Rainbow Agar Research Articles

Performance of Chromogenic Agar Media for Isolation of Shiga Toxin–Producing Escherichia coli from Ground Beef

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.

Detection and Isolation of the “Top Seven” Shiga Toxin–Producing Escherichia coli in Ground Beef: Comparison of RapidFinder Kits to the U.S. Department of Agriculture Microbiology Laboratory Guidebook Method

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are often referred to as the "top seven" STEC, and these have been declared to be adulterants in beef by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS). The aim of this work was to compare the methods described in the USDA FSIS Microbiology Laboratory Guidebook (MLG) to a two-stage Applied Biosystems RapidFinder STEC real-time PCR method to test for the top seven STEC in raw ground beef. The specificity of the RapidFinder workflow that targets non-O157 STEC O-antigen genes, stx1, stx2, and eae, and E. coli O157-specific targets was determined with 132 top seven STEC strains and 283 exclusion strains. All inclusion strains were positive, and all exclusion strains gave negative results with the RapidFinder assay. Strains carrying all of the known variants of stx1 and stx2, including stx2f and stx2g, were also detected. For RapidFinder analysis, 375-g ground beef samples spiked with ≥4 CFU of representative STEC strains were enriched in 1 L of tryptic soy broth (TSB) for 10 h at 42 ± 1°C, and for the MLG method, 325-g samples were similarly spiked and enriched in 975 mL of modified TSB for 15 h at 42 ± 1°C. Following DNA extraction, real-time PCR was performed using RapidFinder Express software, and for the MLG method, the BAX Real-Time PCR STEC Suite and the BAX Real-Time E. coli O157:H7 assay were used with the BAX System Q7 software. Following immunomagnetic separation, presumptive colonies from modified Rainbow agar O157 plates were confirmed by the real-time PCR assays. Results of the RapidFinder and BAX assays were similar; all samples were positive after 10 and 15 h of enrichment, respectively. Isolation and confirmation of isolates was possible on all samples, except that two O111:NM strains could not be isolated from a portion of the inoculated samples. Thus, the RapidFinder system can be used for routine and rapid detection of the top seven STEC in beef.

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

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

Overcoming Molecular Sample Processing Limitations: (a) New Platform Technologies, (b) RNA and DNA Extraction Strategies, and (c) Fiber-Optic Biosensors

Overcoming Molecular Sample Processing Limitations: (a) New Platform Technologies, (b) RNA and DNA Extraction Strategies, and (c) Fiber-Optic Biosensors

Hyperspectral imaging using RGB color for foodborne pathogen detection

This paper reports the development of a spectral reconstruction technique for predicting hyperspectral images from RGB color images and classifying food-borne pathogens in agar plates using reconstructed hyperspectral images. The six representative non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) grown on Rainbow agar plates were used for the study. A line-scan pushbroom hyperspectral imaging spectrometer was used to scan full reflectance spectra of pure non-O157 STEC cultures in the visible and near-infrared spectral range from 400 to 1000 nm. RGB color images were generated by simulation from hyperspectral images. Polynomial multivariate least-squares regression analysis was used to reconstruct hyperspectral images from RGB color images. The mean R-squared value for hyperspectral image reconstruction was ∼0.98 in the spectral range between 400 and 700 nm for linear, quadratic, and cubic polynomial regression models. The accuracy of the hyperspectral image classification algorithm based on k-nearest neighbors algorithm of principal component scores was validated to be 92% with the test set (99% with the original hyperspectral images). The results of the study suggested that color-based hyperspectral imaging would be feasible without much loss of prediction accuracy compared to true hyperspectral imaging.

Automatic Counting and Classification of Bacterial Colonies Using Hyperspectral Imaging

Detection and counting of bacterial colonies on agar plates is a routine microbiology practice to get a rough estimate of the number of viable cells in a sample. There have been a variety of different automatic colony counting systems and software algorithms mainly based on color or gray-scale pictures although manual counting is still common. In microbiology, identification of presumptive-positive colonies on agar plates is predominantly done manually, which is laborious and time-consuming. This paper addresses a problem related to automatic colony segmentation and classification that can count the number of colonies according to their types. Hyperspectral imaging was used to develop a colony segmentation algorithm for detecting non-O517 Shiga-toxin producing Escherichia coli (STEC) pathogens on Rainbow agar. Hyperspectral absorbance image analysis in the visible and near-infrared spectral range from 400 to 1000 nm showed that colony morphology including size and texture was dependent on wavelength. The non-O157 STEC colonies showed dome-like absorbance profiles with local absorbance maxima. Touching colonies, causing problems for accurate counting and identification, were separated by optimally tessellating the mesh structure of local maximal points. The 428 nm was determined as the optimal wavelength for non-O157 STEC colony segmentation. The accuracy of the colony segmentation and counting algorithm was over 99 %. The average of the colony classification algorithm using automated colony segments was 92.5 %.

Genetically Marked Strains of Shiga Toxin–Producing O157:H7 and Non-O157 Escherichia Coli: Tools for Detection and Modeling

Shiga toxin-producing E. coli (STEC) is an important group of foodborne pathogens in the United States and worldwide. Nearly half of STEC-induced diarrheal disease in the United States is caused by serotype O157:H7, while non-O157 STEC account for the remaining illnesses. Thus, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service has instituted regulatory testing of beef products and has a zero-tolerance policy for regulatory samples that test positive for STEC O157:H7 and six other non-O157 STEC (serogroups O26, O45, O103, O111, O121, and O145). In this study, positive control (PC) strains for the detection of STEC O157:H7 and the six USDA-regulated non-O157 STEC were constructed. To ensure that the food testing samples are not cross-contaminated by the PC sample, it is important that the STEC-PC strains are distinguishable from STEC isolated from test samples. The PC strains were constructed by integrating a unique DNA target sequence and a gene for spectinomycin (Sp) resistance into the chromosomes of the seven STEC strains. End-point and real-time PCR assays were developed for the specific detection of the PC strains and were tested using 93 strains of E. coli (38 STEC O157:H7, at least 6 strains of each of the USDA-regulated non-O157 STEC, and 2 commensal E. coli) and 51 strains of other bacteria (30 species from 20 genera). The PCR assays demonstrated high specificity for the unique target sequence. The target sequence was detectable by PCR after 10 culture passages (∼100 generations), demonstrating the stability of the integrated target sequence. In addition, the strains were tested for their potential use in modeling the growth of STEC. Plating the PC strains mixed with ground beef flora on modified rainbow agar containing Sp eliminated the growth of the background flora that grew on modified rainbow agar without Sp. Thus, these strains could be used to enumerate and model the growth of STEC in the presence of foodborne background flora.

Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in 375 Grams of Beef Trim Enrichments across Multiple Commercial PCR Detection Platforms

Although serotype O157:H7 remains the pathogenic Shiga toxin–producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

Comparison of eight different agars for the recovery of clinically relevant non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro, alfalfa sprouts and raw milk

The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not all have been thoroughly tested for recovering STECs from food. Using E. coli strains representing ten clinically relevant O serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh produce – bagged baby spinach, alfalfa sprouts, cilantro, and raw milk – we evaluated the performance of 8 different agars. Performance was highly dependent upon strain used and the presence of inhibitors, but not necessarily dependent on food matrix. Tellurite resistant-negative strains, O91:–, O103:H6, O104:H21, O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to CHROMagar STEC and mRB agars improved overall performance; however, this also reversed the inhibition of non-target bacteria provided by original formulations. Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB agars difficult. Study results support a strategy using inclusive agars (e.g. L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7, CHROMagar STEC) to allow for recovery of the most STECs while increasing the probability of recovering STEC in high bacterial count matrices.

Light scattering sensor for direct identification of colonies of Escherichia coli serogroups O26, O45, O103, O111, O121, O145 and O157.

BackgroundShiga-toxin producing Escherichia coli (STEC) have emerged as important foodborne pathogens, among which seven serogroups (O26, O45, O103, O111, O121, O145, O157) are most frequently implicated in human infection. The aim was to determine if a light scattering sensor can be used to rapidly identify the colonies of STEC serogroups on selective agar plates.Methodology/Principal FindingsInitially, a total of 37 STEC strains representing seven serovars were grown on four different selective agar media, including sorbitol MacConkey (SMAC), Rainbow Agar O157, BBL CHROMagarO157, and R&F E. coli O157:H7, as well as nonselective Brain Heart Infusion agar. The colonies were scanned by an automated light scattering sensor, known as BARDOT (BActerial Rapid Detection using Optical scattering Technology), to acquire scatter patterns of STEC serogroups, and the scatter patterns were analyzed using an image classifier. Among all of the selective media tested, both SMAC and Rainbow provided the best differentiation results allowing multi-class classification of all serovars with an average accuracy of more than 90% after 10–12 h of growth, even though the colony appearance was indistinguishable at that early stage of growth. SMAC was chosen for exhaustive scatter image library development, and 36 additional strains of O157:H7 and 11 non-O157 serovars were examined, with each serogroup producing unique differential scatter patterns. Colony scatter images were also tested with samples derived from pure and mixed cultures, as well as experimentally inoculated food samples. BARDOT accurately detected O157 and O26 serovars from a mixed culture and also from inoculated lettuce and ground beef (10-h broth enrichment +12-h on-plate incubation) in the presence of natural background microbiota in less than 24 h.ConclusionsBARDOT could potentially be used as a screening tool during isolation of the most important STEC serovars on selective agar plates from food samples in less than 24 h.

Non-O157 Shiga Toxin-Producing Escherichia coli in U.S. Retail Ground Beef

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.

Advances in detection methods for Shiga toxin-producing Escherichia coli (STEC).

Advances in detection methods for Shiga toxin-producing Escherichia coli (STEC).

Method for the detection of priority Shiga toxin-producing Escherichia coli in beef trim.

A method has been developed for the detection in beef trim of priority Shiga toxin-producing E. coli (STEC) strains, defined as E. coli possessing the virulence factors stx1 and/or stx2 and intimin (eae), with O serogroups O26, O45, O103, O111, O121, O145, or O157. The method is based on recovery of the target bacteria by overnight enrichment in a broth optimized for recovery of O157 and non-O157 STEC, followed by screening using multiplex PCR techniques targeting (i) stx1, stx2, and eae (STE PCR) and (ii) gene sequences associated with the seven priority O serogroups (Poly O PCR), and then direct plating of broth samples positive in both STE and Poly O PCR onto Rainbow agar. Colonies on agar media were screened batchwise for STEC by the STE PCR, and presumptive isolates were characterized using a multiplex PCR and cloth-based hybridization array system targeting key virulence and O serogroup-specific markers. Using one representative strain of each priority O serogroup individually inoculated in beef trim samples, the method exhibited a limit of detection approaching 1 to 2 viable STEC cells per 65 g. None of the uninoculated trim samples produced positive results with either of the screening PCR procedures or on analysis of colonies recovered on plating media. STEC-negative samples were readily identified by screening PCR within 24 h, with a turnaround time of fewer than 4 days for confirmation of positives. The inclusivity and exclusivity characteristics of the screening PCR techniques were verified using a total of 65 different priority STEC strains: 24 nonpriority STEC, 15 non-STEC bacteria, and only those strains bearing the targeted characteristics produced screening PCR-positive results.

Detection by Hyperspectral Imaging of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 on Rainbow Agar

The U.S. Department of Agriculture, Food Safety Inspection Service has determined that six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) are adulterants in raw beef. Isolate and phenotypic discrimination of non-O157 STEC is problematic due to the lack of suitable agar media. The lack of distinct phenotypic color variation among non-O157serogroups cultured on chromogenic agar poses a challenge in selecting colonies for confirmation. In this study, visible and near-infrared hyperspectral imaging and chemometrics were used to detect and classify non-O157 STEC serogroups grown on Rainbow agar O157. The method was first developed by building spectral libraries for each serogroup obtained from ground-truth regions of interest representing the true identity of each pixel and thus each pure culture colony in the hyperspectral agar-plate image. The spectral library for the pure-culture non-O157 STEC consisted of 2,171 colonies, with spectra derived from 124,347 of pixels. The classification models for each serogroup were developed with a k nearest-neighbor classifier. The overall classification training accuracy at the colony level was 99%. The classifier was validated with ground beef enrichments artificially inoculated with 10, 50, and 100 CFU/ml STEC. The validation ground-truth regions of interest of the STEC target colonies consisted of 606 colonies, with 3,030 pixels of spectra. The overall classification accuracy was 98%. The average specificity of the method was 98% due to the low false-positive rate of 1.2%. The sensitivity ranged from 78 to 100% due to the false-negative rates of 22, 7, and 8% for O145, O45, and O26, respectively. This study showed the potential of visible and near-infrared hyperspectral imaging for detecting and classifying colonies of the six non-O157 STEC serogroups. The technique needs to be validated with bacterial cultures directly extracted from meat products and positive identification of colonies by using confirmatory tests such as latex agglutination tests or PCR.

Hyperspectral Imaging for Differentiating Colonies of Non-0157 Shiga-Toxin Producing Escherichia Coli (STEC) Serogroups on Spread Plates of Pure Cultures

Direct plating on solid agar media has been widely used in microbiology laboratories for presumptive-positive pathogen detection, although it is often subjective and labour-intensive. Rainbow agar is a selective and differential chromogenic medium used to isolate pathogenic Escherichia coli (E. coli) strains. However, it is challenging to differentiate colonies of the six representative pathogenic non-0157 Shiga-toxin producing E. coli (STEC) serogroups (026, 045, 0103, 0111, 0121 and 0145) on Rainbow agar due to the phenotypic differences and the presence of background microflora. Therefore, there is a need for a method or technology to objectively, rapidly and accurately perform high-throughput screening of non-0157 STEC colonies on agar plates. In this paper, we report the development of a visible-near infrared hyperspectral imaging technique and prediction model for differentiating colony types of the six non-0157 STEC serogroups in spread plates of pure cultures inoculated on Rainbow agar. The prediction model was based on supervised linear classification of factor scores obtained by principal component analysis (PCA). Both PCA- MD (Mahalanobis distance) and PCA- kNN ( k-nearest neighbour) classifiers were used for model development. From the 24 hyperspectral images measured from two replicates, 51,173 spectral samples were collected from 1421 colonies. Chemometric preprocessing methods and other operating parameters, such as scatter correction, first derivative, moving average, sample size and number of principal components (PCs), were compared with a classification and regression tree (CART) method, configured as a classification tree and followed by brute-force searching from candidates selected by the CART. The number of PCs, scatter correction and moving average were selected as the most important elements to consider before selecting a set of candidate models. Cross-validation ( CV), such as hold out and k-fold CV, was used to validate the prediction performance of candidate models. Serogroups 0111 and 0121 consistently showed over 99% classification accuracy regardless of the classification algorithms. However, the classification accuracies of serogroups 026, 045, 0103 and 0145 showed varying results from 84% up to 100%, depending on which preprocessing treatment and prediction model were adopted. The best overall mean classification accuracy of 95.06% was achieved with PCA- kNN ( k=3), six PCs, five-pixel sample size defined around each colony centre, standard normal variate and detrending, first derivative with 11-point gaps and moving average with 11-point gaps. Future studies will focus on automating colony segmentation, further improving detection accuracy of the developed models, expanding the spectral library to include background microflora from ground beef and developing prediction models to detect the target bacteria in the presence of background microflora.

Isolation of Shiga Toxin–Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 from Ground Beef Using Modified Rainbow Agar and Post–Immunomagnetic Separation Acid Treatment

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (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 (1 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.

Detection and Isolation of Low Levels of E. coli O157:H7 in Cilantro by Real‐Time PCR, Immunomagnetic Separation, and Cultural Methods with and without an Acid Treatment

Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real-time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (~0.02 CFU/g) and slightly higher (~0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow(®) agar, R&F(®) E. coli O157 Chromogenic medium, TC-SMAC and CHROMagar™ 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.

The Effect of Regions of Interest and Spectral Pre-Processing on the Detection of Non-0157 Shiga-Toxin Producing Escherichia Coli Serogroups on Agar Media by Hyperspectral Imaging

Foodborne infection caused by Shiga toxin-producing Escherichia coli (STEC) is a major worldwide health concern. The best known and highly virulent STEC serogroup is E. coli 0157:H7, which can be easily identified when cultured on sorbitol-MacConkey (SMAC) agar. Recently, six non-0157 STEC serogroups have been found to cause human illnesses. These non-0157 serogroups ferment sorbital and form pink colonies; therefore SMAC agar cannot be used to differentiate non-0157 serogroups from each other and other flora growing on the plate. This study investigated the potential of visible and near infrared hyperspectral imaging and chemometrics to spectrally differentiate six representative non-0517 STEC serogroups (026, 045, 0103, 0111, 0121 and 0145) grown as spots on Rainbow agar media. Mahalanobis distance classifiers were developed with spectra obtained from ground truth regions of interest (ROIs) of each serogroup colony. The ROIs were selected as a doughnut-like open-ellipse to only include the leading edge of growth and as a closed-ellipse covering the entire colony. For each ROI type, the Mahalanobis distance classifiers were developed with log (1/Reflectance), first derivative and standard normal variate and detrending (SNVD) pre-processing treatments. Serogroups 045 and 0121 were consistently classified over 98% accurate, regard less of the classification algorithm used. The lowest classification accuracies were from classifiers developed with only log (1/ R) ROI spectra. First derivative and SNVD spectra helped to increase the detection accuracies of the other serogroups. The classification accuracy for serogroups 026, 0111, 0103 and 0145 with the closed-ellipse and open-ellipse classification algorithms showed varying results from 8% to 87% and 57% to 100%, respectively. The lower accuracies with closed ellipse spectra were due to greater spectral variation in the centre pixels on a per-pixel basis. Practical implications of this study are the demonstrated potential of hyperspectral imaging for presumptive-positive screening of non-0157 serogroups on Rainbow agar and the extensibility of the developed sampling methods and classification models for future research to identify the target bacteria in the presence of background flora grown on spread plates.

Comparison of Methods for Detection and Isolation of Cold- and Freeze-Stressed Escherichia coli O157:H7 in Raw Ground Beef

A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at ≤0.5 and ≤2 CFU/g, and samples were then enriched immediately or were stored at 4°C for 72 h or at −20°C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42°C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35°C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42°C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35°C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+nat35°C, 3.7 times more likely with an initial inoculum of ≤2.0 CFU/g than with ≤0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.

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

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