应用PCR—SSCP快速鉴定结构分枝杆菌复合群

通过聚合酶链反应（ＰＣＲ）－单链的权象多态性（ＳＳＣＰ）技术分析结构分枝杆菌和非结构分枝杆菌临床株的１６Ｓ ｒＤＮＡ基因。６０例临床标本中,２０例为阳性,与传统方法比较无差异。其中分型：１８例为结核支杆菌,２例为结核分枝杆菌和非结核分枝杆菌双重感染。２０例阴性标本中,ＰＣＲ－ＳＳＣＰ又检查出５例阳性。２０例对照标本中,３种传统方法与ＰＣＲ－ＳＳＣＰ法均检测为阴性。全部试验３ｄ执行结果。结核分枝杆菌     

Technical Factors Affecting an Immunoelectrophoretic Reference System for Analysis of Mycobacterial Antigens

The length of immunoelectrophoretic separation patterns obtained with a reference mycobacterial antigen was reduced significantly when a continuous buffer system was used in place of the recommended discontinuous system. Varying the supporting medium by using 1% concentrations of different agar preparations in the discontinuous system also affected the separation length and resolution of the reference pattern. Measurement of the electrical resistivity of the agar medium provided an explanation of these differences on the basis of current density or field intensity applied per slide. Variation in the reference pattern obtained in differently designed electrophoresis chambers also was attributed to differences in field intensity. To avoid these obvious alterations in the separation pattern of the reference antigen, it was suggested that separation procedures described originally for the reference system be used without modification.     

Morpho-Functional Analysis Using Procrustes Superimposition by Static Reference

In conventional geometric morphometric analyses of limb long bones, differences in the evolutionary capacity of articular surfaces and non-articular structures often remain unrecognised. It can be shown that areas of high spatial variance dominate shape data, which is problematic for the functional interpretation of limb long bone shape. We herein introduce Procrustes superimposition by static reference (PSSR), a novel analysis strategy that aims to facilitate morpho-functional inference. This procedure exploits the spatial constraint of some reference structures (in our case, articular surfaces) for the superimposition of other subareas (e.g. muscle attachment sites) in relation to that static reference. PSSR allows for the transformation of raw scan data, enabling researchers to extract geometric models of two- and three-dimensional substructures that cannot effectively be integrated with landmarks. As we demonstrate by a simple model analysis for one muscle attachment site, this procedure can yield measures of direct functional relevance. Multivariate analysis of an extensive set of subareas indicates how this type of data relates to conventional shape coordinates. The shape evolution of xenarthran humeri, which has previously been subject to a detailed study (Milne et al., J Zool 278(1):48–56, 2009), serves as a test case. The concept of a variance-based separation of landmark subsets expands mathematical methods by incorporating knowledge about evolutionary constraints. PSSR could therefore find application far beyond the intuitive case study of long bone shape.     

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium.     

Specificity of Acquired Resistance Produced by Immunization with Mycobacterial Cells and Mycobacterial Fractions

Mice were immunized intraperitoneally with 5.0 mg of living and 5.0 mg of heat-killed H37Ra cells of the attenuated strain Mycobacterium tuberculosis and challenged intraperitoneally with Listeria monocytogenes and Klebsiella pneumoniae. The period of protection provided by the living and heat-killed H37Ra cells against both heterologous infections was the same. When mice were immunized intraperitoneally with graded doses of living and heat-killed H37Ra and challenged intraperitoneally with listeria or klebsiella, the lowest immunizing dose providing protection against both klebsiella and listeria challenge was the same for living and heat-killed cells. Living and heat-killed cells also immunized equally effectively when the routes of immunization and challenge were different. Mice also were immunized intraperitoneally with mycobacterial ribosomal fraction, mycobacterial cell walls, and several nonspecific agents (Escherichia coli endotoxin, mineral oil emulsion, and Freund's incomplete adjuvant). The mice were challenged intraperitoneally with listeria or klebsiella at varying times after immunization. The mycobacterial components and all the nonspecific agents provided transitory protection lasting no longer than 4 days after immunization. Only the mycobacterial cell walls and the endotoxin provided protection against listeria challenge. It was concluded that the protection provided by the mycobacterial ribosomal fraction is specific for tuberculosis infection, since this fraction provided no protection against listeria infection and only transitory protection against klebsiella. It was also concluded that the mycobacterial component providing protection against heterologous infections is heat stable and probably is found in the cell wall.     

Tuberculin Activity of Mycobacterial Fractions Obtained by Chromatography

Commercial purified protein derivatives (PPD), old tuberculin (OT), the bacillary extract, and the culture filtrate of Mycobacterium tuberculosis H37Ra were submitted to Sephadex G-25 and diethylaminoethyl (DEAE)-cellulose chromatography. The ability of the fractions obtained to elicit delayed dermal hypersensitivity in M. tuberculosis H37Ra-infected guinea pigs was studied. Skin tests with Sephadex fractions in M. tuberculosis H37Ra-infected guinea pigs showed that the tuberculin activity was localized in the first fraction. All other Sephadex fractions were nonessential and nonspecifically irritating. Fractions from chromatography of Sephadex G-25 fraction 1 on DEAE-cellulose columns showed that all but the first were able to elicit delayed hypersensitivity reactions. There was a variability in the capacity to elicit the tuberculin reaction according to the fraction injected and the stage of tuberculous infection in guinea pigs. Compared to the others, the seven lots of commercial PPD were variable in composition and content. They contained both essential and nonessential materials for the tuberculin reaction. Sephadex fraction 1 would appear to be a better tuberculin as it excludes nonessential nonspecifically irritating elements and contains the complement able to elicit the tuberculin reaction. Its methodological simplicity would be economically advantageous.     

Serological Study of Bacterial Flagellar Hooks

Bacterial hooks were partially purified from flagella isolated from Salmonella SJ25, by treatment with heat to depolymerize flagellar filaments and with n-butanol and calcium chloride to remove membranes. Antihook serum was obtained from a rabbit inoculated with a preparation of hooks. The serum contained antibodies directed against the flagellar filament and cell membrane. These antibodies could be removed from the serum by absorption with purified flagellar filaments and cells of a nonflagellated mutant strain. It was shown by electron microscopy that anti-SJ25-hook antibody reacts with hooks from a number of strains of Salmonella which differed from SJ25 in H and O antigens, flagellar shape, and motility. Hooks possessed by various strains of Salmonella have a common antigenicity. In addition, anti-SJ25-hook cross-reacted with hooks from Escherichia coli W3110 but did not react at all which those from strains of Serratia, Proteus, Aerobacter, and Klebsiella. It is well known that bacteria stop moving upon addition of antiflagella serum to the medium. However, the addition of purified antihook was found to have little effect on motility. At physiological ionic strength and pH, flagellin (Salmonella) can polymerize into flagellar filaments only in the presence of seeds. It was shown that a crude preparation of hooks was able to initiate in vitro polymerization of flagellin.     

Structural Basis of Mycobacterial Inhibition by Cyclomarin A

Cyclomarin A (CymA) was identified as a mycobactericidal compound targeting ClpC1. However, the target was identified based on pulldown experiments and in vitro binding data, without direct functional evidence in mycobacteria. Here we show that CymA specifically binds to the N-terminal domain of ClpC1. In addition we have determined the co-crystal structure of CymA bound to the N-terminal domain of ClpC1 to high resolution. Based on the structure of the complex several mutations were engineered into ClpC1, which showed reduced CymA binding in vitro. The ClpC1 mutants were overexpressed in mycobacteria and two showed resistance to CymA, providing the first direct evidence that ClpC1 is the target of CymA. Phe⁸⁰ is important in vitro and in cells for the ClpC1-CymA interaction and this explains why other bacteria are resistant to CymA. A model for how CymA binding to the N-terminal domain of ClpC1 leads to uncontrolled proteolysis by the associated ClpP protease machinery is discussed.     

Metabolism of acetylene by Nocardia rhodochrous.

A Nocardia rhodochrous strain capable of utilizing acetylene as its sole source of carbon and energy exhibited slow growth on low concentrations of acetaldehyde. Resting cells incubated with acetylene formed a product identified as acetaldehyde, but attempts to demonstrate acetylene hydrase activity in cell-free extracts were unsuccessful. Acetaldehyde dehydrogenase in N. rhodochrous was found to be NAD+ linked and nonacylating, converting acetaldehyde to acetate. Specific activities of acetaldehyde dehydrogenase, acetothiokinase, and isocitrate lyase were enhanced in cells grown on acetylene and ethanol as compared with cells grown on alternate substrates. These results suggest that acetylene is catabolized via acetaldehyde to acetate and eventually to acetyl coenzyme A. Acetylene oxidation in N. rhodochrous appears to be constitutive and is not inhibited in the presence of either ethylene, nitrous oxide, or methane.     

Mycetoma of thumb caused by Nocardia transvalensis

A case of mycetoma of thumb due to Nocardia transvalensis is described. Four earlier isolations of this rarely repoted etiological agent of mycetoma are reviewed. The patient in the present case was successfully treated with cotrimoxazole given orally.     

Bioconversion of steroid glycosides by Nocardia restricta

The bioconversion of steroid alkaloid tomatine by Nocardia restricta yields the conjugate with lactic acid. We studied the bioconversion of some steroid glycosides without a nitrogen atom in the molecule to determine the effect of the nitrogen atom. The glycosides were of three different types: sterol glycosides, bufadienolide rhamnoside and steroid saponine. The results of bioconversions showed that Nocardia restricta converts steroid glycosides differently according to the sugar bound to the steroid aglycone. It can be concluded that in the absence of a nitrogen atom in the steroid molecule no conjugation with lactic acid by Nocardia restricta occurs.     

Degradation of Crystal violet by Nocardia corallina

Crystal Violet (BV3), a typical triphenylmethane dye, was degraded by growing cells of Nocardia corallina IAM 12121, although their growth was inhibited at the initial stage of incubation. The dye was degraded at a low concentration, below 5 mol dm^–3. The growth of the cells was completely inhibited at a dye concentration of 7 mol dm^–3. A degradation product of BV3 was identified as 4,4-bis(dimethylamino) benzophenone (Michler's ketone; MK) by gas chromatography-mass spectrometry. The product was obtained in a reasonable yield since it was not further metabolized by N. corallina IAM 12121.Correspondence to: C. Yatome     

Kinetics of phenol removal by Nocardia species

Summary Phenol removal by pure cultures of Nocardia corallina was studied in a batch reacting system by estimating both the active biomass and the phenol concentration in the liquid during the experiments. A simple kinetic model was tested and the relative constants determined at 37°C.     

Serological Diagnosis of Paracoccidioidomycosis: High Rate of Inter-laboratorial Variability among Medical Mycology Reference Centers

Background

Serological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded.

Methodology/Principal findings

We compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the “reference” titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the “reference” titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center''s criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient''s treatment. Surprisingly, all centers exhibited a high rate of “major” discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better.

Conclusion

There are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients'' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.