Enterohaemorrhagic Escherichia coli gains a competitive advantage by using ethanolamine as a nitrogen source in the bovine intestinal content.

The bovine gastrointestinal tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients that promote the carriage of these pathogens by the ruminant would help to develop ecological strategies to reduce their survival in the bovine gastrointestinal tract. In this study, we show for the first time that free ethanolamine (EA) constitutes a nitrogen source for the O157:H7 EHEC strain EDL933 in the bovine intestinal content because of induction of the eut (ethanolamine utilization) gene cluster. In contrast, the eut gene cluster is absent in the genome of most species constituting the mammalian gut microbiota. Furthermore, the eutB gene (encoding a subunit of the enzyme that catalyses the release of ammonia from EA) is poorly expressed in non-pathogenic E. coli. Accordingly, EA is consumed by EHEC but is poorly metabolized by endogenous microbiota of the bovine small intestine, including commensal E. coli. Interestingly, the capacity to utilize EA as a nitrogen source confers a growth advantage to E. coli O157:H7 when the bacteria enter the stationary growth phase. These data demonstrate that EHEC strains take advantage of a nitrogen source that is not consumed by the resident microbiota, and suggest that EA represents an ecological niche favouring EHEC persistence in the bovine intestine.

Serotyping, stx2 subtyping, and characterization of the locus of enterocyte effacement island of shiga toxin-producing Escherichia coli and E. coli O157:H7 strains isolated from the environment in France.

Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx(1), and 24 were positive for stx(2) (10 were positive for stx(2vh-a) or stx(2vh-b), 19 were positive for stx(2d), and 15 were positive for stx(2e)). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.

Morphological and immune response alterations in the intestinal mucosa of the mouse after short periods on a low-magnesium diet.

The importance of Mg for the immune function is well recognized; however, there is no information available about the effect of Mg intake on the modulation of local immune response in the intestine. Thus, in the present study the hypothesis that short periods of Mg deprivation can affect intestinal mucosa and local immune response was tested. For this purpose, OF1 female mice were fed a semipurified diet (1000 mg Mg/kg diet). For 3 d before immunization and 1 d after, half of the animals were fed a Mg-deficient diet (30 mg Mg/kg diet), three immunizations per os were performed every 3 weeks with Escherichia coli producing the CS31A capsule-like protein (1010 or bacteria per animal). Mice were killed 10 d after the last immunization. The level of specific anti CS31A immunoglobulin (Ig) G and IgA in the serum and secretory IgA in the intestinal secretions and faeces were measured by ELISA. The results indicated that administration of a high dose of immunogen with a low-Mg diet led to lower specific IgA levels in the intestinal mucus and serum. Administration of a low dose of immunogen with a low-Mg diet led to lower IgA and IgG levels in the serum and secretory IgA coproantibodies. To assess alterations of intestinal mucosa caused by a low-Mg diet for a short period, histological and scanning electron microscopy analyses were performed on samples from mice (not submitted to the vaccination protocol) after 3 d on the Mg-deficient diet. These analyses showed several alterations, suggesting perturbations in the growth of the intestinal mucosa. These changes were accompanied by modifications in the expression of several genes involved in cell growth and stress response. From this present work, it may be concluded that short periods of Mg deprivation can affect the intestinal mucosa and local immune response of the intestine.

Conserved structural features in class I major fimbrial subunits (Pilin) in gram-negative bacteria. Molecular basis of classification in seven subfamilies and identification of intrasubfamily sequence signature motifs which might Be implicated in quaternary structure.

Type 1 and P-pili are prototype members of Class I fimbriae produced by Gram-negative bacteria. Despite common structural characteristics, the low level of amino acid sequence conservation among the Class I major fimbrial subunits (pilins) indicates considerable evolutionary distance between members of this superfamily. We highlight here structural relatedness between Class I pilins from their two-dimensional sequence analysis using hydrophobic cluster analysis (HCA) and secondary structure predictions (PHD program). We present evidence that all members of the Class I pilin family have clear structural relatedness and suggest that classification based on phylogenetic analysis of Class I pilins into seven subfamilies correlates with differences in structural properties of the amino acid sequences. Using a sensitive alignment process (HCA), we identified 29 residues in topohydrophobic positions which probably play a prominent role in folding. The most striking aspects that distinguish the different pilin subfamilies are (i) large variation in the length of the loops connecting the structurally conserved regions and (ii) intrasubfamily sequence signature motifs located on regions predicted to be in the beta-conformation. We suggest that these "intrasubfamily sequence signature motifs" are part of interactive surfaces which participate in subunit-subunit interactions. These motifs prove highly useful in characterizing and classifying new Class I fimbriae that have not yet been described and whose sequence diverges appreciably from those of characterized groups. (After the submission of our manuscript, the experimental structure of Class I pilus subunits was published. In light of these actual pilin structures, a comparison has been made between the predicted results and the crystal structure in the Note Added in Proof.)

Epidemiological study of pap genes among diarrheagenic or septicemic Escherichia coli strains producing CS31A and F17 adhesins and characterization of Pap(31A) fimbriae.

The association of the pap operon with the CS31A and F17 adhesins was studied with 255 Escherichia coli strains isolated from calves, lambs, or humans with diarrhea. The three classes of PapG adhesin with different receptor binding preferences were also screened. The pap operon was associated with 50 and 36% of human strains that produced CS31A and ovine strains that produced F17, respectively. Among the bovine isolates, the pap operon was detected in 61% of the CS31A-positive isolates and 72% of the strains that produce both CS31A and F17. The class II adhesin gene was present in bovine (20%) and ovine (71%) isolates. Both class II and III adhesins were genetically associated with 36% of the human strains. The highest prevalence of the pap operon was observed among E. coli strains that produce additional adhesins involved in the binding of bacteria to intestinal cells. Among the bovine isolates, the reference strain for CS31A and F17c was found to be positive for the pap operon. Phenotypic and genotypic characterizations were undertaken. Pap(31A) appeared as fine and flexible fimbriae surrounding the bacteria but did not mediate adhesion to calf intestinal villi. Pap(31A) production was optimal with bacteria cultured on minimal growth media and repressed by addition of exogenous leucine. The deduced amino acid sequence of the PapA(31A) structural subunit showed 57 to 97% identity with the different P-related structural subunits produced by E. coli strains isolated from pigs with septicemia or humans with urinary tract infections. None of the three papG allelic variants was detected, but a homologous papG gene was present in the chromosome of strain 31A.

Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes.

The enzymatic properties of two endoglucanases from Fibrobacter succinogenes, EGB and EGC, were analysed. EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E. coli as well as its detection in F. succinogenes cultures. Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies. One of the polyclonal antipeptide antisera was used to purify EGB. EGC was purified by affinity chromatography with Ni-NTA resin. The endo mode of action of the two enzymes on carboxymethyl-cellulose was different. The values of K(m) and V(max) were respectively 13.6 mg/ml and 46 micromol/min mg protein for EGB, and 7 mg/ml and 110 micromol/min mg protein for EGC. The reactivity of the antipeptide and the anti-EGC sera with F. succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational modification of the two enzymes in F. succinogenes cultures. Expression of endB and endC genes in F. succinogenes was confirmed by RT-PCR.

Association of genes encoding P fimbriae, CS31A antigen and EAST 1 toxin among CNF1-producing Escherichia coli strains from cattle with septicemia and diarrhea.

Fifty-six CNF1-producing Escherichia coli strains isolated from cattle with diarrhea or septicemia were screened by PCR for the detection of pap, sfa, afa, clpG, or f17 adherence factor and EAST 1 toxin genes. All the isolates were pap-positive, in accordance with the close association of pap, CNF1 and alpha-hemolysin genes observed on human and porcine E. coli. Only the gene encoding the P adhesin of class III (PrsG) was detected. Genes encoding CS31A antigen (71%) and S fimbriae (34%) (but not Afa or F17) were detected among the bovine isolates. E. coli producing both CNF1 and plasmid-encoded CS31A is a new example of association between bacterial clones and plasmid-mediated virulence factors. The EAST 1 toxin-encoding gene was detected on 66% of the CNF1-producing isolates but was linked to CS31A rather than to CNF1. These results suggest a close association between EAST 1 toxin and the adherence factor CS31A among pathogenic bovine E. coli.

A study of relationships among F17 a producing enterotoxigenic and non-enterotoxigenic Escherichia coli strains isolated from diarrheic calves.

We investigated the clonal relationships among 41 enterotoxigenic (ETEC) or non-enterotoxigenic (NETEC) Escherichia coli strains producing the F17 a fimbriae isolated from diarrheic calves in France or Belgium in the early 1980s. Twenty-three of the 26 ETEC strains were highly clonally related, most of them with a O101:K32:H9-serotype. The NETEC strains were also divided in clonal subgroups, most of them with O101:H-serotype. The F17 a positive ETEC strains are no longer isolated from diarrheic calves in these countries. It is postulated that the use of a vaccine including O101, K32 and H9 antigens in addition to K99 (F5) explains the strongly reduced isolation of the O101:K32:H9, K99 (F5) E. coli clone.

The central variable V2 region of the CS31A major subunit is involved in the receptor-binding domain.

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.

An Escherichia coli CS31A fibrillum chimera capable of inducing memory antibodies in outbred mice following booster immunization with the entero-pathogenic coronavirus transmissible gastroenteritis virus.

CS31A fibrillae are thin, flexible, heteropolymeric proteinaceous appendages exposed as a capsule-like material around the cell surface of certain Escherichia coli strains. Two antigenic peptides of the S spike glycoprotein (TGEV-S) amino acids (aa) 363-371 and 521-531 of the transmissible gastroenteritis virus (TGEV) were tandemly introduced in the loop-structured, variable region aa 202-218 of the major ClpG subunit protein composing the bulk of CS31A. The resulting hybrid fibrillae with a 25 aa heterologous peptide were produced at the cell surface. Using a monoclonal antibody (Mab) specific for the TGEV epitopes, purified hybrid fibrillae were analysed in Western blotting under native conditions, which showed that the two viral epitopes were recognized immunologically as an integral part of the hybrid fibrillae, and therefore that they were antigenically active. The immunogenicity of the fusion construct was evaluated with live recombinant bacteria, purified hybrid ClpG monomers, and purified chimeric CS31A polymers. Whatever the form of hybrid used as antigen, intraperitoneally immunized outbred mice elicited serum anti-TGEV peptides antibodies (Abs) with significant titres and capable of recognizing native TGEV particles, indicating that the epitopes are exposed in an immunogenic conformation in all cases. However, virus neutralization titres were only obtained after immunization with either purified polymers or monomers. Furthermore, 4 months after an ultimate immunization with 20 micrograms of hybrid fibrillae mice developed a strong anamnestic Ab response against the two TGEV peptides following booster inoculation with virions. We conclude that CS31A fibrillae carrying a combination of TGEV epitopes as insert can induce an immunological memory in outbred animals infected with TGEV, and therefore that hybrid CS31A fibrillae may prove efficient as components of a subunit vaccine.

Rapid and specific detection of F17-related pilin and adhesin genes in diarrheic and septicemic Escherichia coli strains by multiplex PCR.

The F17-related adhesins are prevalent in Escherichia coli strains isolated from calves with diarrhea or septicemia and from lambs with nephropathy. The F17 family includes the F17a, F17b, F17c, and F111 fimbriae produced by bovine E. coli strains and the G agglutinin produced by human uropathogenic E. coli strains. An easy and inexpensive multiplex PCR method was developed to detect all the F17-related fimbriae and to identify four subtypes of structural subunit genes and two distinct subfamilies of adhesin genes by only two runs of amplification. A strict correlation was observed between the phenotypic assays and the multiplex PCR method when 166 pathogenic E. coli strains isolated from intestinal content of calves or lambs were tested. Genes encoding the F17c structural subunit and the subfamily II adhesins were prominent among the bovine and ovine isolates, and the capsule-like CS31A antigen was strictly associated with the F17c fimbriae. The F17b subtype fimbriae were prominent among the bovine isolates producing the CNF2 toxin, whereas the F17a subtype fimbriae were associated with the bovine isolates producing neither the CS31A antigen nor the CNF2 toxin. Five bacterial strains possessed two distinct and complete F17-related fimbrial gene clusters, and two of them produced two F17-related fimbriae at the bacterial cell surface. The related fimbrial gene clusters are probably organized in mosaic operons consisting of F17-related pilin and adhesin genes, and horizontal gene transfer may occur among E. coli strains isolated form different animal species.

Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.

Characterization of 20K fimbria, a new adhesin of septicemic and diarrhea-associated Escherichia coli strains, that belongs to a family of adhesins with N-acetyl-D-glucosamine recognition.

Bovine septicemic Escherichia coli 31A agglutinates bovine, rabbit, and human erythrocytes and adheres in vitro to the brush border of bovine or ovine intestinal epithelial cells and to the human colon carcinoma Caco-2 cell line. The adhesion and hemagglutination of E. coli 31A are mediated by a chromosome-encoded fimbrial adhesin serologically distinct from known fimbrial adhesins found in enterotoxigenic and septicemic bovine E. coli strains. By electron microscopy studies the fimbriae designated 20K were observed as fine flexible filaments (diameter, 3 nm) and the purified major fimbrial subunit appeared with an apparent molecular mass of 20,000 Da. Western blot (immunoblot) analysis, N-terminal sequence alignment, and amino acid composition revealed a high homology with the N-acetyl-D-glcosamine-specific G fimbria of human uropathogenic E. coli and with fimbriae belonging to the F17 family produced by bovine enterotoxigenic and invasive E. coli strains. Immunological study revealed that 20K fimbria was closely related to G fimbria and represents a serological variant of F17 fimbria. Hemagglutination and adhesion inhibition assays demonstrated that 20K, G, and F17 fimbriae bind to an N-acetyl-D-glucosamine-containing receptor, but each probably binds to different oligosaccharide sequences or different receptors on host tissues. 20K fimbriae were produced by a limited group of clonally related strains with the unusual m-inositol-positive phenotype and appeared highly associated with the plasmid-mediated virulence factor. An examination of natural occurrence of 20K fimbriae among a large collection of human and animal pathogenic E. coli showed that 20K fimbria is the prominent adhesin among bovine septicemic E. coli isolated from European countries.

Molecular characterization and adhesive properties of CF29K, an adhesin of Klebsiella pneumoniae strains involved in nosocomial infections.

We previously described a CS31A-related protein, CF29K, expressed by Klebsiella pneumoniae strains involved in nosocomial infections. In this study, we cloned and sequenced cf29A, the structural gene of the CF29K protein, and showed that CF29K is an antigenic subtype of CS31A. The CF29K protein was found to be identical to the CS31A-L protein on the basis of biochemical and immunological properties. In contrast, the CS31A-H protein presented a different apparent molecular mass during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a different limited degradation pattern with endopeptidase V8, and a specific conformational epitope. We cloned and sequenced the CS31A-L structural gene and confirmed that CF29K and CS31A-L are identical, but their major subunits differ from ClpG (the CS31A-H subtype major subunit) by one amino acid at position 89 of the mature protein, which is a lysine in CF29K instead of the asparagine in ClpG. Site-directed mutagenesis experiments demonstrated that the biochemical and immunological differences between CS31A-H and CF29K or CS31A-L were dependent only on the amino acid at position 89 of the mature protein. To study the adhesive properties of CS31A-H and CF29K in the same Escherichia coli reference strain, we performed transcomplementation experiments with the cloned CS31A major-subunit structural genes or cloned cf29A gene and the clp accessory genes of the CS31A operon. We showed that CS31A-L, CF29K, and CS31A-H were involved in adhesion to Caco-2 and Int-407 cells but not to HEp-2 cells. Nevertheless, K. pneumoniae strains and corresponding E. coli transconjugants producing CF29K adhered to cultured Caco-2, Int-407, and HEp-2 cells, indicating the expression of another R-plasmid-encoded adhesin that mediated adhesion to HEp-2 cells. The carbohydrate part of the eucaryotic receptor of CF29K and CS31A-H adhesins was investigated by adhesion inhibition experiments with Int-407 cells. Although CS31A and CF29K belong to the K88 adhesin family, the receptor does not contain N-acetyl-D-galactosamine residues but contains N-acetylneuraminic acid and N-acetyl-D-glucosamine.

Hydrophobic cluster analysis and secondary structure predictions revealed that major and minor structural subunits of K88-related adhesins of Escherichia coli share a common overall fold and differ structurally from other fimbrial subunits.

The structural relatedness of K88-related major and minor subunits was deduced from their sequences by hydrophobic cluster analysis (HCA) and secondary structure predictions produced by the profile neural network prediction program (PHD) on multiple sequence alignments. Although the weak residue identity between major and minor subunits is evidence of a high evolutionary distance, an overall structural similarity was observed In addition, clear amphipathic conformations were conserved in predicted secondary structure. On the basis of this predicted structural similarity, a schematic 2D model of ClpG subunit was developed.

Pilins of fimbrial adhesins of different member species of Enterobacteriaceae are structurally similar to the C-terminal half of adhesin proteins.

The structural relatedness of pilins and the C-terminal half of adhesin proteins in different member species of Enterobacteriaceae was deduced from their two-dimensional sequence analysis using the hydrophobic cluster analysis (HCA) and secondary structure predictions from the profile network Hei-Delberg program (PHD). Despite a large evolutionary distance between the two protein families, we show that pilins and the C-terminal domain of adhesins have a similar folding that can serve as modules for pilus assembly.

Self-transmissible R plasmids encoding CS31A among human Escherichia coli strains isolated from diarrheal stools.

The CS31A antigen was first described for septicemic and enterotoxigenic bovine E. coli strains. In our study, of 597 human Escherichia coli strains isolated from diarrheagenic stools of hospitalized patients, 30 (5%) hybridized with the CS31A DNA probe. These CS31A-positive E. coli strains diffusely adhered to Caco-2 and/or HEp-2 cells and produced a major surface protein of either 30 or 30.5 kDa according to the strain. These proteins were antigenically related to the two forms of the CS31A antigen, namely, CS31A-L and CS31A-H. Genes encoding CS31A were located on 140-kb conjugative R plasmids. E. coli transconjugants expressed major surface proteins similar to those of the wild-type strains and adhered to Caco-2 and/or HEp-2 cells. An association of CS31A and another adhesive factor of the Dr family was found in 70% of wild-type strains, since 21 strains hybridized with the diffuse adhesion DNA probe corresponding to the accessory gene (daaC) of the F1845 adhesin. Comparison of the restriction patterns of the 140-kb R plasmids of the CS31A-positive E. coli strains showed these plasmids to be similar. Hybridization experiments indicated that the genes encoding CS31A and resistance to penicillin were located together on either of two 20- or 27-kb EcoRI restriction fragments in four E. coli strains. We reported a similar linkage between these genes in Klebsiella pneumoniae strains which produced CF29K, a CS31A-like antigen. These results suggest a horizontal transfer between E. coli and K. pneumoniae strains.

CS31A capsule-like antigen as an exposure vector for heterologous antigenic determinants.

CS31A is a multimeric surface protein surrounding certain enterotoxigenic and septicemic bovine Escherichia coli strains. The possibility of using CS31A as a carrier of foreign antigenic determinants was investigated. Introduction of heterologous viral epitopes into the CS31A major subunit, ClpG, was successfully performed in the V3 region of the molecule which encodes for an immunodominant linear epitope. E. coli K-12 strains producing the hybrid CS31A molecules or the purified chimeric proteins were used for mice immunization. By using the C epitope derived from the S protein of the porcine transmissible gastroenteritis virus, significant antiviral antibody titers were elicited and seroneutralization of the virus was demonstrated, confirming that the molecular environment in V3 is favorable for antigen presentation. These results indicate that synthesis of CS31A hybrid proteins by the wild-type strain 31A could become a powerful tool for the development of oral vaccines directed against mucosal pathogens.

[Factors and markers of virulence of Escherichia coli strains isolated from diarrhea in calves aged 4-45 days in Algeria]. / Facteurs et marqueurs de virulence de souches Escherichia coli isolées de diarrhées chez des veaux âgés de 4 à 45 jours en Algérie.

The study involved 492 strains of Escherichia coli isolated from the feces of 44 diarrhoeal calves and 4 healthy calves in 7 wilayates in Algeria (Tipaza, Ain Defla, Bejaïa, Borj Bou Arreridj, Bouira, Medea and Algiers). The authors looked for the surface proteins K99, CS31A, Vir, F17 (FY), 20K and certain factors or markers of virulence such as the production of colicins, particularly colicin V, the aerobactin siderophore, alpha hemolysin and enterohemolysin. They also studied the frequency of certain 0 serogroups and evaluated the resistance of the E. coli strains to 10 antibiotics. The results showed that the majority of diarrhoeal calves were colonized by Escherichia coli expressing one or more factors of virulence and that the stocks which produce the antigen CS31A are usually resistant to 4 or 6 antibiotics.