Blood Microbiota and Cancer: Cell Wall-Deficient L-Forms of Bacteria and Fungi as Cancer-Promoting Environment

In recent years, valuable experience and insights have been gained into L-forms (cell-wall-deficient variants) of bacteria and fungi and their disease-trigger potential in cases with chronic infections, autism spectrum disorders, autoimmune and neurodegenerative diseases. Based on the concept of “internal” blood microbiota, consisting of L-forms and its relevance to health and disease, the current study aims to outline the profile of dysbiotic disorders in three cancer patients (with endometrial cancer, breast cancer and acute myeloid leukemia), all in a phase before chemotherapy. Venous blood samples from the patients and from one control healthy person, were microbiologically studied. The used novel methodology of blood microbiota assessment was based on the following phases: isolation of L-forms, development and propagation, cultivation and conversion of L-forms into classical bacteria and fungi, as well as their identification with MALDI-TOF method. From the patients were isolated L-forms of opportunistic bacteria (Enterococcus faecalis, Esherichia coli, Enterobacter cloacae and Pseudomonas oryzihabitans) and fungi such as Rhodotorula mucilaginosa, Aspergillus fumigatus and Mucorales. In conclusion, the common feature found for the three cancer patients was the isolation from the blood of highly associated communities consisting of morphologically indistinguishable L-bodies, which through reversion in broth, were identified as distinct bacterial and fungal species. Unlike classic bacteria or fungi causing sepsis and bacteremia/fungemia, the presence of L-forms in blood is hidden, it does not demonstrate clinical signs nor it can be detected by conventional methods. It should be noted, however, that the dysbiotic blood microbiota shows unique and individual characteristics for the concrete cancer patient, correlates to the common state of the organism and tumor localization in the body, as well as it outlines the cancer promoting role of L-forms in processes of malignization, cancer genesis and progression.

Novel Approach to Microbiological Study of Chronic Inflammations at Upper Respiratory Tract: Research of Blood L-Form Microbiota

Background: The recognition of human blood microbiota, consisting of cell wall-deficient microbes (L-forms), is a major challenge today in the field of microbiology. There are accumulating data confirming the concept of “internal” blood L-form microbiota and its significance for health and diseases. Finding out whether the blood microbiota can be of diagnostic and prognostic importance for detection and evaluation of chronic infections anywhere in the body is a major objective. In the context of chronically infected upper respiratory tract (URT), the aim of the current study was to understand whether a local infection can be a source for entry of bacteria and fungi in the blood. Methods: Blood samples from six persons with chronic inflammations in URT diagnosed with hypertrophied adenoids, chronic sinusitis, nasal polyps, chronic naso-pharyngitis and one control healthy person were studied. Blood microbiota assessment methodology that be used, included three phases: 1) isolation of L-form cultures from blood-development and propagation;2) cultivation directed to conversion of L-forms into bacterial and fungal cultures;3) isolation of pure classical bacterial and fungal cultures and their identification by MALDI-TOF method. Results: From the patients were isolated L-forms of opportunistic bacteria (Streptococcus mitis, Roseomonas mucosa, Dermacoccus nishinomiyaensis, Enterococcus faecalis, Acinetobacter johnsonii, Pseudomonas putida, Staphylococcus aureus, Pseudomonas luteola, Enterobacter cloacae) and fungi such as Rhodotorula mucilaginosa, Aspergillus niger, Aspergillus fumigatus and Mucorales. Conclusion: The novel innovative methodology for assessment of blood L-form microbiota was successfully applied for detection of microbes responsible for chronic infections at URT.

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconiosis workers

To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers＇ pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods： A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results： No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% （29/31） in resistant strains, mainly concentrated in codon 531 （51.6%, 16/31） and 526 （32.26%, 10/31）. Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn＇t reported by internal and overseas scholars. Conclusion： The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.

Detecting katG Drug Resistant Genetic Mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms by PCR-SSCP

Objective：To study the relationship between mutation in the katG gene and drug resistance of INH in Mycobacterium tuberculosis L-forms among patients of pneumoconiosis complicated with pulmonary tuberculosis, and to explore the clinical application of PCR-SSCP. Methods： A total of 52 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected. Mutation in the katG genes was detected by PCR-SSCP and traditional antimicrobial susceptibility test （AST）. Results： The results by AST showed that there were 40 persisters in 52 clinical isolated strains. The drug resistant rate was 76.92%（40/52）, and the gene mutation rate of katG was 57.70%（30/52）by PCR-SSCP, the difference was no quite significance （X^2 = 2.8507, P 〉 0.05）. The coincidence rate of two methods was 75.00% （30/40）. Conclusion： The detectionrate of katG resistant strains in Mycobacterium tuberculosis L-forms was high by PCR-SSCP. The combined application of PCR-SSCP and traditional antimicrobial susceptibility test can improve the detecting rate.

Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

Objective： To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCR- SSCP to clinical application. Methods： A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97 pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP, and the results were compared with those analyzed by traditional antimicrobial susceptibility test（AST）. Results： The gene muta- tion rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% （30/52）, 65.38% （32/52） and 40.38% （21/52）. The rate of reversion was 78.85%（41/52） and the result of drag-resistant genes was invariable. The results of AST showed that there were 40 （76.92%） multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21 （40.38%） and that of two-drug resistant was 19（36.54%）, but only 12（23.08%） strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene （P 〉 0.05）. Conclusion： PCR-SSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.

SBR法处理工业废水中有机负荷对污泥膨胀的影响

活性污泥中丝状菌与絮状菌的竞争生长使污泥的性状发生变化.有机负荷对污泥膨胀的影响一直有两种完全相反的说法,我们对此作了专门研究,试验结果表明:当反应器中溶解氧(DO)充足时,低有机负荷易引起污泥膨胀,提高有机负荷能有效的控制膨胀;高负荷下,引起污泥膨胀的原因往往是DO浓度不足,而提高DO浓度则能使污泥膨胀得到控制.这一结果也解释了高有机负荷发生污泥膨胀的实质原因.

污泥膨胀的诱因及控制措施

分析了活性污泥膨账的原因,基质浓度、营养物、pH值等指标中某一个或多个发生变化均可导致活性污泥发生膨胀.据此.可采取一定措施对它进行预防或消除。

用PVA包埋法固定化细胞去除洗衣粉废水中LAS的研究

经富集培养得到降解LAS的细菌菌系,在24小时内可将40—50mg/L的LAS分解90％以上。用PVA-硼酸化法包埋该菌系得到的固定化细胞,在处理洗衣粉废水中LAS时的最适pH为5.35—7.70,最适温度为25℃—35℃。在1L反应器中的间歇式连续运行表明:PVA小球/废水(V/V)=30％,进水LAS 40—70mg/L,HRT=3h条件下,LAS去除率可达90％以上。

基于电子鼻的带鱼货架期预测模型

为探索通过气味分析判断海产品贮藏品质的方法,利用电子鼻对带鱼在不同贮藏温度与贮藏时间下的挥发性气味变化进行了分析,对所获数据进行了主成分分析与货架期分析,并与理化品质指标值挥发性盐基氮(totalvolatile basicnitrogen,TVBN)相联系,建立了带鱼在273～283K下的货架期预测模型。结果表明:电子鼻18个金属传感器能很好地将贮藏于273与283K下的带鱼随着贮藏时间变化的气味进行区分。贮藏于不同温度条件下的带鱼的TVBN值与菌落总数值均随着贮藏时间的增加而增长,且均符合一级化学动力学模型(R2>0.9)。基于电子鼻货架期分析获得的273～283K下的气味变化结果与该温度下理化品质指标变化具有较好的对应关系,采用Arrhenius动力学模型推导公式求得带鱼在(273～283K)温度段内TVBN的Q10(温差为10K的货架寿命之比)值,对照该温度段下电子鼻货架期分析获得的气味变化货架期分析值,得到带鱼在该温度段内的Q10货架期预测模型,经验证,其预测误差小于20%。可根据获得的货架期预测模型对带鱼在273～283K条件下的货架期进行预测。

混菌发酵对干玉米秸喂牛饲用价值的影响

干玉米秸混菌二次发酵处理后,粗纤维含量下降.用4mol/L盐酸不溶灰法进行肉牛消化试验,测得相近营养水平条件下喂给牛只等量的未处理及发酵处理的玉米秸时,饲粮干物质、有机物及粗纤维的表观消化率在干玉米秸混菌处理后有显著提高(P<0.01).

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。

海洋石油降解菌株选育及组合降解优化

[目的]研究海洋石油降解菌株的选育及组合降解优化。[方法]从唐山市曹妃店港区石油污染海水中筛选得到3种菌株,分别为芽孢杆菌属、鞘氨醇杆菌属和假单胞菌属,并命名为B1、S1和P1,对其进行紫外诱变和组合降解优化试验,分别测定降解效率。[结果]菌株B1、S1和P1经10 d降解的降解率分别为63.89%、67.33%和67.25%;对其进行紫外诱变,得到诱变菌株yB1y、S1和yP1,其降解率分别为70.34%7、7.05%和72.81%,均比诱变前有所提高;yS1与yP1y、B1与yP1、yB1与yS1等比例组成的混合菌群的降解率分别为78.21%7、3.65%和72.23%。yS1与产生物表面活性剂的菌株yPa1混合,其降解率达到了81.45%。[结论]利用各菌株的优势互补可构建出降解能力高、能对石油烃进行全面降解的优势混合菌群。

从细菌的生化特性看生物脱氮与生物除磷的关系

目前一般的污水生物脱氮除磷机理认为生物反硝化脱氮与生物除磷是两个相互独立、相互竞争的生理过程,并且以这两个生理过程区分反硝化菌和聚磷菌。对生物脱氮除磷工艺中的细菌组成和生化特性的研究发现:硝酸盐还原性和超量吸磷只是两种并不冲突的细菌的生化特性,生物脱氮与生物除磷可以相互结合。即同时拥有这两种生化特性的细菌可以同时进行反硝化吸磷和脱氮生化反应。

菌藻对碳酸盐颗粒的泥晶化作用研究─以滇西保山地区下石炭统研究为例

碳酸盐颗粒泥晶化由真菌类和藻类的穿孔所引起，颗粒泥晶化可划分为四个阶段６种类型，泥晶套和泥晶铸型分别代表泥晶化的成长、成熟阶段。丰富的泥晶化颗粒为浅滩标志；泥晶化的深度与沉积速度成反比；泥晶化均匀程度与颗粒翻转次数成正比；颗粒泥晶化类型组合与风暴沉积有关；暴露环境出现溶蚀孔洞或负鲕。菌藻的泥晶化作用可加速海水及成岩压实作用对颗粒的破碎和细化并产生内碎屑和灰泥；鲕粒泥晶化后转变为辐射鲕和假鲕。

生物防腐过程中pH值与腐败菌的消长规律研究

根据腐败菌不适应酸性环境的特点，在防腐底物中添加适量的碳源及防腐菌，使之迅速繁殖、产酸、降低ｐＨ，抑制腐败菌的生长繁殖，进而达到防腐之目的。试验证明，当ｐＨ值降到４．０以下时即可达防腐效果。

洗精处理的细菌学分析

人类精液中可能存在各种微生物,人工授精引起生殖道感染已有报道,要实现更高一级的授精技术,如宫腔内授精,配子输卵管内移植,体外授精等必须从细菌学角度去证实精子经过处理后不含任何引起盆腔感染的微生物。作者收集20例不孕妇女的丈夫与有生育能力的健康供精者精液标本,按工作习惯分随意收集与注意清洁处理的特定收集两组,比较洗精处理前后定量细菌培养菌落数。培养结果,洗精处理前,除特定收集精液标本2份需氧与厌氧菌阴性外,其余18份需氧与厌氧菌均为阳性,每份标本阳性菌落数范围1～4个,总菌落数40,菌落计数均>10~2cfu(集落形成单位)/ml;57.5%>10~3cfu/ml;30％>10~5cfu/ml。洗精后特定收集组无论需氧与厌氧菌为阴性,随意收集组需氧菌培养无一例阳性,而厌氧培养7例阳性,但菌落均<10~2cfu/ml。对比洗精处理前显著减少(P<0.01),7例特定收集标本经洗精处理后沉淀物电子显微镜超薄切片与扫描检查均未见有任何微生物附着精子任何部位,也未见有游离微生物。因而认为从注意无菌操作收集精液开始,经过洗精处理是可以有效去除人精液微生物的。

一株犬种布鲁氏菌的鉴定及毒力测定

对分离到的一株疑似犬种布鲁氏菌进行生物特性和基因型鉴定,并用宿主实验动物(比格犬)测定了最小感染量和脾含菌量。形态与染色特性、培养特性、血清学特性、生化特性鉴定以及Multi-PCR和16S rRNA测序结果表明,该分离株为犬种布鲁氏菌,其对比格犬的最小感染剂量为10~5 CFU/m L,对应的脾含菌量为4×10~5~3×10~6 CFU/g。

注射用阿莫西林钠克拉维酸钾细菌内毒素检查的方法研究

目的:注射用阿莫西林钠克拉维酸钾为一抗生素复方制剂,厂家内控质量标准中控制细菌内毒素的方法为动力学浊度法,内毒素限值为0.29EU/mg。USP23,USP24及BP1998均未收载本品的注射剂,而中国药典1995年版1998增补已收载,但控制细菌内毒素的方法为家兔热原检查法,剂量为20mg/kg。为了在国内开展本品的细菌内毒素检查,我们对本品进行了细菌内毒素检查凝胶法的方法研究。方法:按中国药典1995年版1998增补本细菌内毒素检查进行干扰实验和结果判断。结果:本品在0.12至2.0mg/ml的浓度范围内,对细菌内毒素与鲎试剂的反应无干扰作用,确定本品细菌内毒素限值为0.25EU/mg。结论:按0.25EU/mg限值我们对两个国家进口的5批样品进行了检验,并与家兔热原检查法进行了对比,结果一致,都为阴性,认为本品可用细菌内毒素检查凝胶法代替家兔热原检查法。

质粒谱分析泌尿道感染复发与细菌L型检出

为探讨泌尿道感染复发的病原菌 ,采用一般培养及细菌L型培养对 85 0例泌尿道感染者的尿标本进行检测 ,并进行追踪观察及细菌质粒谱分析 .结果 :12 8例复发 (15 % ) ,初诊患者普通细菌培养阳性率为5 8 3% ,复发为 30 4% (P <0 0 1) .细菌L型培养初诊仅为 5 6 %复发高达 32 0 % (P <0 0 1) ,质粒谱分析复发后细菌原型及L型均与初诊分离的细菌有较高同源型 .提示初诊患者不必做细菌L型培养 ,而复发者应做 .证明复发者病原菌多来自初诊治疗不彻底 .