Influence of reduced levels or suppression of sodium nitrite on the outgrowth and toxinogenesis of psychrotrophic Clostridium botulinum Group II type B in cooked ham.

Outgrowth and toxinogenesis of Clostridium botulinum Group II (non-proteolytic) type B were studied in cooked ham prepared with different NaNO2 (ranging from 0 to 80 mg/kg) and sodium chloride (NaCl, ranging from 12 to 19 g/kg) incorporation rates. Cured ground pork batters were inoculated with a cocktail of 3 strains of C. botulinum Group II type B at 3.5 log10 CFU/g, portioned and samples of 50 g were vacuum packed then cooked and cooled based on thermal processing employed by the meat processing industry. These cooked ham model samples were stored under reasonably foreseeable conditions of use and storage i.e. for 14 days at 4 °C, followed by a cold chain break for 1 h at 20 °C then up to 33 days at 8 °C. Storage times and temperatures were used to mimic those commonly encountered along the supply chain. Enumeration of C. botulinum and detection of the botulinum neurotoxin type B (BoNT/B) were performed in triplicate at different storage times. Under these experimental conditions, incorporation rates of NaNO2 ≥ 30 mg/kg prevented the outgrowth and toxinogenesis of C. botulinum Group II type B in the cooked ham model, regardless of the NaCl concentrations tested. In contrast, total removal of nitrite allowed outgrowth and toxin production during storage of the processed meat product. Results showed that the maximum ingoing amount of nitrite (i.e. 150 mg/kg) that may be added according to the EU legislation (Regulation (EC) No 1333/2008) can be reduced in cooked ham while still ensuring control of C. botulinum Group II type B. According to the multiple factors that could affect C. botulinum behavior in processing meat products, outgrowth and toxin production of C. botulinum should be evaluated on a case by case basis, depending on the recipe, manufacturing process, food matrix and storage conditions.

Reduced susceptibilities to biocides and resistance to antibiotics in food-associated bacteria following exposure to quaternary ammonium compounds.

AIMS: Our aim was to assess the effects of step-wise exposure to didecyl dimethyl ammonium chloride (DDAC) on the antimicrobial (antibiotics and biocides) susceptibilities of food-associated bacterial strains. METHODS AND RESULTS: Adaptive responses of bacterial strains were investigated by exposing the strains daily to increasing subinhibitory concentrations of DDAC for 7 days. Following adaptation to DDAC, a threefold increase in the minimum inhibitory concentration (MIC) values for this biocide was observed in 48% of the Escherichia coli and Listeria monocytogenes strains, and 3% of the Salmonella strains. Reduced susceptibility to other biocides was found with the most important increase in MIC for benzalkonium chloride (BC) and a commercial biocide formulation (Galox Horizon) containing DDAC and glutaraldehyde, for all species except Salmonella. Increase in antibiotic MIC values was more pronounced in E. coli in terms of antibiotic numbers and of magnitude (from 4- to 32-fold increase) and, to a lesser extent, in Salmonella strains. Most of these strains had acquired resistance to ampicillin, cefotaxime, ceftazidime, chloramphenicol and ciprofloxacin. CONCLUSIONS: The effects of exposure to DDAC on biocides and antibiotics susceptibilities depend upon the bacteria species. SIGNIFICANCE AND IMPACT OF THE STUDY: Extensive use of DDAC at subinhibitory concentrations may lead to the development of antibiotic-resistant bacteria and may represent a public health issue.

Quantitative real-time PCR assays for sensitive detection of Canada goose-specific fecal pollution in water sources.

Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.

Survival and spread of Shiga toxin-producing Escherichia coli in alpine pasture grasslands.

AIMS: To determine the fate of Shiga toxin-producing Escherichia coli (STEC) strains defecated onto alpine grassland soils. METHODS AND RESULTS: During the summers of 2005 and 2006, the field survival of STEC was monitored in cowpats and underlying soils in four different alpine pasture units. A most probable number (MPN)-PCR stx assay was used to enumerate STEC populations. STEC levels ranged between 3.9 and 5.4 log(10) CFU g(-1) in fresh cowpats and slowly decreased until their complete decay (inactivation rates k < 0.04 day(-1)). PFGE typing of STEC strains isolated from faecal and soil samples assessed the persistence of various clonal types for at least 2 months in cowpats and their vertical dispersal down through the soil at a depth up to at least 20 cm. STEC cells counts in soil were always below 2 log(10) CFU g(-1), regardless of the pasture unit investigated. The soil became rapidly free of detectable STEC once the cowpat had decomposed. The eight STEC strains isolated during this study belonged to six distinct serotypes and tested positive for the gene(s) stx2, including the stx2g and stx2 NV206 variants. CONCLUSIONS: STEC were able to persist in cowpats and disseminate down through the soil but were unable to establish. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information concerning the ecology of STEC in alpine pasture grasslands and may have implications for land and cattle management.

Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed.

Our ability to identify and eliminate fecal contamination of water, now and in the future, is essential to reduce incidences of waterborne disease. Bacterial source tracking is a recently developed approach for identifying sources of fecal pollution. PCR primers designed by Bernhard and Field [Bernhard, A.E., Field, K.G., 2000a. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66(10), 4571-4574] and Dick et al. [Dick, L.K., Bernhard, A.E., Brodeur, T.J., Santo Domingo, J.W., Simpson, J.M., Walters, S.P., Field, K.G., 2005. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71(6), 3184-3191] for the detection of human (HF183), pig (PF163) and ruminant (CF128) specific Bacteroidales 16s rRNA genetic markers were tested for their suitability in detecting fecal pollution in Saskatchewan, Canada. The sensitivity and specificity of these primers were assessed by testing eight raw human sewage samples and 265 feces from 12 different species in Saskatchewan. The specificity of each primer set was > or =94%. The accuracy of HF183 and PF163 to distinguish between the different species was 100%, whereas CF128 cross-reacted with 22% of the pig feces. Occurrence of the host-specific Bacteroidales markers and the conventional indicator Escherichia coli in relation to several enteropathogens was investigated in 70 water samples collected from different sites along the Qu'Appelle River (Saskatchewan, Canada). Human and ruminant fecal markers were identified in 41 and 14% of the water samples, respectively, whereas the pig marker was never detected in the river water. The largest concentrations in E. coli counts were concomitant to the simultaneous detection of HF183 and CF128. Thermotolerant Campylobacter spp., Salmonella spp. and Shiga toxin genes (stx1 and stx2)-positive E. coli (STEC) were detected in 6, 7 and 63% of the water samples, respectively. However, none of the stx positive water samples were positive for the E. coli O157:H7 gene marker (uidA). Odds ratios analysis suggests that CF128 may be predictive for the presence of Salmonella spp. in the river investigated. None of the fecal indicators were able to confidently predict the presence of thermotolerant Campylobacter spp. and STEC.

Assessment of the microbial quality of irrigation water in a prairie watershed.

AIMS: To assess levels of faecal contamination in the Qu'Appelle River (Saskatchewan, Canada) and its suitability for irrigation, by using the Colilert-18/Quanti-Tray technology. METHODS AND RESULTS: Various sites located along the Qu'Appelle River were sampled weekly from May to August 2005-2007. A total of 594 freshwater samples were collected and analysed for enumeration of Escherichia coli using the Colilert-18. The false-positive rate for E. coli detection using Colilert-18 was at most 1.5%. Throughout the irrigation period (June to August), up to 85% of the water samples collected from one of the irrigation water-pumping sites exceeded the recommended limit of 100 CFU per 100 ml. Spikes in E. coli counts were generally concomitant with the sudden rise in river flows. A sub-sample of confirmed E. coli isolates were typed by randomly amplified polymorphic DNA (RAPD). RAPD analysis revealed a high degree of genetic diversity among E. coli isolates. A significant association between RAPD patterns and the month of E. coli isolation was demonstrated. CONCLUSIONS: Colilert-18 provides an effective means for assessing microbial quality of irrigation water. SIGNIFICANCE AND IMPACT OF THE STUDY: Qu'Appelle River is subject to variability of faecal contamination during irrigation times and monitoring throughout irrigation season is important for ensuring safe production practices.

Long-term survival of Shiga toxin-producing Escherichia coli in cattle effluents and environment: an updated review.

Shiga toxin-producing Escherichia coli (STEC) are one of the most important emergent foodborne pathogens. STEC are common as colonizers in the intestine of healthy cattle and are spread into the environment by fecal shedding or following the surface application of farm effluent on soil. The bacteria can be transmitted to humans through food, such as inadequately cooked ground beef or unpasteurized milk. During the last decade, a wide variety of environmentally related exposures have emerged as new routes of transmission. Major outbreaks due to the consumption of raw fruits and vegetables or accidental ingestion of soil or water contaminated by STEC have been increasingly reported. STEC survival in cattle effluents, soil, plants and water is discussed in the light of new knowledge regarding both biotic and abiotic factors which may affect their survival or enhance their dissemination in the environment. The ability to persist in cattle production environments contributes to the contamination and recontamination of cattle, as well as for human infection. Consequently, effective control strategies must be considered on cattle farms, in order to limit entry of STEC cells into the environment.

Persistence of Shiga toxin-producing Escherichia coli O26 in various manure-amended soil types.

AIMS: To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions. METHODS AND RESULTS: Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10(6) CFU g(-1)) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20 degrees C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0 x 08 g H2O g(-1) dry soil. At 4 and 20 degrees C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20 degrees C) was significantly associated with the highest STEC count decline in all soils tested. CONCLUSIONS: The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.

Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry.

AIMS: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. METHODS AND RESULTS: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 10(6) CFU ml(-1) in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8.5 to 9.5 units during the last 50 days. CONCLUSION: STEC O26 strains were able to survive in cow slurry for an extended period. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26.

Growth of Shiga-toxin producing Escherichia coli (STEC) and bovine feces background microflora in various enrichment protocols.

Cattle are an important reservoir for STEC and eating food contaminated with fecal material is a frequent source of human STEC infection. It is thus essential to reliably determine the prevalence of STEC contamination in cattle. Currently, different enrichment protocols are used before the detection of Shiga-Toxin producing Escherichia coli (STEC) in fecal samples. However, there have not been any studies performed that have compared the effectiveness of these various enrichment protocols for the growth of non-O157 STEC in fecal samples. The objective of this present study was to characterize the effects of different enrichment factors on the simultaneous growth of the feces background microflora (BM) and two non-O157 STEC strains. The different factors studied were the basal medium (TSB and EC), the effect of novobiocin in the broth (N+ or N-) and the incubation temperature (37 or 40 degrees C). The BM and STEC growth data were simultaneously fitted by using a competitive growth model. The STEC final levels obtained after 24h were higher for the protocols with novobiocin and/or EC compared to the others. However, novobiocin inhibited the growth of one STEC strain. We observed that the addition of novobiocin into broths is not advisable for optimal growth conditions. Moreover, given high BM and low STEC levels often observed in feces, predictions made with the growth model highlighted that false negative results could more likely appear with protocols using TSB without novobiocin than with protocols using EC. In conclusion, the use of EC broth in enrichment protocols seems to be more appropriate for detecting non-O157 STEC from bovine fecal samples. This can help avoid false negative results that cause an underestimation of the STEC prevalence in cattle.

Growth and survival of non-O157:H7 Shiga-toxin-producing Escherichia coli in cow manure.

AIMS: The main objective of this study was to evaluate the behaviour of non-O157:H7 Shiga-toxin-producing Escherichia coli (STEC) strains in cow manure. METHODS AND RESULTS: A mixture of eight green-fluorescent-protein-labelled STEC strains was inoculated around 10(6)-10(7) CFU g(-1) into four manure heaps. Two heaps were regularly turned and the two others remained unturned. STEC counts and physical parameters (temperature, pH, moisture content and oxido-reduction potential) were monitored for 1000 manure samples. The highest mean pH values were obtained near the surface at the base of all manure heaps. At the surface, the moisture content decreased from 76.5% to 42% in turned heaps. Temperatures reached 65 degrees C near the main body of all manure heaps, and only 35 degrees C near the superficial parts located at the base of them. These two sites (the centre and the base) were associated with D values for the STEC counts of 0.48 and 2.39 days, respectively. We were able to detect STEC strains during 42 days in turned manure heaps and during at least 90 days in unturned ones. CONCLUSIONS: These results emphasize the long-term survival of non-O157:H7 STEC in cow manure. SIGNIFICANCE AND IMPACT OF THE STUDY: Good management practices (e.g. turning) should be respected in order to minimize the risk of environmental contamination by STEC.

Dissemination and persistence of Shiga toxin-producing Escherichia coli (STEC) strains on French dairy farms.

Some Shiga toxin-producing Escherichia coli strains (STEC), and in particular E. coli O157:H7, are known to cause severe illness in humans. STEC have been responsible for large foodborne outbreaks and some of these have been linked to dairy products. The aim of the present study was to determine the dissemination and persistence of STEC on 13 dairy farms in France, which were selected out of 151 randomized dairy farms. A total of 1309 samples were collected, including 415 faecal samples from cattle and 894 samples from the farm environment. Bacteria from samples were cultured and screened for Shiga toxin (stx) genes by polymerase chain reaction (PCR). STEC isolates were recovered from stx-positive samples after colony blotting, and characterized for their virulence genes, serotypes and XbaI digestion patterns of total DNA separated by pulsed-field gel electrophoresis (PFGE). Stx genes were detected in 145 faecal samples (35%) and 179 (20%) environmental samples, and a total of 118 STEC isolates were recovered. Forty-six percent of the STEC isolates were positive for stx1, 86% for stx2, 29% for intimin (eae-gene) and 92% for enterohemolysin (ehx), of which 16% of the STEC strains carried these four virulence factors in combination. Furthermore, we found that some faecal STEC strains belonged to serotypes involved in human disease (O26:H11 and O157:H7). PFGE profiles indicated genetic diversity of the STEC strains and some of these persisted in the farm environment for up to 12 months. A large range of contaminated samples were collected, in particular from udders and teats. These organs are potential sources for contamination and re-contamination of dairy cattle and constitute an important risk for milk contamination.