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.

Growth in colonies with type 1 morphology and the presence of pili are characteristics that have been associated with virulence of gonococci for humans. To determine whether the presence of pili per se might be responsible for colony type 1 morphology, the relationship ofpili to colony type was examined in various species ofNeisseria. Short pili (175 to 210 nm in length) were seen only on nonpathogenic neisseria, whereas long pili (up to 4,300 nm) were seen on organisms ofboth nonpathogenic and pathogenic species. Although long pili, similar to those found on organisms from high-domed, type 1 colonies of gonococci, were observed on organisms from high-domed, type 1 colonies of nonpathogenic Neisseria species, they were also observed on low-convex, type 4 colonies of meningococci and nonpathogenic neisseria. Among meningococci there was no difference in the morphology of colonies consisting of organisms with many long pili and colonies consisting of organisms that completely lacked pili. Thus, there was no consistent relationship of pili to colonial morphology. Unless the pili of N. gonorrhoeae are unique among Neisseria species in their influence on colonial morphology, it is likely that factors other than pili determine colony type 1 morphology of gonococci. Whether these same factors, either alone or in conjunction with pili, are also responsible for gonococcal virulence warrants further investigation.

BackgroundDifferent strategies of colonization or infection by E. coli result in formation of certain adhesion patterns which help also in classifying intestinal E. coli into pathotypes. Little is known about adhesion patterns and host- and tissue adaption of commensal E. coli and about E. coli originating in clinically healthy hosts carrying pathotype-specific virulence-associated genes.FindingsAdhesion pattern of E. coli (n = 282) from humans and from 18 animal species were verified on intestinal human Caco-2 and porcine IPEC-J2 cells and, furthermore, for comparison on human urinary bladder 5637, porcine kidney PK-15 epithelial and HEp-2 cells. The analysis was carried out on 150,000 images of adhesion assays.Adhesion patterns were very diverse; 88 isolates were completely non-adherent, whereas 194 adhered to at least one cell line with the dominant adhesion patterns “diffusely distributed” and “microcolony formation”. Adhesion patterns “chains” and “clumps” were also visible. Chain formation was mediated by the presence of epithelial cells. Clump formation was very specific on only the 5637 cell line. All enteropathogenic (eae+) E. coli (EPEC; n = 14) were able to form microcolonies which was cell line specific for each isolate. Most EPEC formed microcolonies on intestinal IPEC-J2 and Caco-2 but several also on urinary tract cells. Shigatoxin-producing (stx+) E. coli (n = 10) showed no specific adhesion patterns.ConclusionsE. coli isolates were highly diverse. Commensal and pathogenic isolates can adhere in various forms, including diffuse distribution, microcolonies, chains and clumps. Microcolony formation seems to be a global adhesion strategy also for commensal E. coli.

The nature and distribution of surface appendages morphologically distinct from pili were examined in three strains of gonococci. As reported previously by others, large appendages were seen emanating from colony type 1 gonococci when negatively stained preparations were examined by electron microscopy. Whereas the diameter of pili was 4 to 6 nm, the appendages varied from 8 to 40 nm in diameter with bulbous enlargements of up to 130 nm. However, in contrast to previous findings, the appendages were not limited to virulence-associated colonial types but occurred with almost equal frequency on organisms from colonial types 1 and 4. Thus, the presence of these appendages on gonococci was unrelated to colonial morphology, and there is currently no reason to associate them with gonococcal virulence. The large appendages were seldom seen if the organisms were suspended in isotonic fluid rather than in distilled water, which was used in previous studies. This suggests that the large appendages may occur infrequently in the isotonic environment of tissues or in slightly hypertonic purulent exudates. Nevertheless, we did observe small appendages or cell wall blebs on the surface of gonococci suspended in isotonic medium and noted similar structures as spheres free in the surrounding milieu. These findings suggest that the material of which the large appendages consist may form small appendages or cell wall blebs and be shed into the milieu under isotonic conditions. Such cell wall blebs have been demonstrated on meningococci and been shown to consist of endotoxin.