Refine
Year of publication
Document Type
Way of publication
- Open Access (4)
Keywords
- CRISPR/Cas9 (2)
- Pan troglodytes ellioti (2)
- multiplexing (2)
- 3D Printing (1)
- ANCA (1)
- Bushmeat hunting (1)
- Cell Biology (1)
- Core areas (1)
- CytoBead technology (1)
- DNA damage response (DDR) (1)
Institute
- FG Multiparameterdiagnostik (122)
- FG Biochemie (3)
- FG Ökologie (3)
- FG Zellbiologie (2)
- FG Anorganische Chemie (1)
- FG Molekularbiologie (1)
- FG Molekulare Zellbiologie (1)
- FG Nanobiotechnologie (1)
BTU
Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics.
Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche, which is based on the expression of colonization factors. Escherichia coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal (exVAGs) infection. Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization. We developed new screening tools to genotypically and phenotypically characterize E. coli isolates originating in humans, domestic pigs, and 17 wild mammal and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA) and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes. inVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and the European hedgehog ( Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host, and tissue, though it was also unspecific. Occurrence of the following VAGs was associated with a higher rate of adhesion to one or more cell lines: afa-dra, daaD, tsh, vat, ibeA, fyuA, mat, sfa-foc, malX, pic, irp2, and papC. In summary, we established new screening methods which enabled us to characterize large numbers of E. coli isolates. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.
We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars.
Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation.
In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.