract of all pigs, and the high contamination of tonsils (66.7%) and intestinal tract (100%) might represent an occupational health hazard. There was no statistical difference between the traditional method for isolation of Y. enterocolitica [International Organization for Standardization, 1994. Microbiology-General Guidance for the Detection of Presumptive Pathogenic Yersinia enterocolitica (ISO 10273). International Organization for Standardization, Genève, Switzerland (16 pp.)] and the BUGS'n BEADS detection method for virulent Y. enterocolitica. Likewise, there was no statistical difference between the traditional method for isolation of Campylobacter spp. [Nordic Committee on Food Analysis, 1990. Campylobacter jejuni/coli. Detection in Food. Method No. 119, 2nd ed. Nordic Committee on Food Analysis, Esbo (7 pp.)] and the BUGS'n BEADS detection method or the VIDAS CAM method for detection of Campylobacter spp.

Contribution of Composition, Physicochemical Characteristics and Polyphosphates to the Microbial Safety of Pasteurized Cheese Spreads.

Pasteurized process cheese spread was manufactured with moisture contents of 52, 54, 56 and 60%. Three different types of phosphate emulsifier were used, disodium ortho-phosphate and two commercially-available polyphosphates, S9 and S9H. Pasteurized, processed cheese spreads were inoculated with approximately 1 × 104 Clostridium botulinum spores/gram cheese in the cook kettle, held 3 min at 80°C, hot-filled into glass containers, and incubated at 30°C. Samples were analyzed over 30 weeks for growth of C. botulinum and toxigenesis. Toxin was first detected in 60% moisture cheese with disodium ortho-phosphate as the emulsifier at 8 weeks and in 60% moisture cheese with the test polyphosphates as the emulsifier when tested at 20 weeks. None of the other cheese formulations were toxic at 20 weeks. Toxin production correlated statistically to time, moisture, pH and phosphate type.

Enhanced Recovery and Isolation of Salmonella and Listeria Using a Novel Culture-Transfer-Inoculation Device.

A novel culture-transfer-inoculation device (CTID), comprised of a fiber-foam matrix suspended in the enrichment medium and thereafter used in subsequent transfers and inoculations, was used in the analysis of foods for Salmonella using the Food and Drug Administration (FDA) method and for Listeria using the FDA and United States Department of Agriculture (USDA) procedures. In these studies, the food sample was introduced into the enrichment medium including the CTID. Following incubation, transfer from the primary enrichment culture and subsequent transfers were made by both conventional procedures and by the CTID. Four-hundred-and-twenty-five samples were analyzed for Salmonella of which 115 were positive. Using conventional transfer and inoculation techniques, 87 positive results were observed; whereas using the CTID-modified technique, 112 positive results were obtained. The false-negative rate for the conventional transfer method was 21.7%. A 2.6% false-negative rate was observed with the CTID. The difference between conventional and CTID results were statistically significant (p<0.0160). One-hundred samples were analyzed for Listeria using the FDA method with 32 positive results. Using the CTID 42 positives were detected from the FDA enrichments. One-hundred-and-ninety-eight samples were analyzed using the USDA method with 77 positive results. With the CTID, the same samples yielded 86 positives. No false-negatives resulted through the use of the CTID while a 10.5% to 23.8% rate was observed with the conventional transfer method. The difference between conventional USDA and CTID-modified USDA results were statistically significant (p<0.0086). These data indicate that the fiber-foam matrix of the CTID provides a micro-environment enhancing the sensitivity of the culture methods for the detection of Salmonella and Listeria .

Comparison of Resistance to Microbial Contamination of Conventional and Modified Water Dispensers.

Water dispensers with traditional, open reservoirs were tested comparatively against modified, closed reservoirs and modified caps to determine their failure to resist contamination from exterior surfaces of water bottles and aerosols. Bottle rims and caps were surface inoculated with Escherichia coli (SLR 51), Staphylococcus aureus (SLR 717), and Pseudomonas fluorescens (ATCC 13525) at levels approximating 1 × 107 to 1 × 108 cells for a high level and 1 × 103 cells for a low level. The bottles contained 2,000 ml of water. After mounting the bottles on the appropriate water dispenser type, all water was withdrawn from the units 24 h later. Levels of test organisms recovered ranged from 5.0 × 103 to 4.5 × 105 CFU/ml for the high inoculum and 0.39 to 0.84 CFU/ml for the low inoculum in the traditional water dispensing unit. No test organisms (detection limit <0.01 CFU/ml) were recovered from the modified water dispenser for either high or low inoculum level when the unit was sanitized between trials. Test microorganisms were recovered at levels of ≈ 0.06 CFU/ml to ≈0.8 CFU/ml after 3-5 repetitions of very high level (3 × 108 CFU/ml) inoculation with E. coli and S. aureus without sanitization between trials. An aerosol of 2.02 × 109 CFU E. coli per ml was generated in a chamber directly over the water dispensers without bottles mounted for one trial. The modified water dispenser reduced aerosol contamination by 100- to 1,000-fold.

Fluorometric Analysis of Alkaline Phosphatase Inactivation Correlated to Salmonella and Listeria Inactiviation.

The correlation between alkaline phosphatase (ALP) inactivation and bacterial pathogen inactivation in raw milk was studied using a newly Association of Official Analytical Chemists-approved fluorometric assay (AOAC 991.24). Fresh, raw milk was inoculated with Listeria monocytogenes Scott A and Salmonella senftenberg 775W at levels of approximately 1 × 104 and 1 × 106 CFU/g milk, respectively. Milk was heat treated to target temperatures of 63 ± 0.5°C, 65 ± 0.5°C, 67 ± 0.5°C, 68 ± 0.5°C, or 71 ± 0.5°C in five trials. The D values calculated for S. senftenberg 775W ranged from 4.6 at 63°C to 0.17 at 71 °C with z values of 5.0 to 6.7. The D values calculated for L. monocytogenes Scott A ranged from 8.4 at 63°C to 0.19 at 71°C with z value of 4.8 to 6.1. Concomitantly, ALP concentration was monitored using a fluorometric assay. The inactivation rates of the test microbes were greater than or equal to that of alkaline phosphatase over the temperature range tested. The fluorometric assay exhibited excellent accuracy, precision, reproducibility, and repeatability under the test conditions. Viable test pathogens were isolated from milk samples with ALP levels corresponding to legal pasteurization requirements of < 500 milli-Units/L ALP activity assayed fluorometrically (=1.0µg phenol per mL/15 min) when inoculated with 1 × 106 CFU/g milk.

Potential for the Low-Temperature Pasteurization of Dairy Fluids Using Membrane Processing.

Raw milk, reconstituted skim milk, skim milk, sweet whey, and acid whey were membrane processed on different units from several manufacturers using various membranes with pore sizes ranging from nanofiltration through microfiltration. The bulk fluids were inoculated with either Staphylococcus aureus 196E, Salmonella typhimurium var. Hillfarm, and/or Pseudomonas fragi 4973. In addition, indigenous microorganisms were present. The permeate and retentate streams were monitored for bacterial numbers. Percent total solids of the permeate streams was determined. Temperatures and pressures were controlled. In no cases were the bacteria completely retained while concomitantly allowing permeated solids to equal the solids in the original bulk fluid. Findings indicated different membranes of same molecular weight cut-off exhibited dissimilar bacterial retention characteristics. Unit design/configuration appeared to play a role in retention of bacteria. Spiral wound microfiltration and ultrafiltration membranes reduced bacterial loads in the permeate by 98.9 to 99.99% while allowing 5 to 6% of the solids in the bulk fluid to pass through the membrane. The bulk fluid does not appear to affect the bacterial retention. The different wheys, milks, and reconstituted skim milk showed similar reductions in bacterial numbers when microfiltered or ultrafiltered through the same type of membrane. All three test microbes demonstrated similar declines during membrane processing. It appeared that bacterial morphology and size did not affect the bacterial retention characteristics. Results indicated that low-temperature membrane processing will not eliminate all microorganisms in the permeate nor did all milk components pass through the membrane into the permeate.

Comparison of Salmonella Bio-EnzaBead Immunoassay Method and Conventional Culture Procedure for Detection of Salmonella in Foods.

A rapid enzyme immunoassay screening procedure (EIA) utilizing two monoclonal antibodies specific for salmonellac was compared to the standard culture method (BAM/AOAC) on 1,289 samples representing 26 food types. The samples consisted of 760 artificially inoculated, 150 naturally contaminated, and 379 uninoculated food samples. There were 594 samples positive by the EIA (optical densities greater than 0.2 at 405 nm), of which 568 were confirmed culturally from M-broth. A total of 570 samples was positive by the BAM/AOAC procedure. Of the foods tested, there was no significant difference between the two methods, with the exception of cake mix and raw shrimp. The EIA was significantly better for detecting Salmonella in cake mix, while the culture procedure was more productive for shrimp. The method employed a 24 ± 2-h preen-richment, an 18-h selective enrichment, and a 6-h M-broth post-enrichment. The EIA assay required an additional 2 h for a total of 48 h, compared to a minimum of 4 d by BAM/AOAC.