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Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens., Competing Interests: The authors have declared that no competing interests exist.

Escherichia coli sequence type 131 (ST131) is a major cause of urinary and bloodstream infections. Its association with extended-spectrum β-lactamases (ESBLs) significantly complicates treatment. Its best-described component is the rapidly expanding H 30Rx clade, containing allele 30 of the type 1 fimbrial adhesin gene fimH This lineage appears to have emerged in the United States and spread around the world in part due to the acquisition of the ESBL-encoding bla CTX-M-15 gene and resistance to fluoroquinolones. However, non- H 30 ST131 sublineages with other acquired CTX-M-type resistance genes are also emerging. Based on whole-genome analyses, we describe here the presence of an ( fimH ) H 27 E. coli ST131 sublineage that has recently caused an outbreak of community-acquired bacteremia and recurrent urinary tract infections (UTIs) in Denmark. This sublineage has acquired both a virulence plasmid (pAA) that defines the enteroaggregative E. coli (EAEC) diarrheagenic pathotype and multiple genes associated with extraintestinal E. coli (ExPEC); combined, these traits have made this particular ST131 sublineage successful at colonizing its human host and causing recurrent UTI. Moreover, using a historic World Health Organization (WHO) E. coli collection and publicly available genome sequences, we identified a global H 27 EAEC ST131 sublineage that dates back as far as 1998. Most H 27 EAEC ST131 isolates harbor pAA or pAA-like plasmids, and our analysis strongly implies a single ancestral acquisition among these isolates. These findings illustrate both the profound plasticity of this important pathogenic E. coli ST131 H 27 sublineage and genetic acquisitions of EAEC-specific virulence traits that likely confer an enhanced ability to cause intestinal colonization. IMPORTANCE E. coli ST131 is an important extraintestinal pathogenic lineage. A signature characteristic of ST131 is its ability to asymptomatically colonize the gastrointestinal tract and then opportunistically cause extraintestinal infections, such as cystitis, pyelonephritis, and urosepsis. In this study, we identified an ST131 H 27 sublineage that has acquired the enteroaggregative diarrheagenic phenotype, spread across multiple continents, and caused multiple outbreaks of community-acquired ESBL-associated bloodstream infections in Denmark. The strain's ability to both cause diarrhea and innocuously colonize the human gastrointestinal tract may facilitate its dissemination and establishment in the community., (Copyright © 2020 Boll et al.)

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management., Competing Interests: The authors declare that they have no conflict of interest.

Shiga toxin (Stx) is the key virulence factor in Shiga toxin producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with life-threatening complications. Stx comprises two toxin types, Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, which are variable in sequences, toxicity and host specificity. Here, we report the identification of a novel Stx2 subtype, designated Stx2k, in E. coli strains widely detected from diarrheal patients, animals, and raw meats in China over time. Stx2k exhibits varied cytotoxicity in vitro among individual strains. The Stx2k converting prophages displayed considerable heterogeneity in terms of insertion site, genetic content and structure. Whole genome analysis revealed that the stx 2k -containing strains were genetically heterogeneous with diverse serotypes, sequence types, and virulence gene profiles. The nine stx 2k -containing strains formed two major phylogenetic clusters closely with strains belonging to STEC, enterotoxigenic E. coli (ETEC), and STEC/ETEC hybrid. One stx 2k -containing strain harbored one plasmid-encoded heat-stable enterotoxin sta gene and two identical copies of chromosome-encoded stb gene, exhibiting STEC/ETEC hybrid pathotype. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of STEC strains. Given the wide distribution of the Stx2k-producing strains in diverse sources and their pathogenic potential, Stx2k should be taken into account in epidemiological surveillance of STEC infections and clinical diagnosis., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Aims: This systematic review aimed to estimate the effect of tobacco smoking cessation on the risk for periodontitis compared to the risk among never-smokers and to evaluate the effect of tobacco smoking cessation on the clinical outcomes of nonsurgical periodontal treatment., Methods: Electronic searches were performed in PubMed, Scopus, and Embase. Search strategy included MeSH and free terms: periodontitis, periodontal diseases, smoking, tobacco use, tobacco, tobacco products, cigarette, pipe, and cigar. Only original prospective longitudinal observational and interventional studies that investigated the association between smoking cessation and periodontitis onset or progression were included. Meta-analyses were conducted to summarize the evidence., Results: A total of 2743 articles were identified in electronic searches; out of which only six were included in the meta-analysis. Pooled estimates showed that the risk of periodontitis incidence or progression among those who quit smoking was not significantly different from the risk for never-smokers (risk ratio [RR] = 0.97; 95% confidence interval [CI] = 0.87% to 1.08%). Smokers had approximately 80% higher risk of periodontitis than quitters (RR = 1.79; 95% CI = 1.36% to 2.35%) and never-smokers (RR = 1.82; 95% CI = 1.43% to 2.31%). Periodontal therapy resulted in up to 0.2 mm (95% CI = -0.32% to -0.08%) higher gain in attachment level and extra 0.32 mm (95% CI = 0.07% to 0.52%) reduction in pocket depth among quitters over nonquitters after short follow-up (12-24 months)., Conclusions: Few studies on the topic were identified. Smoking cessation reduced the risk for periodontitis onset and progression, and improved the outcomes of nonsurgical periodontal therapy., Implications: This review provides the first quantitative evidence of the impact of smoking cessation on the risk for periodontitis onset and progression. The findings have demonstrated that the risk for periodontitis becomes comparable to that of never-smokers and that nonsurgical periodontal treatment outcomes improve after smoking cessation. Dental professionals ought to consider smoking cessation interventions as a relevant component of the periodontal therapy., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The use of culture methods to detect Escherichia coli diversity does not provide sufficient resolution to identify strains present at low levels. Here, we target the hypervariable gnd gene and describe a database containing 534 distinct partial gnd sequences and associated O groups for use with culture-independent E. coli community analysis., (Copyright © 2019 Cookson et al.)

An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a large outbreak of bloody diarrhea and the hemolytic uremic syndrome in 2011. We previously developed an ampicillin (Amp)-treated C57BL/6 mouse model to measure morbidity (weight loss) and mortality of mice orally infected with the prototype Stx-EAEC strain C227-11. Here, we hypothesized that mice fed C227-11 cured of the pAA plasmid or deleted for individual genes on that plasmid would display reduced virulence compared to animals given the wild-type (wt) strain. C227-11 cured of the pAA plasmid or deleted for the known pAA-encoded virulence genes aggR , aggA , sepA , or aar were fed to Amp-treated C57BL/6 mice at doses of 10 10 -10 11 CFU. Infected animals were then either monitored for morbidity and lethality for 28 days or euthanized to determine intestinal pathology and colonization levels at selected times. The pAA-cured, aggR , and aggA mutants of strain C227-11 all showed reduced colonization at various intestinal sites. However, the aggR mutant was the only mutant attenuated for virulence as it showed both reduced morbidity and mortality. The aar mutant showed increased expression of the aggregative adherence fimbriae (AAF) and caused greater systemic effects in infected mice when compared to the C227-11 wt strain. However, unexpectedly, both the aggA and aar mutants displayed increased weight loss compared to wt. The sepA mutant did not exhibit altered morbidity or mortality in the Amp-treated mouse model compared to wt. Our data suggest that the increased morbidity due to the aar mutant could possibly be via an effect on expression of an as yet unknown virulence-associated factor under AggR control.

Hybrid E. coli pathotypes are representing emerging public health threats with enhanced virulence from different pathotypes. Hybrids of Shiga toxin-producing and enterotoxigenic E. coli (STEC/ETEC) have been reported to be associated with diarrheal disease and hemolytic uremic syndrome (HUS) in humans. Here, we identified and characterized four clinical STEC/ETEC hybrids from diarrheal patients with or without fever or abdominal pain and healthy contact in Sweden. Rare stx2 subtypes were present in STEC/ETEC hybrids. Stx2 production was detectable in stx2a and stx2e containing strains. Different copies of ETEC virulence marker, sta gene, were found in two hybrids. Three sta subtypes, namely, sta1, sta4 and sta5 were designated, with sta4 being predominant. The hybrids represented diverse and rare serotypes (O15:H16, O187:H28, O100:H30, and O136:H12). Genome-wide phylogeny revealed that these hybrids exhibited close relatedness with certain ETEC, STEC/ETEC hybrid and commensal E. coli strains, implying the potential acquisition of Stx-phages or/and ETEC virulence genes in the emergence of STEC/ETEC hybrids. Given the emergence and public health significance of hybrid pathotypes, a broader range of virulence markers should be considered in the E. coli pathotypes diagnostics, and targeted follow up of cases is suggested to better understand the hybrid infection.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens causing severe gastroenteritis, which may lead to hemolytic uremic syndrome. The Locus of Enterocyte Effacement (LEE), a Pathogenicity Island (PAI), is a major determinant of intestinal epithelium attachment of a group of STEC strains; however, the virulence repertoire of STEC strains lacking LEE, has not been fully characterized. The incidence of LEE-negative STEC strains has increased in several countries, highlighting the relevance of their study. In order to gain insights into the basis for the emergence of LEE-negative STEC strains, we performed a large-scale genomic analysis of 367 strains isolated worldwide from humans, animals, food and the environment. We identified uncharacterized genomic islands, including two PAIs and one Integrative Conjugative Element. Additionally, the Locus of Adhesion and Autoaggregation (LAA) was the most prevalent PAI among LEE-negative strains and we found that it contributes to colonization of the mice intestine. Our comprehensive and rigorous comparative genomic and phylogenetic analyses suggest that the accumulative acquisition of PAIs has played an important, but currently unappreciated role, in the evolution of virulence in these strains. This study provides new knowledge on the pathogenicity of LEE-negative STEC strains and identifies molecular markers for their epidemiological surveillance.

The U.S. Food and Drug Administration Escherichia coli Identification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin-producing E. coli (STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. coli strains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxA and eae genes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx 2a , stx 2c , and stx 2d genes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxA and eae and accurately subtyped the eae alleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stx and eae subtyping, and ehxA detection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx 2a , stx 2c , and stx 2d subtypes.

Aim: Even though the association between diabetes and periodontitis is taken for granted, results on this association are conflicting within the literature. This systematic review assessed whether poorly controlled diabetes was associated with periodontitis onset or progression., Methods: Electronic searches were performed in PubMed, Scopus and Embase databases. Hand search was carried out in the reference list of all articles included. Gray literature was investigated with a Google Scholar search. Prospective longitudinal studies on the association between diabetes and periodontitis were considered for this review. Studies should have presented at least two measurements of periodontal conditions over time. Data on study design, crude and adjusted estimates were collected. We used meta-analysis to estimate the pooled effect of hyperglycemia in people with diabetes on periodontitis onset or progression. Meta-regression and subgroup analyses were employed to investigate potential sources of heterogeneity between studies., Results: Thirteen studies matched the inclusion criteria, comprising 49,262 individuals, including 3197 diagnosed with diabetes. Meta-analyses of adjusted estimates showed that diabetes increased the risk of incidence or progression of periodontitis by 86% (RR 1.86 [95% CI 1.3-2.8]). However, there is scarce information on the association between diabetes and periodontal destruction., Conclusions: This study provides evidence that diabetes is associated with increased risk of periodontitis onset and progression in adults. Upcoming prospective longitudinal studies ought to overcome methodological caveats identified in this review.

Sensitive, probe-based detection of multiple DNA targets is limited by the competitive reannealing of the antiparallel duplex DNA helix with the complementary DNA strand. To address this, we developed Cliffhanger primers, which create single-stranded DNA overhangs on PCR amplicons while simultaneously increasing the multiplex PCR efficacy and allowing PCR amplification using crude lysates of human faecal samples. A multiplex PCR that targeted eight genes from diarrhoeagenic Escherichia coli plus an internal control was performed and compared to a routine method that consisted of culture followed by multiplex PCR with fragment length separation. A total of 2515 clinical faecal samples from patients with diarrhoea were tested using both methods, and there was a significant increase in clinical sensitivity and negative predictive value with the Cliffhanger method for seven out of eight genes. All Cliffhanger-only positive samples were confirmed by Sanger sequencing of the PCR amplicon. Notably, the Cliffhanger method reduced the total sample turn-around time in the laboratory from 20 hours to 6 hours. Hence, use of Cliffhanger primers increased assay robustness, decreased turn-around time and increased PCR efficacy. This increased the overall clinical sensitivity without the loss of specificity for a heavily multiplexed PCR assay., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: UVS financial competing interests: Current ownership of stocks, previous paid employment and previous consulting for Anapa Biotech A/S. Inventor of WO 2011/137911 “Method for generating a double stranded nucleic acid with a single stranded overhang” which is fully entrusted to Anapa Biotech A/S. Non-financial competing interests: None. NDM financial competing interests: Previous paid employment for Anapa Biotech A/S. Non-financial competing interests: None. FS financial and non-financial competing interests: None. AF-M financial and non-financial competing interests: None. GL financial competing interests: Previous paid employment and previous consulting for Anapa Biotech A/S. Inventor of WO 2009/112032 “Target amplification and sequencing with primers comprising triplex forming monomer units” and WO 2011/137911, which are fully entrusted to Anapa Biotech A/S. Non-financial competing interests: None. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Context: The study systematically reviewed articles on the association between tobacco smoking and periodontitis, as it has been hypothesized that smoking affects the course of periodontitis through impairment of immunological and vascular mechanisms., Evidence Acquisition: Searches of articles indexed in PubMed, Scopus, and Embase were performed up to and including May 2017. Search strategy included MeSH and free terms: periodontitis, periodontal diseases, smoking, tobacco use, tobacco, tobacco products, cigarette, pipe, and cigar. Only original prospective longitudinal studies that investigated the association between smoking and periodontitis incidence or progression were included. Results were shown as combined risk ratio. Meta-regression and subgroup analyses were used to explore potential sources of heterogeneity. Analyses were conducted in August 2017., Evidence Synthesis: Twenty-eight studies were included in the review; of these, only 14 presented data that could be included in the meta-analysis. Pooled adjusted risk ratios estimate that smoking increases the risk of periodontitis by 85% (risk ratio=1.85, 95% CI=1.5, 2.2). Meta-regression demonstrated that age explained 54.2% of the variability between studies, time of follow-up explained 13.5%, loss to follow-up 10.7%, criteria used to assess the periodontal status explained 2.1%, and severity of periodontitis explained 16.9%., Conclusions: Smoking has a detrimental effect on the incidence and progression of periodontitis. Tobacco smoking, therefore, is important information that should be assessed along with other risk factors for periodontitis., (Copyright © 2018 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights reserved.)

In Escherichia coli , more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli ., (Copyright © 2018 American Society for Microbiology.)

Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized pathogen associated with acute and persistent diarrhea worldwide. While EAEC strains are considered highly heterogeneous, aggregative adherence fimbriae (AAFs) are thought to play a pivotal role in pathogenicity by facilitating adherence to the intestinal mucosa. In this study, we optimized an existing multiplex PCR to target all known AAF variants, which are distinguished by differences in their pilin subunits. We applied the assay on a collection of 162 clinical Danish EAEC strains and interestingly found six, by SNP analysis phylogenetically distinct, strains harboring the major pilin subunits from both AAF/III and AAF/V. Whole-genome and plasmid sequencing revealed that in these six strains the agg3A and agg5A genes were located on a novel pAA plasmid variant. Moreover, the plasmid also encoded several other virulence genes including some not previously found on pAA plasmids. Thus, this plasmid endows the host strains with a remarkably high number of EAEC associated virulence genes hereby likely promoting strain pathogenicity.

Enteroaggregative Escherichia coli (EAEC) has been associated with persistent diarrhea, reduced growth acceleration, and failure to thrive in children living in developing countries and with childhood diarrhea in general in industrialized countries. The clinical implications of an EAEC carrier-status in children in industrialized countries warrants clarification. To investigate the pathological significance of an EAEC carrier-state in the industrialized countries, we designed a 1-year dynamic cohort study and performed follow-up every second month, where the study participants submitted a stool sample and answered a questionnaire regarding gastrointestinal symptoms and exposures. Exposures included foreign travel, consumption of antibiotics, and contact with a diseased animal. In the capital area of Denmark, a total of 179 children aged 0-6 years were followed in a cohort study, in the period between 2009 and 2013. This is the first investigation of the incidence and pathological significance of EAEC in Danish children attending daycare facilities. Conventional microbiological detection of enteric pathogens was performed at Statens Serum Institute, Copenhagen, Denmark, and at Hvidovre Hospital, Copenhagen, Denmark. Parents completed questionnaires regarding gastrointestinal symptoms. The EAEC strains were further characterized by serotyping, phylogenetic analysis, and susceptibility testing. EAEC was detected in 25 (14%) of the children during the observational period of 1 year. One or more gastrointestinal symptoms were reported from 56% of the EAEC-positive children. Diarrhea was reported in six (24%) of the EAEC positive children, but no cases of weight loss, and general failure to thrive were observed. The EAEC strains detected comprised a large number of different serotypes, confirming the genetic heterogeneity of this pathotype. EAEC was highly prevalent (n = 25, 14%) in Danish children in daycare centers and was accompanied by gastrointestinal symptoms in 56% of the infected children. No serotype or phylogenetic group was specifically linked to children with disease.

Objectives: A consensus has existed on not to treat verocytotoxin-producing Escherichia coli (VTEC)-infected individuals with antibiotics because of possible subsequent increased risk of developing haemolytic uraemic syndrome (HUS). The aim of this systematic review is to clarify the risk associated with antibiotic treatment during acute VTEC infection and in chronic VTEC carrier states., Methods: A systematic search in PubMed identified 1 meta-analysis, 10 clinical studies and 22 in vitro/in vivo studies., Results: Four clinical studies found an increased risk of HUS, four studies found no altered risk of HUS and two studies found a protective effect of antibiotics. In vitro and clinical studies suggest that DNA synthesis inhibitors should be avoided, whereas evidence from in vitro studies indicates that certain protein and cell wall synthesis inhibitors reduce the release of toxins from VTEC isolates. Overall, these studies provide a more nuanced view of the diversity of responses by VTEC strains to antibiotics., Conclusions: Based on these data, as well as data from the Danish cohort of registered VTEC infections, we propose that antibiotic treatment with protein and cell wall synthesis inhibitors can be considered when specific criteria regarding patient group, serotype, virulence profile and duration of disease are met., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Accurate and rapid typing of pathogens is essential for effective surveillance and outbreak detection. Conventional serotyping of Escherichia coli is a delicate, laborious, time-consuming, and expensive procedure. With whole-genome sequencing (WGS) becoming cheaper, it has vast potential in routine typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-based in silico serotyping of E. coli applicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). All E. coli isolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific SerotypeFinder CGE tool. SerotypeFinder was evaluated on 682 E. coli genomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen genes wzx, wzy, wzm, and wzt and the flagellin genes fliC, flkA, fllA, flmA, and flnA were detected in 569 and 508 genome sequences, respectively. SerotypeFinder for WGS-based O and H typing predicted 560 of 569 O types and 504 of 508 H types, consistent with conventional serotyping. In combination with other available WGS typing tools, E. coli serotyping can be performed solely from WGS data, providing faster and cheaper typing than current routine procedures and making WGS typing a superior alternative to conventional typing strategies., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The O serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and enhancing phylogenetic studies. In particular, the identification of strains of the same O serogroup is essential in outbreak investigations and surveillance. In a previous study, we analyzed the O-antigen biosynthesis gene cluster in all known E. coli O serogroups (A. Iguchi et al., DNA Res, 22:101-107, 2015, http://dx.doi.org/10.1093/dnares/dsu043). Based on those results, we have arranged 162 PCR primer pairs for the identification or classification of O serogroups. Of these, 147 pairs were used to identify 147 individual O serogroups with unique O-antigen biosynthesis genes, and the other 15 pairs were used to identify 15 groups of strains (Gp1 to Gp15). Each of these groups consisted of strains with identical or very similar O-antigen biosynthesis genes, and the groups represented a total of 35 individual O serogroups. We then used the 162 primer pairs to create 20 multiplex PCR sets. Each set contained six to nine primer pairs that amplify products of markedly different sizes. This genetic methodology (E. coli O-genotyping PCR) allowed for comprehensive, rapid, and low-cost typing. Validation of the PCR system using O-serogroup references and wild strains showed that the correct O serogroups were specifically and accurately identified for 100% (182/182) and 90.8% (522/575) of references and wild strains, respectively. The PCR-based system reported here might be a promising tool for the subtyping of E. coli strains for epidemiological studies as well as for the surveillance of pathogenic E. coli during outbreaks., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In 2011, a Shiga toxin (Stx) type 2a-producing enteroaggregative E. coli (EAEC) strain of serotype O104:H4 caused a large lethal outbreak in Northern Europe. Until recently, the pathogenic mechanisms explaining the high virulence of the strain have remained unclear. Our laboratories have shown that EAEC genes encoded on the pAA virulence plasmid, particularly the AggR-regulated AAF/I fimbriae, enhance inflammation and enable the outbreak strain to both adhere to epithelial cells and translocate Stx2a across the intestinal epithelium, possibly explaining the high incidence of the life threatening post-diarrheal sequelae of hemolytic uremic syndrome. Epidemiologic evidence supports a model of EAEC pathogenesis comprising the concerted action of multiple virulence factors along with induction of inflammation. Here, we suggest a model for the pathogenesis of the O104:H4 outbreak strain that includes contributions from EAEC alone, but incorporating additional injury induced by Stx2a.

Of 23 unique Escherichia coli strains from 10 men with febrile urinary tract infections (UTIs) and their female sex partners, 6 strains (all UTI causing) were shared between partners. Molecularly, the 6 shared strains appeared more virulent than the 17 nonshared strains, being associated with phylogenetic group B2, sequence types ST73 and ST127, and multiple specific virulence genes. This indicates that UTIs are sometimes sexually transmitted., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Escherichia coli is the leading causative agent of acute bovine mastitis. Here, we report the complete genome sequence of E. coli O70:H32 strain 1303, isolated from an acute case of bovine mastitis, and E. coli Ont:Hnt strain ECC-1470, isolated from a persistent infection., (Copyright © 2015 Leimbach et al.)

To identify possible explanations for the recent global emergence of Escherichia coli sequence type (ST) 131 (ST131), we analyzed temporal trends within ST131 O25 for antimicrobial resistance, virulence genes, biofilm formation, and the H30 and H30-Rx subclones. For this, we surveyed the WHO E. coli and Klebsiella Centre's E. coli collection (1957 to 2011) for ST131 isolates, characterized them extensively, and assessed them for temporal trends. Overall, antimicrobial resistance increased temporally in prevalence and extent, due mainly to the recent appearance of the H30 (1997) and H30-Rx (2005) ST131 subclones. In contrast, neither the total virulence gene content nor the prevalence of biofilm production increased temporally, although non-H30 isolates increasingly qualified as extraintestinal pathogenic E. coli (ExPEC). Whereas virotype D occurred from 1968 forward, virotypes A and C occurred only after 2000 and 2002, respectively, in association with the H30 and H30-Rx subclones, which were characterized by multidrug resistance (including extended-spectrum-beta-lactamase [ESBL] production: H30-Rx) and absence of biofilm production. Capsular antigen K100 occurred exclusively among H30-Rx isolates (55% prevalence). Pulsotypes corresponded broadly with subclones and virotypes. Thus, ST131 should be regarded not as a unitary entity but as a group of distinctive subclones, with its increasing antimicrobial resistance having a strong clonal basis, i.e., the emergence of the H30 and H30-Rx ST131 subclones, rather than representing acquisition of resistance by diverse ST131 strains. Distinctive characteristics of the H30-Rx subclone-including specific virulence genes (iutA, afa and dra, kpsII), the K100 capsule, multidrug resistance, and ESBL production-possibly contributed to epidemiologic success, and some (e.g., K100) might serve as vaccine targets., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: E.coli ST131 is a globally disseminated clone of multi-drug resistant E. coli responsible for that vast majority of global extra-intestinal E. coli infections. Recent global genomic epidemiological studies have highlighted the highly clonal nature of this group of bacteria, however there appears to be inconsistency in some phenotypes associated with the clone, in particular capsule types as determined by K-antigen testing both biochemically and by PCR., Results: We performed improved quality assemblies on ten ST131 genomes previously sequenced by our group and compared them to a new reference genome sequence JJ1886 to identify the capsule loci across the drug-resistant clone H30Rx. Our data shows considerable genetic diversity within the capsule locus of H30Rx clone strains which is mirrored by classical K antigen testing. The varying capsule locus types appear to be randomly distributed across the H30Rx phylogeny suggesting multiple recombination events at this locus, but that this capsule heterogeneity has little to no effect on virulence associated phenotypes in vitro., Conclusions: Our data provides a framework for determining the capsular genetics of E. coli ST131 and further beyond to ExPEC strains, and highlights how capsular mosaicism may be an important strategy in becoming a successful globally disseminated human pathogen.

Escherichia coli strains such as Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli, enterotoxigenic, attaching, and effacing E. coli, and enteroinvasive E. coli cause diarrhea in humans. Although other serotypes exist, the most commonly reported STEC in outbreaks is O157:H7. A cross-sectional study was conducted to isolate and characterize non-sorbitol-fermenting (NSF) E. coli O157:H7 from urban and periurban livestock settings of Morogoro, Tanzania. Human stool, cattle feces, and soil and water samples were collected. Observations and questionnaire interview studies were used to gather information about cattle and manure management practices in the study area. E. coli were isolated on sorbitol MacConkey agar and characterized by conventional biochemical tests. Out of 1049 samples, 143 (13.7%) yielded NSF E. coli. Serological and antimicrobial tests and molecular typing were performed to NSF E. coli isolates. These procedures detected 10 (7%) pathogenic E. coli including STEC (n=7), enteropathogenic E. coli (EPEC) (n=2), and attaching and effacing E. coli (A/EEC) (n=1) strains. The STEC strains had the ability to produce VT1 and different VT2 toxin subtypes that caused cytopathic effects on Vero cells. The prevalence of STEC in cattle was 1.6%, out of which 0.9% was serotype O157:H7 and the overall prevalence of diarrheagenic E. coli in cattle was 2.2%. The serotypes O157:H7, O142:H34, O113:H21, O+:H-, O+:H16, and O25:H4 were identified. One ESBL-producing isolate showed the MLST type ST131. To our knowledge, this is the first finding in Tanzania of this recently emerged worldwide pandemic clonal group, causing widespread antimicrobial-resistant infections, and adds knowledge of the geographical distribution of ST131. Cattle manure was indiscriminately deposited within residential areas, and there was direct contact between humans and cattle feces during manure handling. Cattle and manure management practices expose humans, animals, and the environment to pathogenic E. coli and other manure-borne pathogens. Therefore, there is a need to improve manure management practices in urban and periurban areas to prevent pathogen spread and associated human health risks.

To help assess the clinical and public health risks associated with different Shiga toxin-producing Escherichia coli (STEC) strains, an empirical classification scheme was used to classify STEC into five "seropathotypes" (seropathotype A [high risk] to seropathotypes D and E [minimal risk]). This definition is of considerable value in cases of human infection but is also problematic because not all STEC infections are fully characterized and coupled to reliable clinical information. Outbreaks with emerging hybrid strains continuously challenge our understanding of virulence potential and may result in incorrect classification of specific pathotypes; an example is the hybrid strain that caused the 2011 outbreak in Germany, STEC/EAggEC O104:H4, which may deserve an alternative seropathotype designation. The integration of mobile virulence factors in the stepwise and parallel evolution of pathogenic lineages of STEC collides with the requirements of a good taxonomy, which separates elements of each group into subgroups that are mutually exclusive, unambiguous, and, together, include all possibilities. The concept of (sero)-pathotypes is therefore challenged, and the need to identify factors of STEC that absolutely predict the potential to cause human disease is obvious. Because the definition of hemolytic-uremic syndrome (HUS) is distinct, a basic and primary definition of HUS-associated E. coli (HUSEC) for first-line public health action is proposed: stx2 in a background of an eae- or aggR-positive E. coli followed by a second-line subtyping of stx genes that refines the definition of HUSEC to include only stx2a and stx2d. All other STEC strains are considered "low-risk" STEC.

Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.

Enteroaggregative Shiga-toxin-producing Escherichia coli strains were responsible for a massive outbreak in Europe in 2011, and had been previously isolated from French patients. The objective of this study was to investigate the presence of enteroaggregative E. coli (EAEC) in slaughterhouse effluents (wastewater, slurry, sludge and effluents), and in river waters near these slaughterhouses. A total of 10,618 E. coli isolates were screened by PCR for the presence of EAEC-associated genetic markers (aggR, aap and aatA). None of these markers was detected in E. coli isolated from slaughterhouse samples. A unique enteroaggregative E. coli (EAEC) O126:H8 was detected in river water sampled upstream from slaughterhouse effluent discharge. These results confirmed that animals might not be reservoirs of EAEC, and that further studies are required to evaluate the role of the environment in the transmission of EAEC to humans., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The attaching and effacing Escherichia coli (AEEC) are characterized by the presence of a type III secretion system encoded by the locus of enterocyte effacement (LEE). Enterohemorrhagic E. coli (EHEC) are often identified as isolates that are LEE+ and carry the Shiga toxin (stx)-encoding phage, which are labeled Shiga toxin-producing E. coli; whereas enteropathogenic E. coli (EPEC) are LEE+ and often carry the EPEC adherence factor plasmid-encoded bundle-forming pilus (bfp) genes. All other LEE+/bfp-/stx- isolates have been historically designated atypical EPEC. These groups have been defined based on the presence or absence of a limited number of virulence factors, many of which are encoded on mobile elements. This study describes the comparative analysis of the genomes of 114 LEE+ E. coli isolates. Based on a whole-genome phylogeny and analysis of type III secretion system effectors, the AEEC are divided into five distinct genomic lineages. The LEE+/stx+/bfp- genomes were primarily divided into two genomic lineages, the O157/O55 EHEC1 and non-O157 EHEC2. The LEE+/bfp+/stx- AEEC isolates sequenced in this study separated into the EPEC1, EPEC2, and EPEC4 genomic lineages. A multiplex PCR assay for identification of each of these AEEC genomic lineages was developed. Of the 114 AEEC genomes analyzed, 31 LEE+ isolates were not in any of the known AEEC lineages and thus represent unclassified AEEC that in most cases are more similar to other E. coli pathovars than to text modification AEEC. Our findings demonstrate evolutionary relationships among diverse AEEC pathogens and the utility of phylogenomics for lineage-specific identification of AEEC clinical isolates.

We report the draft genome sequences of three enteropathogenic Escherichia coli (EPEC) isolates that display the O157 serogroup but do not have the Shiga toxin genes (stx), which are characteristic of O157 enterohemorrhagic E. coli (EHEC). E. coli strain RN587/1 has the O157:H8 serotype and possesses the EAF plasmid characteristic of typical EPEC (J. B. Kaper, J. P. Nataro, and H. L. Mobley, Nat. Rev. Microbiol. 2:123-140, 2004). The other two isolates, strains C844-97 and C639-08, are both O157:H45 and possess the locus of enterocyte effacement (LEE) pathogenicity island; however, they do not contain the EAF plasmid or the stx-carrying phage.

We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.

Verocytotoxin-producing Escherichia coli (VTEC) strains of serotype O128ab:H2 were isolated from blood and stool of a 27-year-old male presenting diarrhea-associated hemolytic-uremic syndrome complicated by bacteremia. This report once again illustrates the pathogenic potential of a non-O157 VTEC strain carrying a virulence profile previously associated with mild disease.

Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin-producing E. coli (STEC) that do not encode the locus of enterocyte effacement (LEE-negative STEC) often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage-encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx(1) and/or stx(2), as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

In 1991, multiresistant Escherichia coli O78:H10 strains caused an outbreak of urinary tract infections in Copenhagen, Denmark. The phylogenetic origin, clonal background, and virulence characteristics of the outbreak isolates, and their relationship to nonoutbreak O78:H10 strains according to these traits and resistance profiles, are unknown. Accordingly, we extensively characterized 51 archived E. coli O78:H10 isolates (48 human isolates from seven countries, including 19 Copenhagen outbreak isolates, and 1 each of calf, avian, and unknown-source isolates), collected from 1956 through 2000. E. coli O78:H10 was clonally heterogeneous, comprising one dominant clonal group (61% of isolates, including all 19 outbreak isolates) from ST10 (phylogenetic group A) plus several minor clonal groups (phylogenetic groups A and D). All ST10 isolates, versus 25% of non-ST10 isolates, were identified by molecular methods as enteroaggregative E. coli (EAEC) (P < 0.001). Genes present in >90% of outbreak isolates included fimH (type 1 fimbriae; ubiquitous in E. coli); fyuA, traT, and iutA (associated with extraintestinal pathogenic E. coli [ExPEC]); and sat, pic, aatA, aggR, aggA, ORF61, aaiC, aap, and ORF3 (associated with EAEC). An outbreak isolate was lethal in a murine subcutaneous sepsis model and exhibited characteristic EAEC "stacked brick" adherence to cultured epithelial cells. Thus, the 1991 Copenhagen outbreak was caused by a tight, non-animal-associated subset within a broadly disseminated O78:H10 clonal group (ST10; phylogenetic group A), members of which exhibit both ExPEC and EAEC characteristics, whereas O78:H10 isolates overall are phylogenetically diverse. Whether ST10 O78:H10 EAEC strains are both uropathogenic and diarrheagenic warrants further investigation.

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

The incidence of verocytotoxin-producing Escherichia coli (VTEC) was investigated by PCR in all human stools from Universitair Ziekenhuis Brussel (UZB) and in selected stools from six other hospital laboratories in the Brussels-Capital Region, Belgium, collected between April 2008 and October 2010. The stools selected to be included in this study were those from patients with hemolytic-uremic syndrome (HUS), patients with a history of bloody diarrhea, patients linked to clusters of diarrhea, children up to the age of 6 years, and stools containing macroscopic blood. Verocytotoxin genes (vtx) were detected significantly more frequently in stools from patients with the selected conditions (2.04%) than in unselected stools from UZB (1.20%) (P = 0.001). VTEC was detected most frequently in patients with HUS (35.3%), a history of bloody diarrhea (5.15%), or stools containing macroscopic blood (1.85%). Stools from patients up to the age of 17 years were significantly more frequently vtx positive than those from adult patients between the ages of 18 and 65 years (P = 0.022). Although stools from patients older than 65 years were also more frequently positive for vtx than those from patients between 18 and 65 years, this trend was not significant. VTEC was isolated from 140 (67.9%) vtx-positive stools. One sample yielded two different serotypes; thus, 141 isolates could be characterized. Sixty different O:H serotypes harboring 85 different virulence profiles were identified. Serotypes O157:H7/H- (n = 34), O26:H11/H- (n = 21), O63:H6 (n = 8), O111:H8/H- (n = 7), and O146:H21/H- (n = 6) accounted for 53.9% of isolates. All O157 isolates carried vtx2, eae, and a complete O island 122 (COI-122); 15 also carried vtx1. Non-O157 isolates (n = 107), however, accounted for the bulk (75.9%) of isolates. Fifty-nine (55.1%) isolates were positive for vtx1, 36 (33.6%) were positive for vtx2, and 12 (11.2%) carried both vtx1 and vtx2. Pulsed-field gel electrophoresis revealed wide genetic diversity; however, small clusters of O157, O26, and O63:H6 VTEC that could have been part of unidentified outbreaks were identified. Antimicrobial resistance was observed in 63 (44.7%) isolates, and 34 (24.1%) showed multidrug resistance. Our data show that VTEC infections were not limited to patients with HUS or bloody diarrhea. Clinical laboratories should, therefore, screen all stools for O157 and non-O157 VTEC using selective media and a method for detecting verocytotoxins or vtx genes.

Background: Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood., Methods: We characterized 121 EAEC strains isolated in 2008 as part of a case-control study of moderate to severe acute diarrhea among children 0-59 months of age in Bamako, Mali. We applied multiplex polymerase chain reaction and comparative genome hybridization to identify potential virulence factors among the EAEC strains, coupled with classification and regression tree modeling to reveal combinations of factors most strongly associated with illness., Results: The gene encoding the autotransporter protease SepA, originally described in Shigella species, was most strongly associated with diarrhea among the EAEC strains tested (odds ratio, 5.6 [95% confidence interval, 1.92-16.17]; P = .0006). In addition, we identified 3 gene combinations correlated with diarrhea: (1) a clonal group positive for sepA and a putative hemolysin; (2) a group harboring the EAST-1 enterotoxin and the flagellar type H33 but no other previously identified EAEC virulence factor; and (3) a group carrying several of the typical EAEC virulence genes., Conclusion: Our data suggest that only a subset of EAEC strains are pathogenic in Mali and suggest that sepA may serve as a valuable marker for the most virulent isolates.

Specificity analysis for stx or Stx subtypes in Escherichia coli showed that the PCR assays we tested did not detect stx(1d) and stx(2f), and some also missed stx(2b) and stx(2g). 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.

The Escherichia coli strain causing a large outbreak of haemolytic uraemic syndrome and bloody diarrhoea in Germany in May and June 2011 possesses an unusual combination of pathogenic features typical of enteroaggregative E. coli together with the capacity to produce Shiga toxin. Through rapid national and international exchange of information and strains the known occurrence in humans was quickly assessed.We describe simple diagnostic screening tools to detect the outbreak strain in clinical specimens and a novel real-time PCR for its detection in foods.

Shiga toxin-producing Escherichia coli (STEC) O26 is one of the top five enterohemorrhagic E. coli (EHEC) O groups most often associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. STEC O26 is considered to have evolved from enteropathogenic (EPEC) O26 strains through the acquisition of Shiga toxin (Stx)-encoding genes. Our PCR data identified several STEC-like strains expressing all features of STEC except Stx production and carrying remnants of Stx phages that were probably derivatives of EHEC O26. EHEC and EPEC O26 strains phenotypically resemble O26 EHEC-like and apathogenic E. coli O26 strains and are therefore undistinguishable by cultural methods. A clear discrimination between the different O26 groups is required for diagnostics in patients and for control of food safety. To develop an assay for specific detection of EHEC and EHEC-like O26 strains, we used a high-throughput PCR approach for selection of discriminative genetic markers among 33 tested genes mostly encoding type III secretion system effector proteins. The genes ECs1822, nleH1-2, nleA, nleC, nleH1-1, nleG, nleG2, nleG6-1, nleG6-2, espJ, espM2, nleG8-2, espG, ent (or espL2), nleB, nleE, efa1, and espB were detected at different frequencies in O26 EHEC, EHEC-like, and EPEC strains, indicating the possible role of these genes in virulence of human pathogenic O26 strains. The espK and espN genes were detected only in EHEC and EHEC-like O26 strains. espK was present in 99.14% of EHEC and 91.14% of EHEC-like O26 strains and was hence the best candidate as a genetic marker for characterizing these pathogroups. These data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. The results indicate that a combination of molecular detection tools for O26 wzx (wzx(O26)), eae-beta, stx, espK, and arcA genotyping is highly discriminative for clear identification of EHEC and EHEC-like E. coli O26 strains. This simple diagnostic test might be applicable in hospital service laboratories or public health laboratories to test strains isolated from stools of patients suffering from diarrhea.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.

The burden of diseases caused by food-borne pathogens remains largely unknown. Importantly data indicating trends in food-borne infectious intestinal disease is limited to a few industrialised countries, and even fewer pathogens. It has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. Evidence for such a downward trend is limited. This prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. In this review evidence is presented to indicate that the microbiological safety of food remains a dynamic situation heavily influenced by multiple factors along the food chain from farm to fork. Sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. In addition the pathogen populations relevant to food safety are not static. Food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonisation site in a new host. Although food production practices change, the well-recognised food-borne pathogens, such as Salmonella spp. and Escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce, and even generate new public health challenges, for example antimicrobial resistance. In addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. Current understanding of the trends in food-borne diseases for bacterial, viral and parasitic pathogens has been reviewed. The bacterial pathogens are exemplified by those well-recognized by policy makers; i.e. Salmonella, Campylobacter, E. coli and Listeria monocytogenes. Antimicrobial resistance in several bacterial food-borne pathogens (Salmonella, Campylobacter, Shigella and Vibrio spp., methicillin resistant Staphylcoccus aureas, E. coli and Enterococci) has been discussed as a separate topic because of its relative importance to policy issues. Awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on Norovirus, Hepatitis A, rotaviruses and newly emerging viruses such as SARS. Many food-borne parasitic pathogens are known (for example Ascaris, Cryptosporidia and Trichinella) but few of these are effectively monitored in foods, livestock and wildlife and their epidemiology through the food-chain is poorly understood. The lessons learned and future challenges in each topic are debated. It is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. Such collaboration is essential to monitor changing trends in the well-recognised diseases and detect emerging pathogens. It will also be necessary understand the multiple interactions these pathogens have with their environments during transmission along the food chain in order to develop effective prevention and control strategies., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

At least 11 linked outbreaks of gastroenteritis with a total of 260 cases have occurred in Denmark in mid January 2010. Investigations showed that the outbreaks were caused by norovirus of several genotypes and by enterotoxigenic Escherichia coli. Lettuce of the lollo bionda type grown in France was found to be the vehicle.

Nationwide, CTX-M-producing clinical Escherichia coli isolates from the Norwegian ESBL study in 2003 (n=45) were characterized on strain and plasmid levels. Bla(CTX-M) allele typing, characterization of the genetic environment, phylogenetic groups, pulsed field gel electrophoresis (PFGE), serotyping and multilocus sequence typing were performed. Plasmid analysis included S1-nuclease-PFGE, polymerase chain reaction-based replicon typing, plasmid transfer and multidrug resistance profiling. Bla(CTX-M-15) (n=23; 51%) and bla(CTX-M-14) (n=11; 24%) were the major alleles of which 18 (78%) and 6 (55%), respectively, were linked to ISEcp1. Thirty-two isolates were of phylogenetic groups B2 and D. Isolates were of 29 different XbaI-PFGE-types including six regional clusters. Twenty-three different O:H serotypes were found, dominated by O25:H4 (n=9, 20%) and O102:H6 (n=9, 20%). Nineteen different STs were identified, where ST131 (n=9, 20%) and ST964 (n=7, 16%) were dominant. Bla(CTX-M) was found on > or =100 kb plasmids (39/45) of 10 different replicons dominated by IncFII (n=39, 87%), FIB (n=20, 44%) and FIA (n=19, 42%). Thirty-nine isolates (87%) displayed co-resistance to other classes of antibiotics. A transferable CTX-M phenotype was observed in 9/14 isolates. This study reveals that the majority of CTX-M-15-expressing strains in Norway are part of the global spread of multidrug-resistant ST131 and ST-complex 405, associated with ISEcp1 on transferrable IncFII plasmids.

The successful Escherichia coli O15:K52:H1 clonal group provides a case study for the emergence of multiresistant clonal groups of Enterobacteriaceae generally. Accordingly, we tested the hypotheses that, over time, the O15:K52:H1 clonal group has become increasingly (i) virulent and (ii) resistant to antibiotics. One hundred archived international E. coli O15:K52:[H1] clinical isolates from 100 unique patients (1975 to 2006) were characterized for diverse phenotypic and molecular traits. All 100 isolates derived from phylogenetic group D and, presumptively, sequence type ST393. They uniformly carried the F16 papA allele and papG allele II (P fimbria structural subunit and adhesin variants), iha (adhesin-siderophore), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iutA (aerobactin receptor), and kpsM II (group 2 capsule); 85% to 89% of them contained a complete copy of the pap operon and ompT (outer membrane protease). Slight additional virulence profile variation was evident, particularly within a minor diarrhea-associated subset (biotype C). However, in contrast to the clonal group's fairly stable virulence profiles over the past 30+ years, during the same interval the clonal group members' antimicrobial resistance profiles increased by a mean of 2.8 units per decade (P < 0.001). Moreover, the numbers of virulence genes and resistance markers were positively associated (P = 0.046), providing evidence against antimicrobial resistance and virulence being mutually exclusive in these strains. Thus, the O15:K52:H1 clonal group has become increasingly resistant to antimicrobials while maintaining (or expanding) its virulence potential, a particularly concerning trend if other emerging multiresistant enterobacterial clonal groups follow a similar pattern.

Enteroaggregative Escherichia coli (EAEC) pathogenesis is thought to comprise intestinal colonization followed by the release of enterotoxins and cytotoxins. Here, we use a polymerase chain reaction (PCR) to determine the prevalence of 10 genes encoding serine protease autotransporter toxins (SPATEs) in a collection of clinical EAEC isolates. Eighty-six percent of EAEC strains harbored genes encoding one or more class I cytotoxic SPATE proteins (Pet, Sat, EspP, or SigA). Two Class II, non-cytotoxic, SPATE genes were found among EAEC strains: pic and sepA, each originally described in Shigella flexneri 2a. Using a multiplex PCR for five SPATE genes (pet, sat, sigA, pic, and sepA), we found that most of the Shigella isolates also harbored more than one SPATE, whereas members of most other E. coli pathotypes rarely harbored a cytotoxic SPATE gene. SPATEs may be relevant to the pathogenesis of both EAEC and Shigella spp.

Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized TnphoA mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a TnphoA insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the aggR gene. Cloning of the complete Hda gene cluster and expression in E. coli DH5alpha resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.

Background: There is hardly any information regarding oral health status of handicapped primary school pupils in Tanzania. Determination of their oral health status could help in planning sustainable intervention programmes for this disadvantaged group., Objectives: To determine caries and periodontal status and treatment needs of handicapped primary school pupils in Dar es Salaam, Tanzania., Design: A descriptive cross-sectional study., Setting: Uhuru Mchanganyiko and Buguruni special schools, Dar es Salaam., Results: The sample consisted of 179 (55.8%) males and 142 (44.2%) females aged between 7 and 22 years. Majority (71%) were deaf followed by blind (17.8%) and mentally retarded (8.7%). Six (1.9%) pupils were both deaf and blind, while one (0.3%) pupil was blind and mentally retarded. Forty one (12.8%) pupils had at least one decayed deciduous tooth, with the mean (dmfs) ranging from 0.25 to 3.24. The deaf had the highest mean decayed surfaces, followed by the mentally retarded and the blind. There was only one (0.3%) pupil who had a filled deciduous tooth. Thirty three (10.3%) pupils had decayed permanent teeth and 31 (9.7%) had missing permanent teeth. None of the decayed permanent teeth were restored. The blind had the lowest mean deciduous surfaces (DS) scores of between 0 and 1.0. In the mentally retarded group the mean DS ranged from 0.25 to 1.75. About 73.5% of the studied group had bleeding of the gums, with the blind having the highest mean bleeding index scores (p < 0.001) and about 82.8% of the pupils had calculus, with highest mean scores mainly among the blind (p = 0.008)., Conclusion: The caries prevalence among handicapped primary school pupils was quite low. However, there was relatively high level of gingival bleeding and calculus. Regarding treatment needs, 23% required dental fillings mainly of one and two surface restorations and 82% required scaling and polishing. Despite these treatment needs these pupils had not received any dental attention.