Comparison of Common Enrichment Broths Used in Diagnostic Laboratories for Shiga Toxin—Producing Escherichia coli

Acute gastroenteritis caused by Shiga toxin-producing Escherichia coli (STEC) affects more than 4 million individuals in Canada. Diagnostic laboratories are shifting towards culture-independent diagnostic testing; however, recovery of STEC remains an important aspect of surveillance programs. The objective of this study was to compare common broth media used for the enrichment of STEC. Clinical isolates including O157:H7 as well as non-O157 serotypes were cultured in tryptic soy (TSB), MacConkey (Mac), and Gram-negative (GN) broths and growth was compared using culture on sheep’s blood agar and real-time PCR (qPCR). In addition, a selection of the same isolates was spiked into negative stool and enriched in the same three broths, which were then evaluated using culture on CHROMagarTM STEC agar and qPCR. TSB was found to provide the optimal enrichment for growth of isolates with and without stool. The results from this study suggest that diagnostic laboratories may benefit from enriching STEC samples in TSB as a first line enrichment instead of GN or Mac.

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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.

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Real-time PCR for differentiation of F18 variants among enterotoxigenic and Shiga toxin-producing Escherichia coli from piglets with diarrhoea and oedema disease

The Veterinary Journal ◽

10.1016/j.tvjl.2013.07.021 ◽

2013 ◽

Vol 198 (2) ◽

pp. 538-540 ◽

Cited By ~ 8

Author(s):

Jae-Won Byun ◽

Byeong Yeal Jung ◽

Ha-Young Kim ◽

John M. Fairbrother ◽

Myoung-Heon Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Oedema Disease

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Comparison of a New Enrichment Procedure for Shiga Toxin–Producing Escherichia coli with Five Standard Methods

Journal of Food Protection ◽

10.4315/0362-028x-68.8.1593 ◽

2005 ◽

Vol 68 (8) ◽

pp. 1593-1599 ◽

Cited By ~ 13

Author(s):

MICHAEL A. GRANT

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Target Cells ◽

Macconkey Agar ◽

Standard Methods ◽

E Coli ◽

Canadian Health ◽

Health Products

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin–producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g−1. Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 × 108 CFU ml−1 and 1.80 × 106 CFU ml−1 after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.

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Immunomagnetic Capture of Big Six Shiga Toxin–Producing Escherichia coli Strains in Apple Juice with Detection by Multiplex Real-Time PCR Eliminates Interference from the Food Matrix

Journal of Food Protection ◽

10.4315/0362-028x.jfp-19-134 ◽

2019 ◽

Vol 82 (9) ◽

pp. 1512-1523

Author(s):

ODBERT A. TRIPLETT ◽

JIEKUN XUAN ◽

STEVEN FOLEY ◽

RAJESH NAYAK ◽

WILLIAM H. TOLLESON

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Internal Control ◽

Shiga Toxin ◽

Real Time Pcr ◽

Apple Juice ◽

Dna Amplification ◽

Probability Of Detection ◽

Solid Agar ◽

Big Six

ABSTRACT Having reliable methods for detecting Shiga toxin–producing Escherichia coli (STEC) in foods is an important food safety goal. The majority of STEC outbreaks have involved either the O157:H7 serotype or one of six non-O157 serogroups, O26, O45, O103, O111, O121, and O145, termed “The Big Six.” We have compared detection by PCR of the Shiga toxin genes stx1a and stx2a from STEC bacteria isolated from unclarified apple juice by simple centrifugation with the use of an immunocapture technique to minimize contaminants (such as pectin and polyphenols that may copurify with DNA) that may interfere with DNA amplification efficiencies and limit sensitivity. An internal control for successful immunocapture, DNA extraction, and PCR amplification was generated by introducing the pmRaspberry plasmid into an stx null strain, yielding an E. coli O45 pmRaspberry derivative that can be added to food samples directly. Using serial dilutions of a representative Big Six STEC in apple juice, our immunocapture method resulted in a 50% probability of detection value of 3.34, 2.25, and 4.25 CFU for detection by multiplex real-time PCR, growth on solid agar, and multiplex endpoint PCR, respectively. The time to result was 6.5 h, 9.5 h, and 1.5 days for immunocapture of Big Six STECs and detection by multiplex real-time PCR, endpoint PCR, and growth on solid agar, respectively. A set of 52 Big Six STEC isolates and 30 non–Big Six STEC strains was used to establish the inclusivity and exclusivity of the method. Finally, the ability to detect Big Six STEC contamination reliably was confirmed at 4.5 and 45 CFU/25-mL portions of refrigerated apple juice.

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P184 Detection of non-O157 and viable but non-culturable shiga toxin-producing Escherichia coli by real-time PCR

International Journal of Antimicrobial Agents ◽

10.1016/s0924-8579(09)70403-0 ◽

2009 ◽

Vol 34 ◽

pp. S86

Author(s):

L. Chui ◽

N. Zelyas ◽

H. Tabor ◽

M. Gilmour

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr

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Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta, Canada

Journal of Clinical Microbiology ◽

10.1128/jcm.03288-14 ◽

2015 ◽

Vol 53 (3) ◽

pp. 1019-1023 ◽

Cited By ~ 15

Author(s):

Linda Chui ◽

Laura Patterson-Fortin ◽

Julie Kuo ◽

Vincent Li ◽

Valerie Boras

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Sensitivity And Specificity ◽

Shiga Toxin ◽

Real Time Pcr ◽

Content Type ◽

Enzyme Immunoassays ◽

Southern Alberta

Two immunoassays (Shiga Toxin Chek and Shiga Toxin Quik Chek) and real-time PCR were used to detect Shiga toxin-producingEscherichia coli. For enriched culture, the sensitivity and specificity of the three methods ranged from 80.0% to 98.2% and 98.0% to 100.0%, respectively. STEC isolates were identified in 2.6% of the 784 samples.

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