Detection of Live <em>Escherichia coli</em> O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Food and Drug Administration

The nucleotide sequence of the 12.6 kb region between the mutS and rpoS genes of Salmonella enterica serovar Typhimurium LT2 (S. typhimurium) was compared to other enteric bacterial mutS-rpoS intergenic regions. The mutS-rpoS region is composed of three distinct segments, designated HK, O and S, as defined by sequence similarities to contiguous ORFs in other bacteria. Inverted chromosomal orientations of each of these segments are found between the mutS and rpoS genes in related ENTEROBACTERIACEAE: The HK segment is distantly related to a cluster of seven ORFs found in Haemophilus influenzae and a cluster of five ORFs found between the mutS and rpoS genes in Escherichia coli K-12. The O segment is related to the mutS-rpoS intergenic region found in E. coli O157:H7 and Shigella dysenteriae type 1. The third segment, S, is common to diverse Salmonella species, but is absent from E. coli. Despite the extensive collinearity and conservation of the overall genetic maps of S. typhimurium and E. coli K-12, the insertions, deletions and inversions in the mutS-rpoS region provide evidence that this region of the chromosome is an active site for horizontal gene transfer and rearrangement.

Evolution of multi-gene segments in the mutS-rpoS intergenic region of Salmonella enterica serovar Typhimurium LT2.

Deficiencies in the MutS protein disrupt methyl-directed mismatch repair (MMR), generating a mutator phenotype typified by high mutation rates and promiscuous recombination. How such deficiencies might arise in the natural environment was determined by analysing pathogenic strains of Escherichia coli. Quantitative Western immunoblotting showed that the amount of MutS in a wild-type strain of the enterohaemorrhagic pathogen E. coli O157 : H7 decreased about 26-fold in stationary-phase cells as compared with the amount present during exponential-phase growth. The depletion of MutS in O157 : H7 is significantly greater than that observed for a laboratory-attenuated E. coli K-12 strain. In the case of stable mutators, mutS defects in strains identified among natural isolates were analysed, including two E. coli O157 : H7 strains, a diarrhoeagenic E. coli O55 : H7 strain, and a uropathogenic strain from the E. coli reference (ECOR) collection. No MutS could be detected in the four strains by Western immunoblot analyses. RNase T2 protection assays showed that the strains were either deficient in mutS transcripts or produced transcripts truncated at the 3' end. Nucleotide sequence analysis revealed extensive deletions in the mutS region of three strains, ranging from 7.5 to 17.3 kb relative to E. coli K-12 sequence, while the ECOR mutator contained a premature stop codon in addition to other nucleotide changes in the mutS coding sequence. These results provide insights into the status of the mutS gene and its product in pathogenic strains of E. coli.

Molecular analysis of mutS expression and mutation in natural isolates of pathogenic Escherichia coli.

A new inorganic-organic nanocomposite of polyacrylamide (PAM) and gamma-zirconium phosphate (gamma-ZrP) was prepared by intercalative polymerization. Intercalation of acrylamide (AM) monomer in gamma-ZrP was investigated by sonochemical and refluxing methods. High-intensity ultrasound does not induce the polymerization of AM but enhances greatly the intercalation rate. On the other hand, ultrasound also enhanced hydrolyzation of -CONH2 and shrinkage of PAM gel. The interlayer distance of AM-intercalated gamma-ZrP is 16.4 A. The polymerization and intercalation of AM occurred nearly at the same time by refluxing method. The same gamma-ZrP/PAM nanocomposites were obtained with (NH4)2S2O8 treatment.

Preparation of polyacrylamide and gamma-zirconium phosphate nanocomposites by intercalative polymerization.

Phosphoprotein phosphatases encoded by the prpA and prpB genes function in signal transduction pathways for degradation of misfolded proteins in the extracytoplasmic compartments of Escherichia coli. In order to trace the evolution of prp genes and assess their roles in other enteric pathogens, the structure and distribution of these genes among closely related Shigella subgroups were studied. PCR amplification, probe hybridization studies and DNA sequencing were used to determine the prp genotypes of 58 strains from the four Shigella subgroups, Dysenteriae, Boydii, Sonnei and Flexneri. It was found that the prp alleles among Shigella subgroups were extremely susceptible to gene inactivation and that the mutations involved in prp allele inactivation were varied. They included IS insertions, gene replacement by an IS element, a small deletion within the gene or large deletion engulfing the entire gene region, and base substitutions that generated premature termination codons. As a result, of 58 strains studied, only eight (14 %) possessed intact prpA and prpB genes. Of the Shigella strains examined, 76 % (44/58) showed at least one of the prp alleles inactivated by one or more IS elements, including IS1, IS4, IS600 and IS629. Phylogenetic analysis revealed that IS elements have been independently acquired in multiple lineages of Shigella, suggesting that loss of functional alleles has been advantageous during Shigella strain evolution.

Structure and distribution of the phosphoprotein phosphatase genes, prpA and prpB, among Shigella subgroups.

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.

Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker.

Although a variety of methodologies are available for detection of Salmonella, sensitive, specific, and efficient methods are urgently needed for differentiation of live Salmonella cells from dead cells in food and environmental samples. Propidium monoazide (PMA) can preferentially penetrate the compromised membranes of dead cells and inhibit their DNA amplification, however, such inhibition has been reported to be incomplete by some studies. In the present study, we report an efficient qPCR assay targeting a conserved region of the invA gene of Salmonella in conjunction with PMA treatment for detection of DNA from live Salmonella cells in food samples.

Baoguang Li

Center for Food Safety and Applied Nutrition,
Division of Molecular Biology,
Center for Food Safety and Applied Nutrition, Division of Molecular Biology

Food and Drug Administration

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Baoguang Li

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Food Safety and Applied Nutrition,Division of Molecular Biology,Center for Food Safety and Applied Nutrition, Division of Molecular Biology

Food and Drug Administration

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Center for Food Safety and Applied Nutrition,
Division of Molecular Biology,
Center for Food Safety and Applied Nutrition, Division of Molecular Biology