THE TUBERCULOUS DISEASES Originating from Different Species of Acid-Fast Bacilli

Disease due to the typical human type tubercle bacillus is rapidly diminishing as a result of public health measures and specific chemotherapy. Lesions in man resulting from other kinds of acid-fast bacilli are now being recognized in increasing numbers. Some of these bacilli had been seen before but were confused with typical M. hominis, others were considered to be harmless saprophytes, while others could not be found with the methods used. Special culture media, different conditions for cultivation, new physical, chemical and biological tests, and inoculation into a variety of animal hosts are now available. With their use more than a dozen different strains of human type tubercle bacilli, and more than a score of other species of acid-fast bacilli may now be distinguished. A simple chemical test readily separates the human type tubercle bacilli from all other kinds of acid-fast bacilli. The differentiation of the different human and animal pathogenic acid-fast bacilli from the avirulent saprophytes and other harmless mycobacteria presents great difficulties, but methods are becoming available which usually make this possible. Since the distinction may be of great therapeutic and epidemiologic importance, the effort should be made.     

A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture

Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1 x 10(7) bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1 x 10(4) bacilli/ml while auramine-O method showed upto 1 x 10(2) bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1 x 10(3) cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens.     

Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis. In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.     

肿瘤患者感染三种常见革兰阴性杆菌耐药性分析

目的分析肿瘤患者临床分离的革兰阴性杆菌分布及其主要菌株的耐药性,为指导临床合理用药提供依据。方法对2014年1月~2015年12月恶性肿瘤患者感染分离的1478株革兰阴性杆菌及其主要菌株的耐药性进行回顾性分析。结果分离的1478株革兰阴性杆菌中,以大肠埃希菌(568/1478)38.43%、肺炎克雷伯菌(338/1478)22.87%、铜绿假单胞菌(216/1478)14.61%3种为主。这3种革兰阴性杆菌均呈多药耐药性;除亚胺培南对革兰阴性杆菌有较好的抗菌活性外,其它药物均显示有一定的耐药性,其中对派拉西林的耐药率在91%以上,头孢他啶、头孢噻肟、复方新诺明等药物的耐药率在70%以上。结论肿瘤患者感染革兰阴性杆菌耐药现象严重,尤其是感染产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌和肺炎克雷伯菌呈多重耐药,实验室应加强对革兰阴性杆菌耐药性的监测,以指导临床合理选用抗菌药物。     

Resuscitation of dormant Mycobacterium tuberculosis by phospholipids or specific peptides

The presence of dormant tubercle bacilli presents a major problem for tuberculosis treatment. The culture supernatant of Mycobacterium tuberculosis was previously shown to resuscitate dormant bacilli in vitro. Here we report identification of active components as phospholipids and a tuberculosis protein Rv1174c. Remarkably, dormant bacilli from a one year old culture which failed to form any colonies could be resuscitated with peptides derived from Rv1174c and formed 10(5-7) colonies/ml. This finding represents the first unambiguous demonstration of resuscitation of dormant tubercle bacilli in vitro and may have implication for the study of mycobacterial dormancy and the design of novel strategies for improved treatment of tuberculosis.     

亚胺培南临床用药与非发酵菌群分离率和耐药性的关系

目的了解亚胺培南临床用药和非发酵菌群分离率与耐药性变迁的关系,有利于非发酵菌感染的预防与治疗。方法统计分析浙江省人民医院2000～2003年临床分离非发酵菌的菌株分布、与亚胺培南的临床用药的相关性及耐药率。结果4年来非发酵菌的分离以铜绿假单胞菌(30．7％)、乙酸钙-鲍曼不动复合杆菌(19．％)及嗜麦芽糖寡养单胞菌(19．1％)为主．其分离率与亚胺培南用药存在相关性。对各菌属的主要菌种进行耐药性分析,嗜麦芽糖寡养单胞菌、脑膜脓毒性金黄杆菌和洋葱伯克霍尔德菌的耐药率极高,铜绿假单胞菌和乙酸钙-鲍曼不动复合杆菌的耐药率呈现明显升高的趋势。结论非发酵菌的检出率与亚胺培南使用关系密切,耐亚胺培南的菌株迅速升高。临床应合理使用抗生素,并加强对耐药菌株的监控。     

The Staining Of Acid Fast Bacilli In Sections Of Glycol Methacrylate Embedded Tissues

Acid-fast bacilli can be stained in tissue embedded in glycol methacrylate. Modification of the Ziehl-Neelsen technique, along with changes in the formula of the plastic embedding medium, allow production of 1 to 2 micron sections which retain their integrity throughout the procedure, and within which the bacilli are clearly visible.     

慢性支气管炎患者下呼吸道感染病原菌分布及耐药性分析

目的:研究老年慢性支气管炎患者合并下呼吸道感染病原菌分布以及耐药性。方法:选取2009年1月到2013年12月我院收治的老年慢性支气管炎患者合并下呼吸道感染患者261例,采集所有患者的痰液,然后进行病原菌鉴定和药敏试验。结果:261例患者中,144例革兰阴性杆菌感染(55.2%),51例革兰阳性杆菌感染(19.5%),66例真菌感染(25.3%),其中混合感染者36例(13.8%)。革兰阴性杆菌以肺炎克雷伯菌最多(18.4%),革兰阳性杆菌以金黄色葡萄球菌最多(9.2%)。革兰阴性杆菌对亚胺培南的耐药性最低,其次是头孢哌酮和阿米卡星,对氨苄西林耐药率最高。金黄色葡萄球菌和表皮葡萄球菌对青霉素的耐药率均为100.0%,均对万古霉素敏感,其次是对环丙沙星敏感。结论:老年慢性支气管炎患者合并下呼吸道感染以革兰阴性杆菌感染为主,真菌和混合感染也占一定的比例,应该引起注意。     

A Modification of the Fite Formaldehyde (Fite I) Method for Staining Acid-Fast Bacilli in Paraffin Sections

For maximal demonstration of acid-fast bacilli in tissue sections it is necessary to avoid sequences of reagents which, first, affect the integrity of the complex upon which acid-fastness depends, and, second, extracts it from those bacillary elements which have been made vulnerable by age or other factors. The greatest damage occurs during dewaxing of paraffin sections by xylene and alcohols; the older and more decrepit bacilli being especially affected. In the technic presented, which is a modified combination of two processes devised by Fite, sections are deparaffinized by a protective mixture of rectified turpentine and heavy liquid petrolatum (2:1) and blotted to water. Staining is with Fite's new fuchsin (magenta III) solution, overnight at room temperature. The sections are then treated with reagent grade formaldehyde, which turns the color of the bacilli deep blue-black, followed by an aqueous sulfuric acid decolorizer, the potassium permanganate-oxalic acid sequence, and a modified Van Gieson counterstain, nuclear staining with hematoxylin being omitted. For total demonstration of all stainable bacilli, restorative treatment in the turpentine-oil mixture before staining is sometimes required, most frequently with leprosy material but also with some tuberculosis lesions.     

Levels of cell wall enzymes in endospores and vegetative cells of Bacillus subtilis

Vegetative bacilli and refractile endospores of Bacillus subtilis 168 were disrupted by homogenization with glass beads and fractionated by differential centrifugation. Most of the protein of endospores was particulate, whereas for bacilli most was soluble. Alanine racemase activity was sixfold higher in extract of endospores than in extract of bacilli and was particulate, whereas the enzyme from bacilli was soluble. The specific activities of seven other enzymes involved in peptidoglycan and teichoic acid biosynthesis were higher in extracts of bacilli than in those of endospores. The results suggest that restoration of activities of these seven enzymes to vegetative levels occurs during germination and outgrowth.     

Novel cost-effective method of screening soils for the presence of mosquito-pathogenic bacilli

AIMS: The aim was to simplify the cumbersome conventional process of isolating virulent bacilli, which involves isolating all bacilli strains from a source followed by screening for strains that are effective for bio-control of mosquito vectors. METHODS: A new simplified technique involving eight steps was devised for screening soil samples for the presence of mosquito-pathogenic bacilli before isolating individual strains. RESULTS: Using the new technique, we obtained eight bacilli strains (KSD1-8) showing pathogenic activity against mosquito larvae from three out of 10 soil samples screened. These strains were characterized, identified and the main bioassay tests were performed with three most promising strains (KSD-4, KSD-7 and KSD-8), and their pathogenic activity against Anopheles stephensi Liston, Culex quinquefasciatus, Say and Aedes aegypti Linnaeus compared well with commercial reference strains of B. thuringiensis israelensis and B. sphaericus. SIGNIFICANCE AND IMPACT OF THE STUDY: The new technique of screening soil samples for the presence of virulent pathogenic strains of bacilli against mosquito larvae proved quick, efficient and cost effective.     

乳粉中嗜热菌污染研究进展

嗜热菌是乳粉中的常见污染菌,是影响乳粉品质的一个重要因素。本文综述乳粉中嗜热菌的种类、污染源对乳品工业的危害及其控制手段的研究进展,为国内相关领域研究提供参考。     

Role of cytokines and nitric oxide in the induction of tuberculostatic macrophage functions

The aim of this study was to determine phenotypic differences when BCG invades macrophages. Bacilli prepared from the same BCG primary seed, but produced in different culture media, were analysed with respect to the ability to stimulate macrophages and the susceptibility to treatment with cytokines and nitric oxide (NO). Tumour necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) were assayed by enzyme-linked immunosorbent assay (ELISA), whereas NO levels were detected by Griess colorimetric reactions in the culture supernatant of macrophages incubated with IFN-gamma, TNF or NO and subsequently exposed to either BCG-I or BCG-S. We found that BCG-I and BCG-S bacilli showed different ability to simulate peritoneal macrophages. Similar levels of IL-6 were detected in stimulated macrophages with lysate from two BCG samples. The highest levels of TNF and IFN-gamma were observed in macrophages treated with BCG-S and BCG-I, respectively. The highest levels of NO were observed in cultures stimulated for 48 h with BCG-S. We also found a different susceptibility of the bacilli to exogenous treatment with IFN-gamma and TNF which were capable of killing 60 and 70% of both bacilli, whereas NO was capable of killing about 98 and 47% of BCG-I and BCG-S, respectively. The amount of bacilli proportionally decreased with IFN-gamma and TNF, suggesting a cytokine-related cytotoxic effect. Moreover, NO also decreased the viable number of bacilli. Interestingly, NO levels of peritoneal macrophages were significantly increased after cytokine treatment. This indicates that the treatment of macrophages with cytokines markedly reduced bacilli number and presented effects on NO production. The results obtained here emphasize the importance of adequate stimulation for guaranteeing efficient killing of bacilli. In this particular case, the IFN-gamma and TNF were involved in the activation of macrophage bactericidal activity.     

High resolution shadowing of Mycobacterium leprae.

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

Antimycobacterial activity of lecithin-cholesterol liposomes in the presence of phospholipase A2.

Tubercle bacilli were preincubated with lecithin-cholesterol liposomes to be subsequently exposed to phospholipase A2. After further incubation in the environment of acidic buffer, viable units in the final mixture were enumerated by inoculating the serial dilutions of an aliquot onto Kirchner agar medium containing horse serum in 5%. Another aliquot was used for lipid analyses to confirm hydrolysis of lecithin. In addition to this bactericidal type of experiments, bacteriostatic tests were also conducted with Kirchner semi-solid agar medium, into which liposome-treated bacilli were inoculated with the enzyme at a time. Various natural and synthetic lecithins different in constituent fatty acids were employed. The results indicated that toxic fatty acids released from lecithin acted to kill the bacilli or to inhibit their growth.     

High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

Inhibition of growth of tubercle bacilli by ali-esterase inhibitors in various culture media

Summary The effect of the potent ali-esterase inhibitor E 600 (paraoxon) on the growth of tubercle bacilli is potentiated by the addition of Tween 80 to the medium. This may be due to a change in the permeability of the bacteria for the inhibitor. Growth of tubercle bacilli from sputa is also inhibited by E 600 in the haemolysed human blood medium of Price. Cultures of tubercle bacilli in media with E 600 begin to grow after some time, when the inhibitor has broken down. The results obtained by the method of Price indicate that growth of the cultures does not originate from a few resistant cells but from the whole population which may have regained the ali-esterase activity essential to their growth.     

肾综合征出血热少尿期的肺部感染菌群分布及其耐药分析

目的了解肾综合征出血热少尿期的肺部细菌感染对临床治疗的意义。方法送检的所有痰液标本按常规方法进行细菌分离鉴定,药敏试验为K-B法,ESBLs以双纸片协同试验进行,MRS检测为纸片筛选法。结果痰液标本338份,细菌培养检出率68.9%,革兰阴性菌占优势,以铜绿假单胞菌、大肠埃希菌、肺炎克雷伯菌为主。对碳青霉烯类、头孢哌酮/他唑巴坦均分别低于9.09%、12.5%,头孢吡肟为27.4%,其余抗生素的耐药率均高于31.3%。3种主要产ESBLs菌对碳青霉烯类、头孢哌酮/他唑巴坦均分别低于15.8%、17.2%,其余抗生素的耐药率均高于55.2%。革兰阳性菌主要以金黄色萄葡球菌为主。结论肺部感染以革兰阴性菌占优势,碳青霉烯类、头孢哌酮/他唑巴坦是抗感染治疗的首选药物。     

Automated identification of tubercle bacilli in sputum. A preliminary investigation

OBJECTIVE: To use an automated method to detect tubercle bacilli in sputum specimens. STUDY DESIGN: Using fluorescence microscopy, tubercle bacilli were identified on auramine-stained sputum specimens. Images were then captured with a digital camera and enhanced through imaging processing techniques. The bacilli were recognized using neural network classifiers. RESULTS: This preliminary investigation demonstrated a sensitivity of 94.1% for the identification of individual bacilli. As there are usually fairly numerous tubercle bacilli in the sputum of patients with active pulmonary tuberculosis, the overall diagnostic accuracy of sputum smear-positive patients can be expected to be very high. CONCLUSION: Potential benefits of automated screening for TB bacilli are: rapid, accurate, inexpensive diagnosis; the ability to screen larger numbers of people; increased resources to monitor patients; and reduction in health risks to staff.