Evaluation of an optical microbiological method for rapidly estimating populations of aerobic bacteria, coliforms, and Escherichia coli from ground pork

The BioSys optical methods for estimating populations of aerobic bacteria, coliforms, and Escherichia coli from ground pork were evaluated. Ground pork samples were analyzed immediately, after temperature abuse at 25 degrees C for various periods of time, or after temperature abuse and dilution by mixing with pork that was prepared by grinding whole muscles that had the outer portion excised using a sterile scalpel. Each ground pork sample was tested using standard methods such as aerobic plate counts (APC), violet red bile (VRB) agar plate counts (coliforms), and three-tube most probable numbers (MPN--E. coli). Each sample was tested using the BioSys for total viable counts (TVC) by placing 2 ml of ground pork homogenate (25 g into 225 ml of sterile 1% buffered peptone water) into 8 ml of nutrient medium containing brom-cresol purple in a test vial and monitoring at 35 degrees C. Coliforms were enumerated by placing 5 ml of ground pork homogenate into 5 ml of coliform medium (CM) in a test vial and monitoring at 35 degrees C. E. coli were enumerated by placing 5 ml of ground pork homogenate into 5 ml of double-strength CM with 2% dextrose in a test vial and monitoring at 42 degrees C. The correlation coefficients for the regression lines comparing APC to BioSys TVC detection times (DT), VRB to BioSys coliform DT, and MPN to BioSys E. coli DT were -0.95, -0.94, and -0.93, and the line equations were logl0 CFU/ml = 8.94 - 0.40(DT), log10 CFU/ml = 8.77 - 0.43(DT), and log10 CFU/ml = 8.96 - 0.81(DT), respectively. These methods may allow pork producers to monitor equipment surfaces and products in less than 16 h and obtain microbiological results prior to shipment.     

Presence and level of Campylobacter, coliforms, Escherichia coli, and total aerobic bacteria recovered from broiler parts with and without skin

This study was undertaken to determine if broiler chicken parts without skin are less contaminated with Campylobacter than those with skin. Samples were taken in a commercial plant from defeathered carcasses before evisceration. Bacterial counts from rinse of aseptically removed meat samples were lower than those from stomached skin samples. No Campylobacter were recovered from meat collected from the breasts or thighs, and only 2 of 10 drumstick meat samples had detectable levels of Campylobacter. However, 9 of 10 breast skin, 10 of 10 thigh skin, and 8 of 10 drumstick skin samples were positive for Campylobacter, with between 2 and 3 log10 CFU/g of Campylobacter. Breasts, thighs, and drumsticks were removed from broiler carcasses following evisceration before entering the chill tank. There was a significant difference (50 to 90%) in the levels of Campylobacter on breasts, thighs, and drumsticks with and without skin. Similar trends were noted for coliform, Escherichia coli, and total aerobic bacterial counts from samples collected in the plant. Broiler part samples were also collected at retail outlets. These samples were either skin on and skinned in the laboratory or skin off at purchase. Aseptic removal of skin from broiler breasts, thighs, and drumsticks did not cause change in Campylobacter, coliform, E. coli, or total aerobic counts recovered from the skinned part. Likewise, parts purchased without skin did not have different bacterial counts than paired parts purchased with the skin on. Consumers should not expect to significantly lower the number of bacteria present on a chicken breast, thigh, or drumstick by removing the skin.     

Enumeration of total aerobic bacteria and Escherichia coli in minced meat and on carcass surface samples with an automated most-probable-number method compared with colony count protocols

An automated most-probable-number (MPN) system for the enumeration of total bacterial flora and Escherichia coli was compared with plate count agar and tryptone-bile-glucuronide (TBX) and ColiID (in-house method) agar methodology. The MPN partitioning of sample aliquots was done automatically on a disposable card containing 48 wells of 3 different volumes, i.e., 16 replicates per volume. Bacterial growth was detected by the formation of fluorescent 4-methylumbilliferone. After incubation, the number of fluorescent wells was read with a separate device, and the MPN was calculated automatically. A total of 180 naturally contaminated samples were tested (pig and cattle carcass surfaces, n = 63; frozen minced meat, n = 62; and refrigerated minced meat, n = 55). Plate count agar results and MPN were highly correlated (r = 0.99), with log MPN = -0.25 + 1.05 x log CFU (plate count agar) (n = 163; range, 2.2 to 7.5 log CFU/g or cm2). Only a few discrepancies were recorded. In two samples (1.1%), the differences were > or = 1.0 log; in three samples (1.7%), the differences were > or = 0.5 log. For E. coli, regression analysis was done for all three methods for 80 minced meat samples, which were above the limit of detection (1.0 log CFU/g): log MPN = 0.18 + 0.98 x log CFU (TBX), r = 0.96, and log MPN = -0.02 + 0.99 x log CFU (ColiID), r = 0.99 (range, 1.0 to 4.2 log CFU/g). Four discrepant results were recorded, with differences of > 0.5 but < 1.0 log unit. These results suggest that the automated MPN method described is a suitable and labor-saving alternative to colony count techniques for total bacterial flora and E. coli determination in minced meat or on carcass surfaces.     

Comparison of methods for recovery and enumeration of Campylobacter from freshly processed broilers.

Most traditional Campylobacter detection and enumeration procedures are difficult and time consuming. Estimations of Campylobacter populations by the most probable number (MPN) method are especially laborious. The objective of this collaborative study, performed in duplicate in Agricultural Research Service and Food Safety Inspection Service laboratories, was to compare two MPN procedures (utilizing different selective enrichment broths and plating media) to the direct plating technique for enumeration of Campylobacter from freshly processed (postchill, postdrip) broiler chicken carcasses. Results obtained from the direct plating of carcass rinse samples on Campy-cefex agar were not significantly different (P > 0.05) from an MPN procedure employing Hunt's Campylobacter selective enrichment broth followed by recovery on modified Campylobacter charcoal differential agar. However, both of these procedures provided significantly (P < 0.05) better recovery than a second MPN procedure using Rosef's selective enrichment broth followed by plating on Mueller-Hinton blood agar with antibiotics. The direct plating method offers a more simple, less expensive, more rapid alternative to traditional MPN procedures for estimating Campylobacter populations associated with freshly processed broiler carcasses.     

食品安全微生物学指示菌国内外标准应用的比较分析

菌落总数、大肠菌群、大肠埃希菌、肠杆菌科作为食品安全微生物限量的指示菌在国内外标准中的应用不尽相同.本文通过比较我国与欧盟、澳大利亚、新西兰、加拿大和香港地区的相关食品指示菌标准,为制定我国的食品安全微生物标准提供技术依据.     

Rapid and miniaturized method for detection of hygiene indicators,Escherichia coli and coliforms,in dairy products

Current investigation was aimed to develop colorimetric tests for rapid detection of Escherichia coli/coliforms. These test (s) for E. coli and coliforms were developed using the modified E. coli selective medium (M-ECSM) and coliform selective medium, respectively. The selective media contain a combination of group specific marker enzymes and selective agents. The marker enzymes were screened using chromogenic substrates wherein β-D-glucuronidase and glutamate decarboxylase were found specific for E. coli while β-D-galactosidase for coliforms. The selectivity of the media was achieved using different concentrations of ampicillin and gentamicin. The optimized test procedures enabled sensitive detection of 0.35 ± 0.10 log cfu/ml of E. coli and 0.57 ± 0.15 log cfu/ml of coliforms at 37°C within 14.30 ± 0.45 and 12.15 ± 0.30 hr, respectively. M-ECSM selectively inhibited major Enterobacteriaceae contaminants (Salmonella, Shigella, and Yersinia) up to 6 log cfu/ml. Moreover, better selectivity of M-ECSM was reported against tested commercial chromogenic media. Field evaluation of developed test (s) reported prevalence of E. coli/coliforms as 57.29/88.54% in 96 raw milk and 16.28/51.16% in 43 pasteurized milk samples. Further, test components were vacuum dried in the form of miniaturized point-of-need test for field application in dairy farms and industries with minimal infrastructural requirements.     

Comparison of a new enrichment procedure for Shiga toxin-producing Escherichia coli with five standard methods

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g(-1). Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 x 10(8) CFU ml(-1) and 1.80 x 10(6) CFU ml(-1) after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.     

Comparison of a new digital imaging technique for yeast cell counting and viability assessments with traditional methods

In this study, a new rapid automated yeast cell counter was assessed. The cell counter (Aber Countstar) uses bright‐field microscopy and a dye‐exclusion method. This study's aim was to determine whether this method could be effectively employed in an automated slide based counter to assess viability and to compare this method with results from traditional microscopy and from a radio‐frequency impedance‐based instrument (Aber Compact Lab Yeast Analyser). Excellent correlations were observed between methods. The instrument performed well over a range of yeast concentrations (R² = 0.9913 correlation between automated and manual live cell concentrations). Cell diameters compared well with manual recordings. The instrument was also able to track decreasing cell viability in conjunction with the haemocytometer at viabilities over 20%. The radio‐frequency impedance based instrument exhibited the smallest deviation between 10 repeats, followed by the cell counter (SD: ± 1.08 × 10⁷; ± 5.97 × 10⁷, respectively). Manual counts using a haemocytometer exhibited the largest error between repeats (SD: ± 2.63 × 10⁸) and also required substantially more time (2.28 min) compared with the cell counter (7 sec). The automated cell counter successfully reduced inter‐operator errors, a major hindrance with manual analyses. Tests carried out at a brewery in the UK demonstrated that the cell counter provides consistent counts for assorted yeast strains. External tests highlighted the instrument's ease of use and consistency among different strains of brewing yeast and various stages in the brewing process. Copyright © 2015 The Institute of Brewing & Distilling     

Presence of faecal coliforms, Escherichia coli and diarrheagenic E. coli pathotypes in ready-to-eat salads, from an area where crops are irrigated with untreated sewage water

Consumption of ready-to-eat (RTE) salads has increased worldwide. Consequently, the number of outbreaks caused by food-borne pathogens, including diarrheagenic E. coli pathotypes (DEPs), associated with the consumption of RTE-salads has increased. DEPs include enterotoxigenic (ETEC), typical and atypical enteropathogenic (tEPEC, aEPEC), enteroinvasive (EIEC), enteroaggregative (EAEC), diffuse adherent (DAEC) and Shiga toxin-producing (STEC) E. coli. In less-developed areas of the world, fresh crops continue to be irrigated with untreated sewage water. The aims of this study were to evaluate the microbiological quality and prevalence of DEPs in RTE-salads of raw vegetables, purchased from restaurants at Pachuca-City, Hidalgo, Mexico, where most locally consumed vegetables are irrigated with untreated sewage water. A total of 130 salads were purchased from restaurants of three categories: A) national chain restaurants and B) local restaurants, both with the H distinctive (a recognition that the Secretary of Tourism grants to restaurants that manage supplies with high levels of hygiene); and C) local small inexpensive restaurants without H distinctive. A total of 6 restaurants were included, 2 per category (A(1-2), B(1-2), C(1-2)). Each sample was tested for the presence of faecal coliforms (FC) and E. coli by standard procedures. E. coli strains were further characterized for the presence of DEPs loci by two multiplex polymerase chain reactions. Among the 130 salad samples 99% (129) were contaminated with FC; 85% (110/129) harboured E. coli and 7% (8/110) DEPs. The amount of positive salad samples for FC and E. coli was similar between restaurants and categories. The FC mean (571 FC/g) of all samples was significantly higher (p<0.001) than the E. coli mean (63 E. coli/g). A weak correlation of 7.7% (r(2)=0.077, p=0.003) between median FC and E. coli MPN (most probable number) per sample was found. Of the 8 salad samples contaminated with DEPs, 2 were spinach salads from restaurant A(2) and 3 were (Mixed salad) samples from each C restaurant. Three samples harboured non-O157 STEC strains, 2 EIEC, 1 ETEC and 2 samples had non-O157 STEC and EIEC strains, simultaneously. A significant difference (p=0.008) between the prevalence of E. coli vs. DEPs was observed. Independently of the restaurants' overall hygienic status, most RTE-salads had a poor microbiological quality and some harboured DEPs that have been associated with illness in Mexico. Health authorities should focus on implementing DEPs screening in raw vegetables and enforcing the legislation that forbids irrigation with untreated sewage water of both root and leafy vegetables.     

食品和水中大肠埃希氏菌验证方法的比较

目的探讨IMViC检测实验培养基在大肠菌群验证实验中的适用性,优选相对快速确证为大肠菌群的培养基。方法比较IMViC检测实验培养基与国标法中验证实验所用培养基测定大肠埃希氏菌以及食品、生活饮用水、天然饮用矿泉水中大肠菌群不同时段的结果,对比所有样本确证为大肠菌群所需时间。结果测定大肠埃希氏菌以及食品、生活饮用水、天然饮用矿泉水中大肠菌群时, IMViC检测实验培养基6 h测定结果与国标法最终结果均无显著性差异(P0.05)。结论 IMViC检测实验培养基适用于大肠菌群验证试验,可根据实验室检测需求选择适用于大肠菌群快速检测实验的培养基。     

Comparison of the traditional three-tube most probable number method with the Petrifilm, SimPlate, BioSys optical, and Bactometer conductance methods for enumerating Escherichia coli from chicken carcasses and ground beef

A study was conducted to compare commonly used methods, such as Petrifilm and SimPlate, and the rapid microbiological methods BioSys optical and Bactometer conductance to the standard most probable number (MPN) procedure for enumerating Escherichia coli from poultry carcasses and ground beef. Broiler carcasses and ground beef were evaluated in each of three replicate trials. Five groups of carcasses or ground beef were sampled and analyzed using Petrifilm, SimPlate, BioSys optical, and Bactometer conductance measurements after temperature abuse at 37 degrees C for 0 (Petrifilm and SimPlate only), 2, 4, 6, or 8 h. The correlation coefficients for the regression lines comparing the standard E. coli MPN procedure to Petrifilm and SimPlate for chicken and ground beef, respectively, were as follows: 0.95, 0.94, 0.93, and 0.91. The correlation coefficients for the regression lines comparing the standard E. coli MPN procedure to BioSys optical and Bactometer conductance measurements for chicken and ground beef, respectively, were -0.91, -0.90, -0.93, and -0.96. Although Petrifilm and SimPlate performed well, E. coli could not be enumerated from 16.7 and 10% of samples, respectively, using these methods. The BioSys optical and Bactometer conductance methods performed very well when compared with Petrifilm and SimPlate. Using rapid methods (BioSys optical and Bactometer conductance), results were obtained in 1 to 11 h rather than the 48 h required to conduct Petrifilm or SimPlate or the 5 days required to conduct the MPN procedure. These methods may allow processors to test products and obtain results before shipping, avoiding the cost and loss of reputation associated with a recall or foodborne illness outbreak.     

Comparative analysis of a modified ecolite method, the colicomplete method, and a most-probable-number method for detecting Escherichia coli in orange juice

The Ecolite High Volume Juice (HVJ) presence-absence method for a 10-ml juice sample was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual most-probable-number (MPN) method for analysis of artificially contaminated orange juices. Samples were added to Ecolite-HVJ medium and incubated at 35 degrees C for 24 to 48 h. Fluorescent blue results were positive for glucuronidase- and galactosidase-producing microorganisms, specifically indicative of about 94% of Escherichia coli strains. Four strains of E. coli were added to juices at concentrations of 0.21 to 6.8 CFU/ ml. Mixtures of enteric bacteria (Enterobacter plus Klebsiella, Citrobacter plus Proteus, or Hafnia plus Citrobacter plus Enterobacter) were added to simulate background flora. Three orange juice types were evaluated (n = 10) with and without the addition of the E. coli strains. Ecolite-HVJ produced 90 of 90 (10 of 10 samples of three juice types, each inoculated with three different E. coli strains) positive (blue-fluorescent) results with artificially contaminated E. coli that had MPN concentrations of <0.3 to 9.3 CFU/ml. Ten of 30 E. coli ATCC 11229 samples with MPN concentrations of <0.3 CFU/ml were identified as positive with Ecolite-HVJ. Isolated colonies recovered from positive Ecolite-HVJ samples were confirmed biochemically as E. coli. Thirty (10 samples each of three juice types) negative (not fluorescent) results were obtained for samples contaminated with only enteric bacteria and for uninoculated control samples. A juice manufacturer evaluated citrus juice production with both the Ecolite-HVJ and Colicomplete methods and recorded identical negative results for 95 20-ml samples and identical positive results for 5 20-ml samples artificially contaminated with E. coli. The Ecolite-HVJ method requires no preenrichment and subsequent transfer steps, which makes it a simple and easy method for use by juice producers.     

Effects of recovery, plating, and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries

Effects of different recovery and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries were studied. Strawberries were spot or dip inoculated with 7 to 8 log CFU per strawberry of each pathogen, air dried for 2 h, and stored for 1, 3, and 7 days at 4 degrees C. The inoculated samples were stomached or washed with phosphate-buffered saline (PBS; pH 7.2) or with modified PBS (pH 8.4). Bacterial levels were determined using a direct selective plating, thin agar layer plating, or membrane-transferring plating (MTP) with tryptic soy agar and sorbital MacConkey agar (E. coli O157:H7) or modified Oxford agar (L. monocytogenes). Under most test conditions, washing with PBS followed by MTP had significantly higher (P < 0.05) recovery for both bacteria compared with other tested methods. Within a 7-day storage period for spot-inoculated strawberries, a stomaching step resulted in an injury of 0.9 to 1.4 log CFU for E. coli O157: H7 and 1.4 to 1.7 log CFU for L. monocytogenes. When a washing step was used instead, this resulted in an injury of only 0.2 to 0.6 log CFU for E. coli O157:H7 and 0.2 to 0.7 log CFU for L. monocytogenes. Both bacteria could survive on strawberry surfaces, but their recovered levels decreased with the increase of storage time at 4 degrees C for both spot and dip inoculation methods. Dip inoculation generally had a lower recovery than spot inoculation. An ideal protocol to recover and enumerate E. coli O157:H7 and L. monocytogenes from strawberries involved shaking and washing samples with 100 ml of PBS for 15 min at 22 degrees C coupled with a MTP enumeration method.     

Comparison of recovery methods for freeze-injured Listeria monocytogenes,Salmonella Typhimurium,and Campylobacter coli in cell suspensions and associated with pork surfaces

Cells injured as a result of freezing, heating, and acidification treatments may not grow during conventional microbiological procedures owing to the presence of selective agents, compounds, or dyes in the media, impairing the cell's ability to repair itself and grow. Injured cells can be recovered by combining selective and nonselective media into a single system. With such combinations, the diffusion of the selective compounds or dyes is controlled, allowing for the resuscitation of injured cells of interest while also inhibiting the growth of undesirable background microflora. In this study, Listeria monocytogenes, Salmonella Typhimurium, and Campylobacter coli suspended in buffer or associated with pork surfaces were subjected to a freeze-thaw cycle (-15 degrees C for 24 h, 4 degrees C for 4 h). Following treatments, freeze-injured cells were plated on appropriate media for the overlay (OV), thin agar layer (TAL), and Lutri plate (LP) recovery methods. The levels of L. monocytogenes and Salmonella Typhimurium recovered from cell suspensions and pork surfaces by the TAL, OV, and LP methods following freeze treatments were not statistically different (P > 0.05) from recovery levels associated with nonselective media. Conversely, levels of pathogens on selective media were significantly reduced compared with those for the other methods employed. The TAL method's recovery of C. coli was not significantly different from that achieved with the nonselective media. Overall, the results presented in this study demonstrate that the TAL method not only was easier to perform, but also allowed improved isolation of single colonies for further characterization. This study may provide researchers with better methods to determine the effectiveness of industry-employed chilling processes in reducing pathogenic bacteria associated with red meat surfaces.     

Evaluation of dry medium (Sanita-Kun) for enumeration of coliforms and Escherichia coli in milk, ice cream, ham, and codfish fillet

This study was performed to compare the efficiency and accuracy of Sanita-Kun dry medium with those of conventional culture and Petrifilm dry medium methods for enumerating coliforms and Escherichia coli in milk, ice cream, ham, and codfish fillet. All 3 methods showed high correlations each other and no significant differences in enumerating coliforms and E. coli. In addition, the Sanita-Kun method was easier to count microbial load than the Petrifilm method. Therefore, we concluded that the Sanita-Kun method can be used as an alternative to conventional culture and Petrifilm methods for the analysis of coliforms and E. coli in milk, ice cream, ham, and codfish fillet products.     

Comparison of methods for fluorescent detection of viable, dead, and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers

The influence of treating Escherichia coli O157:H7 cells labeled with an enhanced green fluorescent protein (EGFP) plasmid with 20 microg/ml active chlorine, 100 mg/ml hydrogen peroxide, and 80 mg/ml acetic acid on fluorescence intensity was determined. In addition, fluorescent staining methods to differentiate viable and dead E. coli O157:H7 cells on the cuticle of Red Delicious cv. apples following treatment with water or 200 microg/ml active chlorine were evaluated. Suspensions of E. coli O157:H7 EGFP+ cells were exposed to chemical treatment solutions for 0, 30, 60, 120, or 300 s before populations (log10 cfu/ml) were determined by surface plating, and fluorescence intensities of suspensions and individual cells were measured using spectrofluorometry and confocal scanning laser microscopy (CSLM), respectively. The relative fluorescence intensity of suspensions and individual cells changed upon exposure to various treatments. Results indicate that the use of EGFP to tag E. coli O157:H7 may not be appropriate for investigations seeking to microscopically differentiate viable and dead cells on produce following surface treatment with sanitizers. SYTOX Orange and SYTOX Green nucleic acid stains fluorescently labeled dead E. coli O157:H7 cells attached to apple cuticles more intensely than did propidium iodide. A cross-signal occurred between CSLM photomultipliers when examining tissues treated with SYTOX Orange to detect dead cells and antibody labeled with Alexa Fluor 488 to detect total (dead and viable) cells. Because of the possibility of cross-signal resulting in an overestimation of the number of dead cells on apples and, perhaps, other produce treated with these stains, SYTOX Green is preferred to detect dead cells and antibody labeled with Alexa Fluor 594 is preferred to detect the total number of cells on apple surfaces following treatment with sanitizers. The performance of SYTOX Green in combination with Alexa Fluor 594 to detect dead and total cells of E. coli O157:H7 on other produce remains to be determined.     

Characterization of pathogenic Escherichia coli strains linked to an outbreak associated with kimchi consumption in South Korea, 2012

Pathogenic Escherichia coli (PEc) is a leading cause of both foodborne and waterborne illness. In September 2012, a major foodborne outbreak with PEc occurred, affecting approximately 1,200 students and food handlers from 7 schools in Gyeonggi province, South Korea caused by contaminated kimchi. For detection of PEc in kimchi, real-time polymerase chain reaction (RT-PCR) and traditional culture methods were used. EAEC and ETEC genotypes were identified in samples from individuals with the illness and in kimchi using conventional PCR. Bacteria in stool samples were genetically similar to bacteria from kimchi (98% homology). PEc from kimchi was identified as the causative agent of a foodborne outbreak in South Korea. A significant link between kimchi and individuals with foodborne illnesses after consuming kimchi was demonstrated.     

Comparing the mannitol-egg yolk-polymyxin agar plating method with the three-tube most-probable-number method for enumeration of Bacillus cereus spores in raw and high-temperature, short-time pasteurized milk

The U.S. Food and Drug Administration's Bacteriological Analytical Manual recommends two enumeration methods for Bacillus cereus: (i) standard plate count method with mannitol-egg yolk-polymyxin (MYP) agar and (ii) a most-probable-number (MPN) method with tryptic soy broth (TSB) supplemented with 0.1% polymyxin sulfate. This study compared the effectiveness of MYP and MPN methods for detecting and enumerating B. cereus in raw and high-temperature, short-time pasteurized skim (0.5%), 2%, and whole (3.5%) bovine milk stored at 4°C for 96 h. Each milk sample was inoculated with B. cereus EZ-Spores and sampled at 0, 48, and 96 h after inoculation. There were no differences (P > 0.05) in B. cereus populations among sampling times for all milk types, so data were pooled to obtain overall mean values for each treatment. The overall B. cereus population mean of pooled sampling times for the MPN method (2.59 log CFU/ml) was greater (P < 0.05) than that for the MYP plate count method (1.89 log CFU/ml). B. cereus populations in the inoculated milk samples ranged from 2.36 to 3.46 and 2.66 to 3.58 log CFU/ml for inoculated milk treatments for the MYP plate count and MPN methods, respectively, which is below the level necessary for toxin production. The MPN method recovered more B. cereus, which makes it useful for validation research. However, the MYP plate count method for enumeration of B. cereus also had advantages, including its ease of use and faster time to results (2 versus 5 days for MPN).     

Comparison of rapid enzyme-linked immunosorbent assay and immunomagnetic separation methods for detection of Escherichia coli O157 in fecal, hide, carcass, and ground beef samples

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coil O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.     

Comparison of four commercial DNA extraction kits for PCR detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in fresh, minimally processed vegetables

Four commercial DNA extraction methods, PrepMan Ultra (Applied Biosystems), InstaGene Matrix (BioRad), DNeasy Tissue kit (Qiagen), and UltraClean (MoBio), were tested for PCR detection of Listeria monocytogenes, Escherichia coli O157: H7, Salmonella, and Staphylococcus aureus in fresh, minimally processed vegetables. For comparative purposes, sensitivity assays with specific PCRs were carried out after DNA extraction with the four methods in green pepper, broccoli, and onion artificially inoculated with the four pathogens separately. As confirmed by statistical analysis, the DNeasy Tissue kit rendered the highest sensitivity values in the three matrices assayed for Salmonella, L. monocytogenes, and E. coli O157:H7 and in onion for S. aureus. Despite being the most expensive of the methods compared, the DNeasy Tissue Kit can be successfully applied for any of the four most commonly studied pathogens, thus saving time and overall reducing the cost of the analysis.