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Uropathogenic Escherichia coli attach to tissues using pili type 1. Each pilus is composed by thousands of coiled FimA domains followed by the domains of the tip fibrillum, FimF-FimG-FimH. The domains are linked by non-covalent β-strands that must resist mechanical forces during attachment. Here, we use single-molecule force spectroscopy to measure the mechanical contribution of each domain to the stability of the pilus and monitor the oxidative folding mechanism of a single Fim domain assisted by periplasmic FimC and the oxidoreductase DsbA. We demonstrate that pilus domains bear high mechanical stability following a hierarchy by which domains close to the tip are weaker than those close to or at the pilus rod. During folding, this remarkable stability is achieved by the intervention of DsbA that not only forms strategic disulfide bonds but also serves as a chaperone assisting the folding of the domains.

Classification of pathogenic Escherichia coli (E. coli) has traditionally relied on detecting specific virulence associated genes (VAGs) or combinations thereof. For E. coli isolated from faecal samples, the presence of specific genes associated with different intestinal pathogenic pathovars will determine their classification and further course of action. However, the E. coli genome is not a static entity, and hybrid strains are emerging that cross the pathovar definitions. Hybrid strains may show gene contents previously associated with several distinct pathovars making the correct diagnostic classification difficult. We extended the analysis of routinely submitted faecal isolates to include known virulence associated genes that are usually not examined in faecal isolates to detect the frequency of possible hybrid strains. From September 2012 to February 2013, 168 faecal isolates of E. coli routinely submitted to the Norwegian Institute of Public Health (NIPH) from clinical microbiological laboratories throughout Norway were analysed for 33 VAGs using multiplex-PCR, including factors associated with extraintestinal pathogenic E. coli (ExPEC) strains. The strains were further typed by Multiple Locus Variable-Number Tandem-Repeat Analysis (MLVA), and the phylogenetic grouping was determined. One isolate from the study was selected for whole genome sequencing (WGS) with a combination of Oxford Nanopore's MinION and Illumina's MiSeq. The analysis showed a surprisingly high number of strains carrying ExPEC associated VAGs and strains carrying a combination of both intestinal pathogenic E. coli (IPEC) and ExPEC VAGs. In particular, 93.5% (101/108) of isolates classified as belonging to an IPEC pathovar additionally carried ExPEC VAGs. WGS analysis of a selected hybrid strain revealed that it could, with present classification criteria, be classified as belonging to all of the Enteropathogenic Escherichia coli (EPEC), Uropathogenic Escherichia coli (UPEC), Neonatal meningitis Escherichia coli (NMEC) and Avian pathogenic Escherichia coli (APEC) pathovars. Hybrid ExPEC/IPEC E. coli strains were found at a very high frequency in faecal samples and were in fact the predominant species present. A sequenced hybrid isolate was confirmed to be a cross-pathovar strain possessing recognised hallmarks of several pathovars, and a genome heavily influenced by horizontal gene transfer.

To establish infection, enteric pathogens integrate environmental cues to navigate the gastrointestinal tract (GIT) and precisely control expression of virulence determinants. During passage through the GIT, pathogens encounter relatively high levels of oxygen in the small intestine before transit to the oxygen-limited environment of the colon. However, how bacterial pathogens sense oxygen availability and coordinate expression of virulence traits is not resolved. Here, we demonstrate that enterohemorrhagic Escherichia coli O157:H7 (EHEC) regulates virulence via the oxygen-responsive small RNA DicF. Under oxygen-limited conditions, DicF enhances global expression of the EHEC type three secretion system, which is a key virulence factor required for host colonization, through the transcriptional activator PchA. Mechanistically, the pchA coding sequence (CDS) base pairs with the 5′ untranslated region of the mRNA to sequester the ribosome binding site (RBS) and inhibit translation. DicF disrupts pchA cis-interactions by binding to the pchA CDS, thereby unmasking the pchA RBS and promoting PchA expression. These findings uncover a feed-forward regulatory pathway that involves distinctive mechanisms of RNA-based regulation and that provides spatiotemporal control of EHEC virulence.

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.

The emergence of E.coli strains displaying patterns of virulence genes from different pathotypes shows that the current classification of E.coli pathotypes may be not enough, the study aimed to compare the phylogenetic groups and urovirulence genes of uropathogenic Escherichia coli (UPEC) and diarrheagenic E.coli (DEC) strains to extend the knowledge of E.coli classification into different pathotypes. A total of 173 UPEC and DEC strains were examined for phylogenetic typing and urovirulence genes by PCR amplifications. In contrast to most reports, phylogenetic group A was the most prevalent in both UPEC and DEC strains, followed by B2 group. Amplification assays revealed that 89.32% and 94.29% of UPEC and DEC strains, respectively, carried at least one of the urovirulence genes, 49.5% and 31.4% of UPEC and DEC strains, respectively, carried ≥ 2 of the urovirulence genes, fim H gene was the most prevalent (66.9% and 91.4%) in UPEC and DEC strains respectively. Twenty different patterns of virulence genes were identified in UPEC while 5 different patterns in DEC strains. Strains with combined virulence patterns of four or five genes were belonged to phylogenetic group B2. Our finding showed a closer relationship between the DEC and UPEC, so raised the suggestion that some DEC strains might be potential uropathogens. These findings also provide different insights into the phylogenetic classification of E. coli as pathogenic or commensals where group A can be an important pathogenic type as well as into the classification as intestinal or extra- intestinal virulence factors.

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases. We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains. Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies.

Abstract Escherichia coli is the most researched microbial organism in the world. Its varied impact on human health, consisting of commensalism, gastrointestinal disease, or extraintestinal pathologies, has generated a separation of the species into at least eleven pathotypes (also known as pathovars). These are broadly split into two groups, intestinal pathogenic E. coli (InPEC) and extraintestinal pathogenic E. coli (ExPEC). However, components of E. coli’s infinite open accessory genome are horizontally transferred with substantial frequency, creating pathogenic hybrid strains that defy a clear pathotype designation. Here, we take a birds-eye view of the E. coli species, characterizing it from historical, clinical, and genetic perspectives. We examine the wide spectrum of human disease caused by E. coli, the genome content of the bacterium, and its propensity to acquire, exchange, and maintain antibiotic resistance genes and virulence traits. Our portrayal of the species also discusses elements that have shaped its overall population structure and summarizes the current state of vaccine development targeted at the most frequent E. coli pathovars. In our conclusions, we advocate streamlining efforts for clinical reporting of ExPEC, and emphasize the pathogenic potential that exists throughout the entire species. A schematic characterization of the disease manifestations, genomic flexibility, population dynamics, and vaccine targets of Escherichia coli, a multi-faceted bacterium with pathogenic potential throughout the entire species.

Urinary Tract Infection (UTI) is one of the most common bacterial infectious diseases which causes considerable morbidity and costly health problems. Uropathogenic Escherichia coli (UPEC), the most common pathogen causing UTI, is a highly heterogeneous group of extraintestinal pathogenic E. coli (ExPEC) which may carry a variety of virulence factors and belonging to different phylogenetic backgrounds. The current study aimed to investigate the frequency and association between various virulence factors (VFs) and phylogenetic groups of UPEC and commensal isolates. UPEC and commensal E. coli strains isolated from UTI and feces of healthy humans were compared for the presence of VFs and phylogenetic groups. Association between virulence genes was investigated and cluster analysis was employed. According to the results, among a 30 virulence markers tested, the pathogenicity-associated island (PAI), papAH, papEF, fimH, fyuA, and traT genes prevalence were statistically significant in UPEC isolates. A strong association was found between the B2 and D phylogenetic groups and clinical isolates of UPEC; while, commensal isolates were mostly associated with phylogenetic group A. The aggregated VFs scores were more than twice higher in the UPEC isolates in comparison with the commensal isolates. Interestingly, the B2 group in both UPEC and commensal isolates had the highest VF scores. A strong positive association was found between several virulence genes. The clustering results demonstrated that UPEC or commensal E. coli isolates were highly heterogeneous due to different composition of their virulence gene pool and pathogenicity islands. Genetic structure and VFs of UPEC strains vary from region to region; therefore, to control the UTI, the epidemiological aspects and characterization of the UPEC isolates need to be investigated in different regions. Since UPEC isolates are generally originate from the commensal strains, it may be feasible to reduce the UTI burden by interfering the intestinal colonization, particularly in the highly pathogenic clonal lineages such as B2.

Background Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. Materials and methods A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes ( sxt1 , stx2 , eae and ehlyA ) simultaneously. Results Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1 , stx2 , eae and ehlyA genes simultaneously. Conclusion It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.