Cloning of genes encoding coli-surface (CS) antigens in enterotoxigenic Escherichia coli

Abstract Sequences encoding the CS6 antigen of colonisation factor antigen (CFA)IV were cloned on a 3kb Cla I fragment. The recombinant plasmid pDEP5 coded for surface expression of CS6 measured by ELISA and production of CS6 polypeptides was detected in E. coli minicells. The genes for the CS1, CS2 and CS3 components of colonisation factor antigen CFA/II were cloned together on a length of DNA corresponding to about 17kb. CS3 was always expressed but production of either CS1 or CS2 depended on the serotype and biotype of the host strain. Separate subclones were obtained that expressed CS3 or CS1 and CS2.     

Serologically distinct fimbriae on enterotoxigenic Escherichia coli of serotype O6:K15:H16 or H-

Abstract Serologically distinct, surface-associated, mannose-resistant haemagglutinins have been reported on enterotoxigenic Escherichia coli of serotype O6:K15:H16 or H- of human origin. Using immune electron microscopy, these haemagglutinins, termed the CS1 and CS2 antigens, have been identified as serologically distinct fimbriae, the presence of which correlates with the rhamnose fermentation phenotype of strains. The CS1 and CS2 fimbriae are morphologically indistinguishable from common type fimbriae. In contrast, using the same technique, no labelling of fimbriae was obtained with specific antibodies to another protein surface antigen termed the CS3 antigen, which is common to most strains of this serotype and also found on certain enterotoxigenic E. coli of serotype O8:H9. The morphological nature of the CS3 antigen was not disclosed.     

Expression of CS fimbriae in Escherichia coli strains of human and animal origin belonging to O-serovar 6, K-serovar 15 or H-serovar 16

Abstract A non-conjugative CS-fimbriae-associated plasmid pCS001 was mobilized into rifampicin-resistant mutants of strains of Escherichia coli of O-serovar 6 or K-serovar 15 or H-serovar 16. By a variety of serological procedures only the production of CS3 fimbriae was detected in transoconjugants. The finding extend previous observations that only strains of E. coli of serotype O6:K15:H16 or H− and of appropriate biotype are able to express either CS1 or CS2 fimbriae, as even a recipient of serotype O6:K15:H31 possessing a rhamnose-positive fermentation phenotype did not express either of these two fimbriae. The results indicate that expression of CS1 or CS2 fimbriae probably involves chromosomal determinants only found in strains of serotype O6:K15:H16 or H−.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.     

Chromosomal mapping of genes encoding mannose-sensitive (type I) and mannose-resistant F8 (P) fimbriae of Escherichia coli O18:K5:H5

DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pil) exist in a single copy on the chromosome of E. coli O18:K5 strain 2980. In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined. The pil genes were mapped close to the gene clusters thr and leu controlling the biosynthesis of threonine and leucine, respectively. The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E. coli chromosomal linkage map.     

Elimination of ColE1 (pBR322 and pBR329) plasmids in Escherichia coli by α-santonin

alpha-Santonin, a compound extracted from the flower heads of Arthemisia maritima plant, was effective in elimination of small, multicopy, relaxed plasmids (pBR322 and pBR329 with CoLE1 origin of replication in Escherichia coli, whereas plasmids of IncF1, H1 and X group were totally refractory under similar conditions, suggesting that this agent was specific in curing the CoLE1 group of plasmids.     

Characterization of the second long polar (LP) fimbriae of Escherichia coli O157:H7 and distribution of LP fimbriae in other pathogenic E. coli strains

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Magnesium ions are required for HEp-2 cell adhesion by enteroaggregative strains of Escherichia coli O126:H27 and O44:H18

Enteroaggregative strains of Escherichia coli, belonging to serotypes O44:H18 and O126:H27, were used to show that magnesium ions were essential for the adhesion of these enteroaggregative strains to HEp-2 cells. The removal of Mg²⁺ ions from culture media was correlated with the inability of strains to produce an outer membrane-associated protein of 18 kDa and a pellicle. It was concluded that magnesium ions were directly involved with the expression of an 18 kDa outer membrane-associated protein by strains of E. coli O126:H27 and O44:H18, and that the outer membrane-associated protein was involved in both HEp-2 adhesion and pellicle formation.     

Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166

Entertoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I. Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae. Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae. CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe. Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes. The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production. Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae.(ABSTRACT TRUNCATED AT 250 WORDS)