Highlights • We developed and tested a new oral ETEC vaccine in mice, alone and with an adjuvant. • The vaccine contains E. coli overexpressing CFA/I, CS3, CS5 and CS6 and B-subunit. • Oral ...immunization induced intestinal and serum antibody responses to all antigens. • These responses were further enhanced by dmLT, a nontoxic E. coli adjuvant. • The vaccine, both with and without dmLT, was stable and well tolerated.
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.
Enterotoxigenic
(ETEC) strains producing heat-labile toxin (LT) and/or heat-stable toxin (STa) are a top cause of children's diarrhea and travelers' diarrhea. Holotoxin-structured GM
-binding LT is a ...strong immunogen and an effective adjuvant, and can serve a carrier or a platform for multivalent vaccine development. However, the significance of peptide domains or epitopes of LT particularly enzymatic LT
subunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we identified B-cell epitopes
from LT
subunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes identified from LT
subunit were individually fused to a modified chicken ovalbumin carrier protein, and each epitope-ovalbumin fusion was used to immunize mice. Data showed all 11 LT
epitopes were immunogenic; epitope 7 (
SPHPYEQEVSA
) induced greater titers of anti-LT antibodies which neutralized LT enterotoxicity more effectively. To examine these epitopes for the significance in LT enterotoxicity, we constructed LT mutants by substituting each of 10 epitopes at the toxic A1 domain of LT
subunit with a foreign epitope and examined LT mutants for enterotoxicity and GM
-binding activity. Data showed that LT mutants exhibited no enterotoxicity but retained GM
-binding activity. The results from this study indicated that while not all immunodominant LT
epitopes were neutralizing, LT mutants with an individual epitope substituted lost enterotoxicity but retained GM
-binding activity. These results provided additional information to understand LT immunogenicity and enterotoxicity and suggested the potential application of LT platform for multivalent vaccines against ETEC diarrhea and other diseases.
No vaccine is licensed for enterotoxigenic
(ETEC) strains, which remain a leading cause of diarrhea in children from developing countries and international travelers. GM
-binding heat-labile toxin (LT) which is a key virulence factor of ETEC diarrhea is a strong vaccine antigen and a self-adjuvant. LT can also serve a backbone or platform for MEFA (multiepitope fusion antigen), a newly developed structural vaccinology technology, to present heterogeneous epitopes (by replacing LT epitopes) and to mimic epitope antigenicity for development of broadly protective vaccines. Data from this study identified neutralizing LT epitopes and demonstrated that substitution of LT epitopes eliminated LT enterotoxicity without altering GM
-binding activity, suggesting LT is potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens.
Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) are intestinal pathogens that cause food and water-borne disease in humans. Using ...biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA), and indole-3-acetic acid (IAA), as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE), which encodes virulence factors that cause "attaching and effacing" (A/E) lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling.
The empirical and pragmatic nature of diagnostic microbiology has given rise to several different schemes to subtype
.coli, including biotyping, serotyping, and pathotyping. These schemes have proved ...invaluable in identifying and tracking outbreaks, and for prognostication in individual cases of infection, but they are imprecise and potentially misleading due to the malleability and continuous evolution of
. Whole genome sequencing can be used to accurately determine
subtypes that are based on allelic variation or differences in gene content, such as serotyping and pathotyping. Whole genome sequencing also provides information about single nucleotide polymorphisms in the core genome of
, which form the basis of sequence typing, and is more reliable than other systems for tracking the evolution and spread of individual strains. A typing scheme for
based on genome sequences that includes elements of both the core and accessory genomes, should reduce typing anomalies and promote understanding of how different varieties of
spread and cause disease. Such a scheme could also define pathotypes more precisely than current methods.
Abstract The prevalence of diarrheagenic Escherichia coli (DEC) in children under 5 years was studied in children with diarrhea and controls in South India. Four polymerase chain reaction (PCR) ...“schemes” were used to detect genes of the 6 pathotypes of DEC. In 394 children with diarrhea, 203 (52%) DEC infections were found. Among the 198 controls, 126 (63%) DEC infections were found. Enteroaggregative E. coli was the most common pathotype by multiplex PCR both in cases (58, 14.7%) and controls (47, 23.7%), followed by enteropathogenic E. coli seen in 10% cases and 8% of controls. Enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) were found in 4.1%, 2.0%, 1.0%, and 0.5% of cases, respectively. ETEC was found in 2.5% of controls, but EHEC, EIEC, and DAEC were not detected. Overall, no single assay worked well, but by discounting genes with a pathogenicity index of less than 1, it was possible to use the PCR assays to identify DEC in 75/394 (19%) cases and 12/198 (6.1%) controls, while mixed infection could be identified in 8/394 (2%) cases and 2/198 (1%) controls.
•We provide an overview APEC's virulence factors.•Critically review attempts to develop a vaccine against APEC.•We analyze the only commercially available vaccine against APEC.
Avian pathogenic ...Escherichia coli (APEC) is one of the most economically devastating pathogens affecting the poultry industry. This group of extra-intestinal E. coli causes a variety of clinical conditions including airsacculitis and cellulitis. The economic impact of APEC is mainly due to mortality, slower growth rates, and carcass downgrading. In commercial broiler operations, APEC infections are controlled indirectly by vaccination against other respiratory diseases and minimizing stress conditions, and directly by administration of antimicrobial agents to suppress the infection in already infected flocks. The fact that most APEC strains possess some common virulence factors suggests that an effective vaccine against APEC is a viable option. The most important virulence factors that have been investigated over the years include type I and P fimbriae, aerobactin iron-acquisition system, and serum resistance traits. Despite the potential for developing an efficacious vaccine to combat this economically important poultry disease, several obstacles hinder such efforts. Those obstacles include the cost, vaccine delivery method and timing of vaccination as the birds should be immune to APEC by 21 days of age. Herein, we review the various attempts to develop an effective vaccine against the respiratory form of APEC diseases in poultry. We also discuss in-depth the potentials and limitations of such vaccines.
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.
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.
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