Levels Of Shiga Toxin-producing Escherichia Coli Research Articles

The Use of Antimicrobial Washes to Inactivate Shiga Toxin-Producing Escherichia coli from In-Shell Pecans and Wash Water Contaminated by Different Inoculation Routes

In-shell pecans are typically harvested after falling from trees to the ground, presenting a potential route of contamination of foodborne pathogens from soil contact. In-shell pecans are often subjected to various processing or washing steps prior to being shelled. This study determined Shiga toxin-producing Escherichia coli (STEC) reductions after treatment with antimicrobial washes on direct and soil-inoculated in-shell pecans and evaluated the cross-contamination potential of the spent pecan washes after treatment. Pecans were directly and soil-inoculated with an STEC cocktail (O157:H7, O157:NM, O121, O26). Direct inoculation was achieved by spraying the STEC cocktail on the pecans. For soil-inoculation pecans, autoclaved soil was sprayed with the STEC cocktail, homogenized for 2 min, and used to coat in-shell pecans. Inoculated pecans were washed in treatments of 2% lactic acid (LA), 1,000 ppm free chlorine (sodium hypochlorite; NaClO), hot water (HW; 85 ± 2 °C), or ambient water (C [control]; 18 ± 2 °C) for 2, 5, and 10 min and diluted to enumerate STEC populations. After treatments, 100 mL of the spent wash was vacuum filtered through a 0.45-µm membrane and plated on selective agar. HW significantly reduced STEC populations from pecans with and without soil regardless of treatment time (p < 0.05), NaClO reduced STEC populations more than the ambient control wash on directly inoculated pecans, but there were no significant differences between STEC reductions from ambient water (C), LA, and NaClO treatments on soil-inoculated pecans (p > 0.05). Larger STEC populations were enumerated from ambient water wash compared to the antimicrobial washes (p < 0.05). The HW, LA, and NaClO treatments were effective at maintaining the quality of the wash water, with STEC levels being generally at or below the detection limit (<1 CFU/100 mL), while HW was the most effective at reducing STEC from in-shell pecans with and without a soil coating (>5-log CFU/mL reductions).

Viability of Shiga Toxin–Producing Escherichia coli, Salmonella spp., and Listeria monocytogenes during Preparation and Storage of Fuet, a Traditional Dry-Cured Spanish Pork Sausage

The primary objective of this study was to monitor viability of Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Listeria monocytogenes during preparation and storage of fuet. Regarding methodology, coarse-ground pork (ca. 35% fat) was mixed with salt (2.5%), dextrose (0.3%), starter culture (ca. 7.0 log CFU/g), celery powder (0.5%), and ground black pepper (0.3%) and then separately inoculated with a multistrain cocktail (ca. 7.0 log CFU/g) of each pathogen. The batter was stuffed into a ca. 42-mm natural swine casing and fermented at 23 ± 2°C and ca. 95% ± 4% relative humidity to ≤pH 5.3 (≤48 h). Sausages were then dried at 12 ± 2°C and ca. 80% ± 4% relative humidity to a water activity (aw) of 0.89 (within 33 days) or aw 0.86 (within 60 days). A portion of each batch of fuet was subjected to high-pressure processing (HPP; 600 MPa for 3 min) before chubs were vacuum packaged and stored for 30 days at 20 ± 2°C. The results revealed that pathogen numbers remained relatively unchanged after fermentation (≤0.35 log CFU/g reduction), whereas reductions of ca. 0.8 to 3.2 log CFU/g were achieved after drying fuet to aw 0.89 or 0.86. Regardless of whether fuet was or was not pressure treated, additional reductions of ca. 2.2 to ≥5.3 log CFU/g after drying were achieved following 30 days of storage at 20°C. For non-HPP-treated fuet dried to aw 0.89 and stored for 30 days at 20°C, total reductions of ≥5.3 log CFU/g in levels of STEC or Salmonella spp. were achieved, whereas levels of L. monocytogenes were reduced by ca. 3.6 log CFU/g. Total reductions of ≥5.3 log CFU/g in levels of all three pathogens were achieved after drying non-HPP-treated fuet to aw 0.86. For fuet dried to aw 0.89 or 0.86, that were pressure treated and then stored for 30 days at 20°C, total reductions of >6.2 log CFU/g in levels of all three pathogens were achieved. In conclusion, the processing parameters tested herein, with or without application of HPP, validated that reductions of ≥2.0 or ≥5.0 log CFU/g in levels of STEC, Salmonella spp., and L. monocytogenes were achieved during preparation and storage of fuet.

Inactivation of Shiga Toxin–Producing Escherichia coli and Listeria monocytogenes within Plant versus Beef Burgers in Response to High Pressure Processing

We evaluated high pressure processing to lower levels of Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes inoculated into samples of plant or beef burgers. Multistrain cocktails of STEC and L. monocytogenes were separately inoculated (∼7.0 log CFU/g) into plant burgers or ground beef. Refrigerated (i.e., 4°C) or frozen (i.e., -20°C) samples (25 g each) were subsequently exposed to 350 MPa for up to 9 or 18 min or 600 MPa for up to 4.5 or 12 min. When refrigerated plant or beef burger samples were treated at 350 MPa for up to 9 min, levels of STEC were reduced by ca. 0.7 to 1.3 log CFU/g. However, when refrigerated plant or beef burger samples were treated at 350 MPa for up to 9 min, levels of L. monocytogenes remained relatively unchanged (ca. ≤0.3-log CFU/g decrease) in plant burger samples but were reduced by ca. 0.3 to 2.0 log CFU/g in ground beef. When refrigerated plant or beef burger samples were treated at 600 MPa for up to 4.5 min, levels of STEC and L. monocytogenes were reduced by ca. 0.7 to 4.1 and ca. 0.3 to 5.6 log CFU/g, respectively. Similarly, when frozen plant and beef burger samples were treated at 350 MPa up to 18 min, reductions of ca. 1.7 to 3.6 and ca. 0.6 to 3.6 log CFU/g in STEC and L. monocytogenes numbers, respectively, were observed. Exposure of frozen plant or beef burger samples to 600 MPa for up to 12 min resulted in reductions of ca. 2.4 to 4.4 and ca. 1.8 to 3.4 log CFU/g in levels of STEC and L. monocytogenes, respectively. Via empirical observation, pressurization did not adversely affect the color of plant burger samples, whereas appreciable changes in color were observed in pressurized ground beef. These data confirm that time and pressure levels already validated for control of STEC and L. monocytogenes in ground beef will likely be equally effective toward these same pathogens in plant burgers without causing untoward effects on product color.

Phage Biocontrol Improves Food Safety by Significantly Reducing the Level and Prevalence of Escherichia coli O157:H7 in Various Foods

Management of Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, in food products is a major challenge for the food industry. Several interventions, such as irradiation, chemical disinfection, and pasteurization, have had variable success controlling STEC contamination. However, these interventions also indiscriminately kill beneficial bacteria in foods, may impact organoleptic properties of foods, and are not always environmentally friendly. Biocontrol using bacteriophage-based products to reduce or eliminate specific foodborne pathogens in food products has been gaining attention due to the specificity, safety, and environmentally friendly properties of lytic bacteriophages. We developed EcoShield PX, a cocktail of lytic bacteriophages, that specifically targets STEC. This study was conducted to examine the efficacy of this bacteriophage cocktail for reducing the levels of E. coli O157:H7 in eight food products: beef chuck roast, ground beef, chicken breast, cooked chicken, salmon, cheese, cantaloupe, and romaine lettuce. The food products were challenged with E. coli O157:H7 at ca. 3.0 log CFU/g and treated with the bacteriophage preparation at ca. 1 × 106, 5 × 106, or 1 × 107 PFU/g. Application of 5 × 106 and 1 × 107 PFU/g resulted in significant reductions (P < 0.05) in E. coli O157:H7 levels of up to 97% in all foods. When bacteriophages (ca. 1 × 106 PFU/g) were used to treat lower levels of E. coli O157:H7 (ca. 1 to 10 CFU/10 g) on beef chuck roast samples, mimicking the levels of STEC found under real-life conditions in food processing plants, the prevalence of STEC in the samples was significantly reduced (P < 0.05) by ≥80%. Our results suggest that this STEC-targeting bacteriophage preparation can result in significant reduction of both the levels and prevalence of STEC in various foods and, therefore, may help improve the safety and reduce the risk of recalls of foods at high risk for STEC contamination.

Viability of Shiga Toxin–producing Escherichia Coli, Salmonella, and Listeria monocytogenes Within Plant Versus Beef Burgers During Cold Storage and Following Pan Frying

The viability of Shiga toxin-producing Escherichia coli (STEC), Salmonella, and Listeria monocytogenes within plant- and beef-based burgers was monitored during storage and cooking. When inoculated (ca. 3.5 log CFU/g) into 15-g portions of plant- or beef-based burgers, levels of STEC and Salmonella decreased slightly (≤0.5-log decrease) in both types of burgers when stored at 4°C, but increased ca. 2.4 and 0.8 log CFU/g, respectively, in plant-based burgers but not beef-based burgers (≤1.2-log decrease), after 21 days at 10°C. For L. monocytogenes, levels increased by ca. 1.3 and 2.6 log CFU/g in plant burgers after 21 days at 4 and 10°C, respectively, whereas pathogen levels decreased slightly (≤0.9-log decrease) in beef burgers during storage at 4 and 10°C. Regarding cooking, burgers (ca. 114 g each) were inoculated with ca. 7.0 log CFU/g STEC, Salmonella, or L. monocytogenes and cooked in a sauté pan. Cooking plant- or beef-based burgers to 62.8°C (145°F), 68.3°C (155°F), or 73.9°C (165°F) delivered reductions ranging from ca. 4.7 to 6.8 log CFU/g for STEC, ca. 4.4 to 7.0 log CFU/g for L. monocytogenes, and ca. 3.5 to 6.7 log CFU/g for Salmonella. In summary, the observation that levels of all three pathogens increased by ca. 1.0 to ca. 2.5 log CFU/g in plant-based burgers when stored at an abusive temperature (10°C) highlights the importance of proper storage (4°C) to lessen risk. However, because all three pathogens responded similarly to heat in plant-based as in beef-based burgers, well-established cooking parameters required to eliminate STEC, Salmonella, or L. monocytogenes from ground beef should be as effective for controlling cells of these same pathogens in a burger made with plant-sourced protein.

Meat Bars: A Survey To Assess Consumer Familiarity and Preparation Parameters and a Challenge Study To Quantify Viability of Shiga Toxin–Producing Escherichia coli Cells during Processing and Storage

Meat bars are dried snacks containing a mixture of meat, berries, and nuts. To explore consumer awareness of meat bars, we conducted two online, nationally representative surveys and established that 70.8% (743 of 1,050) of U.S. citizens were unfamiliar with this product. When asked to check all answers that applied, most of the 545 respondents (who were recruited based on their familiarity with meat bars) preferred beef (n = 385) as the protein source, followed by chicken (n = 293), pork (n = 183), and turkey (n = 179). Most meat bars were purchased from grocery stores (n = 447), followed by online orders (n = 130) and outdoor stores (n = 120). When asked specifically whether they made their own meat bars, 17.8% of respondents (97 of 545) replied "yes," the majority (52 of 97, 54%) of which obtained recipes online. Some 69.1% (67 of 97) measured the internal temperature of the meat during dehydration, but only 10.3% (10 of 97) confirmed the internal temperature by using a thermometer. Given the paucity of information available on the fate of pathogenic or spoilage bacteria associated with meat bars, as another component of this study, batter was prepared with or without encapsulated citric acid (ECA; 0.74%) added to a formulation of ground beef (65%; 90% lean, 10% fat), chopped pecans (15%), golden flaxseed flour (9.7%), chopped cranberries (5.0%), chopped sunflower seeds (3.1%), sea salt (1.1%), black pepper (0.8%), and celery powder (0.35%). Batter was inoculated (ca. 6.5 log CFU/g) with Shiga toxin-producing Escherichia coli (STEC), portioned by hand (40 ± 0.1 g each), and then dried in a commercial dehydrator. Regardless of the drying treatment, inclusion of ECA in the batter resulted in a pH decrease from ca. 5.5 to ca. 4.7 to 5.0 in the finished product. Without ECA, when meat bars were dried at 62.8°C for 6 h, 71.1°C for 4 h, or 62.8°C for 2 h and then 71.1°C for 2 h, levels of STEC decreased by ca. 6.2, 6.3, or 5.2 log CFU/g, respectively. With ECA, STEC decreased by ca. 6.0, 6.6, or 6.0 log CFU/g in meat bars dried at 62.8°C for 6 h, 71.1°C for 4 h, or 62.8°C for 2 h and then 71.1°C for 2 h, respectively. Our results confirmed that a ≥5.0-log reduction in STEC could be achieved in meat bars formulated with or without ECA under all dehydration conditions tested.

English

Healthy ruminants appear to be the main reservoir of Shiga toxin-producing Escherichia coli (STEC). Importantly, this pathogen is shed in faeces of sheep and can cause outbreaks of human illness ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing E. coli (STEC) have been reported worldwide. The manure of ruminants when used as agricultural fertilizer can serve as a vehicle for STEC contamination of fruits, vegetables, water and soil. The aim of the present study was to evaluate whether the use of probiotic strains of Ruminobacter amylophilus, Ruminobacter succinogenes, Succinovibrio dextrinosolvens, Bacillus cereus sub toyoi, Lactobacillus acidophilus and Enterococcus faecium, supplemented to the daily oral food ration provided to sheep, together with composting of their feces, may be used as a strategy to reduce STEC levels on a farm. The first stage of the present study was performed during a six-week period with a total of 160 sheep distributed among four groups comprised of 40 sheep each. Group A did not receive either STEC or probiotic, Group B received probiotic alone, Group C received STEC plus probiotic and Group D received STEC alone. After the sheep were inoculated, samples of their feces were collected and the number of STEC and E. coli were counted. In the second stage of the study, after the six-week period, all fecal material was composted into four separate heaps. A possible protective effect of the probiotic strains against colonization by STEC was observed. It was also observed that composting was very efficient at eliminating or decreasing the STEC population. Although the number of STEC isolates was effectively decreased in all compost heaps, the Group C derived compost heap was found to have a lower amount of STEC than the Group D derived compost heap. These findings suggest that the use of probiotics, such as lactic bacteria, together with composting manure may be an efficient strategy to decrease the STEC population on a farm. Key words: Composting, Escherichia coli, probiotic, Shiga-like-toxin

Short communication: Behavior of different Shiga toxin-producing Escherichia coli serotypes (O26:H11, O103:H2, O145:H28, O157:H7) during the manufacture, ripening, and storage of a white mold cheese

Ruminants are healthy carriers of Shiga toxin-producing Escherichia coli (STEC). If good hygienic and agricultural practices at the farm level, especially during the milking process, are not adequately followed, milk and dairy products made with raw milk could become contaminated. Sporadic cases and rare food outbreaks have been linked with dairy products. Consequently, understanding STEC behavior in cheeses would help to evaluate risks for human health. The behavior of 4 different STEC strains belonging to the serotypes O26:H11, O103:H2, O145:H28, and O157:H7 were monitored during the manufacture, ripening, and storage of a white mold soft cheese. These strains, originating from dairy products, were inoculated individually in raw milk from cow at 102 cfu/mL. During the first 24 to 36h of the manufacturing stage, the STEC level increased by 2 to 3 log10 cfu/g. Over the course of the ripening stage, the concentration of the non-O157 STEC remained relatively constant, whereas a decrease of the E. coli O157:H7 concentration was observed. During the storage stage, the level of the different non-O157 STEC strains decreased slowly in the core and in the rind of cheeses. The non-O157 STEC level reached between 3.1 and 4.1 log10 cfu/g at d 56. Interestingly, the concentration of the E. coli O157:H7 strain decreased dramatically: the strains remained detectable only after enrichment. During ripening and storage, STEC levels were generally higher in rinds than in cheese cores. In contrast to what was seen in cheese cores, the E. coli O157:H7 strain remained enumerable in rinds during these steps. These results highlight that STEC can grow during the manufacture and survive during the ripening and storage of a white mold soft cheese.

Effect of Exposure Time and Organic Matter on Efficacy of Antimicrobial Compounds against Shiga Toxin–Producing Escherichia coli and Salmonella

Several antimicrobial compounds are in commercial meat processing plants for pathogen control on beef carcasses. However, the efficacy of the method used is influenced by a number of factors, such as spray pressure, temperature, type of chemical and concentration, exposure time, method of application, equipment design, and the stage in the process that the method is applied. The objective of this study was to evaluate effectiveness of time of exposure of various antimicrobial compounds against nine strains of Shiga toxin-producing Escherichia coli (STEC) and four strains of Salmonella in aqueous antimicrobial solutions with and without organic matter. Non-O157 STEC, STEC O157:H7, and Salmonella were exposed to the following aqueous antimicrobial solutions with or without beef purge for 15, 30, 60, 120, 300, 600, and 1,800 s: (i) 2.5% lactic acid, (ii) 4.0% lactic acid, (iii) 2.5% Beefxide, (iv) 1% Aftec 3000, (v) 200 ppm of peracetic acid, (vi) 300 ppm of hypobromous acid, and (vii) water as a control. In general, increasing exposure time to antimicrobial compounds significantly (P ≤ 0.05) increased the effectiveness against pathogens tested. In aqueous antimicrobial solutions without organic matter, both peracetic acid and hypobromous acid were the most effective in inactivating populations of STEC and Salmonella, providing at least 5.0-log reductions with exposure for 15 s. However, in antimicrobials containing organic matter, 4.0% lactic acid was the most effective compound in reducing levels of STEC and Salmonella, providing 2- to 3-log reductions with exposure for 15 s. The results of this study indicated that organic matter and exposure time influenced the efficacy of antimicrobial compounds against pathogens, especially with oxidizer compounds. These factors should be considered when choosing an antimicrobial compound for an intervention.

Thermal Inactivation of Shiga Toxin‐Producing Escherichia coli Cells within Veal Cordon Bleu

AbstractVeal cutlets were surface inoculated with ca. 6.6 cfu/g of an eight‐strain rifampicin‐resistant cocktail of Shiga toxin‐producing Escherichia coli (STEC) (O26:H11, O45:H2, O103:H2, O104:H4, O111:H‐, O121:H19, O145:NM and O157:H7). Cutlets were mechanically tenderized and cordon bleu was prepared by adding slices of ham and cheese between two cutlets prior to batter/breading and cooking. Fully assembled cordon bleu were cooked in preheated (191.5C) extra virgin olive oil (45 mL) on a griddle. Cooking for 4, 5 or 6 min per side reduced STEC levels by ca. 1.3, 2.2 or 3.4 log cfu/g, respectively, whereas cooking for 7–10 min per side resulted in reductions of ca. ≥6.2 log cfu/g. These data validated that cooking tenderized veal cordon bleu for at least 7 min per side in 45 mL of olive oil on a griddle maintained at ca. 191.5C is sufficient to achieve an internal cordon bleu temperature of 69.0 ± 3.3C and a ≥5‐log reduction of STEC.Practical ApplicationsVeal cordon bleu is a popular meal prepared by placing a slice of cheese and a slice of cured pork between two cutlets and then coating/breading the resulting turnover‐type product. We evaluated time and temperature cooking regimens for lethality toward Shiga toxin‐producing Escherichia coli (STEC) inoculated onto mechanically tenderized multicomponent veal cordon bleu. Our results confirmed that a 5‐log reduction of STEC was achieved by cooking veal cordon bleu to an internal temperature of 69.0 ± 3.3C for at least 7 min per side on a griddle maintained at 191.5C. Reductions of ca. 1.5–3.5 log cfu/g were also achieved following cooking for 4 to 6 min per side. These data establish guidelines for cooking veal cordon bleu, which, in turn, may decrease the potential risk of STEC illnesses associated with this product if pathogens are present and if these products are undercooked or mishandled.

Immersion in Antimicrobial Solutions Reduces Salmonella enterica and Shiga Toxin–Producing Escherichia coli on Beef Cheek Meat

The objective of this study was to determine the effect of immersing beef cheek meat in antimicrobial solutions on the reduction of O157:H7 Shiga toxin-producing Escherichia coli (STEC), non-O157:H7 STEC, and Salmonella enterica. Beef cheek meat was inoculated with O157:H7 STEC, non-O157:H7 STEC, and S. enterica on both the adipose and muscle surfaces. The inoculated cheek meat was then immersed in one of seven antimicrobial solutions for 1, 2.5, or 5 min: (i) 1% Aftec 3000 (AFTEC), (ii) 2.5% Beefxide (BX), (iii) 300 ppm of hypobromous acid (HOBR), (iv) 2.5% lactic acid (LA2.5), (v) 5% lactic acid (LA5), (vi) 0.5% levulinic acid and 0.05% sodium dodecyl sulfate (LEV-SDS), or (vii) 220 ppm of peroxyacetic acid (POA). Inoculated cheek meat was also immersed in 80 °C tap water (HW) for 10 s. In general, increasing immersion duration in antimicrobial solutions did not significantly (P ≥ 0.05) increase effectiveness. Immersion in HW for 10 s was the most effective intervention, reducing STEC and S. enterica by 2.2 to 2.3 log CFU/cm2 on the adipose surface and by 1.7 to 1.8 log CFU/cm2 on the muscle surface. Immersion for 1 min in AFTEC, BX, LA2.5, LA5, or POA was also effective as an intervention, reducing STEC and S. enterica by 0.8 to 2.0 log CFU/cm2 on the adipose surface and by 0.6 to 1.4 log CFU/cm2 on the muscle surface. Immersion for 1 min in HOBR or LEV-SDS was not an effective intervention because STEC and S. enterica reductions ranged from 0.1 to 0.4 log CFU/cm2, which were not significantly different (P ≥ 0.05) from the reductions obtained when cheek meat was immersed in room temperature tap water. We conclude that immersion of cheek meat in HW for 10 s and immersion for 1 min in AFTEC, BX, LA2.5, LA5, or POA effectively reduced levels of STEC and S. enterica.

Evaluating the effectiveness of transport media on Shiga toxin-producing E. coli serotypes (2014)

Introduction One of the key issues involved in accurately testing beef and the environment for the presence of specific bacteria, particularly pathogens such as Shiga toxin-producing Escherichia coli (STEC), is maintaining the viability of the microorganisms when transporting samples from the field to the laboratory. This process may take up to three days when considering collection, shipping and laboratory preparation times. Allowing the target bacteria to increase or decrease in numbers during transit is undesirable, so samples must be kept chilled and the media used for transport must offer a stable but non-nutritive environment. Three commonly used non-selective transport media were evaluated for their ability to maintain original STEC levels during transport. Holding temperature may vary during shipping, so this study evaluated two separate temperatures as co-variables.

Survival of Escherichia coli O26:H11 exceeds that of Escherichia coli O157:H7 as assessed by simulated human digestion of contaminated raw milk cheeses

Shiga toxin producing Escherichia coli (STEC) are an important cause of human foodborne outbreaks. The consumption of raw milk dairy products may be an important route of STEC infection. For successful foodborne transmission, STEC strains must survive stress conditions met during gastrointestinal transit in humans. The aim of this study was to evaluate the survival of two STEC strains of serotypes O157:H7 and O26:H11 during simulated human digestion in the TNO gastro-Intestinal tract Model (TIM) of contaminated uncooked pressed cheeses. The survival of cheese microflora during in vitro gastrointestinal transit was also determined for the first time. The level of STEC increased from 2log10CFU/ml to 4log10CFU/g during the first 24h of cheese making and remained stable at around 4log10CFU/g during cheese ripening and conservation. During transit through the artificial stomach and duodenum, levels of STEC decreased: 0.2% of E. coli O157:H7 and 1.8% of E. coli O26:H11 were recovered at 150min in the gastric compartment, compared with 14.3% for the transit marker. Bacterial resumption was observed in the jejunum and ileum: 35.8% of E. coli O157:H7 and 663.2% of E. coli O26:H11 were recovered at 360min in the ileal compartment, compared with 12.6% for the transit marker. The fate of STEC was strain-dependent, the survival of E. coli O26:H11 being 13 times greater than that of E. coli O157:H7 at the end of digestion in the cumulative ileal deliveries. These data provide a better understanding of STEC behavior during gastrointestinal transit in humans after ingestion of contaminated cheese.

Behavior of different Shiga toxin-producing Escherichia coli serotypes in various experimentally contaminated raw-milk cheeses.

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness. The public health implication of the presence of STEC in dairy products remains unclear. Knowledge of STEC behavior in cheeses would help to evaluate the human health risk. The aim of our study was to observe the growth and survival of experimentally inoculated STEC strains in raw-milk cheeses manufactured and ripened according to five technological schemes: blue-type cheese, uncooked pressed cheese with long ripening and with short ripening steps, cooked cheese, and lactic cheese. Cheeses were contaminated with different STEC serotypes (O157:H7, O26:H11, O103:H2, and O145:H28) at the milk preparation stage. STEC growth and survival were monitored on selective media during the entire manufacturing process. STEC grew (2 to 3 log(10) CFU · g(-1)) in blue-type cheese and the two uncooked pressed cheeses during the first 24 h of cheese making. Then, STEC levels progressively decreased in cheeses that were ripened for more than 6 months. In cooked cheese and in lactic cheese with a long acidic coagulation step (pH < 4.5), STEC did not grow. Their levels decreased after the cooking step in the cooked cheese and after the coagulation step in the lactic cheese, but STEC was still detectable at the end of ripening and storage. A serotype effect was found: in all cheeses studied, serotype O157:H7 grew less strongly and was less persistent than the others serotypes. This study improves knowledge of the behavior of different STEC serotypes in various raw-milk cheeses.

Survival and spread of Shiga toxin-producingEscherichia coliin alpine pasture grasslands

To determine the fate of Shiga toxin-producing Escherichia coli (STEC) strains defecated onto alpine grassland soils. 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. STEC were able to persist in cowpats and disseminate down through the soil but were unable to establish. This study provides useful information concerning the ecology of STEC in alpine pasture grasslands and may have implications for land and cattle management.

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 °C). The BM and STEC growth data were simultaneously fitted by using a competitive growth model. The STEC final levels obtained after 24 h 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.