An active fraction extracted from Lactobacillus acidophilus La5 cell-free spent medium (LAla-5AF) was incorporated in a dairy matrix and tested to assess its antivirulent effect against enterohemorrhagic Escherichia coli (EHEC). Mice in experimental groups were fed for 4 days with yogurt supplemented with LAla-5AF. On the fifth day, mice were challenged with a single dose (10 7 CFU per mouse) of E. coli O157:H7. The clinical manifestations of the infection were significantly less severe in mice fed the yogurt supplemented with LAla-5AF. EHEC attachment and colonization was attenuated by LAla-5AF. Tumor necrosis factor alpha production was down-regulated, which might indicate a protective effect in the kidney during EHEC infection. To investigate the mechanisms associated with the in vivo effects observed, LAla-5AF was tested by reverse transcription real-time PCR to confirm its effects on the expression of several virulence genes of EHEC O157. The results showed that these fractions were able to down-regulate several virulence genes of EHEC, including stxB2, qseA, luxS, tir, ler, eaeA , and hlyB .

Salmonella Typhimurium pathogenesis relies mainly on the expression of genes of two pathogenicity islands, Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Each island has its own pattern of expression and regulation. Success in suppression of the responsible key activator of each island would be an effective way of controlling Salmonella , especially with the emerging problem of antibiotic-resistant strains. Probiotics have been shown to inhibit several foodborne pathogens, and their mode of action may partly involve down-regulation of virulence genes. To investigate whether probiotics played a role in the regulation of the pathogenicity islands SPI1 and SPI2 in Salmonella , two reporter strains were constructed in which the general regulator of SPI1, hilA , and the response regulator of SPI2, ssrB , were fused with luxCDABE genes. These constructs were used to screen the effect of probiotics on the expression of each gene. Molecules secreted by Bifidobacterium bifidum were able to down-regulate both genes.

The technological feasibility of producing fermented sausages using the bacteriocin-producing Lactobacillus pentosus 31-1, isolated from a traditional Chinese fermented meat product (Xuanwei ham), was evaluated. Strain 31-1 was used both as a single starter and in coculture for manufacture of fermented sausages. The microbiological and physicochemical properties (color, texture, and sensory quality) and the production of bacteriocin during ripening of these products were compared with those characteristics of sausages produced with a commercial meat starter. Challenge tests were performed using Listeria innocua or Staphylococcus aureus as target strains. The addition of L. pentosus 31-1 can significantly reduce L. innocua and S. aureus populations during all ripening phases. Free amino acid and free fatty acid analysis suggested that strain 31-1 might have proteolytic and lipolytic activity. The use of this strain resulted in a final product with a brighter surface and better texture and sensory profiles. A maximum bacteriocin (pentocin 31-1) concentration of 640 AU/g was detected in homogenized sausages with added L. pentosus 31-1. The bacteriocin-producing strain L. pentosus 31-1 could be used as a novel functional starter culture or coculture for sausage fermentation.

Quorum sensing (QS) is a signalling mechanism through which bacteria cellular functions are modified to promote access to nutrients and more favorable environmental niches. The frequent occurrence of Pseudomonas spp. in fresh and spoiled meat may involve enhanced gene expression regulated by QS. Several Pseudomonas spp. produce different N -acyl homoserine lactone (AHL) signal molecules. Meat spoilage during aerobic, refrigerated storage is often associated with the presence of Pseudomonas fragi . As with other Pseudomonas species in natural habitats, the dominance and activities of P. fragi in meat may be regulated by QS. In this study, five biosensor strains were used to detect AHL production on three different media by 72 different P. fragi strains isolated from fresh and spoiled meat. Positive and negative AHL-producing strains were used to verify the assays. None of the strains produced detectable quantities of AHLs, even when concentrated cell-free culture supernatants were assayed, nor did exogenous lactones increase biofilm formation in P. fragi strains. However, all isolates produced furanosyl borate diesters (type II autoinducers; AI-2) when tested using the bioluminescent biosensor strain of Vibrio harveyi (BB170). The production of AI-2 was presumed to be of metabolic origin even though Pseudomonas spp. have not been shown to harbor the luxS gene. Thus, the efficient development of P. fragi in fresh meat is not regulated by an AHL-mediated QS system. The mechanism of AI-2 production and its possible role in spoilage dynamics needs further study.

In the present study, nanoalumina filters were used as a sample preparation step for the concentration of a norovirus surrogate (murine norovirus 1) from food, and this was coupled with a two-step, real-time reverse transcriptase PCR for quantification. The nanoalumina medium was provided in a syringe-filter format, and its binding and elution capacities were tested with different buffers. Among the binding buffers tested (0.1 M Tris-HCl [pH 7.0] with 0.1% Tween 80, 0.1% 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate, or 1 M NaCl), no significant differences were found in the capture capacity of the nanoalumina filters, which was found to be as high as 99.8% of murine norovirus 1 present in the buffer. Elution of 50% of captured viral particles from the filters was possible by using glycine buffer. The desorption capacity of the binding buffers was tested on different inoculated food surfaces. Recoveries of up to 100% from lettuce, raspberries, strawberries, or mussels were obtained with 0.1 M Tris-HCl (pH 7.0) containing 1 M NaCl by using orbital shaking or pipetting. The latter method was more efficient and gave higher recoveries than did orbital shaking. The combination of an efficient desorption-binding-elution buffer with the high concentration capacity of the nanoalumina medium allowed the detection of 10 1 PFU from inoculated produce and 10 5 PFU from inoculated mussels.

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 scenario analyses were performed to indicate possible outcomes of staphylococcal intoxication under the scenarios generated based on these data gaps. Staphylococcal growth was predicted during holding before and after cooking, and the highest estimated concentration (4.59 log CFU/g for the 99.9th percentile value) of S. aureus was observed in raw pork initially contaminated with S. aureus and held before cooking. The estimated probability for staphylococcal intoxication was very low, using currently available data. However, scenario analyses revealed an increased possibility of staphylococcal intoxication when increased levels of initial contamination in the raw meat, and longer holding time both before and after cooking the meat occurred.

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 and yeasts were unaffected. Conjugation of rose bengal and phloxine B with poly(vinyl amine) resulted in an enhanced bactericidal effect during both dark and light incubation. This was explained by electrostatic interaction of the polymer with the cell surface, which resulted in closer contact of the photoactive dye and cell. No killing effect was observed for yeasts incubated with dye conjugates. Filter paper treated with dye–poly(vinyl amine) conjugates showed high photodynamic bactericidal activity against the bacterial strains, but not against the yeasts. The extent of bacterial killing depended on the nature and concentration of the dye conjugate and the type of microorganism. The presented data suggest that a photodynamic approach for constructing “self-decontaminating” materials has potential.

In this work, the feasibility of using a large-volume immunocapture system as a sample pretreatment before detection of Campylobacter was studied. Real-time PCR was used for detection of captured cells after immunocapture. This immunocapture system is able to process high-volume samples by recirculation, increasing the possibility of capturing cells in low numbers. After 30 min of recirculation, the sample is concentrated from 250 ml to 200 μl. In this study, different parameters were compared in order to improve cell capture. The analysis of inoculated chicken skin showed that detection of Campylobacter at levels of 10 3 CFU/25 g was possible after 8 h of enrichment. The low recovery of Campylobacter cells (<1%) makes this separation method qualitative rather than quantitative. The detection limit of the entire protocol was increased due to the low cell recovery of the sample pretreatment. Therefore, this immunoseparation is able to recover cells present in high concentration after enrichment but not cells present in low concentration. Isolation of Campylobacter cells is achievable using this separation method rather than rapid detection.

Under conditions of stress, cells of Campylobacter assume a coccoid shape that may be an evolutionary strategy evolved by the organism to enable survival between hosts. However, the physiology of Campylobacter as it devolves from spiral to coccoid-shaped morphology is poorly understood. In this study, conditions influencing the survival of Campylobacter jejuni ATCC 35921 in broth were determined. Cells in late log phase were resuspended in broth at 4 or 60°C. The culturability of these cold- or heat-stressed cell suspensions was determined by spread plate counts and the activity of cells by the direct viable count technique and 5-cyano-2,3-ditolyltetrazolium chloride staining. C. jejuni changed form completely from culturable to viable but nonculturable cells (VBNC) within 25 days at 4°C, and 15 min at 60°C. Light microscopy of C. jejuni VBNC cells showed that the spiral-shaped cells became coccoid, and transmission electron microscopy of C. jejuni VBNC cells showed that the outer membrane was lost in aging cell suspensions. Furthermore, a limited proteomic study was carried out to compare C. jejuni proteins that exhibited increased or decreased synthesis on exposure to 60°C.

The internalization and persistence of a bioluminescent Escherichia coli O157:H7 Ph1 was investigated in growing spinach plants that had been either biologically or mechanically damaged. In control (undamaged) plants cultivated in soil microcosms inoculated with E. coli O157:H7 Ph1, the bacterium was recovered from surface-sterilized root tissue but not from leaves. Mechanical disruption of the seminal root and root hairs of the plants did not result in the internalization of the pathogen into the aerial leaf tissue. When imprints of the root tissue were made on plates of tryptic soy agar plus ampicillin, no colonies of E. coli O157:H7 were observed around damaged tissue. The roots of growing plants were exposed to the northern root-knot nematode, Meloidogyne hapla , in the presence of E. coli O157:H7. Although this treatment caused knot formation on the roots, it did not enhance the internalization of the bacterium into the plant vascular system. Coinoculation of intact leaves with E. coli O157:H7 and the phytopathogen Pseudomonas syringae DC3000 resulted in localized necrosis, but the persistence of the human pathogen was not affected. The mechanical disruption of roots does not result in the internalization of E. coli O157:H7 into the aerial tissue of spinach, and there does not appear to be any effect of P. syringae in terms of enhancing the persistence of E. coli O157:H7 in spinach leaves.

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 σ 28 promoter and a promoterless luxCDABE cassette carried on the plasmid pRYluxCDABE, which resulted in strains with quantifiable luminescence linked to flaA σ 28 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 σ 28 promoter activity. Two nonprobiotic lactic acid bacterial strains, Lactococcus lactis and Streptococcus thermophilus , were less inhibitory.

Primers designed to amplify a Campylobacter jejuni cadF gene sequence were used in an SYBR Green I real-time PCR assay as an alternative to conventional bacteriological methods for the rapid detection of C. jejuni in foods. Twenty-five grams of chicken skin (breast and thigh) was contaminated by adding approximately 1, 10, or 50 CFU of C. jejuni ATCC 35560. Twenty-five grams of pork and 25-ml aliquots of milk were also inoculated with 1 and 10 CFU of the pathogen. The samples were incubated in Bolton broth for different periods at 37 and 42°C under microaerophilic conditions. Using a commercial robotic DNA purification system, DNA was extracted and purified from 1-ml aliquots of the enrichment cultures before and after centrifugation of the 250-ml enrichment broth at 15,900 × g for 10 min at 4°C. The DNA was used as the template in a real-time PCR assay. C. jejuni was detected after 12 h of enrichment from samples inoculated with about 50 CFU/25-g sample. After centrifugation, an enrichment step of 8 h was sufficient to allow detection of pathogen in samples inoculated with 10 CFU/25 g. However, 24 h of enrichment was necessary to detect pathogen in samples inoculated with approximately 1 CFU/25 g. The real-time PCR protocol developed in this study significantly reduced the detection time of C. jejuni in foods.

In this study, we investigated the effect of starvation on cryotolerance of Escherichia coli O157:H7 grown in tryptic soy broth (TSB) and Luria-Bertani broth (LB). Starved cells (cells suspended in water at 37°C for 6 h) and control cells (cells in TSB or LB) were frozen at −18°C for up to 240 h in their respective growth media. The E. coli grown in TSB showed a greater starvation effect (the difference in percent survival of starved and control cells) and cryotolerance. The starved E. coli grown in TSB showed a 30% increase in their ability to survive frozen storage for 24 h at −18°C. The corresponding increase in survival for LB-grown E. coli was only 3.8%. Cryotolerance induced by starvation of TSB- and LB-grown E. coli was correlated with the expression of genes involved in general stress response pathways, such as uspA, grpE , and rpoS . The expression of uspA, grpE , and rpoS was quantified by measuring the green fluorescence generated from autofluorescent E. coli harboring p uspA :: gfp , p grpE :: gfp , and p rpoS :: gfp gene fusions. The results obtained in this study indicate that uspA, grpE , and rpoS were induced on starvation when E. coli was grown in TSB, and their expression correlated well with subsequent induction of cryotolerance developed at −18°C. In contrast, cells grown in LB and subsequently exposed to starvation conditions showed no increase in expression of uspA, grpE , or rpoS , and, as expected, these cells did not exhibit increased cryotolerance at −18°C. Knowledge of molecular mechanisms involved in cross-protection might make it possible to devise strategies to limit their effects and lead to ways to predict the survival of foodborne pathogens in stressful environments.

A rapid, specific, and sensitive method for detecting Salmonella spp. in pasteurized milk, ground beef, and alfalfa sprouts was developed. The method combined immunomagnetic separation with a real-time PCR assay based on the double-stranded DNA binding dye SYBR Green I. The primers used produced a product with a melting temperature of 87 ± 0.5°C during the PCR assay by amplifying a 284-bp sequence from the invasive gene ( invA ) of Salmonella . The method was successful in detecting 20 Salmonella strains, but the expected PCR product was not formed by any of 11 other bacterial strains. To test this combined method for the monitoring of Salmonella , Salmonella enterica serotype Newport was inoculated into 52 samples each of pasteurized milk, ground beef, and alfalfa sprouts. Following a 10-h nonselective enrichment step in buffered peptone water, cells were removed by immunomagnetic separation and DNA extracted using the High Pure PCR template preparation kit. The DNA produced was used as a template in the real-time PCR assay. When spiked pasteurized milk, ground beef, and alfalfa sprout samples were analyzed by this protocol, an initial inoculum of 1 CFU/ml, 25 CFU/25 g, and 1.5 CFU/25 g, respectively, was detectable within 13 h. These results indicate that the combination of immunomagnetic separation and real-time PCR assay was a highly specific and sensitive method for the rapid detection of Salmonella .

A novel method of DNA extraction and purification was developed and was used in conjunction with a multiplex real-time PCR assay for the simultaneous detection of Salmonella and Listeria monocytogenes in a raw meat sample. The PCR used primers targeting the invA gene of Salmonella and the hlyA gene of L. monocytogenes , and PCR products were detected with a LightCycler on the basis of fluorescence from SYBR Green and melting temperature. The assay allowed the detection of 3 Listeria cells and 4 Salmonella cells per g of the original sausage within 10 h, including an enrichment period of 6 to 8 h.

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein ( gfp ) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37°C were compared with the levels of expression of the uspA and grpE genes. D 52 -values (times required for decimal reductions in count at 52°C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37°C, for 24 h at 10°C, and for 2 days at 5°C, respectively. In all cases, these increases were significant ( P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5°C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37°C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.

A multiplex SYBR Green I–based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein ( fimI ) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene ( hly ) of L. monocytogenes . These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75°C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green–based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes , respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1 μl of culture to enable direct detection with the LightCycler.

A novel method for assessing the effect of fermented milk and its components on the course of Salmonella infection in live mice is described. Following a period of feeding with whole fermented milk (group W), a cell-free fraction of fermented milk (group S), or saline (group C, control), mice were challenged by oral gavage with a bioluminescent strain of Salmonella Enteritidis. Colonization of the gastrointestinal tract and subsequent infection could be followed by bioluminescent imaging of live mice with a cooled slow-scan CCD camera. Each group of mice was fed for 7 days with the appropriate product. On the eighth day, all mice were orally infected with 10 6 Salmonella cells. On the sixth day after infection, mice in groups W and C showed evidence of disease. No bioluminescent signal was observed for any of the mice in group S. The physical condition of the mice in groups W and S was normal, but some deterioration in the health of the mice in the control group (group C) occurred. On the eighth day after infection, a weak bioluminescence signal was observed for the mice in group W, but still no signal was observed for any of the mice in group S. Mice in both of these groups appeared normal, but the mice in group C showed strong evidence of infection and marked deterioration in their physical condition accompanied by a bioluminescence signal. This method allows the assessment of the effects of potential nutraceutical agents on the course of infection by foodborne pathogens in live animals in real time.

The effects of storage temperature (4, 22, and 30°C), pH (4.0 to 8.5), and sodium chloride concentration (0.25 to 7.5%, wt/vol) on the survival of a strain of Campylobacter jejuni ATCC 35921 with an autobioluminescent phenotype (generated from the fusion of luxCDABE genes from Xenorhabdus luminescens to the C. jejuni flaA promoter) in Mueller-Hinton broth under aerobic conditions were determined. At comparable pHs, autobioluminescent C. jejuni cells die most rapidly at 30°C and most slowly at 4°C. For example, at pH 5.5, a 1.2-log decrease in the cell count was observed after 4 days of storage at 4°C, while 3.7- and 4.8-log reductions in cell numbers were observed after 4 days of storage at 22 and 30°C, respectively. At 4°C, C. jejuni was sensitive to NaCl concentrations of ≥2.5%. However, the degree of inactivation at this storage temperature was also significantly lower than that observed at 22°C, which, in turn, was substantially lower than that observed at 30°C. In the presence of different NaCl concentrations at 22 and 30°C, a strong correlation ( r = 0.98) between plate count and bioluminescence output was obtained. However, at low pHs (4.0 and 4.5) and/or a low storage temperature (4°C), bioluminescence did not correlate well with plate count. Despite these limitations, an autobioluminescent phenotype of C. jejuni proved to be a useful tool for studying the behavior of C. jejuni exposed to environmental stresses.

Five DNA extraction protocols for the detection of Campylobacter spp. by polymerase chain reaction (PCR) were compared. A method involving Triton X-100 produced template DNA of sufficient quality to allow the detection of Campylobacter jejuni at levels of 100 CFU/ml in pure culture. Primers were designed on the basis of the cadF gene sequence. With a SYBR Green I real-time PCR assay, these primers amplified only sequences present in C. jejuni to produce a product with a melting temperature of 81.5°C. None of the strains of Campylobacter coli , Campylobacter lari , or Campylobacter fetus tested produced this product during the PCR assay. Other noncampylobacter species tested were shown not to possess the cadF sequence. The real-time PCR combined with a rapid, simple Triton X-100 DNA extraction protocol made it possible to detect <10 CFU of C. jejuni per ml of chicken rinse within 14 h.