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To study the gastrointestinal survival and enterotoxin production of the food-borne pathogen Bacillus cereus, an in vitro simulation experiment was developed to mimic gastrointestinal passage in 5 phases: (i) the mouth, (ii) the stomach, with gradual pH decrease and fractional emptying, (iii) the duodenum, with high concentrations of bile and digestive enzymes, (iv) dialysis to ensure bile reabsorption, and (v) the ileum, with competing human intestinal bacteria. Four different B. cereus strains were cultivated and sporulated in mashed potato medium to obtain an inoculum of 7.0 log spores/ml. The spores showed survival and germination during the in vitro simulation of gastrointestinal passage, but vegetative outgrowth of the spores was suppressed by the intestinal bacteria during the final ileum phase. No bacterial proliferation or enterotoxin production was observed, despite the high inoculum levels. Little strain variability was observed: except for the psychrotrophic food isolate, the spores of all strains survived well throughout the gastrointestinal passage. The in vitro simulation experiments investigated the survival and enterotoxin production of B. cereus in the gastrointestinal lumen. The results obtained support the hypothesis that localized interaction of B. cereus with the host's epithelium is required for diarrheal food poisoning. [ABSTRACT FROM AUTHOR]

Degradation of the quorum-sensing signal molecule N-acyl-ʟ-homoserine lactone (AHL) in cocultures was verified with Bacillus cereus and Yersinia enterocolitica in culture medium and in pork extract. Results showed evidence of microbial interaction when the AHL-degrading bacterium and AHL-producing bacterium were cocultured in a food-simulating condition. [ABSTRACT FROM AUTHOR]

This study intends to establish and apply methods evaluating both viral capsid and genome integrities of human noroviruses (NoVs), which thus far remain nonculturable. Murine norovirus 1 (MNV-1) and human NoV GII.4 in phosphate-buffered saline suspensions were treated with heat, UV light, or ethanol and detected by reverse transcription-quantitative PCR (RT-qPCR), long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR. For MNV-1 heated at 60°C for 2 and 30 min, limited reductions of genomic copies (<0.3-log) were obtained by RT-qPCR and long-range RT-qPCR, while the cell-binding pretreatments obtained higher reductions (>1.89-log reduction after 60°C for 30 min by binding long-range RT-qPCR). The human NoV GII.4 was found to be more heat resistant than MNV-1. For both MNV-1 and human NoV GII.4 after UV treatments of 20 and 200 mJ/cm², no significant difference (P > 0.05) was observed between the dose-dependent reductions obtained by the four detection methodologies. Treatment of 70% ethanol for 1 min was shown to be more effective for inactivation of both MNV-1 and human NoV GII.4 than the heat and UV treatments used in this study. Subsequently, eight raspberry and four shellfish samples previously shown to be naturally contaminated with human NoVs by RT-qPCR (GI and GII; thus, 24 RT-qPCR signals) were subjected to comparison by this method. RT-qPCR, long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR detected 20/24, 14/24, 24/24, and 23/24 positive signals, respectively, indicating the abundant presence of intact NoV particles. [ABSTRACT FROM AUTHOR]

Significant interest in studying the lipooligosaccharide (LOS) of Campylobacter jejuni has stemmed from its potential role in postinfection paralytic disorders. In this study we present the results of PCR screening of five LOS locus classes (A, B, C, D, and E) for a collection of 116 C. jejuni isolates from chicken meat (n = 76) and sporadic human cases of diarrhea (n = 40). We correlated LOS classes with clonal complexes (CC) assigned by multilocus sequence typing (MLST). Finally, we evaluated the invasion potential of a panel of 52 of these C. jefuni isolates for Caco-2 cells. PCR screening showed that 87.1% (101/116) of isolates could be assigned to LOS class A, B, C, D, or E. Concordance between LOS classes and certain MLST CC was revealed. The majority (85.7% [24/28]) of C.jejuni isolates grouped in CC-21 were shown to express LOS locus class C. The invasion potential of C. jejuni isolates possessing sialylated LOS (n = 29; classes A, B, and C) for Caco-2 cells was significantly higher (P < 0.0001) than that of C. jejuni isolates with nonsialylated LOS (n = 23; classes D and E). There was no significant difference in invasiveness between chicken meat and human isolates. However, C. jejuni isolates assigned to CC-206 (correlated with LOS class B) or CC-21 (correlated with LOS class C) showed statistically significantly higher levels of invasion than isolates from other CC. Correlation between LOS classes and CC was further confirmed by pulsed-field gel electrophoresis. The present study reveals a correlation between genotypic diversity and LOS locus classes of C. jejuni. We showed that simple PCR screening for C.jejuni LOS classes could reliably predict certain MLST CC and add to the interpretation of molecular-typing results. Our study corroborates that sialylation of LOS is advantageous for C. jejuni fitness and virulence in different hosts. The modulation of cell surface carbohydrate structure could enhance the ability of C. jejuni to adapt to or survive in a host. [ABSTRACT FROM AUTHOR]

Campylobacterjejuni is one of the most important causes of human diarrhea worldwide. In the present work, multilocus sequence typing was used to study the genotypic diversity of 145 C. jejuni isolates from 135 chicken meat preparations sampled across Belgium. Isolates were further typed by pulsed-field gel electrophoresis, and their susceptibilities to six antimicrobials were determined. Fifty-seven sequence types (ST5) were identified; 26.8% of the total typed isolates were ST-SO, ST-45, or ST-257, belonging to clonal complex CC-21, CC-45, or CC-257, respectively. One clonal group comprised 22% (32/145) of all isolates, originating from five different companies and isolated over seven sampling months. Additionally, 53.1% of C. jejuni isolates were resistant to ciprofloxacin, and 48.2% were resistant to tetracycline; 28.9% (42/145) of all isolates were resistant to both ciprofioxacin and tetracycline. The correlation between certain C. jejuni clonal groups and resistance to ciprofloxacin and tetracycline was notable. C. jejuni isolates assigned to CC-21 (n = 35) were frequently resistant to ciprofloxacin (65.7%) and tetracycline (40%); however, 90% (18/20) of the isolates assigned to CC-45 were pansusceptible. The present study demonstrates that certain C. jejuni genotypes recur frequently in the chicken meat supply. The results of molecular typing, combined with data on sample sources, indicate a possible dissemination of C. jejuni clones with high resistance to ciprofloxacin and/or tetracycline. Whether certain clonal groups are common in the environment and repeatedly infect Belgian broiler flocks or whether they have the potential to persist on farms or in slaughterhouses needs further investigation. [ABSTRACT FROM AUTHOR]

From February to November 2007, chicken meat preparations (n = 656) were sampled at 11 processing companies across Belgium. All samples were tested for Campylobacter by enrichment culture and by direct plating according to standard methods. Almost half (48.02%) of the samples were positive for Campylobacter spp. The mean Campylobacter count was 1.68 log10 CFU/g with a standard deviation of ± 0.64 log10 CFU/g. The study revealed a statistically significant variation in Campylobacter contamination levels between companies; processors with a wider frequency distribution range of Campylobacter counts provided chicken meat preparations with higher Campylobacter incidences and concentrations. There was no significant difference between the counts of Campylobacter spp. in various preparation types. However, the Campylobacter counts and incidences in chicken wings were the highest and portioned-form products (legs, wings, and breasts) showed a higher probability of being Campylobacter positive compared to minced-form products (sausages, burgers, and minced meat). The proportion of Campylobacter-positive samples was significantly higher in July than in other months. Recovery of Campylobacter spp. recovery by direct plating was higher (41.0%) compared to detection after enrichment (24.2%). Statistical modeling of the survey data showed that the likelihood of obtaining a positive result by enrichment culture increases with an increase in the Campylobacter concentration in the sample. In the present study, we provide the first enumeration data on Campylobacter contamination in Belgian chicken meat preparations and address proposals for improving Campylobacter monitoring programs. [ABSTRACT FROM AUTHOR]

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml-1) than the in-house ELISA and had a dynamic range of approximately 10 pg ml-1 to approximately 30,000 pg ml-1. iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEE in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42°C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42°C than at 37°C and were strain dependent. [ABSTRACT FROM AUTHOR]

Strawberries are an important fruit in Belgium in both production and consumption, but little information is available about the presence of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in these berries, the risk factors in agricultural production, and possible specific mitigation options. In 2012, a survey was undertaken of three soil and three soilless cultivation systems in Belgium. No Salmonella spp. were isolated. No STEC was detected in the strawberry samples (0 of 72), but STEC was detected by PCR in 11 of 78 irrigation water and 2 of 24 substrate samples. Culture isolates were obtained for 2 of 11 PCR-positive irrigation water samples and 2 of 2 substrate samples. Multivariable logistic regression analysis revealed elevated generic E. coli numbers (the odds ratio [OR] for a 1 log increase being 4.6) as the most important risk factor for STEC, together with the berry-picking season (elevated risk in summer). The presence of generic E. coli in the irrigation water (≥1 CFU per 100 ml) was mainly influenced by the type of irrigation water (collected rainfall water stored in ponds was more often contaminated than groundwater pumped from boreholes [OR = 5.8]) and the lack of prior treatment (untreated water versus water subjected to sand filtration prior to use [OR = 19.2]). The follow-up study in 2013 at one of the producer locations indicated cattle to be the most likely source of STEC contamination of the irrigation water. [ABSTRACT FROM AUTHOR]

Fresh produce is known to carry nonpathogenic epiphytic microorganisms. During agricultural production and harvesting, leafy greens can become contaminated with antibiotic-resistant pathogens or commensals from animal and human sources. As lettuce does not undergo any inactivation or preservation treatment during processing, consumers may be exposed directly to all of the (resistant) bacteria present. In this study, we investigated whether lettuce or its production environment (irrigation water, soil) is able to act as a vector or reservoir of antimicrobial-resistant Escherichia coli. Over a 1-year period, eight lettuce farms were visited multiple times and 738 samples, including lettuce seedlings (leaves and soil), soil, irrigation water, and lettuce leaves were collected. From these samples, 473 isolates of Escherichia coli were obtained and tested for resistance to 14 antimicrobials. Fiftyfour isolates (11.4%) were resistant to one or more antimicrobials. The highest resistance rate was observed for ampicillin (7%), followed by cephalothin, amoxicillin-clavulanic acid, tetracycline, trimethoprim, and streptomycin, with resistance rates between 4.4 and 3.6%. No resistance to amikacin, ciprofloxacin, gentamicin, or kanamycin was observed. One isolate was resistant to cefotaxime. Among the multiresistant isolates (n = 37), ampicillin and cephalothin showed the highest resistance rates, at 76 and 52%, respectively. E. coli isolates from lettuce showed higher resistance rates than E. coli isolates obtained from soil or irrigation water samples. When the presence of resistance in E. coli isolates from lettuce production sites and their resistance patterns were compared with the profiles of animal-derived E. coli strains, they were found to be the most comparable with what is found in the cattle reservoir. This may suggest that cattle are a potential reservoir of antimicrobial-resistant E. coli strains in plant primary production. [ABSTRACT FROM AUTHOR]

Chlorine was assessed as a reconditioning agent and wash water disinfectant in the fresh-cut produce industry. Artificial fresh-cut lettuce wash water, made from butterhead lettuce, was used for the experiments. In the reconditioning experiments, chlorine was added to artificial wash water inoculated with Escherichia coli O157 (6 log CFU/ml). Regression models were constructed based on the inactivation data and validated in actual wash water from leafy vegetable processing companies. The model that incorporated chlorine dose and chemical oxygen demand (COD) of the wash water accurately predicted inactivation. Listeria monocytogenes was more resistant to chlorine reconditioning in artificial wash water than Salmonellaspp. and Escherichia coli O157. During the washing process with inoculated lettuce (4 log CFU/g), in the absence of chlorine, there was a rapid microbial buildup in the water that accumulated to 5.4 ± 0.4 log CFU/100 ml after 1 h. When maintaining a residual concentration of 1 mg/liter free chlorine, wash water contamination was maintained below 2.7, 2.5, and 2.5 log CFU/100 ml for tap water and artificial process water with COD values of 500 and 1,000 mg O2/liter, respectively. A model was developed to predict water contamination during the dynamic washing process. Only minor amounts of total trihalomethanes were formed in the water during reconditioning. Total trihalomethanes accumulated to larger amounts in the water during the wash water disinfection experiments and reached 124.5 ± 13.4 μg/liter after 1 h of execution of the washing process in water with a COD of 1,000 mg O2/liter. However, no total trihalomethanes were found on the fresh-cut lettuce after rinsing. [ABSTRACT FROM AUTHOR]

The anti-norovirus (anti-NoV) effect of grape seed extract (GSE) was examined by plaque assay for murine norovirus 1 (MNV-1), cell-binding reverse transcription-PCR for human NoV GII.4, and saliva-binding enzyme-linked immunosorbent assay for human NoV GII.4 P particles, with or without the presence of interfering substances (dried milk and lettuce extract). GSE at 0.2 and 2 mg/ml was shown to reduce the infectivity of MNV-1 (<3-log PFU/ml) and the specific binding ability of NoV GII.4 to Caco-2 cells (<-log genomic copies/ml), as well as of its P particles to salivary human histo-blood group antigen receptors (optical density at 450 nm of <0.8). These effects were decreased as increasing concentrations of dried milk (0.02 and 0.2%) or lettuce extract were added. Under an electron microscope, human NoV GII.4 virus-like particles showed inflation and deformation after treatment with GSE. Under conditions that simulated applications in the food industry, the anti-NoV effect of GSE using MNV-1 as a target organism was shown to be limited in surface disinfection (>1 -log PFU/ml, analyzed in accordance with EN 13697:2001). However, a 1.5- to 2-log PFU/ml reduction in MNV-1 infectivity was noted when 2 mg of GSE/ml was used to sanitize water in the washing bath of fresh-cut lettuce, and this occurred regardless of the chemical oxygen demand (0 to 1,500 mg/ml) of the processing water [ABSTRACT FROM AUTHOR]

In this study, the inactivating properties of liquid hydrogen peroxide (L-H2O2), vaporized hydrogen peroxide (V-H2O2), UV light, and a combination of V-H2O2 and UV light were tested on murine norovirus 1 (MNV-1) and bacteriophages (ΦX174 and B40-8) as models for human noroviruses. Disinfection of surfaces was examined on stainless steel discs based on European Standard EN 13697 (2001). For fresh-produce decontamination, a mixture of the viruses was inoculated onto shredded iceberg lettuce and treated after overnight incubation at 2°C. According to our results, L-H2O2 2.1%) was able to inactivate MNV-1 and ΦX174 on stainless steel discs by approximately 4 log10 units within 10 min of exposure, whereas for B40-8, 15% of L-H2O2 was needed to obtain a similar reduction in 10 min. Only a marginal reduction (≤1 log10 unit after 5 min of exposure) by V-H2O2 (2.52%) was achieved for the tested model viruses, although in combination with UV light, a 4-log10-unit decrease within 5 min of treatment was observed on stainless steel discs. Similar trends were observed for the decontamination of shredded iceberg lettuce, but the viral decline was reduced. These results demonstrated that both L-H2O2 and a combination of V-HΦO2 and UV light can be used for norovirus inactivation on surfaces; V-H2O2 (2.52%) in combination with UV light is promising for decontamination of fresh produce with much less consumption of water and disinfectant. [ABSTRACT FROM AUTHOR]

Mesophilic (37°C) and thermophilic (52°C) anaerobic digestion of pig slurry induced at least a 4-log decrease in murine norovirus 1, used as a surrogate virus for porcine norovirus, after 13 and 7 days, respectively. Bacteroides fragilis phage B40-8, employed as a universal viral model, was lowered by 2.5 log after 7 days. The viral titer declined due to temperature and matrix effects. [ABSTRACT FROM AUTHOR]

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag°. The antiviral action of biogenic Ag° was examined in water spiked with an Enterobacter aerogenes-infectiiig bacteriophage (UZ1). Addition of 5.4 mg liter-1 biogenic Ag° caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg°) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter-1 ionic Ag+ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag° were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag° was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag° m-2 on the filter (135 mg biogenic Ag° kg-1 filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag° and its promising opportunities for continuous water disinfection. [ABSTRACT FROM AUTHOR]

The correlation between the detection of murine norovirus 1 RNA by real-time reverse transcription-PCR and the infectivity by plaque assay before and after heat exposure (80°C) was examined. No correlation was found in the current study. Moreover, heat inactivation had a much stronger detrimental effect on virus infectivity than on the integrity of the viral genome. [ABSTRACT FROM AUTHOR]

In this study, a number of Listeria monocytogenes strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical L. monocytogenes strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical L. monocytogenes strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of inlA, inlB, and ami, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical L. monocytogenes strains. Significant differences in inlA and inlB expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of inlA and inlB possibly play a role in the virulence capacities of L. monocytogenes strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific L. monocytogenes strains. [ABSTRACT FROM AUTHOR]

A correction to the article "Inactivation of Murine Norovirus 1, Coliphage ΦX714, and Bacteroides fragilis Phage B40-8 on Surfaces and Fresh-Cut Iceberg Lettuce by Hydrogen Peroxide and UV Light" that was published in the 2011 issue is presented.