Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104:H4

Escherichia coli biotype O104:H4 recently caused the deadliest E. coli outbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impact in vivo colonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as ‘Generally Recognized as Safe’ (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks.     

Shiga toxin-producing Escherichia coli O104:H4: a new challenge for microbiology

In 2011, Germany experienced the largest outbreak with a Shiga toxin-producing Escherichia coli (STEC) strain ever recorded. A series of environmental and trace-back and trace-forward investigations linked sprout consumption with the disease, but fecal-oral transmission was also documented. The genome sequences of the pathogen revealed a clonal outbreak with enteroaggregative E. coli (EAEC). Some EAEC virulence factors are carried on the virulence plasmid pAA. From an unknown source, the epidemic strains acquired a lambdoid prophage carrying the gene for the Shiga toxin. The resulting strains therefore possess two different mobile elements, a phage and a plasmid, contributing essential virulence genes. Shiga toxin is released by decaying bacteria in the gut, migrates through the intestinal barrier, and is transported via the blood to target organs, like the kidney. In a mouse model, probiotic bifidobacteria interfered with transport of the toxin through the gut mucosa. Researchers explored bacteriophages, bacteriocins, and low-molecular-weight inhibitors against STEC. Randomized controlled clinical trials of enterohemorrhagic E. coli (EHEC)-associated hemolytic uremic syndrome (HUS) patients found none of the interventions superior to supportive therapy alone. Antibodies against one subtype of Shiga toxin protected pigs against fatal neurological infection, while treatment with a toxin receptor decoy showed no effect in a clinical trial. Likewise, a monoclonal antibody directed against a complement protein led to mixed results. Plasma exchange and IgG immunoadsoprtion ameliorated the condition in small uncontrolled trials. The epidemic O104:H4 strains were resistant to all penicillins and cephalosporins but susceptible to carbapenems, which were recommended for treatment.     

Role of Type 1 Fimbriae in the Adhesion of Escherichia coli to Salivary Mucin and Secretory Immunoglobulin A

Saliva is known to modulate the adhesion of bacteria in the oral cavity. The present work was performed to assess the effect of salivary components on the adhesion of Escherichia coli to a model oral surface. Several genetically engineered E. coli strains were used to examine the role of type 1 fimbriation in the interaction of these strains with salivary components in solution or adsorbed to hydroxyapatite. High (MG1) and low (MG2) molecular weight salivary mucins, and secretory immunoglobulin A (sIgA), were found to interact with the surface of E. coli, and these interactions were independent of the expression of fimbriae or capsule. In contrast, fimbriated strains of E. coli adhered to a greater extent to saliva-coated synthetic hydroxyapatite (HAP) than did nonfimbriated strains. Testing of salivary components separated by gel filtration chromatography revealed that only high-molecular-weight components promoted adhesion of E. coli to HAP. Additional studies found that purified MG2 and sIgA promoted the adhesion of E. coli to HAP. Expression of type 1 fimbriae enhanced adhesion, while mannose inhibited adhesion of fimbriated strains, to saliva-coated HAP and to HAP coated with MG2 and sIgA. We conclude that salivary MG2 and sIgA may provide receptors for the adhesion of type 1 fimbriated E. coli to oral surfaces. Received: 10 February 1996 / Accepted: 11 March 1996     

Correlation between In Vivo Biofilm Formation and Virulence Gene Expression in Escherichia coli O104:H4

The emergence of novel pathogens poses a major public health threat causing widespread epidemics in susceptible populations. The Escherichia coli O104:H4 strain implicated in a 2011 outbreak in northern Germany caused the highest frequency of hemolytic uremic syndrome (HUS) and death ever recorded in a single E. coli outbreak. Therefore, it has been suggested that this strain is more virulent than other pathogenic E. coli (e.g., E. coli O157:H7). The E. coli O104:H4 outbreak strain possesses multiple virulence factors from both Shiga toxin (Stx)-producing E. coli (STEC) and enteroaggregative E. coli (EAEC), though the mechanism of pathogenesis is not known. Here, we demonstrate that E. coli O104:H4 produces a stable biofilm in vitro and that in vivo virulence gene expression is highest when E. coli O104:H4 overexpresses genes required for aggregation and exopolysaccharide production, a characteristic of bacterial cells residing within an established biofilm. Interrupting exopolysaccharide production and biofilm formation may therefore represent effective strategies for combating future E. coli O104:H4 infections.     

Presence of enterohemorrhagic Escherichia coli ST678/O104:H4 in France prior to 2011

Two isolates of enterohemorrhagic Escherichia coli (EHEC) O104:H4 were isolated in France in 2004 and 2009. Both were characterized and compared to the strain which caused the German outbreak in 2011 and to other O104:H4 strains. This suggests that different O104:H4 EHEC strains were present several years prior to the 2011 outbreak.     

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.     

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.     

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.     

Long polar fimbriae and tissue tropism in Escherichia coli O157:H7

In vitro organ culture has demonstrated the human intestinal tropism of enterohaemorrhagic Escherichia coli O157:H7 for follicle associated epithelium overlying Peyer's patches of the terminal ileum. Long polar (LP) fimbriae are considered to mediate the attachment of Salmonella enterica serovar Typhimurium to Peyer's patch epithelium and, as homologous genes have been identified in O157:H7, we hypothesised that LP fimbriae in O157:H7 may perform the same function. However, mutation of LP fimbriae in O157:H7 strain 85/170 resulted in the novel phenotype of proximal and distal small intestinal colonisation with attaching/effacing lesion formation, while retaining adhesion to follicle associated epithelium. Application of whole genome DNA array technology did not identify changes in known fimbrial genes that could explain the change in tropism, but highlighted several genes that require further investigation. LP fimbrial genes are the first genes to be identified outside the locus of enterocyte effacement pathogenicity island that influence O157:H7 human intestinal tissue tropism.     

Rapid genomic-scale analysis of Escherichia coli O104:H4 by using high-resolution alternative methods to next-generation sequencing

Two technologies, involving DNA microarray and optical mapping, were used to quickly assess gene content and genomic architecture of recent emergent Escherichia coli O104:H4 and related strains. In real-time outbreak investigations, these technologies can provide congruent perspectives on strain, serotype, and pathotype relationships. Our data demonstrated clear discrimination between clinically, temporally, and geographically distinct O104:H4 isolates and rapid characterization of strain differences.     

Adhesion and colonization of enterohemorrhagic Escherichia coli O157:H7 in cecum of mice

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157:H7 adhere to intestinal epithelial cells of mice and cause F-actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system-related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild-type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F-actin accumulation beneath the attached bacteria.     

F1C Fimbriae Play an Important Role in Biofilm Formation and Intestinal Colonization by the Escherichia coli Commensal Strain Nissle 1917

Bacterial biofilm formation is thought to enhance survival in natural environments and during interaction with hosts. A robust colonizer of the human gastrointestinal tract, Escherichia coli Nissle 1917, is widely employed in probiotic therapy. In this study, we performed a genetic screen to identify genes that are involved in Nissle biofilm formation. We found that F1C fimbriae are required for biofilm formation on an inert surface. In addition, these structures are also important for adherence to epithelial cells and persistence in infant mouse colonization. The data suggest a possible connection between Nissle biofilm formation and the survival of this commensal within the host. Further study of the requirements for robust biofilm formation may improve the therapeutic efficacy of Nissle 1917.     

Characteristics of the Shiga-toxin-producing enteroaggregative Escherichia coli O104:H4 German outbreak strain and of STEC strains isolated in Spain

A Shiga-toxin-producing Escherichia coli (STEC) strain belonging to serotype O104:H4, phylogenetic group B1 and sequence type ST678, with virulence features common to the enteroaggregative E. coli (EAEC) pathotype, was reported as the cause of the recent 2011 outbreak in Germany. The outbreak strain was determined to carry several virulence factors of extraintestinal pathogenic E. coli (ExPEC) and to be resistant to a wide range of antibiotics. There are only a few reports of serotype O104:H4, which is very rare in humans and has never been detected in animals or food. Several research groups obtained the complete genome sequence of isolates of the German outbreak strain as well as the genome sequences of EAEC of serotype O104:H4 strains from Africa. Those findings suggested that horizontal genetic transfer allowed the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli (STEAEC) O104:H4 strain responsible for the outbreak in Germany. Epidemiologic investigations supported a linkage between the outbreaks in Germany and France and traced their origin to fenugreek seeds imported from Africa. However, there has been no isolation of the causative strain O104:H4 from any of the samples of fenugreek seeds analyzed. Following the German outbreak, we conducted a large sampling to analyze the presence of STEC, EAEC, and other types of diarrheagenic E. coli strains in Spanish vegetables. During June and July 2011, 200 vegetable samples from different origins were analyzed. All were negative for the virulent serotype O104:H4 and only one lettuce sample (0.6%) was positive for a STEC strain of serotype O146:H21 (stx1, stx2), considered of low virulence. Despite the single positive case, the hygienic and sanitary quality of Spanish vegetables proved to be quite good. In 195 of the 200 samples (98%), <10 colony-forming units (cfu) of E. coli per gram were detected, and the microbiological levels of all samples were satisfactory (<100 cfu/g). The samples were also negative for other pathotypes of diarrheagenic E. coli (EAEC, ETEC, tEPEC, and EIEC). Consistent with data from other countries, STEC belonging to serotype O157:H7 and other serotypes have been isolated from beef, milk, cheese, and domestic (cattle, sheep, goats) and wild (deer, boar, fox) animals in Spain. Nevertheless, STEC outbreaks in Spain are rare.     

Rectal Administration of Escherichia coli O157:H7: Novel Model for Colonization of Ruminants

Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening complications. Because healthy cattle are reservoirs for the bacterium, ruminant infection models have applications in analyzing the relationship between cattle and this human pathogen and in testing interventions to reduce or prevent bovine colonization with this bacterium. Current approaches often do not reliably mimic natural, long-term bovine colonization with E. coli O157:H7 in older calves and adult animals (ages that enter our food chain). Based on the recent identification of the bovine rectoanal junction mucosa as a site of E. coli O157:H7 colonization, we developed a novel rectal swab administration colonization model. We compared this method with oral dosing and direct contact transmission (Trojan) methods. E. coli O157:H7 carriage status was determined by fecal or rectoanal mucosa swab culture. High (~10¹⁰ CFU) and low (~10⁷ CFU) oral doses of E. coli O157:H7 in sheep and cattle resulted in variable infection with the bacterium. Some animals became colonized with the bacteria and remained culture positive for several weeks, and some animals did not become colonized and rapidly cleared the bacteria in a few days. Pen mates of E. coli O157:H7 culture-positive Trojan cattle had a low infection rate and variable colonization status. However, rectal swab administration of E. coli O157:H7 to cattle resulted in consistent long-term colonization in all animals. The surprising ease with which long-term infections resulted from a single application of bacteria to the rectoanal mucosa also strongly supported this location as a site of E. coli O157:H7 colonization in cattle.     

Comparative Genomic Analysis of Two Novel Sporadic Shiga Toxin-Producing Escherichia coli O104:H4 Strains Isolated 2011 in Germany

A large outbreak of gastrointestinal disease occurred in 2011 in Germany which resulted in almost 4000 patients with acute gastroenteritis or hemorrhagic colitis, 855 cases of a hemolytic uremic syndrome and 53 deaths. The pathogen was an uncommon, multiresistant Escherichia coli strain of serotype O104:H4 which expressed a Shiga toxin characteristic of enterohemorrhagic E. coli and in addition virulence factors common to enteroaggregative E. coli. During post-epidemic surveillance of Shiga toxin-producing E. coli (STEC) all but two of O104:H4 isolates were indistinguishable from the epidemic strain. Here we describe two novel STEC O104:H4 strains isolated in close spatiotemporal proximity to the outbreak which show a virulence gene panel, a Shiga toxin-mediated cytotoxicity towards Vero cells and aggregative adherence to Hep-2 cells comparable to the outbreak strain. They differ however both from the epidemic strain and from each other, by their antibiotic resistance phenotypes and some other features as determined by routine epidemiological subtyping methods. Whole genome sequencing of these two strains, of ten outbreak strain isolates originating from different time points of the outbreak and of one historical sporadic EHEC O104:H4 isolate was performed. Sequence analysis revealed a clear phylogenetic distance between the two variant strains and the outbreak strain finally identifying them as epidemiologically unrelated isolates from sporadic cases. These findings add to the knowledge about this emerging pathogen, illustrating a certain diversity within the bacterial core genome as well as loss and gain of accessory elements. Our results do also support the view that distinct new variants of STEC O104:H4 repeatedly might originate from yet unknown reservoirs, rather than that there would be a continuous diversification of a single epidemic strain established and circulating in Germany after the large outbreak in 2011.     

Escherichia coli O157:H7 Colonization at the Rectoanal Junction of Long-Duration Culture-Positive Cattle

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.     

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4

Horizontal dissemination of the genes encoding extended spectrum beta-lactamases (ESBLs) via conjugative plasmids is facilitating the increasingly widespread resistance of pathogens to beta-lactam antibiotics. However, there is relatively little known about the regulatory factors and mechanisms that govern the spread of these plasmids. Here, we carried out a high-throughput, transposon insertion site sequencing analysis (TnSeq) to identify genes that enable the maintenance and transmission of pESBL, an R64 (IncI1)-related resistance plasmid that was isolated from Escherichia coli O104:H4 linked to a recent large outbreak of gastroenteritis. With a few exceptions, the majority of the genes identified as required for maintenance and transmission of pESBL matched those of their previously defined R64 counterparts. However, our analyses of the high-density transposon insertion library in pESBL also revealed two very short and linked regions that constitute a previously unrecognized regulatory system controlling spread of IncI1 plasmids. In addition, we investigated the function of the pESBL-encoded M.EcoGIX methyltransferase, which is also encoded by many other IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in expanding the plasmid''s host range. Collectively, our work illustrates the power of the TnSeq approach to enable rapid and comprehensive analyses of plasmid genes and sequences that facilitate the dissemination of determinants of antibiotic resistance.