Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains

Abstract

Escherichia coli O157:H7 and other pathogenic E. coli strains are enteric pathogens associated with food safety threats and which remain a significant cause of morbidity and mortality worldwide. In the current study, we investigated whether enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC) strains can be rapidly and specifically differentiated with multiplex PCR (mPCR) utilizing selected biomarkers associated with each strain’s respective virulence genotype. Primers were designed to amplify multiple intimin (eae) and long polar fimbriae (lpfA) variants, the bundle-forming pilus gene bfpA, and the Shiga toxin-encoding genes stx1 and stx2. We demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (lpfA1-3, lpfA2-2, stx1, stx2, and eae-γ) and EPEC O127:H6 E2348/69 (eae-α, lpfA1-1, and bfpA) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic E. coli collection revealed that the mPCR assay was successful in predicting the correct pathotype of EPEC and EHEC clones grouped in the distinctive phylogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The assay detection threshold was 2 × 104 CFU per PCR reaction for EHEC and EPEC. mPCR was also used to screen Argentinean clinical samples from hemolytic uremic syndrome and diarrheal patients, resulting in 91% sensitivity and 84% specificity when compared to established molecular diagnostic procedures. In conclusion, our mPCR methodology permitted differentiation of EPEC, STEC and EHEC strains from other pathogenic E. coli; therefore, the assay becomes an additional tool for rapid diagnosis of these organisms.

Detection of Zoonotic Pathogens and Characterization of Novel Viruses Carried by Commensal Rattus norvegicus in New York City

“…In this study, one protozoan and eight bacterial agents commonly associated with mild to severe gastrointestinal disease in humans were identified in rats across multiple sampling sites ( Table 1 ) ( 14 , 15 , 34 ). Widespread prevalence of pathogens, including atypical EPEC and S. moniliformis (which can cause severe vomiting in humans), was observed in rats in NYC, along with sporadic infection with several highly pathogenic bacteria, such as Clostridium difficile and Salmonella enterica .…”

Facile synthesis of a highly SERS active nanosilver sol using microwaves and its application in the detection of E. coli using Victoria blue B as a molecular probe

“…In recent years, SERS has drawn increasing attention due to its capability for trace-level molecule detection and it is extremely attractive for chemical and biological detection. 24 Although these are faster than the other traditional approaches, they are complex procedures and need a well equipped laboratory. 15 Precious metal silver nanoparticles have become the main SERS substrate because of their simple preparation, stability and high SERS activity.…”

Differentiation of Human and Migratory Water Fowl by Multiplex Escherichia coli Differential Amplification Technique (MECDAT) in South Punjab, Pakistan

“…The survival studies in bird's intestinal tract of pathogenic bacteria show that they can survive and can be transferred to humans from food, animals through faecal contact [54]. Data clearly suggest that migratory birds can carry E. coli O157:H7 and 10 disseminates to a large area and distances in short time [54,55]. This all suggests strict monitoring of all possible food contamination to be reinforced [53,56,57].…”