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

ABSTRACT 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. HIGHLIGHTS

Download Full-text

Evaluation of CHROMagar STEC and STEC O104 Chromogenic Agar Media for Detection of Shiga Toxin-Producing Escherichia coli in Stool Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.03121-12 ◽

2013 ◽

Vol 51 (3) ◽

pp. 894-900 ◽

Cited By ~ 30

Author(s):

Malika Gouali ◽

Corinne Ruckly ◽

Isabelle Carle ◽

Monique Lejay-Collin ◽

François-Xavier Weill

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Chromogenic Agar ◽

Agar Media ◽

Stool Specimens

Download Full-text

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†

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-110 ◽

2012 ◽

Vol 75 (9) ◽

pp. 1548-1554 ◽

Cited By ~ 29

Author(s):

GLENN E. TILLMAN ◽

JAMIE L. WASILENKO ◽

MUSTAFA SIMMONS ◽

TODD A. LAUZE ◽

JOSEPH MINICOZZI ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Acid Treatment ◽

Agar Medium ◽

Ground Beef ◽

Immunomagnetic Separation ◽

The United States ◽

Treatment Target ◽

Potassium Tellurite ◽

Treatment Step

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.

Download Full-text

Efficacy of Enrichment Broths in the Recovery of Freeze-Injured Escherichia coli O157:H7 in Inoculated Ground Beef by PCR

Journal of Food Protection ◽

10.4315/0362-028x-66.10.1911 ◽

2003 ◽

Vol 66 (10) ◽

pp. 1911-1915 ◽

Cited By ~ 12

Author(s):

W. C. LIONBERG ◽

L. RESTAINO ◽

E. W. FRAMPTON ◽

W. M. BARBOUR

Keyword(s):

Escherichia Coli ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Automated System ◽

Escherichia Coli O157 ◽

Pcr Assay ◽

E Coli ◽

Cultural Isolation ◽

Peptone Water ◽

Chromogenic Agar

Escherichia coli O157:H7 strains ATCC 35150 and ATCC 43894 and five pooled isolates from beef and pork freeze injured at −25°C in beef infusion were used to inoculate ground beef. Samples (25 g each) were added to 225 ml of buffered peptone water with vancomycin, cefsulodin, and cefixime (BPW-VCC), 225 ml of modified EC broth plus novobiocin (mEC+n), and 225 ml of R&F enrichment broth (R&F-EB) and aerobically incubated at 41 to 42°C. After 6, 7, 8, and 24 h of incubation, levels of E. coli O157:H7 recovered from each broth by a PCR assay with the BAX automated system as well as by conventional enrichment with the use of nonaerated mEC+n incubated at 35°C for 24 h were compared with levels recovered by cultural isolation with immunomagnetic separation and plating on BCM E. coli O157:H7 chromogenic agar. For ground beef inoculated with a mean of 4.23 ± 1.00 total cells (74% freeze injured) per 25 g, after 6 h the PCR assay identified 72.7, 57.6, and 66% of the samples for R&F-EB, BPW-VCC, and mEC+n, respectively, as presumptive positive, whereas the recovery rates after 7 and 8 h exceeded 90%, with the rate for R&F-EB being 100%. For ground beef inoculated with a mean of 1.50 ± 0.56 total cells (80% freeze injured) per 25 g, after 6 h the PCR assay identified 47.6, 19.1, and 9.5% of the samples for R&F-EB, BPW-VCC, and mEC+n, respectively, as presumptive positive. These values increased to 81.0, 61.9, and 52.4% after 7 h and to 95.2, 61.9, and 71.4% after 8 h. After 24 h, only 55 to 60% of the samples at both inoculum levels tested positive by PCR with conventional enrichment and incubation, whereas >95% of the samples tested positive with R&F-EB aerated at 41 to 42°C. Culture results for R&F-EB and mEC+n after 7 and 8 h of incubation were closely correlated with presumptive positive PCR results.

Download Full-text

Recovery Rate of Cells of the Seven Regulated Serogroups of Shiga Toxin-Producing Escherichia coli from Raw Veal Cutlets, Ground Veal, and Ground Beef from Retail Stores in the Mid-Atlantic Region of the United States

Journal of Food Protection ◽

10.4315/jfp-20-290 ◽

2020 ◽

Author(s):

YangJin Jung ◽

Anna C. S. Porto-Fett ◽

Salina Parveen ◽

Joan Meredith ◽

Bradley A Shoyer ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Ground Beef ◽

Immunomagnetic Separation ◽

The United States ◽

Specific Gene ◽

Atlantic Region ◽

Recovery Rates ◽

Related Data ◽

The U.S

A total of 482 veal cutlet, 555 ground veal, and 540 ground beef samples were purchased from retail establishments in the Mid-Atlantic region of the U.S. over a non-contiguous, two-year period between 2014 and 2017. Samples (325 g each) were individually enriched and screened via real-time PCR for all seven regulated serogroups of Shiga toxin-producing Escherichia coli (STEC). Presumptive STEC positive samples were subjected to serogroup-specific immunomagnetic separation and plated onto selective media. Up to five isolates typical for STEC from each sample were analyzed via multiplex PCR for both the virulence genes (i.e., eae , stx 1 and/or stx 2 , and ehxA ) and serogroup-specific gene(s) for the seven regulated STEC serogroups. The recovery rates of non-O157 STEC from veal cutlets (3.94%, 19 of 482 samples) and ground veal (7.03%, 39 of 555 samples) were significantly higher (P < 0.05) than that from ground beef (0.93%, 5 of 540 samples). In contrast, only a single isolate of STEC O157:H7 was recovered; this isolate originated from one (0.18%) of 555 samples of ground veal. Recovery rates for STEC were not associated with state, season, packaging type, or store type (P > 0.05), but were associated with brand and fat content (P < 0.05). Pulsed-field subtyping of the 270 viable/confirmed STEC isolates from the 64 total samples testing positive revealed 78 pulsotypes (50 to 80% similarity) belonging to 39 pulsogroups, with ≥90% similarity among pulsotypes within pulsogroups. Also, multiple isolates from the same sample displayed an indistinguishable pulsotype for 43 of 64 (67.7%) samples testing positive. These findings support related data from regulatory sampling exercises over the past decade and confirm that recovery rates for the regulated STEC serogroups are appreciably higher for raw veal compared to raw beef samples as was also observed herein for meat purchased at food retailers in the Mid-Atlantic region of the U.S.

Download Full-text

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-296 ◽

2017 ◽

Vol 80 (5) ◽

pp. 829-836 ◽

Cited By ~ 6

Author(s):

Pina M. Fratamico ◽

Lori K. Bagi ◽

Aisha Abdul-Wakeel

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Microbiology Laboratory ◽

Department Of Agriculture ◽

E Coli ◽

The U.S

ABSTRACT 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.

Download Full-text

Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

Microorganisms ◽

10.3390/microorganisms8111801 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1801

Author(s):

Michael Bording-Jorgensen ◽

Brendon D. Parsons ◽

Gillian A.M. Tarr ◽

Binal Shah-Gandhi ◽

Colin Lloyd ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Gene Detection ◽

Culture Positivity ◽

Hands On ◽

Culture Independent ◽

Chromogenic Agar ◽

Stool Samples

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

Download Full-text

Detection of Escherichia coli O157:H7 in ground beef using immunomagnetic separation and multiplex PCR

Food Control ◽

10.1016/j.foodcont.2008.06.002 ◽

2009 ◽

Vol 20 (4) ◽

pp. 357-361 ◽

Cited By ~ 34

Author(s):

Belgin Sarimehmetoglu ◽

Mihriban Hatun Aksoy ◽

Naim Deniz Ayaz ◽

Yildiz Ayaz ◽

Ozlem Kuplulu ◽

...

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Escherichia Coli O157

Download Full-text

Determination of the Sensitivity of a RapidEscherichia coli O157:H7 Assay for Testing 375-Gram Composite Samples

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.4149-4151.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 4149-4151 ◽

Cited By ~ 16

Author(s):

Wan-Ling Tsai ◽

Cynthia E. Miller ◽

Edward R. Richter

Keyword(s):

Escherichia Coli ◽

Sample Size ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Enzyme Linked Immunosorbent Assay ◽

Elisa System ◽

Composite Ground ◽

Single Size ◽

Composite Samples

ABSTRACT Both 25-g single-size ground beef samples and 375-g composite ground beef samples were tested by a method combining an immunomagnetic separation (IMS) technique with a sandwich enzyme-linked immunosorbent assay (ELISA) system (IMS-ELISA). The results demonstrated that IMS-ELISA could detect the target, Escherichia coliO157:H7, at the level of 10−1 CFU/g of sample in either the 25- or 375-g sample size.

Download Full-text