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.2013 Apr 26;8(4):e59242.
doi: 10.1371/journal.pone.0059242. Print 2013.

Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method

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Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method

Peter Schierack et al. PLoS One..

Abstract

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.

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Conflict of interest statement

Competing Interests:The authors have the following conflicts: Dirk Roggenbuck is a shareholder of GA Generic Assays GmbH and Medipan GmbH. The remaining authors declare that they have no competing financial interests. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Adhesion rates ofE.coli to IPEC-J2.
IPEC-J2 cells were incubated withE. coli from domestic pigs (DP, n = 104) or wild boars (WB, n = 93) over 2 h or 6 h. Adhesion was quantified after removing non-adherent bacteria by washing. There were significant differences in adhesion following 2 h and 6 h incubation periods. Additionally, there was a significant difference in adhesion betweenE. coli from domestic pigs and wild boar after 6 h incubation. All: isolates of domestic pigs and wild boars together, *: p<0.05 between the groups connected by the beam. Solid squares represent the sample mean.
Figure 2
Figure 2. Associations between adhesion rates and a gene.
IPEC-J2 cells were incubated withE. coli over 2 h or 6 h. Adhesion was quantified after removing non-adherent bacteria by washing. AllE. coli isolates were grouped being positive (pos) or negative (neg) for a gene. Median of adhesion rates of one group is represented by one dot. There were three significant associations between adhesion and a specific gene (sfa/foc,iha,sat).
Figure 3
Figure 3. Cluster analysis with virulence-associated genes (VAGs).
Isolates from domestic pigs (n = 104) and from wild boars (n = 93) were clustered according to 9 VAGs typical forE. coli isolated from diarrheic pigs (iVAGs) and 37 VAGs typical forE. coli isolated from extraintestinal disease (eVAGs). Using the manhattan distance measure the distance between strains was computed including a hierarchical cluster analysis using a single linkage method (“friends of friends”) clustering strategy. The minimal difference in the number (N) of genes between clusters of strains to other clusters is described. All isolates of one cluster have identical VAG pattern. Distance of 1 (δN = 1) means that two clusters differed at least in one gene. Clusters which included at least four isolates, which were not different in more than 2 genes, were used to associate virulence gene profiles with adhesion rates or a probiotic effect. There were no associations between a specific cluster and adhesion or a probiotic effect. *: Clustered isolates are identified by strain numbers.
Figure 4
Figure 4. Association of adhesion and a probiotic effect.
X-axis: IPEC-J2 cells were incubated withE. coli over 2 h or 6 h. Adhesion was quantified after removing non-adherent bacteria by washing. Y-axis: EPEC infection rates were determined in an EPEC inhibition assay:E. coli were incubated over 2 h with IPEC-J2 cells. Non-adherent bacteria were removed by washing. Subsequently, IPEC-J2 cells were incubated with EPEC. After 6 h non-adherent bacteria were removed by washing. The EPEC infection rate was calculated in relation to EPEC mono-infection (no domestic pig or wild boarE. coli pre-incubation = 100%). Conclusively, a number below 100% indicates a probiotic effect = reduction of EPEC. Isolates from domestic pigs had a higher probiotic effect ( = higher reduction of the EPEC infection rate (p<0.05)). There were no significant associations between the adhesion capabilities of strains and their probiotic effects. *: Mean of EPEC infection rate.
Figure 5
Figure 5. Reduction of EPEC infection rate is associated with the reduction of microcolonies.
X-axes: EPEC infection rate is the number of all EPEC bacteria present on IPEC-J2 cells and was determined in an EPEC inhibition assay:E. coli (n = 197, domestic pig and wild boarE. coli together) were incubated over 2 h with IPEC-J2 cells. Non-adherent bacteria were removed by washing. Subsequently, IPEC-J2 cells were incubated with EPEC. After 6 h non-adherent bacteria were removed by washing. The EPEC infection rate was calculated in relation to EPEC mono-infection (no domestic pig or wild boarE. coli pre-incubation = 100%). Y-axes: EPEC adhesion events, single bacteria, small microcolonies, medium size microcolonies and large colonies were calculated in relation to EPEC mono-infection (no domestic pig or wild boarE. coli pre-incubation = 100%). Adhesion event: one EPEC formation (including single bacteria as well as microcolonies) which presumably based on one initial adherent EPEC bacterium, single bacterium: one bacterium with no contact to other bacteria, small microcolony: one microcolony consisting of 2–10 bacteria, medium size microcolony: one microcolony consisting of 11–20 bacteria, large microcolony: one microcolony consisting of more than 20 bacteria. If anE. coli isolate reduced the EPEC infection rate than preferentially adhesion events and numbers of microcolonies were reduced.
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References

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This work was supported by InnoProfile IP 03 IP 611 funded by the Bundesministerium für Bildung und Forschung (BMBF, Germany) and by Collaborative Research Group (SFB) 852 "Nutrition and intestinal microbiota - host interactions in the pig" funded by the Deutsche Forschungsgemeinschaft (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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