El Tor型霍乱弧菌L型诱导及其意义的探讨

本文报道儿种体内外因素可诱导 El Tor 型霍乱弧菌产生 L 型。羧苄青霉素纸片法和倾注平板法可诱导 El Tor 型霍乱弧菌产生长丝体、圆球体和巨形体。El Tor 型霍乱弧菌分型噬菌体可诱导产生长丝体。El Tor 型霍乱弧菌陈旧肉汤培养物中可出现许多圆球体样物。上述因素诱导的 L 型均不稳定,很容易回复为原菌。作者还用鳝鱼和鲫鱼的胆汁诱导 El Tor 型霍乱弧菌形成稳定 L 型,并对 EL Tor 型霍乱弧菌 L 型的流行病学意义及其与霍乱弧菌越冬的关系进行了讨论。     

免疫酶技术鉴定 El Tor 型霍乱弧菌稳定 L 型

细菌稳定 L 型的形态、培养特性以及生化反应常与原菌不同,其菌落在盐水中不能乳化,故不能通过玻片凝集测定其抗原。对于这种一时不能回复为原菌的 L 型很难进行鉴定。本文采用免疫酶技术对由鳝鱼和鲫鱼胆汁诱导的 El Tor 型霍乱弧菌稳定 L 型进行了鉴定。实验证明 L 型的细胞壁可有不同程度的缺失,稳定 L 型仍可能有少量“O”抗原存在。PAP 法比较敏感,即使少量抗原亦可以检出。     

用单克隆抗体ELISA研究和分析霍乱弧菌抗原结果

本文用ELISA以12株单克隆抗体(McAb)对55株EI Tor弧菌和用遗传学方法突变的28株霍乱弧菌的菌体抗原进行了研究,同时与四种常规分型方法进行对比试验。用12株McAb可将55株El Tor弧菌分成11种抗原决定簇和18个McAb型,将遗传学方法的突变株分成9种抗原决定簇和10个McAb型。其中一株McAb2B_1H_1F_3能识别所有55株EI Tor弧菌和大部分突变株霍乱弧菌,表明有种的特异性;其余11株McAb与各型霍乱弧菌的抗原反应,均有差异,与这些菌株的反应百分比各不相同(21.0％～100％),表明可能存在着亚型和亚群。文中着重讨论了用McAb研究分析抗原的重要意义和McAb用于诊断的价值。     

El Tor型CTXΦ对O1群不同霍乱弧菌的转染性研究

研究丝状噬菌体CTXΦ对O1群不同霍乱弧菌的水平转移效率及菌株的噬菌体免疫能力。利用带有氯霉素抗性基因遗传标记的CTXETΦ感染颗粒对O1群的4株不同霍乱弧菌进行体外和体内转染实验,根据氯霉素抗性筛选转染子,通过Southern Blot等方法进行验证并判断CTXΦ基因组的存在形式,计算比较不同菌株的转染率,分析转染及噬菌体免疫机制。带有遗传标记的CTXETΦ对古典型霍乱弧菌1119的体内转染率高于体外;体内转染实验中,古典菌株1119的转染率远高于其它3株El Tor型霍乱弧菌;在El Tor型霍乱弧菌中,不含rstR基因的IEM101的转染率高于另外两株带有rstR基因的霍乱弧菌2~3个数量级。古典型霍乱弧菌比El Tor型菌株对CTXETΦ噬菌体颗粒更易感,TCP菌毛的表达和rstR基因介导的噬菌体免疫影响CTXΦ在霍乱弧菌中的水平转移。     

枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究

为便于枯草芽孢杆菌工业化生产应用,对Spizizen创立的枯草芽孢杆菌DNA转化方法进行改进.用GMI和GMII溶液处理枯草芽孢杆菌野生型菌株BS501a、营养缺陷型突变株DBl342和非营养缺陷型突变株WB800,用改进的方法制备感受态细胞,用7.5kb质粒pSBPTQ进行转化,并研究RNA、酵母粉、水解酪蛋白、培养方法对枯草芽孢杆菌质粒转化的影响.结果表明,该方法适用于不同基因型枯草芽孢杆菌的质粒转化,营养缺陷型突变株DBl342的转化率为750 CFU/μg/DNA,非营养缺陷型突变株WB800转化率为1 070 CFU/xg DNA,野生型菌株BS501a转化率为270 CFU/μg/DNA.根据影响转化效率的因素,推测在该方法中,枯草芽孢杆菌质粒转化原理:一定生物量的枯草芽孢杆菌在外界营养条件和钙、镁离子作用下,细胞壁和细胞膜形成缺陷,使外源DNA转入枯草芽孢杆菌细胞内.     

El Tor型霍乱弧菌分泌性蛋白初步分析

摘要：【目的】利用高通量蛋白质组分析技术,初步探讨El Tor型霍乱弧菌的分泌性蛋白组成,为进一步的功能研究打下基础。【方法】选取El Tor型霍乱弧菌流行株N16961和非流行株92-3,用双相电泳和激光辅助解析-飞行时间串联质谱（Matrix Assisted Laser Desorption Ionization-Time of Flight,MALDI-TOF）技术对培养上清的全部蛋白质组份进行鉴定和分析。【结果】从N16961株培养上清中可检测到206个蛋白点,并鉴定出49种蛋白;从92-3株培养上清中可检测到236个蛋白点,并鉴定出42种蛋白,两株菌共鉴定蛋白68种,其中经预测含有信号肽的蛋白占总数的55.88%（38/68）。按功能不同将所鉴定出的蛋白分为10类,其中代谢酶、蛋白折叠/伴侣蛋白、蛋白合成以及信号传导相关蛋白占全部培养上清蛋白数的36.76%,转运蛋白占14.71%,鞭毛蛋白占11.76%,降解酶占10.29%,外膜蛋白占5.88%,毒素占1.47%,在所鉴定出的蛋白中有11种具有信号肽的假想蛋白和2种功能未知的蛋白为首次被实验证实可出现在霍乱弧菌培养上清中,占19.12%。【结论】初步获得了El Tor型霍乱弧菌流行株和非流行株的分泌性蛋白谱及其组成特征,并提示与霍乱弧菌致病关系密切的鞭毛蛋白在胞外释放机制、红血球凝集素/蛋白酶在非流行株中的高表达特征等值得高度关注。     

大连市两种来源的副溶血性弧菌分子分型

目的分析食品来源、患者来源及2种来源的副溶血性弧菌之间的PFGE图谱的关系,从分子流行病学角度探讨2种来源的副溶血性弧菌的关联。方法收集患者和食品2种来源的副溶血性弧菌178株,经限制性内切酶SfiI酶切,用脉冲场凝胶电泳方法进行电泳,凝胶成像仪获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析。结果食品来源的96株菌,有13株降解,83株菌被限制性内切酶SfiI酶切出83个PFGE types(PT),聚类分析发现各菌株间相似系数为55.6%~97.4%,按带型相似系数为85%标准划分为1~67共67个克隆群。患者来源的82株菌,有5株降解,77株菌被限制性内切酶SfiI酶切出46个PFGE types(PT),聚类分析发现各菌株间相似系数为64.1%~100.0%,按带型相似系数为85.0%标准划分为A~O群共15个克隆群。将2种来源的共160株副溶血性弧菌的PFGE图谱进行聚类分析,各菌株间相似系数为41.7%~100.0%,按带型相似系数为85.0%标准划分为79个克隆群。结论多数食品来源菌株间相似系数较低,患者来源菌株间相似系数高,而食品和患者来源菌株间相似系数较低,只有少数食品来源与患者来源菌株相似系数较高。     

血红铆钉菇氨基酸缺陷型菌株的紫外诱变选育

通过原生质体去细胞壁技术,以血红铆钉菇原生质体为材料,利用紫外线对其进行诱变处理,筛选出6株氨基酸营养缺陷型突变株,经稳定性试验确认1株突变株性状可以稳定遗传,利用生长谱法对缺陷型进行了鉴定、分析。结果表明,该菌株为L-半胱氨酸缺陷型菌株,为营养缺陷型突变株的筛选奠定了基础。     

对流免疫电泳对El Tor型霍乱弧菌与不凝集弧菌抗原相关性的初步探讨

本文对37株NAG 8株El Tor弧菌的抗原性进行了初步分析,用对流免疫电泳不同程度地出现有交叉沉淀线,这说明它们之间有血清学相关性;通过血清吸收试验,发现“H”抗原吸收后的血清大部分仍出现沉淀线,而用同一菌株的可溶性抗原吸收后的血清与5种NAG可溶性抗原进行对流免疫电泳,除少数仍有沉淀线外,大部分均消失,因而证明它们之间的抗原有相关性,而此相关部分并非弧菌共同的H抗原,以对流电泳与糖发酵反应双盲试验结果也证明二者之间亦有一定联系。本文还介绍了用去氧胆酸钠裂解细菌的方法、原理和注意事项。     

紫外诱变筛选海洋红酵母S8的尿嘧啶缺陷型菌株

本课题组从海南天然海域筛选到一株高产类胡萝卜素的海洋红酵母菌株S8,该菌株对鱼无毒害,并与鱼共生,欲将其应用于盐诱导表达外源蛋白的海洋红酵母工程菌的构建。本研究利用紫外诱变筛选的方法处理S8菌株,通过统计其UV致死率、5-氟乳清酸致死率等筛选S8的尿嘧啶营养缺陷型突变株。研究结果表明,供试菌株通过紫外线诱变、5-氟乳清酸致死和回复突变率的实验筛选,共获得16株稳定的尿嘧啶缺陷型突变株,突变菌株在基本培养基中培养了8d仍不能生长。选择了其中的一株ST5进行了产胡萝卜素能力的测定,结果表明,在同样的培养条件下,野生型S8菌株细胞生物产量可达87.55g/L,类胡萝卜素含量可达520μg/g,突变株ST5的细胞生物产量为85.45g/L,类胡萝卜素含量为512μg/g;ST5的产胡萝卜素能力方面与野生型S8无明显差异。因此,尿嘧啶缺陷型菌株ST5可为下一步海洋红酵母工程菌的构建提供受体菌。     

A 14-kilodalton inner membrane protein of Vibrio cholerae biotype e1 tor confers resistance to group IV choleraphage infection to classical vibrios.

Choleraphage phi 149 differentiates the two biotypes, classical and el tor, of Vibrio cholerae. This phage cannot replicate in V. cholerae biotype el tor cells because the concatemeric DNA intermediates produced are unstable and cannot be chased to mature phage DNA. A V. cholerae biotype el tor gene coding for a 14,000-Da inner membrane protein which destabilizes the concatemeric DNA intermediates by hindering their binding to the cell membrane has been identified. Presumably, a 22,000-Da V. cholerae biotype el tor protein might also have a role in conferring phage phi 149 resistance to cells belonging to the biotype el tor. A nucleotide sequence homologous to the 1.2-kb V. cholerae biotype el tor DNA coding for both the 14,000- and 22,000-Da proteins is present in all strains of classical vibrios but is not transcribed. The nucleotide sequence of the gene coding for the 14,000-Da protein has been determined.     

Hemolysin from Vibrio cholerae eltor: cloning and expression of genes in Escherichia coli]

A 6.56-kb V. cholerae eltor DNA fragment encoding hemolysin synthesis was cloned in pUC18. The resultant recombinant plasmid pES4H (9.25 kb) was mapped by restriction analysis and shown to express in different E. coli strains as well as in nonhemolytic V. cholerae strains. Application of the cloned fragment as a molecular probe revealed homologous sequences in all V. cholerae strains tested independently on their biotypes, hemolytic activity and presence of vct-genes in their genomes while none of other Vibrio species and related microorganisms contained such sequences. A recombinant E. coli strain, a V. cholerae eltor hemolysin producer, was constructed. The simultaneous expression of hemolytic and toxinogenic properties by the same V. cholerae strains is discussed.     

Prevalence of major virulence genes among various Vibrio cholerae El-TOR strains for evaluating their epidemiological significance

Specific oligonucleotide primers were chosen for identifying the fragments of the four major virulence genes of V. cholerae eltor (ctxA, tcpA, toxR, and hap) using the polymerase chain reaction (PCR). In order to estimate the efficiency of complex PCR testing of V. cholerae for evaluation of their epidemiological significance, a collection of 80 V. cholerae eltor strains with known virulence was selected, whose most important specific features had been studied previously. The hap was appropriate species-specific gene making it possible to detect V. cholerae strains regardless of their virulence. The most complete and objective data for evaluating the epidemic significance can be obtained by detecting the presence of three virulence genes (ctxA, tcpA, and toxR) in their chromosome. The prevalence of the above four genes in various V. cholerae strains isolated from the environment during epidemic and non-epidemic periods was studied.     

Construction of a genetic map of the chromosome of Vibrio cholerae based on conjugational crossings

The results of cholera vibrio chromosomal mapping using Vibrio cholerae classica and V. cholerae eltor donor strains obtained with the help of various R. plasmids, are summarized in the paper. A genetic map of V. cholerae chromosome was established showing the order of 35 gene markers. The relationship between the genetic structures of cholera eltor and classical vibrio biotypes is discussed.     

Testing the biological activity of Vibrio cholerae on cell subcultures

Testing the supernatants of ctx(+) strains of V. cholerae eltor and V. cholerae O139 on cell subcultures confirmed the possibility of the synthesis of hemolysin by V. cholerae under the condition of growing them in tripton medium lacking FeCl3. At the same time ctx(+) strains of V. cholerae of both serogroups retained, simultaneously with hemolysin production, their capacity for the synthesis of cholera toxin.     

Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139

Abstract The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V. cholerae O1 of the E1 Tor biotype. Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V. cholerae tested carried cholera toxin genes and other gene associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the E1 Tor biotype of V. cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.     

Comparative study of the diagnostic value of new cholera eltor bacteriophages ctx+, ctx-, and KhDF-3, 4, and 5

The comparative evaluation of the diagnostic value of new cholera eltor bacteriophages ctx+ and ctx-, as well as monophages X[symbol: see text]-3, 4, 5, demonstrated their high activity and specificity. Using of these bacteriophages epidemic potential of 95% Vibrio cholerae eltor strains ctx+ and 84.5% of V. cholerae eltor stains ctx- was determined. Commercial monophages X[symbol: see text]-3, 4, 5 were inferior to bacteriophages ctx+ and ctx- in their diagnostic value: only 55% of strains having gene ctxAB were found to be epidemically dangerous, i.e. 45% of strains capable of causing the disease were not detected. On the basis of the results obtained in this investigation cholera eltor bacteriophages ctx+ and ctx- were recommended for introduction into practical use, while further production of cholera diagnostic monophages X[symbol: see text]-3, 4, 5 was recommended to be stopped.     

A study of the prevalence of regulatory genes controlling virulence gene expression among Vibrio choleraeeltor biovariant strains varying in their pandemic potential

The evolution of the genome of the pathogenic agent of the seventh cholera pandemia Vibrio cholerae eltor biovariant was thought to occur by acquiring not only structural genes of virulence but also regulatory systems as a result of horizontal transfer events. The polymerase chain reaction revealed the presence of the following regulatory genes that control the virulence gene expression in the chromosome of pre-pandemic and pandemic strains of cholera vibrios eltor: toxR, toxT, tcpP, tcpH, luxS, luxO, crp, vicH, pepA. The avirulent V. cholerae strain ATCC14033 isolated in 1910 (hypothetical predecessor of the cholera eltor agent) was shown to be lacking the regulatory genes toxT, tcpP, tcpHlocalized in the pathogenicity island VPI-1, and to be capable of realizing positive control over the expression of the virulence genes involved in the ToxR regulon. The virulent strains isolated from cholera patients during the local cholera outbreak in Indonesia in 1937 did not differ from the strains that caused cholera eltor pandemic in 1961. The strains had identical content of the regulatory genes tested. Only one strain of the four isolates studied contained no tcpPgene. Two key regulatory genes, toxR and toxT, were sequenced in all the isolates. The toxR nucleotide sequence of three pre-pandemic strains was shown to be indistinguishable from that of the pandemic isolates. On the other hand, the clinical strain MAK757 isolated prior to the emergence of the epidemic demonstrated an altered nucleotide sequence in its toxR gene. Experiments with the intra-intestinal challenge of suckling rabbits were indicative of similar virulence levels for the pre-pandemic and pandemic clinical strains. These results may serve as the evidence of the in vivo activity of the pre-pandemic strains of the toxT, tcpH, and tcpP positive regulatory genes that acquired in V. cholerae during the evolutionary process.     

Characterization of Vibrio cholerae eltor in the city of Kazan in 2001

Information on V. cholerae eltor isolated in the focus of cholera in Kazan in 2001 at different periods of the outbreak is presented. The identity of strains isolated from patients, vibriocarriers and environmental objects, including their antibioticograms (sensitivity to cyprofloxacin and resistance to trimethoprim--sulfamethoxazole, streptomycin, furazolidone and nalidixic acid, which may be regarded as markers), is shown. Variable tandem repetitions in the DNA of 30 isolates strains of different origin have been determined. The results of this determination make it possible to classify all these strains as one genotype, which confirms the suggestion on the circulation of one subclone of the infective agent of cholera in the focus. As revealed in this investigation, the isolated strains are labile with respect to diagnostic phage eltor, while ctx+ strains are resistant to phage eltor ctx+.