The Role of Long Polar Fimbriae in Escherichia coli O104:H4 Adhesion and Colonization

A renewed interest in Shiga toxin-producing Escherichia coli (STEC) strains was sparked due to the appearance of an outbreak in 2011, causing 3,816 diarrheal cases and some deaths in Europe. The causative strain was classified as enteroaggregative E. coli of serotype O104:H4 that had acquired Shiga toxin genes. The ability of STEC O104:H4 to cause disease relies greatly on the bacteria’s capacity to colonize, persist, and produce Shiga toxin. However, not much is known about the colonization factors of this strain. Because long polar fimbriae (lpf) lpf1 and lpf2 operons encode important colonization factors in other STEC isolates and E. coli O104:H4 possesses both loci, we hypothesized that Lpf is required for adhesion and colonization. In this study, isogenic lpfA1 and lpfA2 major fimbrial subunit mutants were constructed. To determine their role in O104:H4’s virulence, we assessed their ability to adhere to non-polarized and polarized intestinal epithelial cells. The ΔlpfA1 showed decreased adherence in both cell systems, while the ΔlpfA2 only showed a decrease in adherence to polarized Caco-2 cells. We also tested the O104:H4 mutants’ ability to form biofilm and found that the ΔlpfA1 was unable to form a stable biofilm. In an in vivo murine model of intestinal colonization, the ΔlpfA1 had a reduced ability to colonize the cecum and large intestine, consistent with the in vitro data. Further, we tested the lpfA1 mutants’ ability to compete against the wild type. We found that in the in vitro and in vivo models, the presence of the wild type O104:H4 facilitates increased adherence of the ΔlpfA1 to levels exceeding that of the wild type. Overall, our data demonstrated that Lpf1 is one of the factors responsible for O104:H4 intestinal adhesion and colonization.     

Escherichia coli O104 in Feedlot Cattle Feces: Prevalence,Isolation and Characterization

Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzx_O104 gene, then colonies were tested individually to identify wzx_O104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzx_O104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzx_O104 has been shown to be present in E. coli O8/O9/O9a, wzx_O104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdD_O8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzx_O104 and negative for wbdD_O8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar’s test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzx_O104 and samples that were positive for wzx_O104, but negative for wbdD_O8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzx_O104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdD_O8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzx_O104 and negative for wbdD_O8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7.     

An Investigation of the Diversity of Strains of Enteroaggregative Escherichia coli Isolated from Cases Associated with a Large Multi-Pathogen Foodborne Outbreak in the UK

Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.     

Molecular and serotyping characterization of shiga toxogenic Escherichia coli associated with food collected from Saudi Arabia

Shiga toxin-producing Escherichia coli (STEC) strains are considered as one of the major food-borne disease agents in humans worldwide. STEC strains, also called verotoxin-producing E. coli strains. The objectives of the present study were serotyping and molecular characterization of shiga toxigenic E. coli associated with raw meat and milk samples collected from Riyadh, Saudi Arabia. A total of 540 milk samples were collected from 5 dairy farms and 150 raw meat samples were collected from different abattoirs located in Riyadh, Saudi Arabia. E. coli were recovered from 86 milk samples (15.93%), serotyping of E. coli isolates revealed, 26 (4.81%) strains O157: H7, 23 (4.26%) strains O111, 20 (3.70%) strains O113: H21, 10 (1.85%) strains O22: H8 and 7 (1.3%) strains O172: H21. Meanwhile, 17 (11.33%) strains of E. coli were recovered from raw meat samples, serotyping of E. coli isolates revealed, 6 (4%) strains O157: H7, 5 (3.33%) strains O111 and 4 (2.67%) strains O174: H2 and only two (1.33%) strains were identified as O22: H8. Shiga toxin2 was detected in 58 (67.44%) serotypes of E. coli recovered from milk samples and 16 (94.12%) serotypes of E. coli recovered from meat samples, while intimin gene was detected in 38 (44.186%) serotypes of E. coli recovered from milk samples and in 10 (58.82%) serotypes of E. coli recovered from meat samples. The results of this study revealed the efficiency of combination between serotyping and molecular typing of E. coli isolates recovered from food of animal origin for rapid detection and characterization of STEC.     

Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid,and Prophage Heterogeneity,Including Shiga Toxin Encoding Phage stx2

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.     

Presence and Characterization of Shiga Toxin-Producing Escherichia coli and Other Potentially Diarrheagenic E. coli Strains in Retail Meats

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx₁ and stx₂, 2 positive for stx₁, and 10 positive for stx₂. The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx₂ genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.Escherichia coli is an important component of the intestinal microflora of humans and warm-blooded mammals. While E. coli typically harmlessly colonizes the intestinal tract, several E. coli clones have evolved the ability to cause a variety of diseases within the intestinal tract and elsewhere in the host. Those strains that cause enteric infections are generally called diarrheagenic E. coli strains, and their pathogenesis is associated with a number of virulence attributes, which vary according to pathotype (54). Currently, diarrheagenic E. coli strains are classified into six main pathotypes based on their distinct virulence determinants and pathogenic features, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC)/Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusively adherent E. coli (DAEC) (37).Among diarrheagenic E. coli strains, STEC strains are distinguished by the ability to cause severe life-threatening complications, such as hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (30). Other symptoms of STEC infection include watery diarrhea, bloody diarrhea, and hemorrhagic colitis (HC). STEC strains that cause HC and HUS are also called EHEC. Although individuals of all ages are at risk of STEC infection, children younger than 5 years of age and the elderly are more likely to suffer from severe complications (51). Outbreaks and sporadic cases of STEC infections have been reported frequently worldwide.The pathogenesis of STEC infection in humans is not fully understood. The major virulence factors implicated in STEC infection are potent Shiga toxins, which are classified into two groups: Stx1 and Stx2 (23). Additional factors that contribute to virulence have also been described, including intimin (encoded by the eae gene), an outer membrane protein involved in the attachment of E. coli to the enterocyte, and EHEC hemolysin (encoded by EHEC hlyA), which acts as a pore-forming cytolysin and causes damage to cells (41).The first STEC O157 infections were reported in 1982, when E. coli O157:H7 was involved in outbreaks associated with two fast food chain restaurants in the United States (44). Since then, ever-increasing numbers of cases and outbreaks due to STEC O157 have been reported worldwide. Although non-O157 STEC strains have also been associated with human cases and outbreaks, few laboratories have been looking for them, and their potential in causing human infections may be underestimated (2). Recently, though, the significance of non-O157 STEC strains as human pathogens has become more recognized. In the United States alone, there were 23 reported outbreaks of non-O157 STEC infection between 1990 and 2007 (10).Shiga toxin-producing E. coli can be transmitted through different routes, including food and water, person-to-person contact, and animal-to-person contact (9). Most human infections are caused by consumption of contaminated foods (16). Domestic and wild ruminant animals, in particular cattle, are considered the main reservoir of STEC and the main source for contamination of the food supply. Retail meats derived from animals could potentially act as transmission vehicles for STEC and other diarrheagenic E. coli strains. However, there is limited information about STEC contamination in retail meats, and fewer data exist about the presence of other diarrheagenic E. coli strains in retail meats. In the present study, we investigated 7,258 E. coli isolates from four types of meat samples (beef, chicken, pork, and turkey) collected during 2002 to 2007 to assess STEC contamination of retail meats. In addition, the presence of other potentially diarrheagenic E. coli strains was examined by detecting specific virulence determinants among E. coli isolates collected in 2006.     

Isolation and Characteristics of Shiga Toxin 2f-Producing Escherichia coli among Pigeons in Kyushu,Japan

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx _2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx _2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA _O26, irp ₂, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.     

Spread of a Distinct Stx2-Encoding Phage Prototype among Escherichia coli O104:H4 Strains from Outbreaks in Germany, Norway, and Georgia

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.     

Free-Ranging Frigates (Fregata magnificens) of the Southeast Coast of Brazil Harbor Extraintestinal Pathogenic Escherichia coli Resistant to Antimicrobials

Seabirds may be responsible for the spread of pathogenic/resistant organisms over great distances, playing a relevant role within the context of the One World, One Health concept. Diarrheagenic E. coli strains, known as STEC (shiga toxin-producing E. coli), and the extraintestinal pathogenic E. coli (ExPEC and the subpathotype APEC), are among the E. coli pathotypes with zoonotic potential associated with the birds. In order to identify health threats carried by frigates and to evaluate the anthropic influence on the southern coast of Brazil, the aim of this work was to characterize E. coli isolated from free-ranging frigates in relation to virulence genotypes, serotypes, phylogenetic groups and antimicrobial resistance. Cloacal and choanal swabs were sampled from 38 Fregata magnificens from two oceanic islands and one rescue center. Forty-three E. coli strains were recovered from 33 out of the 38 birds (86.8%); 88.4% of strains showed some of the virulence genes (VGs) searched, 48.8% contained three or more VGs. None of the strains presented VGs related to EPEC/STEC. Some of the isolates showed virulence genotypes, phylogenetic groups and serotypes of classical human ExPEC or APEC (O2:H7, O1:H6, ONT:H7, O25:H4). Regarding antimicrobial susceptibility, 62.8% showed resistance, and 11.6% (5/43) were multidrug-resistant. The E. coli present in the intestines of the frigates may reflect the environmental human impact on southeast coast of Brazil; they may also represent an unexplored threat for seabird species, especially considering the overlap of pathogenic potential and antimicrobial resistance present in these strains.     

Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104:H4 outbreak strains

Background

An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates.

Methodology/Principal Findings

Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 ‘positive’ E. coli O104:H4 outbreak and 32 ‘negative’ non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity.

Conclusions/Significance

Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.     

Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains

Background

Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.

Methods

The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.

Results

The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.

Conclusions

This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics of E. coli infections.     

Detection of Hemolysin Variants of Shiga Toxin-Producing Escherichia coli by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar

The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.     

Lability of the pAA Virulence Plasmid in Escherichia coli O104:H4: Implications for Virulence in Humans

Background

Escherichia coli O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. The strain displays “stacked-brick” aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I) encoded on the pAA plasmid. The AAF/I-mediated augmented intestinal adherence might facilitate systemic absorption of Shiga toxin, the major virulence factor of EHEC, presumably enhancing virulence of the outbreak strain. However, the stability of pAA in the outbreak strain is unknown. We therefore tested outbreak isolates for pAA, monitored pAA loss during infection, and determined the impact of pAA loss on adherence and clinical outcome of infection.

Methodology/Principal Findings

E. coli O104:H4 outbreak isolates from 170 patients (128 with hemolytic uremic syndrome [HUS] and 42 with diarrhea without HUS) were tested for pAA using polymerase chain reaction and plasmid profiling. pAA-harboring bacteria in stool samples were quantified using colony blot hybridization, and adherence to HCT-8 cells was determined. Isolates from 12 (7.1%) patients lacked pAA. Analyses of sequential stool samples demonstrated that the percentages of pAA-positive populations in the initial stools were significantly higher than those in the follow-up stools collected two to eight days later in disease (P≤0.01). This indicates a rapid loss of pAA during infections of humans. The pAA loss was associated with loss of the aggregative adherence phenotype and significantly reduced correlation with HUS (P  = 0.001).

Conclusions/Significance

The pAA plasmid can be lost by E. coli O104:H4 outbreak strain in the human gut in the course of disease. pAA loss might attenuate virulence and diminish the ability to cause HUS. The pAA instability has clinical, diagnostic, epidemiologic, and evolutionary implications.     

Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.     

Development of a Robust Method for Isolation of Shiga Toxin-Positive Escherichia coli (STEC) from Fecal,Plant, Soil and Water Samples from a Leafy Greens Production Region in California

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the “Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.     

Diversity of Shiga Toxin-Producing Escherichia coli (STEC) O26:H11 Strains Examined via stx Subtypes and Insertion Sites of Stx and EspK Bacteriophages

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx_1a subtype, while human strains carried mainly stx_1a or stx_2a. The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB. Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx-negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx-positive or stx-negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx-negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.     

Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin‐producing E. coli O157:H7 and O104:H4

Haemolytic uraemic syndrome caused by Shiga toxin‐producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx‐related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx‐related events and enhance Stx absorption during STEC infection.     

Persistent Colonization of Sheep by Escherichia coli O157:H7 and Other E. coli Pathotypes

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10⁷ or 10¹⁰ CFU/strain/animal. The other strains were given only at 10¹⁰ CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10⁷ or 10¹⁰ CFU. One of the ETEC strains also persisted when inoculated at 10¹⁰ CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10⁷ CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.     

Genome sequence analyses of two isolates from the recent <Emphasis Type="Italic">Escherichia coli</Emphasis> outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EAHEC)

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).     

Comparative Genomic Analysis of Escherichia coli O157:H7 Isolated from Super-Shedder and Low-Shedder Cattle

Cattle are the primary reservoir of the foodborne pathogen Escherichia coli O157:H7, with the concentration and frequency of E. coli O157:H7 shedding varying substantially among individual hosts. The term ‘‘super-shedder” has been applied to cattle that shed ≥10⁴ cfu E. coli O157:H7/g of feces. Super-shedders have been reported to be responsible for the majority of E. coli O157:H7 shed into the environment. The objective of this study was to determine if there are phenotypic and/or genotypic differences between E. coli O157:H7 isolates obtained from super-shedder compared to low-shedder cattle. From a total of 784 isolates, four were selected from low-shedder steers and six isolates from super-shedder steers (4.01–8.45 log cfu/g feces) for whole genome sequencing. Isolates were phage and clade typed, screened for substrate utilization, pH sensitivity, virulence gene profiles and Stx bacteriophage insertion (SBI) sites. A range of 89–2473 total single nucleotide polymorphisms (SNPs) were identified when sequenced strains were compared to E. coli O157:H7 strain Sakai. More non-synonymous SNP mutations were observed in low-shedder isolates. Pan-genomic and SNPs comparisons did not identify genetic segregation between super-shedder or low-shedder isolates. All super-shedder isolates and 3 of 4 of low-shedder isolates were typed as phage type 14a, SBI cluster 3 and SNP clade 2. Super-shedder isolates displayed increased utilization of galactitol, thymidine and 3-O-β-D-galactopyranosyl-D-arabinose when compared to low-shedder isolates, but no differences in SNPs were observed in genes encoding for proteins involved in the metabolism of these substrates. While genetic traits specific to super-shedder isolates were not identified in this study, differences in the level of gene expression or genes of unknown function may still contribute to some strains of E. coli O157:H7 reaching high densities within bovine feces.