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Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli.

ABSTRACT: Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences of K88-positive and F18-positive porcine ETEC strains and examined the phylogenetic distribution of clinical porcine ETEC strains and their plasmid-associated genetic content. The genomes of porcine ETEC strains UMNK88 and UMNF18 were both found to contain remarkable plasmid complements containing known virulence factors, potential novel virulence factors, and antimicrobial resistance-associated elements. The chromosomes of these strains also possessed several unique genomic islands containing hypothetical genes with similarity to classical virulence factors, although phage-associated genomic islands dominated the accessory genomes of these strains. Phylogenetic analysis of 78 clinical isolates associated with neonatal and porcine diarrhea revealed that a limited subset of porcine ETEC lineages exist that generally contain common toxin and fimbrial profiles, with many of the isolates belonging to the ST10, ST23, and ST169 multilocus sequencing types. These lineages were generally distinct from existing human ETEC database isolates. Overall, most porcine ETEC strains appear to have emerged from a limited subset of E. coli lineages that either have an increased propensity to carry plasmid-encoded virulence factors or have the appropriate ETEC core genome required for virulence.

SUBMITTER: Shepard SM

PROVIDER: S-EPMC3256668 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli.

Shepard Sara M SM Danzeisen Jessica L JL Isaacson Richard E RE Seemann Torsten T Achtman Mark M Johnson Timothy J TJ

Journal of bacteriology 20111111 2

Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences ...[more]

PMID: 22081385

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Project description:This study aimed to characterize in silico enterotoxigenic Escherichia coli F4- and F18-positive isolates (n = 90) causing swine postweaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance, prophage content, and metal tolerance rates. F4 strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 sequence types (STs). F18 strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23, and ST42. The highest rates of antimicrobial resistance were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and ampicillin. No resistance was found toward ciprofloxacin, cefotaxime, ceftiofur, and colistin. Genes conferring tolerance to copper (showing the highest diversity), cadmium, silver, and zinc were predicted in all genomes. Enterotoxin genes (ltcA, 100% F4, 62% F18; astA, 100% F4, 38.1% F18; sta, 18.8% F4, 38.1% F18; stb, 100% F4, 76.2% F18) and fimbria-encoding genes typed as F4ac and F18ac were detected in all strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. A highly diverse variety of plasmids was found; IncFII, IncFIB, and IncFIC were prevalent among F4 isolates, while IncI1 and IncX1 were dominant among F18 strains. Interestingly, F4 isolates from the early 1990s belonged to the same clonal group detected for most of the F4 strains from 2018 to 2019 (ONT:H10-A-ST100-CH27-0). The small number of single-nucleotide polymorphism differences between the oldest and recent F4 ST100 isolates suggests a relatively stable genome. Overall, the isolates analyzed in this study showed remarkably different genetic traits depending on the fimbria type.IMPORTANCE Diarrhea in the postweaning period due to enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by zinc oxide administration (to be discontinued by 2022). In addition, a breeding program has been implemented that aims to reduce the prevalence of this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among diarrhea-associated isolates by whole-genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC ST100 isolates from China, the United States, and Spain. High rates of AMR and high diversity of virulence genes were detected. The characterization of diarrhea-related ETEC is important for understanding the disease epidemiology and pathogenesis and for implementation of new strategies aiming to reduce the impact of the disease in pig production.

Project description:Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Project description:BackgroundEnterotoxigenic Escherichia coli K88 (E. coli K88) are considered as a major cause of diarrhea and death in newly weaned piglets. Oral passive immunization with chicken egg yolk immunoglobulins (IgY) have attracted considerable attention for treatment of gastrointestinal infection due to its high specificity. In this study it was estimated the protective effect of anti-K88 fimbriae IgY against E. coli K88 adhesion to piglet intestinal mucus in vitro and to investigate the potential use of IgY for controlling E. coli-induced diarrhea in weaned piglets in vivo.ResultsE. coli K88 was incubated with IgY for 24 h, and the bacterial growth profiles showed that specific IgY with a concentration higher than 5 mg/mL was observed to significantly inhibit the growth of E. coli K88 compared to nonspecific yolk powder in a liquid medium. Moreover, pretreatment with 50 mg/mL of IgY was found to significantly decrease the adhesion ability of E. coli K88 to porcine jejunal and ileal mucus, further supported by the observations from our immunofluorescence microscopic analysis. In vivo, administration of IgY successfully protected piglets from diarrhea caused by E. coli K88 challenge. Additionally, IgY treatment efficiently alleviated E. coli-induced intestinal inflammation in piglets as the gene expression levels of inflammatory cytokines TNF-α, IL-22, IL-6 and IL-1β in IgY-treated piglets remained unchanged after E. coli K88 infection. Furthermore, IgY significantly prevented E. coli K88 adhering to the jejunal and ileal mucosa of piglets with E. coli infection and significantly decreased E. coli and enterotoxin expression in colonic contents.ConclusionOutcome of the study demonstrated that IgY against the fimbrial antigen K88 was able to significantly inhibit the growth of E. coli K88, block the binding of E. coli to small intestinal mucus, and protect piglets from E. coli-induced diarrhea. These results indicate that passive immunization with IgY may be useful to prevent bacterial colonization and to control enteric diseases due to E. coli infection. The study has great clinical implication to provide alternative therapy to antibiotics in E coli induced diarrhea.

Dataset Information

Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli.

Publications

Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli.

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