Raw Beef Samples Research Articles

Detection of E. coli O157:H7 in raw ground beef by Pathatrix™ immunomagnetic-separation, real-time PCR and cultural methods

Detection of Escherichia coli O157:H7 by conventional cultural methods can be difficult in food matrices with a high background flora such as raw ground beef. Raw ground beef samples, artificially contaminated separately with five strains of E. coli O157:H7 at low (~0.2 cfu/g) and high (~2 cfu/g) levels, were enriched by two enrichment protocols; buffered peptone water (BPW) at 37 °C for 5h and 24h and modified buffered peptone water with pyruvate (mBPWp) for 5h at 37 °C followed by adding selective agents and incubating at 42 °C to 24h. Detection of added E. coli O157:H7 by real-time PCR (RTiPCR) and recovery on isolation agars was performed before and after PATHATRIX™ immunomagnetic separation (IMS). RTiPCR detection and cultural recovery of inoculated E. coli O157:H7 after 5h enrichment were poor at 0.21-0.24 cfu/g. The addition of IMS after 5h enrichment did not improve RTiPCR detection but markedly improved recovery by culturing. By extending enrichment to 24h, RTiPCR detection improved to 76% for either enrichment protocol without IMS. When 24h enrichment was followed by IMS, RTiPCR detection was also further improved. Cultural recovery after 24h enrichment was 56% and 84% without IMS and 100% and 92% after IMS for BPW and mBPWp respectively. Extended enrichment to 24h followed by IMS was found to be sensitive and reliable for detection and cultural recovery from raw ground beef using either enrichment method.

Identification and Molecular Characterization of Antimicrobial-Resistant Shiga Toxin–ProducingEscherichia coliIsolated from Retail Meat Products

Ten (2.7%) Shiga toxin-producing Escherichia coli (STEC) were isolated from 370 samples of raw minced beef, mutton, pork, and chicken from the Jilin region of China; and additional 10 E. coli O157:H7 isolates were previously isolated from different Jilin regions. Seventeen of the isolates were multiresistant, exhibiting resistance to ampicillin, ciprofloxacin, tetracycline, sulfamethoxazole-trimethoprim, gentamycin, and streptomycin. Class 1 integrons were detected in nine (45.0%) of the STEC isolates and consisted of serogroups O157, O62, O113, O149, and O70. Integrons containing amplicons of a 0.5-1.5 or 1.0 kb gene cassette were found in seven (77.8%) of the integron-containing isolates. Sequencing analysis revealed that these gene cassettes encode genes conferring resistance to trimethoprim (dfrA1) and streptomycin (aadA1). The 0.5 kb cassette described here was found to encode a putative transporter peptide in the STEC. Seventeen isolates contained plasmids with different bands, and transfer by conjugation between strains of E. coli demonstrated that class 1 integrons located on mobile plasmids could contribute to the emergence and dissemination of antimicrobial resistance to ampicillin, gentamycin, streptomycin, and sulfamethoxazole-trimethoprim amongst STEC. These data revealed the high prevalence of antimicrobial-resistant STEC isolates in Jilin's surrounding regions, providing important and useful surveillance information reflecting antimicrobial selection pressure.

Discrimination of the Geographical Origin of Beef by 1H NMR-Based Metabolomics

The geographical origin of beef is of increasing interest to consumers and producers due to "mad cow" disease and the implementation of the Free Trade Agreement (FTA). In this study, (1)H NMR spectroscopy coupled with multivariate statistical analyses was used to differentiate the geographical origin of beef samples. Principal component analysis (PCA) and orthogonal projection to latent structure-discriminant analysis (OPLS-DA) showed significant separation between extracts of beef originating from four countries: Australia, Korea, New Zealand, and the United States. The major metabolites responsible for differentiation in OPLS-DA loading plots were succinate and various amino acids including isoleucine, leucine, methionine, tyrosine, and valine. A one-way ANOVA was performed to statistically certify the difference in metabolite levels. The data suggest that NMR-based metabolomics is an efficient method to distinguish fingerprinting difference between raw beef samples, and several metabolites including various amino acids and succinate can be possible biomarkers for discriminating the geographical origin of beef.

Validation of an Improved Method for Detection of Campylobacter jejuni in Foods

Campylobacter jejuni ATCC 29428 and 33560 were inoculated separately into beef muscle, ground beef, and chicken skin to yield approximately 10 to 100 CFU/g of food sample. The samples were stored at 4 degrees C for 10 d. On days 0, 3, 7, and 10, enrichment cultures in Bolton broth supplemented with antibiotics, with and without blood supplementation were made for each sample, for 24 and 48 h following the Food and Agricultural Products Center (FAPC) and the Food and Drug Administration (FDA) protocols. Enumeration of the organisms in the enrichment cultures was done on Campylobacter Karmali selective agar after 24 and 48 h of enrichment to compare the extent of growth in both protocols. There were no significant differences between counts recovered using the FDA and the FAPC methods for detection of Campylobacter jejuni for either strain in any of the food products tested (P > 0.05). No significant differences were observed in performance of enrichment broth supplemented with and without blood (P > 0.05). After 48 h of enrichment, the counts recovered were similar for all products. The organisms were detectable on all days of storage in raw chicken skin, beef, and ground beef samples after both 24 and 48 h of enrichment. The results from the FAPC method for detection of C. jejuni from food were not different from the FDA method. While in the proposed method incubation at 37 degrees C was adequate for the strains tested it is recommended that both enrichment temperatures be used for naturally contaminated samples to ensure detection of all strains that might be present.

한국형 우육포의 제조공정 중 연화제가 육포품질 및 미생물학적 안전성에 미치는 영향

The physical quality and microbial safety of Korean beef jerky was evaluated at various steps during its preparation. Microbial counts in raw beef demonstrated mesophillic bacteria at 4.20 Log CFU/g, psychrotrophic bacteria at 3.85 Log CFU/g, anaerobic bacteria at 4.90 Log CFU/g, and yeast and molds at 1.92 Log CFU/g. Spore-forming bacteria and coliforms were not detected in raw beef samples. Spices and spiced meats showed similar trends in microbial counts, demonstrating minimal microbial contamination during these stages of preparation. The final beef jerky product exhibited counts of mesophillic bacteria at 1.15-1.66 Log CFU/g, psychrotrophic bacteria at 1.15-1.66 Log CFU/g, and anaerobic bacteria at 0.81-1.72 Log CFU/g. Spore-forming bacteria, yeast and molds, and coliforms were not detected in beef jerky. Significant differences from added ingredients occurred for instron textural profile analysis traits for hardness. In general, Korean beef jerky with humectant and tenderizer had lower hardness than control (without humectant and tenderizer). Also, the sample added with 0.01% protease from Streptomyces griseus had lower hardness than all samples. All samples had 0.7l to 0.72 water activities, and the color and pH were not shown in significant changes of all samples.

Prevalence of methicillin-resistant Staphylococcus aureus in meat

Recently the isolation of methicillin-resistant Staphylococcus aureus (MRSA) strains from several food-producing animals has been reported. During slaughtering of MRSA-positive animals, contamination of carcasses with MRSA may occur and consequently the meat of these animals may get contaminated. The aim of this study was to estimate the prevalence of MRSA in raw meat samples from the retail trade. Samples of raw beef, pork, veal, lamb/mutton, chicken, turkey, fowl and game were collected from the retail trade. A detection method including a two-step enrichment in Mueller–Hinton broth + 6.5% NaCl and phenol red mannitol broth containing ceftizoxime and aztreonam, followed by isolation on MRSA ID agar (bioMérieux) was evaluated and subsequently applied for the detection of MRSA in samples of raw meats. MRSA strains were isolated from 264 (11.9%) of 2217 samples analyzed. Isolation percentages for the meat species were: beef (10.6%), veal (15.2%), lamb and mutton (6.2%), pork (10.7%), chicken (16.0%), turkey (35.3%), fowl (3.4%) and game (2.2%). The majority (85%) of the isolated strains belonged to spa-types of pulsed-field gel electrophoresis (PFGE) non-typeable (NT)-MRSA, corresponding to the multilocus sequence type ST398, a type also recently isolated in the Netherlands from pigs. However, a smaller part of these strains were found to be of other ST's, possibly of human origin. Further studies are needed to elucidate transmission routes of MRSA in relation to meat and other foods and to provide the tools for preventing the spread of MRSA. At present the high prevalence of MRSA in meat has not been shown to contribute significantly to the dissemination of MRSA to humans and the possible health hazard for consumers of the presence of MRSA in foods should be further elucidated.

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) strains in ground beef in São Paulo, Brazil

This study aims to assess the prevalence of Shiga toxin-producing Escherichia coli (STEC) in ground beef collected in two cities located in the State of Sao Paulo, Brazil. A total of 250 samples of raw ground beef were collected in local grocery stores during the period of March to December 2002 in the cities of Ribeirao Preto (114 samples) and Campinas (136 samples), Sao Paulo State, Brazil. The samples were processed according to standard methods. The resulting 591 E.coli colonies were screened for STEC by hybridization assays using the specific DNA probes, stx1,stx2 and eae. Further characterization of STEC isolates included the search for the ehxA sequence, detection of enterohemolysin and expression of Shiga toxin using the Vero cell assay. STEC isolates belonging to serotypes O93:H19, ONT:HNT, ONT:H7, and O174:HNT we recovered from four samples (3.5%) collected in Ribeirao Preto. All samples from Campinas were negative for STEC. Three of the strains carried stx2 and ehxA sequences while one harbored stx1,stx2 and ehxA sequences. Considering that among foods of animal origin, ground beef is an important vehicle for STEC transmission, these data emphasize the need of a closer surveillance of these microorganisms. They can survive in unfavorable conditions specially when the products are refrigerated or frozen for long periods of time and can be the cause of outbreaks affecting a great number of consumers.

Predicting Pathogen Growth during Short-Term Temperature Abuse of Raw Pork, Beef, and Poultry Products: Use of an Isothermal-Based Predictive Tool

A computer-based tool (available at: www.wisc.edu/foodsafety/meatresearch) was developed for predicting pathogen growth in raw pork, beef, and poultry meat. The tool, THERM (temperature history evaluation for raw meats), predicts the growth of pathogens in pork and beef (Escherichia coli O157:H7, Salmonella serovars, and Staphylococcus aureus) and on poultry (Salmonella serovars and S. aureus) during short-term temperature abuse. The model was developed as follows: 25-g samples of raw ground pork, beef, and turkey were inoculated with a five-strain cocktail of the target pathogen(s) and held at isothermal temperatures from 10 to 43.3°C. Log CFU per sample data were obtained for each pathogen and used to determine lag-phase duration (LPD) and growth rate (GR) by DMFit software. The LPD and GR were used to develop the THERM predictive tool, into which chronological time and temperature data for raw meat processing and storage are entered. The THERM tool then predicts a Δ log CFU value for the desired pathogen-product combination. The accuracy of THERM was tested in 20 different inoculation experiments that involved multiple products (coarse-ground beef, skinless chicken breast meat, turkey scapula meat, and ground turkey) and temperature-abuse scenarios. With the time-temperature data from each experiment, THERM accurately predicted the pathogen growth and no growth (with growth defined as Δ log CFU ≥ 0.3) in 67, 85, and 95% of the experiments with E. coli O157:H7, Salmonella serovars, and S. aureus, respectively, and yielded fail-safe predictions in the remaining experiments. We conclude that THERM is a useful tool for qualitatively predicting pathogen behavior (growth and no growth) in raw meats. Potential applications include evaluating process deviations and critical limits under the HACCP (hazard analysis critical control point) system.

Evaluation of Immunomagnetic Separation and PCR for the Detection of Escherichia coli O157 in Animal Feces and Meats

Series of animal feces and meat samples artificially contaminated with strains of Escherichia coli O157 isolated from different sources were tested by both an immunomagnetic separation (IMS)–based method and a PCR method using primers specific for a portion of the rfbE gene of E. coli O157. IMS is laborious and time consuming but ends up with the isolation of the pathogen. PCR is fast and less laborious, but it can only be used for screening purposes, so a further culture step is required to isolate the organism. For both fecal and meat samples, the IMS method was found to be more sensitive than the PCR. Furthermore, the detection efficiency of the PCR was influenced by the origin of the fecal sample and the type of meat. For sheep feces, the efficiency of the PCR appeared to be systematically lower than for cattle feces. And the efficiency of the PCR in detecting E. coli O157 in spiked samples of raw minced beef and dry-fermented sausages was systematically lower than in samples of filet americain. Based on this study, it can be concluded that both for animal feces and meat, IMS can be used more successfully to detect E. coli O157 than PCR, because IMS showed to be more sensitive and the outcome was not influenced by the type of animal feces or meat.

Prevalence of Arcobacter spp. in Raw Milk and Retail Raw Meats in Northern Ireland

A 1-year study was undertaken to determine the prevalence of Arcobacter spp. in raw milk and retail raw meats on sale in Northern Ireland. Retail raw poultry samples (n = 94), pork samples (n = 101), and beef samples (n = 108) were obtained from supermarkets in Northern Ireland, and raw milk samples (n = 101) were kindly provided by the Milk Research Laboratory, Department of Agriculture and Rural Development, Belfast, Northern Ireland. Presumptive arcobacters were identified by previously described genus-specific and species-specific PCR assays. Arcobacter spp. were found to be common contaminants of retail raw meats and raw milk in Northern Ireland. Poultry meat (62%) had the highest prevalence, but frequent isolations were made from pork (35%), beef (34%), and raw milk (46%). Arcobacter butzleri was the predominant species isolated from retail raw meats and was the only species isolated from raw milk samples. Arcobacter cryaerophilus was detected less frequently, and Arcobacter skirrowii was detected only as a cocontaminant. To our knowledge, this is the first report of Arcobacter spp. prevalence in a diverse range of products of animal origin in Northern Ireland.

Sustained Decrease in the Rate of Escherichia coli O157:H7–Positive Raw Ground Beef Samples Tested by the Food Safety and Inspection Service

Sustained Decrease in the Rate of Escherichia coli O157:H7–Positive Raw Ground Beef Samples Tested by the Food Safety and Inspection Service

Current epidemiological status of enterohaemorrhagic Escherichia coli O157:H7 in Africa

Enterohaemorrhagic E。关口 i (EHEC ) 从人的痢疾的疾病，出血性的大肠炎(HC ) ，和溶血的尿毒症的症候群(HUS ) 被孤立。在 20 年以后自从对 E 的第一描述。关口 i O157：在美国的 H7， EHEC 成为了世界范围的一个重要公共健康问题。给 E 的最近的爆发的大小和严厉。关口 i O157：H7 感染，有迫切需要减少这病原体引起的人的危险。牛被看作 E 的主要水库。关口 i O157 : H7 因为从牛过时的人或物的煮得太生的肉产品的消费经常与许多人的感染联系了。属于 serogroup EHEC O157 的 EHEC 紧张最经常与人的疾病被联系。他们有 flagellar (H7 ) 抗原或 arenon 能动(H-) 。然而，从人，动物和食物来源的另外的 serotypes 的 EHEC 紧张被报导了。EHEC 紧张生产一个或二主要 shiga 毒素(ST1 和 ST2 ) 。ST1 与 Shigella dysenteriae 类型 1 生产的 shiga 毒素几乎相同，而 ST2 是 antigenicallydistinct。通常在英国孤立的 O157 EHEC 的紧张仅仅生产 ST2。详细描述两毒素的紧张经常被发现更少，并且 ST1 制片人是相对稀罕的。词法上， E.coli O157 : H7 与另外的 E.coli serotypes 难区分。文化上 E。关口 i O157 : H7distinctly 别在 24 小时孵化以后使山梨糖醇和 gulucuronide 发酵。

Effect of Refrigerating Delayed Shipments of Raw Ground Beef on the Detection of Salmonella Typhimurium

In eight separate trials, four groups of raw ground beef samples were inoculated with 0.04 to 0.3 CFU/g of Salmonella Typhimurium (DT 104). Each group consisted of four 25-g samples (three inoculated and one uninoculated). After inoculation, these samples were shipped by overnight courier in shipping containers with ice packs from the U.S. Department of Agriculture (USDA), Eastern Regional Research Center, in Wyndmoor, Pa., to the U.S. Food Safety and Inspection Service (FSIS), Eastern Laboratory, in Athens, Ga. A total of 128 samples (32 in each of four groups) were shipped. A temperature data logger was placed inside each shipping container to record the temperature during shipping and storage. The first group of ground beef samples was analyzed within approximately 1 h of arrival. The second group of samples was left in the original containers, with a gel ice pack, for 24 h before processing. The third and fourth groups of samples were removed from the original shipping containers and stored at room temperature (21 ± 2°C) for 6 h and then in a refrigerator at 4 ± 2°C for 24 and 48 h, respectively, before analysis. The samples were analyzed for the presence of Salmonella according to the USDA/FSIS Microbiological Laboratory Guidebook, chapter 4.02. There was no significant difference in the presence and levels of Salmonella in ground beef among the four test groups. These data show that it is acceptable to process the late-arriving ground beef samples for the detection of Salmonella if they are kept in a refrigerator (4 ± 2°C) for 24 to 48 h or when the shipments arrive late (24 h in the container with ice pack).

Sensory shelf-life predictions by survival analysis accelerated storage models

An Arrhenius model and survival analysis are introduced to analyze data based on consumers acceptance or rejection of samples stored at different times and different temperatures. Sample data comprises 60 consumers who observed raw minced beef samples stored at 2 °C, 9 °C and 19 °C, with seven different storage times for each temperature, answering yes or no as to whether they would accept the samples. An activation energy of 15.3 kcal/mol was estimated from this censored data set, and shelf-life predictions for temperatures outside the tested temperature range were obtained. In the present study it was found that experimental storage at three temperatures was necessary to obtain satisfactory shelf-life estimations for temperatures outside the tested range.

Food Safety and Inspection Service Regulatory Testing Program for Escherichia coli O157:H7 in Raw Ground Beef

We analyzed raw ground beef testing data to determine whether a decrease in the rate of Escherichia coli O157:H7–positive raw ground beef samples has occurred since the inception of Food Safety and Inspection Service (U.S. Department of Agriculture) regulatory actions and microbiological testing concerning this commodity and pathogen. A main effects log-linear Poisson regression model was constructed to evaluate the association between fiscal year and the rate of E. coli O157: H7–positive raw ground beef samples while controlling for the effect of season for the subset of test results obtained from fiscal year (FY)2000 through FY2003. Rate ratios were used to compare the rate of E. coli O157:H7–positive raw ground beef samples between sequential years to identify year-to-year differences. Of the 26,521 raw ground beef samples tested from FY2000 through FY2003, 189 (0.71%) tested positive for E. coli O157:H7. Year-to-year comparisons identified a 50% reduction in the rate of positive ground beef samples from FY2002 to FY2003 when controlling for season (95% CI, 10 to 72% decrease; P = 0.02). This decrease was the only significant year-to-year change in the rate of E. coli O157:H7–positive raw ground beef samples but was consistent in samples obtained from both federally inspected establishments and retail outlets. We believe this decrease is attributed to specific regulatory actions by Food Safety and Inspection Service and subsequent actions implemented by the industry, with the goal of reducing E. coli O157:H7 adulteration of raw ground beef. Continued monitoring is necessary to confirm that the decrease in the rate of E. coli O157:H7 in raw ground beef samples we observed here represents the beginning of a sustained trend.

Effect of Food Matrix and Cell Growth on PCR-Based Detection of Escherichia coli O157:H7 in Ground Beef

The purpose of this work was (i) to investigate the feasibility of a previously reported upstream processing method for PCR template preparation to facilitate the detection of Escherichia coli O157:H7 from ground beef and (ii) to assess the impact of cell growth (no growth in the matrix versus growth in the matrix) on molecular detection limits. Two food matrices (autoclaved and raw ground beef) were evaluated in all studies. For no-growth experiments, 10-g meat samples were inoculated with 102 to 107 CFU/g E. coli O157:H7 and then homogenized. The homogenates were processed to remove large particulates and inhibitors using a two-phase upstream processing method consisting of two sequential centrifugation steps, the second of which used titanous hydroxide to facilitate bacterial immobilization. After upstream processing, sample concentrates were extracted for DNA isolation and amplified by PCR. For growth experiments, 10-g meat samples were inoculated at 1 CFU of E. coli O157:H7 per gram, allowed to grow to 102 to 107 CFU/g, and then processed for PCR assay. Cell recoveries after upstream processing ranged from 15.9 to 77.6% and were not facilitated by the use of titanous hydroxide, as compared with a saline control (P > 0.05). Bacterial cell recovery and PCR detection limits were similar when comparing autoclaved ground beef and raw ground beef, but cell recoveries were highly variable for raw ground beef samples in which E. coli O157:H7 cells were allowed to grow before processing for detection. Overall, PCR detection limits approximated 103 CFU/g of ground beef for all treatments. These results indicate that use of model food systems may not always provide an accurate replication of real-world conditions when evaluating PCR detection limits.

Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis

The development of rapid methods for detection of viable Listeria monocytogenes is crucial to prevent listeriosis and product recalls. While immunomagnetic separation has been used for isolating Listeria spp., lack of specificity and pathogenicity determination render this method unsatisfactory. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody, monoclonal antibody (MAb)-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from a variety of samples and tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue staining, and by an alkaline phosphatase (AP)-based cytotoxicity assay. The two-step method was used to test uninoculated hotdogs, bologna, and raw beef, chicken, and pork samples, following selective enrichment in half-Fraser broth. Pure culture studies proved the assay to be specific for L. monocytogenes, while a similar assay with Dynal® Anti-Listeria immunomagnetic beads was positive for L. monocytogenes, L. ivanovii, and L. seeligeri. Detection and confirmation of cytopathogenicity of Listeria cells from food samples after 24-h selective enrichment were completed in 2–4 h. Isolates were further analyzed by the CAMP test for hemolytic activity and RiboPrinter® for genomic patterns. Using immunoseparation and cytotoxicity as a two-step rapid method, viable L. monocytogenes could be isolated, detected, and confirmed as cytopathogenic in 28 h or less.

Prevalence of Listeria monocytogenes in milk, meat and foodstuff of animal origin and the phenotype of antibiotic resistance of isolated strains

In 2000–2002 samples of raw materials (milk and beef, 518 samples), ready-to-cook foods and final products from milk (30 and 200 samples) and from meat (105 and 110 samples) and swabs from surfaces in two meat-processing plants (41 samples) were examined for the presence of Listeria monocytogenes (L. monocytogenes). 70 isolates were tested using the standard microdilution method for the susceptibility to 12 antimicrobial drugs, minimum inhibitory concentration (MIC) characteristics (MIC<sub>m</sub>, MIC<sub>50</sub>, MIC<sub>90</sub>) were determined. L. monocytogenes was isolated from raw milk samples (15 samples). It was not isolated from any of the semi-finished and final milk products except for one sample of pasteurized milk. Furthermore L. monocytogenes was isolated from samples of raw beef, meat emulsion, fermented dry meat products and from swabs from production equipment. In vitro testing of susceptibility showed the considerable effectiveness of examined antimicrobial substances with streptomycin and norfloxacin being least effective (MIC<sub>m</sub> = 4 μg/ml). Apart from the intermediate resistance (MIC = 1–2 μg/ml) and resistance (MIC = 4 μg/ml) to clindamycin (37 and 5 strains, respectively), all strains were susceptible to the tested substances. While the presence of L. monocytogenes in foodstuffs, in particular, are serious, current tests of susceptibility of L. monocytogenes isolates indicate low probability untreatable infections as a result of resistant strains from foods or transfer of resistance to other microorganisms in the CzechRepublic.

Isolation of shiga toxigenic Escherichia coli from raw beef in Palestine

Shiga toxigenic Escherichia coli (STEC) isolated from raw beef samples in northern Palestine during a 1-year period were characterized for virulence genes by a polymerase chain reaction (PCR) assay and screened for their antibiotic resistance. STEC was identified in 44 (14.7%) of 300 raw beef samples. Twelve (27.3%) of the STEC isolates were serotype O157. Nine of those were isolated during summer. The majority of STEC isolates (70.5%) harbored both stx 1 and stx 2 genes, while the others harbored either stx 1 or stx 2. High levels of resistance against different antimicrobial agents were detected. Resistance to at least three drugs was found in 55% of the isolates.

Development of Measurement System for Three-Dimensional Structure of Ice Crystals in Raw Beef Samples

Development of Measurement System for Three-Dimensional Structure of Ice Crystals in Raw Beef Samples