A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli

Gonzales, Tina K.; Kulow, Megan; Park, Dong-Jin; Kaspar, Charles W.; Anklam, Kelly; Pertzborn, Kelly M.; Kerrish, Kristen D.; Ivanek, Renata; Döpfer, Dörte

doi:10.1016/j.mcp.2011.08.004

Cited by 26 publications

(25 citation statements)

References 49 publications

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“…Prophage polymorphisms are effective targets when combined with an open-array PCR platform for subtyping E. coli O157:H7 (38). Using a similar approach, 10 prophage regions that vary in O157 genomes were used to design a set of 48 prophage gene markers to characterize the polymorphic prophage pattern(s) (PPP) of E. coli O157:H7.…”

Section: Resultsmentioning

confidence: 99%

Evolution of the Stx2-Encoding Prophage in Persistent Bovine Escherichia coli O157:H7 Strains

Park

Stanton

Ciezki

et al. 2013

Appl Environ Microbiol

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e Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx 2 subtypes, and the levels of stx 2 transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx 2a by IS1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx 2c transcription and induction of the prophage and stx 2a ::IS1203v in Stx2a-encoding prophage generated a truncated stx 2a mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx 2a ::IS1203v). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.

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Section: Resultsmentioning

confidence: 99%

Evolution of the Stx2-Encoding Prophage in Persistent Bovine Escherichia coli O157:H7 Strains

Park

Stanton

Ciezki

et al. 2013

Appl Environ Microbiol

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“…Thus, our data confirm that O157 infections more frequently present with HUS, although the large number of non-O157 cases that were complicated by fever and bloody diarrhea and required hospitalization also clearly emphasizes the need for timely identification of non-O157 STEC. Given the higher risk of HUS with O157, the stx-testing algorithm of a laboratory can incorporate testing of stx-positive GN broths either by plating on selective medium or by real-time PCR assay (31), in order to provide next-day notification of treating physicians regarding the presence of this pathogen.…”

Section: Discussionmentioning

confidence: 99%

“…The assay incorporates an independent primer mix for same-day confirmation of stx positivity and allows reporting of preliminary stx positivity to treating clinicians within 24 to 36 h after specimen submission. It also can be used as part of a realtime PCR panel for testing of community-acquired diarrhea that includes other pathogens such as Salmonella, Shigella, and Campylobacter and as part of an algorithm that allows identification of the most common STEC serotypes encountered in a given population (31).…”

Section: Discussionmentioning

confidence: 99%

A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California

et al. 2013

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dRapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) of all serotypes from patients with diarrhea is critical for medical management and for the prevention of ongoing transmission. In this prospective study, we assessed the performance of a multiplex, real-time PCR assay targeting stx 1 and stx 2 for the detection of O157 and non-O157 STEC in diarrheal stool samples enriched in Gram-negative broth. We show that the assay is 100% sensitive (95% confidence interval [CI], 89.1% to 100%) and 98.5% specific (95% CI, 90.6% to 99.9%) based on a panel of 40 known STEC-positive specimens and 65 known negative specimens. During a 2-year postvalidation period, the assay detected more positive samples from patients in northern California than did culture and PCR testing performed at a public health reference laboratory, with a positive predictive value of 95.6% (95% CI, 87.6% to 99.1%). Serotyping data showed an incidence rate of 51.2% for non-O157 STEC strains, with 5.8% of patients (1/17) with non-O157 strains and 42.9% (6/14) with O157 strains (P ‫؍‬ 0.03) developing hemolytic-uremic syndrome. The findings from this study underscore the recommendations of the CDC for laboratories to test all diarrheal stool samples from patients with acute community-acquired diarrhea for non-O157 STEC in addition to the O157 serotype by using a sensitive assay. Additionally, a survey of 17 clinical laboratories in northern California demonstrated that nearly 50% did not screen all stool specimens for the presence of Shiga toxins, indicating that many clinical microbiology laboratories still do not routinely screen all stool specimens for the presence of Shiga toxins as recommended in the 2009 CDC guidelines. Shiga toxin-producing Escherichia coli (STEC) infections are a major cause of bacterial gastroenteritis throughout the world and can be complicated by hemorrhagic colitis and hemolyticuremic syndrome (HUS) (1, 2). Although previously the majority of reported cases were attributed to E. coli O157:H7 and many recent studies indicate higher incidence rates of HUS associated with E. coli O157:H7 (3, 4), non-O157 STEC serotypes are emerging as important etiological agents of both sporadic cases and community outbreaks of diarrhea (3-8). Most notably, in May and June 2011, a large outbreak of E. coli O104:H4-associated diarrheal illness in Germany led to HUS in over 800 patients, many of whom were adults, and ultimately resulted in 54 deaths (5, 9). This outbreak and others have underscored the fact that severe disease and HUS can occur in cases related to non-O157 or O157 E. coli strains and that adults as well as children are susceptible to these complications (8-10). Timely laboratory identification of STEC has important implications for outbreak containment and patient management, including prompt parenteral hydration, monitoring for development of HUS, and avoidance of antibiotics and antidiarrheal agents, which can exacerbate disease (1). Importantly, a number of studies have shown that STECrelated illnesses...

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“…Three strains of E. coli O157:H7 were selected for the experimental inoculation challenges based on their differences in genotypes, origins, and their potential for colonization (45,47). These strains had distinct genotypes as determined by pulsed-field gel electrophoresis (47,48) and a genetic marker profile analysis using realtime PCR (22). Strain FRIK 47 was of human origin and had observed pathogenicity in human (45).…”

Section: Methodsmentioning

confidence: 99%

Differences in Colonization and Shedding Patterns after Oral Challenge of Cattle with Three Escherichia coli O157:H7 Strains

Kulow

Gonzales

Pertzborn

et al. 2012

Appl Environ Microbiol

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Experimental oral challenge studies with three different genotypes of Escherichia coli O157:H7 were conducted in cattle to determine the genotype-specific variability in shedding frequencies and concentrations and the frequency and extent of contamination of the environment. The results indicated that the E. coli O157:H7 genotype and ecological origin maybe important factors for the occurrence and concentration in the cattle host. Four groups of six young Holstein steers each were orally challenged with 10 6 CFU of one of three E. coli O157:H7 strains: FRIK 47 (groups 1 and 2), FRIK 1641 (group 3), and FRIK 2533 (group 4). Recto-anal mucosal swabs (RAMS) and environmental samples were taken on alternate days over 30 days. The numbers of E. coli O157:H7 cells and generic E. coli cells per sample were determined. Also, the presence and absence of 28 gene targets were determined for 2,411 isolates using high-throughput real-time PCR. Over the study period, strains FRIK 47, FRIK 1641, and FRIK 2533 were detected in 52%, 42%, and 2% of RAMS, respectively. Environmental detection of the challenge strains was found mainly in samples of the hides and pen floors, with strains FRIK 47, FRIK 1641, and FRIK 2533 detected in 22%, 27%, and 0% of environmental samples, respectively. Based on the panel of 28 gene targets, genotypes of enterohemorrhagic E. coli (EHEC) and generic E. coli from the experimental samples were clustered into three subgroups. In conclusion, the results suggested that the type and intensity of measures to control this pathogen at the preharvest level may need to be strain specific.

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A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli

Cited by 26 publications

References 49 publications

Evolution of the Stx2-Encoding Prophage in Persistent Bovine Escherichia coli O157:H7 Strains

Evolution of the Stx2-Encoding Prophage in Persistent Bovine Escherichia coli O157:H7 Strains

A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California

Differences in Colonization and Shedding Patterns after Oral Challenge of Cattle with Three Escherichia coli O157:H7 Strains

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