Studies on the ribosomal RNA operons of Listeria monocytogenes

Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves. A fimbrial component with an M(r) of 17,200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E. coli strain 4787 of serogroup O115. This fimbrial component of F165 antigen was named F165(2). Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic. The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae. The amino acid sequence was determined for approximately 40% of the molecule. For the first 33 residues, the F165(2) sequence was identical to that of F1B fimbriae and very similar to that of F1C. Fimbriae F165(2) could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera.     

Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup O166

Enterotoxigenic Escherichia coli (ETEC) of serogroup O166 gave mannose-resistant haemagglutination (MRHA) with bovine and human erythrocytes. The strains did not react with antisera prepared against the known colonization factors CFA/I, CFA/II, CFA/III, CFA/IV and PCFO159:H4. Strain E7476 of serotype O166:H27, which produced heat-stable enterotoxin (ST), was examined initially. It produced fimbriae about 7 nm in diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular mass 15.5 and 17.0 kDa were seen. When variants of strain E7476 were isolated, loss of ST and MRHA together was associated with loss of a 98 MDa plasmid, while loss of ST alone correlated with plasmid deletion. An absorbed anti-strain E7476 antiserum reacted specifically with the 15.5 and 17.0 kDa polypeptides in Western immunoblotting and bound to the intact fimbriae by immuno-electron microscopy. When this antiserum was used in an ELISA to examine other strains of serogroup O166, a positive reaction was obtained with all the ST- and MRHA-positive strains. One strain of serotype O71:H27 and two strains of serotype O98:H- also reacted with the absorbed anti-strain E7476 antiserum. The antiserum did not react with ETEC carrying known colonization factors. E. coli K12 and a number of E. coli of different serotypes carrying a plasmid coding for ST transferred from strain E7476, all gave MRHA and reacted with the absorbed anti-strain E7476 antiserum. The term putative colonization factor O166 (PCFO166) is proposed to describe the adhesive factor(s) on ETEC of serogroup O166 because of the similarity of properties with those of known colonization factors.     

Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E. coli strain from a diseased animal

Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E. coli fimbrial adhesins. This strain produces two fimbriae: F165(1) and F165(2). F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon. Data from this study suggest that these two operons are part of two PAIs. PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E. coli chromosome. PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes. PAI II(4787) is found between the proA and yagU at 6 min of the E. coli chromosome.     

Incidence of enteritis of Enterobacteriaceae isolates possessing human colonization factor antigen. New human colonization factors

Of 462 Enterobacteriaceae strains including 435 Escherichia coli isolated from 250 patients, 298 haemagglutinating (HA) cultures were classified into 36 different HA groups. Sixteen of them belonged to Evan's I or II groups, although none possessed CF I or CF II antigen detectable by slide agglutination. Seventy-seven strains showed 4+ mannose resistant (MR) HA with human (53), bovine (2), chicken (6), guinea pig (7) or human and guinea pig (9) erythrocytes. These strains were significantly more frequent in patients under one year of age. Eighty-eight percent of the typable strains belonged to E. coli serogroups O1, O2, O4, O6, O18. HA positivity and fimbrial structures were correlated in 2 isolates (15/1, O18a, c:-K77: H-; 12/2/1 O1: K1: H .). Fimbriae of the two strains exhibited adhesive properties. Their fimbrial antigens differed serologically from each other and from those of the reference strains H 10407 and PB 176. Forty-nine of 4+ human MRHA strains showed variable reactions in the two sera for the new fimbrial antigens.     

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.     

The possession of three novel coli surface antigens by enterotoxigenic Escherichia coli strains positive for the putative colonization factor PCF8775

A possible colonization factor, E8775, has previously been described for enterotoxigenic Escherichia coli strains of serogroups O25, O115 and O167. Re-examination of these strains by immunodiffusion has revealed that the antigenic nature of this factor, renamed putative colonization factor (PCF) 8775, is more complex than was first thought. All the strains of serogroup O25 tested possessed two antigenic components, termed CS4 and CS6, and gave mannose-resistant haemagglutination (MRHA) of human and bovine erythrocytes. Spontaneous variants possessing CS6 only did not give MRHA. Strains of serogroups O115 and O167 had the antigenic components CS5 and CS6, and gave MRHA of human, bovine and guinea-pig erythrocytes. Using immune electron microscopy, the components CS4 and CS5 were identified as fimbriae. No fimbriae were associated with CS6.     

Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli.

The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.     

Isolation and characterization of a new fimbrial antigen (CS1541) from a porcine enteroxigenic Escherichia coli O8: KX105 strain

Abstract A fimbrial antigen (CS1541) was extracted and purified from the porcine enterotoxigenic Escherichia coli strain 1541P (O8:K-:H9). CS1541 fimbriae appeared as long thin filaments 3–5 mm in diameter. CS1541 antigen consisted of two peptide bands of about 18 and 19 kDa as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. It was expressed both at 37°C and 15°C and did not demonstrate hemagglutinating properties. It was antigenically distinct from the fimbrial antigens K88, K99, F41, FY(Att25), F165, type 1, CFA-I, and CFA-II complex but demonstrated serological cross-reactions with the 987P fimbrial antigen.     

Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis.

The aggregative pattern of adherence (AA) exhibited by enteroaggregative Escherichia coli upon HEp-2 cells is a plasmid-associated property which correlates with aggregative adherence fimbria I (AAF/I) expression and human erythrocyte hemagglutination. By using cloning and mutagenesis strategies, two noncontiguous plasmid segments (designated regions 1 and 2) required for AA expression have previously been identified in enteroaggregative E. coli 17-2. TnphoA mutagenesis was performed on clones containing region 1, and 16 TnphoA mutants which were negative for the AA phenotype were analyzed. The TnphoA insertion site for each mutant was determined by junctional DNA sequencing. All 16 mutations occurred within a 4.6-kb span in region 1. Nucleotide sequence analysis of the region revealed four contiguous open reading frames, designated aggDCBA, in the same span. AA-negative TnphoA insertions into all open reading frames except aggB were obtained. On the basis of mutational analysis and protein homology data, it is inferred that aggA, aggC, and aggD are involved in biogenesis of AAF/I, encoding a major fimbrial subunit, outer membrane usher, and periplasmic fimbrial chaperone, respectively. By immunogold electron microscopy, polyclonal antiserum raised against the aggA gene product decorated AAF/I fimbriae, affirming that AggA encodes an AAF/I subunit.     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

Prevalence of CS31A and F165 surface antigens in Escherichia coli isolates from animals in France, Canada and India

Bovine and porcine enterotoxigenic and non-enterotoxigenic Escherichia coli isolates from France, Canada, and India were characterized with respect to serogroup and production of fimbrial antigens CS31A and F165. Of 231 bovine isolates from the 3 countries, 20.5% produced CS31A alone, 17.7% produced F165 alone, and 17.3% produced both CS31A and F165. On the other hand, of 84 porcine isolates from Canada, 1.2% produced CS31A alone, 14.3% produced F165 alone, and no isolate produced both CS31A and F165. CS31A was found together with F5 (K99) in 7 of 16 bovine enterotoxigenic E. coli isolates of serogroups 08, 09, 020, and 023, but was not found in any of 20 F4 (K88)- or 5 F6 (987P)-positive porcine enterotoxigenic E. coli isolates. F165 was not found in enterotoxigenic E. coli. Among non-enterotoxigenic isolates, CS31A and F165 were mainly associated with serogroups 08, 09, 011, 015, 017, 023, 025, 078, 0101, 0115, 0117, 0141, and 0153.     

The role of capsular antigens in serum resistance and in vivo virulence of Escherichia coli

Abstract To identify critical microbial determinant(s) in serum resistance and in vivo virulence, 5 strains of Escherichia coli with different combinations of K and O types were examined along with their unencapsulated mutants. All 3 E. coli strains possessing the K1 capsule were considerably more resistant to normal human serum in vitro and more virulent in newborn rats than their isogenic mutants lacking the K1 capsule. In contrast, two K5 encapsulated strains and their unencapsulated mutants were essentially the same for their degree of serum resistance and in vivo virulence. These findings suggest that for the demonstration of serum resistance and in vivo virulence, a capsule is required for E. coli strains encapsulated with K1, whereas certain E. coli strains encapsulated with K5 may not need a capsule.     

Expression of antigenically distinct fimbriae with hemagglutination and HeLa cell adherence properties by an enteroaggregative Escherichia coli strain belonging to the enteropathogenic serogroup

Abstract An enteroaggregative Escherichia coli (EAggEC) strain (DS92), isolated from a case of infantile diarrhea, was shown to express mannose-resistant hemagglutination and HeLa cell adhering properties when grown at 37°C but not at 28°C. Cellular adherence properties of DS92, which belonged to enteropathogeci serogroup 0125, were shown to correlate well with the expression of fimbriae that were encoded by a 112 kb plasmid. The fimbriae of the EAggEC strain DS92 were composed of 20 kDa subunit proteins and were serologically distinct from fimbrial or non-fimbrial cell surface antigen(s) of other diarrheagenic E. coli strains including the reference EAggEC strain 17-2. Interestingly, the 20-kDa fimbrial protein was found to be antigenically related to 18- and 14.5-kDa cell surface proteins of two other locally isolated EAggEC strains belonging to the enteropathogenic serogroup 086.     

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.     

Aerobactin production and dulcitol fermentation in clonal groups of Escherichia coli O1: K1 strains from urinary tract infections

Abstract 70 urinary Escherichia coli O1:K1 strains were characterized for O1 antigen factors, mannose-resistant hemagglutination of human erythrocytes, flagellar and fimbrial antigens, dulcitol fermentation and aerobactin production. On the basis of their O1 and H antigens the strains could be assigned to 6 distinct groups. The most prevalent groups were: O1abcd: H⁻ :F9 (33 strains; pattern II), O1abc: H⁻ :F11 (9 strains; pattern IV), and O1abc: H7: F11 (19 strains; pattern V). Strains with patterns IV and V, both expressing fimbrial antigen F11, fermented dulcitol and produced aerobactin, whereas strains with pattern II were negative for both characteristics.     

Urea metabolism in the cyanobacterium Anabaena cycadeae: regulation of urea uptake and urease by ammonia

A total of 160 Escherichia coli positive for F165 fimbrial antigen and isolated from diarrheic and septicemic animals, were examined for the presence of the pap, afa, and sfa/foc operons or related nucleotide sequences using colony hybridization. Most isolates shared DNA sequences with the pap operon sequences alone or in association with afa or sfa. Thus, our results indicate that F165-positive E. coli from diseased animals share DNA sequences with operons coding for adhesins important in human extra-intestinal disease and that multiple adhesin systems are often found in single isolates. However, 20% of the F165-positive isolates did not show any homology with the probes representing the three adhesin systems, suggesting that one of the operons responsible for F165 production could be different from the pap, sfa/foc, and afa operons.     

occurrence of mannose- resistant adhesins MRHA in E.Coli strains isolated from children with diarrhea]

The study was aimed at determination of the frequency of occurrence of mannose-resistant adhesins in E. coli strains isolated from children with diarrhoea. It was also of interest whether their presence is associated with the serological type or other virulence factors. The material used in this study consisted of 1022 strains of E. coli (EPEC, ETEC and EIEC) and 3431 isolates from sick children and 960 from healthy children (non-EPEC-ETEC-EIEC). Enterotoxigenicity and entero-invasiveness of strains was evaluated by biological tests performed on animals and in tissue culture. Production of MRHA adhesins was determined by the test of mannose-resistant active hemagglutination, and of colonization factors antigens CFA by application of agglutination and agar gel immunodiffusion tests. Most frequently MRHA adhesins were produced by ETEC strains-80% of strains. All of them appeared to be a colonization factor antigen CFA/I. EPEC strains produced various MRHA adhesins only by 12.6% of strains. Production of MRHA adhesins by EIEC strains was not detected. Frequency of occurrence of MRHA adhesins in E. coli strains which were non-EPEC-ETEC-EIEC was dependent from the isolation source. MRHA adhesins were most frequently found in strains isolated from sporadic cases of light diarrhoea in ambulatory treated children (49%), much less among isolates from children hospitalized because of severe diarrhoea (33%), and from healthy children in 9% of isolates only. These results may indicate the potential role of MRHA adhesins in pathogenesis of diarrhoea in children.     

Comparative study of inhibition of mannose-resistant haemagglutination caused by CFA/I, CFA/II, K88 and K99-positive Escherichia coli strains

Abstract We have studied the inhibition of mannose-resistant haemagglutination (MRHA) caused by Escherichia coli strains with CFA/I, CFA/II, K88, K99 and by other faecal E. coli lacking these colonisation antigens, by means of 30 sugar compounds and by enzymatic treatment of erythrocytes with neuraminidase, α-mannosidase, β-galactosidase, trypsin and pronase, and with formaldehyde. Inhibition of MRHA by sugars was effective only in K88-positive strains with d (+)glucosamine, mucic acid and bovine submaxillary mucin. Enzymatic treatment and the formolisation of erythrocytes gave different results on MRHA activity in strains possessing each colonisation antigen type. Results suggest that the erythrocyte receptor for CFA/I and CFA/II may possibly be sialoglycoprotein in which N -acetylneuraminic acid (NANA) plays an important role, because MRHA activity in these strains was inhibited by treatment of erythrocytes with neuraminidase and pronase. On the other hand, erythrocyte receptors for K88 and K99, like receptors for haemagglutinins of faecal E. coli lacking these colonization antigens, may have other glycoconjugate structures in which proteins and NANA are not essential. Our observations also suggest that the nature (or structure) of the receptor for a specific colonisation antigen on diverse erythrocyte types may be different.     

F1651 fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils

The F165(1) fimbrial system has been associated with the resistance of Escherichia coli O115:K"V165" to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs). One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F165(1) fimbriae. In order to confirm whether or not the F165(1) fimbriae are involved in this inhibition, we evaluated the effect of F165(1)-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F165(1)-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA. Incubation of pNs with pathogenic E. coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2-) release from the phagocytes, and by the chemiluminescence assay. Similarly, incubation of pNs with the F165(1)-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101. In contrast, addition of purified F165(1) fimbriae to the pNs had no effect on the oxidative response.