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The Bacillus cereus group of bacteria, which causes foodborne diseases, can be detected by culture on selective media. However, the presence of competing flora is the most common factor preventing the accurate enumeration of B. cereus on selective agars. In this study, we improved the selectivity of mannitol–yolk–polymyxin B agar (MYPA) and its modified version containing trimethoprim (mMYPA) developed in our previous study by supplementation with ceftazidime (16 μg/mL). Ceftazidime-supplemented MYPA (C-MYPA16) and mMYPA (C-mMYPA16) were evaluated for bacteria recovery and selectivity with three types of ready-to-eat vegetables. Four B. cereus and one Bacillus thuringiensis strains were mixed and artificially inoculated into vegetable salad, radish sprouts, and sprout mix and then recovered on MYPA, mMYPA, C-MYPA16, and C-mMYPA16. In all tested vegetables, mMYPA, C-MYPA16, and C-mMYPA16 culture resulted in similar recovery of B. cereus and B. thuringiensis (P > 0.05), whereas radish sprout and sprout mix colonies grown on MYPA were undistinguishable. C-mMYPA16 was the most selective medium because it eliminated most of the competing flora, especially that in sprouts, without negatively affecting the recovery of B. cereus and B. thuringiensis. Our results indicate that supplementation of mMYPA with ceftazidime may improve the selectivity of this medium for B. cereus and B. thuringiensis in food testing. Enumeration of bacteria in the B. cereus group in ready-to-eat vegetables is difficult. Addition of ceftazidime to MYPA improved selectivity for B. cereus group. These new media are useful for detection of bacteria in ready-to-eat vegetables. [ABSTRACT FROM AUTHOR]

This study was carried out to evaluate the prevalence of mycobiota and aflatoxin (AF) B1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. For this purpose, 100 sesame seed samples were collected directly from the fields of major sesame-producing areas in rainfed and irrigated zones. The agar plate method was used for isolation of mycobiota, and thin-layer chromatography was used to determine AFB1 concentrations. Seed samples were then stored for 12 months. After 12 months, the seeds were again analyzed for mycobiota and AFB1 for comparison. All samples were positive for fungal growth under fresh and stored conditions. Twenty-one fungal species of 10 genera were isolated. Aspergillus flavus was the most prevalent contaminant found in fresh and stored sesame seeds from rainfed and irrigated zones, followed by Aspergillus niger, Alternaria alternata, and Fusarium oxysporum. The least prevalent fungi were Aspergillus ochraceus and Cladosporium oxysporum. Analysis revealed that 92% of fresh and 99% of stored seed samples were contaminated with AFB1. In the rainfed zone, 88% of fresh and 100% of stored seed samples were contaminated with AFB1, with mean concentrations of 15.74 and 33.8 ppb, respectively. In the irrigated zone, 96% of fresh and 98% of stored seed samples were contaminated with AFB1, with mean concentrations of 20.5 and 27.56 ppb, respectively. AFB1 concentrations >20 ppb were found in 20% of fresh and 100% of stored seeds samples from the rainfed zone and in 28% of fresh and 60% of stored samples from the irrigated zone and thus were not fit for human consumption as per the maximum limit (20 ppb) assigned by the U.S. Food and Drug Administration and the Food and Agriculture Organization of the United Nations. This report is the first on the mycobiota and AFB1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. These baseline data are an initial step in the effort to deal with this significant food safety issue. Fresh and stored sesame seeds were positive for fungal growth. The leading contaminant in fresh and stored sesame seeds was Aspergillus flavus. Overall, 92% of fresh and 99% of stored seed samples were contaminated with AFB1. In the rainfed zone, 88% of fresh and 100% of stored samples were contaminated with AFB1. In the irrigated zone, 96% of fresh and 98% of stored samples were contaminated with AFB1. [ABSTRACT FROM AUTHOR]

Thermophilic spore-forming bacteria are found ubiquitously in natural environments and, therefore, are present in a number of agricultural food products. Spores produced by these bacteria can survive harsh environmental conditions encountered during food processing and have been implicated in food spoilage. During research efforts to develop a standardized method for enumerating spores in dairy powders, the dairy powder–associated thermophilic sporeformer Anoxybacillus flavithermus was discovered growing in uninoculated control plates of tryptic soy agar (TSA) supplemented with 1% (w/v) starch, after incubation at thermophilic (55°C) growth temperatures. This article reports the investigation into the source of this thermophilic sporeformer in TSA medium components and characterization of the bacterial isolates collected. Aqueous solutions of tryptic soy broth powder from four suppliers and four agar–agar powders (two manufacturing lots from one supplier [agar A_1 and agar A_2] and two from separate suppliers [agar B and agar C]) were subjected to two different autoclave cycle times (121°C for 15 min or 121°C for 30 min) and then prepared as TSA. After incubation at 55°C for 48 h, bacterial growth was observed only in media prepared from both lots of agar A agar–agar powder, and only when they were subjected to a 15-min autoclave cycle, implicating these powders as a source of the sporeformer contamination. Genetic characterization of 49 isolates obtained indicated the presence of five unique rpoB allelic types of the thermophilic sporeformer Geobacillus spp. in agar–agar powder from agar A. These results not only highlight the importance of microbiological controls but also alert researchers to the potential for survival of thermophilic sporeformers such as Anoxybacillus and Geobacillus in microbiological media used for detection and enumeration of these same thermophilic sporeformers in products such as dairy powders. Anoxybacillus flavithermus and Geobacillus spp. were contaminants in a growth medium. The source of the contaminant was the agar–agar powder medium component. Longer autoclave sterilization durations eliminated observable bacterial growth. Robust media controls should be implemented when culturing thermophilic sporeformers. [ABSTRACT FROM AUTHOR]

The performance of three chromogenic agar media for detection of the "top seven" Shiga toxin–producing Escherichia coli (STEC) in beef was compared. Samples of retail ground beef were inoculated with STEC O26, O45, O103, O111, O121, O145, or O157 at geometric mean (±standard error of the mean) levels of 0, 48 (±1), 420 (±1), 4,100 (±1), or 45,000 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against the seven STEC serogroups: pool I, O26, O45, and O121; pool II, O103, O111, and O145; and pool III, O157. After immunomagnetic separation, 50 μL of washed bead suspensions in buffered peptone water were spiral plated onto modified Rainbow Agar O157 (mRBA), CHROMagar STEC (CS), or modified Possé differential medium (mPossé2) and incubated at 37°C for 18 h. Up to six isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC on the respective media, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, mRBA had the highest sensitivity (99.2%), correctly detecting a significantly higher proportion of STEC serogroups than either CS (79.4%; P < 0.05) or mPossé2 (91.7%; P < 0.05). mRBA also had the highest negative predictive value (90.0%), correctly identifying a significantly higher proportion of true-negative samples compared with CS (25.7%; P < 0.05) and mPossé2 (46.2%; P < 0.05). However, mRBA also had the lowest analytical specificity of 83.2% (P < 0.05), yielding the lowest proportion of colonies tested that were STEC positive (3,548 of 4,263) compared with 97.7% (3,607 of 3,693) for mPossé2 and 98.0% (2,875 of 2,935) for CS. Reduced specificity results in more work and higher expense due to the increased number of colonies that must be tested. Further improvements in agar culture media for non-O157 STEC isolation are needed. The performance of various chromogenic agars for STEC isolation was compared. Modified Rainbow Agar O157 was the most sensitive but also the least specific. CHROMagar STEC and modified Possé differential medium were the most specific. Lower specificity suggests that more work and higher expense will be incurred. Improvements in agar culture media for non-O157 STEC isolation are still needed. [ABSTRACT FROM AUTHOR]

A modified polymyxin–egg yolk–mannitol–bromothymol blue agar (mPEMBA) was developed by supplementing polymyxin–egg yolk–mannitol–bromothymol blue agar (PEMBA) with trimethoprim to improve the selectivity for and recoverability of Bacillus cereus from naturally and artificially contaminated food samples. The number of B. cereus in mPEMBA was significantly higher than in PEMBA, indicating better recoverability (P < 0.05) in red pepper powder (PEMBA 0.80 ± 0.22 log CFU/g versus mPEMBA 1.95 ± 0.17 log CFU/g) and soybean paste (PEMBA 2.19 ± 0.18 log CFU/g versus mPEMBA 3.09 ± 0.13 log CFU/g). In addition, mPEMBA provided better visual differentiation of B. cereus colonies than PEMBA, which is attributable to the reduced number of competing microflora. We conclude that the addition of trimethoprim to PEMBA could generate a synergistic effect to improve selectivity for B. cereus. [ABSTRACT FROM AUTHOR]

The isolation and quantification of non-0157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces are challenging. The primary objective of this study was to evaluate the performance of selected agar media in an attempt to identify an optimal medium for the detection and quantification of non-0157 STEC in cattle feces. Comparison studies were performed using CHROMagar STEC, Posse differential agar (Posse), Posse modified by the reduction or addition of antimicrobials, STEC heart infusion washed blood agar with mitomycin C (SH1BAM), and SHIBAM modified by the addition of antimicrobials. Fourteen STEC strains, two each belonging to serogroups 026, 045, 0103, O il 1, 0121, 0145, and 0157, were used to test detection in inoculated fecal suspensions at concentrations of 10² or 10³ CFU/g. One STEC strain from each of these seven serogroups was used to estimate the concentration of recovered STEC in feces inoculated at 10³, 104, or 105 CFU/g. Significantly more suspensions (P < 0.05) were positive for STEC when plated on Posse containing reduced concentrations of novobiocin and potassium tellurite compared with SHIBAM, but not SHIBAM modified by containing these same antimicrobials at the same concentrations. Numerically, more suspensions were positive for STEC by using this same form of modified Posse compared with Posse, but this difference was not statistically significant. More suspensions were positive for STEC cultured on CHROMagar STEC compared with those on Posse (P < 0.05) and on modified Posse (P = 0.05). Most inoculated fecal suspensions below 104 CFU/g of feces were underestimated or not quantifiable for the concentration of STEC by using CHROMagar STEC or modified Posse. These results suggest that CHROMagar STEC performs better than Posse or SHIBAM for detection of STEC in bovine feces, but adjustments in the concentrations of novobiocin and potassium tellurite in the latter two media result in significant improvements in their performance. [ABSTRACT FROM AUTHOR]

The objectives of this study were (i) to assess the efficacy of quaternary ammonium chloride-based wet foam (WF) and dry foam (DF) sanitizer systems (600 ppm) for reducing Listeria innocua (a nonpathogenic surrogate of Listeria monocytogenes) or a 100.0 µg/ml rifampin-resistant Salmonella Typhimurium LT2 (a nonpathogenic surrogate of Salmonella enterica serovar Typhimurium) on niche and transfer point areas of an unwashed retail deli sheer as compared with traditional chlorine (Cl-) treatment (200 ppm) and (ii) to compare sanitizer surface contact times (10 and 15 min) for pathogen surrogate control. Turkey frankfurter slurries inoculated with L. innocua or Salmonella Typhimurium were used to inoculate seven high-risk sites on a commercial sheer. After 30 min of bacterial attachment, slicers were dry wiped to remove excess food matter, followed by a randomly assigned sanitizer treatment. Surviving pathogen surrogate cells were enumerated on modified Oxford's agar not containing antimicrobic supplement (L. innocua) or on tryptic soy agar supplemented with 100 µg/ml rifampin (Salmonella Typhimurium LT2). Replicate-specific L. innocua and Salmonella Typhimurium reductions were calculated as log CFU per square centimeter of control minus log CFU per square centimeter of enumerated survivors for each site. For both organisms, all sanitizer treatments differed from each other, with Cl- producing the least reduction and WF the greatest reduction. A significant (P < 0.05) site-by-treatment interaction was observed. The results of the study indicate that quaternary ammonium chloride sanitizers (600 ppm) applied by both WF and DF were more effective at reducing L. innocua and Salmonella Typhimurium than a traditional Cl sanitizer (200 ppm) on unwashed sheer surfaces. [ABSTRACT FROM AUTHOR]

The present study was conducted to compare Campylobacter counts obtained by three selective media: modified charcoal cefoperazonedeoxycholate agar (mCCDA), Campy Food agar (CFA), and a novel agar RAPID' Campylobacter agar. Analysis of 12 artificially and 36 naturally contaminated samples indicated no significant differences in Campylobacter counts obtained with all three selective media. Lin's concordance correlation coefficient (CCC) and the Bland-Altman plot revealed a high level of agreement between Campylobacter counts when evaluating RAPID versus mCCDA and CFA plates. RAPID agar was the only medium tested that could effectively suppress the growth of the background microflora with naturally contaminated samples. Results of this study clearly indicated that RAPID agar is highly selective without loss of sensitivity for recovering Campylobacter. Results obtained are in agreement with those for other commonly used media; therefore, RAPID medium is suitable for Campylobacter enumeration in poultry meat samples. [ABSTRACT FROM AUTHOR]

The detection ability and selectivity of Karmali agar was improved by supplementation of an extended-spectrum β-lactamase inhibitor, potassium clavulanate. The optimum concentration of potassium clavulanate (0.5 µg/ml) in Karmali agar was determined by inoculation of 50 Campylobacter and 30 extended-spectrum β-lactamase-producing E. coli strains onto normal and modified Karmali agar containing various concentrations of the agent. Eighty retail carcasses were rinsed with 400 ml of buffered peptone water. The rinse samples were enriched in 2 x blood-free Bolton enrichment broth at 42°C for 48 h and then were streaked onto normal and modified Karmali agar containing 0.5 µg/ml potassium clavulanate. The suspicious colonies were subcultured on Columbia blood agar and confirmed by colony PCR. In chicken carcass samples, the modified Karmali agar showed a significantly greater isolation rate than normal Karmali agar (42.5 versus 21.3%; P < 0.05). Furthermore, the selectivity of the modified Karmali agar was also significantly higher (P < 0.05) than that of the normal Karmali agar, as seen by comparison of the number of contaminated agar plates (83.8 versus 97.5%) and the growth index (1.67 versus 2.91) of the non-Campylobacter colonies. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are the leading cause of STEC-associated infections in humans in the United States. In the United States, these organisms are considered adulterants in raw nonintact beef products and in intact beef destined to be made into or used in nonintact raw beef products. The objective of this study was to provide an estimate of the burden of the six serogroups of non-O157 STEC in ground beef obtained from retail stores across the United States. A convenience sample of commercial ground beef products (n = 1,129) were purchased from retail stores in 24 states from October 2011 to May 2012. The samples had various lean/fat proportions, muscle group of origin (chuck, round, sirloin, or not specified), and packaging types. For each ground beef sample, 25 g was inoculated in 225 ml of modified tryptic soy broth, stomached for 1 min, and then incubated at 41 °C for 18 ± 2 h. These enrichment cultures were then screened for stx, eae, and O group genes using a commercially available, closed-platform PCR-based method. The potential positive samples were subjected to immunomagnetic separation and plated on modified Rainbow agar. Morphologically typical colonies were subjected to latex agglutination and PCR determination of stx and eae genes. Nine (0.8%) of the ground beef samples were potentially positive for at least one STEC serogroup after PCR screening. The serogroups detected by PCR assay were O26 (four samples), O103 (four samples), O145 (three samples), O45 (two samples), and O121 (one sample). No STEC isolates belonging to these serogroups were recovered from the sample cultures. The current research provides updated surveillance data for non-O157 STEC isolates among commercial ground beef products and information regarding the potential sources of contamination from different parts of beef trims destined for ground beef production. [ABSTRACT FROM AUTHOR]

Florida Tomato Good Agricultural Practices (T-GAPs) mandate the removal of dirt and debris from tomatoes during harvest but do not provide any specific regulations or guidance; thus, the current practice of using cloths needs to be evaluated. This study examined Salmonella transfer from inoculated green tomatoes to uninoculated cloths and from inoculated cloths to uninoculated tomatoes, upon single and multiple touches. Tomatoes were spot inoculated with a rifampin-resistant Salmonella cocktail (107 CFU per tomato) and were touched with cloth (clean, dirty-dry, dirty-wet) at 0, 1, or 24 h postinoculalion. Salmonella was enumerated on tryptic soy agar, followed by enrichments when necessary. The transfer direction was then reversed by touching freshly inoculated cloths with uninoculated tomatoes. Transfer coefficients (TCs) were then calculated. Salmonella TCs from inoculated tomato and cloth were highest when the inoculum was wet (0.44 ± 0.13 to 0.32 ± 0.12), regardless of the condition of the cloth. Although Salmonella TCs from inoculated tomato to uninoculated cloth decreased significantly when the inoculum was dried (0.17 ± 0.23 to 0.01 ± 0.00), low levels of Salmonella were detected on cloth even after 24 h of drying. Inoculated dirty cloth did not transfer more Salmonella compared with inoculated clean cloth, and Salmonella survival was not higher on dirty cloth. When inoculated clean cloth (wet) was touched with 25 tomatoes, significantly higher levels of Salmonella were transferred to the first, second, and fourth tomatoes (0.03 ± 0.10 to 0.09 ± 0.02). However, inoculated dirty-wet (below limit of detection) and dirty-dry (0.00 to 0.04 ± 0.01) cloths transferred similar levels of Salmonella to all 25 tomatoes. Results indicate a low risk of potential Salmonella contamination when the same cloth is used multiple times for debris removal, especially under high moisture levels. Results also show that the use of dirty cloths did not increase the risk of Salmonella cross-contamination. [ABSTRACT FROM AUTHOR]

A sublethally injured bacterial cell has been defined as a cell that survives a stress such as heating, freezing, acid treatment, or other antimicrobial intervention but can repair the cellular damage exerted by the stressor and later regain its original ability to grow. Consequently, sublethally injured cells are not likely to be included in conventional enumeration procedures, which could result in unrealistically low counts unless efforts are made to encourage recovery of the injured cells before enumeration. The objective of this study was to evaluate the use of the thin agar layer (TAL) method for the recovery of pressure-injured and heat-injured Listeria monocytogenes in a tryptic soy broth with 0.6% yeast extract system. Pressure injury consisted of treatment of a culture of mixed L. monocytogenes strains with high hydrostatic pressure at 400 or 600 MPa for 1 s, 2 min, 4 min, or 6 min at a process temperature of 12 ± 2°C. Heat injury consisted of treatment of a culture of mixed L. monocytogenes strains at 60 ± 1°C for 3, 6, or 9 min. Growth media were tryptic soy agar (TSA) with 0.6% yeast extract, modified Oxford medium (MOX), and TAL, which consisted of a 7-ml layer of TSA overlaid onto solidified MOX. Counts of viable L. monocytogenes on TAL were higher than those on MOX in the heat-injury experiment but not in the pressure-injury experiment. Therefore, the effectiveness of the TAL method may be specific to the type of injury applied to the microorganism and should be investigated in a variety of cellular injury scenarios. [ABSTRACT FROM AUTHOR]

Sodium nitrite exerts an inhibitory effect on the growth of Listeria monocytogenes. The objective of this study was to investigate the effects of various nitrite concentrations from a vegetable source with and without high hydrostatic pressure (HHP) on the recovery and growth of L. monocytogenes on ready-to-eat restructured ham. A preconverted celery powder was used as the vegetable source of nitrite. Targeted concentrations of natural nitrite investigated were 0, 50, and 100 mg/kg. HHP treatments evaluated were 400 MPa for 4 min and 600 MPa for 1 or 4 min at 12 ± 2°C (initial temperature of the pressurization fluid). Viable L. monocytogenes populations were monitored on modified Oxford medium and thin agar layer medium through 98 days of storage at 4 ± 1°C. Populations on both media did not differ. The HHP treatment at 600 MPa for 4 min resulted in L. monocytogenes populations below the detection limit of our sampling protocols throughout the storage period regardless of the natural nitrite concentration. The combination of HHP at 400 MPa for 4 min or 600 MPa for 1 min with natural nitrite resulted in initial inhibition of viable L. monocytogenes. Ham formulations that did not contain natural nitrite allowed faster growth of L. monocytogenes than did those with nitrite, regardless of whether they were treated with HHP. The results indicate that nitrite from a vegetable source at the concentrations used in this study resulted in slower growth of this microorganism. HHP treatments enhanced the inhibitory effects of natural nitrite on L. monocytogenes growth. Thus, the combination of natural nitrite plus HHP appears to have a synergistic inhibitory effect on L. monocytogenes growth. [ABSTRACT FROM AUTHOR]

We performed a simultaneous immunomagnetic separation (IMS) assay on Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O111, and O157 with immunobeads coated with O26, O111, or O157 antibodies that were simultaneously added to an aliquot of food culture. We also compared the usefulness of CHROMagar STEC medium against various selective isolation agars designed to test for the three serogroups. Samples of sliced beef, ground beef. and radish sprout were artificially contaminated with STEC O26, O111, and O157 strains after incubation in enrichment broth and were subjected to conventional and simultaneous IMS assays. Simultaneous IMS did not affect the sensitivity of target cell detection. For STEC O26, O111, and O157 inoculated into the enriched samples of sliced beef and radish sprout, the detection ability of CHROMagar STEC was similar to or exceeded that of other isolation agars. However for STEC O157 inoculated into ground beef cultures, cefixime tellurite sorbitol MacConkey agar (CT-SMAC) was the superior detection medium. The performance of simultaneous IMS combined with CT-SMAC and CHROMagar STEC detection is similar to that of the Japanese standard method for isolating E. coli O26, O111, and O157. However, the proposed approach involves the same time, materials, and labor costs as the standard E. coli O157 reference procedure but allows detection of three E. coli serotypes rather than a single strain. [ABSTRACT FROM AUTHOR]

Quality control procedures during food processing may involve direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured E. coli B-41560 and O157:H7 strains. Bacteria were propagated in tryptic soy broth (TSB), ground beef slurry, and infant milk formula to a density of 106 to 108 CFU/ml and then were stressed for 6 min either in lactic acid (pH 4.5) or heat shocked for 3 min at 60°C. Samples were pour plated in basal layers of either tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), or violet red bile agar (VRB) and were resuscitated for 4 h prior to addition of agar overlays. Other stressed bacteria were plated directly onto Petrifilm and Easygel. Results indicate that selective and nonselective agar overlays recovered significantly higher numbers (greater than 1 log) of acid-and heat-injured E. coli OI57:H7 from TSB, ground beef, and infant milk formula compared with direct plating onto selective media, Petrifilm, or Easygel, while no significant differences among these media combinations were observed for stressed E. coli B-41560. Nonstressed bacteria from TSB and ground beef were also recovered at densities significantly higher in nonselective TSA-TSA and in VRB-VRB and SMAC-SMAC compared with Petrifilm and Easygel. These data underscore the need to implement food safety measures that address sublethally injured pathogens such as E. coli O157:H7 in order to avoid underestimation of true densities for target pathogens. [ABSTRACT FROM AUTHOR]

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically important foodborne pathogens. Unlike E. coli O157:H7, these foodborne pathogens have no unique biochemical characteristics to readily distinguish them from other E. coli strains growing on plating media. In this study, a chromogenic agar medium was developed in order to differentiate among non-O157 STEC strains of serogroups O26, O45, O103, O111, O121, and O145 on a single agar medium. The ability of this chromogenic agar medium to select and distinguish among these pathogens is based on a combination of utilization of carbohydrates, β-galactosidase activity, and resistance to selective agents. The agar medium in combination with immunomagnetic separation was evaluated and successfully allowed for the detection and isolation of these six serogroups from artificially contaminated fresh beef. The agar medium in combination with immunomagnetic separation also allowed successful detection and isolation of naturally occurring non-O157 STEC strains present in cattle feces. Thirty-five strains of the top six non-O157 STEC serogroups were isolated from 1,897 fecal samples collected from 271 feedlot cattle. This chromogenic agar medium could help significantly in routine screening for the top six non-0157 STEC serogroups from beef cattle and other food. [ABSTRACT FROM AUTHOR]

This study developed models to predict the growth probabilities and kinetic behavior of Salmonella enterica strains on cutting boards. Polyethylene coupons (3 by 5 cm) were rubbed with pork belly, and pork purge was then sprayed on the coupon surface, followed by inoculation of a five-strain Salmonella mixture onto the surface of the coupons. These coupons were stored at 13 to 35°C for 12 h, and total bacterial and Salmonella cell counts were enumerated on tryptic soy agar and xylose lysine deoxycholate (XLD) agar, respectively, every 2 h, which produced 56 combinations. The combinations that had growth of <0.5 log CFU/cm² of Salmonella bacteria recovered on XLD agar were given the value 1 (growth), and the combinations that had growth of >=0.5 log CFU/cm² were assigned the value 0 (no growth). These growth response data from XLD agar were analyzed by logistic regression for producing growth/no growth interfaces of Salmonella bacteria. In addition, a linear model was fitted to the Salmonella cell counts to calculate the growth rate (log CFU per square centimeter per hour) and initial cell count (log CFU per square centimeter), following secondary modeling with the square root model. All of the models developed were validated with observed data, which were not used for model development. Growth of total bacteria and Salmonella cells was observed at 28, 30, 33, and 35°C, but there was no growth detected below 20°C within the time frame investigated. Moreover, various indices indicated that the performance of the developed models was acceptable. The results suggest that the models developed in this study may be useful in predicting the growth/no growth interface and kinetic behavior of Salmonella bacteria on polyethylene cutting boards. [ABSTRACT FROM AUTHOR]

Selective agar is inadequate for supporting recovery of injured cells. During risk assessment of certain foods, both injured and noninjured cells must be enumerated. In this study, a new method (agar spray method) for recovering sublethally heat-injured microorganisms was developed and used for recovery of heat-injured Salmonella Typhimurium and Listeria monocytogenes. Molten selective agar was applied as an overlay to presolidified nonselective tryptic soy agar (TSA) by spray application. Heat- injured cells (55°C for 10 min in 0.1% peptone water or 55°C for 15 min in sterilized skim milk) were inoculated directly onto solidified TSA. After a 2-h incubation period for cell repair, selective agar was applied to the TSA surface with a sprayer, and the plates were incubated. The recovery rate for heat-injured Salmonella Typhimurium and L. monocytogenes with the spray method was compared with the corresponding rates associated with TSA alone, selective media alone, and the conventional overlay method (selective agar poured on top of resuscitated cells grown on TSA and incubated for 2 h). No significant differences (P > 0.05) were found in pathogen recovery obtained with TSA, the overlay method, and the spray method. However, a lower recovery rate (P < 0.05) was obtained for isolation of injured cells on selective media. Overall, these results indicate that the agar spray method is an acceptable alternative to the conventional overlay method and is a simpler and more convenient approach to recovery and detection of injured cells. [ABSTRACT FROM AUTHOR]

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coil (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (l to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef. [ABSTRACT FROM AUTHOR]

Mannitol-yolk-polymyxin B agar (MYPA) was modified by supplementation with trimethoprim. The ability of the supplemented medium to select for and recover Bacillus cereus from pure cultures and food samples with high background microflora was compared with MYPA. For evaluation of the modified MYPA (mMYPA) in food samples with high background microflora, B. cereus was experimentally spiked into red pepper powder, fermented soybean paste, vegetable salad, and radish sprouts, and then it was recovered on MYPA and mMYPA for comparison. In all food samples, there was no difference in recoverability (P > 0.05) between mMYPA (red pepper powder, 3.34 ± 0.24 log CFU/g; fermented soybean paste, 3.52 ± 0.47 log CFU/g; vegetable salad, 3.51 ± 0.23 log CFU/g; radish sprouts, 3.32 ± 0.40 log CFU/g) and MYPA (red pepper powder, 3.18 ± 0.20 log CFU/g; fermented soybean paste, 3.33 ± 0.43 log CFU/g; vegetable salad, 3.36 ± 0.19 log CFU/g; radish sprouts, 3.33 ± 0.31 log CFU/g). However, mMYPA exhibited better selectivity than MYPA, because additional trimethoprim made the differentiation of suspected colonies easier by inhibiting competing flora. The addition of trimethoprim to conventional media could be a useful option to improve selectivity in foods with high background microflora. [ABSTRACT FROM AUTHOR]

Agar contact microbiological sampling techniques, based on a transfer of the microorganisms present on a surface to a culture medium, are widely used to assess and control surface cleanliness and to evaluate microbial contamination levels. The effectiveness of these techniques depends on many environmental parameters that influence the strength of attachment of the bacteria to the surface. In the present study, stainless steel and high density polyethylene surfaces were inoculated with known concentrations of Staphylococcus epidermidis. Following an experimental design, the surfaces were sampled with different types of replicate organism direct agar contact plates and Petrifilm; results indicated that recovery rates were influenced by the presence of egg white albumin or Tween 80 in the inoculum solutions or by the introduction of surfactants into the contact agar of the microbiological sampling techniques. The techniques yielded significantly different results, depending on sampling conditions, underlining the need for a standardization of laboratory experiments to allow relevant comparisons of such techniques. [ABSTRACT FROM AUTHOR]

Cronobacter spp. (Enterobacter sakazakii) are emerging opportunistic pathogens for all age groups, and are of particular concern when it comes to infants. Prior to contaminating food, the organism may be exposed to a variety of stresses, leading to a generation of sublethally injured cells that may not be detected by selective media unless a protracted recovery period is included in the isolation procedure. This study evaluated the efficacy of the thin agar layer (TAL) method for the recovery of Cronobacter cells that had been exposed to various stress conditions. Five strains of C. sakazakii and C. muytjensii were exposed to starvation, heat, cold, acid, alkaline, chlorine, or ethanol, with or without further exposure to desiccation stress. The recovery of the stressed cells was determined on tryptone soy agar (TSA; nonselective control medium), violet red bile glucose agar (VRBGA; selective agar), Druggan-Forsythe-Iversen (DFI; selective agar), and TAL media (viz., VRBGA overlaid with TSA, and DFI overlaid with TSA). Regardless of stress type. there were no significant differences among the recoveries of stressed desiccated Cronobacter spp. cultures on TSA, DFI+TSA, and VRBGA+TSA, but there was significantly less recovery on VRBGA. The recovery of prestressed desiccated Cronobacter spp. on DFI+TSA was similar to that on TSA, whereas the recovery on VRBGA+TSA was lower. DFI+TSA performed better than VRBGA+TSA did in differentiating Cronobacter spp. within mixed bacterial cultures. The results of this study suggest the use of the TAL method DFI+TSA as an improved method for the direct recovery of stressed Cronobacter spp. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the effect of water activity (aw; 0.995, 0.98, 0.96, 0.94, 0.92, and 0.90), temperature (5, 18, 25, and 30°C), incubation time (7 to 35 days), and their interactions on mycelial growth and alternariol (AOH) and alternariol monomethyl ether (AME) production. Two Alternaria alternata strains isolated from soybeans in Argentina were grown on 2% soybean extract agar. Maximum growth rates were obtained at the highest aw (0.995) and 25°C, with growth decreasing as the water availability of the medium was reduced. Maximum amount of AOH was produced at 0.98 aw and 25°C for both strains. Maximum AME production was obtained for both strains at 30°C but different aw values, 0.92 and 0.94, for the strains RC 21 and RC 39, respectively. The concentrations of both toxins varied considerably depending on the aw and temperature interactions assayed. The two metabolites were produced from 5 to 30°C and at aw values of 0.92 to 0.995. Although at 5 and 18°C little mycotoxin was produced at aw lower than 0.94. Two-dimensional profiles of aw by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soybeans. All the conditions of aw and temperature that resulted in maximum production of both toxins are those found during soybean development in the field. Thus, field conditions are likely to be conducive to optimum A. alternata growth and toxin production. [ABSTRACT FROM AUTHOR]

Bacillus species may be resistant to processing and sanitation procedures, making their control an important issue in the food industry. The objective of this study was to develop a rapid method for the differentiation of Bacillus cells at the strain level using infrared microspectroscopy and multivariate pattern recognition techniques. Aliquots (10 ml) of vegetative cells (∼10² CFU/ml) from four strains of each of three Bacillus species (B. cereus, B. mycoides, and B. thuringiensis) were filtered onto hydrophobic grid membranes. The membranes were placed on tryptic soy agar and incubated at 42°C for 24 h and then removed from the agar and dried, and the biomass of individual vegetative colonies was directly measured by attenuated total reflectance infrared (ATR-IR) microspectroscopy. Soft independent modeling of class analogy models generated from second derivative transformed spectra in the 1,300 to 900 cm-1 region exhibited clusters that permitted accurate strain-level classification of all isolates. Major discrimination was related to the signal from phosphate-containing compounds, likely phospholipids. Results indicate that a simple ATR-IR microspectroscopy technique combined with multivariate analysis could provide the food industry with a rapid and reagent-free screening procedure to complement more elaborate molecular identification methods. [ABSTRACT FROM AUTHOR]

A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9-specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains beating the 09 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples. [ABSTRACT FROM AUTHOR]

An automated most-probable-number (MPN) system (TEMPO, bioM6rieux, Marcy l'Etoile, France) for enumeration of Enterobacteriaceae (EB) was compared with methods involving violet red bile glucose agar (VRBG) (International Organization for Standardization [ISO] method 21528-2) (ISO-VRBG) and Petrifilm (PF-EB). The MPN partitioning (three different volumes with 16 replicates of each) is done automatically in a disposable card. Bacterial growth is indicated by acid production from sugars, lowering the pH of the medium, and quenching the fluorescence of 4-methylumbelliferone. After incubation, the number of nonfluorescent wells is read in a separate device, and the MPN is calculated automatically. A total of 411 naturally contaminated foods were tested, and 190 were in the detection range for all methods. For these results, the mean (±standard deviation) counts were 2.540 ± 1.026, 2.547 ± 0.995, and 2.456 ± 1.014 log CFU/g for the ISO-VRBG, PF-EB, and automated MPN methods, respectively. Mean differences were -0.084 ± 0.460 log units for the automated MPN results compared with the ISO-VRBG and 0.007 ± 0.450 for the PF-EB results compared with the ISO-VRBG results. The automated MPN method tends to yield lower numbers and the PF-EB method tends to yield higher numbers than does the ISO-VRBG method (difference not significant; Kruskal-Wallis test, P = 0.102). Thus, the average difference was highest between the automated MPN method and the PF-EB method (-0.091 ± 0.512 log units). Differences between the automated MPN and ISO-VRBG results of > 1 log unit were detected in 3.4% of all samples. For 3.9% of the samples, one comparison yielded differences of < 1 log CFU/g and the other yielded > 1 but <2 log CFU/g, which means that the differences are possibly >1 log CFU/g. For the ISO-VRBG method, confirmation of isolates was necessary to avoid a bias due to the presence of oxidase-positive glucose-fermenting colonies. The automated MPN system yielded results comparable to those of the confirmed Enterobacteriaceae ISO-VRBG method but required only 24 h of analysis time. [ABSTRACT FROM AUTHOR]

A study was done to compare Nissui Compact Dry Yeast and Mold plates (CDYM), 3M Petrifilm Yeast and Mold count plates (PYM), dichloran-rose bengal chloramphenicol (DRBC) agar, and dichloran 18% glycerol (DGIS) agar for enumerating yeasts and molds naturally occurring in 97 foods (grains, legumes, raw fruits and vegetables, nuts, dairy products, meats, and miscellaneous processed foods and dry mixes). Correlation coefficients for plates incubated for 5 days were DG18 versus DRBC (0.93), PYM versus DRBC (0.81), CDYM versus DG18 (0.81). PYM versus DG18 (0.801, CDYM versus DRBC (0.79), and CDYM versus PYM (0.75). The number of yeasts and molds recovered from a group of foods (n = 32) analyzed on a weight basis (CFU per gram) was not significantly different (α = 0.05) when samples were plated on DRBC, DG18. PYM, or CDYM. However, the order of recovery from foods (n = 65) in a group analyzed on a unit or piece basis, or a composite of both groups (n = 97), was DRBC > DG18 - CDYM > PYM. Compared with PYM, CDYM recovered equivalent, significantly higher (α = 0.05) or significantly lower (α = 0.05) numbers of yeasts and molds in 51.5, 27.8, and 20.6%, respectively, of the 97 foods tested: respective values were 68.8, 15.6, and 15.6% in the small group (n = 32) and 43.1, 33.8, and 23.1% in the large group (n = 65) of foods. The two groups contained different types of foods, the latter consisting largely (73.8%) of raw fruits (n = 16) and vegetables (n = 32). Differences in efficacy of the four methods in recovering yeasts and molds from foods in the two groups are attributed in part to differences in genera and predominant mycoflora. While DG18 agar, CDYM, and PYM appear to be acceptable for enumerating yeasts and molds in the foods analyzed in this study, overall. DRBC agar recovered higher numbers from the 97 test foods, thereby supporting its recommended use as a general purpose medium for mycological analysis. [ABSTRACT FROM AUTHOR]

This study was designed to evaluate the occurrence of Bacillus cereus spores with at damaged exosporium and the consequences of such damages on spore adhesion. The analysis of nine strains sporulated under optimal conditions (Spo8- agar, 30°C) revealed that damaged exosporia were systematically found in one strain (B. cereus D17) and occasionally in two others (B. cereus ATCC 14579T and B. cereus D6). The prevalence of spores with damaged exosporia increased when sporulation occurred under less favorable conditions (SpoS-broth or high temperature); for example, more than 50% of the B. cereus ATCC 14579T spores were damaged when sporulation occurred at 40°C on SpoS-agar or at 30°C in SpoS-broth. Furthermore, when subjected to shear stresses by circulation of spore suspensions through it peristaltic pump, the exosporium of a significant amount of spores became partially or totally shorn off (for example, 40% of the B. cereus ATCC 14579T spores). The ability of damaged spores to adhere to inert surfaces and to resist cleaning under shear stress was significantly affected when compared with intact spores, resulting in a decreased number of adhering spores (P ≤ 0.004) and enhanced resistance to cleaning (P ≤ 0.008). This study provides evidence that, under various conditions, the exosporium of B. cereus spores can be partly or wholly damaged, thereby of affecting the ability of spores to contaminate the surfaces of food processing lines. The presence of spores devoid of exosporium will be of importance in determining the risk associated with B. cereus spheres adherent to food processing line surfaces. [ABSTRACT FROM AUTHOR]

This study investigated flow-through immunocapture (FTI), using the Pathatrix device, followed by plating on xylose lysine desoxycholate (XLD) agar (FTI-XLD) or analysis by real-time PCR (FTI-PCR) for the detection of Salmonella on smooth tomato surfaces and in potato salad and ground beef within 8 h. Food samples were inoculated with an appropriate dilution of a five-serovar Salmonella cocktail and enriched for 5 h. Following enrichment, samples were analyzed by the FTI-XLD and FTI-PCR methods. Food samples were also analyzed by a modified U.S. Food and Drug Administration Bacterio-logical Analytical Manual (BAM) Salmonella culture method for comparison. Salmonella inoculated at 100 CFU per tomato or 100 CFU/25 g was detected by the FTI-XLD method in 6, 8, and 4 of 10 samples for tomatoes, potato salad, and ground beef, respectively. Salmonella inoculated at 100 CFU per tomato or 100 CFU/25 g was detected by the FTI-PCR method in 8, 9, and 9 of 10 samples for tomatoes, potato salad, and ground beef, respectively. The FTI-PCR method achieved significantly higher (P < 0.05) detection of Salmonella on tomatoes, whereas the FTI-XLD method achieved significantly lower (P < 0.05) detection of Salmonella in ground beef when compared with the modified BAM Salmonella culture method; however, all other comparisons to the modified BAM method were not significantly different. The FTI-XLD method demonstrated the ability to isolate presumptive Salmonella colonies up to 48 h faster than did the modified BAM Salmonella culture method. [ABSTRACT FROM AUTHOR]

Shells of agar-filled and whole eggs were inoculated with 10³ to 104 CFU of Salmonella enterica serovar Enteritidis per eggshell. The agar-filled eggs were used to study bacterial eggshell penetration, and the whole egg results were used to characterize contamination of the egg contents. In each group, half of the eggs were stored for 21 days at 20°C and 60% relative humidity (RH), and the other half was stored for 24 h at 6°C and then for 20 days at 20°C. The latter conditions resulted in condensation on the eggshell for 30 mm from the moment the eggs were placed in the 20°C chamber. Taking into account the ages at which hens were studied (39, 53, and 67 weeks), an average of 62% of the eggshells with condensate were penetrated compared with 43% for the control group; this difference was significant (P < 0.01). No significant difference in whole egg contamination was found; 18% of the control eggs were contaminated compared with 22% of the condensate eggs. Whole egg contamination was significantly higher for eggs from the hens at an older age (67 weeks). This difference probably was not due to a higher penetration potential because differences were not observed for the corresponding agar-filled eggs. Condensation on the eggshell seemed to encourage bacterial penetration of the eggshell but had a smaller impact on whole egg contamination. [ABSTRACT FROM AUTHOR]

Risk studies have identified cross-contamination during beef fabrication as a knowledge gap, particularly as to how and at what levels Escherichia coli O157:H7 transfers among meat and cutting board (or equipment) surfaces. The objectives of this study were to determine and model transfer coefficients (TCs) between E. coli O157:H7 on beef tissue and high-density polyethylene (HDPE) cutting board surfaces. Four different transfer scenarios were evaluated: (i) HDPE board to agar, (ii) beef tissue to agar, (iii) HDPE board to beef tissue to agar, and (iv) beef tissue to HDPE board to agar. Also, the following factors were studied for each transfer scenario: two HDPE surface roughness levels (rough and smooth), two beef tissues (fat and fascia), and two conditions of the initial beef tissue inoculation with E. coli O157:H7 (wet and dry surfaces), for a total of 24 treatments. The TCs were calculated as a function of the plated inoculum and of the cells recovered from the first contact. When the treatments were compared, all of the variables evaluated interacted significantly in determining the TC. An overall TC-per-treatment model did not adequately represent the reduction of the cells on the original surface after each contact and the interaction of the factors studied. However, an exponential model was developed that explained the experimental data for all treatments and represented the recontamination of the surfaces with E. coli O157:H7. The parameters for the exponential model for cross-contamination with E. coli O157:H7 between beef tissue and HDPE surfaces were determined, allowing for the use of the resulting model in quantitative microbial risk assessment. [ABSTRACT FROM AUTHOR]

The effects of acidified sodium chlorite (ASC) against Listeria monocytogenes on the surface of cooked roast beef were investigated. L. monocytogenes, strain V7, serotype 1/2a, was inoculated at numbers of 6.0 log CFU/g onto 5-g cubes of cooked regular or spicy roast beef. The samples were allowed to air dry for 1 h. The cooked roast beef samples were dipped into ASC or sprayed with ASC solutions of 250, 500, 750, or 1,000 ppm, then placed in bags with or without a vacuum and refrigerated at 4°C. L. monocytogenes counts were determined after 0, 7, 14, 21, and 28 days of storage by spread plating roast beef samples onto Oxford agar plates that were incubated at 37°C for 48 h. At day 28, the number of L. monocytogenes on the ≥500 ppm ASC-treated spicy roast beef samples had count reductions that were >4.0 log CFU/g, whereas the same concentrations of ASC-treated regular roast beef samples had approximately a 2.5 log CFU/g reduction in L. monocytogenes counts when compared with the untreated samples. No significant differences (P > 0.05) were observed in L. monocytogenes counts between the vacuum- or nonvacuum-packaged ASC-treated cooked roast beef samples. Sensory evaluation showed no significant differences (P > 0.05) between ASC-treated and untreated roast beef. ASC can be used as a processing aid in the form of a dip or spray treatment to control L. monocytogenes on the surface of cooked roast beef. [ABSTRACT FROM AUTHOR]

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58°C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies. [ABSTRACT FROM AUTHOR]

A chromogenic agar, R&F Enterobacter sakazakii chromogenic plating medium (ESPM), was developed for isolating presumptive colonies of E. sakazakii from foods and environmental sources. ESPM contains two chromogenic substrates (5-bromo-4-chloro-3-indoxyl-α-D-glucopyranoside and 5-bromo-4-chloro-3-indoxyl-β-D-cellobioside), three sugars (sorbitol, D-arabitol, and adonitol), a pH indicator, and inhibitors (bile salts, vancomycin, and cefsulodin), which all contribute to its selectivity and differential properties. On ESPM, 79 pure culture strains of E. sakazakii (10 clinical isolates and others from food and environmental sources) yielded blue-black (three strains were blue-gray) raised colonies, 1 to 2 mm in diameter with and without halos after 24 h at 35°C. Other enteric organisms plus Pseudomonas aeruginosa yielded white, yellow, green, or clear colonies with and without clear halos. Of these genera, only Shigella sonnei and one Pantoea strain produced blue-black to blue-gray colonies. ESPM was used to isolate E. sakazakii from a variety of foods: corn, wheat, and rice flours; powdered infant formula; dairy products (dried milk, whey, and caseinates); cereals; and environmental sources. Most false-positive results on ESPM were eliminated by observing acid production on either sucrose or melibiose after 6 h at 35°C on a R&F E. sakazakii screening medium (ESSM) biplate. In an analysis of 240 samples, the number of samples positive for E. sakazakii by the ESPM-ESSM method and the U.S. Food and Drug Administration protocols (violet red bile glucose agar and tryptic soy agar) were 27 and 16, respectively, with sensitivity and specificity values of 100.0 and 96.9% versus 59.3 and 43.7%, respectively. These data support the fact that E. sakazakii confirmation should be based on more than one confirmation system. Both the API 20E and Biolog Microlog3 4.20 systems should be used for confirmation of E. sakazakii isolates. [ABSTRACT FROM AUTHOR]

Objectives of this research were to investigate the detection frequency of presumptive Alicyclobacillus strains, also known as thermoacidophilic or acidothermophilic bacteria, on oranges entering juice-processing facilities and to compare results from three common isolation agars (acidified potato dextrose agar, Ali agar, and K agar). A total of 1,575 fruits were sampled from three points (ungraded fruits, graded sound fruits, and graded defective fruits) at two juice-processing facilities during two harvest seasons. Buffer used to rinse individual fruits was assayed for the presence of thermoacidophilic bacteria using an enrichment procedure. Isolates were considered presumptive Alicyclobacillus if they were gram-positive, sporogenous, rod-shaped bacteria, with growth at 45°C and no or slight growth at 25°C on a low pH medium (pH 3.7) coupled with lack of growth on a neutral pH medium (pH 7.0) at both temperatures and a lack of growth in Sulfobacillus broth medium (pH 2.0). More than one third of all fruits sampled at the two facilities were contaminated with presumptive Alicyclobacillus strains. Therefore, incoming fruits are a substantial means by which these organisms gain entrance to the processing facility. Significantly (P « 0.05) more detection was observed with a mineral-containing medium (All agar) than with nonmineral containing media (K agar and acidified potato dextrose agar). [ABSTRACT FROM AUTHOR]

Proteins and genes involved in the recovery of heat-injured Salmonella Enteritidis were investigated. Salmonella Enteritidis cells cultured overnight in tryptic soy broth (TSB; nonselective medium) were suspended in citric acid-disodium hydrogen phosphate buffer (pH 6). After heat treatment at 55°C for 15 min, the culturable counts measured by tryptic soy agar (TSA; nonselective medium) decreased from 108 to 107 CFU/ml. On the other hand, culturable counts measured by desoxycholate-hydrogen sulfite-lactose (DHL) agar (selective medium) were decreased from 108 to 104 CFU/ml by the same treatment. The results suggest that 99.9% of Salmonella Enteritidis detected on TSA were injured but recoverable. When injured Salmonella Enteritidis was incubated in TSB, the culturable count measured by TSA did not increase for 2 h, whereas that by DHL agar increased after incubation for 30 min. After incubation for 2 h, the culturable count measured by DHL agar reached a similar level with that by TSA, indicating that Salmonella Enteritidis had recovered. The two-dimensional polyacrylamide gel electrophoresis analysis revealed that elongation factor G (FusA) and pyruvate kinase (PykF) specifically increased in the cells just after heat treatment and in the recovery cells. The levels of transcription of 86 stress-inducible genes were also investigated by reverse transcription PCR. Nineteen heat-inducible (clpB, clpX, degP, dnaJ, fkpA, frsJ, gapA, hflB, hslJ, hslU, hslV, htpG, htrA, lon, mopA, mopB, mreB, rpoE, and ppiD), and 12 oxidative-stress and DNA damage-inducible (ahpC, ahpF, fldB, fur, grxA, dinF, katG, mutM, recA, soxR, trxC, and zwf) genes were transcribed extensively during recovery in TSB. The results obtained in this study will be used to develop the media or culture conditions that will promote recovery for the detection of food poisoning bacteria, including injured cells from food products. [ABSTRACT FROM AUTHOR]

Four sampling devices, a sterile environmental sponge (ES), a sterile cotton-tipped swab (CS), a sterile calcium alginate fiber-tipped swab (CAS), and a one-ply composite tissue (CT), were evaluated for quantitative recovery of Listeria monocytogenes from a food-grade stainless steel surface. Sterile 304-grade stainless steel plates (6 by 6 cm) were inoculated with approximately 106 CFU/cm2 L. monocytogenes strain Scott A and dried for 1 h. The ES and CT sampling devices were rehydrated in phosphate buffer solution. After plate swabbing, ES and CT were placed in 40 ml of phosphate buffer solution, stomached for 1 mm and hand massaged for 30 s. Each CS and CAS device was rehydrated in 0.1% peptone before swabbing. After swabbing, CS and CAS were vortexed in 0.1% peptone for 1 mm. Samples were spiral plated on modified Oxford agar with modified Oxford agar Rodac Contact plates used to recover any remaining cells from the stainless steel surface. Potential inhibition from CT was examined in both phosphate buffer solution and in a modified disc-diffusion assay. Recovery Was 2.70, 1.34, and 0.62 log greater using CT compared with ES, CS, and CAS, respectively, with these differences statistically significant (P < 0.001) for ES and CT and for CAS, CS, and CT (P < 0.05). Rodac plates were typically overgrown following ES, positive after CS and CAS, and negative after CT sampling. CT was noninhibitory in both phosphate buffer solution and the modified disc-diffusion assay. Using scanning electron microscopy, Listeria cells were observed on stainless steel plates sampled with each sampling device except CT. The CT device, which is inexpensive and easy to use, represents a major improvement over other methods in quantifying L. monocytogenes on stainless steel surfaces and is likely applicable to enrichment of environmental samples. [ABSTRACT FROM AUTHOR]

Selective culture media, such as Nash and Snyder medium (NS), dichloran-chloramphenicol peptone agar (DCPA), modified Czapek-Dox agar (MCz), Czapek Dox iprodione dichloran agar (CZID), potato dextrose iprodione dichloran agar (PDID), or malachite green agar (MGA 2.5), have been developed for isolating and enumerating Fusarium spp. from natural samples. However, some of these culture media are not very selective because they allow the growth of many other fungal species. In this study, a comparison of the selective efficacy of these culture media, using different strains of Fusarium spp. (F. anthophilum, F. culmorum, F. dlamini, F. graminearum, F. napiforme, F. nygamai, F. oxysporum, F. proliferatum, F. semitectum, F. solani, F. subglutinans, and F. verticillioides) and natural samples has been carried out. Among the six recommended selective culture media assayed, no statistical differences were detected in colony counts of the Fusarium spp. strains tested, although the colony diameters in MGA 2.5 were significantly lower than in NS, MCz, DCPA, CZID, and PDID media. With natural samples, MGA 2.5 performs as a potent selective medium for Fusarium spp., whereas the other recommended selective media allow the growth of many other different fungal species including Zygomycetes and yeasts. [ABSTRACT FROM AUTHOR]

The ability to detect and isolate Shiga toxin-producing Escherichia coli (STEC) remains a major challenge for food microbiologists. Although methods based on nucleic acids and antibodies have improved detection of STECs in foods, isolation of these bacteria remains arduous. STEC isolation is necessary for matching food, environmental, and clinical isolates during outbreak investigations and for distinguishing between pathogenic and nonpathogenic organisms. STEC heart infusion washed blood agar with mitomycin-C (SHIBAM) is a modification of washed sheep blood agar prepared by adding mitomycin-C and optimizing both the washed blood and base agar to better isolate STECs. Most STEC isolates produce a zone of hemolysis on SHIBAM plates and are easily distinguishable from background microbiota. Here, we present data supporting the use of SHIBAM to isolate STECs from fresh produce. SHIBAM was tested for accuracy in identifying STECs (365 of 410 STEC strains were hemolytic, and 63 of 73 E. coli strains that did not produce Shiga toxin were not hemolytic) and for recovery from artificially inoculated fresh produce (11 of 24 romaine lettuce samples and 6 of 24 tomato samples). STEC recovery with SHIBAM agar was greatly improved when compared with recovery on Levine's eosin-methylene blue agar as a reference method. [ABSTRACT FROM AUTHOR]

In this study, three different selective media, modified cefoperazone charcoal deoxycholate agar (mCCDA), Karmali agar, and Preston agar, were compared for isolating Campylobacter jejuni from artificially contaminated ground beef and fresh-cut vegetables that have different levels of background microflora. Concurrently, an automated enzyme-linked immunosorbent assay method for detecting Campylobacter spp. (VIDAS Campylobacter) was evaluated by comparing it with the culture methods. Food samples inoculated with C. jejuni were enriched in Bolton broth at 42°C for 44 h and then streaked onto the three different selective media, followed by incubation under microaerobic conditions at 42°C for 48 h. The enriched Bolton broth (1 ml) was used in the VIDAS Campylobacter assay. No statistical differences in sensitivities were observed between the three selective media for ground beef and fresh-cut vegetables, but the selectivity of Preston agar was better (P < 0.05) than those of mCCDA and Karmali agar. The VIDAS Campylobacter assay showed a recovery rate similar (P > 0.05) to those of all of the medium combinations in ground beef. However, more positive samples (P < 0.05) were detected with the VIDAS Campylobacter than with the selective agars, except for the combinations of mCCDA plus Preston agar or mCCDA plus Karmali agar plus Preston agar in fresh-cut vegetables. [ABSTRACT FROM AUTHOR]