Viability of acid-adapted Escherichia coli O157:H7 in ground beef treated with acidic calcium sulfate

The effects of lactic acid, acetic acid, and acidic calcium sulfate (ACS) on viability and subsequent acid tolerance of three strains of Escherichia coli O157:H7 were determined. Differences in tolerance to acidic environments were observed among strains, but the level of tolerance was not affected by the acidulant to which cells had been exposed. Cells of E. coli O157:H7 adapted to grow on tryptic soy agar acidified to pH 4.5 with ACS were compared to cells grown at pH 7.2 in the absence of ACS for their ability to survive after inoculation into ground beef treated with ACS, as well as untreated beef. The number of ACS-adapted cells recovered from ACS-treated beef was significantly (alpha = 0.05) higher than the number of control cells recovered from ACS-treated beef during the first 3 days of a 10-day storage period at 4 degrees C, suggesting that ACS-adapted cells might be initially more tolerant than unadapted cells to reduced pH in ACS-treated beef. Regardless of treatment of ground beef with ACS or adaptation of E. coli O157:H7 to ACS before inoculating ground beef, the pathogen survived in high numbers.     

Thermal inactivation of Escherichia coli O157:H7 in ground beef supplemented with sodium lactate

A study was conducted to investigate the antimicrobial effect of sodium lactate (NaL) (0, 1.5, 3.0, and 4.5%) on the survival of Escherichia coli O157:H7 in 93% lean ground beef. Samples inoculated with a mixture of four strains of E. coli O157:H7 (10(7) to 10(8) CFU/g) were subjected to immersion heating in a water bath stabilized at 55, 57.5, 60, 62.5, or 65 degrees C. Results of statistical analysis indicated that the heating temperature was the only factor affecting the decimal reduction times (D-values) of E. coli O157:H7 in 93% lean ground beef. The change in temperature required to change the D-value (the z-value) was determined as 7.6 degrees C. The thermal resistance of this organism was neither affected by the addition of NaL nor by the interactions between NaL and temperature. Adding NaL to ground beef to reduce the thermal resistance of E. coli O157:H7 is therefore not recommended.     

Presence of Escherichia coli O157:H7 in ground beef and ground baby beef meat

A total of 114 beef and baby beef samples were examined. The samples included ground baby beef, mixed ground baby beef and pork, and chopped and shaped meat. The samples were analyzed from 30 different grocery stores in Zagreb, Croatia. The object of this study was to evaluate the prevalence of Escherichia coli O157:H7 in the samples that can enhance the potential risk of outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. The results in all tested samples of E. coli O157:H7 were negative. A single sample was positive in a latex agglutination test using antiserum to O157:H7. It was identified as Proteus vulgaris at the Pasteur Institute, Paris, France. This result correlates positively with cross-contamination with Yersinia enterocolitica 09, Brucella abortus, Salmonella type N, and Pseudomonas maltophila.     

Carvacrol and cinnamaldehyde facilitate thermal destruction of Escherichia coli O157:H7 in raw ground beef

The heat resistance of a four-strain mixture of Escherichia coli O157:H7 in raw ground beef in both the absence and presence of the antimicrobials carvacrol and cinnamaldehyde was tested at temperatures ranging from 55 to 62.5 degrees C. Inoculated meat packaged in bags was completely immersed in a circulating water bath, cooked for 1 h to an internal temperature of 55, 58, 60, or 62.5 degrees C, and then held for predetermined lengths of time ranging from 210 min at 55 degrees C to 5 min at 62.5 degrees C. The surviving bacteria were enumerated by spiral plating onto tryptic soy agar overlaid with sorbitol MacConkey agar. Inactivation kinetics of the pathogens deviated from first-order kinetics. D-values (time for the bacteria to decrease by 90%) in the control beef ranged from 63.90 min at 55 degrees C to 1.79 min at 62.5 degrees C. D-values determined by a logistic model ranged from 43.18 min (D1, the D-value of a major population of surviving cells) and 89.84 min (D2, the D-value of a minor subpopulation) at 55 degrees C to 1.77 (D1) and 0.78 min (D2) at 62.5 degrees C. The thermal death times suggested that to achieve a 4-D reduction, contaminated processed ground beef should be heated to an internal temperature of 60 degrees C for at least 30.32 min. Significantly increased sensitivity to heat (P < 0.05) was observed with the addition and/or increasing levels of carvacrol or cinnamaldehyde from 0.5 to 1.0%. The observed thermal death times may facilitate the design of acceptance limits at critical control points for ground beef at lower times and temperatures of heating.     

Reduction of Escherichia coli O157:H7 and Salmonella in ground beef using lactic acid bacteria and the impact on sensory properties

Studies were conducted to determine whether four strains of lactic acid bacteria (LAB) inhibited Escherichia coli O157: H7 and Salmonella in ground beef at 5 degrees C and whether these bacteria had an impact on the sensory properties of the beef. The LAB consisted of frozen concentrated cultures of four Lactobacillus strains, and a cocktail mixture of streptomycin-resistant E. coli O157:H7 and Salmonella were used as pathogens. Individual LAB isolates at 10(7) CFU/ml were added to tryptic soy broth containing a pathogen concentration of 10(5) CFU/ml. Samples were stored at 5 degrees C, and pathogen populations were determined on days 0, 4, 8, and 12. After 4 days of storage, there were significant differences in numbers of both pathogens exposed to LAB isolates NP 35 and NP 3. After 8 and 12 days of storage, all LAB reduced populations of both pathogens by an average of 3 to 5 log cycles. A second study was conducted in vacuum-packaged fresh ground beef. The individual LAB isolates resulted in an average difference of 1.5 log cycles of E. coli O157:H7 after 12 days of storage, and Salmonella populations were reduced by an average of 3 log cycles. Following this study, a mixed concentrated culture was prepared from all four LAB and added to ground beef inoculated with pathogen at 10(8) CFU/g. After 3 days of storage, the mixed culture resulted in a 2.0-log reduction in E. coli O157:H7 compared with the control, whereas after 5 days of storage, a 3-log reduction was noted. Salmonella was reduced to nondetectable levels after day 5. Sensory studies on noninoculated samples that contained LAB indicated that there were no adverse effects of LAB on the sensory properties of the ground beef. This study indicates that adding LAB to raw ground beef stored at refrigeration temperatures may be an important intervention for controlling foodborne pathogens.     

Inactivation of Escherichia coli O157:H7 in packaged ground beef by allyl isothiocyanate

Commercial allyl isothiocyanate (AIT) was examined for its ability to reduce numbers of Escherichia coli O157:H7 inoculated in fresh ground beef packaged under nitrogen and stored refrigerated or frozen. A five-strain cocktail of E. coli O157:H7 containing 3 or 6 log10 cfu/g was inoculated into 100 g ground beef and formed into 10x1-cm patties. A 10-cm diameter filter paper disk treated with AIT suspended in sterile corn oil was placed on top of a single patty. One patty and paper disk were placed in a bag of Nylon/EVOH/PE with O2 permeability of 2.3 cm3 m(-2) 24 h atm at 23 degrees C. The bags were back-flushed with 100% nitrogen, heat-sealed and stored at 10, 4 and -18 degrees C for 8, 21 or 35 days, respectively. During storage, the AIT levels in the package headspaces were determined by gas liquid chromatography, and mesophilic bacteria and E. coli O157:H7 were counted. The mesophilic aerobic bacteria in ground beef patties were largely unaffected by the addition of AIT. At an initial population of 3 log10 cfu/g, E. coli O157:H7 was reduced by AIT to undetectable levels after 18 days at 4 degrees C or 10 days at -18 degrees C. In samples inoculated with 6 log10 cfu/g, a >3 log10 reduction of E. coli O157:H7 was observed after 21 days at 4 degrees C, while a 1 log10 reduction was observed after 8 and 35 days at 10 and -18 degrees C, respectively. The final AIT concentrations in the headspaces after storage at 10, 4, and -18 degrees C were 444, 456, and 112 microg/ml at 8, 21, and 35 days, respectively. Results showed that AIT can substantially reduce numbers of E. coli O157:H7 in fresh ground beef during refrigerated or frozen storage.     

Antibacterial effect of lactoferricin B on Escherichia coli O157:H7 in ground beef.

The antibacterial activity of lactoferricin B on enterohemorrhagic Escherichia coli O157:H7 in 1% peptone medium and ground beef was studied at 4 and 10 degrees C. In 1% peptone medium, 50 and 100 microg of lactoferricin B per ml reduced E. coli O157:H7 populations by approximately 0.7 and 2.0 log CFU/ml, respectively. Studies comparing the antibacterial effect of lactoferricin B on E. coli O157:H7 in 1% peptone at pH 5.5 and 7.2 did not reveal any significant difference (P > 0.5) at the two pH values. Lactoferricin B (100 microg/g) reduced E. coli O157:H7 population in ground beef by about 0.8 log CFU/g (P < 0.05). No significant difference (P > 0.5) was observed in the total plate count between treatment and control ground beef samples stored at 4 and 10 degrees C. The antibacterial effect of lactoferricin B on E. coli O157:H7 observed in this study is not of sufficient magnitude to merit its use in ground beef for controlling the pathogen.     

Chlorine inactivation of Escherichia coli O157:H7 in water

Six human isolates of Escherichia coli O157:H7 and E. coli (ATCC 11229) were used to determine the concentrations of free chlorine and exposure times required for inactivation. Free chlorine concentrations of 0.25, 0.5, 1.0, and 2.0 ppm at 23 degrees C were evaluated, with sampling times at 0, 0.5, 1.0, and 2.0 min. Results revealed that five of six E. coli O157:H7 isolates and the E. coli control strain were highly susceptible to chlorine, with >7 log10 CFU/ml reduction of each of these strains by 0.25 ppm free chlorine within 1 min. However, comparatively, one of the seven strains was unusually tolerant to chlorine at 23 degrees C for 1 min, with a 4-, 5.5-, 5.8-, and >5.8-log CFU/ml reduction at free chlorine concentrations (ppm) of 0.25, 0.5, 1.0, and 2.0. respectively. Based on these studies most isolates of E. coli O157:H7 have no unusual tolerance to chlorine; however, one strain was exceptional in being recovered after 1-min of exposure of 10(7) CFU/ml to 2.0 ppm of free chlorine. This isolate may be a useful reference strain for future studies on chlorine tolerance of E. coli O157:H7.     

Magnetic nanoparticle-antibody conjugates for the separation of Escherichia coli O157:H7 in ground beef

The immunomagnetic separation with magnetic nanoparticle-antibody conjugates (MNCs) was investigated and evaluated for the detection of Escherichia coli O157:H7 in ground beef samples. MNCs were prepared by immobilizing biotin-labeled polyclonal goat anti-E. coli antibodies onto streptavidin-coated magnetic nanoparticles. For bacterial separation, MNCs were mixed with inoculated ground beef samples, then nanoparticle-antibody-E. coli O157:H7 complexes were separated from food matrix with a magnet, washed, and surface plated for microbial enumeration. The capture efficiency was determined by plating cells bound to nanoparticles and unbound cells in the supernatant onto sorbitol MacConkey agar. Key parameters, including the amount of nanoparticles and immunoreaction time, were optimized with different concentrations of E. coli O157:H7 in phosphate-buffered saline. MNCs presented a minimum capture efficiency of 94% for E. coli O157:H7 ranging from 1.6 x 10(1) to 7.2 x 10(7) CFU/ml with an immunoreaction time of 15 min without any enrichment. Capture of E. coli O157:H7 by MNCs did not interfere with other bacteria, including Salmonella enteritidis, Citrobacter freundii, and Listeria monocytogenes. The capture efficiency values of MNCs increased from 69 to 94.5% as E. coli O157:H7 decreased from 3.4 x 10(7) to 8.0 x 10(0) CFU/ml in the ground beef samples prepared with minimal steps (without filtration and centrifugation). An enrichment of 6 h was done for 8.0 x 10(0) and 8.0 x 10(1) CFU/ml of E. coli O157:H7 in ground beef to increase the number of cells in the sample to a detectable level. The results also indicated that capture efficiencies of MNCs for E. coli O157:H7 with and without mechanical mixing during immunoreaction were not significantly different (P > 0.05). Compared with microbeads based immunomagnetic separation, the magnetic nanoparticles showed their advantages in terms of higher capture efficiency, no need for mechanical mixing, and minimal sample preparation.     

Thermal inactivation of Escherichia coli O157:H7 in beef treated with marination and tenderization ingredients

Internalization of Escherichia coli O157:H7 in nonintact beef products during mechanical tenderization or during injection of marination and tenderization ingredients is of concern if such products are undercooked. This study tested organic acids (0.2% citric acid and 0.3% acetic acid), potassium and calcium salts (1.8% potassium lactate, 0.63% calcium lactate, 0.86% calcium ascorbate, and 0.23% calcium chloride), and sodium chloride (2.5%) for their influence on thermal destruction of E. coli O157:H7 in ground beef serving as a model system. Ground beef batches (700 g; 5% fat) were mixed with equal volumes (22 ml) of each treatment solution or distilled water and portions (30 g) of treated ground beef were extruded in test tubes (2.5 by 10 cm). A five-strain mixture of E. coli O157:H7 (0.3 ml; 7 log CFU/g) was introduced at the center of the sample with a pipette. After overnight storage (4 degrees C), simulating product marination, samples were heated to 60 or 65 degrees C internal temperature, simulating rare and medium rare doneness of beef, in a circulating water bath. At 65 degrees C, treatments with citric and acetic acid showed greater (P < 0.05) reduction (4 to 5 log CFU/g) of E. coli O157:H7 than all the other ingredients and the control (3 to 4 log CFU/g). Sodium chloride reduced weight losses (16 to 18% compared with 20 to 27% by citric or acetic acid) and resulted in a 4-log reduction in counts during cooking to 65 degrees C. Ingredients such as citric or acetic acid may improve thermal inactivation of E. coli O157:H7 internalized in nonintact beef products, while sodium chloride may reduce cooking losses in such products.     

Inactivation of Escherichia coli O157:H7 in ground beef by single-cycle and multiple-cycle high-pressure treatments

The effect of single- and multiple-cycle high-pressure treatments on the survival of Escherichia coli CECT 4972, a strain belonging to the O157:H7 serotype, in ground beef was investigated. Beef patties were inoculated with 10(7) CFU/g E. coli O157:H7, and held at 4 degrees C for 20 h before high-pressure treatments. Reduction of the E. coli O157:H7 population by single-cycle treatments at 400 MPa and 12 degrees C ranged from 0.82 log CFU/g for a 1-min cycle to 4.39 log CFU/g for a 20-min cycle. Multiple-cycle treatments were very effective, with four 1-min cycles at 400 MPa and 12 degrees C reducing the E. coli O157:H7 population by 4.38 log CFU/g, and three 5-min cycles by 4.96 log CFU/g. The color parameter L* increased significantly with high-pressure treatments in the interior and the exterior of beef patties, whereas a* decreased in the interior, and b* increased in the exterior-changes that might diminish consumer acceptance of the product. Kramer shear force and energy were generally higher in pressurized than in control ground beef. Maximum values for these texture parameters, which corresponded to tougher patties, were reached after one 10-min cycle in the case of single-cycle treatments or two 5-min cycles in the case of multiple-cycle treatments. High-pressure treatments had no significant effect on Warner-Bratzler shear force.     

Evaluation of consumer-style cooking methods for reduction of Escherichia coli O157:H7 in ground beef

The objective of this study was to evaluate the thermal inactivation of Escherichia coli O157:H7 in ground beef cooked to an internal temperature of 71.1 degrees C (160 degrees F) under conditions simulating consumer-style cooking methods. To compare a double-sided grill (DSG) with a single-sided grill (SSG), two different cooking methods were used for the SSG: for the one-turnover (OT-SSG) method, a patty was turned once when the internal temperature reached 40 degrees C, and for the multiturnover (MT-SSG) method, a patty was turned every 30 s. Patties (100 g, n = 9) inoculated with a five-strain mixture of E. coli O157: H7 at a concentration of 10(7) CFU/g were cooked until all three temperature readings (for two sides and the center) for a patty were 71.1 degrees C. The surviving E. coli O157:H7 cells were enumerated on sorbitol MacConkey (SMAC) agar and on phenol red agar base with 1% sorbitol (SPRAB). The order of the cooking methods with regard to the cooking time required for the patty to reach 71.1 degrees C was as follows: DSG (2.7 min) < MT-SSG (6.6 min) < OT-SSG (10.9 min). The more rapid, higher-temperature cooking method was more effective (P < 0.01) in destroying E. coli O157:H7 in ground beef. E. coli O157:H7 reduction levels were clearly differentiated among treatments as follows: OT-SSG (4.7 log10 CFU/g) < MT-SSG (5.6 log10 CFU/g) < DSG (6.9 log10 CFU/g). Significantly larger numbers of E. coil O157:H7 were observed on SPRAB than on SMAC agar. To confirm the safety of ground beef cooked to 71.1 degrees C, additional patties (100 g, n = 9) inoculated with lower concentrations of E. coli O157:H7 (10(3) to 10(4) CFU/g) were tested. The ground beef cooked by the OT-SSG method resulted in two (22%) of nine samples testing positive after enrichment, whereas no E. coli O157:H7 was found for samples cooked by the MT-SSG and DSG methods. Our findings suggest that consumers should be advised to either cook ground beef patties in a grill that cooks the top and the bottom of the patty at the same time or turn patties frequently (every 30 s) when cooking on a grill that cooks on only one side.     

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.     

Distribution patterns of Escherichia coli O157:H7 in ground beef produced by a laboratory-scale grinder

This study determined the distribution patterns of Escherichia coli O157:1H7 in ground beef when a contaminated beef trim was introduced into a batch of uncontaminated beef trims prior to grinding in a small-scale laboratory grinder. A beef trim (15.3 +/- 2 g) was inoculated with a rifampicin-resistant strain of E. coli O157:H7 (E. coli O157:H7rif) and introduced into a stream of noncontaminated beef (322 +/- 33 g) prior to grinding. Seven inoculum levels (6, 5, and 4 total log CFU [high]; and 3, 2, 1, and 0 total log CFU [low]) were studied in triplicate. E. coli O157:H7rif was not detected in 3.1 to 43% of the ground beef inoculated with the high levels or in 3.4 to 96.9% of the ground beef inoculated with the low levels. For all inoculum levels studied, the five ground beef fractions (each 7.8 +/- 0.6 g) with the highest pathogen levels accounted for 59 to 100% of the total pathogens detected. For all inoculum levels, there was a linear relationship between the quantity of ground beef containing E. coli O157:H7rif and the inoculum level. The quantity of E. coli O157:H7rif in the beef remaining in the grinder was proportional to the inoculum level and was related to the location in the grinder. Different components of the grinder accumulated E. coli O157:H7rif in different quantities, with the most significant accumulation being in the nut (collar) that attaches the die to the blade. This study determined specific distribution patterns of E. coli O157:H7rif after the grinding of a contaminated beef trim along with uncontaminated trims, and the results indicate that the grinding operation should be regarded as a means of distribution of microbial contamination in risk analyses of ground beef operations.     

Quantifying the robustness of a broth-based Escherichia coli O157:H7 growth model in ground beef

The robustness of a microbial growth model must be assessed before the model can be applied to new food matrices; therefore, a methodology for quantifying robustness was developed. A robustness index (RI) was computed as the ratio of the standard error of prediction to the standard error of calibration for a given model, where the standard error of calibration was defined as the root mean square error of the growth model against the data (log CFU per gram versus time) used to parameterize the model and the standard error of prediction was defined as the root mean square error of the model against an independent data set. This technique was used to evaluate the robustness of a broth-based model for aerobic growth of Escherichia coli 0157:H7 (in the U.S Department of Agriculture Agricultural Research Service Pathogen Modeling Program) in predicting growth in ground beef under different conditions. Comparison against previously published data (132 data sets with 1,178 total data points) from experiments in ground beef at various experimental conditions (4.8 to 45 degrees C and pH 5.5 to 5.9) yielded RI values ranging from 0.11 to 2.99. The estimated overall RI was 1.13. At temperatures between 15 and 40 degrees C, the RI was close to and smaller than 1, indicating that the growth model is relatively robust in that temperature range. However, the RI also was related (P < 0.05) to temperature. By quantifying the predictive accuracy relative to the expected accuracy, the RI could be a useful tool for comparing various models under different conditions.     

Rapid detection of Escherichia coli O157:H7 in ground beef using a fiber-optic biosensor.

A portable fiber-optic biosensor was used to detect Escherichia coli O157:H7 in seeded ground beef samples. The principle of the system is a sandwich immunoassay using cyanine 5 dye-labeled polyclonal anti-E. coli O157:H7 antibodies for generation of a specific fluorescent signal. Signal acquisition is effected by launching light from a 635-nm diode laser into a dual tapered 600-microm silica fiber. Fluorescent molecules within approximately 100 nm of the fiber surface are excited by the evanescent field, and a portion of the emission recouples into the fiber. A photodiode allows for quantitation of the collected emission light at wavelengths of 670 to 710 nm. Biotin-avidin interactions are used to attach polyclonal antibodies specific for E. coli O157:H7 to the final 7.5 cm of the fiber probe. The biosensor was able to detect E. coli O157:H7 to 3 to 30 CFU/ml in seeded ground beef samples. The reaction was highly specific. Signals with Listeria monocytogenes, Salmonella Typhimurium, or E. coli nonO157:H7 were 2 to 3% of those observed with a similar concentration of E. coli O157:H7. Assays were conducted at or near real-time with results obtained within 20 min of sampling.     

Quantitative detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction

A method for quantitative detection of Escherichia coli O157:H7 based on the polymerase chain reaction (PCR) was developed. The method used the NIH Image 1·61 software program to quantitatively analyse the intensity of the fluorescent image of the amplified PCR product. Based on the PCR with SLT1 and SLT2 primers used separately, a log-linear relationship between the numbers of cfu of E. coli O157:H7 inoculated into ground beef and the intensity of the PCR products was achieved with and without enrichment. Without enrichment, 150 cfu of E. coli O157:H7 per gram of ground beef were detected. In contrast, the detection limit decreased to 1·2 cfu g⁻¹ of ground beef using SLT1 and SLT2 primers after 4·5 h of enrichment using modified EC broth with 20 μg ml⁻¹ of novobiocin.