scholarly journals Polymorphic Amplified Typing Sequences and Pulsed-Field Gel Electrophoresis Yield Comparable Results in the Strain Typing of a Diverse Set of BovineEscherichia coliO157:H7 Isolates

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Indira T. Kudva ◽  
Margaret A. Davis ◽  
Robert W. Griffin ◽  
Jeonifer Garren ◽  
Megan Murray ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Gel Electrophoresis ◽  
Turnaround Time ◽  
Pulsed Field Gel Electrophoresis ◽  
Strain Typing ◽  
Nucleotide Polymorphisms ◽  
O157 Strain ◽  
Single Nucleotide ◽  
Pulsed Field ◽  
Quick Turnaround Time

Polymorphic amplified typing sequences (PATS), a PCR-basedEscherichia coliO157:H7 (O157) strain typing system, targets insertions-deletions and single nucleotide polymorphisms atXbaI andAvrII restriction enzyme sites, respectively, and the virulence genes (stx1,stx2,eae,hlyA) in the O157 genome. In this study, the ability of PATS to discriminate O157 isolates associated with cattle was evaluated. An in-depth comparison of 25 bovine O157 isolates, from different geographic locations across Northwest United States, showed that about 85% of these isolates shared the same dendogram clade by PATS and pulsed-field gel electrophoresis (PFGE), irrespective of the restriction enzyme sites targeted. The Pearson’s correlation coefficient,r, calculated at about 0.4, 0.3, and 0.4 forXbaI-based,AvrII-based and combined-enzymes PATS and PFGE similarities, respectively, indicating that these profiles shared a good but not high correlation, an expected inference given that the two techniques discriminate differently. Isolates that grouped differently were better matched to their locations using PATS. Overall, PATS discriminated the bovine O157 isolates without interpretive biases or sophisticated analytical software, and effectively complemented while not duplicating PFGE. With its quick turnaround time, PATS has excellent potential as a convenient tool for early epidemiological or food safety investigations, enabling rapid notification/implementation of quarantine measures.

scholarly journals Interpretation of band differences to distinguish strains of Serratia marcescens by pulsed-field gel electrophoresis of XbaI DNA digests

2000 ◽  
Vol 125 (1) ◽  
pp. 63-70 ◽  
Author(s):  
H. M. AUCKEN ◽  
T. BOQUETE ◽  
M. E. KAUFMANN ◽  
T. L. PITT
Keyword(s):  
Serratia Marcescens ◽  
Restriction Enzyme ◽  
Gel Electrophoresis ◽  
Epidemiological Studies ◽  
Pulsed Field Gel Electrophoresis ◽  
Phage Typing ◽  
Dice Coefficient ◽  
Index Profile ◽  
Pulsed Field ◽  
Index Strain

The number of band differences in DNA macrorestriction profiles required to distinguish unrelated strains from an index strain varies in an outbreak with the species and restriction enzyme used. In order to define this difference for epidemiological studies of Serratia marcescens, we produced DNA fingerprints from 57 isolates of the organism using the restriction enzyme XbaI and pulsed-field gel electrophoresis (PFGE). The isolates were selected on the basis of their epidemiology, serotype and phage-typing patterns to include 28 unrelated strains and 29 representatives from 2 distinct outbreaks. One of the outbreaks was prolonged, lasting for several years. Electrophoretic profiles consisting of 20 or more clearly resolved bands were obtained for all isolates. Twenty-six of the unrelated strains had unique profiles with over 10 band differences from all other strains, while 27 of the outbreak representatives could be assigned to the appropriate outbreak with confidence. The majority of the outbreak isolates had none or 2 band differences from the index profile, although 3 isolates differed by 5–7 bands. The 2 exceptions among the unrelated strains differed by 4 bands, and 3 phage typing reactions, and were isolated from London and Berlin 3 years apart, while the 2 exceptions among the outbreak collection had clearly unique profiles with over 20 band differences from each other and the outbreak profiles. Cluster analysis using Dice coefficient and UPGMA gave cut-off values of 75–78% similarity overall for related isolates, while the closest similarity for unrelated strains was 70%. The results of this study together with those of the 6 previous reports of PFGE for S. marcescens (which used either enzymes XbaI or SpeI) confirm that this technique is of value for this species and that with XbaI at least, most epidemiologically related strains will only differ by 3–4 bands. However, on occasion up to 7 band differences can be found within an apparent outbreak, which may be suggestive of genetic drift.

Enrichment, Amplification, and Sequence-Based Typing of Salmonella enterica and Other Foodborne Pathogens

2016 ◽  
Vol 80 (1) ◽  
pp. 15-24 ◽  
Author(s):  
TOM EDLIND ◽  
JEFFREY D. BREWSTER ◽  
GEORGE C. PAOLI
Keyword(s):  
Foodborne Pathogens ◽  
Gel Electrophoresis ◽  
Pure Culture ◽  
Salmonella Enterica ◽  
Cost Effective ◽  
Pulsed Field Gel Electrophoresis ◽  
Alternative Methods ◽  
Strain Typing ◽  
Isolation And Identification ◽  
Pulsed Field

ABSTRACT Detection of Salmonella enterica in foods typically involves microbiological enrichment, molecular-based assay, and subsequent isolation and identification of a pure culture. This is ideally followed by strain typing, which provides information critical to the investigation of outbreaks and the attribution of their sources. Pulsed-field gel electrophoresis is the “gold standard” for S. enterica strain typing, but its limitations have encouraged the search for alternative methods, including whole genome sequencing. Both methods typically require a pure culture, which adds to the cost and turnaround time. A more rapid and cost-effective method with sufficient discriminatory power would benefit food industries, regulatory agencies, and public health laboratories. To address this need, a novel enrichment, amplification, and sequence-based typing (EAST) approach was developed involving (i) overnight enrichment and total DNA preparation, (ii) amplification of polymorphic tandem repeat–containing loci with electrophoretic detection, and (iii) DNA sequencing and bioinformatic analysis to identify related strains. EAST requires 3 days or less and provides a strain resolution that exceeds serotyping and is comparable to pulsed-field gel electrophoresis. Evaluation with spiked ground turkey demonstrated its sensitivity (with a starting inoculum of ≤1 CFU/g) and specificity (with unique or nearly unique alleles relative to databases of >1,000 strains). In tests with unspiked retail chicken parts, 3 of 11 samples yielded S. enterica–specific PCR products. Sequence analysis of three distinct typing targets (SeMT1, SeCRISPR1, and SeCRISPR2) revealed consistent similarities to specific serotype Schwarzengrund, Montevideo, and Typhimurium strains. EAST provides a time-saving and cost-effective approach for detecting and typing foodborne S. enterica, and postenrichment steps can be commercially outsourced to facilitate its implementation. Initial studies with Listeria monocytogenes and Shiga toxigenic Escherichia coli suggest that EAST can be extended to these foodborne pathogens.

scholarly journals Lactobacillus helveticus : Strain Typing and Genome Size Estimation by Pulsed Field Gel Electrophoresis

1997 ◽  
Vol 34 (3) ◽  
pp. 180-185 ◽  
Author(s):  
Sylvie Lortal ◽  
Annette Rouault ◽  
Stéphane Guezenec ◽  
Michel Gautier
Keyword(s):  
Genome Size ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Lactobacillus Helveticus ◽  
Strain Typing ◽  
Size Estimation ◽  
Pulsed Field

scholarly journals Produce Isolates of the Escherichia coli Ont:H52 Serotype That Carry both Shiga Toxin 1 and Stable Toxin Genes

2006 ◽  
Vol 72 (4) ◽  
pp. 3062-3065 ◽  
Author(s):  
Steven R. Monday ◽  
Christina Keys ◽  
Patricia Hanson ◽  
Yuelian Shen ◽  
Thomas S. Whittam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Nucleotide Polymorphism ◽  
Shiga Toxin ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Pulsed Field ◽  
Clonal Group ◽  
Toxin Genes

ABSTRACT Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group.

Emergence of Enterohemorrhagic Escherichia coli Serovar O157 Strains in Clade 8 with Highly Similar Pulsed-Field Gel Electrophoresis Patterns

2011 ◽  
Vol 74 (8) ◽  
pp. 1324-1327 ◽  
Author(s):  
EIJI YOKOYAMA ◽  
YOSHIKI ETOH ◽  
SACHIKO ICHIHARA ◽  
KAZUMI HORIKAWA ◽  
NORIKO KONISHI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Enterohemorrhagic Escherichia Coli ◽  
Common Source ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Pulsed Field ◽  
Genetic Features ◽  
Insertion Sites

Enterohemorrhagic Escherichia coli serovar O157 (O157) strains with highly similar pulsed-field gel electrophoresis (PFGE) patterns were isolated in Japan during 2007 and 2008. Several genetic features related to O157 evolution were investigated to indicate whether homoplasy might have contributed to the highly similar PFGE patterns in these strains. The O157 strains were classified in lineage I/II, as defined by a lineage-specific polymorphism assay-6 with an atypical allele in Z5935 (code: 231111). Analysis of the insertion sites of stx2 phage in these strains showed that the sites were “occupied” in yehV and “intact” in wrbA, indicating that the strains were derived from “Cluster 1” of “Subgroup C.” When a specific single-nucleotide polymorphism in ECs2357 in clade 8 strains was investigated, all of the strains in the present study were confirmed to be clade 8 strains. These results indicated that the O157 strains in this study had common genetic features, suggesting that the highly similar PFGE patterns of these strains were not due to homoplasy. Because no common source of these strains could be identified in 2007 to 2008 in Japan, these strains may have emerged from a unique O157 clade 8 clone and then spread by dissemination in Japan.

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