Applicability of a Multiplex PCR to Detect the Seven Major Shiga Toxin–Producing<i>Escherichia coli</i>Based on Genes That Code for Serogroup-Specific O-Antigens and Major Virulence Factors in Cattle Feces

Bai, Jianfa; Paddock, Zachary Dean; Shi, Xiaorong; Shubo, Li; An, Baoyan; Nagaraja, T. G.

doi:10.1089/fpd.2011.1082

Cited by 89 publications

(74 citation statements)

References 34 publications

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“…Although both O26 and O103 IMS beads appear to be relatively specific for the respective serogroups, colonies from O111 beads were often positive for the O103 gene (Table 1). This supports previous findings reported by Bai et al (2012). The detection of other STEC serogroups is likely due to nonspecific binding on the beads and subsequent use of a nondifferential medium.…”

Section: Discussionsupporting

confidence: 91%

“…This issue should be further investigated in order to characterize STEC risk associated with cattle production systems. Paddock et al (2012) and Bai et al (2012) concluded that multiplex PCR for STEC serogroups is most applicable for confirming putative isolates. Our results support their findings that culture-based methods using specific IMS beads followed by PCR confirmation of serogroup and virulence genes identifies a greater number of O-positive samples than using PCR directly.…”

Section: Discussionmentioning

confidence: 99%

“…It may be that PCR detected genes carried by bacteria other than the targeted STEC serogroups. Methods for isolation and detection of non-O157 STEC in feces are still under development (Possé et al, 2008;Bai et al, 2012;Hedge et al, 2012;Paddock et al, 2012;Wylie et al, 2013); specific potential needs are to develop a selective medium to phenotypically identify STEC serogroups, improve specificity of IMS beads, validate molecular techniques for faster and more sensitive detection, and standardize protocols to accurately identify STEC.…”

Section: Discussionmentioning

confidence: 99%

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Prevalence of Shiga Toxin–Producing Escherichia coli and Associated Virulence Genes in Feces of Commercial Feedlot Cattle

Cernicchiaro

Cull

Paddock

et al. 2013

Foodborne Pathogens and Disease

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The objective of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) serogroups and associated virulence genes in feces of commercial feedlot cattle. During March to May 2011, fecal samples were collected from individual cattle (n = 960) in 10 cohorts (cattle subpopulations within a feedlot) comprising 17,148 total steers that originated from 48 backgrounding operations in six U.S. states. Fecal samples were enriched in E. coli broth and subjected to two detection protocols: (1) an 11-gene multiplex polymerase chain reaction (PCR) that identifies seven O serogroups (O26, O45, O103, O111, O121, O145, and O157) and four virulence genes (stx1, stx2, eae, and ehxA) applied to extracted total DNA (''direct PCR''); and (2) cultural procedures that involve immunomagnetic separation (IMS) with O26, O103, and O111 beads, plating on a nondifferential MacConkey agar, followed by the multiplex PCR of pooled colonies (''culture-based method''). Generalized linear mixed models were used to adjust prevalence estimates for clustering. Based on direct PCR detection, O157 (49.9%) was the most prevalent O serogroup followed by O26 (20.3%), O103 (11.8%), O121 (10.7%), O45 (10.4%), O145 (2.8%), and O111 (0.8%). Cumulative adjusted prevalence estimates were 22.3, 24.6, and 0.01% for O26, O103, and O111 serogroups, respectively, based on culture-based methods. However, prevalence varied significantly by cohort ( p-values < 0.05) for O26, O121, and O157 based on direct PCR, and for O26, O103, and O111 serogroups based on culture-based methods. Results of this study indicate that all seven STEC serogroups were identified in feedlot cattle feces, with O157, O26, and O103 being the most prevalent serogroups. A substantial proportion of serogroup-positive samples did not harbor Shiga toxin genes; thus, additional elucidation of the potential human health risk is required. Further evaluation of diagnostic methods for non-O157 STEC is needed given their impact on prevalence estimation.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Prevalence of Shiga Toxin–Producing Escherichia coli and Associated Virulence Genes in Feces of Commercial Feedlot Cattle

Cernicchiaro

Cull

Paddock

et al. 2013

Foodborne Pathogens and Disease

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“…Up to 10 target colonies were picked per plate and heated at 958C in 50 ll of water for use as DNA template. Colonies were tested with the 11-plex PCR assay (4,9,53). Colonies positive for one of the EHEC-7 serogroup genes plus stx and eae were confirmed as an EHEC-7 strain, and the sample was counted as positive.…”

Section: Methodsmentioning

confidence: 99%

Prevalence and Level of Enterohemorrhagic Escherichia coli in Culled Dairy Cows at Harvest

Stromberg

Lewis

Aly

et al. 2016

Journal of Food Protection

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The primary objective of this study was to determine the prevalence and level of enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, and O145 (collectively EHEC-6) plus EHEC O157 in fecal, hide, and preintervention carcass surface samples from culled dairy cows. Matched samples (n =300) were collected from 100 cows at harvest and tested by a culture-based method and two molecular methods: NeoSEEK STEC (NS) and Atlas STEC EG2 Combo. Both the culture and NS methods can be used to discriminate among the seven EHEC types (EHEC-7), from which the cumulative prevalence was inferred, whereas the Atlas method can discriminate only between EHEC O157 and non-O157 EHEC, without discrimination of the serogroup. The EHEC-7 prevalence in feces, hides, and carcass surfaces was 6.5, 15.6, and 1.0%, respectively, with the culture method and 25.9, 64.9, and 7.0%, respectively, with the NS method. With the Atlas method, the prevalence of non-O157 EHEC was 29.1, 38.3, and 28.0% and that of EHEC O157 was 29.1, 57.0, and 3.0% for feces, hides, and carcasses, respectively. Only two samples (a hide sample and a fecal sample) originating from different cows contained quantifiable EHEC. In both samples, the isolates were identified as EHEC O157, with 4.7 CFU/1,000 cm2 in the hide sample and 3.9 log CFU/g in the fecal sample. Moderate agreement was found between culture and NS results for detection of EHEC O26 (κ =0.58, P < 0.001), EHEC O121 (κ = 0.50, P < 0.001), and EHEC O157 (κ = 0.40, P < 0.001). No significant agreement was observed between NS and Atlas results or between culture and Atlas results. Detection of an EHEC serogroup in fecal samples was significantly associated with detection of the same EHEC serogroup in hide samples for EHEC O26 (P =0.001), EHEC O111 (P =0.002), EHEC O121 (P < 0.001), and EHEC-6 (P = 0.029) based on NS detection and for EHEC O121 (P < 0.001) based on detection by culture. This study provides evidence that non-O157 EHEC are ubiquitous on hides of culled dairy cattle and that feces are an important source of non-O157 EHEC hide contamination.

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“…EIA or PCR methods to detect the toxin itself or the cognate genes are available (13). However, this provides little epidemiologic information, and neither does detection of other virulence factors such as the intimin gene, eae, or the hemolysin gene, ehxA (14)(15)(16). The current method employed by the USDA for food safety uses a multiplex PCR for stx 1 , stx 2 , and eae detection before using other genetic markers, such as variations in the wzx gene, to screen for O26, O45, O103, O111, O121, or O145 (17).…”

mentioning

confidence: 99%

Hemolytic Uremic Syndrome following Infection with O111 Shiga Toxin-Producing Escherichia coli Revealed through Molecular Diagnostics

2014

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We report a case of hemolytic uremic syndrome in a 69-year-old woman due to Shiga toxin-producing Escherichia coli, possibly serotype O111, to illustrate the potentially deleterious implications of a Campylobacter enzyme immunoassay (EIA) result and the increasing importance of molecular testing when conventional methods are limited. CASE REPORTA 69-year-old female presented to our hospital in July 2013 with a two-day history of abdominal cramping and hematochezia after a camping trip. During the trip the patient reported consuming fresh produce and store-bought bottled water but no meat, poultry, or eggs. Reportedly, none of the other 40 people on the camping trip were ill. Examination revealed an afebrile and tachycardic (pulse rate 118 beats/minute) woman with a diffusely tender abdomen on deep palpation. Initial stool studies were positive for lactoferrin and Campylobacter by enzyme immunoassay (EIA) (Meridian Biosciences Inc., Cincinnati, OH) and negative for Clostridium difficile PCR and Giardia antigen while Shiga toxin testing (EIA; Meridian Biosciences) was pending. Azithromycin was empirically initiated for presumed infectious colitis due to Campylobacter spp. The next day, the Shiga toxin test returned positive and the patient developed worsening bloody diarrhea and leukocytosis (white cell count 24 ϫ 103/mm 3 ) and lactic acidosis. An abdominal-pelvic computed tomography (CT) scan revealed severe bowel wall thickening throughout the length of the colon. She was transferred to the intensive care unit for further monitoring and management. Ciprofloxacin and metronidazole were added for severe colitis. Thrombocytopenia developed (platelet count 68 ϫ 10 3 /mm 3 ), after which all antimicrobial therapy was discontinued as hemolytic uremic syndrome (HUS) was suspected. The patient went on to develop hemolytic anemia with schistocytosis, acute renal failure, and altered mental status changes. The patient was supported with plasmapharesis and hemodialysis therapy. An additional stool sample was obtained 2 days after admission which was Campylobacter EIA negative and also Shiga toxin negative in broth culture. The initial broth was sent to the Virginia Division of Consolidated Laboratory Services (DCLS) and resulted in no culturable bacteria but was PCR positive for stx 1 and negative for stx 2 and uidA genes (Table 1). We developed and utilized two multiplex real-time PCRs targeting Shiga toxin-producing Escherichia coli (STEC)-associated genes: one panel for eae, stx 1 , stx 2 , and rfbE O157 and a second panel for wzx or clustered regularly interspaced short palindromic repeat (CRISPR) genes of relevant non-O157 serotypes. These assays confirmed the presence of stx 1 and detected both the wzx gene of O111 and eae (Table 1). All multiplex real-time results were confirmed in singleplex. The wzx amplicon was sequenced and revealed a perfect match with 50 available O111-specific wzx sequences within NCBI (representing both STEC and non-STEC sources). Molecular testing results for Campylobacter spp. (16S) and C...

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Applicability of a Multiplex PCR to Detect the Seven Major Shiga Toxin–ProducingEscherichia coliBased on Genes That Code for Serogroup-Specific O-Antigens and Major Virulence Factors in Cattle Feces

Cited by 89 publications

References 34 publications

Prevalence of Shiga Toxin–Producing Escherichia coli and Associated Virulence Genes in Feces of Commercial Feedlot Cattle

Prevalence of Shiga Toxin–Producing Escherichia coli and Associated Virulence Genes in Feces of Commercial Feedlot Cattle

Prevalence and Level of Enterohemorrhagic Escherichia coli in Culled Dairy Cows at Harvest

Hemolytic Uremic Syndrome following Infection with O111 Shiga Toxin-Producing Escherichia coli Revealed through Molecular Diagnostics

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