Fresh and retail eggs were exposed to luminescent S. enteritidis cultures containing from 104 to ... more Fresh and retail eggs were exposed to luminescent S. enteritidis cultures containing from 104 to 109 CFU/ml at either room temperature (approximately 21°C) for 3 days or 40°C for 16 h. The entry of S. enteritidis through egg shell was evidenced by luminescence in the eggs which was visualized using an Image Quantifier. The rate of contamination of the eggs increased with increasing inoculum size. Scanning electron microscopy was used to confirm the position of S. enteritidis cells in the eggs. The survival rate of the Salmonella cells in liquid eggs and whole shell eggs during storage at 4°C was investigated. Although S. enteritidis did not grow in eggs during storage at 4°C for up to 8 weeks, cells were able to survive. Under these storage conditions, the count was reduced by 1.7 to 2.5 log cycles per g in liquid egg and 0.8 to 1.4 log cycle per g in whole shell eggs. Similar trends were observed using both plate count and luminescence to monitor survival.
The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, ph... more The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, phloxine B, and malachite green (oxalate salt) at concentrations of 5 to 5,000 μg/ml against two gram-negative strains (Escherichia coli LJH 128 and Salmonella Typhimurium C1058), two gram-positive strains (Bacillus sp. C578 and Listeria monocytogenes LJH 375), and yeast (Saccharomyces cerevisiae C1172) was investigated. Incubation of the investigated bacteria with acriflavine neutral under illumination resulted in a significant reduction in cell numbers compared with dark incubation. Rose bengal caused a significant killing effect for bacteria incubated both in the dark and under illumination. Malachite green was active against gram-positive bacteria under illumination and did not affect gram-negative bacteria or yeasts. Incubation with phloxine B resulted in a significant decline in cell numbers for gram-positive bacteria, both in the dark and under illumination; gram-negative bacteria a...
Response surface analysis was used to determine the effects and interactions of water activity (0... more Response surface analysis was used to determine the effects and interactions of water activity (0.965 to 0.995), pH (5.8 to 8.0), temperature (6 to 38°C), glucose concentration (0 to 1.8%), and starch concentration (0 to 0.625%) on the growth of and toxin production by a psychrotrophic strain of Bacillus cereus in brain heart infusion broth. Growth was measured by monitoring optical density and plate counts, toxin production was assayed by an immunological method (BCET-RPLA toxin assay) and cytotoxicity with Vero and HEp-2 cells. Regressions were performed using response surface techniques, on Growth, LnGrowth, RPLA, LnRPLA, Vero, LnVero, and HEp-2; quadratic predictive equations for growth and toxin production were obtained. The results indicate the factors that had the greatest influence on both growth and toxin production were water activity and temperature. Predicted values obtained from the model were in good agreement with experimental values.
Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens
Proper sample preparation is a precondition for advanced laboratory diagnostics. The newer method... more Proper sample preparation is a precondition for advanced laboratory diagnostics. The newer methods that claim less time to reach a result have a major limitation with how much volume they can take prior to the rapid analysis for the target molecule. Among these approaches, the use of signal amplification techniques has received attention. Another key issue is automation, where the key drivers are miniaturization and multiple testing. Quantification by real-time PCR is an established technique. Recent developments in real-time PCR have made it possible to carry out high-throughput source tracing for routine purposes in the food industry. The genomics technologies have reached a stage where they are used on a routine basis in many laboratories. These technologies are expected to move towards culture-independent detection and characterization techniques based on the purification of total DNA from diagnostic samples and subsequent metagenomic analysis. The emphasis of future testing developments should be on the use of metagenomics (gene-based) rather than the phenotypic methods used today. The food safety risk analysis tool as first described by the Food and Agriculture Organization/World Health Organization states that it should be the role of the official bodies to use risk analysis to determine realistic and achievable risk levels of foodborne hazards. However, by moving towards online testing, it may also be appropriate to change the risk assessment concept towards online product risk assessments based on the microbiological testing.
Microbial Decontamination in the Food Industry, 2012
Abstract: Despite advances in dairy production and processing methods, outbreaks of illness assoc... more Abstract: Despite advances in dairy production and processing methods, outbreaks of illness associated with milk and milk products continue to occur. At the same time there has been a resurgence of interest by consumers in organic, ‘slow’ and ‘raw’ foods. To offset issues related to food safety and to comply with the demand for these fresh foods, there is an urgent need to develop novel processing concepts that provide effective public health protection with minimal treatments to retain nutrient and flavour characteristics of the product. In the first part of this chapter all milk and dairy pathogens of relevance are discussed with particular focus on their relative risk ranking. The second part of the chapter reviews the features and potential of conventional and most promising emerging preservation techniques for pathogen mitigation in milk and dairy products, highlighting the advantages of combined decontamination strategies and indicating hurdle processing as the most efficacious approach.
Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of human... more Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of humans and animals and are thought to have positive effects on human health. Therefore, there is an increasing interest in using these microorganisms as probiotics to be incorporated into either fermented dairy products or tablets. However, convincing scientific data that support claims of their health benefits are scarce. The effect of cell-free extracts of milk fermented by 10 probiotic bacteria (five Bifidobacterium strains and five Lactobacillus strains) on the expression of the flaA gene of Campylobacter jejuni was assessed using a fusion between the flaA σ28 promoter and a promoterless luxCDABE cassette carried on the plasmid pRYluxCDABE, which resulted in strains with quantifiable luminescence linked to flaA σ28 promoter activity. Cell-free extracts of milk fermented by all of the tested probiotic strains inhibited the growth of the C. jejuni and down-regulated flaA σ28 promoter activi...
A significant feature of Enterobacter sakazakii is the ability to form biofilms enabling its pers... more A significant feature of Enterobacter sakazakii is the ability to form biofilms enabling its persistence in manufacturing and neonatal environments, and implicating it as the primary cause of outbreaks of neonatal meningitis. Conventional methods of studying E. sakazakii are time consuming, inaccurate and damaging to cells, preventing further analysis. A novel method for detecting biofilm formation has been utilized that takes advantage of a dual lux/gfp reporter system cloned into cells for simultaneous quantification of bacterial metabolic activity and cell numbers respectively. To evaluate the effectiveness and accuracy of the novel method compared to the conventional method, strains of bacteria were allowed to form biofilms and were measured using both methods. Biofilm formation of E. sakazakii strains over 2 days was measured and peak formation and changes in biofilm development was determined for each strain. The lux reporter was utilized to determine metabolic activity of cel...
With the advent of antimicrobial resistance in animal pathogens, novel methods to combat infectio... more With the advent of antimicrobial resistance in animal pathogens, novel methods to combat infectious diseases are being sought. Among these, probiotics have been proposed as a means of promoting animal health but problems with their use has been reported. Research has demonstrated that bioactive molecules produced during the growth of certain probiotics interfere with bacterial cell-to-cell communication, which consequently results in an attenuation of virulence in a number of pathogens, including E. coli. The objective of this study was to determine the efficacy of the bioactive molecules, termed proteobiotics, produced by Lactobacillus acidophilus in preventing enterotoxigenic E, coli (ETEC) infection in pigs, which is the etiological agent for enteric colibacillosis, a common disease of nursing and young pigs. To achieve this, piglets were fed a preparation of the bioactive at four levels: 0, 0.5×, 1.0× and 2.0× for 7 days prior to challenge with E. coli K88. There were 36 pigs (1...
The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and an... more The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and animal sources. In this study, enhancement of the growth and aerotolerance of C. jejuni ATCC 35921 in nutrient broth no. 2 (NB2) was investigated. The efficiency of recovery of C. jejuni in NB2 containing FBP (0.025% [each] ferrous sulfate, sodium metabisulfite, and sodium pyruvate), 5% laked horse blood, hemin, Oxyrase, or activated charcoal in an aerobic atmosphere was compared with that obtained under microaerophilic incubation. The shortest lag time (λ) for cells grown aerobically was observed with NB2 supplemented with FBP, 5% laked horse blood, 0.01 g/liter of hemin, or 0.15 U/ml of Oxyrase. The efficacy of these media to resuscitate C. jejuni cells in late exponential phase, as well as cells subjected to stress induced by cold, heat, starvation, or acid, was determined in aerobic or microaerobic atmospheres. The λ of cells grown aerobically in NB2 containing both FBP and blood was s...
Foodborne illness contracted at food service operations is an important public health issue in Ko... more Foodborne illness contracted at food service operations is an important public health issue in Korea. In this study, the probabilities for growth of, and enterotoxin production by, Staphylococcus aureus in pork meat–based foods prepared in food service operations were estimated by the Monte Carlo simulation. Data on the prevalence and concentration of S. aureus as well as compliance to guidelines for time and temperature controls during food service operations were collected. The growth of S. aureus was initially estimated by using the U.S. Department of Agriculture's Pathogen Modeling Program. A second model based on raw pork meat was derived to compare cell number predictions. The correlation between toxin level and cell number as well as minimum toxin dose obtained from published data was adopted to quantify the probability of staphylococcal intoxication. When data gaps were found, assumptions were made based on guidelines for food service practices. Baseline risk model and s...
Recombinant bacteriophages specific for Salmonella spp. and containing bacterial luciferase genes... more Recombinant bacteriophages specific for Salmonella spp. and containing bacterial luciferase genes were constructed. The phage caused the host cells to luminesce when mixed with Salmonella spp. and the luminescence could be detected using a photon-counting charge-coupled device (CCD) camera, a luminometer, or X-ray film. The initial assay system was capable of detecting Salmonella isolates from group B and group D. Certain isolates from group C could also be detected. With 6 h of preincubation, as few as 10 CFU of Salmonella cells per ml in the original sample could be detected. The minimum time required for the detection of 108 CFU/ml was 1 to 3 h with no preincubation, depending on bacteriophage adsorption temperatures. The phage-based assay could be carried out on Petrifilm E. coli Count Plates and the light emission detected within 24 h. The system allowed Salmonella cells to be detected in whole eggs by direct addition of recombinant bacteriophages into the eggs followed by visu...
Fresh and retail eggs were exposed to luminescent S. enteritidis cultures containing from 104 to ... more Fresh and retail eggs were exposed to luminescent S. enteritidis cultures containing from 104 to 109 CFU/ml at either room temperature (approximately 21°C) for 3 days or 40°C for 16 h. The entry of S. enteritidis through egg shell was evidenced by luminescence in the eggs which was visualized using an Image Quantifier. The rate of contamination of the eggs increased with increasing inoculum size. Scanning electron microscopy was used to confirm the position of S. enteritidis cells in the eggs. The survival rate of the Salmonella cells in liquid eggs and whole shell eggs during storage at 4°C was investigated. Although S. enteritidis did not grow in eggs during storage at 4°C for up to 8 weeks, cells were able to survive. Under these storage conditions, the count was reduced by 1.7 to 2.5 log cycles per g in liquid egg and 0.8 to 1.4 log cycle per g in whole shell eggs. Similar trends were observed using both plate count and luminescence to monitor survival.
The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, ph... more The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, phloxine B, and malachite green (oxalate salt) at concentrations of 5 to 5,000 μg/ml against two gram-negative strains (Escherichia coli LJH 128 and Salmonella Typhimurium C1058), two gram-positive strains (Bacillus sp. C578 and Listeria monocytogenes LJH 375), and yeast (Saccharomyces cerevisiae C1172) was investigated. Incubation of the investigated bacteria with acriflavine neutral under illumination resulted in a significant reduction in cell numbers compared with dark incubation. Rose bengal caused a significant killing effect for bacteria incubated both in the dark and under illumination. Malachite green was active against gram-positive bacteria under illumination and did not affect gram-negative bacteria or yeasts. Incubation with phloxine B resulted in a significant decline in cell numbers for gram-positive bacteria, both in the dark and under illumination; gram-negative bacteria a...
Response surface analysis was used to determine the effects and interactions of water activity (0... more Response surface analysis was used to determine the effects and interactions of water activity (0.965 to 0.995), pH (5.8 to 8.0), temperature (6 to 38°C), glucose concentration (0 to 1.8%), and starch concentration (0 to 0.625%) on the growth of and toxin production by a psychrotrophic strain of Bacillus cereus in brain heart infusion broth. Growth was measured by monitoring optical density and plate counts, toxin production was assayed by an immunological method (BCET-RPLA toxin assay) and cytotoxicity with Vero and HEp-2 cells. Regressions were performed using response surface techniques, on Growth, LnGrowth, RPLA, LnRPLA, Vero, LnVero, and HEp-2; quadratic predictive equations for growth and toxin production were obtained. The results indicate the factors that had the greatest influence on both growth and toxin production were water activity and temperature. Predicted values obtained from the model were in good agreement with experimental values.
Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens
Proper sample preparation is a precondition for advanced laboratory diagnostics. The newer method... more Proper sample preparation is a precondition for advanced laboratory diagnostics. The newer methods that claim less time to reach a result have a major limitation with how much volume they can take prior to the rapid analysis for the target molecule. Among these approaches, the use of signal amplification techniques has received attention. Another key issue is automation, where the key drivers are miniaturization and multiple testing. Quantification by real-time PCR is an established technique. Recent developments in real-time PCR have made it possible to carry out high-throughput source tracing for routine purposes in the food industry. The genomics technologies have reached a stage where they are used on a routine basis in many laboratories. These technologies are expected to move towards culture-independent detection and characterization techniques based on the purification of total DNA from diagnostic samples and subsequent metagenomic analysis. The emphasis of future testing developments should be on the use of metagenomics (gene-based) rather than the phenotypic methods used today. The food safety risk analysis tool as first described by the Food and Agriculture Organization/World Health Organization states that it should be the role of the official bodies to use risk analysis to determine realistic and achievable risk levels of foodborne hazards. However, by moving towards online testing, it may also be appropriate to change the risk assessment concept towards online product risk assessments based on the microbiological testing.
Microbial Decontamination in the Food Industry, 2012
Abstract: Despite advances in dairy production and processing methods, outbreaks of illness assoc... more Abstract: Despite advances in dairy production and processing methods, outbreaks of illness associated with milk and milk products continue to occur. At the same time there has been a resurgence of interest by consumers in organic, ‘slow’ and ‘raw’ foods. To offset issues related to food safety and to comply with the demand for these fresh foods, there is an urgent need to develop novel processing concepts that provide effective public health protection with minimal treatments to retain nutrient and flavour characteristics of the product. In the first part of this chapter all milk and dairy pathogens of relevance are discussed with particular focus on their relative risk ranking. The second part of the chapter reviews the features and potential of conventional and most promising emerging preservation techniques for pathogen mitigation in milk and dairy products, highlighting the advantages of combined decontamination strategies and indicating hurdle processing as the most efficacious approach.
Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of human... more Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of humans and animals and are thought to have positive effects on human health. Therefore, there is an increasing interest in using these microorganisms as probiotics to be incorporated into either fermented dairy products or tablets. However, convincing scientific data that support claims of their health benefits are scarce. The effect of cell-free extracts of milk fermented by 10 probiotic bacteria (five Bifidobacterium strains and five Lactobacillus strains) on the expression of the flaA gene of Campylobacter jejuni was assessed using a fusion between the flaA σ28 promoter and a promoterless luxCDABE cassette carried on the plasmid pRYluxCDABE, which resulted in strains with quantifiable luminescence linked to flaA σ28 promoter activity. Cell-free extracts of milk fermented by all of the tested probiotic strains inhibited the growth of the C. jejuni and down-regulated flaA σ28 promoter activi...
A significant feature of Enterobacter sakazakii is the ability to form biofilms enabling its pers... more A significant feature of Enterobacter sakazakii is the ability to form biofilms enabling its persistence in manufacturing and neonatal environments, and implicating it as the primary cause of outbreaks of neonatal meningitis. Conventional methods of studying E. sakazakii are time consuming, inaccurate and damaging to cells, preventing further analysis. A novel method for detecting biofilm formation has been utilized that takes advantage of a dual lux/gfp reporter system cloned into cells for simultaneous quantification of bacterial metabolic activity and cell numbers respectively. To evaluate the effectiveness and accuracy of the novel method compared to the conventional method, strains of bacteria were allowed to form biofilms and were measured using both methods. Biofilm formation of E. sakazakii strains over 2 days was measured and peak formation and changes in biofilm development was determined for each strain. The lux reporter was utilized to determine metabolic activity of cel...
With the advent of antimicrobial resistance in animal pathogens, novel methods to combat infectio... more With the advent of antimicrobial resistance in animal pathogens, novel methods to combat infectious diseases are being sought. Among these, probiotics have been proposed as a means of promoting animal health but problems with their use has been reported. Research has demonstrated that bioactive molecules produced during the growth of certain probiotics interfere with bacterial cell-to-cell communication, which consequently results in an attenuation of virulence in a number of pathogens, including E. coli. The objective of this study was to determine the efficacy of the bioactive molecules, termed proteobiotics, produced by Lactobacillus acidophilus in preventing enterotoxigenic E, coli (ETEC) infection in pigs, which is the etiological agent for enteric colibacillosis, a common disease of nursing and young pigs. To achieve this, piglets were fed a preparation of the bioactive at four levels: 0, 0.5×, 1.0× and 2.0× for 7 days prior to challenge with E. coli K88. There were 36 pigs (1...
The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and an... more The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and animal sources. In this study, enhancement of the growth and aerotolerance of C. jejuni ATCC 35921 in nutrient broth no. 2 (NB2) was investigated. The efficiency of recovery of C. jejuni in NB2 containing FBP (0.025% [each] ferrous sulfate, sodium metabisulfite, and sodium pyruvate), 5% laked horse blood, hemin, Oxyrase, or activated charcoal in an aerobic atmosphere was compared with that obtained under microaerophilic incubation. The shortest lag time (λ) for cells grown aerobically was observed with NB2 supplemented with FBP, 5% laked horse blood, 0.01 g/liter of hemin, or 0.15 U/ml of Oxyrase. The efficacy of these media to resuscitate C. jejuni cells in late exponential phase, as well as cells subjected to stress induced by cold, heat, starvation, or acid, was determined in aerobic or microaerobic atmospheres. The λ of cells grown aerobically in NB2 containing both FBP and blood was s...
Foodborne illness contracted at food service operations is an important public health issue in Ko... more Foodborne illness contracted at food service operations is an important public health issue in Korea. In this study, the probabilities for growth of, and enterotoxin production by, Staphylococcus aureus in pork meat–based foods prepared in food service operations were estimated by the Monte Carlo simulation. Data on the prevalence and concentration of S. aureus as well as compliance to guidelines for time and temperature controls during food service operations were collected. The growth of S. aureus was initially estimated by using the U.S. Department of Agriculture's Pathogen Modeling Program. A second model based on raw pork meat was derived to compare cell number predictions. The correlation between toxin level and cell number as well as minimum toxin dose obtained from published data was adopted to quantify the probability of staphylococcal intoxication. When data gaps were found, assumptions were made based on guidelines for food service practices. Baseline risk model and s...
Recombinant bacteriophages specific for Salmonella spp. and containing bacterial luciferase genes... more Recombinant bacteriophages specific for Salmonella spp. and containing bacterial luciferase genes were constructed. The phage caused the host cells to luminesce when mixed with Salmonella spp. and the luminescence could be detected using a photon-counting charge-coupled device (CCD) camera, a luminometer, or X-ray film. The initial assay system was capable of detecting Salmonella isolates from group B and group D. Certain isolates from group C could also be detected. With 6 h of preincubation, as few as 10 CFU of Salmonella cells per ml in the original sample could be detected. The minimum time required for the detection of 108 CFU/ml was 1 to 3 h with no preincubation, depending on bacteriophage adsorption temperatures. The phage-based assay could be carried out on Petrifilm E. coli Count Plates and the light emission detected within 24 h. The system allowed Salmonella cells to be detected in whole eggs by direct addition of recombinant bacteriophages into the eggs followed by visu...
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