细胞壁缺陷白喉棒状杆菌致病作用的观察及其毒力基因检测

目的比较白喉棒状杆菌亲代细菌型及其稳定L型动物致病性的差异,了解白喉棒状杆菌稳定L型变异的特点,探讨其变异的性质及其与细胞壁缺陷突变的关系。方法用氨苄青霉素在非高渗培养基内人工诱导产毒性白喉棒状杆菌为稳定L型。采用白喉棒状杆菌稳定L型纯培养物及其代谢产物皮内感染家兔,观察局部感染部位皮肤或全身的病理改变。采用微量法提取白喉棒状杆菌稳定L型的染色体DNA,用Tox基因特异性引物进行PCR扩增以检测毒素蛋白结构基因,并进行序列测定和分析。结果白喉棒状杆菌在氨苄青霉素作用下可发生细胞壁缺陷而成为L型,白喉棒状杆菌稳定L型不能引起动物局部或全身发生异常表现,该稳定L型的传代培养物可仍然保留同其亲代细菌型一致的Tox基因及其核苷酸序列。结论提示细胞壁缺失将导致白喉棒状杆菌与产生毒素蛋白有关结构基因在宿主菌细胞内的表达受到抑制,以致使白喉棒状杆菌稳定L型丧失了产生外毒素致病的作用。     

聚合酶链反应-单链构型多态性检测细胞壁缺陷型淋病奈瑟菌cppB基因变异

目的：检测与分析淋病奈瑟菌L型的cppB基因,探讨细胞壁缺陷对淋病奈瑟菌cppB基因的影响。方法：用青霉素诱导淋病奈瑟菌成为L型并获得稳定L型纯培养物,用cppB基因特异性引物以聚合酶链反应(PCR)检测稳定L型纯培养物的cppB基因和进行单链构型多态性(SSCP)分析。结果：淋病奈瑟菌的细菌型及其L型都具有cppB基因扩增产物,但PCR—SSCP分析可见异常泳动DNA带型(细菌型有2条带、L型有3条带)。结论：细胞壁缺陷淋病奈瑟菌仍然具有cppB基因,但其碱基序列可以发生改变。     

细胞壁缺陷型淋病奈瑟菌cppB基因的检测

目的：了解细胞壁缺陷对淋病奈瑟菌隐蔽性质粒B基因（cppB）的影响。方法：用青霉素诱导林病奈瑟菌成L型,以非高渗液体培养基传代培养并获得L型纯培养物,用cppB基因特异性引物以聚合酶链反应（PCR）检测不同代次稳定L型纯培养物的cppB基因。结果：淋病奈瑟菌细菌型及其传1-4代的L型培养物具有cppB基因,传5代后的L型培养物不能检出cppB基因。结论：细胞壁缺陷可造成淋病奈瑟菌隐蔽性质粒丢失,导致cppB基因PCR检测漏诊。     

细菌稳定L型返祖性的研究

营养琼脂培养的白喉杆菌、葡萄球菌、伤寒杆菌及甲型副伤寒杆菌的稳定L型经硬琼脂,20%血清琼脂,血琼脂及12%,20%,30%明胶培养基传代培养或注射动物体内均不能返祖。但L型—细菌型接触培养具有促沙门氏菌L型返祖作用,可能同细菌型的某种“促返祖因子”传递给L型有关。     

杀伤血管内皮生长因子受体 1 阳性细胞的靶向毒素

白喉毒素 (diphtheria toxin DT) 是棒状白喉杆菌被β噬菌体感染后分泌的一种外毒素. 它可以阻断真核细胞的蛋白质合成,杀死细胞. 血管内皮生长因子 (VEGF) 的 R82A, K84A, H86A 突变体可以和肿瘤血管上高表达的 VEGF 受体 1 (VEGFR-1) 特异性结合. 首先从白喉杆菌中提取基因组 DNA,扩增出白喉毒素 C 区、 T 区基因. 并运用点突变技术,制成 VEGF 的 R82A, K84A, H86A 突变体. 利用这个可以和肿瘤血管上特异性受体相结合的 VEGF 的突变体,代替白喉毒素上的受体结合区,制成了针对 VEGFR-1 的靶向融合毒素——— DT391-mVEGF. 以去除了受体结合区的 DT391 为阴性对照,细胞实验表明,融合毒素对 VEGFR-1 阳性的肿瘤细胞有特异性杀伤作用.     

细菌L型的生物学特性与致病作用

L型是细菌因变异而产生的细胞壁缺陷型。由于细胞壁缺损,使生物学性状发生改变,对低渗敏感,用常规培养法不易检出,因此对其性状认识不足,常造成临床的漏诊与误诊。L型是否有致病性争论已久。过去一般认为有致病性的细菌变为L型后即不致病。目前已有一些资料表明L型亦具有一定的致病性。本文就细菌L型的类型、生物学特性、致病作用等加以综述。     

非高渗透压培养基培养细菌L型的研究

本文用青霉素在高渗透压的L型培养基上,诱导金黄色葡萄球菌和白喉杆菌形成L型后,直接种入无血清亦无渗透压保护剂的非高渗透压培养基中,传代培养和观察其形态、培养及代谢特性。结果表明,金黄色葡萄球菌L型和白喉杆菌L型在非高渗透压培养基中,传代培养后,不能在高渗透压的L型培养基上生长形成菌落。和丧失了对多种糖发酵的能力。     

云贵川湖泊芽孢杆菌分离、鉴定及抗动物病原菌活性研究

为从湖泊沉积物中筛选出对动物病原菌具有抑菌活性的芽孢杆菌Bacillus,采用纯培养方法对云贵川地区9个湖泊样品中的芽孢杆菌进行分离、16S rRNA基因序列测定和发酵,以4株常见动物病原菌作为对象,对芽孢杆菌次级代谢产物进行活性检测,筛选有抗菌活性的菌株,并对活性菌株的发酵产物进行液相色谱-质谱联用技术分析。结果显示,在9个湖泊样品中共分离出47株芽孢杆菌,筛选出13株有抗菌活性的菌株,且其产生的脂肽类抗生素对动物病原菌显示出较好的抗菌活性。本研究不仅扩大了芽孢杆菌物种资源信息库,为后续开展芽孢杆菌次生代谢产物研究奠定理论依据。同时也为开发新型动物致病菌抗生素提供了新的途径。     

细胞壁缺陷结核分支杆菌致细胞病变作用的观察

目的：探讨细胞壁缺陷结核分支杆菌的致细胞病变作用。方法：用利福平诱导结核分支杆菌形成稳定L型后感染Vero细胞,直接在显微镜下和抗酸染色观察细胞病变情况以及结核分支杆菌同宿主细胞的关系。结果：Vero细胞受结核分支杆菌L型感染72h后形成空泡、变圆、脱落和裂解,结核分支杆菌稳定L型细胞粘附或侵入细胞内。结论：细胞壁缺陷结核分支杆菌仍然能够引起Vero细胞发生病变但致细胞病变的作用较其亲代细菌型明显减弱,L型能够粘附于宿主细胞表面或进入宿主细胞内生长繁殖,引起缓慢的细胞病变。     

嗜酸乳杆菌发酵上清液对常见肠道致病菌及其L型抑菌作用的实验研究

目的探讨嗜酸乳杆菌(L.acidophilus)对肠道致病菌及其L型菌的抑菌作用,为临床开发L.acidophilus活菌制剂提供实验依据。方法采用肉汤稀释法,检测L.acidophilus 36h发酵上清液对肠道致病菌及其L型菌吸光度值的影响,计算抑菌率,探讨乳杆菌的抑菌活性。结果3个浓度的L.acidophilus发酵上清液对3种常见肠道致病菌(致病性大肠埃希菌、伤寒沙门菌、痢疾志贺菌)及其L型菌均有不同程度抑菌作用,其中原液浓度抑菌作用最强。结论 L.acidophilus是一种可用于治疗多种肠道致病菌及其L型菌感染引起肠道疾病的微生态制剂,其代谢产物发挥抑菌作用是其机制之一。     

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.     

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.     

金葡菌L型感染在卵巢癌中检测的意义

目的探讨金葡菌L型感染在卵巢癌中检测的意义。方法应用革兰染色、免疫组织化学染色技术检测HO-8910PM细胞株以及97例卵巢乳头状癌、23例卵巢乳头状瘤石蜡包埋组织中细菌L型的感染情况。结果（1）HO-8910PM细胞与金葡菌L型体外共培养后在肿瘤细胞的胞浆及胞核中检测到L型的阳性表达。（2）卵巢乳头状癌和乳头状瘤组织中,细菌L型感染率分别为25.8%（25/97）和13.0%（3/23）。L型检出率与卵巢癌的临床分期、病理分级及腹腔淋巴结转移的差异均有显著性（P〈0.005）,与组织学类型无关（P〉0.05）。结论金葡菌L型可以进入细胞内,并能够在一定程度上促进卵巢癌的发生、发展。     

从医院使用的新洁尔灭溶液中分离出细菌L型的报告

本文报道,作者采用高渗和等渗牛肉汤试管及平板培养法,从某医院正在使用的新洁尔灭器械浸泡液中分离出4株细菌L型,其中金黄色葡萄球菌L型1株,表皮葡萄球菌L型2株,类白喉杆菌L型1株。上述细菌经形态观察,细胞壁染色,返祖鉴定等一系列细菌L型鉴定程序;并通过电镜观察,菌体细胞图像分析。其结果均提示,细菌L型与原菌之间存在明显差异。作者认为,新洁尔灭器械浸泡液,消毒灭菌的不彻底性,是造成术后感染的重要因素。     

STREPTOCOCCAL L-FORMS IV. : Comparison of the Metabolic Rates of a Streptococcus and Derived L-Form.

Panos, Charles (University of Illinois College of Medicine, Chicago, and Albert Einstein Medical Center, Philadelphia, Pa.). Streptococcal L-forms. IV. Comparison of the metabolic rates of a Streptococcus and derived L-form. J. Bacteriol. 84:921-928. 1962.-Glycolytic rates of hexoses, amino sugars, pentoses, two-carbon compounds, and certain intermediates of glycolysis and the adaptive response to glucose of a group A Streptococcus and its derived L-form were compared. It was found that removal of the streptococcal cell wall did not result in the loss of the homolactic characteristic of the parent coccus or in a marked increase in the metabolism of certain glycolytic intermediates by the L-form. It was shown that (i) a major difference exists between the coccus and its L-form in the metabolism of glucosamine and N-acetylglucosamine; (ii) apparently, a loss of selectivity and internal control occurred in the transformation to the L-form; and (iii) this form, unlike the parent coccus, displayed an adaptive response to glucose. These data were not the result of an internal loss of essential cofactors or enzymes by diffusion from within the L-form. Nor could they be accounted for by dry-weight differences due to loss of the streptococcal cell wall. Finally, it was observed that the sonically disintegrated L-form in 0.5 m NaCl was capable of a glycolytic activity of 46% of that of the total intact culture. These data suggest that the conversion of a streptococcus to the L-form is accompanied by an alteration in carbohydrate metabolism as well as the loss of the cell wall. Previously reported data are in agreement with these findings and support the conclusion that the resulting form is not merely a bacterial cell without a rigid cell wall.     

Envelope structure in three different L-forms ofProteus mirabilis

One A-type, stable and two different B-type, unstable L-forms were obtained from a strain ofProteus mirabilis and studied by electron microscopy and by chemical analysis for the presence of peptidoglycan. The wall of the parent bacterium is characterized by a profile of three superimposed dense lines and a content of 11.07 nmoles of muramic acid (MUR) and of 7.85 nmoles of diaminopimelic acid (DAP) per mg of dry weight. The stable, A-type L-form has completely lost the cell wall of the bacterium and is enveloped only by the plasma membrane to which very small quantities of peptidoglycan components are associated (MUR: 0.041 nmoles/mg; DAP: 0.075 nmoles/mg). The two B-type, unstable L-forms have the same wall structure in only two dense lines, but they differ in their peptidoglycan content. The first one does not contain more peptidoglycan components than the A-type, L-form (MUR: 0.022 nmoles/mg; DAP: 0.016 nmoles/mg), whereas the peptidoglycan content of the second one (MUR: 2.6 nmoles/mg; DAP: 1.65 nmoles/mg) is about one fifth of the content of muramic acid and diaminopimelic acid of the bacterial cell wall.     

慢性肾盂肾炎L型细菌感染及耐药分析

目的了解L型细菌在慢性肾盂肾炎的感染及耐药状况。方法对71例患者清洁中段尿做普通细菌培养(B型)、L型细菌培养(L型)及耐药分析。结果细菌阳性率为77.5%,其中单独L型阳性率为49.3%、B型与L型混合感染为15.5%,而B型阳性率仅为12.7%。主要是大肠埃希菌,其次是葡萄球菌;青霉素及头孢噻肟均有较高的耐药率(88.9%及73.6%)。结论L型细菌在慢性肾盂肾炎感染中占主导,β-内酰胺类药物有较高的耐药性,临床治疗应据药敏结果合理选择及时调整抗生素。     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Quantitation of bacillus subtilis L-form growth parameters in batch culture

Several methods were used to monitor the growth of a stable L-form in batch culture. The end of the exponential growth phase was determined with greatest accuracy by the amounts of deoxyribonucleic acid per milliliter of culture. Optical density and viable count data were not as reliable because the L-forms began to lyse at the end of exponential growth. Lysis was detected visually, by phase-contrast observations of wet mounts, and by release of ultraviolet-absorbing material into culture supernatant fluids.