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1.  Genome Sequence of Escherichia coli O157:H7 Strain 2886-75, Associated with the First Reported Case of Human Infection in the United States 
Genome Announcements  2014;2(1):e01120-13.
First identified in 1982 as a human pathogen, enterohemorrhagic Escherichia coli of the O157:H7 serotype is a major cause of food-borne acquired human infections. Here, we report the genome sequence of the first known strain of this serotype isolated in the United States.
doi:10.1128/genomeA.01120-13
PMCID: PMC3886948  PMID: 24407635
2.  Whole-Genome Draft Sequences of 26 Enterohemorrhagic Escherichia coli O157:H7 Strains 
Genome Announcements  2013;1(2):e00134-12.
First identified in 1982, Escherichia coli O157:H7 is the dominant enterohemorrhagic serotype underlying food-borne human infections in North America. Here, we report the genomes of twenty-six strains derived from patients and the bovine reservoir. These resources enable detailed whole-genome comparisons and permit investigations of genotypic and phenotypic plasticity.
doi:10.1128/genomeA.00134-12
PMCID: PMC3593324  PMID: 23516226
3.  Conducting composite materials from the biopolymer kappa-carrageenan and carbon nanotubes 
Summary
Conducting composite films containing carbon nanotubes (CNTs) were prepared by using the biopolymer kappa-carrageenan (KC) as a dispersant. Rheological studies indicated that 0.5% w/v was the appropriate KC concentration for dispersing CNTs. Our results showed that multiwalled nanotubes (MWNTs) required less sonic energy than single-walled nanotubes (SWNTs) for the dispersion process to be complete. Films prepared by vacuum filtration exhibited higher conductivity and improved mechanical characteristics compared to those prepared by evaporative casting. All composite films displayed sensitivity to water vapour, but MWNT films were more sensitive than SWNT films.
doi:10.3762/bjnano.3.48
PMCID: PMC3388366  PMID: 23016146
biopolymers; carbon nanotubes; carrageenan; composite materials; conductivity; mechanical; rheology
4.  Specificity of PCR and Serological Assays in the Detection of Escherichia coli Shiga Toxin Subtypes ▿  
Applied and Environmental Microbiology  2011;77(18):6699-6702.
Specificity analysis for stx or Stx subtypes in Escherichia coli showed that the PCR assays we tested did not detect stx1d and stx2f, and some also missed stx2b and stx2g. Most of the serological assays examined did not detect Stx2c, Stx2e, Stx2f, and Stx2g, and some strain-to-strain variation in reactivity was observed for Stx2b.
doi:10.1128/AEM.00370-11
PMCID: PMC3187163  PMID: 21803918
5.  Virulence Characterization of Shiga-Toxigenic Escherichia coli Isolates from Wholesale Produce▿  
The 13 Shiga-toxigenic Escherichia coli (STEC) strains isolated from wholesale spinach and lettuce consisted mostly of serotypes that have not been implicated in illness. Among these strains, however, were two O113:H21 that carried virulence genes common to this pathogenic serotype (stx2, ehxA, saa, and subAB), suggesting that their presence in ready-to-eat produce may be of health concern.
doi:10.1128/AEM.01872-10
PMCID: PMC3019700  PMID: 21057025
6.  Characterization of a gne::IS629 O Rough:H7 Escherichia coli Strain from a Hemorrhagic Colitis Patient▿  
Applied and Environmental Microbiology  2010;76(15):5290-5291.
Shiga-toxigenic Escherichia coli strains that are O rough:H7 due to gne::IS629 were thought to be rare and to have unknown pathogenic potential. Recently, an O rough:H7 strain caused by gne::IS629 was isolated from a hemorrhagic colitis patient, suggesting that these strains are pathogenic and may not be as rare as anticipated.
doi:10.1128/AEM.00740-10
PMCID: PMC2916453  PMID: 20543034
7.  Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7 Escherichia coli Strain ▿  
The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6.
doi:10.1128/AEM.02046-09
PMCID: PMC2813021  PMID: 19948859
8.  PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species 
BMC Microbiology  2010;10:90.
Background
The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species.
Results
In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well.
Conclusion
This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses.
doi:10.1186/1471-2180-10-90
PMCID: PMC2856557  PMID: 20331883
9.  Microbiological Quality of Bagged Cut Spinach and Lettuce Mixes▿  
Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log10 CFU/g and a broad range of <4 to 8.3 log10 CFU/g. Most probable numbers (MPN) of ≥11,000 /g coliforms were found in 55 samples, and generic Escherichia coli bacteria were detected in 16 samples, but no E. coli count exceeded 10 MPN/g.
doi:10.1128/AEM.02258-07
PMCID: PMC2258584  PMID: 18156327
10.  Genetic Diversity among Clonal Lineages within Escherichia coli O157:H7 Stepwise Evolutionary Model 
Emerging Infectious Diseases  2007;13(11):1701-1706.
Molecular characterization and subtyping show genetic diversities within clonal complexes.
Escherichia coli O157:H7 variants were examined for trait mutations and by molecular subtyping to better define clonal complexes postulated on the O157:H7 evolution model. Strains of β-glucuronidase–positive, sorbitol-negative O157:H7 isolated in United States and Japan were identical to A5 clonal strain and shared sequence type (ST)–65 by multilocus sequence typing (MLST); thus, they belong in A5. However, these strains exhibited pulsed-field gel electrophoresis (PFGE) profile differences that suggested genomic divergence between populations. Sorbitol-fermenting O157 (SFO157) strains from Finland, Scotland, and Germany were identical to A4 clonal strain and belong in A4. Some SFO157 strains, isolated years apart and from different countries, had identical PFGE profiles, suggesting a common origin. Despite similarities, some Finnish and Scottish and all of the German strains have ST-75 (“German clone”), whereas others have ST-76, a new variant (“Scottish clone”). MLST of strains in other clonal complexes also discriminated strains thought to be identical and showed that genetic differences will further distinguish clonal populations into subclones.
doi:10.3201/eid1311.070381
PMCID: PMC3375798  PMID: 18217554
Enterohemorrhagic E. coli O157:H7; clonal complexes; genetic diversity; molecular evolution; research
11.  Produce Isolates of the Escherichia coli Ont:H52 Serotype That Carry both Shiga Toxin 1 and Stable Toxin Genes 
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.
doi:10.1128/AEM.72.4.3062-3065.2006
PMCID: PMC1448988  PMID: 16598020
12.  Identifying New PCR Targets for Pathogenic Bacteria Using Top-Down LC/MS Protein Discovery 
We have investigated the use of a top-down liquid chromatography/mass spectrometric (LC/MS) approach for the identification of specific protein biomarkers useful for differentiation of closely related strains of bacteria. The sequence information derived from the protein biomarker was then used to develop specific polymerase chain reaction primers useful for rapid identification of the strains. Shiga-toxigenic Escherichia coli (STEC) strains were used for this evaluation. The expressed protein profiles of two closely related serotype 0157:H7 strains, the predominant strain implicated in illness worldwide, and the nonpathogenic E. coli K-12 strain were compared with each other in an attempt to identify new protein markers that could be used to distinguish the 0157:H7 strains from each other and from the E. coli K-12 strain. Sequencing of a single protein unique to one of the 0157:H7 strains identified it as a cytolethal distending toxin, a potential virulence marker. The protein sequence information enabled the derivation of genetic sequence information for this toxin, thus allowing the development of specific polymerase chain reaction primers for its detection. In addition, the top-down LC/MS technique was able to identify other unique biomarkers and differentiate nearly identical 0157:H7 strains, which exhibited identical phenotypic, serologic, and genetic traits. The results of these studies demonstrate that this approach can be expanded to other serotypes of interest and provide a rational approach to identifying new molecular targets for detection.
PMCID: PMC2291713  PMID: 16030320
Biomarkers; mass spectrometry; LC/MS
13.  A 12-Base-Pair Deletion in the Flagellar Master Control Gene flhC Causes Nonmotility of the Pathogenic German Sorbitol-Fermenting Escherichia coli O157:H− Strains 
Journal of Bacteriology  2004;186(8):2319-2327.
An atypical, Stx2-producing, pathogenic Escherichia coli O157:H− strain has been isolated with increasing frequency from hemolytic uremic syndrome patients in Germany. The lack of the H7 antigen coupled with the strain's ability to ferment sorbitol and express β-glucuronidase have complicated its detection and identification. In this study, we have determined that the loss of motility in these German sorbitol-fermenting (SF) O157 strains is due to a 12-bp in-frame deletion in flhC that is required for transcriptional activation of genes involved in flagellum biosynthesis. Either complementation with a functional flhC or repair of this mutation restored H7 antigen expression and motility. PCR analysis of several nonmotile E. coli O157 strains from various geographical sources confirmed that the 12-bp flhC deletion is found only in the cluster of German SF O157 strains, providing a potentially useful marker by which these atypical strains can be identified. The loss of motility via mutations in the flhDC operon that we observed in the German SF O157 strains is consistent with a similar phenomenon currently observed in a significant subset of other important gram-negative pathogens.
doi:10.1128/JB.186.8.2319-2327.2004
PMCID: PMC412172  PMID: 15060034
14.  Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic Escherichia coli Groups 
The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx1 and stx2 was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.
doi:10.1128/AEM.67.7.3110-3114.2001
PMCID: PMC92988  PMID: 11425729
15.  Isogenic Strain of Escherichia coli O157:H7 That Has Lost both Shiga Toxin 1 and 2 Genes 
An Escherichia coli O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit β-glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the +93 gusA mutation, the cluster I ehxA gene for enterohemolysin, and the eae gene for γ-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of XbaI-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stx-producing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both stx genes.
doi:10.1128/CDLI.8.4.711-717.2001
PMCID: PMC96132  PMID: 11427416
16.  Genetic Analysis for Virulence Factors in Escherichia coli O104:H21 That Was Implicated in an Outbreak of Hemorrhagic Colitis 
Isolates of enterohemorrhagic Escherichia coli (EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers. By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (stx2) and to express Stx2. They did not have the eaeA gene for γ-intimin, which is typically found in O157:H7, or the α- or β-intimin derivatives, which are common in other EHEC and enteropathogenic E. coli serotypes. Results of the multiplex PCR also indicated that the ehxA gene for enterohemolysin was absent from O104:H21. This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set of ehxA-specific primers, which indicated the presence of ehxA. To resolve this discrepancy, the ehxA region in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced. Comparison of the sequences showed that the upstream primer binding site in the ehxA gene of O104:H21 was not identical to that of O157:H7. Specifically, there were several base mutations, including an A-to-G substitution at the 3′ end of the primer binding site. These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the ehxA primers used in the multiplex PCR. Comparison of the ehxA sequences of O104:H21 strains with those of other Stx-producing E. coli strains showed that they more closely resembled those of O8:H19 strains, which have cluster II ehxA genes, than those of O157:H7 strains, which have cluster I ehxA sequences. By modifying the upstream ehxA primer, the multiplex PCR was able to detect ehxA genes in both O157:H7 and O104:H21 strains.
doi:10.1128/JCM.39.1.24-28.2001
PMCID: PMC87673  PMID: 11136742
17.  Identification of a Rough Strain of Escherichia coli O157:H7 That Produces No Detectable O157 Antigen 
Journal of Clinical Microbiology  1998;36(8):2339-2341.
MA6, an O157:H7-like strain, did not react with most anti-O157 kits examined; however, it had the rfbE gene that is essential for O157 expression and carried O157:H7 virulence factors. Lipopolysaccharide analysis showed that MA6 is a rough strain that does not produce the O157 antigen, but genetically, it belongs in the O157:H7 clonal group.
PMCID: PMC105044  PMID: 9666018

Results 1-17 (17)