Shiga Toxin-producing Escherichia Coli Cells Research Articles

Fate of salmonella and shiga-toxin producing Escherichia coli on wheat grain during tempering

Outbreaks of Salmonella and Shiga-toxin producing Escherichia coli (STEC) linked to wheat flour led to increased interest in characterizing the fate of Salmonella and STEC on wheat during processing. Tempering is the stage of wheat processing where water is added to toughen the bran prior to milling, which has the potential to influence pathogen behavior on the kernels. This study aimed to quantify changes in the numbers of STEC and Salmonella inoculated onto soft red winter wheat, and to observe potential changes in the distribution of the pathogens on the surface of kernels during tempering. Lab-scale tempering experiments were conducted to quantify the water activity of and bacterial populations on wheat grain at various time points during 16 h of tempering. The highest water activity observed throughout 16 h of tempering was 0.88. There was no significant change (p > 0.05) in numbers of Salmonella, STEC, or native mesophiles. Using confocal microscopy, observation of Salmonella and STEC cells expressing mCherry on wheat kernels showed an even distribution of inoculated cells, though the localization of cells on kernels did not change significantly after tempering. Even though the environment was not favorable for pathogen replication on grain, the population remained stable, suggesting that disinfection of the kernels prior to milling could reduce food safety concerns in flour.

Serotype-dependent adhesion of Shiga toxin-producing Escherichia coli to bovine milk fat globule membrane proteins.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that can cause severe symptoms for humans. Raw milk products are often incriminated as vehicule for human STEC infection. However, raw milk naturally contains molecules, such as the milk fat globule membrane and associated proteins, that could inhibit pathogen adhesion by acting as mimetic ligands. This study aimed to: (i) evaluate the capability of STEC cells to adhere to bovine milk fat globule membrane proteins (MFGMPs), (ii) highlight STEC surface proteins associated with adhesion and (iii) evaluate the variation between different STEC serotypes. We evaluated the physicochemical interactions between STEC and milk fat globules (MFGs) by analyzing hydrophobic properties and measuring the ζ-potential. We used a plate adhesion assay to assess adhesion between MFGMPs and 15 Escherichia coli strains belonging to three key serotypes (O157:H7, O26:H11, and O103:H2). A relative quantitative proteomic approach was conducted by mass spectrometry to identify STEC surface proteins that may be involved in STEC-MFG adhesion. The majority of E. coli strains showed a hydrophilic profile. The ζ-potential values were between -3.7 and - 2.9 mV for the strains and between -12.2 ± 0.14 mV for MFGs. Our results suggest that non-specific interactions are not strongly involved in STEC-MFG association and that molecular bonds could form between STEC and MFGs. Plate adhesion assays showed a weak adhesion of O157:H7 E. coli strains to MFGMPs. In contrast, O26:H11 and O103:H2 serotypes attached more to MFGMPs. Relative quantitative proteomic analysis showed that the O26:H11 str. 21,765 differentially expressed five outer membrane-associated proteins or lipoproteins compared with the O157:H7 str. EDL933. This analysis also found strain-specific differentially expressed proteins, including four O26:H11 str. 21,765-specific proteins/lipoproteins and eight O103:H2 str. PMK5-specific proteins. For the first time, we demonstrated STEC adhesion to MFGMPs and discovered a serotype effect. Several outer membrane proteins-OmpC and homologous proteins, intimin, Type 1 Fimbriae, and AIDA-I-that may be involved in STEC-MFG adhesion were highlighted. More research on STEC's ability to adhere to MFGMs in diverse biological environments, such as raw milk cheeses and the human gastrointestinal tract, is needed to confirm the anti-adhesion properties of the STEC-MFG complex.

Synergistic Effects of Bacteriophage vB_Eco4-M7 and Selected Antibiotics on the Biofilm Formed by Shiga Toxin-Producing Escherichia coli.

Apart from antibiotic resistance of pathogenic bacteria, the formation of biofilms is a feature that makes bacterial infections especially difficulty to treat. Shiga toxin-producing Escherichia coli (STEC) strains are dangerous pathogens, causing severe infections in humans, and capable of biofilm production. We have reported previously the identification and characterization of the vB_Eco4-M7 bacteriophage, infecting various STEC strains. It was suggested that this phage might be potentially used in phage therapy against these bacteria. Here, we tested the effects of vB_Eco4-M7 alone or in a phage cocktail with another STEC-infecting phage, and/or in a combination with different antibiotics (ciprofloxacin and rifampicin) on biofilm formed by a model STEC strain, named E. coli O157:H7 (ST2-8624). The vB_Eco4-M7 phage appeared effective in anti-biofilm action in all these experimental conditions (2–3-fold reduction of the biofilm density, and 2–3 orders of magnitude reduction of the number of bacterial cells). However, the highest efficiency in reducing a biofilm’s density and number of bacterial cells was observed when phage infection preceded antibiotic treatment (6-fold reduction of the biofilm density, and 5–6 orders of magnitude reduction of the number of bacterial cells). Previous reports indicated that the use of antibiotics to treat STEC-caused infections might be dangerous due to the induction of Shiga toxin-converting prophages from bacterial genomes under stress conditions caused by antibacterial agents. We found that ciprofloxacin was almost as efficient in inducing prophages from the E. coli O15:H7 (ST2-8624) genome as a classical inducer, mitomycin C, while no detectable prophage induction could be observed in rifampicin-treated STEC cells. Therefore, we conclude the latter antibiotic or similarly acting compounds might be candidate(s) as effective and safe drug(s) when used in combination with phage therapy to combat STEC-mediated infections.

Growth medium- and strain-dependent bactericidal efficacy of blue light against Shiga toxin-producing Escherichia coli on food-grade stainless steel and plastic

Shiga toxin-producing Escherichia coli (STEC) are a major group of human pathogens and may persist on both abiotic and biotic surfaces. In this report, two blue-light prototypes were used to evaluate the antimicrobial efficacy against STEC on food processing surfaces (stainless steel and polyoxymethylene plastic). Investigation using a light-bulb prototype (Prototype 1 at 405 nm, 26 mW/cm2) showed significant antimicrobial effects in nutrient deficient condition but not in nutrient rich condition, demonstrating that the presence of organic matters from rich nutrient medium was thought to be light-absorptive and reduce the bactericidal efficacy of blue light, as evident from the lack of bacterial reduction when suspended in cooked meat broth. An advanced (surface-mounted-diode) light panel, Prototype 2 with high light intensity (405 nm; 50 mW/cm2) was able to inactivate a cocktail of seven STEC strains (from seven major serotypes O26, O45, O103, O111, O121, O145 and O157) on type 304 stainless steel (1.66 log10 CFU) and polyoxymethylene plastic (4.25 log10 CFU) at light dosages of 720 and 45 J/cm2, respectively when cells were illuminated in a nutrient-deficient medium (M9 broth). Post-treatment, no STEC cells were recoverable from plastic, both when tested on plates (agar or petrifilms) and by polymerase chain reaction (PCR). In contrast, surviving colonies were identified on samples taken from stainless steel, albeit only four strains could be detected by PCR analysis – those belonging to serotypes O26, O45, O103 and O157 – which indicated that the susceptibility of STEC to blue light varied across the tested strains.

Attachment of Shiga Toxin-Producing Escherichia coli (STEC) to Pre-Chill and Post-Chill Beef Brisket Tissue.

Shiga toxin-producing Escherichia coli (STEC) has caused numerous foodborne illness outbreaks where beef was implicated as the contaminated food source. Understanding how STEC attach to beef surfaces may inform effective intervention applications at the abattoir. This simulated meat processing conditions to measure STEC attachment to adipose and lean beef tissue. Beef brisket samples were warmed to a surface temperature of 30 °C (warm carcass), while the remaining samples were maintained at 4 °C (cold carcass), prior to surface inoculation with an STEC cocktail (O26, O45, O103, O111, O121, O145, and O157:H7). Cocktails were grown in either tryptic soy broth (TSB) or M9 minimal nutrient medium. Loosely and firmly attached cells were measured at 0, 3, 5, and 20 min and 1, 3, 8, 12, 24 and 48 h. TSB-grown STEC cells became more firmly attached throughout storage and a difference in loosely versus firmly attached populations on lean and adipose tissues was observed. M9-grown STEC demonstrated a 0.2 log10 CFU/cm2 difference in attachment to lean versus adipose tissue and variability in populations was recorded throughout sampling. Future research should investigate whether a decrease in intervention efficacy correlates to an increase in firmly attached STEC cells on chilled carcasses and/or subprimals, which has been reported.

A Validation System for Selection of Bacteriophages against Shiga Toxin-Producing Escherichia coli Contamination

Shiga toxin-producing Escherichia coli (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) contaminated with cow feces (often due to the use of natural fertilizers in agriculture). Since the use of antibiotics against STEC is controversial, other methods for protection of food against contaminations by these bacteria are required. Here, we propose a validation system for selection of bacteriophages against STEC contamination. As a model system, we have employed a STEC-specific bacteriophage vB_Eco4M-7 and the E. coli O157:H7 strain no. 86-24, bearing Shiga toxin-converting prophage ST2-8624 (Δstx2::cat gfp). When these bacteria were administered on the surface of sliced cucumber (as a model vegetable), significant decrease in number viable E. coli cells was observed after 6 h of incubation. No toxicity of vB_Eco4M-7 against mammalian cells (using the Balb/3T3 cell line as a model) was detected. A rapid decrease of optical density of STEC culture was demonstrated following addition of a vB_Eco4M-7 lysate. However, longer incubation of susceptible bacteria with this bacteriophage resulted in the appearance of phage-resistant cells which predominated in the culture after 24 h incubation. Interestingly, efficiency of selection of bacteria resistant to vB_Eco4M-7 was higher at higher multiplicity of infection (MOI); the highest efficiency was evident at MOI 10, while the lowest occurred at MOI 0.001. A similar phenomenon of selection of the phage-resistant bacteria was also observed in the experiment with the STEC-contaminated cucumber after 24 h incubation with phage lysate. On the other hand, bacteriophage vB_Eco4M-7 could efficiently develop in host bacterial cells, giving plaques at similar efficiency of plating at 37, 25 and 12 °C, indicating that it can destroy STEC cells at the range of temperatures commonly used for vegetable short-term storage. These results indicate that bacteriophage vB_Eco4M-7 may be considered for its use in food protection against STEC contamination; however, caution should be taken due to the phenomenon of the appearance of phage-resistant bacteria.

Three Dimensional Vero Cell-Platform for Rapid and Sensitive Screening of Shiga-Toxin Producing Escherichia coli.

Shiga-toxin producing Escherichia coli (STEC) is a serious public health concern. Current Vero cell assay, although sensitive, is lengthy and requires 48–72 h to assess STEC presence in a sample. In this study, we investigated if Vero cells in a three-dimensional (3D) platform would provide improved sensitivity for rapid screening of STEC. Vero cells (epithelial kidney cell line) were grown as a monolayer (2D) or in a collagen-matrix (3D) and exposed to Shiga-toxin (Stx) preparation or STEC cells that were pre-exposed to antibiotics (mitomycin C, ciprofloxacin, or polymyxin B) for toxin induction. Lactate dehydrogenase (LDH) release from Vero cells was used as a biomarker for cytotoxicity. Modified tryptic soy broth (mTSB) as enrichment broth containing mitomycin C (2 μg/ml) or ciprofloxacin (100 ng/ml) significantly induced Stx production, which was further confirmed by the dot-immunoblot assay. The 3D Vero platform detected STEC after 6 h post-infection with cytotoxicity values ranging from 33 to 79%, which is considerably faster than the traditional 2D platform, when tested with STEC. The cytotoxicity for non-Stx producing bacteria, Salmonella, Listeria, Citrobacter, Serratia, and Hafnia was found to be below the cytotoxicity cutoff value of 15%. The detection limit for the 3D Vero cell assay was estimated to be 107 CFU/ml for bacteria and about 32 ng/ml for Stx in 6 h. STEC-inoculated ground beef samples (n = 27) resulted in 38–46% cytotoxicity, and the bacterial isolates (n = 42) from ground beef samples were further confirmed to be stx1 and stx2 positive in a multiplex PCR yielding a very low false-positive result. This 3D cell-based screening assay relies on mammalian cell pathogen interaction that can complement other molecular techniques for the detection of cell-free Stx or STEC cells from food samples for early detection and prevention.

Improving the Enrichment and Plating Methods for Rapid Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in Dairy Compost

A culture method to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) was optimized in this study. The finished dairy compost with 30% moisture content was inoculated with a cocktail of six non-O157 STEC serovars at initial concentrations of 1 to 100 CFU/g. Afterward, non-O157 STEC cells in the inoculated dairy compost were enriched by four methods, followed by plating onto cefixime-tellurite sorbitol MacConkey agar supplemented with 5 mg/liter novobiocin (CTNSMAC) and modified Rainbow agar containing 5 mg/liter novobiocin, 0.05 mg/liter cefixime trihydrate, and 0.15 mg/liter potassium tellurite (mRBA). Immunomagnetic bead separation (IMS) was used to compare the cell concentration of individual non-O157 STEC serotypes after enrichment. There was no significant difference (P > 0.05) between CTN-SMAC and mRBA for non-O157 STEC enumeration. The single-step selective enrichment recovered ca. 0.54 log CFU/g more cells (ca. 0.41 log CFU/g for compost-adapted cells) (P < 0.05) compared with the two-step enrichment. Furthermore, the duration of the process to detect non-O157 STEC from dairy compost by selective enrichment, followed by IMS, was optimized. Among six non-O157 STEC serotypes, serotypes O111, O45, and O145 reached the highest cell density after enrichment in dairy compost, and the cell populations reached 7.3, 7.4, and 7.8 log CFU/g within 16 h of incubation, respectively. In contrast, without an enrichment step, the IMS detection limit of individual non-O157 STEC serovars ranged from 3.15 to 4.15 log CFU/g in dairy compost. These results demonstrate that low levels of non-O157 STEC can be detected within 2 days from dairy compost by using a culture method with an optimized enrichment procedure followed by IMS.

Formalin-fixed cells as an internal standard approach for the detection and quantitative assessment of Shiga toxin-producing Escherichia coli (STEC)

Several Shiga toxin-producing Escherichia coli (STEC) are considered adulterants in raw, non-intact ground meat, and numerous detection assays for these microorganisms have been investigated. The objective of this research was to determine the efficacy of formalin-fixed STEC cells as a potential means to calibrate various detection assays. Eight and ten percent formalin-fixed cells were compared with quantitative PCR (qPCR), and the results suggest that the ten percent formalin fixed cell populations were more stable for quantitative assays. These results were based on the approximation of cells/mL by means of phase contrast microscopy. Fixed cells were spiked into ground beef samples, and the recovery rate for each STEC serotype was determined by qPCR. Additionally, the log cells/mL of spiked samples were determined by qPCR and flow cytometry. The log cells/mL evaluated for each assay were determined to not be statistically significant, which suggests that formalin fixed cells could be used across a wide range of detection platforms (immuno-based versus molecular). Commercial detection kits could potentially incorporate formalin-fixed cells as a more standardized positive control to calibrate detection platforms simultaneously as well as among laboratories.

Growth of Stressed Strains of Four Non-O157 Shiga Toxin-Producing Escherichia coli Serogroups in Five Enrichment Broths

The purpose of this study was to evaluate (i) the behavior of several strains of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O103, O111, and O145) exposed to different stress conditions and (ii) the growth dynamics of stressed and nonstressed non-O157 STEC cells in five enrichment media. STEC strains were exposed to acid, cold, and freeze stresses. Lethal and sublethal injuries were determined by plating in parallel on selective and nonselective agar media. Freeze stress (8 days, -20°C) caused the most lethal (95.3% ± 2.5%) injury, as well as the most sublethal (89.1% ± 8.8%) injury in the surviving population. Growth of stressed and nonstressed pure cultures of non-O157 STEC on modified tryptic soy broth, buffered peptone water (BPW), BPW with sodium pyruvate, Brila, and STEC enrichment broth (SEB) was determined using total viable counts. To compare growth capacities, growth after 7 and 24 h of enrichment was measured; lag phases and maximum growth rates were also calculated. In general, growth on BPW resulted in a short lag phase followed by a high maximum growth rate during the enrichment of all tested strains when using all three stress types. Furthermore, BPW ensured the highest STEC count after 7 h of growth. Supplementing the medium with sodium pyruvate did not improve the growth dynamics. The two selective media, Brila and SEB, were less efficient than BPW, but Brila’s enrichment performance was remarkably better than that of SEB. This study shows that irrespective of the effect of background flora, BPW is still recommended for resuscitation of non-O157 STEC.

Thermal Inactivation of Shiga Toxin–Producing Escherichia coli Cells within Cubed Beef Steaks following Cooking on a Griddle

Thermal inactivation of Shiga toxin-producing Escherichia coli (STEC) cells within knitted/cubed beef steaks following cooking on a nonstick griddle was quantified. Both faces of each beef cutlet (ca. 64 g; ca. 8.5 cm length by 10.5 cm width by 0.75 cm height) were surface inoculated (ca. 6.6 log CFU/g) with 250 μl of a rifampin-resistant cocktail composed of single strains from each of eight target serogroups of STEC: O26:H11, O45:H2, O103:H2, O104:H4, O111:H(2), O121:H19, O145:NM, and O157:H7. Next, inoculated steaks were (i) passed once through a mechanical tenderizer and then passed one additional time through the tenderizer perpendicular to the orientation of the first pass (single cubed steak; SCS) or (ii) passed once through a mechanical tenderizer, and then two tenderized cutlets were knitted together by passage concomitantly through the tenderizer two additional times perpendicular to the orientation of the previous pass (double cubed steak; DCS). SCS and DCS were individually cooked for up to 3.5 min per side in 30 ml of extra virgin olive oil heated to 191.5°C (376.7°F) on a hard-anodized aluminum nonstick griddle using a flat-surface electric ceramic hot plate. Regardless of steak preparation (i.e., single versus double cubed steaks), as expected, the longer the cooking time, the higher the final internal temperature, and the greater the inactivation of STEC cells within cubed steaks. The average final internal temperatures of SCS cooked for up 2.5 min and DCS cooked for up to 3.5 min ranged from 59.8 to 94.7°C and 40.3 to 82.2°C, respectively. Cooking SCS and DCS on an aluminum griddle set at ca. 191.5°C for 0.5 to 2.5 min and 1.0 to 3.5 min per side, respectively, resulted in total reductions in pathogen levels of ca. 1.0 to ≥6.8 log CFU/g. These data validated that cooking SCS (ca. 0.6 cm thick) or DCS (ca. 1.3 cm thick) on a nonstick aluminum griddle heated at 191.5°C for at least 1.25 and 3.0 min per side, respectively, was sufficient to achieve a 5.0log reduction in the levels of the single strains from each of the eight target STEC serogroups tested.

Growth characteristics of Shiga toxin-producing Escherichia coli (STEC) stressed by chlorine, sodium chloride, acid, and starvation on lettuce and cantaloupe

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen causing serious illnesses and hospitalizations in the United States. Bacteria that are exposed to environmental stresses during food processing may exhibit different growth patterns in the subsequent growth environment. The purpose of this study was to examine the effect of environmental stresses on the growth of O15H and non-O157 STEC in lettuce or cantaloupe. Strains of O157:H7 and non-O157 STEC (O26:H11, O103:H1, O104:H4, and O145:NM) were subjected to four selected stresses including 2 ppm of chlorine, aw of 0.97 (osmotic stress), and pH 5 (acid stress) at 22 °C for 24 h, or starvation (lack of nutrients) at 22 °C for 15 d. A cocktail mix of stressed or non-stressed (control) O157 or non-O157 at 3 log CFU/g (control or stressed) was inoculated on lettuce or cantaloupe and incubated at 10 and 22 °C for four weeks. While there were significant differences (p < 0.05) in the growth of stressed and unstressed cells of non-O157 STEC, no difference was observed in the growth of stressed and unstressed O157 STEC cells. Osmotic-stressed non-O157 STEC had significantly higher cell populations than control with 2 log difference (9.0 vs. 6.8 log CFU/g) at 10 °C on lettuce and 1 log difference (9.3 vs. 8.3 log CFU/g) at 22 °C on cantaloupe after 4 weeks. Acid-stressed non-O157 STEC had significantly higher cell populations than control at 10 °C after 4 weeks with >1 log difference (7.7 vs. 6.3 log CFU/ml) on cantaloupe. Starvation-stressed non-O157 STEC showed significantly higher cell populations than control with 1 log difference (8.4 vs. 7.2 log CFU/g) at 22 °C on cantaloupe after 4 weeks. The results indicated that osmotic, acid, or starvation stress may enhance the growth of non-O157 STEC on lettuce or cantaloupe and lead to a greater safety risk.

Thermal inactivation kinetics of Shiga toxin-producing Escherichia coli in buffalo Mozzarella curd

The use of raw milk in the processing of buffalo Mozzarella cheese is permitted, but the heat treatment used for stretching the curd must ensure that the final product does not contain pathogens such as Shiga toxin-producing Escherichia coli (STEC) that may be present on buffalo dairy farms. This study carried out challenge tests at temperatures between 68°C and 80°C for 2 to 10min to simulate curd temperatures during the stretching phase. Curd samples were inoculated with 2 STEC strains (serotypes O157 and O26), and their inactivation rates were assessed in the different challenge tests. The curd samples were digested with papain to ensure a homogeneous dispersion of bacteria. The STEC cells were counted after inoculation (range 7.1–8.7 log cfu/g) and after heat treatments using the most probable number (MPN) technique. A plot of log MPN/g versus time was created for each separate experiment. The log linear model with tail was used to provide a reasonable fit to observed data. Maximum inactivation rate (kmax, min−1), residual population (log MPN/g), decimal reduction time (min), and time for a 4D (4-log10) reduction (min) were estimated at each temperature tested. A 4D reduction of the O26 STEC strain was achieved when curd was heated at 68°C for 2.6 to 6.3min or at 80°C for 2.1 to 2.3min. Greater resistance was observed for the O157 strain at 68°C because kmax was 1.48min−1. The model estimates can support cheesemakers in defining appropriate process criteria needed to control possible STEC contamination in raw milk intended for the production of Mozzarella.

Mathematical modeling and numerical analysis of the growth of non-O157 Shiga toxin-producing Escherichia coli in spinach leaves

This study was conducted to investigate the growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) in spinach leaves and to develop kinetic models to describe the bacterial growth. Six serogroups of non-O157 STEC, including O26, O45, O103, O111, O121, and O145, were used in the growth studies conducted isothermally at 4, 8, 15, 20, 25, 30, and 35°C. Both STEC and background microflora were enumerated to develop kinetic models. Growth of STEC in spinach leaves was observed at elevated temperatures (15–35°C), but not at 4 and 8°C.This study considered the dynamic interactions between the STEC cells and the background microflora. A modified Lotka–Volterra and logistic equation was used to simulate the bacterial growth. In combination with an unconstrained optimization procedure, the differential growth equations were solved numerically to evaluate the dynamic interactions between the STEC cells and the background microflora, and to determine the kinetic parameters by fitting each growth curve to the growth equations. A close agreement between the experimental growth curves and the numerical analysis results was obtained.The analytical results showed that the growth of STEC in spinach leaves was unhindered when the population was low, but the growth was suppressed by the background microflora as the STEC population approached the maximum population density. The effect of temperature on the growth of both STEC and background microflora was also evaluated. Secondary models, evaluating the effect of temperature on growth rates, were also developed. The estimated apparent minimum growth temperature for STEC was 11°C in commercial spinach leaves. The methodology and results of this study can be used to examine the dynamic interactions and growth between different bacteria in foods, and to conduct risk assessments of STEC in spinach leaves.

Inactivation of Shiga Toxin-Producing Escherichia coli (STEC) and Degradation and Removal of Cellulose from STEC Surfaces by Using Selected Enzymatic and Chemical Treatments

Some Shiga toxin-producing Escherichia coli (STEC) strains produce extracellular cellulose, a long polymer of glucose with β-1-4 glycosidic bonds. This study evaluated the efficacies of selected enzymatic and chemical treatments in inactivating STEC and degrading/removing the cellulose on STEC surfaces. Six cellulose-producing STEC strains were treated with cellulase (0.51 to 3.83 U/15 ml), acetic and lactic acids (2 and 4%), as well as an acidic and alkaline sanitizer (manufacturers' recommended concentrations) under appropriate conditions. Following each treatment, residual amounts of cellulose and surviving populations of STEC were determined. Treatments with acetic and lactic acids significantly (P < 0.05) reduced the populations of STEC, and those with lactic acid also significantly decreased the amounts of cellulose on STEC. The residual amounts of cellulose on STEC positively correlated to the surviving populations of STEC after the treatments with the organic acids (r = 0.64 to 0.94), and the significance of the correlations ranged from 83 to 99%. Treatments with cellulase and the sanitizers both degraded cellulose. However, treatments with cellulase had no influence on the fate of STEC, and those with the sanitizers reduced STEC cell populations to undetectable levels. Thus, the correlations between the residual amounts of cellulose and the surviving populations of STEC caused by these two treatments were not observed. The results suggest that the selected enzymatic and chemical agents degraded and removed the cellulose on STEC surfaces, and the treatments with organic acids and sanitizers also inactivated STEC cells. The amounts of cellulose produced by STEC strains appear to affect their susceptibilities to certain sanitizing treatments.

Role of cellulose in protecting Shiga toxin-producing Escherichia coli against osmotic and chlorine stress.

This study was undertaken to determine the role of cellulose in protecting Shiga toxin-producing Escherichia coli (STEC) against osmotic and chlorine treatments. STEC cells producing cellulose (19B and 49B) and their respective cellulose-deficient counterparts (19D or 49D) were subjected to osmotic (1, 2, and 3 M NaCl) or chlorine (25, 50, and 100 μg/ml sodium hypochlorite) treatments. The survival of STEC cells was determined at different treatment intervals. Populations of 19B cells were significantly higher (P < 0.05) than those of 19D cells at all sampling intervals for the chlorine treatments, at 24- to 48-h intervals for the 1 M NaCl treatment, and at 9- to 48-h intervals for the 2 M NaCl treatment. Significant differences in populations of 49B and 49D cells were observed after 9, 36, and 48 h of treatment with 2 M NaCl and after 3, 12, 36, and 48 h of treatment with 3 M NaCl (P < 0.05). Populations of 49B cells were higher than those of 49D cells (P < 0.05) also after 5 to 10 min of treatment with 50 μg/ml sodium hypochlorite and 3 to 10 min of treatment with 100 μg/ml sodium hypochlorite. The protective effects conferred by cellulose may explain the greater survival of cellulose-producing STEC under adverse environmental conditions.

Selection and characterization of cellulose-deficient derivates of shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is known to have several defense mechanisms, one of which is the production of extracellular substances including cellulose. The goal of this study was to prepare pairs of STEC cultures for use in future studies designed to address the role of cellulose in protecting the cells of STEC for survival under adverse environmental conditions. Cells of STEC deficient in cellulose production were separated from cellulose-proficient wild-type cells. The identities of the two types of cells were confirmed using serotyping and pulsed-field gel electrophoresis (PFGE). Selected growth characteristics of the two types of cells were determined using three phenotype microarray plates, PM9, PM10, and PM11. The cellulose-deficient and cellulose-proficient cells in each STEC pair shared the same serotype and PFGE profile. The deficiency in cellulose production did not significantly (P > 0.05) affect the growth characteristics of STEC cells under 191 of the 210 tested growth conditions. Significant differences in growth between the two types of cells were observed only in the presence of two antibiotics, a short chain fatty acid, and high concentrations of osmolytes, as well as under extreme acidic and alkaline pH. These results suggest that deficiency in cellulose production did not alter the serological property, PFGE profile, and growth characteristics of selected STEC strains under optimal growth conditions. The STEC strains and their cellulose-deficient derivates could be useful for studying the role of cellulose in protecting the cells of STEC for survival under adverse environmental conditions.

Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing Escherichia coli Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics

Healthy ruminants are the main reservoir of Shiga toxin-producing Escherichia coli (STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic Saccharomyces cerevisiae CNCM I-1077 or Lactobacillus acidophilus BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition, S. cerevisiae did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.

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.

Influence of Selective Media on Successful Detection of Shiga Toxin–ProducingEscherichia coliin Food, Fecal, and Environmental Samples

Shiga toxin-producing Escherichia coli (STEC) strains have caused a large number of human illness outbreaks worldwide. In most cases, the infection was traced to consumption of meats or vegetables contaminated with cattle feces. To combat this public health problem, pre- and post-harvest control strategies are continuously implemented to assure food safety. Thus, rapid, reliable, and sensitive methods for STEC detection must be available to provide confidence not only in the meats or vegetables entering the food chain but also in testing humans with illnesses. As a result, enrichment for STEC has been a critical step in any successful protocol for their detection. The base media commonly used for STEC enrichment include sorbitol MacConkey agar, tryptic soy broth (TSB), E. coli broth, enterohemorrhagic E. coli broth, buffered peptone water (BPW), and brain heart infusion broth. In addition to bile salts, antibiotics (e.g., tellurite, cefixime, novobiocin, vancomycin, cefsulodin, and acriflavin) are used at different concentrations to enrich for STEC. In most published reports, however, the reasons for choosing the selective medium were not provided. Thus, this review was intended to evaluate the base media and antibiotics commonly used for STEC detection. The efficacy of a detection method will certainly depend on the choice of the base medium, selective agents, and their concentrations. The interactions among these factors are also expected to affect sensitivity of the detection method, especially when the test sample contains a small number of STEC cells. Because sensitivity of detection is expected to decline when testing for stressed or injured STEC cells, as is the case in environmental samples, a pre-enrichment step in TSB or BPW without antibiotics may be necessary. Future research should focus on identifying possible antibiotic combinations that effectively inhibit most background bacteria without affecting pathogenic STEC strains in the test sample.