Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae

To express high quantities of colonization factor antigen I (CFA/I) derived from enterotoxigenic Escherichia coli (ETEC) for use in ETEC vaccines, the entire CFA/I operon consisting of four genes (cfa-A, -B, -C, -E) was cloned into plasmid expression vectors that could be maintained either with or without antibiotic selection. Expression from the powerful tac promoter was under the control of the lacIq repressor present on the plasmids. Fimbriae were expressed on the surface of both a non-toxigenic E. coli K12 strain and a non-toxigenic strain of Vibrio cholerae following induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). It was found that the recombinant E. coli strains expressed up to 16-fold higher levels of CFA/I fimbriae compared to a reference strain which had previously been shown to be among the highest natural producers of the CFA/I fimbriae among tested wild type ETEC strains. Oral immunization with formalin-killed recombinant E. coli bacteria over-expressing CFA/I induced significantly higher serum IgA and IgG+M antibodies responses compared to the reference strain. Oral immunization with formalin-killed recombinant V. cholerae bacteria also induce strong CFA/I-specific serum IgA and IgG+M responses. We conclude that our constructs may be useful as candidate strains in an oral killed CF-ETEC vaccine.     

Epidemiology and properties of heat-stable enterotoxin-producing Escherichia coli serotype O169:H41.

Enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 organisms have become the most prevalent ETEC in Japan since the first outbreak in 1991. It was assumed that the outbreaks were due to clonal spread of this new ETEC serotype. The relationship of 32 strains isolated from 6 outbreaks were examined for biotype, antibiotic susceptibility, enterotoxigenicity, protein banding pattern, lipopolysaccharide banding pattern, plasmid analysis, and ribotyping. Further, the strains were examined by haemagglutination, surface hydrophobicity, and the ability to adhere to HEp-2 cells. The present study suggests that the outbreaks were caused by multiple clones of STp-producing O169:H41 since they showed differences in ribotype and outer membrane protein banding patterns. The strains did not agglutinate human or bovine red blood cells in a mannose-resistant manner. They adhered to HEp-2 cells in a manner resembling enteroaggregative E. coli. Five strains were examined by dot-blot tests for the colonization factor antigens CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, PCFO159, PCFO166 and CFA/III. Although four strains expressed CS6, no structure for CS6 was identified. A strain that the anti-CS6 MAbs did not react with could adhere to HEp-2 cells in mannose resistant manner; thus, it is unlikely that CS6 play an important role in the adhesion to the cells. Electron microscopy studies of the O169:H41 strains suggested that curly fimbriae, a possible new colonization factor, may be playing an important role in the adhesion of the bacteria to HEp-2 cells. In conclusion, outbreaks due to ETEC O169:H41 were caused by multiple clones, and the strains should be examined in detail for a possible new colonization factor.     

Fimbriae as a pathologic factor of bacteria and a carrier in conjugate vaccines

Fimbriae play important role as pathogenic factors in many bacteria by their adhesive properties. Adhesin is located at the tip of fimbriae but also in other parts of fimbriae. Recent findings on structure of fimbriae genes and their expression for the biosynthesis and formulation of complete fimbriae have been described. Special attention was focused on the participation of fimbriae in the mechanism of pathogenesis and their specificity towards tissue receptors. Most recent studies have been performed on E. coli and Klebsiella and those data predominate in this work. Fimbriae can be used for the construction of vaccine as a proteinous carrier for haptenic carbohydrate epitopes. In conjugates fimbriae express distinct immunogenic, adjuvant and protective properties.     

Prime-boost vaccine regimen confers protective immunity to human-derived enterotoxigenic Escherichia coli

Development of effective vaccines against diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains is still a priority for those living at or traveling to endemic regions. In this work, we evaluated the protective role of an anti-ETEC vaccine regimen based on parenteral priming with a DNA vaccine, pRECFA, followed by oral boosting with a recombinant attenuated Salmonella Typhimurium vaccine strain, HG3, both encoding the same antigen, the structural subunit (CfaB) of the ETEC CFA/I fimbriae. The DNA-priming Salmonella-boosting protocol enhanced both murine anti-CfaB serum IgG and fecal IgA antibody responses and increased the ability of serum antibodies to inhibit the adhesive properties of the CFA/I fimbriae expressed by live bacteria, as compared to mice immunized with only one vaccine type. Addition of a mucosal adjuvant (LTR192G) to the Salmonella vaccine strain further enhanced the synergic effects of the vaccine regimen on the induced CfaB-specific antibody responses. DBA/2 dams submitted to the prime-boost regimen transferred complete passive protection to suckling neonates challenged with a virulent ETEC strain. Detection of milk anti-CfaB IgA antibodies and protection conferred by vaccinated dams to neonates born from non-vaccinated dams indicated that secretion of antigen-specific IgA is the immune response induced by the protective vaccine regimen. These results demonstrate that priming with a DNA vaccine and boosting with a Salmonella strain enhances both quantitatively and qualitatively the antibody responses to the CfaB antigen and represents an alternative for either active or passive immunization approach to ETEC-associated diarrhea.     

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.     

徐州市小肠结肠耶氏菌的病原性实验研究

目的：研究徐州地区从腹泻病人、家养动物中分离的16株、5种血清型的小肠结肠耶氏菌的病原性。方法：用生化生物分型，自凝性（AA）试验、钙依赖性（CD）试验，PCR检测毒力基因来检验不同菌株的致病性。用ATB Expression自动鉴定系统对致病菌株做药敏试验。结果：仅2株狗源性菌与1株人源性菌为致病菌，他们均为生物3型／血清型O：3，二者耐药模型完全一致。结论：徐州地区，小肠结肠耶氏菌生物3型／血清型O：3是致病菌型，他在狗和人群之间分布关系密切，其他型别菌株可能不致病或潜在致病。     

DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC)

The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.     

Infection with colonization factor antigen I-expressing enterotoxigenic Escherichia coli boosts antibody responses against heterologous colonization factors in primed subjects.

Enterotoxigenic Escherichia coli (ETEC) adhere to the intestinal mucosa by a number of fimbrial colonization factors (CFs) that have been claimed to induce only type-specific immunity. However, adult Bangladeshi patients infected with CFA/I-expressing bacteria, developed significant plasma IgA antibody responses, as determined by enzyme-linked immunosorbent assay, not only against the homologous fimbriae but also against several heterologous CFs, i.e. CS1, CS2, CS4 and PCFO166 fimbriae. In contrast, North American volunteers, who had probably not been infected by ETEC previously, responded with serum IgA against CFA/I fimbriae but not against any other CFs after symptomatic infection with CFA/I-expressing ETEC. Thus, infection with CFA/I-expressing bacteria may boost immune responses against CFs with a related amino acid sequence in previously primed subjects.     

Construction and evaluation of Bordetella pertussis live attenuated vaccine strain BPZE1 producing Fim3

Pertussis or whooping cough is currently the most prevalent vaccine-preventable childhood disease despite >85% global vaccination coverage. In recent years incidence has greatly increased in several high-income countries that have switched from the first-generation, whole-cell vaccine to the newer acellular vaccines, calling for improved vaccination strategies with better vaccines. We have developed a live attenuated pertussis vaccine candidate, called BPZE1, which is currently in clinical development. Unlike other pertussis vaccines, BPZE1 has been shown to provide strong protection against infection by the causative agent of pertussis, Bordetella pertussis, in non-human primates. BPZE1 is a derivative of the B. pertussis strain Tohama I, which produces serotype 2 (Fim2) but not serotype 3 fimbriae (Fim3). As immune responses to fimbriae are likely to contribute to protection, we constructed a BPZE1 derivative, called BPZE1f3, that produces both serotypes of fimbriae. Whereas nasal vaccination of mice with BPZE1 induced antibodies to Fim2 but not to Fim3, vaccination with BPZE1f3 elicited antibodies to both Fim2 and Fim3 at approximately the same level. In mice, both BPZE1 and BPZE1f3 provided equal levels of protection against clinical isolates that either produce Fim2 alone, both Fim2 and Fim3, or no fimbriae. However, vaccination with BPZE1f3 provided significantly stronger protection against Fim3-only producing B. pertussis than vaccination with BPZE1, indicating that immune responses to fimbriae contribute to serotype-specific protection against B. pertussis infection.     

Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates

Enterotoxigenic Escherichia coli (ETEC) are the most common cause of bacterial diarrhea in young children in developing countries and in travelers. Efforts to develop an ETEC vaccine have intensified in the past decade, and intestinal colonization factors (CFs) are somatic components of most investigational vaccines. CFA/I and related Class 5 fimbrial CFs feature a major stalk-forming subunit and a minor, antigenically conserved tip adhesin. We hypothesized that the tip adhesin is critical for stimulating antibodies that specifically inhibit ETEC attachment to the small intestine. To address this, we compared the capacity of donor strand complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, and CFA/I fimbriae to elicit anti-adhesive antibodies in mice, using hemagglutination inhibition (HAI) as proxy for neutralization of intestinal adhesion. When given with genetically attenuated heat-labile enterotoxin LTR192G as adjuvant by intranasal (IN) or orogastric (OG) vaccination, dscCfaE exceeded CFA/I fimbriae in eliciting serum HAI titers and anti-CfaE antibody titers. Based on these findings, we vaccinated Aotus nancymaae nonhuman primates (NHP) with dscCfaE alone or admixed with one of two adjuvants, LTR192G and cholera toxin B-subunit, by IN and OG administration. Only IN vaccination with dscCfaE with either adjuvant elicited substantial serum HAI titers and IgA and IgG anti-adhesin responses, with the latter detectable a year after vaccination. In conclusion, we have shown that dscCfaE elicits robust HAI and anti-adhesin antibody responses in both mice and NHPs when given with adjuvant by IN vaccination, encouraging further evaluation of an ETEC adhesin-based vaccine approach.     

Duration of carriage and transmission of Yersinia enterocolitica biotype 4, serotype 0:3 in dogs.

Human infections with pathogenic strains of Yersinia enterocolitica have been linked to contact with dogs excreting these microorganisms. This study examines the carriage and transmission of Y. enterocolitica biotype 4, serotype 03 in dogs. Fourteen 6-month-old cross-bred dogs were separated into 5 groups, 2 containing 4 dogs (I and II) and the others 2 dogs (III-V). Each of the 4 dogs in Group I and 2 of the dogs in Group II were inoculated orally with the test strain. Bacteriological examination of faecal samples showed that dogs can be readily infected and can carry the organism for up to 23 days. The two in-contact dogs in Group II started to shed the test organism after 5 days. Subsequent transfer of these dogs to Group III and those in Group III to Group IV showed that Y. enterocolitica biotype 4, serotype 03 can be readily transmitted between dogs. At no time did any of the dogs show clinical signs of infection. Group V served as a negative control for the trial. These findings suggest that dogs can carry Y. enterocolitica biotype 4, serotype 03 asymptomatically and hence might act as a potential source of infection for people.     

Biotype traits and antibiotic susceptibility of Vibrio cholerae serogroup O1 before, during and after the emergence of the O139 serogroup.

Sixty-nine strains of Vibrio cholerae O1 isolated at different times were analysed to investigate if there were any differences among the O1 strains isolated before, during and after the advent of the O139 serogroup. Of the 69 O1 strains examined, 68 belonged to the Ogawa serotype while one belonged to the Inaba serotype. With the exception of one strain all other strains of V. cholerae O1 belonged to the eltor biotype. A single O1 strain isolated before the emergence of the O139 serogroup could not be classified as either eltor or classical biotype because it was resistant to both classical and eltor specific bacteriophages. Marked variations in the susceptibility to antibiotics of V. cholerae O1 isolated during the different periods were observed. In addition, strains of V. cholerae isolated after the epidemic of serogroup O139 in Calcutta showed an expanding R-type with resistance to a variety of drugs as compared to the O1 strains isolated before the advent of the O139 serogroup. From this study, it is clear that there is a substantial mobility in genetic elements of V. cholerae O1 which necessitates a continuous monitoring to keep abreast of the changing traits of the etiologic agent of cholera.     

解脲支原体不同生物群别对小鼠体外受精的影响

目的:观察不同生物型别的解脲支原体(UU)对小鼠体外受精的影响,以探讨其对体外受精致病性的差异。方法:用血清型3型UU代表含有较小基因的生物群1(或Parvo生物型),血清型8型UU代表含有较大基因的生物群2(或T960生物型)。取小鼠成熟卵丘-卵母细胞复合体随机分为实验组与对照组,在体外加入小鼠精子进行受精。对照组精卵在不含UU的HTF培养液中培养,实验组精卵分别在含102～105CCU/ml的8型UU、103～106CCU/ml的3型UUHTF培养液中培养48h。每24h在倒置显微镜下观察记录,通过统计各组的受精率、D2评分、4-细胞期胚胎形成率,评价受精及胚胎发育情况。以观察不同生物群UU对小鼠成熟卵子体外受精的影响。结果:3型UU浓度≤103CCU/ml、8型UU浓度≤102CCU/ml时,受精率、D2评分、4-细胞期胚胎形成率等胚胎发育指标与对照组比较差异均无统计学意义(P>0.05);3型UU浓度≥105CCU/ml、8型UU浓度≥104CCU/ml时,受精率、D2评分、4-细胞期胚胎形成率均较对照组低,且差异有统计学意义(P(0.01);而当3型UU浓度为104CCU/ml、8型UU浓度为103CCU/ml时,受精率与对照组比较差异无统计学意义。结论:UU能够干扰小鼠的体外受精,3型UU≥104CCU/ml、8型UU≥103CCU/ml浓度时就可对小鼠的体外受精造成抑制作用。代表生物群2的血清型8型UU可能较代表生物群1的血清型3型UU具有更强的致病性。     

An interesting problem of strain identification with urinary isolates of Escherichia coli

Twenty-one cultures of Escherichia coli, recovered over a 5-year period from 14 urine specimens from a patient, were examined by multiple typing techniques selected for their reproducibility, their proven strain differentiating ability and for their suitability for use in small laboratories. Six isolates were readily identified as belonging to three distinct strains. Despite the finding that the remaining 15 isolates showed minor variations in resistotype and serotype characters, all 15 isolates belonged to the same full biotype. They were probably representatives of a fourth strain distinct from the other three strains identified, but even complete serological analysis could not resolve the problem of their strain identity.     

Discrimination between multiply-resistant klebsiella strains during a hospital outbreak: use of klebecin-typing and a screening test for plasmids

During an outbreak of hospital infection, 71 multiply-resistant klebsiella strains were classified by two extra tests in addition to biotyping, serotyping and sensitivity testing. These were klebecin sensitivity tests (Edmondson & Cooke, 1979) and screening for plasmid DNA molecules by agarose gel electrophoresis of single colony lysates (Eckhardt, 1978). There were 56 examples of an epidemic strain of serotype K21; ten of these differed from 46 identical strains in some minor character. The other 15, although resembling the epidemic strain in biotype and antibiotic resistance differed from it sufficiently to be considered as unrelated.     

Formulation and characterisation of Bordetella pertussis fimbriae as novel carrier proteins for Hib conjugate vaccines

Haemophilus influenzae type b (Hib) capsular polysaccharide (polyribosylribitol phosphate, PRP) is the active component of conjugate vaccines that have proven successful in preventing invasive Hib disease. Conjugation of PRP to a protein carrier greatly improves its immunogenicity providing protection in infants and subsequent antibody maturation upon boosting. In this study, fimbriae isolated from Bordetella pertussis have been assessed as novel carrier proteins. These proteins are components of some acellular pertussis vaccines and clinical trials have indicated that fimbriae could be important protective antigens against whooping cough. Fimbriae (Fim2 and Fim3) purified from B. pertussis were dissociated in 6 M guanidine hydrochloride, pH 10.5, to produce proteins of defined size and to facilitate the production and characterisation of the conjugates. Both carbodiimide-mediated coupling and reductive amination were used to conjugate PRP to dissociated fimbriae. Efficiency of conjugation was determined by size exclusion chromatography followed by protein and polysaccharide analysis of fractionated components. Immunisation of rabbits with dissociated fimbriae-PRP conjugates (D.fim-PRP) produced high anti-fimbrial and anti-PRP IgG titres. Use of a D.fim-PRP conjugate could protect against Hib disease and may also augment protection against B. pertussis.     

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle.     

我国十省市空肠/结肠弯曲菌的生物型和血清型分布

本文采用Lior法对我国十省市不同来源的301株空肠/结肠弯曲菌进行了生物学分型与血清学分型研究。研究结果表明弯曲菌的生物型分布无明显地区差异,血清学总分型率为86.71%,共分出41个血清型。人源菌与鸡、猪。鸭源菌的血清型分布关系密切,其最常见的5个血清型中均包括LIO46、45和8。建立了3种新血清型LION-1、N-2和N-3。同一宿主可以同时感染2种血清型的弯曲菌。生物型和血清型分布之间未见特殊联系。     

东阳市鼠类中小肠结肠炎耶尔森菌的分子生物学浅探

目的:了解东阳市鼠类中分离的40株小肠结肠炎耶尔森菌的生物分型、血清分型、毒力基因和抗生素敏感性。方法:采用生化微量管、诊断血清凝集法、PCR方法及ATB G-5药敏系统进行相应的检测。结果:40株菌株可以分生物1A型、生物2型和生物3型3个生物型;血清型以O:8型为主、其次O:1,2、O:5和无法分型的;利用PCR方法检测得到ail-、ystA-、ystB+、yadA-、virF-和ail-、ystA-、ystB-、yadA-、virF-两个毒力基因型别;抗生素除对头孢噻吩、阿莫西林和替卡西林有较强的耐药性外,其余抗生素的敏感率较高。结论:东阳市鼠类中分离的小肠结肠炎耶尔森菌,虽然存在多种生物型和血清型,但都是不携带毒力基因的菌株,为非致病菌;对部分抗生素有一定的耐药性,但绝大部分抗生素敏感。     

Excretion of Yersinia spp. associated with consumption of pasteurized milk

Yersinia enterocolitica biotype 1, serotype O.10K was isolated from 19 patients in the paediatric wards of a district general hospital over a period of 3 months. Fifteen cases were patients on the medical ward. Shortly afterwards, Y. enterocolitica biotype 1, serotype O.6, 30 was isolated from a further 17 patients on this ward in 1 month. The same serotypes of Y. enterocolitica were isolated from the pasteurized milk supplied to the ward. Epidemiological evidence indicated that contaminated pasteurized milk was the source of the yersinia organisms excreted by the patients.