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.

Patients with cystic fibrosis (CF) are susceptible to chronic respiratory infections with a number of bacterial pathogens. Among them, the Burkholderia cepacia complex (Bcc) bacteria, consisting of nine related species, have emerged as problematic CF pathogens due to their antibiotic resistance, incidence of nosocomial infection, and person-to-person transmission. Bcc organisms present the clinical microbiologist with a diagnostic dilemma due to the lack of phenotypic biochemical or growth-related characterization tests that reliably distinguish among these organisms. The complex taxonomy of the Bcc species colonizing the CF respiratory tract makes accurate identification problematic. Despite the clinical implications of Bcc identification, a clinical laboratory differentiation of species within the Bcc is lacking. Additionally, no commercial assays are available to further identify the Bcc species. In the current study, secretory proteins present in the cultured supernatants of Burkholderia cenocepacia and Burkholderia multivorans were analyzed by two-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To assess differential expression, protein spots of B. cenocepacia and B. multivorans that were unique or displayed different intensities were chosen for MALDI-TOF MS analysis. In total, 341 protein spots were detected, of which 23 were unique to each species, demonstrating that potential diagnostic candidates between these two members of the Bcc exist.The Burkholderia cepacia complex (Bcc) consists of nine genetically distinct but phenotypically similar Gram-negative bacilli distinguished by their role as important agents of pulmonary disease in cystic fibrosis (CF) patients (12, 16). Associated with increased morbidity and mortality, Bcc bacteria make eradication very difficult due to their ability to establish infection and maintain chronic colonization in the CF lung (6,9,18,22). Furthermore, these organisms are capable of person-to-person spread, presenting the threat of nosocomial acquired infection. Highly variable and unpredictable, clinical outcomes of Bcc infection range from asymptomatic carriage to bacteremic disease and death (12). This variation is speculated to be the result of genomovar heterogeneity and differential expression of a virulence factor(s) during Bcc infection. Although all species of the Bcc have been recovered from CF patients, Burkholderia cenocepacia and Burkholderia multivorans are the most prevalent, accounting for 85% of pulmonary infections (16).Establishing accurate methods of Bcc identification is paramount for the implementation of infection control measures and appropriate treatment of infected and/or exposed CF patients. Furthermore, species identification within the Bcc is vital not only for surgical and clinical management of CF patients but also for the elucidation of the group's epidemiology. Bcc organisms present clinical microbiologists with a diagnostic dilemma due to the l...

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