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.     

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.     

Properties of wild-type strains of enterotoxigenic Escherichia coli which produce colonization factor antigen II, and belong to serogroups other than O6

Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than O6 and produced colonization factor antigen II, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransferring plasmid, NTP165, from a strain of E. coli O168. H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTP165 depended on the recipient strain: a biotype A strain of serotype O6. H16 expressed CS1 and CS3; a biotype C strain of serotype O6. H16 expressed CS2 and CS3; strain K12 and strain E19446 of serotype O139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype O139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lost the plasmid coding for CS antigens produced both CS1 and CS3 after the introduction of NTP165.     

Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 025.H42

Two enterotoxigenic Escherichia coli strains of serotype 0.25.H42 that produced coli surface associated antigens CS4 and CS6 hybridized with a probe containing the cfaD sequence that regulates expression of colonization factor antigen CFA/I. Transformation of a cloned cfaD gene into some derivatives of the strains that were negative for CS4 and CS6 resulted in expression of CS4 but not CS6. By hybridization the sequence that regulated CS4 production in the wild type 025 strains was located on a plasmid that also encoded the CS6 antigen. The structural genes for the CS4 antigen were on a separate plasmid. The 025 strains carried a third plasmid encoding enterotoxin production which was therefore unlinked to regulation sequences or genes encoding CS antigens.     

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae

Abstract Porcine Escherichia coli strains isolated from cases fo postweaning diarrhea or edema disease were analysed for the presence of fedA , the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA -specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.     

Purification and characterization of two extracellular β-glucosidases from Aspergillus nidulans

We have characterized the toxic and adhesive properties of Escherichia coli strains producing the second type of cytotoxic necrotizing factor (CNF2) and belonging to the classic enteropathogenic serogroup O55. Bovine CNF2 strains of serotype O55:H4 express P fimbriae as do pyelonephritic Escherichia coli that cause infections in humans. In contrast, strains of serotype O55:H21 which produce CNF2 from bovine origin possess the Vir surface antigen. One human strain of serotype O55:H- was positive for production of CNF2, but was negative for the two adhesive factors and for mannose-resistant haemagglutination.     

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)     

Occurrence of type-1C fimbriae on Escherichia coli strains isolated from human extraintestinal infections

Two monoclonal antibodies specific for type-1C fimbriae of Escherichia coli were produced. In enzyme-linked immunosorbent assay and immunoblotting the antibodies, which were of the IgG1 isotype, reacted with type-1C, but not with P or type-1 fimbriae of E. coli strain KS71. Immunoblotting and immunoprecipitation of crude fimbrial extracts from 25 strains invariably gave an apparent molecular weight of 17 000 for the type-1C fimbrillin. A total of 313 E. coli strains, isolated from patients with extraintestinal infection or from faeces of healthy children, were screened for the presence of type-1C fimbriae using both the monoclonal and polyclonal antibodies. Of these, 45 (14%) strains had type-1C fimbriae, with the highest frequency (27%) on strains isolated from patients with pyelonephritis. No faecal strain had type-1C fimbriae, and the frequency on the other diagnostic groups ranged from 11 to 15%. Thus, no direct correlation between type-1C fimbriae and bacterial virulence in human extraintestinal infections was found. Type-1C fimbriae were detected on only a few E. coli serotypes, notably on all O6:K2:H1 and O22:K13:H1 strains tested.     

肠毒素性大肠杆菌CS3纤毛抗原在痢疾菌中的表达及其免疫效果研究

首先通过体内外重组的方法,构建了福氏2a痢疾菌T32asd基因缺陷的突变体FaD,作为抗原载体菌;同时,构建包含asd基因的表达质粒pYX102,与FaD一起,构成宿主-载体平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因.将肠毒素性大肠杆菌的CS3菌毛抗原基因克隆至pYX102,构建成重组表达质粒pYX103,ELISA检测结果证实CS3在痢疾菌中可以很好地表达.免疫小鼠后可诱生相应的抗体,虽然口服免疫和注射免疫产生的CS3抗体效价有一定差别,但对痢疾菌的毒株攻击均可提供较好保护.该结果为细菌性腹泻疫苗的研制提供了候选株.     

Expression of plasmids coding for colonization factor antigen II (CFA/II) and enterotoxin production in Escherichia coli

Two plasmids transferred from enterotoxigenic Escherichia coli (ETEC) of serotype O6. H16 and biotypes A and C coded for mannose-resistant haemagglutination (MRHA) and production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Both plasmids were nonautotransferring being mobilized most efficiently by the R plasmid R100-1. They were similar in their genetic properties being incompatible with each other and plasmids of the Inc group FI. The wild-type strains produced the colonization factor antigen II (CFA/II) which was made up of different coli surface antigens (CS). The biotype A strains produced CS1 and CS3 while the biotype C strains produced CS2 and CS3. These three antigens have the ability to cause MRHA. When plasmids coding for MRHA were transferred to K12 strains, the degree of haemagglutination was markedly reduced and only CS3 was produced. When both plasmids were transferred back into biotype A strains, good MRHA was restored and the strains produced CS1 and CS3. In a biotype C strain CS2 and CS3 were formed. The production of the antigens was compared by enzyme-linked immunosorbent assay (ELISA). The strains were also examined by electron microscopy where it was found that CS1 and CS2 were fimbrial antigens while CS3 was not.     

Analysis of fimbrial gene clusters and their expression in enterohaemorrhagic Escherichia coli O157:H7

The sequence of two enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains reveals the possession of at least 16 fimbrial gene clusters, many of the chaperone/usher class. The first part of this study examined the distribution of these clusters in a selection of EHEC/EPEC (enteropathogenic E. coli) serotypes to determine if any were likely to be unique to E. coli O157:H7. Six of the clusters, as determined by the presence of amplified main subunit or usher gene sequences, were detected only in the E. coli O157 and O145 serotypes tested. With the exception of one serotype O103 strain that contained an lpf2 cluster, lpf sequences were only detected in E. coli O157 of the serotypes tested. Expression from each cluster was measured by the construction of chromosomally integrated lacZ promoter fusions and plasmid-based eGFP fusions in E. coli O157:H7. This analysis demonstrated that the majority (11/15) of main fimbrial subunit genes were not expressed under the majority of conditions tested in vitro. One of the clusters showing promoter activity, loc8, has a temperature expression optimum indicating a possible role outside the host. From the presence of pseudogenes in three of the clusters, the lack of FimH-like minor adhesins in the clusters and their limited expression in vitro, it would appear that E. coli O157:H7 has a limited repertoire of expressed functional fimbriae. This restricted selection of fimbriae may be important in bringing about the tropism E. coli O157:H7 demonstrates for the terminal rectum of cattle.     

The fimbriae of human enterotoxigenic Escherichia coli strain 334 are related to CS5 fimbriae

Escherichia coli strain 334 is a human enterotoxigenic strain of serotype O15:H11 which had previously been shown to produce 'attachment pili'. These fimbriae were compared with other colonization factors. From strain 334 a mannose-resistant haemagglutination positive colony 334A and a mannose-resistant haemagglutination negative variant 334C were isolated. By electron microscopy the fimbriae of strain 334A were shown to have a helical structure resembling coli-surface-associated antigen (CS5) fimbriae. An antiserum was raised to strain 334A and absorbed with a fimbriae-negative variant of that strain, 334C. By immuno-electron microscopy this antiserum was shown to coat fimbriae of strain 334A but not CS5 fimbriae produced by strain E17018A. Conversely, CS5 antiserum did not coat the fimbriae produced by strain 334A. No antigenic cross-reaction was detected between these intact fimbriae when anti-strain 334A serum and CS5 antiserum were used in immunodiffusion tests. By enzyme-linked immunosorbent assays (ELISAs) the fimbriae of strain 334A were shown to be antigenically unrelated to most other human ETEC adhesins, namely colonization factor antigens (CFA/I, CFA/III and CFA/IV), coli-surface-associated antigens (CS1, CS2, CS3, CS4, CS6 and CS17) and putative colonization factors (PCFO159:H4 and PCFO166). However, a heated suspension of strain 334A reacted weakly with CS5 antiserum in an ELISA. By SDS-PAGE the fimbriae of strain 334A were shown to consist of subunits of similar size to CS5 subunits, that is about 21.5 kDa. Western immunoblotting revealed that the subunits of 334A and CS5 fimbriae shared common epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the F8 fimbrial antigen from Escherichia coli

Abstract The genetic determinant coding for the P-specific F8 fimbriae was cloned from the chromosome of the Escherichia coli wild-type strain 2980 (O18:K5:H5:F1C, F8). The F8 determinant was further subcloned into the Pst I site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned counterpart was demonstrated. The cloned F8 fimbriae and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepithelial cells. The cloned F8 determinant was well expressed in a variety of host strains.     

Immunogenicity of Vibrio cholerae O1 Fimbriae in Animal and Human Cholera

Parenteral immunization with either formalin-fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non-immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.     

P fimbriae on uropathogenic Escherichia coli O16:K1 and O18 strains

Abstract The fimbrial composition of 12 P-fimbriate uropathogenic Escherichia coli O16 and O18 strains was analysed by immunoprecipitation with 14 fimbria-specific antisera. All the O16 strains possessed a P fimbrial serovariant with an apparent M _r of 17500. One strain had an additional, serologically closely related P fimbria with an apparent M _r of 19 800. Two groups were found among the O18 strains; one possessing a type 1C fimbria and a 19800-Da P fimbria, the other lacking type 1C fimbriae and possessing a P-fimbrial variant with an apparent M _r of 17 800. Fimbriae on strains within the groups were serologically similar by immunoprecipitation assays. Also, the fimbriae on the O16 and O18 strains were mutually cross-reactive. The grouping of the O18 strains by fimbrial serology corresponded to the previous clonal grouping based on other phenotypic characters.     

Biochemical properties of enterotoxic and enterohemorrhagic E-coli strains

The purpose of this paper has been to describe the biological characteristics of thirty strains of E. coli. The E. coli strains were isolated from cases of colibacillosis in animals and from human faeces and cow milk samples. Of the thirty analyzed strains, 19 strains (63%) were found to belong to 7 serogroups: O8, O101, O138, O141, O147, O149 and O157. In 17 strains (57%) fimbriae F4 was discovered and in 1 strain (3%) the presence of fimbriae F5 and F41 was detected. Serological and biochemical researches, based on the analysis of 35 enzymatic reactions, confirmed that all strains, used in this study, belonged to the species E. coli. The strains demonstrated differences in biochemical activity for 12 substrates. It was found that strains of serotype O157: H7 had biochemical homogeneity, except in their rate of sucrose fermentation and their ability to hydrolyze arginine and sorbitol after longer incubation. On the basis of the biochemical activity, O157: H7 strains were affiliated with biotype C. In identifying serotype O157: H7, SMAC medium with sorbitol and liquid and solid media with MUG reagent were very useful.     

Discovery and characterization of a fructosylated capsule polysaccharide and sialylated lipopolysaccharide in a virulent strain of Actinobacillus suis

We are developing a serotyping system for Actinobacillus suis based on its capsule (K) and lipopolysaccharide O-chain (O) structures. Previously, we have shown that less virulent strains of this swine pathogen express a (1→6)-β-D-glucan as both K- and O-chain polysaccharides and were serologically classified as K:1/O:1. Here, we show that representative A. suis strains with a high (H91-0380; serotype K:2/O:2) and intermediate (C84; serotype K:2/O:1) degree of virulence possess a capsule polysaccharide (K:2) composed of an O-acetylated diglycosyl phosphate repeat decorated with fructose: [→4)-3-O-Ac-β-D-GlcpNAc-(1→3)-[β-D-Fruf-(2→2)]-α-D-Galp-(1→PO(4)(-)→]. In addition, the serotype O:2 lipopolysaccharide was shown to express a sialylated O-chain [→3)-β-D-Galp-(1→4)-[Neu5Ac-(2→3)-α-D-Galp-(1→6)]-β-D-Glcp-(1→6)-β-D-GlcpNAc-(1→]. As (1→6)-β-D-glucan is ubiquitous in the environment, low levels of antibodies in the animals are predicted to prevent disease by K:1/O:1 strains. The greater potential associated with K:2/O:2 and K:2/O:1 strains is most likely due to the absence of (1→6)-β-D-glucan as the K antigen and, in the case of K:2/O:2, the presence of sialic acid in the lipopolysaccharide, a nonulosonic acid known to promote evasion of host recognition.     

Characterization of uropathogenic E. coli O25b-B2-ST131, O15:K52:H1, and CGA: Neutrophils apoptosis,serum bactericidal assay,biofilm formation,and virulence typing

Pathogenic and drug-resistant strains of Escherichia coli (E. coli) O25b-B2-ST131, O15:H1-D-ST393, and CGA (clonal group A) clonal groups have spread worldwide. This study aimed at determining E. coli epidemic clonal groups, their virulence factors, biofilm formation, neutrophils apoptosis, and antimicrobial resistance pattern of uropathogenic E. coli. A total of 95 CTX-M-1-producing E. coli clinical isolates were enrolled. E. coli O25b-B2-ST131, CGA, and O15:K52:H1 were identified by serotyping and phylogrouping and allele-specific polymerase chain reaction-based assay. Antibiotic susceptibility, biofilm formation, hemolysis, and human serum bactericidal assay were performed. Neutrophil apoptosis was assayed by flow cytometry. Nine E. coli clonal groups including six O25b-B2-ST131 strains, two CGA, and one O15:K52:H1-D-ST393 strains were detected. One O25b-B2-ST131 isolate was a strong biofilm-producer. Three ST131 isolates had type I fimbriae. Furthermore, all the CGA and O15:K52:H1 and three of ST131 isolates harbored the P fimbriae. The virulence genes ompT, fimH, and traT were detected among all the clonal groups. The apoptosis was induced by O25b-B2-ST131, CGA, and O15:K52:H1 E. coli. There was no significant difference regarding apoptosis induction among clonal groups. Furthermore, the presence of the cdt, usp, and vat genes was significantly associated with the apoptosis of neutrophils by O25b-B2-ST131, CGA, and O15:K52:H1-D-ST393 clonal groups.