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Differentiation of mycobacteria in sputa by duplex polymerase chain reaction for mycobacterial hsp65 gene  

Early differentiation of mycobacteria in sputa is crucial. This study was set to evaluate the usefulness of a newly developed duplex polymerase chain reaction (PCR) for hsp65 gene-based method in differentiating mycobacteria in sputum with a positive acid-fast bacilli (AFB) smear before culturing. One hundred forty-seven sputa with positive AFB smear were included for the analysis. Mycobacterial species were identified using a newly developed duplex PCR for hsp65 gene followed by a nested PCR-direct sequencing and the conventional colony-based method. Final decision of mycobacterial species were made based on 1) results of species identification based on mycobacterial colonies or 2) results of species identification of other sputa from the same patients and clinical findings. The duplex PC...

2

Granulomatous pneumonia in a captive freshwater crocodile (Crocodylus johnstoni) caused by Mycobacterium szulgai.  

A 25-yr-old male freshwater crocodile (Crocodylus johnstoni) was diagnosed with pulmonary mycobacteriosis caused by Mycobacterium szulgai. Necropsy revealed fibrinous exudate in the right pleural cavity and white miliary nodules in the right lung lobe. Histopathologic examination revealed well-demarcated granulomas consisting of multinucleated giant cells and epithelioid cells surrounded by fibrous connective tissue. Atypically, lymphocytes had accumulated in the outer region of fibrous connective tissue. Mycobacterial infection was confirmed by nested polymerase chain reaction targeting the hsp65 gene and by Fite's method for detection of acid-fast bacilli within formalin-fixed, paraffin-embedded lung tissue. Sequence analysis of the DNA amplicon revealed that the species of mycobacterium shared 98% homology with the gene encoding the hsp65 gene of M. szulgai. This is the first report of M. szulgai as the causative agent of mycobacteriosis in a reptile. PMID:20945661

3

Mycobacterium pseudoshottsii Isolated from 24 Farmed Fishes in Western Japan  

Mycobacteria isolated from epizootics of farmed fishes in western Japan were examined for the first time using multigenotypic analysis. By analysis of the sequences of the internal transcribed spacer between the 16S and 23S rRNA genes (ITS) region and the partial 16S rRNA, hsp65 and rpoB genes, M. pseudoshottsii was identified as the causative agent in these infections. Prior to this study, only M. marinum has been known as the causative agent of lethal mycobacterial disease in marine fishes in Japan.   

4

Mycobacterium pseudoshottsii isolated from 24 farmed fishes in western Japan  

Mycobacteria isolated from epizootics of farmed fishes in western Japan were examined for the first time using multi-genotypic analysis. By analysis of the sequences of the internal transcribed spacer between the 16S and 23S rRNA genes (ITS) region and the partial 16S rRNA, hsp65, and rpoB genes, M. pseudoshottsii was identified as the causative agent in these infections. Prior to this study, only M. marinum has been known as the causative agent of lethal mycobacterial disease in marine fishes in Japan.   

5

Mycobacterium heckeshornense Lung Infection that was Diagnosed as Mycobacterium xenopi Disease by DNA-DNA Hybridization (DDH)  

The DNA sequencing analyses of the 16S rRNA gene, rpoB and hsp65 were conducted to characterize six strains that had been identified as Mycobacterium xenopi by DNA-DNA hybridization (DDH) for past ten years in our hospital. The results revealed Mycobacterium heckeshornense infection in one of the six cases. A 47-year-old man, who had been treated for pneumonia, had pulmonary nontuberculous mycobacterial disease. The sputa from the patient were culture positive for mycobacterium in three times. And it was diagnosed as M. xenopiby DDH method. Chest X-ray showed fibrocavitary lesion in right upper lobe was successfully treated with clarithromycin for four weeks.   

6

Occurrence and characterization of multiple novel genotypes of Mycobacterium immunogenum and Mycobacterium chelonae in metalworking fluids.  

Rapidly growing mycobacteria colonize metalworking fluids, leading to contamination of occupational environments and exposure-related respiratory illnesses in machine workers. Lately, it has been emphasized that these fluids are colonizable by a single genotype of a rapidly growing mycobacterium species, Mycobacterium immunogenum. Here, we report on the genotypic diversity of mycobacteria in these fluids, including isolation and characterization of multiple novel genotypes of two distinct species, Mycobacterium chelonae and M. immunogenum. Using agar culturing and Mycobacterium-specific PCR, 13 mycobacterial isolates were recovered from 100 geographically diverse in-use metalworking fluid samples. Based on restriction fragment length polymorphism of PCR products, DNA sequencing (hsp65 gene segment), and phylogenetic analysis of 16S-23S rDNA internal transcribed spacer (ITS) sequences, six isolates were identified as M. immunogenum and seven as M. chelonae; an additional isolate from metalworking fluid diluent water was identified as M. diernhoferi. Genomic DNA macro-restriction fragment pattern analysis, using pulsed-field gel electrophoresis with XbaI and SpeI restriction digestions, showed intraspecies variation among the isolates of M. immunogenum and M. chelonae. Visual and computer-assisted dendrogram analysis of the XbaI macro-restriction patterns revealed three novel genotypes of M. immunogenum and two of M. chelonae, whereas SpeI macro-restriction patterns revealed only two genotypes for each isolate. None of the identified genotypes matched the reportedly dominant one of M. immunogenum from metalworking fluids. Both mycobacterial species are prevalent in metalworking fluids and there is a considerable strain-level genetic diversity within them. PMID:16332331

7

Mycobacterium salmoniphilum infection in burbot Lota lota.  

Burbot Lota lota sampled from lakes Mjosa and Losna in southeastern Norway between 2005 and 2008 were found to be infected with Mycobacterium salmoniphilum at a culture-positive prevalence of 18.6 and 3.3%, respectively. The condition factor (CF) of mycobacteria-affected fish sampled from Mjøsa in 2008 was lower than the average CF of total sampled fish the same year. Externally visible pathological changes included skin ulceration, petechiae, exopthalmia and cataract. Internally, the infections were associated with capsulated, centrally necrotic granulomas, containing large numbers of acid-fast bacilli, found mainly in the mesenteries, spleen, heart and swim bladder. Mycobacterial isolates recovered on Middlebrook 7H10 agar were confirmed as M. salmoniphilum by phenotypical investigation and by partial sequencing of the 16S rRNA, rpoB and Hsp65genes as well as the internal transcribed spacer (ITS1) locus. This study adds burbot to the list of fish species susceptible to piscine mycobacteriosis and describes M. salmoniphilum infection in a non-salmonid fish for the first time. PMID:21797036

8

Avian Mycobacteriosis in Psittacines: A Retrospective Study of 123 Cases.  

One hundred and twenty-three cases of mycobacterioses were diagnosed in psittacine birds from a total of 9,241 submissions for necropsy examination or histopathology made to the California Animal Health and Food Safety Laboratory System between 1990 and 2007. The species affected most commonly were Amazon parrots (Amazona spp.)(n = 32; 26%) and grey-cheeked parakeets Brotogeris pyrrophterus (n = 23; 18.7%). The main gross findings on necropsy examination were enlarged and mottled pale livers and spleens and thickening of the small intestinal wall with numerous pale miliary nodules on the mucosa. Microscopical examination revealed infiltration of foamy macrophages and giant cells containing acid-fast bacteria in various organs. The gene encoding mycobacterial 65 kDa heat shock protein (hsp65) was amplified by nested polymerase chain reaction (PCR) from DNA extracted from 22 cases. The species of Mycobacterium involved was determined by analysis of restriction endonuclease patterns of the PCR products. Mycobacteriumgenavense was detected in 19 cases and Mycobacteriumavium in two cases. One parrotlet (Touit spp.) had a mixed infection of both species of mycobacteria. It is concluded that M. genavense is the primary cause of mycobacteriosis in psittacine birds and the potential for zoonotic disease should be considered, especially for immunocompromised owners. PMID:22884283

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Identificación de micobacterias no tuberculosas: comparación de métodos bioquímicos y moleculares/ Identification of non-tuberculosis mycobacteria: comparison between biochemical and molecular methods  

Abstract in spanish Las infecciones causadas por micobacterias no tuberculosas (MNT) o atípicas constituyen en la actualidad un grave problema de salud, especialmente en pacientes inmunocomprometidos. Estas micobacterias presentan patrones de susceptibilidad a antibióticos particulares y distintos a M. tuberculosis, por lo que la administración del tratamiento adecuado requiere de un método rápido, sencillo y sensible de identificación. La técnica de PRA (Análisis de Restricción de (more) Productos de PCR), basada en la digestión enzimática del producto de amplificación del gen hsp65, ha mostrado ser un método adecuado de identificación de micobacterias. En el presente trabajo se comparó la técnica de PRA con el estándar de identificación de micobacterias representado por las pruebas bioquímicas en 30 aislados provenientes del Laboratorio de Tuberculosis del Instituto de Biomedicina. La técnica de PRA permitió identificar 96% de las cepas analizadas, en comparación con 92.% de cepas identificadas por las técnicas bioquímicas. Los resultados obtenidos fueron idénticos en 18 de 22 cepas, correspondiendo al 82% de los resultados. Se concluye que el PRA es un método rápido, sencillo y económico que produce resultados concordantes con las técnicas tradicionales, con un menor grado de error. Basados en estos resultados se recomienda el uso del PRA en los laboratorios clínicos como método de identificación de rutina para micobacterias. Abstract in english Infections caused by atypical mycobacteria at present constitute a serious health problem, especially in immunocompromised patients. These mycobacteria present particular susceptibility patterns, different from M. tuberculosis, due to which the administration of an adequate treatment requires a fast, simple and sensitive identification method. The PRA technique (PCR Restriction), based on the enzymatic digestion of the amplification product of the hsp65 gene has shown to (more) be an adequate method for the identification of mycobacteria. In this study we compared the PRA technique with the standard mycobacterial identification method, represented by biochemical tests, in 30 isolates from the Tuberculosis Laboratory of the Instituto de Biomedicina. The PRA technique allowed the identification of 96% of the strains analyzed, as compared with 92% of strains identified through biochemical methods. The results obtained were identical in 18 of 22 strains, corresponding to 82% of the results. It is concluded that the PRA technique is a fast, simple and economical method that produces results in concord with traditional techniques, with a lesser degree of error. Based in these results, the use of PRA as routine identification technique for mycobacteria is recommended for clinical laboratories.

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The epidemiology of sepsis in the United States from 1979 through 2000.  

Higher IFN-? responses to mycobacterial antigens were observed in Bos taurus (Holsteins) than in Bos indicus (Zebu) cattle which could due to differences in antigen recognition profiles between the two breeds. The present study was conducted to evaluate mycobacterial antigen recognition profiles of the two breeds. Twenty-three mycobacterial antigens were tested on 46 skin test positive (24 Zebu and 22 Holstein) using enzyme-linked immunospot assay (ELISPOT) and multiple antigen print immunoassay (MAPIA). Herds from which the study cattle obtained were tested for Fasciola antibody. The T cells from both breeds recognized most of the mycobacterial antigens at lower and comparable frequencies. However, antigens such as CFP-10, ESAT-6, Rv0287, Rv0288, MPB87, Acr-2, Rv3616c, and Rv3879c were recognized at higher frequencies in zebu while higher frequencies of T cell responses were observed to Hsp65 in both breeds. Furthermore, comparable antibody responses were observed in both breeds; MPB83 being the sero-dominant antigen in both breeds. The prevalence of Fasciola antibody was 81% and similar in both breeds. This piece of work could not lead to a definitive conclusion if there are differences in mycobacterial recognition profiles between the two breeds warranting for further similar studies using sound sample size from the two breeds. PMID:12700374

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Molecular Detection of Nontuberculous Mycobacteria: Advantages and Limits of a Broad-Range Sequencing Approach  

Abstract The isolation of nontuberculous mycobacteria (NTM) from clinical specimens has become very common in recent years. Such organisms are typically environmental and occasionally pathogenic for humans and animals. Standard diagnosis of mycobacterial infections relies on direct examination and culture. However, molecular tools are now available which allow quicker and more accurate diagnosis. Detection of NTM can be performed directly from clinical samples, although identification is mostly carried out after isolation. Sequencing of genomic targets (such as 16S rRNA, ITS, rpoB or hsp65) allows accurate and rapid identification, but has some technical limitations. A brief summary of the molecular methods available for NTM identification and a discussion of the problems associated with t...

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Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli  

Bacille Calmette-Guerin (BCG)-derived heat shock protein 65 (HSP65) has been demonstrated capable of assisting a fused peptide to generate the peptide-specific cellular immunity. Various HSP65 fusion proteins have been developed as therapeutic cancer vaccines. Purifying a recombinant HSP65 fusion protein with no purification tags for human use is routinely a challenge. Here, we report a scheme for purifying a non-tagged recombinant HSP65-Her2 peptide fusion protein (HSP65-Her2) from Escherichia coli. The HSP65-Her2 is being developed as an immunotherapeutic for the treatment of Her2-positive tumors. After fermentation in a 10-L fermentor, the HSP65-Her2 expressing E. coli were harvested and lysed by sonication. The recombinant HSP65-Her2 was then purified with four successive steps includi...

13

Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli.  

Bacille Calmette-Guerin (BCG)-derived heat shock protein 65 (HSP65) has been demonstrated capable of assisting a fused peptide to generate the peptide-specific cellular immunity. Various HSP65 fusion proteins have been developed as therapeutic cancer vaccines. Purifying a recombinant HSP65 fusion protein with no purification tags for human use is routinely a challenge. Here, we report a scheme for purifying a non-tagged recombinant HSP65-Her2 peptide fusion protein (HSP65-Her2) from Escherichia coli. The HSP65-Her2 is being developed as an immunotherapeutic for the treatment of Her2-positive tumors. After fermentation in a 10-L fermentor, the HSP65-Her2 expressing E. coli were harvested and lysed by sonication. The recombinant HSP65-Her2 was then purified with four successive steps including Butyl-Sepharose FF, DEAE-Sepharose FF, 1% Triton X-114 phase separation and Sephadex G-25. Results showed that HSP65-Her2 was purified up to 97% purity and was able to generate Her2-specific cytotoxic T lymphocytes (CTLs), suggesting that the scheme is efficient for purifying the non-tagged HSP65-Her2 fusion protein with biological activity. PMID:17275328

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Diferenciação de micobactérias por PCR multiplex/ Differentiation of micobacteria by multiplex PCR  

Abstract in portuguese O trabalho visou à otimização de um método baseado na reação em cadeia da polimerase multiplex - para diferenciação de micobactérias de interesse para a saúde pública. A PCR Multiplex baseou-se na amplificação simultânea do genehsp65, presente em todo gênero Mycobacterium, do gene dnaJ, presente apenas em Mycobacterium tuberculosis e Mycobacterium avium e da sequência de inserção IS6110 presente no complexo Mycobacterium tuberculosis, gerando amplicons d (more) e 165pb, 365pb e 541pb, respectivamente. O limite de detecção foi de 1fg para o alvo hsp65, 100pg para o dnaJ e 0,1fg para o IS6110. A PCR multiplex detectou até 100pg de DNA de Mycobacterium tuberculosis. O sistema demonstrou ser específico e sensível na detecção de Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium e Mycobacterium smegmatis. Os resultados obtidos utilizando cepas de referência demonstraram que a PCR multiplex pode ser uma ferramenta rápida, sensível e específica na diferenciação de micobactérias. Abstract in english This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuber (more) culosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of mycobacteria.

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Effect of HSP65 on the expression of adhesion molecules in mice heart endothelial cells.  

This study aims to research the effect of HSP65 on the expression of adhesion molecules in activated mice heart endothelial cells (MHECs), which were from myocardial tissue of newborn animals. We used different concentrations of LPS as potent inducers to stimulate MHECs, adhesion molecule expression in vitro, including intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), E-, and P-selectins, then compared the mRNA and protein levels of adhesion molecules expression with or without HSP65 treatment at different levels. The optimal concentration of LPS to induce MHECs adhesion molecule expression is 100 ng/ml; HSP65 treatment significantly reduced the mRNA and protein levels of MHECs' ICAM-1, VCAM-1, E-, and P-selectins expression (p?HSP65 in inhibiting MHECs activation is 0.8 ng. HSP65 has the inhibitory effect on adhesion molecules expression in activated MHECs. PMID:22160869

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Diagnosis of pulmonary disease caused by Mycobacterium abscessus: a case report.  

Mycobacterium abscessus (M. abscessus) is a non-tuberculous mycobacterium and an important emerging pathogen causing skin, soft tissue and pulmonary infections. The case of a 59-year-old man with a history of pulmonary tuberculosis (TB) and current pulmonary infection due to M. abscessus, complicated with pneumocardial disease and bronchiectasis, is described. Zhiel-Neelsen stain and acid Lowenstein-Jensen culture were both positive for acid-fast bacillus. The patient was initially misdiagnosed and ineffectively treated for pulmonary TB. Antimycobacterial susceptibility tests found the isolate to be resistant to four first-line and seven second-line antituberculosis drugs. The isolate was finally identified as M. abscessus using 16S ribosomal RNA and hsp65 and rpoB gene sequence analysis. Species of mycobacterium should be included in the differential diagnosis when patients do not respond to standard antituberculosis therapy. Molecular methods are useful for rapid and species-specific identification. PMID:21819731

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Microbial heat shock protein 65 attenuates airway hyperresponsiveness and inflammation by modulating the function of dendritic cells.  

Heat shock proteins (HSPs), produced in response to stress, are suppressive in disease models. We previously showed that Mycobacterium leprae HSP65 prevented development of airway hyperresponsiveness and inflammation in mice. Our goal in this study was to define the mechanism responsible for the suppressive effects of HSP. In one in vivo approach, BALB/c mice were sensitized to OVA, followed by primary OVA challenges. Several weeks later, HSP65 was administered prior to a single, provocative secondary challenge. In a second in vivo approach, the secondary challenge was replaced by intratracheal instillation of allergen-pulsed bone marrow-derived dendritic cells (BMDCs). The in vitro effects of HSP65 on BMDCs were examined in coculture experiments with CD4(+) T cells. In vivo, HSP65 prevented the development of airway hyperresponsiveness and inflammation. Additionally, Th1 cytokine levels in bronchoalveolar lavage fluid were increased. In vitro, HSP65 induced Notch receptor ligand Delta1 expression on BMDCs, and HSP65-treated BMDCs skewed CD4(+) T cells to Th1 cytokine production. Thus, HSP65-induced effects on allergen-induced airway hyperresponsiveness and inflammation were associated with increased Delta1 expression on dendritic cells, modulation of dendritic cell function, and CD4(+) Th1 cytokine production. PMID:22933632

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The immunogenetics of asthma and eczema: a new focus on the epithelium  

Background Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the Providencia stuartii null-rhomboid mutant and then deleted from Mycobacterium smegmatis for additional insight in mycobacteria. Methodology/Principal Findings Using in silico analysis we identified in M. tuberculosis genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into P. stuartii null-rhomboid mutant (?aarA), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of P. stuartii). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in M. smegmatis, the ?MSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ?MSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ?MSMEG_4904–?MSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ?MSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin). Conclusions/Significance Mycobacterial rhomboid-encoding genes exhibit differences in complementing aarA whereby it's only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids. PMID:15573132

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The uraA locus and homologous recombination in Mycobacterium bovis BCG.  

Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic markers and as sequences used to target homologous integration of recombinant DNA into the genome. We isolated the Mycobacterium bovis BCG gene encoding orotidine-5...

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Action-Mechanism of Trichoderin A, an Anti-dormant Mycobacterial Aminolipopeptide from Marine Sponge-Derived Trichoderma sp.  

In the course of our search for anti-dormant mycobacterial substances from marine organisms, we previously isolated three new aminolipopeptides, named trichoderins A, A1 and B, from the culture of the marine sponge-derived fungus of Trichoderma sp. and determined their chemical structures. To identify the gene that could confer a resistance to trichoderin A, we prepared transformants of Mycobacterium (M.) smegmatis, which were transformed with the genomic DNA library of M. bovis BCG constructed in the multi-copy shuttle cosmid pYUB145. Then, the transformant of M. smegmatis, which over-expressed a part of genes that coded mycobacterial ATP synthase, was found to exhibit a resistance to trichoderin A. In addition, trichoderin A reduced ATP contents in M. bovis BCG. These findings elucidated that the anti-mycobacterial activity of trichoderins comes from the inhibition of ATP synthesis.   

 
 
 
 
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Severe disseminated mycobacterial infection in a boy with a novel mutation leading to IFN-@cR2 deficiency  

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare syndrome characterized by predisposition to severe, sometimes lethal, disease caused by otherwise poorly virulent mycobacteria. We report here a boy with a recurrent mycobacterial infection from the age of five months. Immunological analyses revealed an inability to respond to IFN-@c, subsequent genetic analyses revealed a novel homozygous mutation, r.679G > A in the IFNGR2 gene, resulting in a G227R substitution, that caused IFN-@cR2 deficiency. This is only the 8th mutation in IFN-@cR2 known so far. The boy eventually died of hepatic coma due to liver failure at the age of five.

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Two different 16S rRNA genes in a mycobacterial strain.  

Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-established method used to identify bacteria, particularly mycobacteria. Unique sequences allow identification of a particular genus and species. If more than one 16S rDNA is present on one mycobacterial genome, their sequences are assu...

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PCR-enzyme-linked immunosorbent assay and partial rRNA gene sequencing: a rational approach to identifying mycobacteria.  

A PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid identification of mycobacterial DNA coding for 16S rRNA was developed. The PCR selectively targeted and amplified part of the 16S rRNA gene from all mycobacteria while simultaneously labelling one strand of the amplified pro...

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Roles of MAP1152 and MAP1156 Proteins in the Immunopathogenesis of Mycobacterium paratuberculosis  

The Mycobacterium avium subsp. paratuberculosis (MAP) K-10 genome is a circular chromosome of about 4.8 Mb encoding 4,344 open reading frames (ORFs). Mycobacterial genomes are characterized by the presence of gene clusters encoding conserved proteins with PE and PPE motifs. Members of these families...

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Development of Luminescent Mycobacterium avium subsp. paratuberculosis for Rapid Screening of Vaccine Candidates in Mice  

Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for...

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Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing  

To detect the site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-TB by DNA sequencing. 50 MDR-TB patients were enrolled in our study after informed written consent. Mycobacterial DNA was extracted from sputum samples by Universal Sample Processing (USP) method and RRDR of rpo B ...

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Attenuated Host Resistance against Mycobacterium bovis BCG Infection in Mice Lacking Osteopontin  

Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tuberculosis. We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection. When infected with Mycobacterium bovis BCG, mice lacking a functional OPN gene ha...

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Mycobacterium sherrisii sp. nov., a slow-growing non-chromogenic species.  

'Mycobacterium sherrisii' is an undescribed species that appears to be emerging, in particular, among HIV-positive patients originating from Africa. To describe 'M. sherrisii', to ensure that the species name is validly published and to define its phylogenetic position, we collected 11 of these strains reported in five previous studies, and subjected them to biochemical identification, cell-wall mycolic acid analysis and sequencing of multiple housekeeping genes. The bacteria formed smooth and generally non-chromogenic colonies after 2-3 weeks of subculture at 24-37 °C; photochromogenic and scotochromogenic pigmentation were exhibited by three and two strains, respectively. The strains were positive for the heat-stable catalase test, but negative in tests for hydrolysis of Tween 80, nitrate reduction, ?-glucosidase and 3-day arylsulfatase. Mycolic acid patterns, obtained by HPLC, resembled a trimodal profile similar to those of type strains of Mycobacterium simiae, Mycobacterium lentiflavum, Mycobacterium triplex and Mycobacterium genavense. The 16S rRNA gene sequences of the 11 strains differed by 4 bp (99.7 % similarity) from that of the type strain of the closest related species, M. simiae ATCC 25275(T). Levels of internal transcribed spacer (ITS) and partial hsp65 and rpoB gene sequence similarity between the two taxa were 95.8 % (271/283 bp), 97.5 % (391/401 bp) and 95.2 % (700/735 bp), respectively. On the basis of these results, we propose the formal recognition of Mycobacterium sherrisii sp. nov. The type strain is 4773(T) (?=?ATCC BAA-832(T)?=?DSM 45441(T)). PMID:20639227

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Direct identification of mycobacteria from smear-positive sputum samples using an improved multiplex polymerase chain reaction assay.  

The rapid identification of mycobacteria from smear-positive sputum samples is very important. To identify the Mycobacterium tuberculosis complex (MTBC) and frequently isolated nontuberculous mycobacteria strains directly from smear-positive sputum samples, an improved multiplex polymerase chain reaction (PCR) assay was developed. Nine pairs of primers targeting the 16S-23S rDNA internal transcribed spacer-1, hsp65, and the early secretory antigen (ESAT-6) gene sequences were developed, and their efficacy was evaluated in comparison to traditional culturing and 16S rRNA gene sequencing methods. A total of 200 smear- and culture-positive sputum specimens collected between November 2005 and May 2006 were used for the analysis. The results of the assay showed an accurate identification rate for acid-fast bacilli (AFB) 3+, AFB 2+, and AFB rare/1+ samples of 98%, 95%, and 53%, respectively. The improved multiplex PCR method saves time and has advantages for identifying mycobacteria from AFB 2+ and 3+ sputum samples. The method is suitable for use in countries with a high MTBC prevalence rate. PMID:22280996

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Diversity of environmental Mycobacterium isolates from hemodialysis water as shown by a multigene sequencing approach.  

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus. PMID:17449684

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Distribución de patrones PRA en aislamientos clínicos del complejo Mycobacterium avium procedentes de España y Suramérica  

Abstract in spanish La infección por el complejo Mycobacterium avium (MAC) es la infección sistémica más frecuente en la fase terminal del SIDA. Las sondas de ADN disponibles en el mercado para la identificación de micobacterias son muy precisas pero extremadamente costosas. Por eso, la mayoría de los laboratorios clínicos de Latinoamérica aún tipifican micobacterias mediante pruebas fenotípicas que son lentas, laboriosas y poco precisas. En este trabajo se aplicó el análisis del (more) polimorfismo de los fragmentos de restricción del gen hsp65 (PRA) a la identificación de MAC en 163 aislamientos clínicos procedentes de España y Suramérica. El genotipo PRA predominante en cada país fue: M. avium tipo I en Argentina (23/42, 55%) y Brasil (48/72, 67%), M. avium tipo II en España (18/26, 69%) y M. avium tipo III en Colombia (10/23, 43%). Este último genotipo, que aún no fue descrito fuera del continente americano, resultó muy infrecuente en los otros tres países del estudio. Se discuten ventajas e inconvenientes de la aplicación del PRA al diagnóstico micobacteriológico. Abstract in english Distribution of PRA patterns of clinical isolates of the Mycobacterium avium complex from Spain and South America Mycobacterium avium complex (MAC) infections are the most frequent systemic infections associated with advanced AIDS. DNA probes for accurate identification of mycobacteria are available but are very expensive in many Latin American settings. Consequently, most Latin American diagnostic laboratories employ inaccurate and outdated tests for mycobacteria identif (more) ication. Therefore, PCR restriction analysis (PRA) of the hsp65 gene was evaluated for the identification of 163 MAC human isolates originated from Spain and South America. The predominant PRA type in each country was: M. avium type I in Argentina (23/42, 55%) and Brazil (48/72, 67%), M. avium type II in Spain (18/26, 69%) and M. avium type III in Colombia (10/ 23, 43%). The Colombia frequency is noteworthy, since the PRA type III was quite infrequent in the other three countries. Furthermore, its presence has not been reported outside the Americas. The advantages and disadvantages of PRA in diagnostic mycobacteriology are discussed

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Sensibilidad a los antimicrobianos de micobacterias de crecimiento rápido mediante el método E-test/ Assessment of in vitro susceptibility to antimicrobials of rapidly growing mycobacteria by E-test  

Abstract in english Background: Rapidly growing mycobacteria (RGM) are considered opportunistic pathogens. An increasing number of post traumatic or surgical infections are caused by these microorganisms. Aim: To determine the antimicrobial susceptibility of RGM using the E-test method. Material and methods: A total of 54 isolates of RGM was obtained from several clinical samples and selected for this study Strains were identified to the species level by phenotypic and biochemical characteri (more) stics, PCR-restriction enzyme analysis of the hsp65 gene (PRA) and sequencing of the 16S rRNA. Susceptibility was investigated by E-test to amikacin, cefoxitin, ciprofioxacin, clarithromycin, imipenem, quinupristin/dalfopristin, linezolid and tigecycline. Results: Twelve different species of RGM were identified: Mycobacterium fortuitum (23 strains), M chelonae (11), M abscessus (10), Msenegalense (2), Malvei (1), Mbrumae (1), Mmageritense (1), mucogenicum (1), M neoaurum (1), Mperegrinum (1), M septicum (1) y M smegmatis (1). All the strains were inhibited by low concentrations of amikacin and tigecycline. Susceptibility to cefoxitin, fluoroquinolones, clarithromycin, imipenem and linezolid was variable. All but two strains were resistant to quinupristin/ dalfopristin. Conclusions: Due to the uneven antimicrobial susceptibility of different species of RGM, an antimicrobial susceptibility test is mandatory for these microorganisms. The E-test method is well suited to determine minimum inhibitory concentrations.

33

Identificación rápida de micobacterias no tuberculosas mediante análisis de patrones de restricción/ Rapid identification of non tuberculous mycobacteria by restriction pattern analysis  

Abstract in english Background: The frequency of diseases caused by non tuberculous mycobacteria has increased in the last years. Their clinical diagnosis is difficult, mainly in immunocompromised patients. The identification of these mycobacteria by traditional methods is based on phenotypic characteristics and the results are obtained two to four weeks after their isolation in primary cultures. Aim: To report a new identification method for non tuberculous mycobacteria. Material and method (more) s: The restriction pattern analysis method was implemented. It is based on the amplification, using polymerase chain reaction (PCR), of a polymorphic region of 440 base pairs that codifies Hsp65 protein, followed by a digestion with BstE II and Hae III restriction enzymes. The results were compared with patterns established for each strain. Results: Sixty four strains of mycobacteria obtained from clinical samples and seven reference mycobacteria, were identified using the traditional methods and restriction pattern analysis. The latter method identified the same strain as the former in 87.5% of cases. In the remainder 12.5% of cases there was no agreement between both methods. In these, the sequencing of a fragment of a gene that codifies 16S ribosomal RNA, confirmed the correct identification by restriction patterns. Conclusions: Restriction pattern analysis is a rapid identification method for non tuberculous mycobaterial strains

34

Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum.  

The pSUM vectors, a family of Mycobacterium/Escherichia coli shuttle cloning vectors derived from the pSU series of vectors are described. They all contain the pACYC184 origin of replication enabling them to replicate in E. coli, the mycobacterial pAL5000 origin of replication and a kanamycin-resistance (KmR) gene which allows selection in both E. coli and mycobacteria. They carry a multiple cloning site (MCS) to facilitate cloning and the lacZ alpha reporter gene for screening inserts in E. coli. A library of total DNA from a strain of Mycobacterium fortuitum was constructed with one of these vectors, pSUM36, and a mycobacterial gentamicin-resistance (GmR) gene was cloned by selecting for GmR in Mycobacterium smegmatis. PMID:8918226

35

Pristinamycin-inducible gene regulation in mycobacteria  

In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction; both proteins and ant...

36

Effects of nasal immunization of multi-target preventive vaccines on atherosclerosis.  

Previous investigations have demonstrated that anti-inflammatory or lipid-lowering treatments could be useful for alleviating morbidity and mortality of atherosclerotic cardiovascular diseases. However, whether a vaccine designed to target inflammation and lipid simultaneously is more powerful to control the process of atherosclerosis remain to be unknown. Here, a vaccine was designed to target heat shock protein-65(Hsp65) and cholesteryl ester transfer protein (CETP) simultaneously and the effects of nasal immunization of multi-target vaccine on high-cholesterol-diet-driven rabbit atherosclerosis lesions were evaluated. Sera, nasal lavages and lung washes were used to ELISA assay for the analysis of IgG and IgA against Hsp65 and CETP. Sera were also used to the analysis of the avidity of combination of anti-Hsp65 and anti-CETP IgG antibodies with corresponding antigen, cytokines IL-10 and IFN-?, and lipoproteins. In addition, aortas were harvested for analysis of atherosclerotic lesions. The results showed that lower and lasting specific anti-Hsp65 IgG and high anti-CETP IgG in sera and protective anti-Hsp65 and anti-CETP IgA in nasal cavity and lung were induced, the avidity of combination of anti-Hsp65 and anti-CETP IgG with antigen were higher, and more protective IL-10 and less adverse IFN-? were produced. In addition, sera TC, and LDL-C were decreased. As a result, the size of aorta atherosclerotic plaques was significantly reduced. We conclude that multifaceted vaccine combining lipid-regulating with anti-inflammation was a potential remedy, especially for atherosclerosis with complicated etiology. PMID:22192848

37

Inactivación de genes de Mycobacterium tuberculosis y su potencial utilidad en la prevención y el control de la tuberculosis  

Abstract in spanish El conocimiento derivado del genoma de Mycobacterium tuberculosis, junto con el desarrollo de sofisticados sistemas para la manipulación genética del bacilo, ofrece la mayor promesa para el desarrollo de herramientas nuevas y más eficientes para prevenir y controlar la tuberculosis. Se han desarrollado métodos más eficientes para la inactivación de genes micobacterianos que se han convertido en el pilar de la genómica funcional micobacteriana. La generación de mut (more) antes mediante la inactivación génica, apoyada directa o indirectamente por el desciframiento del genoma micobacteriano, ha permitido la generación de un número significativo de mutantes de M. tuberculosis. En algunos casos, el análisis de estas mutantes ha establecido relaciones entre los productos génicos y sus funciones en la fisiología y la patogenicidad de la micobacteria. En esta revisión se describen los estudios más representativos basados en dichas mutantes. Abstract in english Gene inactivation in Mycobacterium tuberculosis and its use in tuberculosis control and prevention Availability of the M. tuberculosis genome sequence and the development of sophisticated systems for genetic manipulation of bacilli offer the potential for new and effective tools to prevent and control tuberculosis. Efficient methods to inactivate mycobacterial genes have been developed. These methods have become the cornerstone for the application and development of mycob (more) acterial functional genomics. Specific mutants are generated to establish the role of targetted genes associated with mycobacterial physiology and pathogenesis. Gene inactivation, supported directly or indirectly by the deciphering of the mycobacterial genome, has permitted the generation of large numbers of M. tuberculosis mutants. Analysis of these mutants has (in some cases) established relationships between gene products and their role in mycobacterial physiology and pathogenesis.

38

Reacción en cadena de la polimerasa: una contribución para el diagnóstico de la tuberculosis extrapulmonar y de las micobacteriosis/ Polymerase chain reaction (PCR): a contribution for diagnosis of extrapulmonary tuberculosis and micobacteriosis  

Abstract in spanish Introducción. La Organización Mundial de la Salud define la tuberculosis extrapulmonar como la enfermedad tuberculosa localizada en cualquier órgano diferente a los pulmones y, la micobacteriosis, como la infección bacteriana producida por micobacterias no tuberculosas. El diagnóstico de tuberculosis extrapulmonar y micobacteriosis se hace fundamentalmente por el cultivo y la posterior identificación de la micobacteria por pruebas bioquímicas, así como por bacilos (more) copia para el primer caso. El cultivo de micobacterias presenta limitaciones, especialmente, por el tiempo de incubación que puede tardar hasta 12 semanas y por la sensibilidad para determinar la presencia del microorganismo. Actualmente, se utilizan herramientas moleculares que permiten disminuir el tiempo de diagnóstico. Entre ellas está la reacción en cadena de la polimerasa (PCR) que es una prueba rápida con buena sensibilidad y especificidad para la identificación de patógenos. Objetivo. Implementar la (PCR) como apoyo para el diagnóstico de la tuberculosis extrapulmonar y la micobacteriosis, para disminuir el tiempo en la obtención de resultados y contribuir a la instauración de tratamientos oportunos para estas patologías. Materiales y métodos. El estudio se llevó a cabo entre junio de 2005 y agosto de 2006. Se procesaron 57 muestras clínicas provenientes de 15 centros hospitalarios del país, analizadas por PCR para la secuencia de inserción IS6110 y para el gen hsp65. Se incluyeron datos clínicos del paciente, edad, sexo y prueba de VIH. Resultados. Los resultados de amplificación para PCR IS6110 fueron positivos en 6 pacientes, lo que sugiere tuberculosis extrapulmonar, y para PCR hsp65, en igual número, lo que sugiere un proceso de micobacteriosis. Se confirmó el diagnóstico de tuberculosis meníngea en 4 (6,9%) pacientes, seguida por tuberculosis en piel y tuberculosis miliar, cada una en un (1,7%) paciente. Se identificaron micobacterias no tuberculosas en 6 (10,3%) pacientes, así: 4 (7%) en esputo, 1 (1,75%) en secreción de abdomen y 1 (1,75%) en secreción de lesiones en piel. Un aislamiento de Mycobacterium chelonae de piel se asoció con un tratamiento previo con mesoterapia. No se encontró relación entre el estado de ¿portador de? VIH, con las patologías estudiadas. Discusión. Se presenta la PCR como una buena ayuda diagnóstica, específicamente, por la obtención de resultados en un lapso de 3 días. Abstract in english Introduction: The World Health Organization defines extrapulmonary tuberculosis as the disease located in any type of organ different from the lungs, and mycobacteriosis as the bacterial infection produced by environmental Mycobacteria. The diagnosis of extrapulmonary tuberculosis and mycobacteriosis is achieved fundamentally by culture and later identification of bacteria by biochemical tests, as well as by sputum smear for the first case. Culture presents limitations sp (more) ecially related with the time of incubation that can last up to 12 weeks and with the sensitivity to the presence of the microorganism. Currently, molecular tools allow a decrease in the time of diagnosis. Among these tools, the polymerase chain reaction (PCR) is a rapid test with good sensitivity and specificity for the identification of pathogenic Mycobacteria. Objective: To implement PCR as support to the diagnosis of extrapulmonary tuberculosis and mycobacteriosis to decrease the time in obtaining results and to contribute to the implementation of appropriate treatments for these diseases. Materials and methods: The study was carried out between June 2005 and August 2006. 57 clinical samples of 15 hospitals throughout the country were analyzed by PCR for the sequence of insertion of IS6110 and for the gene hsp65. Clinical information of the patients, age, sex, and VIH test were included. Results: Results of amplification for PCR IS6110 were positive in 6 patients suggesting extrapulmonary tuberculosis and for PCR hsp65 in an equivalent number, suggesting a process of mycobacteriosis. The study contributed to the diagnosis of meningeal tuberculosis in 4 (6.9%) patients, followed by skin tuberculosis and miliar tuberculosis each one with 1 (1.7%) patient. We identified ¿non- Mycobacteryum tuberculosis? ¿lNMT? in 6 (10.3%) cases, which were differentiated as follows: 4 (7%) patients in sputum, 1 (1.75 %) patient in secretion of abdomen and 1 (1,75 %) patient in secretion of skin injuries. Mycobacterium chelonae isolation of the skin was associated with cosmetic mesotherapy. No relation was found between VIH status and the studied pathologies. Discussion: This study shows PCR as a valuable diagnostic technique for patients, specifically for obtaining results within three days.

39

Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series.  

A series of Escherichia coli-mycobacteria shuttle plasmids for the isolation and study of gene regulatory sequences was constructed. These pJEM vectors contain an efficient transcription terminator and multiple cloning sites and allow either operon or gene fusions to lacZ. By constructing operon fusions with pJEM15, we assessed various previously characterized mycobacterial promoters in the fast-growing species Mycobacterium smegmatis and the slow-growing species M. bovis BCG. Our results suggest that M. smegmatis and M. bovis BCG RNA polymerases do not share the same specificity. To isolate new mycobacterial promoters, an M. tuberculosis DNA library was generated, using pJEM13, and screened in M. smegmatis. Several Lac+ clones were isolated, and the beta-galactosidase activity was measured. PMID:7961429

40

Analysis of the immune response in infections of the goldfish (Carassius auratus L.) with Mycobacterium marinum  

The rapid doubling time and genetic relatedness of the fish pathogen Mycobacterium marinum to Mycobacterium tuberculosis has rendered the former an attractive model for investigating mycobacterial host-pathogen interactions. We employed the M. marinum-goldfish infection model to investigate the in vivo immune responses to this pathogen in the context of a natural host. Histological analysis revealed mycobacterial infiltrates in goldfish kidney and spleen tissues, peaking 28days post infections (dpi). Quantitative gene expression analysis showed significant increases of mRNA levels of pro-inflammatory cytokines (IFN@c, IL-12p40, IL-1@b1) and cytokine receptors (IFNGR1-1, TNFR2) at 7dpi. Conversely, the gene expression levels of key anti-inflammatory cytokines TGF@b and IL-10 were elevated a...

 
 
 
 
41

Differentiation of Mycobacterial Species by hsp65 Duplex PCR Followed by Duplex-PCR-Based Restriction Analysis and Direct Sequencing?  

Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and dif...

42

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.  

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L.lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-? - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

43

Mycobacterium algericum sp. nov., a novel rapidly growing species related to M. terrae complex and associated with goat lung lesions  

A previously undescribed, rapid growing, non-chromogenic Mycobacterium novel species isolated from a goat lung lesion in Algeria is reported. Biochemical and molecular tools were used for its complete description and showed its affiliation to the M. terrae complex. 16S rRNA, rpoB and hsp65 sequences...

44

Mycobacterium tuberculosis Chaperonin 60.1 Is a More Potent Cytokine Stimulator than Chaperonin 60.2 (Hsp 65) and Contains a CD14-Binding Domain  

Much attention has focused on the Mycobacterium tuberculosis molecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells. However, M. tuberculosis is one of a small g...

45

OsmC proteins of Mycobacterium tuberculosis and Mycobacterium smegmatis protect against organic hydroperoxide stress  

Bacterial antioxidants play a critical role in the detoxification of endogenously and host derived oxidative radicals during host-pathogen interactions. Recently, the osmotically induced bacterial protein C (OsmC) is included in the antioxidant category of enzymes as it shows structural and functional relationships with organic hydroperoxide reductase (Ohr) enzyme. A copy of the gene encoding OsmC is conserved across mycobacterial species, including Mycobacterium tuberculosis (Rv2923c) and Mycobacterium smegmatis (MSMEG2421), but its role in protecting these species against oxidative stress is unknown. To determine the role of OsmC in mycobacterial oxidative stress, we overexpressed and purified OsmCs of M. tuberculosis and M. smegmatis and assessed their ability to reduce peroxide substra...

46

beta-Galactosidase reporter system in mycobacteria and its application in rapid antimycobacterial drug screening.  

Pathogenic mycobacteria are generally slow growing organisms and it takes several weeks to evaluate inhibitors of growth. Therefore, for rapid screening of the inhibitors of mycobacterial growth, a beta-galactosidase reporter system has been described which utilises a recombinant Mycobacterium smegmatis mc(2)155 expressing E. coli lacZ gene as the test organism. The assay is based on production of beta-galactosidase in presence of drugs during growth. A correlation between beta-galactosidase production and colony forming ability of mycobacteria was obtained. beta-galactosidase production was inhibited within 6 h by front line standard antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, pyrazinamide and ofloxacin at their reported MICs. The assay was performed on mycobacterial cells permeabilized with chloroform and sodium dodecyl sulfate. PMID:9207204

47

The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol.  

Ethambutol (EMB), a frontline antituberculous drug, targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan. EMB inhibits the polymerization of cell wall arabinan, and results in the accumulation of the lipid carrier decaprenol phosphoarabinose, which suggests that the drug interferes with the transfer of arabinose to the cell wall acceptor. Unfortunately, resistance to EMB has been described in up to 4% of clinical isolates of Mycobacterium tuberculosis and is prevalent among isolates from patients with multidrug-resistant tuberculosis. We used resistance to EMB as a tool to identify genes participating in the biosynthesis of the mycobacterial cell wall. This approach led to the identification of the embCAB gene cluster, recently proposed to encode for mycobacterial arabinosyl transferases. Resistance to EMB results from an accumulation of genetic events determining overexpression of the Emb protein(s), structural mutation in EmbB, or both. Further characterization of these proteins might provide information on targets for new chemotherapeutic agents and might help development of diagnostic strategies for the detection of resistant M. tuberculosis. PMID:9142129

48

Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria.  

Two novel shuttle vectors for mycobacteria are described which have been derived from the expression system pSD5 developed in our laboratory. Plasmid pSD5B is a promoter-selection vector containing a promoterless lacZ gene and allows the identification of mycobacterial promoters by the blue colour of the colonies on solid media containing XGal. Moreover, the chronological order of appearance of blue colonies and intensity of colour provide a qualitative index of transcriptional strengths of the cloned promoters. Plasmid pSD5C has been designed to construct mycobacterial genomic libraries and express the cloned DNA inserts as fusion proteins with maltose binding protein in mycobacteria. Libraries in pSD5C provide feasibility for their screening with either DNA probes or specific antisera for identifying the genes of interest and for isolation of specific genetic loci by complementation of Escherichia coli and mycobacterial mutants. These vectors combine the ease of working in E. coli with the advantage of directly propagating them in mycobacteria without further manipulations. Finally, we demonstrate that these vectors function efficiently both in fast growing Mycobacterium smegmatis and slow growing mycobacteria including Mycobacterium tuberculosis and Mycobacterium bovis BCG. PMID:9185847

49

Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability  

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG{_}6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG{_}6394, solved to 2.9 {angstrom} resolution, revealed an {alpha}/{beta} hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG{_}6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.

50

Disseminated mycobacteriosis affecting a prosthetic aortic valve: first case of Mycobacterium peregrinum type III reported in Colombia/ Micobacteriosis diseminada con compromiso de válvula aórtica protésica: primer caso de Mycobacterium peregrinum de tipo III reportada en Colombia  

Abstract in spanish Las micobacterias de rápido crecimiento son microorganismos pertenecientes a las micobacterias no tuberculosas que tienen amplia distribución ambiental. Aunque usualmente no son patógenas para los humanos, en condiciones desfavorables, pueden causar enfermedad en la población general o en huéspedes inmunocomprometidos, por lo cual se consideran oportunistas. Mycobacterium preregrinum es una micobacteria de rápido crecimiento perteneciente al complejo fortuitum que h (more) a sido reportado como responsable de casos de micobacteriosis en humanos. Se presenta el caso de una micobacteriosis por M. peregrinum de tipo III, el primero reportado en Colombia, en una paciente de 17 años de edad con una endocarditis de una válvula aórtica protésica, implantada inicialmente por estenosis subaórtica congénita con insuficiencia y, posteriormente, por estenosis aórtica relacionada con la válvula inicialmente implantada. Un año después del segundo implante, presentó sintomas respiratorios y pérdida de peso sugestivos de tuberculosis pulmonar. Las coloraciones de Ziehl-Neelsen del esputo fueron positivas aunque la radiografía de tórax no mostró compromiso del parénquima. En el ecocardiograma se encontró una vegetación en la válvula aórtica. En las muestras de sangre y de esputo, se identificó M. peregrinum de tipo III por cultivo, pruebas bioquímicas y análisis molecular del gen hsp65 por PCR-restriction pattern analysis (PRA). La paciente se sometió a cambio de válvula y recibió tratamiento combinado contra la micobacteria, con rápida recuperación. Las muestras tomadas del sistema respiratorio y sanguíneo se tornaron negativas para micobacterias. Abstract in english Rapidly growing mycobacteria are non-tuberculous mycobacteria amply present in the environment. Although they are not usually pathogenic for humans, they are opportunistic in that they can cause disease in people with disadvantageous conditions or who are immunocompromised. Mycobacterium peregrinum, an opportunistic, rapidly growing mycobacteria, belongs to the M. fortuitum group and has been reported as responsible for human cases of mycobacteriosis. A case of M. peregri (more) num type III is herein reported as the first in Colombia. It presented as a disseminated disease involving a prosthetic aortic valve (endocarditis) in a seventeen-year-old girl with a well-established diagnosis of prosthetic aortic valve endocarditis who was referred for a surgical replacement. Due to a congenital heart disease (subaortic stenosis with valve insufficiency), she had two previous aortic valve implantation surgeries. One year after the second implantation, the patient presented with respiratory symptoms and weight lost indicative of lung tuberculosis. A chest X-ray did not show parenchymal compromise but several Ziehl-Neelsen stains were positive. An echocardiography showed a vegetation on the prosthetic aortic valve. In blood and sputum samples, M. peregrinum type III was identified through culture, biochemical tests and hsp65 gene molecular analysis (PRA). The patient underwent a valve replacement and received a multidrug antimycobacterial treatment. Progressive recovery ensued and further samples from respiratory tract and blood were negative for mycobacteria.

51

Silencing Mycobacterium smegmatis by using tetracycline repressors.  

Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as co-repressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of lacZ expression in Mycobacterium smegmatis in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential secA1 gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of secA1 could help identify substrates of the general secretion pathway, we analyzed the main porin of M. smegmatis, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after secA1 silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems. PMID:17483222

52

Pristinamycin-inducible gene regulation in mycobacteria.  

In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria. PMID:19428723

53

A Copper-responsive Global Repressor Regulates Expression of Diverse Membrane-associated Transporters and Bacterial Drug Resistance in Mycobacteria.  

Sequencing of entire bacterial genomes has led to the identification of many membrane-associated transporters, including several multidrug resistance transport proteins, in recent years. However, the regulators and signaling pathways involved in the expression of these genes remain largely unknown. In this study, we have identified Ms2173, a GntR/FadR family transcription factor, as a novel global regulator in Mycobacterium smegmatis. Ms2173 was found to specifically recognize a 15-bp palindromic motif and to broadly regulate expression of 292 genes, including 37 genes that encode membrane-associated transport proteins. Copper ions induced Ms2173 to form inactive proteins lacking DNA-binding activity. Ms2173 was shown to function as a repressor of its target genes. Interestingly, we found that the function of Ms2173 was linked to mycobacterial drug resistance. Compared with the substantially enhanced drug resistance in the Ms2173-deleted mutant strain, the strains overexpressing Ms2173 were more sensitive to anti-tuberculosis drugs than the wild-type strain. Additionally, copper ions could partially counteract the in vivo function of Ms2173. We have thus characterized the first mycobacterial GntR/Fad-like transcription factor that functions as a copper ion-responsive global repressor that we have renamed GfcR. These findings further enhance our understanding of membrane-associated transporter regulation and drug resistance in mycobacteria. PMID:23014988

54

Clinical and laboratory aspects of the diagnosis and management of cutaneous and subcutaneous infections caused by rapidly growing mycobacteria.  

Rapidly growing mycobacteria (RGM) are known to cause pulmonary, extra-pulmonary, systemic/disseminated, and cutaneous and subcutaneous infections. The erroneous detection of RGM that is based solely on microscopy, solid and liquid cultures, Bactec systems, and species-specific polymerase chain reaction (PCR) may produce misleading results. Thus, inappropriate therapeutic measures may be used in dermatologic settings, leading to increased numbers of skin deformity cases or recurrent infections. Molecular tools such as the sequence analyses of 16S rRNA, rpoB and hsp65 or PCR restriction enzyme analyses, and the alternate gene sequencing of the superoxide dismutase (SOD) gene, dnaJ, the 16S-23S rRNA internal transcribed spacers (ITS), secA, recA1, dnaK, and the 32-kDa protein gene have shown promising results in the detection of RGM species. PCR restriction enzyme analyses (PRA) work better than conventional methods at identifying species that are closely related. Recently introduced molecular tools such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), pyrosequencing, DNA chip technology, and Beacon probes-combined PCR probes have shown comparable results in the detection of various species of RGM. Closely related RGM species (e.g., Mycobacterium fortuitum, M. chelonae, and M. abscessus) must be clearly differentiated using accurate molecular techniques because their therapeutic responses are species-specific. Hence, this paper reviews the following aspects of RGM: (i) its sources, predisposing factors, clinical manifestations, and concomitant fungal infections; (ii) the risks of misdiagnoses in the management of RGM infections in dermatological settings; (iii) the diagnoses and outcomes of treatment responses in common and uncommon infections in immunocompromised and immunocompetent patients; (iv) conventional versus current molecular methods for the detection of RGM; (v) the basic principles of a promising MALDI-TOF MS, sampling protocol for cutaneous or subcutaneous lesions and its potential for the precise differentiation of M. fortuitum, M. chelonae, and M. abscessus; and (vi) improvements in RGM infection management as described in the recent 2011 Clinical and Laboratory Standards Institute (CLSI) guidelines, including interpretation criteria of molecular methods and antimicrobial drug panels and their break points [minimum inhibitory concentrations (MICs)], which have been highlighted for the initiation of antimicrobial therapy. PMID:23139042

55

Mycobacterial species as case-study of comparative genome analysis  

The genus Mycobacterium represents more than 120 species including important pathogens of human and cause major public health problems and illnesses. Further, with more than 100 genome sequences from this genus, comparative genome analysis can provide new insights for better understanding the evolutionary events of these species and improving drugs, vaccines, and diagnostics tools for controlling Mycobacterial diseases. In this present study we aim to outline a comparative genome analysis of fourteen Mycobacterial genomes: M. avium subsp. paratuberculosis K—10, M. bovis AF2122/97, M. bovis BCG str. Pasteur 1173P2, M. leprae Br4923, M. marinum M, M. sp. KMS, M. sp. MCS, M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis H37Ra, M. tuberculosis H37Rv, M. tuberculosis KZN 1435 , M. ulcerans Agy99,and M. vanbaalenii PYR—1, For this purpose a comparison has been done based on their length of genomes, GC content, number of genes in different data bases (Genbank, Refseq, and Prodigal). The BLAST matrix of these genomes has been figured to give a lot of information about the similarity between species in a simple scheme. As a result of multiple genome analysis, the pan and core genome have been defined for twelve Mycobacterial species. We have also introduced the genome atlas of the reference strain M. tuberculosis H37Rv which can give a good overview of this genome. And for examining the phylogenetic relationships among these bacteria, a phylogenic tree has been constructed from 16S rRNA gene for tuberculosis and non tuberculosis Mycobacteria to understand the evolutionary events of these species.

56

Isolation of a novel insertion sequence from Mycobacterium fortuitum using a trap vector based on inactivation of a lacZ reporter gene.  

An insertion sequence of Mycobacterium fortuitum has been isolated using a trap vector following insertion in and inactivation of the lacZ reporter gene. The trap vector is a temperature-sensitive (ts) Escherichia coli-mycobacterium shuttle plasmid, pCD4, which contains ts oriM, the kanamycin-resistance gene as a selection marker and a lacZ expression cassette. The ts mutation present in pCD4 functions in mycobacteria and enables screening for transposable elements from the mycobacterial genome that disrupt the lacZ gene by screening for white colonies on X-Gal plates in both mycobacterial as well as E. coli hosts. The vector was used to isolate a novel 1.653 kb insertion sequence from M. fortuitum named IS219. IS219 duplicated host DNA at the target site, had inverted repeats at its ends and contained two ORFs on one strand. One of the predicted proteins showed homology to a putative transposase from Acetobacter pasteurianus. IS219 was present in two copies in the genome of M. fortuitum. The trap vector appears to be useful in trapping insertion sequences from different mycobacteria by screening for the disrupted LacZ phenotype. PMID:10832643

57

Construction and application of mycobacterial reporter transposons.  

The transposon Tn5367, which is a derivative of the mycobacterial insertion sequence IS1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions. A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional (lacZ), or translational ('aph) fusions. Mutants containing lacZ produced varying intensities of blue colour on indicator media. This reporter activity could be used as a quantitative measure of promoter strength. Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the 'aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions. Finally, inclusion of the R6Kgamma origin within Tn5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin. This study demonstrates that transcriptional and translational reporter derivatives of Tn5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria. PMID:10925203

58

The unusual mycobacterial chaperonins: evidence for in vivo oligomerization and specialization of function.  

The pathogen Mycobacterium tuberculosis expresses two chaperonins, one (Cpn60.1) dispensable and one (Cpn60.2) essential. These proteins have been reported not to form oligomers despite the fact that oligomerization of chaperonins is regarded as essential for their function. We show here that the Cpn60.2 homologue from Mycobacterium smegmatis also fails to oligomerize under standard conditions. However, we also show that the Cpn60.2 proteins from both organisms can replace the essential groEL gene of Escherichia coli, and that they can function with E. coli GroES cochaperonin, as well as with their cognate cochaperonin proteins, strongly implying that they form oligomers in vivo. We show that the Cpn60.1 proteins, but not the Cpn60.2 proteins, can complement for loss of the M. smegmatis cpn60.1 gene. We investigated the oligomerization of the Cpn60.2 proteins using analytical ultracentrifugation and mass spectroscopy. Both form monomers under standard conditions, but they form higher order oligomers in the presence of kosmotropes and ADP or ATP. Under these conditions, their ATPase activity is significantly enhanced. We conclude that the essential mycobacterial chaperonins, while unstable compared to many other bacterial chaperonins, do act as oligomers in vivo, and that there has been specialization of function of the mycobacterial chaperonins following gene duplication. PMID:22834700

59

In situ detection of Mycobacterium tuberculosis transcripts in human lung granulomas reveals differential gene expression in necrotic lesions.  

We have used RNA-RNA in situ hybridization to detect the expression of several Mycobacterium tuberculosis genes in tuberculous granulomas in lung tissue sections from tuberculosis patients. The M. tuberculosis genes chosen fall into two classes. Four genes (icl, narX, and Rv2557 and Rv2558) have been implicated in the persistence of the bacterium in the host, and two genes (iniB and kasA) are upregulated in response to isoniazid exposure. Both necrotic and nonnecrotic granulomas were identified in all of the patients. Necrotic granulomas were divided into three zones: an outer lymphocyte cuff containing lymphocytes and macrophages, a transition zone consisting of necrotic material interspersed with macrophages, and a central acellular necrotic region. Transcripts of all of the genes studied were found in nonnecrotic granulomas and in the lymphocyte cuff of necrotic granulomas. Mycobacterial gene expression was associated with CD68-positive myeloid cells. Rv2557 and/or its homologue Rv2558, kasA, and iniB were expressed within the transition zone of necrotic granulomas, whereas icl and narX transcripts were absent from this area. There was no evidence of transcription of any of the genes examined in the central necrotic region, although mycobacterial DNA was present. The differential expression of genes within granulomas demonstrates that M. tuberculosis exists in a variety of metabolic states and may be indicative of the response to different microenvironments. These observations confirm that genes identified in models of persistence or in response to drug treatment in vitro are expressed in the human host. PMID:12379712

60

Transcriptional analysis of and resistance level conferred by the aminoglycoside acetyltransferase gene aac(2prime)-Id from Mycobacterium smegmatis  

ObjectivesTo analyse the correlation between the expression levels of the aac(2prime)-Id gene from Mycobacterium smegmatis mc2155 and the resistance levels to aminoglycosides conferred by the encoded aminoglycoside 2prime-N-acetyltransferase [AAC(2prime)-Id].MethodsExpression levels were studied using a transductional fusion with the lacZ gene. The promoter region was characterized by primer extension analysis and ribonuclease protection assay. The aac(2prime)-Id gene was placed under the control of different mycobacterial promoters; deletions of the promoter region were done. Each of the plasmids was introduced in M. smegmatis mc2155 and the MICs were determined by resazurin assay.ResultsThe aac(2prime)-Id gene is transcribed from two promoters: P1 (weaker) and P2 (stronger) located 38 an...

 
 
 
 
61

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning  

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribu...

62

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning.  

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis. PMID:20650290

63

A Rapid Loop-Mediated Isothermal Amplification Assay Targeting hspX for the Detection of Mycobacterium tuberculosis Complex  

A rapid, simple, and low-cost diagnostic tool for tuberculosis (TB) detection is urgently needed in countries with a high TB burden. Here, we report a novel loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene for the rapid detection of Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti. The specificity of this assay was evaluated using 4 reference strains of Mycobacterium tuberculosis complex (MTC), 22 species of non-tuberculous mycobacteria (NTM), 7 non-mycobacterial species, and 50 clinical M. tuberculosis isolates. All the reference MTC strains and M. tuberculosis clinical isolates were successfully detected by this method, and there were no false-positive results with NTM or non-mycobacterial species, which demonstrates the high specificity of this assay for MTC. The detection limit was 10 copies of MTC genome within 27 min, and the detection speed of this assay was higher than that of any other isothermal methods reported so far. Because of its speed, simplicity, sensitivity, specificity, and inexpensiveness, the TB hspX LAMP assay is a potential gene diagnostic method for TB detection in developing countries with a high TB burden.   

64

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics.  

Mycobacterium vaccae represents an alternative mycobacterial cloning host that has been largely overlooked to date. The main reason for this may be the reported non-transformability of this species, specifically the so-called Stanford strain (NCTC 11659), with expression vectors that use kanamycin resistance as a selection method. However, this strain can be transformed using hygromycin resistance as an alternative selectable phenotype. The present study has shown that in contrast to previous reports, M. vaccae (ATCC 15483) is capable of being transformed with a range of vectors encoding kanamycin resistance as the selectable marker. Thereafter, the expression of the lacZ reporter gene in M. vaccae, Mycobacterium bovis BCG and Mycobacterium smegmatis mc(2)155 was evaluated using a range of characterized mycobacterial promoter sequences (hsp60, hsp70, PAN, 18kDa and 16S rRNA) cloned in the same promoter probe vector. In general, the promoters showed similar levels of activity in the three species, demonstrating that existing expression systems can readily be employed with M. vaccae (ATCC 15483). This was further confirmed by the observation that M. vaccae was capable of stable, in vitro expression of recombinant S1 subunit of pertussis toxin at levels equivalent to those obtained with BCG and M. smegmatis. Analysis of structural and functional stability of a range of vectors demonstrated that the incidence of instability noted for M. vaccae was lower than that recorded for M. smegmatis. Taken together, the results indicate that M. vaccae is an additional cloning host which may prove useful for specific aspects of mycobacterial biology and provide increased flexibility to the field of recombinant protein technology for mycobacteria. PMID:12101288

65

A novel differential expression system for gene modulation in Mycobacteria  

Tuberculosis (TB) remains a major global health problem, and successful genetic manipulation of mycobacteria is crucial for developing new approaches to study the mechanism of pathogenesis of Mycobacterium tuberculosis (M.tb) and to combat TB. In this study, a series of M.tb furA gene operator/promoter (pfurA) mutants were generated aiming at optimization of the promoter activities in mycobacterial strains. Measured by the lacZ gene-fusion reporter system, change of the initial codon GTG to the preferred ATG resulted in a double increase of b-galactosidase activity, while a 6-bp substitution in the conserved FurA binding AT-rich region upstream of furA gene led to 4-6 folds increase of the activity. It is significant that combination of both mutations showed about 10 folds of b-galactosida...

66

Association of IFNGR2 gene polymorphisms with pulmonary tuberculosis among the Vietnamese  

Interferon-? (IFN-?) is a key molecule of T helper 1 (Th1)-immune response against tuberculosis (TB), and rare genetic defects of IFN-? receptors cause disseminated mycobacterial infection. The aim of the present study was to investigate whether genetic polymorphisms found in the Th1-immune response genes play a role in TB. In our study, DNA samples were collected from two series of cases including 832 patients with new smear-positive TB and 506 unrelated individuals with no history of TB in the general population of Hanoi, Vietnam. Alleles of eight microsatellite markers located around Th1-immune response-related genes and single nucleotide polymorphisms near the promising microsatellites were genotyped. A set of polymorphisms within the interferon gamma receptor 2 gene (IFNGR2) showed a ...

67

Tetracycline Resistance and Presence of Tetracycline Resistance Determinants tet(V) and tap in Rapidly Growing Mycobacteria from Agricultural Soils and Clinical Isolates  

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics, since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring; however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TET-resistance genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50% resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate. In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates’ BOX-PCR profiles, suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding, intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and expression merit further research.   

68

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system  

Abstract in english In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy numbe (more) r and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

69

Functional characterization delineates that a Mycobacterium tuberculosis specific protein kinase (Rv3080c) is responsible for the growth, phagocytosis and intracellular survival of avirulent mycobacteria.  

Serine/threonine protein kinases (STPKs) are predominantly involved in growth, development, division, differentiation, and in regulating immune responses in mycobacteria. A wide variety of functions of mycobacterial STPKs persuade mycobacterial growth and further its survival in the hosts. The polymorphic studies have shown that a full length gene of Rv3080c (pknK) is present in the slow growing mycobacteria. The wild type Mycobacterium smegmatis containing only vector (M. smegmatis) and M. smegmatis containing Rv3080c (pknK) cloned in pMV261 vector (M. smegmatis::K) were cultured in different growth media. The studies have shown that M. smegmatis did not differ in the growth and in survival while a substantial reduction in the growth (four-ten-folds) and a significant delay in the colony formation were observed in M. smegmatis::K. In order to look for the stage specific and modulated expression of PknK, the study was comprehended to quantitate pknK transcripts at different phases of cultures. The mycobacterium, containing high copy number of pknK specific RNA was unable to multiply. The study thus highlights that Rv3080c is largely accountable for changing the fate of avirulent mycobacteria and hence the protein can be utilized as an important molecule to target pathogenesis. PMID:22740025

70

Reduced transcript stabilization restricts TNF-alpha expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan.  

Despite the critical role that TNF-alpha plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohn's disease. We found that in MAP infected macrophages, TNF-alpha gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF-alpha transcriptional one could not fully explain the differential TNF-alpha mRNA expression, suggesting that there must be a substantial contribution by post-transcriptional mechanisms.Accordingly, we found reduced TNF-alpha mRNA stability in MAP-infected macrophages. Further comparison of MAP- and MS-infected macrophages revealed that lower TNF-alpha mRNA stability combined with lower mRNA and protein expression in MAP-infected macrophages correlated with lower p38 MAPK phosphorylation. These findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell-wall lipoglycan LM of MAP and MS induced TNF-alpha mRNA transcription,but only the MS-LM induced p38 MAPK-dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 and TNF-alpha mRNA stabilization as a result of their structural cell-wall components such as LM and thereby, restrict TNF-alpha expression in macrophages. PMID:19850884

71

An ArsR-like transcriptional factor recognizes a conserved sequence motif and positively regulates the expression of phoP in mycobacteria.  

Transcriptional regulation plays a critical role during the infection of Mycobacterium tuberculosis, the causative agent of tuberculosis. A two-component system, PhoPR, is clearly involved in the regulation of pathogenic virulence and persistence. However, the regulatory mechanism, as well as the regulator, of the phoPR operon remains uncharacterized in M. tuberculosis and its related species thus far. In the present study, we characterize an ArsR transcriptional factor, corresponding to Rv2034 and Ms6762 in M. tuberculosis and Mycobacterium smegmatis, respectively, as the first regulator of phoP in both mycobacterial species. The interaction between ArsR regulator and target promoters is conserved in these two mycobacterial species, and an inverted repeat sequence motif is successfully mapped out for the recognition of ArsR. Utilizing lacZ reporter genes and overexpression analysis, the ArsR regulator is shown to positively regulate the expression of phoP in M. smegmatis, different from most ArsR family regulators generally as a repressor. The current study establishes a direct link between the ArsR transcriptional factor and the regulation of phoP in mycobacteria. Our findings imply that ArsR may be involved in the pathogenesis of M. tuberculosis through its regulation of the phoPR operon. PMID:21782791

72

A two-step strategy for the complementation of M: tuberculosis mutants  

Abstract in english The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of t (more) he mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level.

73

A novel differential expression system for gene modulation in Mycobacteria.  

Tuberculosis (TB) remains a major global health problem, and successful genetic manipulation of mycobacteria is crucial for developing new approaches to study the mechanism of pathogenesis of Mycobacterium tuberculosis (M.tb) and to combat TB. In this study, a series of M.tb furA gene operator/promoter (pfurA) mutants were generated aiming at optimization of the promoter activities in mycobacterial strains. Measured by the lacZ gene-fusion reporter system, change of the initial codon GTG to the preferred ATG resulted in a double increase of beta-galactosidase activity, while a 6-bp substitution in the conserved FurA binding AT-rich region upstream of furA gene led to 4-6 folds increase of the activity. It is significant that combination of both mutations showed about 10 folds of beta-galactosidase activity higher than that of the prototype pfurA. Furthermore, all of the furA promoters were expressed continuously in vivo during intracellular growth of Mycobacterium bovis BCG, and were induced early upon infection in macrophages. Employing the series of pfurA-based differential expression vectors, M.tb chimeric antigen Ag856A2 known for its excellent immunogenicity, was shown to be expressed at different levels in the recombinant Mycobacterium smegmatis and BCG strains. These results indicated that this differential expression system is feasible to express any target antigen of interest in a modular fashion for the study of gene regulation in mycobacterial strains, and also for the development of different recombinant BCG vaccine candidates against TB or other infectious diseases, which would be beneficial for elicitation of optimal immune response. PMID:18835406

74

Adherence to protease inhibitors, HIV-1 viral load, and development of drug resistance in an indigent population.  

Background & objectives: mRNA is more rapidly destroyed in cells than rRNA or genomic DNA, an assay targeting bacterial mRNA would provide a better guide to mycobacterial viability than amplification tests directed at DNA or rRNA targets. This study was carried out to standardize reverse transcriptase PCR (RT-PCR) targeting 85B gene for the rapid detection of viable Mycobacterium tuberculosis from sputum specimens of suspected TB patients at Chennai, South India and to detect MDR-TB circulating in this population. Methods: Sputum samples from clinically suspected tuberculosis patients (n=301) and 78 controls were included in the study. The sputum samples were collected in sterile diethyl pyrocarbonate (DEPC) treated containers and transported in ice to the laboratory within 2 h to prevent degradation of RNA. RT-PCR targeting 85B gene, mycobacterial culture and phenotypic drug susceptibility testing for the first line drugs streptomycin (S), isoniazid (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z) were performed by BACTEC microMGIT culture system for all the sputum specimens. Results: All the 78 controls were negative for culture and RT-PCR. Among the 301 sputum specimens from patients, 231 (76.8%) were RT-PCR positive and 70 (23.2%) were negative. There were 166 M. tuberculosis isolates, of which 11 (2.9%) were MDR-TB, 33 (8.7%) were polyresistant, 31 (8.2%) were monoresistant and 91 (30.2%) were sensitive to all five first line anti-tuberculous drugs by phenotypic drug susceptibility testing. Monoresistance was higher with Z [20 (20.8%)], followed by S [6 (3%)]. Interpretation & conclusions: RT-PCR targeting 85B gene of M. tuberculosis was a specific, rapid, reliable technique to detect the M. tuberculosis directly from sputum specimens. Our results showed that 2.9 per cent of M. tuberculosis isolates in the study population of Chennai were MDR. PMID:10770537

75

Novel STAT1 Alleles in a Patient with Impaired Resistance to Mycobacteria  

Partial defects in interferon (IFN)-? signaling lead to susceptibility to infections with nontuberculous mycobacteria. The receptors for IFN-? and IFN-? activate components of the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway. Some defects in STAT1 mainly affect IFN-? signaling, thus resulting in mendelian susceptibility to mycobacterial disease (MSMD). MSMD is a severe disease but patients show a favorable response to anti-mycobacterial chemotherapy. Other defects in STAT1 affect both IFN-? and IFN-? signaling resulting in mycobacterial and lethal viral disease. We report here a patient with two novel STAT1 alleles, which in combination results in a recessive trait with partial STAT1 deficiency and mycobacterial disease. Cells from the patient did respond to mycobacterial antigen, but both the expression of STAT1 and phosphorylation of STAT1 in response to IFN-? treatment were reduced. This is the first report of a mutation in the N-terminal part of STAT1 involved in causing mycobacterial disease.

76

First case of Nocardia nova spinal abscess in an immunocompetent patient  

Abstract in english Nocardia are a group of aerobic actinomycetes that are filamentous gram-positive, weakly acid-fast, and cause opportunistic infection in immunocompromised patients. Primary Nocardia infection mostly involves lung, skin and less commonly, the central nervous system (CNS). Among Nocardia CNS infections, spinal infection is extremely rare. We describe the first case of a spinal abscess caused by Nocardia nova in an immunocompetent patient who experienced a penetrating facial (more) injury six months earlier. Nocardia species were isolated from intradural spinal abscesses and identified by 16S rRNA, hsp65 and secA1 sequence analyses. Surgical excision and treatment with amikacin, cefotaxime, and oral erythromycin was successful.

77

First case of Nocardia nova spinal abscess in an immunocompetent patient.  

Nocardia are a group of aerobic actinomycetes that are filamentous gram-positive, weakly acid-fast, and cause opportunistic infection in immunocompromised patients. Primary Nocardia infection mostly involves lung, skin and less commonly, the central nervous system (CNS). Among Nocardia CNS infections, spinal infection is extremely rare. We describe the first case of a spinal abscess caused by Nocardia nova in an immunocompetent patient who experienced a penetrating facial injury six months earlier. Nocardia species were isolated from intradural spinal abscesses and identified by 16S rRNA, hsp65 and secA1 sequence analyses. Surgical excision and treatment with amikacin, cefotaxime, and oral erythromycin was successful. PMID:22552466

78

Clinical, microscopic, and molecular aspects of canine leproid granuloma in the United States.  

Leproid granulomas from seven dogs in the United States were evaluated. Gross characteristics included nodular and ulcerated dermal and subcutaneous lesions primarily on the caudal aspects of the pinnae and to a lesser extent on the muzzle, face, and forelimbs. In all except one dog, there was complete regression of the lesions within 6 months, either with no therapy or after surgical resection. Cytology or histopathology revealed pyogranulomatous inflammation with few to many acid-fast mycobacterial bacilli within macrophages. The organisms could not be cultivated in vitro. DNA sequencing of part of the 16S ribosomal RNA gene region revealed 99-100% homology among fragments from five of these dogs and fragments from dogs in the south Pacific. This syndrome occurs in dogs in North America and the prognosis is excellent, in contrast to the prognosis for rapid-growing or tuberculous mycobacteriosis. PMID:12009061

79

Isolation of a Recombinant Desulfurizing 4,6-Dipropyl Dibenzothiophene in n-Tetradecane  

Rhodococcus erythropolis strain KA2-5-1 is unable to desulfurize 4,6-dipropyl dibenzothiophene (DBT) in the oil phase. The dsz desulfurization gene cluster from R. erythropolis strain KA2-5-1 was transferred into 22 rhodococcal and mycobacterial strains using a transposon–transposase complex. The recombinant strain MR65, from Mycobacterium sp. NCIMB10403, was able to grow on a minimal medium supplemented with 1.0 mM 4,6-dipropyl DBT in n-tetradecane (50%, v/v) as the sole sulfur source. Resting cells of recombinant strain MR65 could desulfurize 68 mg l–1 of sulfur in light gas oil (LGO) containing 126 mg sulfur l–1. Strain MR65 had about 1.5-times the LGO desulfurization activity of R. erythropolis strain KA2-5-1. The application of a recombinant, which is able to utilize 4,6-dipropyl DBT in the oil phase, was effective in enhancing LGO biodesulfurization.   

80

Lung infection caused by Mycobacterium riyadhense confused with Mycobacterium tuberculosis: the first case in Korea.  

A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection. PMID:22779073

 
 
 
 
81

Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid.  

Mycobacterium tuberculosis cell envelope is a treasure house of biologically active lipids of fascinating molecular architecture. Although genetic studies have alluded to an array of genes in biosynthesis of complex lipids, their mechanistic, structural, and biochemical principles have not been investigated. Here, we have dissected the molecular logic underlying the biosynthesis of a virulence lipid phthiocerol dimycocerosate (PDIM). Cell-free reconstitution studies demonstrate that polyketide synthases, which are usually involved in the biosynthesis of secondary metabolites, are responsible for generating complex lipids in mycobacteria. We show that PapA5 protein directly transfers the protein bound mycocerosic acid analogs on phthiocerol to catalyze the final esterification step. Based on precise identification of biological functions of proteins from Pps cluster, we have rationally produced a nonmethylated variant of mycocerosate esters. Apart from elucidating mechanisms that generate chemical heterogeneity with PDIMs, this study also presents an attractive approach to explore host-pathogen interactions by altering mycobacterial surface coat. PMID:15749014

82

The mechanism of upstream activation in the rrnB operon of Mycobacterium smegmatis is different from the Escherichia coli paradigm.  

Mycobacteria are slow-growing bacteria with a generation time of from 2-3 h up to several weeks. Consistent with the low growth rate, mycobacterial species have a maximum of two rRNA operons, rrnA and rrnB. The rrnA operon is present in all mycobacteria and has between two and five promoters, depending on species, whereas the rrnB operon, with a single promoter, is only found in some of the faster-growing species. The promoter region of the rrnB operon of a typical fast grower, Mycobacterium smegmatis, was investigated. By using lacZ reporter gene fusions it was demonstrated that the rrnB operon contains a highly activating region upstream of the core promoter, comparable to other bacterial rrn operons. However, the results suggest that, unlike the situation in, for example, Escherichia coli, the activating mechanism is solely factor dependent, and that no UP element is involved. PMID:15699196

83

MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria  

SummaryMycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteri...

84

Species variation in ATP-dependent protein degradation: protease profiles differ between mycobacteria and protease functions differ between Mycobacterium smegmatis and Escherichia coli.  

We report here that the existence of the potentially broad substrate specificity protease Lon (also called La), is evolutionarily discontinuous within the order Actinomycetales. Lon homologues were identified in the fast-growing species Mycobacterium smegmatis, and the slow-growing species Micobacterium avium and Mycobacterium intracellulare. However, Lon homologues were not detected in the slow-growing species Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium leprae; or in the non-mycobacterial Actinomycetale Corynebacterium glutamica. To characterize the function of the Lon protease within the Actinomycetales, a viable M. smegmatis Deltalon strain was constructed, demonstrating that lon is not essential under certain conditions. Surprisingly, lon was also dispensable in M. smegmatis cells already lacking intact 20S proteasome alpha- and beta-subunit genes (called prcA and prcB, respectively). Creation of the later double deletion strain (prcBA::kan Deltalon) necessitated use of a novel gene deletion strategy that does not require an antibiotic resistance marker. The M. smegmatis prcBA::kan Deltalon double mutants displayed wild type (wt) growth rates and wt stress tolerances. In addition, the M. smegmatis prcBA::kan Deltalon double mutants degraded at wt rates the broad spectrum of truncated proteins induced by treating cells with puromycin. This later result was in sharp contrast to those in Escherichia coli, where either lon or hslUV single mutants are strongly impaired in their degradation of puromycyl peptides (hslV is a prcB homologue). Overall these data suggested that mycobacterial species contain additional ATP-dependent proteases that have broad substrate specificity. Consistent with this suggestion, M. smegmatis and M. tuberculosis each contain at least one homologue of ClpP, the proteolytic subunit common to the ClpAP and ClpXP proteases. PMID:10231573

85

Formulation of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG in cationic liposomes significantly enhances protection against tuberculosis.  

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (?mmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) - D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ?mmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ?mmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ?mmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFN? and TNF? (double positive) or IFN?, TNF? and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFN? and TNF? median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ?mmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNF? and IL-2 than nonadjuvanted BCG or ?mmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFN?, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ?mmaA4BCG/adjuvant formulation may be an effective, safe, and relatively inexpensive alternative to vaccination with conventional BCG. PMID:22442674

86

Transferable plasmid-mediated resistance to linezolid due to cfr in a human clinical isolate of Enterococcus faecalis.  

Nonmutational resistance to linezolid is due to the presence of cfr, which encodes a methyltransferase responsible for methylation of A2503 in the 23S rRNA. The cfr gene was first described in animal isolates of staphylococci, and more recently, it has been identified in Staphylococcus aureus from human clinical infections, including in an outbreak of methicillin-resistant S. aureus. In enterococci, cfr has been described in an animal isolate of Enterococcus faecalis from China. Here, we report an isolate of linezolid-resistant E. faecalis (603-50427X) recovered from a patient in Thailand who received prolonged therapy with the antibiotic for the treatment of atypical mycobacterial disease. The isolate lacked mutations in the genes coding for 23S rRNA and L3 and L4 ribosomal proteins and belonged to the multilocus sequence type (MLST) 16 (ST16), which is commonly found in enterococcal isolates from animal sources. Resistance to linezolid was associated with the presence of cfr on an ~97-kb transferable plasmid. The cfr gene environment exhibited DNA sequences similar to those of other cfr-carrying plasmids previously identified in staphylococci (nucleotide identity, 99 to 100%). The cfr-carrying plasmid was transferable by conjugation to a laboratory strain of E. faecalis (OG1RF) but not to Enterococcus faecium or S. aureus. The cfr gene was flanked by IS256-like sequences both upstream and downstream. This is the first characterization of the potential horizontal transferability of the cfr gene from a human linezolid-resistant isolate of E. faecalis. PMID:22491691

87

Use of the GenoType Mycobacterium CM and AS Assays To Analyze 76 Nontuberculous Mycobacterial Isolates from Greece  

Seventy-six nontuberculous mycobacterial isolates obtained from patients living in Greece were analyzed with the GenoType Mycobacterium CM (for common mycobacteria) and AS (for additional species) assays. GenoType correctly identified all but one of the mycobacterial species. For this species, addit...

88

Mycobacterium tuberculosis modulators of the macrophages cellular events  

A number of mycobacterial macromolecules have been shown to target biological processes within host macrophages; however, the exact mechanism for the majority of these host-pathogen interactions is poorly understood. The following review summarizes current knowledge and expands on a subset of mycobacterial effectors for which a cognate substrate, cellular partner or signaling pathway have been experimentally identified within the human host.

89

Mycobacterium tuberculosis modulators of the macrophage's cellular events.  

A number of mycobacterial macromolecules have been shown to target biological processes within host macrophages; however, the exact mechanism for the majority of these host-pathogen interactions is poorly understood. The following review summarizes current knowledge and expands on a subset of mycobacterial effectors for which a cognate substrate, cellular partner or signaling pathway have been experimentally identified within the human host. PMID:22841679

90

Functional Dissection of the PE Domain Responsible for Translocation of PE_PGRS33 across the Mycobacterial Cell Wall  

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize th...

91

Mycobacterium avium subspecies paratuberculosis is not associated with Type-2 diabetes mellitus  

Background. The role of pathogenic mycobacteria in diabetes has been a focus of speculation since a decade without any meaningful insights into the mechanism of diabetes causation vis a vis mycobacterial factors. Two of our studies based on PCR identification of mycobacterial DNA and d...

92

Human T-cell clones to the 70-kilodalton heat shock protein of Mycobacterium leprae define mycobacterium-specific epitopes rather than shared epitopes.  

The mycobacterial 70-kDa heat shock protein (Hsp70) is a dominant antigen during the human T-cell response to mycobacterial infection despite the conserved sequence with the human homolog. To determine whether this response is pathogen specific, CD4+ T-cell clones were isolated from Mycobacterium le...

93

Methods for Reducing Bacterial Contamination in the BacT/Alert Mycobacterial Culture Detection System  

Two methods were compared for decreasing bacterial contamination in the BacT/Alert mycobacterial culture detection system. Two concentrations, 0.5 ml (standard amount) and 1.0 ml, of mycobacterial antibiotic supplement were evaluated. Contamination rates were 14% and 6% for the standard and the doub...

94

Regulation of pyr gene expression in Mycobacterium smegmatis by PyrR-dependent translational repression.  

Regulation of pyrimidine biosynthetic (pyr) genes by a transcription attenuation mechanism that is mediated by the PyrR mRNA-binding regulatory protein has been demonstrated for numerous gram-positive bacteria. Mycobacterial genomes specify pyrR genes and contain obvious PyrR-binding sequences in the initially transcribed regions of their pyr operons, but transcription antiterminator and attenuation terminator sequences are absent from their pyr 5' leader regions. This work demonstrates that repression of pyr operon expression in Mycobacterium smegmatis by exogenous uracil requires the pyrR gene and the pyr leader RNA sequence for binding of PyrR. Plasmids containing the M. smegmatis pyr promoter-leader region translationally fused to lacZ also displayed pyrR-dependent repression, but transcriptional fusions of the same sequences to a lacZ gene that retained the lacZ ribosome-binding site were not regulated by PyrR plus uracil. We propose that PyrR regulates pyr expression in M. smegmatis, other mycobacteria, and probably in numerous other bacteria by a translational repression mechanism in which nucleotide-regulated binding of PyrR occludes the first ribosome-binding site of the pyr operon. PMID:17601781

95

Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana/ Alterations in recruitment and activation of Rab proteins during mycobacterial infection  

Abstract in spanish En el fagosoma, Mycobacterium spp. altera la activación y reclutamiento de diferentes proteínas "del gen Ras de cerebro de rata", comúnmente conocidas como Rab. En este manuscrito se revisa una serie de reportes que han demostrado que los fagosomas que contienen micobacterias tienen una expresión mayor y sostenida de Rab5, Rab11, Rab14 y Rab22a, y menor o ninguna expresión de Rab7, Rab9 y Rab6. Esto se correlaciona con aumento de la fusión de estos fagosomas con end (more) osomas tempranos y de reciclaje, lo que les permite mantener ciertas características de compartimentos tempranos, permite que las bacterias obtengan acceso a nutrientes y previene la activación de mecanismos contra la micobacteria. La expresión de mutantes constitutivamente activos de las Rab de endosomas tempranos impide la maduración de fagosomas que contienen esferas de látex o micobacterias inactivadas por calor. Mientras que su silenciamiento, mediante ARN de interferencia o mediante dominantes negativos, induce la maduración de fagosomas micobacterianos. Los mecanismos exactos por los que las micobacterias alteran la dinámica de expresión de estas GTPasas, afectando la maduración fagolisosómica, no se han establecido. El problema podría explicarse por defectos en el reclutamiento de las proteínas que interactúan con Rab, como la cinasa-3 del fosfatidilinositol y el antígeno endosómico temprano 1. La identificación de los mecanismos empleados por Mycobacterium spp. para interrumpir el ciclo de activación de las Rab, será esencial para comprender la fisiopatología de la infección micobacteriana y útil como posibles blancos farmacológicos. Abstract in english At the phagosome level, Mycobacterium spp. alters activation and recruitment of several "Ras gene from rat brain" proteins, commonly known as Rab. Mycobacterial phagosomes have a greater and sustained expression of Rab5, Rab11, Rab14 and Rab22a, and lowered or no expression of Rab7, Rab9 and Rab6. This correlates with increased fusion of the phagosomes with early and recycling endosomes acquiring some features of early phogosomes, allowing the bacteria to gain access to n (more) utrients and preventing the activation of anti-mycobacterial mechanisms. The expression of constitutively active mutants of Rab from the early stage endosomes prevents the maturation of phagosomes containing latex beads or heat-inactivated mycobacteria. Silencing of these mutants by interference RNA or dominant negative forms induces the maturation of mycobacterial phagosomes. The mechanisms have not been established by which mycobacteria alter the expression of these GTPases and thereby shift the phagolysosomal maturation. The problem can be explained by alterations in the recruitment of proteins that interact with Rab, such as phosphoinositide 3-kinases and early endosomal antigen 1. Identifying the mechanisms used by Mycobacterium spp. to disrupt the cycle of Rab activation will be essential to understand the pathophysiology of mycobacterial infections and usefully to potential drug targets.

96

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase  

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD+-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.   

97

Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis  

Only a limited number of proteins from Mycobacterium tuberculosis have so far been shown to possess species-specific epitopes as defined by monoclonal antibodies. One such protein is protein antigen b (Pab) of molecular weight 38,000, which binds the monoclonal antibodies HYT 28, HAT 2, HBT 12, HGT 3, TB 71, and TB 72. The gene encoding this protein was isolated from a lambda gt11 M. tuberculosis DNA library. The nucleotide sequence of the recombinant mycobacterial insert was determined, and an open reading frame of 374 amino acids was identified. The amino acid sequence exhibited 30% homology to a phosphate-binding protein, PstS, from Escherichia coli. The pab gene was subcloned into pBR322 in conjunction with the lacZ gene, and deletions were obtained from the 3' end. The anti-Pab monoclonal antibodies were used to probe crude protein lysates of E. coli transformed with the deletion plasmids. The monoclonal antibodies showed two reactivity patterns; one group of antibodies were dependent on the presence of the ultimate 91 amino acids of the protein, whereas another group of antibodies recognized an antigenic domain located on the middle portion of the molecule. None of the antibodies bound to the N-terminal 117-amino-acid peptide.

98

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis.  

In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 ?g ml(-1). When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo. PMID:21081761

99

Differential Trafficking of TLR1 I602S Underlies Host Protection against Pathogenic Mycobacteria.  

We recently identified I602S as a frequent single-nucleotide polymorphism of human TLR1 that greatly inhibits cell surface trafficking, confers hyporesponsiveness to TLR1 agonists, and protects against the mycobacterial diseases leprosy and tuberculosis. Because mycobacteria are known to manipulate the TLR system to their advantage, we hypothesize that the hyporesponsive 602S variant may confer protection by enabling the host to overcome this immune subversion. We report that primary human monocytes and macrophages from homozygous TLR1 602S individuals are resistant to mycobacterial-induced downregulation of macrophage MHC class II, CD64, and IFN-? responses compared with individuals who harbor the TLR1 602I variant. Additionally, when challenged with mycobacterial agonists, macrophages from TLR1 602S/S individuals resist induction of host arginase-1, an enzyme that depletes cellular arginine stores required for the production of antimicrobial reactive nitrogen intermediates. The differences in cell activation mediated by TLR1 602S and TLR1 602I are observed upon stimulation with soluble mycobacterial-derived agonists but not with whole mycobacterial cells. Taken together, these results suggest that the TLR1 602S variant protects against mycobacterial disease by preventing soluble mycobacterial products, perhaps released from granulomas, from disarming myeloid cells prior to their encounter with whole mycobacteria. PMID:23105135

100

A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion.  

Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871-Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host. PMID:15341647

 
 
 
 
101

Mycobacterium smegmatis Lsr2 physically and functionally interacts with a new flavoprotein involved in bacterial resistance to oxidative stress.  

Lsr2, a bacterial histone-like protein, has been shown to be clearly involved in modulating chromatin organization, compaction and global gene expression. However, the regulatory mechanism of its functions remains largely unclear. In this study, using bacterial two-hybrid technique and pull-down assays, the Mycobacterium smegmatis Lsr2 was detected to associate with a hypothetical flavoprotein, Ms4334. A further co-immunoprecipitation assay confirmed the physical interaction between these two proteins in vivo in mycobacteria. Importantly, the Ms4334 protein was also capable of enhancing the inhibitory effect of Lsr2 in vitro on the function of DNA topoisomerase I (MsTopA). Therefore, Lsr2 could physically and functionally interact with Ms4334. Further, the Ms4334 gene was confirmed to encode a new FAD-binding flavoprotein that displayed two characteristic absorption peaks at about 370 and 450 nm in a UV-visible spectra scanning assay. Interestingly, when comparing the growths of wild-type M. smegmatis with the Ms4334-knockout strain in response to H(2)O(2), Ms4334 was found to contribute to mycobacterial resistance to oxidative stress. The findings provided important clues for a further understanding of the regulation mechanism of Lsr2 in mycobacteria. PMID:22952243

102

Characterization of the Basic Replicon of Rhodococcus Plasmid pSOX and Development of a Rhodococcus-Escherichia coli Shuttle Vector†  

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS?, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.

103

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity  

Abstract in english The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. (more) Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

104

sigA is an essential gene in Mycobacterium smegmatis.  

sigA encodes a sigma factor of the sigma70 family, sigmaA, that is found in all mycobacterial species. As sigmaA shows high similarity to the primary sigma factor in Streptomyces coelicolor, it was postulated that sigmaA has the same role in mycobacteria. However, a point mutation in sigA, resulting in the replacement of arginine 522 by histidine, was found responsible for the attenuated virulence of the Mycobacterium bovis strain ATCC 35721. This raised the possibility that sigmaA was an alternative sigma factor specifically required for virulence gene expression. In this work, we show that sigA can not be disrupted in Mycobacterium smegmatis unless an extra copy of the gene is provided at another chromosomal site, which demonstrates that sigA is essential. To characterize the pattern of sigA expression during exponential and stationary phase in M. smegmatis, we measured the beta-galactosidase activity in a strain carrying a sigA-lacZ transcriptional fusion and monitored sigmaA levels using Western blotting. Our results indicate that sigA is expressed throughout the growth of the culture. The essential character of sigA and its pattern of expression corroborate the hypothesis that sigA codes for the primary sigma factor in M. smegmatis and, most likely, in all mycobacteria. PMID:9720877

105

Strong Impact on the Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Community of a PAH-Polluted Soil but Marginal Effect on PAH Degradation when Priming with Bioremediated Soil Dominated by Mycobacteria  

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation. Copyright © 2007 by the American Society for Microbiology

106

Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases  

Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the i...

107

Exposure to antibiotics induces expression of the Mycobacterium tuberculosis sigF gene: implications for chemotherapy against mycobacterial persistors.  

The sigF gene encodes an alternate sigma factor found in Mycobacterium tuberculosis and related pathogenic mycobacteria. Determination of conditions of sigF expression is an important step in understanding the conditional gene regulation which may govern such processes as virulence and dormancy in mycobacteria. We constructed an in-frame translational lacZ-kan fusion within the sigF gene to determine the conditions of sigF expression. This reporter construct was expressed from a multicopy plasmid in a strain of BCG harboring an integrated luciferase reporter gene under the control of the mycobacteriophage L5 gp71 promoter. Antibiotic exposure, in particular, ethambutol, rifampin, streptomycin, and cycloserine treatment, increased the level of SigF reporter specific expression in a dose-dependent fashion. The level of SigF reporter specific expression increased over 100-fold in late-stationary-phase growth compared to that in exponential growth. During the exponential phase, SigF specific expression could be induced by a number of other stresses. Anaerobic metabolism induced SigF by greater than 150-fold, particularly in the presence of metronidazole. Cold shock increased the level of SigF specific expression, while heat shock decreased it. Oxidative stress was also an important inducer of SigF specific expression; a greater induction was seen with cumene hydroperoxide than with hydrogen peroxide. Comparisons of bacterial viability as determined by the luciferase assay or by plating serial dilutions revealed that luciferase gp71-dependent activity was an unreliable predictor of the numbers of CFU during stationary-phase growth and anaerobic metabolism. The induction of sigF following antibiotic exposure suggests that this bacterial transcription factor may control genes which are important for mycobacterial persistence in the host during chemotherapy. PMID:9925509

108

Fatal Granuloma Necrosis without Exacerbated Mycobacterial Growth in Tumor Necrosis Factor Receptor p55 Gene-Deficient Mice Intravenously Infected with Mycobacterium avium  

The pathogenesis of mycobacterial infections is associated with the formation of granulomas in which both antibacterial protection and tissue damage take place concomitantly. We used murine Mycobacterium avium infection to compare the development of granulomatous lesions in intravenously infected tu...

109

Easy differentiation of Mycobacterium mucogenicum from other species of the Mycobacterium fortuitum complex by thin-layer and gas chromatography of fatty esters and alcohols  

The mycolate pattern of a recently recognized mycobacterial pathogen. Mycobacterium mucogenicum (formerly Mycobacterium chelonae-like organism), was established for the first time. The reference strains, together with 31 environmental and clinical isolates belonging to this species, were examined fo...

110

Spoligotyping and Mycobacterium tuberculosis  

We evaluated the clinical usefulness of spoligotyping, a polymerase chain reaction-based method for simultaneous detection and typing of Mycobacterium tuberculosis strains, with acid-fast bacilli-positive slides from clinical specimens or mycobacterial cultures. Overall sensitivity and specificity w...

111

Tuberculosis (TB): Treatment  

... of Mycobacterial and Respiratory Infections View full profile Tuberculosis (TB): Treatment Given the many effective medications available ... Services Directory Make a Donation Locations Calendar Newsroom Tuberculosis Program National Jewish Health is a world-renowned ...

112

Antimicrobial susceptibilities of mycobacteria as determined by differential light scattering and correlation with results from multiple reference laboratories.  

The DAWN Model B laser light scattering instrument (Wyatt Technology Corporation, Santa Barbara, Calif.) was evaluated to assess its potential to provide rapid mycobacterial antimicrobial susceptibility test results. For Mycobacterium tuberculosis there was a clear separation between susceptible and...

113

Osteopontin: A Novel Cytokine Involved in the Regulation of Mycobacterium avium subsp. paratuberculosis Infection in Periparturient Dairy Cattle  

Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by the intracellular bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), have a devastating impact on the dairy industry. ...

114

Osteopontin Immunoreactivity in the Ileum and Ileoceccal Lymph Node of Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis  

Osteopontin (Opn), a highly acidic glycoprotein, promotes cellular adhesion and recruitment and has been shown to be upregulated in the granulomas of mycobacterial infections. Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is associated with granulomatous enteritis. ...

115

Th1-driven immune reconstitution disease in Mycobacterium avium–infected mice  

Following antiretroviral therapy, a significant proportion of HIV+ patients with mycobacterial coinfections develop a paradoxical, poorly understood inflammatory disease termed immune reconstitution inflammatory syndrome (IRIS). Here, we show that Mycobacterium avium–infected T cell–deficient mice i...

116

Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution  

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, a...

117

Acute kidney injury in a patient with nontuberculous mycobacterial infections: a case report  

Nontuberculous mycobacterial infections are an increasingly recognized cause of chronic lung disease in both immunocompromised and immunocompetent patients. Pre-existing lung disease, alcohol abuse, diabetes mellitus, malignancy, and smoking have been identified as important risk factors in nontuber...

118

MIRU-VNTR typing adds discriminatory value to groups of Mycobacterium bovis and Mycobacterium caprae strains defined by spoligotyping  

Abstract The value of Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) as a genotyping technique for Mycobacterium bovis (M. bovis) and Mycobacterium caprae (M. caprae), has been confirmed in different countries and epidemiological scenarios. However, a ...

119

Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro.  

BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ?leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ?leuD strain. The inhibitory effect of BCG ?leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ?leuD/Ag85B treatment was evaluated by Western blotting. BCG ?leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ?leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ?leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ?leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer. PMID:23053076

120

Severe X-linked chronic granulomatous disease in two unrelated females.  

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by mutations of one of the subunits of phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leading to decreased or complete absence of neutrophil oxidative burst. We report the clinical and laboratory findings in two young unrelated females 14 and 9 years of age and natives of Tahiti and Reunion Islands, respectively, with severe X-linked granulomatous disease. In both cases, the infectious pattern was unusual, with convergent symptoms suggesting underlying mycobacterial infection. Functional analysis revealed low residual NADPH oxidase activity with about 5-10% of normal neutrophil population. De novo null mutations affecting the CYBB gene that encodes the gp91 protein were found in both cases in the heterozygous state (in patient 1, p.Arg130X in exon 5, and in patient 2, a novel insertion in exon 6, c.632_633insCATC). Methylation analysis confirmed that phenotype expression was linked to skewed X inactivation and showed that the de novo mutation arose on the maternally inherited chromosome in one case and on the paternally inherited chromosome in the other case. In conclusion, X-linked CGD carriers could therefore be at risk for severe infectious diseases depending on the skewed X inactivation pattern and the infectious context. PMID:17089090

 
 
 
 
121

Phagolysosome maturation of macrophages was reduced by PE_PGRS 62 protein expressing in Mycobacterium smegmatis and induced in IFN-? priming.  

Mycobacterium bovis parasitizes host macrophages and has developed strategies to survive within macrophages. Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence. To further elucidate the molecular pathogenesis of tuberculosis and host response to M. bovis, we explored the mechanisms by which PE_PGRS62 protein increase persistence of mycobacterium within host macrophages. We found that the M. smegmatis strain expressing M. bovis PE_PGRS 62 protein reduced phagolysosome maturation in human macrophages, and significantly decreased the mRNA expression of IL-1? in a dose- and time-dependent. We identified that IFN-? priming of macrophages immediately prior to infection with PE_PGRS62 expressing M. smegmantis, enhanced the maturation of phagolysosomes and induced IL-1? production both that the protein and mRNA levels and further activated the NF-?B pathway. Overall, we demonstrated that PE_PGRS62 protein altered the immune environment of the host cells, which suggested that the pathogenic PE_PGRS62 protein altering the immune mechanism might be involved in the pathogenesis of mycobacterial disease and hence influenced host cell responses to M. bovis infection. PMID:22658664

122

Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates.  

The prevalence of recently described mutation V176F, located in the beginning of the rpoB gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of rpoB mutations among 80 rifampin (RIF)-resistant Mycobacterium tuberculosis strains isolated in Germany during 1997. The most frequent rpoB mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection of rpoB mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS6110 DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical rpoB mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in M. tuberculosis. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance. PMID:11136757

123

Using polymerase chain reaction with primers based on the plcB-plcC intergenic region to detect Mycobacterium tuberculosis in clinical samples/ Deteção de Mycobacterium tuberculosis em amostras clínicas por reação em cadeia da polimerase utilizando primers baseados na região intergênica plcB-plcC  

Abstract in portuguese OBJETIVO: Desenvolver um sistema para o diagnóstico molecular da tuberculose por reação em cadeia da polimerase, do inglês polymerase chain reaction (PCR), pela construção de primers baseados na diferença da organização de uma região intergênica da fosfolipase (phospholipase) C (região plcB-plcC), que diferencia Mycobacterium tuberculosis das outras micobactérias. MÉTODOS: Um produto de PCR com o tamanho esperado (432 pb) foi obtido somente de M. tuberculosi (more) s e M. africanum. Um total de 33 isolados micobacterianos e 273 amostras clínicas de pacientes com suspeita de tuberculose foram examinados. Estes foram submetidos ao estudo comparativo da técnica de PCR contra o cultivo. RESULTADOS: Os dados mostraram 93,8% de exatidão para PCR contra o cultivo (p Abstract in english OBJECTIVE: To develop a system for the molecular diagnosis of tuberculosis by polymerase chain reaction (PCR), constructing primers based on the difference in gene organization of the intergenic region of phospholipase C (plcB-plcC region), which differentiates Mycobacterium tuberculosis from other mycobacteria. METHODS: A PCR product of the expected size (432 bp) was obtained from M. tuberculosis and M. africanum only. A total of 33 mycobacterial isolates and 273 clinica (more) l samples from patients suspected of having tuberculosis were examined. These were used in the comparative study of the PCR technique versus culture. RESULTS: For PCR versus culture, the data showed 93.8% accuracy (p

124

A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1.  

Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed. PMID:12502715

125

PCR detection of polycyclic aromatic hydrocarbon-degrading mycobacteria  

Polymerase chain reaction (PCR) methods based on the 16S rRNA genes of Mycobacterium sp. PYR-1 and Mycobacterium sp. PAH135, known PAH-degrading bacteria, were developed. An efficient mycobacterial cell lysis procedure was used for the PCR assay. The PCR methods were positive with the target species, but negative for the other 45 bacterial species tested including other Mycobacterium spp. The PCR sensitivity for pure cultures was 20 cells for Mycobacterium sp. PAH135 and 200 cells for Mycobacterium sp. PYR-1. The PCR with a simple sample preparation procedure was used to monitor Mycobacterium sp. PYR-1 cell concentrations in soil slurries amended with [{sup 14}C]pyrene. The pyrene mineralization correlated with the Mycobacterium PYR-1 cell concentrations in the soil slurries. When the PCR titer (the maximum dilution for positive PCR results) reached 10{sup -4}-10{sup -5} at 5-1O days incubation, approximately 50% of the [{sup 14}C]pyrene had been mineralized to {sup 14}CO{sub 2}. However, without inoculation with Mycobacterium PYR-1 cells, both the sterile and nonsterile soils had negative PCR results and no pyrene mineralization. 25 refs., 3 figs., 2 tabs.

126

ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish.  

ESX-5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX-5-deficient M. marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro-inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild-type bacteria. Together our experiments indicate that ESX-5-mediated protein secretion is used by M. marinum to establish a moderate and persistent infection. PMID:22256857

127

Interaction of wild type, G68R and L125M isoforms of the arylamine-N-acetyltransferase from Mycobacterium tuberculosis with isoniazid: a computational study on a new possible mechanism of resistance.  

Isoniazid (INH) is a front-line drug used in the treatment of tuberculosis (TB), a disease that remains a major cause of death worldwide. Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase-peroxidase (CP) enzyme. Recent studies have suggested that acetylation of INH by the arylamine-N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) may be a possible cause of inactivation of the drug thus resulting in resistant strains. In this study, computational techniques were applied to investigate the binding of isoniazid to three TBNAT isoforms: wild type, G68R and L125M. Since there is no experimental structure available, molecular dynamics (MD) simulations were initially used for the refinement of TBNAT homology models. Distinct conformations of the models were selected during the production stage of MD simulations for molecular docking experiments with the drug. Finally, each mode of binding was refined by new molecular MD simulations. Essential dynamics (ED) analysis and linear interaction energy calculations (LIE) were used to evaluate the impact of amino acid substitutions on the structural and binding properties of the enzymes. The results suggest that the wild type and the G68R TBNATs have a similar pattern of affinity to INH. On the other hand, the calculated enzyme-INH dissociation constant (KD) was estimated 33 times lower for L125M isoform in comparison with wild type enzyme. This last finding is consistent with the hypothesis that isolated mutations in the tbnat gene can produce M. tuberculosis strains resistant to isoniazid. PMID:22460521

128

Signalling inhibitors against Mycobacterium tuberculosis--early days of a new therapeutic concept in tuberculosis.  

Tuberculosis causes nearly two million deaths per year world-wide. In addition multidrug-resistant mycobacterial strains rapidly emerge so novel therapeutic approaches are needed. Recently, several promising mycobacterial target molecules were identified, which are involved in bacterial or host cell signalling e.g. the serine/threonine protein kinases, PknB and PknG, NAD kinase and the NAD synthetase. Here we describe some early efforts in the development of novel signal transduction inhibitory anti-mycobacterial drugs using a multiple target approach, with special emphasis on the kinase inhibitory field. Initially, we are using the Nested Chemical Library (NCL) technology and pharmacophore modelling. A hit-finding library, consisting of approximately 19000 small molecules with a bias for prototypic kinase inhibitors from our NCL library and commercial sources was virtually screened against these validated target molecules. Protein structures for the virtual screening were taken from the published three dimensional crystal structures of the enzymes. The hits from the virtual screening were subsequently tested in enzymatic assay systems. Potent hits were then tested for biological activity in macrophages, infected with mycobacteria. The final goal of this exercise is not only to identify potent anti-mycobacterial substances, but also a common pharmacophore for the mycobacterial target PknG in combination with PknB, NAD kinase and/or NAD synthetase. This common pharmacophore still needs to be a unique pharmacophore for the mycobacterial target proteins over human off-targets. Such a pharmacophore might then drive the optimization of a completely new profile of an antibiotic agent with activity against latent mycobacteria and resistance mycobacterial strains. PMID:18991635

129

Bases genéticas de la susceptibilidad a enfermedades infecciosas humanas/ Genetic Basis of Susceptibility to Human Infectious Diseases  

Abstract in spanish Los diferentes componentes genéticos juegan un papel importante en la determinación diferencial de la susceptibilidad a las principales enfermedades infecciosas de los humanos, tales como la malaria, la lepra, VIH/SIDA, tuberculosis y enfermedades por micobacterias, entre otras. La genética epidemiológica, incluyendo los estudios de gemelos, proporciona evidencia fuerte de que la variación genética en las poblaciones humanas contribuye a la susceptibilidad a dichas (more) enfermedades. La genética humana de las enfermedades infecciosas ha postulado que un raro grupo de inmunodeficiencias primarias confiere vulnerabilidad a múltiples enfermedades infecciosas (un gen, múltiples infecciones), mientras que las enfermedades infecciosas comunes están asociadas con la herencia poligénica de múltiples genes de susceptibilidad (una infección, múltiples genes). Simultáneamente, se ha determinado, en varias infecciones comunes, la herencia de un gen principal de susceptibilidad, al menos en algunas poblaciones. Este nuevo paradigma (un gen, una infección) desdibuja la distinción entre la genética mendeliana basada en pacientes, y la genética de enfermedades complejas basadas en estudios de población, lo cual nos da una nueva forma de abordaje conceptual para explorar las bases moleculares de la genética de enfermedades infecciosas en los humanos Abstract in english Several genetic factors play an important role in determining differential susceptibility to major infectious diseases in human populations, such as malaria, leprosy, HIV/AIDS, tuberculosis and mycobacterial infections. Genetic epidemiology, including twin studies, provides robust evidence that genetic variation in human populations contributes to susceptibility to infectious disease. The dominant paradigm in the human genetics of infectious diseases postulates that rare (more) monogenic immunodeficiencies confer vulnerability to multiple infectious diseases (one gene, multiple infections), whereas common infections are associated with the polygenic inheritance of multiple susceptibility genes (one infection, multiple genes). In parallel, several common infections have been shown to reflect the inheritance of one major susceptibility gene, at least in some populations. This new point of view (one gene, one infection), distort the distinction between patient-based Mendelian genetics and population-based complex genetics, and provides a unified conceptual frame for exploring the molecular genetic basis of infectious diseases in humans

130

EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator.  

Ethionamide (ETH) is an important second-line antitubercular drug used for the treatment of patients infected with multidrug-resistant Mycobacterium tuberculosis. Although ETH is a structural analogue of isoniazid, only little cross-resistance to these two drugs is observed among clinical isolates. Both isoniazid and ETH are pro-drugs that need to be activated by mycobacterial enzymes to exert their antimicrobial activity. We have recently identified two M. tuberculosis genes, Rv3854c (ethA) and Rv3855 (ethR), involved in resistance to ETH. ethA encodes a protein that belongs to the Flavin-containing monooxygenase family catalysing the activation of ETH. We show here that ethR, which encodes a repressor belonging to the TetR/CamR family of transcriptional regulators, negatively regulates the expression of ethA. By the insertion of the ethA promoter region upstream of the lacZ reporter gene, overexpression of ethR in trans was found to cause a strong inhibition of ethA expression, independently of the presence of ETH in the culture media. Electrophoretic mobility shift assays indicated that EthR interacts directly with the ethA promoter region. This interaction was confirmed by DNA footprinting analysis, which, in addition, identified the EthR-binding region. Unlike other TetR/CamR members, which typically bind 15 bp operators, EthR recognises an unusually long 55 bp region suggesting multimerization of the repressor on its operator. Identification by primer-extension of the ethA transcriptional start site indicated that it is located within the EthR-binding region. Taken together, bacterial two-hybrid experiments and gel filtration assays suggested a dimerization of EthR in the absence of its operator. In contrast, surface plasmon resonance analyses showed that eight EthR molecules bind cooperatively to the 55 bp operator, which represents a novel repression mechanism for a TetR/CamR member. PMID:14651620

131

Differentiation of phylogenetically related slowly growing mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences.  

Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in Mycobacterium kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szulgai. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus Mycobacterium representing 13 species (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii, M. malmoense, M. marinum, M. shimoidei, M. simiae, M. szulgai, M. triplex, M. ulcerans, and M. xenopi). An alignment of these sequences together with additional sequences available in the EMBL database (for M. intracellulare, M. phlei, M. smegmatis, and M. tuberculosis) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for M. simiae, M. genavense, and M. triplex, and distinct branches for M. xenopi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgai revealed ITS sequence similarities of 93 and 88%, respectively. M. marinum and M. ulcerans possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification. PMID:9431937

132

The clinical use of fluoroquinolones for the treatment of mycobacterial diseases.  

Mycobacterial diseases often require prolonged therapy with multidrug regimens. Fluoroquinolones have excellent bactericidal activity against many mycobacteria; achieve effective serum, tissue, and intracellular levels following oral administration; and produce few adverse effects. These properties have led to the increasing use of fluoroquinolones for the treatment of mycobacterial infections. We reviewed clinical studies and reports involving the use of fluoroquinolones for mycobacterial diseases. Ofloxacin, ciprofloxacin, sparfloxacin, and pefloxacin exhibit clinical efficacy against mycobacterial diseases, especially tuberculosis and leprosy. Fluoroquinolones have generally been administered in regimens that include other agents. However, when a fluoroquinolone has been found to be the sole active agent in a multidrug regimen, the ready emergence of resistance to fluoroquinolones has been recognized, just as when they have been used as monotherapy. Therefore, to forestall the emergence of resistance to fluoroquinolones during the treatment of mycobacterial diseases, these drugs should always be used in combination with at least one other active agent, and they should be used only when effective alternative drugs are not available. PMID:9402384

133

Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles  

Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75?ng of mycobacterial ...

134

Haliclonacyclamines, Tetracyclic Alkylpiperidine Alkaloids, as Anti-dormant Mycobacterial Substances from a Marine Sponge of Haliclona sp.  

A new tetracyclic alkylpiperidine alkaloid, 22-hydroxyhaliclonacyclamine B (1), together with two known alkaloids, haliclonacyclamine A (2) and B (3), were isolated from a marine sponge of Haliclona sp. as anti-dormant mycobacterial substances. The chemical structure of 22-hydroxyhaliclonacyclamine B (1) was determined on the basis of spectroscopic study. The compounds 2 and 3 showed strong anti-mycobacterial activity against Mycobacterium smegmatis and M. bovis Bacille de Calmette et Guérin (BCG) under both aerobic condition and hypoxic condition inducing dormant state with minimum inhibitory concentrations (MICs) in the ranges of 1.0—2.5 ?g/ml. In addition, the anti-microbial activity of compound 3 was bactericidal against M. bovis BCG under both aerobic and hypoxic conditions. The 22-hydroxy group in 1 was found to reduce anti-mycobacterial activity, because 22-hydroxyhaliclonacyclamine B (1) exhibited weaker anti-microbial activities against Mycobacterium bacilli with MICs in the ranges of 12.5—50 ?g/ml.   

135

Inductive microRNA-21 impairs anti-mycobacterial responses by targeting IL-12 and Bcl-2  

miR-21 has been shown to regulate multiple mRNAs and cause tumor progression and metastasis. However, whether miR-21-mediated posttranscriptional regulation is involved in antigen presentation and anti-mycobacterial responses remains unclear. Here, we report that miR-21 can be induced after Bacillus Calmette-Guerin (BCG) vaccination by NF-kB activation. miR-21 suppressed IL-12 production by targeting IL-12p35, which impaired anti-mycobacterial T cell responses both in vitro and in vivo. Additionally, miR-21 also promoted dendritic cell (DC) apoptosis by targeting Bcl-2. Therefore, miR-21 may potentially be involved in fine-tuning of the anti-mycobacterial Th1 response and in regulating the efficacy of BCG vaccination.

136

Mycobacterium bovis BCG and purified protein derivative-induced reduction in the CD80 expression and the antigen up-take function of dendritic cells from patients with cervical cancer  

Objective: Clinical diagnostic studies have reported that co-existence of malignancy and tuberculosis in regions with high incidence of tuberculosis may be possible. However, simultaneous occurrence of cervical cancer (CaCx) and tuberculosis has been reported for it rarity. Our analysis of prevalence of mycobacterial infection among the study group of patients with CaCx had revealed co-infection of mycobacterium species including Mycobacterium tuberculosis. Since M. tuberculosis potentially affects the dendritic cells-mediated protective immune response, the main aim of this study was to analyse the effect of the mycobacterial infection on the immunological functions of dendritic cells (DC) in patients with CaCx. Study design: Initially, the prevalence of mycobacterial infection was analys...

137

Inactivation of tesA reduces cell wall lipid production and increases drug susceptibility in mycobacteria.  

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections. PMID:21592957

138

Clostridium difficile Toxins and Enterococcal Translocation in Vivo and in Vitro.  

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections. PMID:11319888

139

Poor immunogenicity of BCG in helminth infected population is associated with increased in vitro TGF-b production  

The only vaccine available against tuberculosis (TB), BCG, so effective in experimental animal models, has been under scrutiny for a long time owing to its variable efficacy against pulmonary tuberculosis in adults. In this study, we evaluated whether anti-helminthic therapy prior to BCG vaccination could increase the immunogenicity of BCG vaccination in helminth infected population. We recruited volunteers with evidence of prior mycobacterial infection and who were asymptomatic carriers of helminths. The subjects were randomized to receive either anti-helminthic drugs or placebo. Three months later, BCG vaccination was administered to volunteers. Mycobacterial antigen-specific cytokine responses were assessed 2 months after vaccination. The results show that peripheral blood mononuclear c...

140

Diagnosis and management of lung infections.  

This article describes contemporary methods of diagnosis and current treatment regimens for most pulmonary infections. Modern techniques used to improve diagnostic yield in pulmonary infection include bronchoscopy, ultrasound- and electromagnetic-guided endoscopy, transthoracic needle biopsy, and samples obtained with thoracoscopy. The spectrum of bacterial, mycobacterial, fungal, and viral pathogens implicated in pulmonary disease is discussed. Treatment strategies and guideline recommendations for antimicrobial selection are described for community-acquired, health care-associated, hospital-acquired, and ventilator-associated pneumonia, and for the most common fungal, mycobacterial, and viral infections. The state-of-the art in topical and aerosolized anti-infective therapy and an algorithm for managing hemoptysis are also presented. PMID:22789595

 
 
 
 
141

Polymerase Chain Reaction-Based Molecular Diagnosis of Cutaneous Infections in Dermatopathology  

Conventional methods, including microscopy, culture, and serologic studies, are a mainstay in the diagnosis of cutaneous infection. However, owing to limitations associated with these techniques, such as low sensitivity for standard microscopy and in the case of culture delay in diagnosis, polymerase chain-reaction based molecular techniques have taken on an expanding role in the diagnosis of infectious processes in dermatopathology. In particular, these assays are a useful adjunct in the diagnosis of cutaneous tuberculosis, atypical mycobacterial infection, leprosy, Lyme disease, syphilis, rickettsioses, leishmaniasis, and some fungal and viral infections. Already in the case of tuberculosis and atypical mycobacterial infection, standardized polymerase chain-reaction assays are commonly u...

142

Mycobacteria isolated from Chesapeake Bay fish  

Abstract Mycobacteriosis in fish can result in ulcers, emaciation, and in some cases death. Mycobacteria have been previously isolated from a variety of Chesapeake Bay fish species, and the current study was designed to identify potential host specificity and location fidelity of mycobacterial isolates. Mycobacteria were isolated from wild fish of the Chesapeake Bay collected from the Upper Bay, the Choptank River, Herring Bay, the Chicamacomico River, the Pocomoke River and the Potomac River in 2003-2006. Mycobacterial isolates were recovered from striped bass, Morone saxatilis, Atlantic menhaden, Brevoortia tyrannus, white perch, Morone americana, summer flounder, Paralichthys dentatus, spot, Leiostomus xanthurus, largemouth bass, Micropterus salmoides, channel catfish, Ictalurus punctat...

143

Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells  

Culture filtrate proteins from Mycobacterium tuberculosis induce protective immunity in various animal models of tuberculosis. Two molecular mass regions (6 to 10 kDa and 24 to 36 kDa) of short-term culture filtrate are preferentially recognized by Th1 cells in animal models as well as by patients with minimal disease. In the present study, the 24- to 36-kDa region has been studied, and the T-cell reactivity has been mapped in detail. Monoclonal antibodies were generated, and one monoclonal antibody, HYB 71-2, with reactivity against a 29-kDa antigen located in the highly reactive region below the antigen 85 complex was selected. The 29-kDa antigen (CFP29) was purified from M. tuberculosis short-term culture filtrate by thiophilic adsorption chromatography, anion-exchange chromatography, and gel filtration, In its native form, CFP29 forms a polymer with a high molecular mass. CFP29 was mapped in two-dimensional electrophoresis gels as three distinct spots just below the antigen 85 complex component MPT59, CFP29 is present in both culture filtrate and the membrane fraction from M. tuberculosis, suggesting that this antigen is released from the envelope to culture filtrate during growth, Determination of the N-terminal amino acid sequence allowed cloning and sequencing of the cfp29 gene. The nucleotide sequence showed 62% identity to the bacteriocin Linocin from Brevibacterium linens, Purified recombinant histidine-tagged CFP29 and native CFP29 had similar T-cell stimulatory properties, and they both elicited the release of high levels of gamma interferon from mouse memory effector cells isolated during the recall of protective immunity to tuberculosis. Interspecies analysis by immunoblotting and PCR demonstrated that CFP29 is widely distributed in mycobacterial species.

144

Rv2131c from Mycobacterium tuberculosis is a CysQ 3'-phosphoadenosine-5'-phosphatase.  

Mycobacterium tuberculosis ( Mtb) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. These metabolites are products of the sulfate assimilation pathway. CysQ, a 3'-phosphoadenosine-5'-phosphatase, is considered an important regulator of this pathway in plants, yeast, and other bacteria. By controlling the pools of 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), CysQ has the potential to modulate flux in the biosynthesis of essential sulfur-containing metabolites. Bioinformatic analysis of the Mtb genome suggests the presence of a CysQ homologue encoded by the gene Rv2131c. However, a recent biochemical study assigned the protein's function as a class IV fructose-1,6-bisphosphatase. In the present study, we expressed Rv2131c heterologously and found that the protein dephosphorylates PAP in a magnesium-dependent manner, with optimal activity observed between pH 8.5 and pH 9.5 using 0.5 mM MgCl 2. A sensitive electrospray ionization mass spectrometry-based assay was used to extract the kinetic parameters for PAP, revealing a K m (8.1 +/- 3.1 microM) and k cat (5.4 +/- 1.1 s (-1)) comparable to those reported for other CysQ enzymes. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for myo-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. Moreover, the ability of the Rv2131c-encoded enzyme to dephosphorylate PAP and PAPS in vivo was confirmed by functional complementation of an Escherichia coli Delta cysQ mutant. Taken together, these studies indicate that Rv2131c encodes a CysQ enzyme that may play a role in mycobacterial sulfur metabolism. PMID:18454554

145

Inter- and Intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains.  

ABSTRACT: BACKGROUND: Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne's disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as 'Sheep' or 'S-type' and 'Cattle' or 'C-type'. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of large sequence polymorphisms by PCR (LSP analysis), single nucleotide polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis. RESULTS: The presence of LSPA4 and absence of LSPA20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III. CONCLUSION: This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains. PMID:23164429

146

ANTIGEN-INDUCED IFN-G, NITRIC OXIDE, AND TNF-A PRODUCTION BY BLOOD MONONUCLEAR CELLS FROM MYCOBACTERIUM BOVIS-INFECTED CATTLE: DIAGNOSTIC IMPLICATIONS  

Bovine tuberculosis in the United States has proven costly to cattle producers as well as government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary ...

147

Comparative Evaluation of the New Version of the INNO-LiPA Mycobacteria and GenoType Mycobacterium Assays for Identification of Mycobacterium Species from MB/BacT Liquid Cultures Artificially Inoculated with Mycobacterial Strains  

The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a...

148

Evaluation of GenoType and LiPA MYCOBACTERIA Assays for Identification of Finnish Mycobacterial Isolates  

Two DNA strip assays, INNO-LiPA MYCOBACTERIA and GenoType Mykobakterien, were evaluated for identification of 81 Finnish mycobacterial isolates. The LiPA assay correctly identified 89.4% of the 66 isolates studied, and the GenoType assay identified 95.1% of 81 isolates. The GenoType assay had a wide...

149

TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4  

TLR9 activation is important for the maintenance of mycobacteria-elicited pulmonary granulomatous responses, hallmarks of protective immune responses following mycobacterial infection. However, the mechanism or mechanisms underlying this effect of TLR9 are not clear. Here, we show that Tlr9-deficien...

150

TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis  

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9?/? as well as TLR2/9 double knockout mice to aerosol infection...

151

Secondary Infections of AIDS Autopsy Cases in Japan with Special Emphasis on Mycobacterium Avium-Intracellulare Complex Infection  

In order to study the frequency of secondary infections of AIDS autopsy cases in Japan, especially the frequency of Mycobacterium aviumintracellulare complex (MAC) infection, retrospective autopsy study was conducted between 1986 and 1997 at the affiliated hospital of Institute of Medical Sciences, University of Tokyo. Secondary infections of various organs from 43 AIDS autopsy cases were examined using histopathology, genetic diagnosis of tuberculosis, Ziehl-Neelsen stain for acid-fast bacilli and immunohistochemistry. Nontuberculous mycobacterial infection (Mycobacterium avium) was observed in 17 cases (40%) out of 43 using polymerase chain reaction (PCR), but M. tuberculosis infection was not observed. Ziehl-Neelsen staining showed a positive reaction in lung and spleen tissues of 7 AIDS autopsy cases. Immunohistochemistry using anti-BCG antibody revealed positivity in 7 AIDS autopsy cases. CD4 counts of 17 AIDS patients with mycobacterial infection were less than 18.7/?l. Other opportunistic infections were also examined by histopathology. Secondary infections were present in every case, and these included cytomegalovirus infection (32 cases), Pneumocystis carinii (15 cases), Candida (16 cases), Aspergillus (12 cases), Cryptococcus (6 cases), Toxoplasma (6 cases), methicillin-resistant Staphylococcus aureus (3 cases), herpes virus (1 case) and Entamoeba histolytica (1 case). Malignant lymphoma was recognized in 14 cases and Kaposi's sarcoma in 6. This is the systemic report on secondary infections of AIDS autopsy cases in Japan. In diagnosis of mycobacterial infections, PCR was more useful than staining for acid-fast bacilli and immunohistochemistry. Secondary infections (especially mycobacterial infection) were closely associated with the low CD4 count.   

152

Relatively alcohol-resistant mycobacteria are emerging pathogens in patients receiving acupuncture treatment  

Acupuncture has been gaining popularity as a form of alternative medicine. In the past, only blood-borne viruses and anecdotal reports of bacterial infections have been associated with acupuncture. We report on four patients with mycobacterial infections complicating acupuncture who were encountered...

153

Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis?  

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membran...

154

A Novel Interaction Linking the FAS-II and Phthiocerol Dimycocerosate (PDIM) Biosynthetic Pathways*S?  

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme c...

155

The PE-PPE Domain in Mycobacterium Reveals a Serine ?/? Hydrolase Fold and Function: An In-Silico Analysis  

The PE and PPE proteins first reported in the genome sequence of Mycobacterium tuberculosis strain H37Rv are now identified in all mycobacterial species. The PE-PPE domain (Pfam ID: PF08237) is a 225 amino acid residue conserved region located towards the C-terminus of some PE and PPE proteins and h...

156

Quantitative analyses of mycobacteria in water: Adapting methods in clinical laboratories  

An outbreak of occupational hot tub lung necessitated quantitative analysis of mycobacteria in water samples. We combined procedures for cultivation of mycobacteria in urine and quantitative analyses of dialysis water. Whirlpool spa water samples were analyzed showing promising results. In conclusion, quantitative mycobacterial culture of water is possible by adapting methods routinely used in clinical laboratories.

157

Mycobacterium abscessus Pulmonary Infection Presenting as a Solitary Pulmonary Nodule  

Nontuberculous mycobacterial pulmonary infection is a rare cause of a solitary pulmonary nodule. All previously reported cases were caused by Mycobacterium avium complex, and a solitary pulmonary nodule caused by other NTM species has been very rarely reported. We describe the first case of Mycobacterium abscessus infection presenting as a solitary pulmonary nodule in a 51yearold asymptomatic adult patient.   

158

Superficial mycobacterial lymphadenitis in Saskatchewan.  

A total of 43 bacteriologically verified cases of superficial mycobacterial lymphadenitis were reported in Saskatchewan between 1981 and 1986; 35 (81%) were due to Mycobacterium tuberculosis. Among the eight cases (19%) due to nontuberculous mycobacteria the agent most frequently isolated was M. avi...

159

Failure of Synthetic Polynucleotides to Affect the Immunogenicity of Mycobacterial Ribonucleic Acid and Ribosomal Protein Preparations  

The synthetic polyribonucleotides adenylic acid (poly A), uridylic acid (poly U), cytidylic acid (poly C), and inosinic acid (poly I), whether single- or double-stranded (poly A:U, poly I:C), cannot replace mycobacterial ribonucleic acid (RNA) in the production of a high immune response in CF-1 mice...

160

Mycobacterium marinum Strains Can Be Divided into Two Distinct Types Based on Genetic Diversity and Virulence  

Mycobacterium marinum causes a systemic tuberculosis-like disease in a large number of poikilothermic animals and is used as a model for mycobacterial pathogenesis. In the present study, we infected zebra fish (Danio rerio) with different strains of M. marinum to determine the variation in pathogeni...

 
 
 
 
161

Acquired predisposition to mycobacterial disease due to autoantibodies to IFN-?  

Genetic defects in the IFN-? response pathway cause unique susceptibility to intracellular pathogens, particularly mycobacteria, but are rare and do not explain mycobacterial disease in the majority of affected patients. We postulated that acquired defects in macrophage activation by IFN-? may cause...

162

Antibodies against BCG antigen 60 in mycobacterial infection.  

A sensitive specific radioimmunoassay was developed to measure antibodies against BCG antigen 60, a prominent antigenic component of BCG bacilli which cross-reacts with similar components in many mycobacterial species including Mycobacterium leprae and M tuberculosis. A lepromatous serum pool had an...

163

Immune Responses Induced in Cattle by Vaccination with a Recombinant Adenovirus Expressing Mycobacterial Antigen 85A and Mycobacterium bovis BCG  

Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked im...

164

Humoral Immune Responses of White-tailed Deer (Odocoileus virginianus) to Mycobacterium bovis BCG Vaccination and Experimental Challenge with M. bovis  

Monitoring serum antibody production kinetics to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and efficacy of intervention strategies in several species. Humoral immun...

165

Mycobacteria isolated from Chesapeake Bay fish.  

Mycobacteriosis in fish can result in ulcers, emaciation, and in some cases death. Mycobacteria have been previously isolated from a variety of Chesapeake Bay fish species, and the current study was designed to identify potential host specificity and location fidelity of mycobacterial isolates. Mycobacteria were isolated from wild fish of the Chesapeake Bay collected from the Upper Bay, the Choptank River, Herring Bay, the Chicamacomico River, the Pocomoke River and the Potomac River in 2003-2006. Mycobacterial isolates were recovered from striped bass, Morone saxatilis, Atlantic menhaden, Brevoortia tyrannus, white perch, Morone americana, summer flounder, Paralichthys dentatus, spot, Leiostomus xanthurus, largemouth bass, Micropterus salmoides, channel catfish, Ictalurus punctatus, common carp, Cyprinus carpio carpio, spotted seatrout, Cynoscion nebulosus, killifish, Fundulus sp., blueback herring, Alosa aestivalis, American gizzard shad, Dorosoma cepedianum and American silver perch, Bairdiella chrysoura. Twenty-nine well-defined mycobacterial groups resulted from gas chromatography dendrogram clustering of isolates. The majority of groups included more than one host species and more than one site of collection. However, four groups contained only striped bass isolates, three of which were similar to M. shottsii. Therefore, multiple Chesapeake Bay fish species are colonized with multiple mycobacterial isolates, of which few appear to be host or location specific. PMID:19909394

166

Determination of molecular species composition of C80 or longer-chain alpha-mycolic acids in Mycobacterium spp. by gas chromatography-mass spectrometry and mass chromatography.  

The molecular species composition of alpha-mycolic acids ranging from C68 to C86 in 13 rapidly growing and 12 slowly growing mycobacterial species was determined by gas chromatography, gas chromatography-mass spectrometry, and mass chromatography. In gas chromatographic analysis, the molecular speci...

167

Alpha/Beta Interferon Impairs the Ability of Human Macrophages To Control Growth of Mycobacterium bovis BCG  

Administration of alpha/beta interferon (IFN-?/?) to mice infected with Mycobacterium tuberculosis has been shown to increase mycobacterial growth. Because IFN-?/? has direct pleiotropic effects on the differentiation and functional activities of macrophages, we evaluated the effect of IFN-?/? on my...

168

Immunohistochemical diagnosis of abdominal and lymph node tuberculosis by detecting Mycobacterium tuberculosis complex specific antigen MPT64  

Background; The aim of this study was to evaluate the diagnostic potential of immunohistochemistry using an antibody to the secreted mycobacterial antigen MPT64, in abdominal and lymph node tuberculosis; Methods, We used formalinfixed histologically diagnosed abdominal tuberculosis (n=33) and cer...

169

Osteopontin Immunoreactivity in Peripheral Blood Mononuclear Cells, Ileum, and Ileocecal Lymph Node of Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis  

Osteopontin (Opn), a highly acidic glycoprotein, plays an early role in initiating the innate immune response to mycobacterial infections by promoting cellular adhesion and recruitment of inflammatory cells from the peripheral blood. The formation of granulomas at the site of Mycobacterium avium s...

170

Osteopontin Expression During the Periparturient Period in Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis Infection  

Investigation of the role of osteopontin (Opn) in Johne’s disease is of interest based upon its ability to influence cytokine expression and to improve host defense against mycobacterial infections. The objective of this study was to characterize Opn expression and secretion by peripheral blood mono...

171

Prior Exposure to Live Mycobacterium bovis BCG Decreases Cryptococcus neoformans-Induced Lung Eosinophilia in a Gamma Interferon-Dependent Manner  

Some common childhood infections appear to prevent the development of atopy and asthma. In some Mycobacterium bovis BCG-vaccinated populations, strong delayed-type hypersensitivity responses to mycobacterial antigens are associated with a reduced risk of atopy. Although BCG exposure decreases allerg...

172

Relief from Zmp1-Mediated Arrest of Phagosome Maturation Is Associated with Facilitated Presentation and Enhanced Immunogenicity of Mycobacterial Antigens?  

Pathogenic mycobacteria escape host innate immune responses by blocking phagosome-lysosome fusion. Avoiding lysosomal delivery may also be involved in the capacity of mycobacteria to evade major histocompatibility complex (MHC) class I- or II-dependent T-cell responses. In this study, we used a genetic mutant of Mycobacterium bovis BCG that is unable to escape lysosomal transfer and show that presentation of mycobacterial antigens is affected by the site of intracellular residence. Compared to infection with wild-type BCG, infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zinc metalloprotease 1 (Zmp1) resulted in increased presentation of MHC class II-restricted antigens, as assessed by activation of mycobacterial Ag85A-specific T-cell hybridomas. The zmp1 deletion mutant was more immunogenic in vivo, as measured by delayed-type hypersensitivity (DTH), antigen-specific lymphocyte proliferation, and the frequency of antigen-specific gamma interferon (IFN-?)-producing lymphocytes of both CD4 and CD8 subsets. In conclusion, our results suggest that phagosome maturation and lysosomal delivery of BCG facilitate mycobacterial antigen presentation and enhance immunogenicity.

173

Innate and adaptive host responses and their genetic control in tuberculosis : studies in Indonesia, a highly TB endemic setting  

Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis. MTB infection does not necessarily progress to TB. Only 5-10% of exposed individuals develop clinical signs and symptoms of TB. Given the impact of mycobacterial exposure and the immunoregulatory consequences for host ...

174

Kinetics of accumulation of gamma delta receptor-bearing T lymphocytes in mice infected with live mycobacteria.  

The kinetics of accumulation of T cells bearing the gamma delta heterodimer form of the T-cell receptor in mice infected with live Mycobacterium bovis BCG or M. tuberculosis was studied. Substantial numbers of gamma delta T cells accumulated in mice given primary mycobacterial infections, although t...

175

Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens  

Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear cells with tuberculin purified protein derivative, Mycobacterium bovis bacillus Calmette-Guérin (BCG), short- and long-term culture filtrates of virulent Mycobacterium tuberculosis H37Rv, and 30-31-kDa secreted mycobacterial protein. These antigens also induced proliferation and production of gamma interferon. The CD4+ cells proliferated and expressed interleukin-2 receptors following stimulation with mycobacterial antigens. Depletion studies after antigen stimulation showed that the cytotoxic effector cells were CD16+ CD56+ and CD4-; the CD4+ cells alone did not mediate non-MHC-restricted cytotoxicity. To evaluate the influence of CD4+ cells on the development of non-MHC-restricted cytotoxicity, blood mononuclear cells were depleted of CD4+ cells before antigen stimulation. When mononuclear cells were incubated with purified protein derivative or short-term culture filtrate in the absence of CD4+ cells, cytotoxic activity was reduced. This reduction was abolished by interleukin-2 but not by gamma interferon. We conclude that several mycobacterial antigens are able to induce non-MHC-restricted cytotoxicity. This study indicates that non-MHC-restricted cytotoxicity following stimulation with mycobacterial antigens is induced by cytokines released by antigen-specific activated CD4+ cells.

176

PCR in the diagnosis of cutaneous tuberculosis/ Técnica de reação em cadeia da polimerase (PCR) no diagnóstico da tuberculose cutânea  

Abstract in portuguese Visando melhorar o diagnóstico laboratorial da Tuberculose Cutânea, foi realizado o estudo da aplicação da técnica de PCR em amostras de tecidos cutâneos macerados, descontaminados (com H2SO4 4% para eliminação da microbiota normal), neutralizados (com NaOH 4%) e armazenadas a -20ºC. Das 37 amostras submetidas ao estudo, 16,22% apresentavam baciloscopias positivas para bacilos álcool-ácidos resistentes (método concentrado) e em 43,24% houve o isolamento do Myc (more) obacterium tuberculosis em meio de cultivo Löwenstein-Jensen. Utilizando-se de primers para a região 16S rDNA do M. tuberculosis, o DNA micobacteriano foi detectado em 24,32% das biópsias. A sensibilidade e especificidade da PCR foram 43,7% e 90,4%, respectivamente. Devido à baixa sensibilidade e resultados divergentes entre as técnicas bacteriológicas e PCR (para a seqüência 16S rDNA), as amostras foram repetidas em um novo PCR com primers para a região IS6110. Tanto a sensibilidade como a especificidade da PCR com primers para IS6110 alcançaram 100% em relação ao cultivo. Os resultados confirmam a eficácia da PCR utilizando primers para a seqüência IS6110 e oferecem a possibilidade da técnica ser aplicada em amostras congeladas enviadas por serviços que não identificam o M. tuberculosis por técnicas de biologia molecular. Abstract in english Seeking to improve the laboratory diagnosis of Cutaneous Tuberculosis, a study was carried out on the application of PCR technique in macerated, decontaminated(with 4% H2SO4 for elimination of normal microbiot), neutralized (with 4% NaOH)biopsies tissues samples stored at -20ºC. Of the 37 samples submitted for study, 16.22% were positive by microscopy for acid-fast bacilli (concentrated method) and in 43.24% the Mycobacterium tuberculosis was isolated in Löwenstein-Jens (more) en medium. Using a M. tuberculosis complex specific primer set (gene sequence for 16S rDNA), the mycobacterial DNA was detected in 24.32% of the biopsies. The sensitivity and specificity of PCR were 43.7% and 90.4%, respectively. Due to low sensitivity and discrepant results between bacteriological techniques and PCR methodology, the samples were repeated in a new PCR with primers for the IS6110 target. The sensitivity and specificity of PCR for the IS6110 target obtained 100% in comparison with the culture method. The results confirm the effectiveness of PCR methodology using primers for the IS6110 gene sequence and permit the PCR method to be applied to frozen cutaneous biopsies sent by services that do not identify the M. tuberculosis by the biology molecular method.

177

Decreased Sensitivity to Changes in the Concentration of Metal Ions as the Basis for the Hyperactivity of DtxR(E175K)  

The metal-ion-activated diphtheria toxin repressor (DtxR) is responsible for the regulation of virulence and other genes in Corynebacterium diphtheriae. A single point mutation in DtxR, DtxR(E175K), causes this mutant repressor to have a hyperactive phenotype. Mice infected with Mycobacterium tuberculosis transformed with plasmids carrying this mutant gene show reduced signs of the tuberculosis infection. Corynebacterial DtxR is able to complement mycobacterial IdeR and vice versa. To date, an explanation for the hyperactivity of DtxR(E175K) has remained elusive. In an attempt to address this issue, we have solved the first crystal structure of DtxR(E175K) and characterized this mutant using circular dichroism, isothermal titration calorimetry, and other biochemical techniques. The results show that although DtxR(E175K) and the wild type have similar secondary structures, DtxR(E175K) gains additional thermostability upon activation with metal ions, which may lead to this mutant requiring a lower concentration of metal ions to reach the same levels of thermostability as the wild-type protein. The E175K mutation causes binding site 1 to retain metal ion bound at all times, which can only be removed by incubation with an ion chelator. The crystal structure of DtxR(E175K) shows an empty binding site 2 without evidence of oxidation of Cys102. The association constant for this low-affinity binding site of DtxR(E175K) obtained from calorimetric titration with Ni(II) is K{sub a} = 7.6 {+-} 0.5 x 10{sup 4}, which is very similar to the reported value for the wild-type repressor, K{sub a} = 6.3 x 10{sup 4}. Both the wild type and DtxR(E175K) require the same amount of metal ion to produce a shift in the electrophoretic mobility shift assay, but unlike the wild type, DtxR(E175K) binding to its cognate DNA [tox promoter-operator (toxPO)] does not require metal-ion supplementation in the running buffer. In the timescale of these experiments, the Mn(II)-DtxR(E175K)-toxPO complex is insensitive to changes in the environmental cation concentrations. In addition to Mn(II), Ni(II), Co(II), Cd(II), and Zn(II) are able to sustain the hyperactive phenotype. These results demonstrate a prominent role of binding site 1 in the activation of DtxR and support the hypothesis that DtxR(E175K) attenuates the expression of virulence due to the decreased ability of the Me(II)-DtxR(E175K)-toxPO complex to dissociate at low concentrations of metal ions.

178

Diagnosis of pulmonary and extrapulmonary tuberculosis using an in-house PCR method in clinical samples from a middle-income resource setting/ Diagnóstico de tuberculosis pulmonar y extrapulmonar mediante un método de PCR casera en muestras clínicas de una región de desarrollo intermedio  

Abstract in spanish Introducción. La prueba de PCR ofrece la posibilidad de obtener resultados rápidos, una prioridad para los programas de control de la tuberculosis. Objetivos.Evaluar el método de PCR basado en la detección del gen IS6110 de las micobacterias en muestras clínicas de pacientes con tuberculosis pulmonar y extrapulmonar, para demostrar su utilidad y confiabilidad en un sitio de desarrollo medio con alta prevalencia de tuberculosis. Materiales y métodos. Se recolectaron (more) 317 muestras pulmonares y 41 de origen extrapulmonar, de 358 pacientes con sospecha clínica de tuberculosis. Todas las muestras se procesaron para detectar bacilos ácido-alcohol resistentes por microscopía, cultivo en medio sólido y PCR. Para eliminar los inhibidores de la PCR se lavaron tres veces antes de realizar la lisis de la pared de la micobacteria. Resultados. La sensibilidad total fue de 96% y la especificidad de 100% para este método "casero" en muestras pulmonares y extrapulmonares. No se encontró inhibición entre las muestras que fueron negativas por PCR pero con cultivo positivo para Mycobacterium tuberculosis. No se hallaron falsos positivos. Conclusiones. La implementación de la PCR "casera" en regiones de desarrollo intermedio como método simple y estrictamente controlado, podría complementar eficientemente las herramientas bacteriológicas tradicionales para el diagnóstico rápido de la tuberculosis, especialmente en muestras paucibacilares y de origen extrapulmonar. Abstract in english Introduction: PCR detection offers a good opportunity to obtain fast results which is a priority in tuberculosis control programs. Objectives: We assayed an in-house PCR method based on the detection of mycobaterial IS6110 gene in clinical samples of patients with pulmonary and extrapulmonary tuberculosis to demonstrate its usefulness and reliability in the setting of a middle-resource region with high tuberculosis prevalence. Materials and methods: Pulmonary (n=317) and (more) extrapulmonary (n=41) samples were collected from 358 patients with clinical suspicion of tuberculosis. All samples were processed to detect acid-fast bacilli by microscopy, culture on solid media and PCR. To remove PCR inhibitors, three washing steps of the decontaminated pellet were included before mycobacterial cell lysis. Results: The overall sensitivity was 96% in clinical samples, and specificity was 100% for our in-house method in pulmonary and extrapulmonary samples. No inhibition was found among samples that were PCR negative, but culture positive for Mycobacterium tuberculosis. No false positives were found. Conclusions: In-house PCR in a middle-income setting region, with simple and strictly controlled methods, could efficiently complement conventional bacteriological tools for the rapid diagnosis of tuberculosis, especially in paucibacillary and extrapulmonary samples.

179

Diagnóstico molecular de histoplasmosis humana en muestras de sangre entera/ Molecular diagnosis of human histoplasmosis in whole blood samples  

Abstract in spanish Se evaluó el uso de sangre entera para el diagnóstico molecular de histoplasmosis utilizando un método artesanal de extracción de ADN fúngico y una PCR anidada que amplifica una porción del gen HcP100 específica de Histoplasma capsulatum. La sangre entera se trató con liticasa, enzima lisante de Trichoderma harzianum y proteinasa K, seguido de una extracción fenólica. Este tratamiento permitió una lisis completa de las células, mostró buen rendimiento en la o (more) btención de ADN y posibilitó la detección de la banda de 210 pb específica de H. capsulatum en la PCR anidada. El límite de detección fue de 0,25-1 levaduras/ml de sangre. El método se evaluó en 31 muestras de sangre de 19 pacientes con diagnóstico microbiológico de histoplasmosis, en 21 muestras de pacientes con otras micosis o infecciones por micobacterias y en 30 controles sanos. La PCR fue positiva en sangre para 17/19 pacientes con histoplasmosis (14/15 inmunocomprometidos y 3/4 sin inmunocompromiso aparente). Las muestras de sangre de los 30 controles sanos y de 20 pacientes con otras patologías fueron negativas, sólo hubo un falso positivo correspondiente a un paciente con infección por Mycobacterium avium-intracellulare. El método presentó 89% de sensibilidad y 96% de especificidad para el diagnóstico de histoplasmosis en sangre entera. Abstract in english To assess the value of using whole blood samples for the molecular diagnosis of histoplasmosis, we applied an in-house DNA extraction method and a nested PCR targeting a 210 bp specific segment of the Histoplasma capsulatum HcP100 gene. A whole blood volume of 2.5-3 milliliters was centrifuged and the cellular pellet was treated with Trichoderma harzianum lyticase and proteinase K prior to applying a conventional phenol DNA extraction. This procedure allowed complete cell (more) lysis, high DNA yield and specific amplification. The PCR detection limit was 0.25-1 yeast cells/ml of blood sample. The method was assessed on 31 blood samples from 19 patients with microbiological diagnosis of histoplasmosis, 30 healthy persons and 21 patients with other mycoses or mycobacterial diseases. Positive results were obtained in samples from 17/19 patients with histoplasmosis (14/15 immunocompromised and 3/4 without known immunological disorder). Blood samples from the 30 healthy controls and 20 patients with other conditions proved negative; the only false positive result was obtained from a patient with Mycobacterium avium-intracellulare infection. With 89% sensitivity and 98% specificity, this molecular method for detection of the agent in blood shows promising for the rapid diagnosis of human histoplasmosis.

180

Biosafety evaluation of the DNA extraction protocol for Mycobacterium tuberculosis complex species, as implemented at the Instituto Nacional de Salud, Colombia/ Evaluación de la bioseguridad del protocolo de extracción de ADN para especies del complejo Mycobacterium tuberculosis implementado en el Instituto Nacional de Sa  

Abstract in spanish Introducción. El trabajo con Mycobacterium tuberculosis se considera un factor de riesgo para el personal de laboratorio que manipula especímenes clínicos y cultivos. Uno de los procesos que requiere de una alta concentración de microorganismos es la extracción de ADN para realizar metodologías moleculares. Se han reportado casos de tuberculosis pulmonar en profesionales que realizan procedimientos moleculares en los que se requiere previa manipulación del microorg (more) anismo en masa, lo cual ha motivado la investigación sobre la bioseguridad del protocolo de extracción, sin que a la fecha haya consenso sobre los riesgos del proceso. Objetivo. Evaluar la bioseguridad del protocolo de extracción de ADN reportado por van Soolingen et al., 2002, mediante la determinación de la viabilidad de M. tuberculosis en cada etapa del proceso. Materiales y métodos. Se realizaron 880 cultivos a partir de 220 aislamientos clínicos de M. tuberculosis que se procesaron para las tres primeras fases de extracción de ADN. A los cultivos positivos se les realizó identificación molecular por PRA hsp65 y caracterización por spoligotyping. Resultados. Se obtuvo crecimiento en uno de los procedimientos realizados. Por caracterización molecular, se determinó que no correspondió al aislamiento analizado originalmente, sino que fue producto de contaminación cruzada. Conclusión. Se determinó que el protocolo de extracción de ADN descrito por van Soolingen et al. (2002) e implementado en el Instituto Nacional de Salud de Colombia, es seguro para el personal de laboratorio y el medio ambiente. Abstract in english Introduction. Manipulating Mycobacterium tuberculosis clinical specimens and cultures represents a risk factor for laboratory personnel. One of the processes that requires high concentrations of microorganisms is DNA extraction for molecular procedures. Pulmonary tuberculosis cases have occurred among professionals in charge of molecular procedures that require manipulation of massive quantities of microorganisms. This has prompted research studies on biosafety aspects of (more) extraction protocols; however, as yet, no consensus has been reached regarding risks associated with the process. Objective. The biosafety was evaluated for the DNA extraction protocol of van Soolingen, et al. 2002 by determining M. tuberculosis viability at each process stage. Materials and methods. Eight hundred eighty cultures were grown from 220 M. tuberculosis clinical isolates that had been processed through the first three DNA extraction stages. Molecular identifications of positive cultures used a PCR isolation of a fragment of the heat shock protein PRA-hsp65 and examination of its restriction enzyme profile (spoligotyping). Results. Growth was seen in one culture with one of the procedures used. The molecular characterization did not correspond to the initially analyzed isolate, and therefore was deduced to be the product of a cross-contamination. Conclusion. The DNA extraction protocol, as described by van Soolingen, et al. 2002 and as implemented at the Instituto Nacional de Salud, was established to be safe for laboratory personnel as well as for the environment.

 
 
 
 
181

Elevated levels of soluble urokinase receptors in serum from mycobacteria infected patients. Still looking for a marker of treatment efficacy  

In search for a serological marker, which may be used to monitor treatment efficacy in patients with extra-pulmonary mycobacterial infections, serum samples were collected prospectively from patients during a 6-months treatment period. The levels of soluble urokinase-type plasminogen activator receptor (suPAR) and soluble tumour necrosis factor receptor II (sTNFrII) were measured and compared with erythrocyte sedimentation rate (SR) and C-reactive protein levels (CRP). sTNFrII levels were elevated at the time of diagnosis and declined in parallel with traditional inflammation markers (SR and CRP). suPAR levels were elevated to more than double (median 7.7 ng/ml, range 5.6-25.8) compared to levels previously reported for patients with pulmonary tuberculosis. The serum suPAR levels however remained high during the entire treatment period. This may reflect that significant inflammatory activity is continuing for more than 6 months in patients with extrapulmonary mycobacterial infections, despite adequate anti-tuberculosis treatment.

182

Urease testing of mycobacteria with BACTEC radiometric instrumentation  

A total of 140 mycobacterial isolates from patients treated at Fitzsimons Army Medical Center or the National Jewish Hospital and Research Center and from animal specimens submitted to the National Veterinary Services Laboratory were tested by using a urease procedure modified for use with a BACTEC model 301. Mycobacterial suspensions were prepared by using Middlebrook 7H10 Tween broth. Of the 98 mycobacteria isolates which were urease positive utilizing standard methodology, all were positive using the radiometric procedures. Similarly, all 42 urease-negative isolates were also negative employing the new methodology. Although maximum radiometric readings were observed at 48 h, all positive strains were readily identified 24 h after inoculation without sacrificing either test sensitivity or specificity. Thus, urease testing of mycobacteria, using the modified BACTEC radiometric methodology, was as sensitive, as specific, and more rapid than conventional methods.

183

Vulvar tuberculosis  

Objective: Vulvar mycobacterial tuberculosis is a rare disease and usually occurs secondary to spread from a primary lung lesion. Case report: A 74-year-old menopausal woman presented with a history of itching and ulceration of the right labia majora for several months. Excisional biopsy of the ulceration revealed mycobacterial tuberculosis infection. Chest radiograph revealed mild peribronchial infiltration without pleural effusion. Acid-fast stain of sputum revealed acid-fast bacilli and culture was positive for mycobacterium tuberculosis. The patient was treated with a 4-drug regimen, i.e., rifampin, isoniazid, ethambutol, and pyrazinamide. Two months later, acid-fast stain of the sputum did not reveal acid-fast bacilli and culture was negative for mycobacterium tuberculosis and the vul...

184

T-Cell Responses and In Vivo Cytotoxicity in the Target Organ and the Regional Lymphoid Tissue During Airborne Infection with the Virulent Mycobacterium tuberculosis MT103 and its Lipid Mutant fadD26  

Abstract To ascertain the in vivo role of mycobacterial lipids phthiocerol dimycocerosates (PDIM) in experimental murine tuberculosis (Tb), airways infection was used to compare the parental virulent clinical isolate MT103 with its mutant fadD26, lacking PDIM. Lungs were assessed as the Tb-target organ and mediastinal lymph nodes as the corresponding lymphoid tissue, in order to quantify: the major T-cell subsets (CD4+/CD8+/gd+) and their activation kinetics, bacillary burden, and in vivo cytotoxicity against inoculated target cells loaded with mycobacterial Ags. After 4 weeks, infection augmented total and activated CD4+ and CD8+ T cells in lungs and nodes mainly with MT103, while gd+ T cells increased earlier in nodes. MT103 bacillary burden was bigger and appeared earlier than the mutan...

185

Primary immunodeficiencies of protective immunity to primary infections  

The vast majority of primary immunodeficiencies (PIDs) predispose affected individuals to recurrent or chronic infectious diseases, because they affect protective immunity to both primary and secondary or latent infections. We discuss here three recently described groups of PIDs that seem to impair immunity to primary infections without compromising immunity to secondary and latent infections. Patients with mutations in IL12B or IL12RB1 typically present mycobacterial disease in childhood with a favorable progression thereafter. Cross-protection between mycobacterial infections has even been observed. Patients with mutations in IRAK4 or MYD88 suffer from pyogenic bacterial diseases, including invasive pneumococcal diseases in particular. These diseases often recur, although not always with...

186

Non-tuberculous mycobacteria and their surface lipids efficiently induced IL-17 production in human T cells  

Interleukin-17 (IL-17) is produced by a subset of CD4+ T helper (Th) lymphocytes known as Th17 cells. In humans, IL-1b, enhanced by IL-6 and IL-23 is crucial for differentiation of these cells. IL-17 evokes inflammation and is involved in host defence against microorganisms, although little is known about its role in diseases caused by non-tuberculous mycobacteria. The genus Mycobacterium contains both obligate and opportunistic pathogens as well as saprophytes, and the mycobacterial cell envelope is unique in its abundance of lipids. Here we investigated IL-17 and IL-23 production in human PBMC in response to intact UV-inactivated mycobacteria and mycobacterial surface lipids from two opportunistic (Mycobacterium avium and Mycobacterium abscessus) and one generally non-pathogenic (Mycobac...

187

Mycobacterium avium subsp. hominissuis Infection in a Captive-Bred Kiang (Equus kiang)  

Equids are considered highly resistant to mycobacterial infections and clinical cases have been described in domestic horses only. Mycobacterium bovis is the most common species reported, although a single report exists of disease due to definitively diagnosed infection with Mycobacterium avium subsp. hominissuis in two domestic horses. This is the first report of a mycobacterial infection in a kiang (Equus kiang), or indeed any wild equid. The animal had chronic loss of condition and serum biochemical changes suggestive of liver disease and chronic infection. Further investigation showed a chronic granulomatous enteritis, lymphadenitis and hepatitis with focal granulomatous pneumonia due to systemic infection with M. avium subsp. hominissuis. The distribution and severity of the lesions s...

188

Heme oxygenase-1 promotes granuloma development and protects against dissemination of mycobacteria  

Non-tuberculous mycobacterial (NTM) infections occur in both immunocompromised and immunocompetent hosts and are an increasingly recognized cause of morbidity and mortality. The hallmark of pulmonary mycobacterial infections is the formation of granuloma in the lung. Our study focuses on the role of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in the regulation of granuloma development and maturation following infection with Mycobacterium avium. We examined the role of HO-1 in regulating monocyte chemoattractant protein-1 (MCP-1) and chemokine receptor 2 (CCR2), two molecules involved in monocyte-macrophage cell trafficking after infection. We showed that RAW 264.7 mouse monocytes exposed to M. avium expressed HO-1 and MCP-1. Inhibition of HO by zinc protoporphyrin-IX led to inhibitio...

189

Polymerase chain reaction-based molecular diagnosis of cutaneous infections in dermatopathology.  

Conventional methods, including microscopy, culture, and serologic studies, are a mainstay in the diagnosis of cutaneous infection. However, owing to limitations associated with these techniques, such as low sensitivity for standard microscopy and in the case of culture delay in diagnosis, polymerase chain-reaction based molecular techniques have taken on an expanding role in the diagnosis of infectious processes in dermatopathology. In particular, these assays are a useful adjunct in the diagnosis of cutaneous tuberculosis, atypical mycobacterial infection, leprosy, Lyme disease, syphilis, rickettsioses, leishmaniasis, and some fungal and viral infections. Already in the case of tuberculosis and atypical mycobacterial infection, standardized polymerase chain-reaction assays are commonly used for diagnostic purposes. With time, additional molecular-based techniques will decrease in cost and gain increased standardization, thus delivering rapid diagnostic confirmation for many difficult-to-diagnose cutaneous infections from standard formalin-fixed paraffin-embedded tissue specimens. PMID:23174494

190

Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages  

Summary Mycobacterium tuberculosis is an intracellular bacterium that can survive within macrophages. Such survival is potentially associated with Coronin-1a (Coro1a). We investigated the mechanism by which Coro1a promotes the survival of M.-tuberculosis in macrophages and found that autophagy was involved in the inhibition of mycobacterial survival in Coro1a knock-down (KD) macrophages. Fluorescence microscopy and immunoblot analyses revealed that LC3, a representative autophagic protein, was recruited to M.-tuberculosis-containing phagosomes in Coro1a KD macrophages. Thin-section electron microscopy demonstrated that bacilli were surrounded by the multiple membrane structures in Coro1a KD macrophages. The proportion of LC3-positive mycobacterial phagosomes colocalized with p62/SQSTM1, ub...

191

An ArsR-like transcriptional factor recognizes a conserved sequence motif and positively regulates the expression of phoP in mycobacteria  

Transcriptional regulation plays a critical role during the infection of Mycobacterium tuberculosis, the causative agent of tuberculosis. A two-component system, PhoPR, is clearly involved in the regulation of pathogenic virulence and persistence. However, the regulatory mechanism, as well as the regulator, of the phoPR operon remains uncharacterized in M. tuberculosis and its related species thus far. In the present study, we characterize an ArsR transcriptional factor, corresponding to Rv2034 and Ms6762 in M. tuberculosis and Mycobacterium smegmatis, respectively, as the first regulator of phoP in both mycobacterial species. The interaction between ArsR regulator and target promoters is conserved in these two mycobacterial species, and an inverted repeat sequence motif is successfully ma...

192

Synthesis of Deoxygenated (? 1?5)-Linked Arabinofuranose Disaccharides as Substrates and Inhibitors of Arabinosyltransferases of Mycobacterium tuberculosis  

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf ?(1?5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The ?(1?5)-linked disaccharides were produced by coupling n-octyl ?-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H37Ra and M. avium complex strains.

193

Origin, Spread and Demography of the Mycobacterium tuberculosis Complex  

The evolutionary timing and spread of the Mycobacterium tuberculosis complex (MTBC), one of the most successful groups of bacterial pathogens, remains largely unknown. Here, using mycobacterial tandem repeat sequences as genetic markers, we show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. Using Bayesian statistics and experimental data on the variability of the mycobacterial markers in infected patients, we estimated the age of the MTBC at 40,000 years, coinciding with the expansion of “modern” human populations out of Africa. Furthermore, coalescence analysis revealed a strong and recent demographic expansion in almost all M. tuberculosis lineages, which coincides with the human population explosion over the last two centuries. These findings thus unveil the dynamic dimension of the association between human host and pathogen populations.

194

Neamphamide B, new cyclic depsipeptide, as an anti-dormant mycobacterial substance from a Japanese marine sponge of Neamphius sp.  

A new cyclic depsipeptide, designated neamphamide B (1), was isolated from a marine sponge of Neamphius sp. collected at Okinawa, Japan in 1993 as an anti-mycobacterial substance against active and dormant bacilli. The planar structure of neamphamide B (1) was determined on the basis of spectroscopic analysis, and stereostructure of amino acid was deduced by chromatographic comparison of the acid hydrolysate of 1 with appropriate amino acid standards after derivatizing with FDAA or GITC. Neamphamide B (1) showed potent anti-mycobacterial activity against Mycobacterium smegmatis under standard aerobic growth conditions as well as dormancy-inducing hypoxic conditions with MIC of 1.56mg/mL. Neamphamide B (1) was also effective to Mycobacterium bovis BCG with MIC in the ranging of 6.25-12.5mg/...

195

Primary cutaneous anaplastic large cell lymphoma successfully treated with local thermotherapy using pocket hand warmers  

ABSTRACT Apart from for cutaneous deep fungal or mycobacterial infections, thermotherapy has been used for various malignant tumors. We report a case of primary cutaneous anaplastic large cell lymphoma, which responded quite well to topical thermotherapy using chemical pocket hand warmers. The treatment resulted in an immediate tumor regression without recurrence. This method is simple and might be a useful tool against solitary cutaneous lymphoma, especially of elderly patients with poor performance status or with various systemic complications.

196

Phosphatidylinositol synthesis in mycobacteria.  

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M. smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [3H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [3H]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [3H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy. PMID:9989274

197

Stereoselective syntheses of heptaprenylphosphoryl b-d-arabino-and b-d-ribo-furanoses  

The stereoselective syntheses of heptaprenylphosphoryl b-d-arabinofuranose and heptaprenylphosphoryl b-d-ribofuranose are described. In the synthesis of the d-arabino product, the stereoselectivity was achieved by the coupling of a suitably protected b-d-arabinofuranosyl phosphate intermediate with an activated form of heptaprenol and subsequent deprotection. In the case of the ribo-analog, the desired b-anomer could be obtained by the more convenient phosphoramidite method. The products were successfully employed in the mycobacterial epimerase assay.

198

Prurigo nodularis due to Mycobacterium tuberculosis.  

Prurigo nodularis (PN) is a rare chronic skin disorder of unknown origin. Here we describe what is believed to be the first case of PN associated with tuberculosis. For the first time, culture and PCR analysis of skin biopsy confirmed the presence of Mycobacterium tuberculosis complex in PN skin lesions. The pruritus and skin lesions resolved following antitubercular therapy. Our case provides further evidence in favour of a link between PN and mycobacterial infection. PMID:19661207

199

Specimen contamination in mycobacteriology laboratory detected by pseudo-outbreak of multidrug-resistant tuberculosis: analysis by routine epidemiology and confirmation by molecular technique.  

A cluster of multidrug-resistant tuberculosis sputum isolates led to the detection of specimen contamination in a hospital mycobacteriology laboratory. Thirteen specimens were smear negative but culture positive from one specimen only; 12 appeared to be contaminated. Each of these specimens was processed in the same batch as one or more smear- and culture-positive isolates. Molecular analysis confirmed the traditional epidemiologic, laboratory, and clinical methods of evaluating presumed mycobacterial contamination. PMID:8815074

200

Bacillus Calmette-Guérin (BCG) complications associated with primary immunodeficiency diseases.  

Primary immunodeficiency diseases (PIDs) are a group of inherited disorders, characterized by defects of the immune system predisposing individuals to variety of manifestations, including recurrent infections and unusual vaccine complications. There are a number of PIDs prone to Bacillus Calmette-Guérin (BCG) complications. This review presents an update on our understanding about the BCGosis-susceptible PIDs, including severe combined immunodeficiency, chronic granulomatous disease, and Mendelian susceptibility to mycobacterial diseases. PMID:22430715

 
 
 
 
201

Multiple strains of Mycobacterium bovis revealed by molecular typing in a herd of cattle  

Mycobacterium bovis isolates from an outbreak of bovine tuberculosis in a herd of cattle in Rio de Janeiro, Brazil, were analysed by spoligotyping and variable-number tandem repeat PCR analysis of the mycobacterial interspersed repetitive unit and exact tandem repeats. Molecular typing revealed a high genetic diversity of strains in the herd. The genetic diversity could be explained by the introduction of infected animals from different sources.

202

Anthelmintic avermectins kill M. tuberculosis, including multidrug resistant clinical strains.  

Avermectins are a family of macrolides known for their anthelmintic activities and traditionally believed to be inactive against all bacteria. Here we report that members of the family, ivermectin, selamectin and moxidectin, are bactericidal against mycobacterial species, including multi- and extensively-drug resistant clinical strains of Mycobacterium tuberculosis. Avermectins are approved for clinical and veterinary uses and have documented pharmacokinetic and safety profiles. We suggest that avermectins could be repurposed for tuberculosis treatment. PMID:23165468

203

Disseminated BCG infection in a patient with severe combined immunodeficiency  

Disseminated mycobacterial infection after bacillus Calmette-Guerin (BCG) accination is a very rare disorder, occurring mostly in patients with immunologic eficiency. We report a case of disseminated BCG infection in a 16-month-old girl with severe combined immunodeficiency. Plain radiographs showed multiple osteolytic lesions in the femora, tibiae, humerus, and phalanges. Abdominal sonography and CT scanning revealed multiple nodules in the spleen, and portocaval lymphadenopathy.

204

Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals  

HIV is the greatest single risk factor for the development of tuberculosis. Diseases caused by M. tuberculosis and mycobacteria are the most common opportunistic infections in HIV-infected persons, which may stem from a functional defect of the CD4+ T-cell-mediated killing of macrophages harboring mycobacteria. Our objective was to investigate the M.tuberculosis-and M. avium-specific cytotoxic capacity of T cells from healthy, bacille Calmette-Guérin-vaccinated, HIV-seropositive individuals. Blood mononuclear cells were obtained from 10 healthy HIV-seropositive and 10 healthy seronegative persons with no history of previous or active mycobacterial infection. Antigen-specific killing of macrophages presenting mycobacterial antigens (purified protein derivative or M. avium culture filtrate) was conducted. The phenotype of the killer cells was determined by a fluorescence-activated cell sorter after antigen stimulation and by using purified CD4+ and CD8+ cell subsets. Substantial, but reduced antigen-specific cytotoxicity was observed in patients with asymptomatic HIV infection. The immunological dysfunction leading to reduced cytotoxic activity in healthy HIV-seropositive subjects could not be explained by a defect in the cytotoxic capacity of the individual CD4+ lymphocyte after antigen stimulation, and it could not be explained by a reduction in the total number of CD4+ cells before antigen stimulation. The antigen-specific cytotoxic activity was, however, closely related to the ability of the CD4+ T cells to respond to mycobacterial antigens. The immunological dysfunction leading to reduced mycobacterial-specific cytotoxic activity in healthy HIV-seropositive subjects is caused either by a reduction in the number of antigen-responsive CD4+ T cells (memory) or by an impairment of their ability to respond to antigenic stimuli.

205

Estudo comparativo entre um sistema de sonda genética e métodos clássicos na identificação das micobactérias/ Gene probes versus classical methods in the identification of mycobacteria  

Abstract in portuguese OBJETIVO: O aparecimento da co-infecção tuberculose/HIV e o aumento de casos de doenças provocadas por micobactérias não-tuberculosas (MNT) exigem repostas laboratoriais rápidas tanto no isolamento como na identificação das micobactérias. O objetivo deste trabalho foi avaliar a identificação das micobactérias através de sonda genética em comparação com os métodos bioquímicos clássicos. MÉTODOS: Entre 2002 e 2004, foram analisadas 178 culturas de micoba (more) ctérias, confirmadas como bacilos álcool-ácido resistentes e obtidas de isolados clínicos de pacientes sintomáticos respiratórios ou com suspeita clínica de tuberculose pulmonar e/ou micobacterioses, atendidos nas Unidades de Saúde da Baixada Santista. RESULTADOS: A sonda genética identificou 137 amostras (77%) como complexo Mycobacterium tuberculosis e 41 (23%) como MNT. A discordância observada de 3% entre os métodos ocorreu apenas no ano de implantação (2002). Ao comparar os métodos, a sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo da sonda genética foram 98%, 93%, 98% e 93%, respectivamente. CONCLUSÕES: Apesar do custo elevado, a identificação de micobactérias pela técnica molecular é mais rápida: máximo de 3 h vs. 28-30 dias para os métodos clássicos. A utilização de sondas genéticas é uma técnica molecular validada, simples e disponível no mercado, com elevada especificidade, sensibilidade e rapidez, o que justifica sua implantação e uso rotineiro em laboratórios de referência, facilitando o diagnóstico e permitindo uma intervenção clínica ágil. Abstract in english OBJECTIVE: The emergence of tuberculosis/HIV co-infection and the increase in the number of cases of infection with nontuberculous mycobacteria (NTM) require rapid laboratory test results in the isolation and identification of mycobacteria. The objective of this study was to evaluate the identification of mycobacteria by means of gene probes in comparison with that obtained using classical biochemical methods. METHODS: Between 2002 and 2004, 178 mycobacterial cultures, al (more) l testing positive for acid-fast bacilli, were analyzed. Samples were obtained from clinical specimens of patients with respiratory symptoms or with clinical suspicion of pulmonary tuberculosis/mycobacteriosis who were treated in the greater metropolitan area of Santos. RESULTS: The gene probe identified 137 samples (77%) as Mycobacterium tuberculosis complex and 41 (23%) as NTM. Discordant results between the methods (3%) were obtained only in the year of implementation (2002). When comparing the methods, the sensitivity, specificity, positive predictive value and negative predictive value of the gene probe method were 98%, 93%, 98% and 93%, respectively. CONCLUSIONS: Despite the cost, the identification of mycobacteria using the molecular technique is faster: maximum 3 h vs. 28-30 days for classical methods. The use of gene probes is a validated molecular technique. It is fast, easy to use and readily available on the market. It has high specificity and sensitivity, which justifies its implementation and routine use in referral laboratories, since it facilitates the diagnosis providing agile clinical interventions.

206

Biochemical and genetic analysis of the four DNA ligases of mycobacteria.  

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining. PMID:14985346

207

Interferon-gamma independent formation of pulmonary granuloma in mice by injections with trehalose dimycolate (cord factor), lipoarabinomannan and phosphatidylinositol mannosides isolated from Mycobacterium tuberculosis.  

The mechanisms by which pulmonary granuloma formation is caused by administration of mycobacterial glycolipids such as trehalose dimycolate (TDM), lipoarabinomannan (LAM) and phosphatidylinositol mannosides (PIM) were investigated. When peritoneal and alveolar macrophages were stimulated with TDM, LAM and PIM in vitro, TDM exhibited the strongest tumour necrosis factor (TNF)-inducing activity. Responsiveness of macrophages from mice defected Toll-like receptor 4 (TLR4) was much higher than that of the wild-type mice. Although PIM and LAM also had a significant activity, LAM rather than PIM stimulated higher TNF-alpha production by alveolar macrophage. When mycobacterial glycolipids were injected as water-in-oil-in-water emulsion into mice via the tail vein, development of pulmonary granuloma in response to glycolipids were related closely to their TNF-inducing activity and TDM exhibited the strongest activity. Granuloma formation was observed not only in mice lacking interleukin (IL)-12 signalling but also interferon (IFN)-gamma knock-out mice. Granuloma formation caused by glycolipids correlated with TNF-alpha levels in lungs. Administration of anti-TNF-alpha monoclonal antibody into TDM-injected IFN-gamma knock-out mice decreased in granuloma formation, suggesting that development of pulmonary granuloma by mycobacterial glycolipids such as TDM is due to IFN-gamma-independent and TNF-alpha-dependent pathway. PMID:16542375

208

In vivo induction of apoptosis in the thymus by administration of mycobacterial cord factor (trehalose 6,6'-dimycolate).  

It is reported that some bacteria or bacterial components cause thymic atrophy via the apoptotic process. The present study demonstrated for the first time in vivo induction of apoptosis in the mouse thymus by mycobacterial cord factor (CF) (trehalose 6,6'-dimycolate). When 300 microg of purified CF from Mycobacterium tuberculosis was intravenously administered to BALB/c mice in the form of water-in-oil-in-water (w/o/w) emulsion, thymic atrophy and pulmonary granulomas were induced with a peak on day 7, whereas, in the form of liposomes, CF induced thymic atrophy on days 14 to 21 in parallel with the development of hepatic granulomas. Thymic atrophy resulted from the depletion of cortical lymphocytes via apoptosis as revealed by DNA fragmentation and karyorrhectic changes. In contrast, mycobacterial sulfatide (2,3,6,6'-tetraacyl trehalose 2'-sulfate) caused neither thymic atrophy nor granuloma formation. Compared to lipopolysaccharide-induced thymocyte apoptosis, CF (w/o/w)-induced thymocyte apoptosis developed more slowly, reached a maximum later, and lasted longer but was less intense. Although serum tumor necrosis factor alpha (TNF-alpha) levels in CF-treated mice were not significantly elevated, administration of anti-TNF-alpha antibody almost completely inhibited thymic atrophy and granuloma formation. Serum corticosterone levels were only slightly elevated by CF administration. The present results indicate that mycobacterial CF induces thymic atrophy via apoptosis, which is closely linked with granuloma formation. PMID:9125563

209

Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles.  

Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 microl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples. PMID:19539667

210

T-cell responses and in vivo cytotoxicity in the target organ and the regional lymphoid tissue during airborne infection with the virulent Mycobacterium tuberculosis MT103 and its lipid mutant fadD26.  

To ascertain the in vivo role of mycobacterial lipids phthiocerol dimycocerosates (PDIM) in experimental murine tuberculosis (Tb), airways infection was used to compare the parental virulent clinical isolate MT103 with its mutant fadD26, lacking PDIM. Lungs were assessed as the Tb-target organ and mediastinal lymph nodes as the corresponding lymphoid tissue, in order to quantify: the major T-cell subsets (CD4+/CD8+/gammadelta+) and their activation kinetics, bacillary burden, and in vivo cytotoxicity against inoculated target cells loaded with mycobacterial Ags. After 4 weeks, infection augmented total and activated CD4+ and CD8+ T cells in lungs and nodes mainly with MT103, while gammadelta+ T cells increased earlier in nodes. MT103 bacillary burden was bigger and appeared earlier than the mutant fadD26, especially in the lung than in mediastinal nodes. At day 14 of MT103 infection, there was no cytotoxicity in lungs and nodes; while with fadD26 there was some in the nodes. At day 21 of MT103 infection, important cytotoxicity was detected only in lungs; while with fadD26 both tissues showed important activity. Interestingly, unlike the infection with fadD26, cytotoxicity under MT103 fell considerably in the target organ (lung) from days 21 to 60, the advanced phase. Although upon airways infection both mycobacteria behaved similarly regarding T cell (CD4/CD8/gammadelta) stimulation kinetics; they differed in the magnitude of these responses, in the bacterial load within tissues, and to trigger in vivo cytotoxicity in lungs and regional lymph nodes. This highlights the relevance of certain mycobacterial lipids to modify crucial effector branches of immunity. PMID:20017806

211

Lingual ulcer as the only sign of recurrent mycobacterial infection in an HIV/AIDS-infected patient.  

The report describes an HIV/AIDS patient seen at a referral center in Mexico City, in whom a mycobacterial infection in the oral mucosa, probably tuberculosis (TB) was identified. The purpose is to describe the clinical and histological findings in an HIV-infected patient, who after being treated successfully for tuberculous lymphangitis 4 years ago, presented with a lingual ulcer as the only suggestive sign of recurrence of mycobacterial infection, probably M. tuberculosis. A 39-year-old man seen in the HIV clinic of the Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán" in Mexico City since 1991 for HIV infection. In 1999 the patient developed tuberculous lymphangitis; he was managed with a 4-drug regimen for 12 months, with improvement of local and systemic symptoms. In May of 2003, the patient presented a painful superficial lingual ulcer, 0.7 cm in diameter, well circumscribed, crateriform with slightly elevated, irregular and indurated borders, of 4 months duration. The histopathological examination showed chronic granulomatous inflammation with giant multinucleated cells, suggestive of mycobacterial infection, and recurrence of TB was considered. Rifampin, isoniazide, pyrazinamide, ethambutol and streptomycin were administered. The lingual lesion improved with partial healing at the first week and total remission at 45 days after the beginning of the antituberculous treatment. In June, 2003, the patient began highly active antiretroviral therapy (HAART) that included two NRTIs and one NNRTI. At 7 months of follow-up, the patient remains free of lingual lesions. The particularity of the present case is that the lingual ulcer was the only sign of infection by mycobacteria, suggestive of TB, in an HIV/AIDS patient that probably represented a recurrence of a previous episode. PMID:15735542

212

The reliability of diagnostic coding and laboratory data to identify tuberculosis and nontuberculous mycobacterial disease among rheumatoid arthritis patients using anti-tumor necrosis factor therapy  

AbstractPurpose Anti-tumor necrosis factor-alpha (anti-TNF) therapies are associated with severe mycobacterial infections in rheumatoid arthritis patients. We developed and validated electronic record search algorithms for these serious infections. Methods The study used electronic clinical, microbiologic, and pharmacy records from Kaiser Permanente Northern California (KPNC) and the Portland Veterans Affairs Medical Center (PVAMC). We identified suspect tuberculosis and nontuberculous mycobacteria (NTM) cases using inpatient and outpatient diagnostic codes, culture results, and anti-tuberculous medication dispensing. We manually reviewed records to validate our case-finding algorithms. Results We identified 64 tuberculosis and 367 NTM potential cases, respectively. For tuberculosis, diagn...

213

Melioidosis: epidemiology, pathophysiology, and management.  

Background Rhomboids are ubiquitous proteins with diverse functions in all life kingdoms, and are emerging as important factors in the biology of some pathogenic apicomplexa and Providencia stuartii. Although prokaryotic genomes contain one rhomboid, actinobacteria can have two or more copies whose sequences have not been analyzed for the presence putative rhomboid catalytic signatures. We report detailed phylogenetic and genomic analyses devoted to prokaryotic rhomboids of an important genus, Mycobacterium. Results Many mycobacterial genomes contained two phylogenetically distinct active rhomboids orthologous to Rv0110 (rhomboid protease 1) and Rv1337 (rhomboid protease 2) of Mycobacterium tuberculosis H37Rv, which were acquired independently. There was a genome-wide conservation and organization of the orthologs of Rv1337 arranged in proximity with glutamate racemase (mur1), while the orthologs of Rv0110 appeared evolutionary unstable and were lost in Mycobacterium leprae and the Mycobacterium avium complex. The orthologs of Rv0110 clustered with eukaryotic rhomboids and contained eukaryotic motifs, suggesting a possible common lineage. A novel nonsense mutation at the Trp73 codon split the rhomboid of Mycobacterium avium subsp. Paratuberculosis into two hypothetical proteins (MAP2425c and MAP2426c) that are identical to MAV_1554 of Mycobacterium avium. Mycobacterial rhomboids contain putative rhomboid catalytic signatures, with the protease active site stabilized by Phenylalanine. The topology and transmembrane helices of the Rv0110 orthologs were similar to those of eukaryotic secretase rhomboids, while those of Rv1337 orthologs were unique. Transcription assays indicated that both mycobacterial rhomboids are possibly expressed. Conclusions Mycobacterial rhomboids are active rhomboid proteases with different evolutionary history. The Rv0110 (rhomboid protease 1) orthologs represent prokaryotic rhomboids whose progenitor may be the ancestors of eukaryotic rhomboids. The Rv1337 (rhomboid protease 2) orthologs appear more stable and are conserved nearly in all mycobacteria, possibly alluding to their importance in mycobacteria. MAP2425c and MAP2426c provide the first evidence for a split homologous rhomboid, contrasting whole orthologs of genetically related species. Although valuable insights to the roles of rhomboids are provided, the data herein only lays a foundation for future investigations for the roles of rhomboids in mycobacteria. PMID:15831829

214

A hybrid multi-compartment model of granuloma formation and T cell priming in Tuberculosis  

Tuberculosis is a worldwide health problem with 2 billion people infected with Mycobacterium tuberculosis (Mtb, the bacteria causing TB). The hallmark of infection is the emergence of organized structures of immune cells forming primarily in the lung in response to infection. Granulomas physically contain and immunologically restrain bacteria that cannot be cleared. We have developed several models that spatially characterize the dynamics of the host–mycobacterial interaction, and identified mechanisms that control granuloma formation and development. In particular, we published several agent-based models (ABMs) of granuloma formation in TB that include many subtypes of T cell populations, macrophages as well as key cytokine and chemokine effector molecules. These ABM studies empha...

215

Three Cases of Fungemia in HIV-Infected Patients Diagnosed Through the Use of Mycobacterial Blood Culture Bottles  

We treated three cases of fungemia in HIV-infected patients. These cases were caused by Candida albicans, Cryptococcus neoformans, and Penicillium marneffei, respectively, and all were diagnosed through the use of mycobacterial blood culture bottles. Although the detection of the etiologic agents of fungal infection is difficult, it has been shown that blood culture media for mycobacteria are more effective for the detection of fungemia than media for aerobes and anaerobes. Some reports have shown that Bactec Myco/F lytic bottles were useful for the diagnosis of fungemia in clinical samples. Here, we report the successful use of BacT MB bottles.   

216

SITVITWEB - A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology  

Among various genotyping methods to study Mycobacterium tuberculosis complex (MTC) genotypic polymorphism, spoligotyping and mycobacterial interspersed repetitive units-variable number of DNA tandem repeats (MIRU-VNTRs) have recently gained international approval as robust, fast, and reproducible typing methods generating data in a portable format. Spoligotyping constituted the backbone of a publicly available database SpolDB4 released in 2006; nonetheless this method possesses a low discriminatory power when used alone and should be ideally used in conjunction with a second typing method such as MIRU-VNTRs for high-resolution epidemiological studies. We hereby describe a publicly available international database named SITVITWEB which incorporates such multimarker data allowing to have a g...

217

Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system.  

The importance of non-tuberculosis mycobacterial biofilm species in medicine, industry and the environment has recently gained attention. Our objectives were to characterize biofilm growth of Mycobacterium phlei M4, as a model of rapidly growing mycobacteria using the minimal biofilm eradication concentration (MBEC) and to compare biocide susceptibility of planktonic and biofilm organisms. Scanning electron microscopy was also carried out to observe biofilm morphology. With the exception of Sporicidin and Virkon the minimum bactericidal concentration values for all biocides tested were lower than the MBEC values. The MBEC assay system was seen to produce multiple and reproducible biofilms of M. phlei and to be a useful tool for susceptibility studies. PMID:11583858

218

Tuberculosis and leprosy in perspective  

Two of humankinds most socially and psychologically devastating diseases, tuberculosis and leprosy, have been the subject of intensive paleopathological research due to their antiquity, a presumed association with human settlement and subsistence patterns, and their propensity to leave characteristic lesions on skeletal and mummified remains. Despite a long history of medical research and the development of effective chemotherapy, these diseases remain global health threats even in the 21st century, and as such, their causative agents Mycobacterium tuberculosis and M. leprae, respectively, have recently been the subject of molecular genetics research. The new genome-level data for several mycobacterial species have informed extensive phylogenetic analyses that call into question previously...

219

Extensive cortical involvement in leptomeningeal carcinomatosis  

We present a 77-year-old previously well patient with facial asymmetry and progressive weakness of the lower extremities. An initial MRI revealed slight contrast enhancement of the meninges. Three consecutive cerebrospinal fluid examinations demonstrated low glucose concentration, marked elevation of total protein and moderate pleocytosis. No tumor cells, fungi, acid-fast bacilli or mycobacterial DNA were found. The patient's level of consciousness deteriorated dramatically, and follow-up MRI showed widespread extensive cortical hyperintensities. The lesions showed restricted diffusion on diffusion-weighted images as well as low values on the corresponding apparent diffusion coefficient maps, the changes consistent with diffuse cytotoxic edema. Neuropathological examination findings were o...

220

Acquired Aplastic Anemia After Nosocomial Serratia marcescens Bone Marrow Infection in an Elderly Patient  

A 76-year-old female patient with a multiple medical history developed refractory pancytopenia during treatment for peptic ulcer, bacteremia, pneumonia, and respiratory failure. Bone marrow biopsy revealed hypocellularity with few hematopoietic cells, which was indicative of aplastic anemia. Common causes including viral infection, mycobacterial infection, environmental exposure, congenital bone marrow disorder, and hematologic malignancy were primarily excluded. Identical Serratia marcescens cultures were isolated from the central venous catheter tip and bone marrow, and the pancytopenia improved gradually with proper antibiotics treatment. This case indicates that nosocomial S. marcescens infection may cause potentially fatal acquired aplastic anemia. Crucial issues in avoiding aplastic ...

 
 
 
 
221

Synthesis and in vivo biological activity of large-ringed calixarenes against Mycobacterium tuberculosis  

A series of large-ringed calix[6,7,8]arene analogues have been synthesised and their affect against Mycobacterium tuberculosis in vivo established. In general, when p-phenylcalixarenes and tert-butylcalixarenes were not functionalised at the lower rim, low biological activities were observed. However on going from partially to fully lower rim pegylated calixarenes the anti-mycobacterial properties improved. The addition of cyanopropoxy groups at the lower rim gave rise to low activities, whereas the addition of acetate moieties interestingly had pro-TB effects. Two upper rim sulfonated calixarenes showed promising properties. In the course of this work, a high yielding procedure to synthesise p-phenylcalix[7]arene was also established.

222

Mycobacterium abscessus activates the NLRP3 inflammasome via Dectin-1–Syk and p62/SQSTM1  

Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin-1/Syk-dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin-1 and Toll-like receptor 2 (TLR2) were required for Mabc-induced mRNA expression of pro-interleukin (IL)-1?, cath...

223

Mycobacterium simiae infection in a patient with Acquired Immunodeficiency Syndrome  

Abstract in english Mycobacterium simiae is usually an environmental contaminant rarely associated with human disease. We report a fatal case of M.simiae infection in a 37 year old, HIV positive, male from whom the organism was isolated from blood culture. The identification of M.simiae was performed using DNA amplification followed by analysis on 3% agarose gel of the amplicon fragments after digestion by restriction endonucleases. The precise identification of mycobacterial isolates to the species level is important, with both epidemiological and therapeutic implications.

224

Lower production of IL-17A and increased susceptibility to Mycobacterium bovis in mice coinfected with Strongyloides venezuelensis  

Abstract in english The presence of intestinal helminths can down-regulate the immune response required to control mycobacterial infection. BALB/c mice infected with Mycobacterium bovis following an infection with the intestinal helminth Strongyloides venezuelensis showed reduced interleukin-17A production by lung cells and increased bacterial burden. Also, small granulomas and a high accumulation of cells expressing the inhibitory molecule CTLA-4 were observed in the lung. These data sugges (more) t that intestinal helminth infection could have a detrimental effect on the control of tuberculosis (TB) and render coinfected individuals more susceptible to the development of TB.

225

Localization of Propionibacterium acnes in granulomas supports a possible etiologic link between sarcoidosis and the bacterium  

Sarcoidosis likely results from the exposure of a genetically susceptible subject to an environmental agent, possibly an infectious one. Mycobacterial and propionibacterial organisms are the most commonly implicated potential etiologic agents. Propionibacterium acnes is the only microorganism, however, found in sarcoid lesions by bacterial culture. To evaluate the pathogenic role of this indigenous bacterium, we screened for the bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger-factor protein (TIG antibody). We examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarco...

226

Photodynamic therapy as an adjunct to systemic treatment in a case with unilateral presumed vascularized choroidal tuberculous granuloma  

Intraocular tuberculosis cannot be diagnosed easily in some clinical circumstances. A 29-year-old otherwise healthy woman who was initially misdiagnosed and treated empirically with systemic steroids and sulfamethoxazole?trimethoprim for toxoplasmosis was referred to us for severe visual loss. We instituted quadruple antituberculosis treatment upon chest medicine consultation as all attempts, including consecutive intravitreal antibiotic injections, could not prevent the enlargement of lesion. Systemic antituberculosis treatment halted the fulminant course but the granuloma became vascularized. Because photodynamic therapy (PDT) has very recently been shown to reduce viable mycobacterial cells in animal experiments we performed PDT, and the vascularized tuberculous granuloma was successful...

227

Specificity of the tuberculin skin test is modified by use of a protein cocktail containing ESAT-6 and CFP-10 in cattle naturally infected with Mycobacterium bovis.  

The mycobacterial immunodominant ESAT-6 and CFP-10 antigens are strongly recognizable in tuberculosis-infected cattle, and they do not elicit a response in cattle without infection. In addition, they are absent in most environmental mycobacterial species, and therefore, their use can be an alternative to purified protein derivative (PPD) tuberculin in the development of a more specific skin diagnostic test in cattle. The aim of the current study was to assess the potential of an ESAT-6 and CFP-10 (E6-C10) protein cocktail in a skin test format in naturally tuberculosis-infected and paratuberculosis-infected cattle. We also included MPB83 as a third component in one of the protein cocktail preparations. The protein cocktail was tested at different dose concentrations (5, 10, and 15 ?g per protein). The best skin response to the E6-C10 protein cocktail was obtained with 10 ?g. Subsequently, this concentration was tested in 2 herds with high and low bovine tuberculosis prevalence, the latter with paratuberculosis coinfection. Our data show that the E6-C10 cocktail allows identification of an important proportion of animals that PPDB is not able to recognize, especially in low-prevalence herds. The protein cocktail did not induce reactions in tuberculosis-free cattle or in paratuberculosis-infected cattle. Addition of MPB83 to the protein cocktail did not make any difference in the skin reaction. PMID:22419675

228

Identification of mycobacteria in peat moss processing plants : application of molecular biology approaches  

Health concerns regarding environmental mycobacteria has led to the development of exposure assessment methods for the evaluation of certain workplaces where the presence of these agents is suspected. Hypersensitivity pneumonitis (HP) has been described in peat moss workers who are regularly exposed to significant levels of bioaerosols in peat moss processing plants. Although mycobacteria have been cultured from peat moss, plant workers exposure to mycobacterial bioaerosols has never been studied. This article presented the results of a study that evaluated the presence of mycobacteria in air samples from peat moss processing plants using molecular biology approaches (cloning-sequencing and polymerase chain reaction (PCR)) and the workers exposure using immunoglobin G (IgG) complexes to mycobacteria. It also compared species detected in air samples and in peat moss. Two peat moss processing plants were chosen among 14 previously studied and a total of 49 clones were sequenced. Real-time PCR was also performed on the same air samples to evaluate the airborne concentration of mycobacteria and estimate exposure levels. The article discussed the materials and methods used in the study, the results of the study, and subsequent discussion of the results. It was concluded that peat moss processing plants workers are exposed to mycobacteria in addition to other biological agents. It was suggested that further studies are needed to confirm the specificity of the mycobacterial IgG. 34 refs., 2 tabs., 1 fig.

229

Chronic Necrotizing Pulmonary Aspergillosis Complicated by a Cavitary Lesion Caused by Pulmonary Mycobacterium-avium Complex Disease  

A 66-year-old woman who had undergone one year?s treatment for pulmonary nontuberculous mycobacterial disease due to Mycobacterium avium (rifampicin, ethambutol, clarithromycin, streptomycin?levofloxacin) five years earlier was admitted to our hospital because of continuous fever and a newly detected abnormal chest shadow, which was like a fungus ball in the right upper lobe on chest computed tomography in the giant cavitary lesion caused by pulmonary Mycobacterium-avium complex (MAC) disease. A diagnosis of chronic necrotizing pulmonary aspergillosis (CNPA) complicated by pulmonary MAC disease was made because Aspergillus niger was isolated from several sputum specimens, anti-aspergillus antibody was positive, and clinical symptoms such as fever, were disclosed with the radiological finding of a fungus ball-like shadow and an infiltration shadow around the cavity. The patient had received various forms of antifungal chemotherapy, but the clinical effect had been poor. Since then, she had been slowly worsening. Although mycetomas, with the typical appearance of a fungus ball on a chest radiograph, have been reported to easily form in cavitary lesions caused by previous pulmonary tuberculosis, we believe, as illustrated by the present case, that they could also form in such lesions caused by pulmonary MAC disease, since the frequency of pulmonary nontuberculous mycobacterial disease has recently been increasing in comparison with that of pulmonary tuberculosis.   

230

Antibacterial activity of Aristolochia brevipes against multidrug-resistant Mycobacterium tuberculosis.  

The increased incidence of Multidrug-Resistant Mycobacterium tuberculosis (MDR-MT) requires the search for alternative antimycobacterial drugs. The main aim of this study was to evaluate the dichloromethane extract from Aristolochia brevipes (Rhizoma) and the compounds isolated from this extract against several mycobacterial strains, sensitive, resistant (monoresistant), and clinical isolates (multidrug-resistant), using the alamarBlue™ microassay. The extract was fractionated by column chromatography, yielding the following eight major compounds: (1) 6?-7-dehydro-N-formylnornantenine; (2) E/Z-N-formylnornantenine; (3) 7,9-dimethoxytariacuripyrone; (4) 9-methoxy-tariacuripyrone; (5) aristololactam I; (6) ?-sitosterol; (7) stigmasterol; and (8) 3-hydroxy-?-terpineol. The structures of these compounds were elucidated by 1H- and 13C- (1D and 2D) Nuclear Magnetic Resonance (NMR) spectroscopy. This study demonstrates that the dichloromethane extract (rhizome) of A. brevipes possesses strong in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (Minimum Inhibitory Concentration value [MIC], 12.5 µg/mL). The most active compound against all mycobacterial strains tested was the compound aristolactam I (5), with MIC values ranging between 12.5 and 25 µg/mL. To our knowledge, this the first report of antimycobacterial activity in this plant. PMID:21876482

231

Dissecting memory T cell responses to TB: concerns using adoptive transfer into immunodeficient mice.  

Several studies have used adoptive transfer of purified T cell subsets into immunodeficient mice to determine the subset of T cells responsible for mediating protection against Mycobacterium tuberculosis. These studies suggested that CD62L(hi) memory CD4(+) T cells from BCG-vaccinated mice are key for protection against tuberculosis. Importantly, we observed that transfer of naïve CD4(+) T cells into Rag1-/- recipients protected against a mycobacterial challenge as well as transfer of BCG-experienced CD4(+) T cells. We found that transfer of total CD4(+) T cells from naïve mice or enriched CD62L(hi)CD4(+) T cells from BCG-vaccinated mice into Rag1-/- recipients induced severe colitis by 3 weeks post cell transfer, whereas transfer of CD62L(lo)CD4(+) T cells from BCG-vaccinated mice did not. Naïve and CD62L(hi)CD4(+) T cells proliferated extensively upon transfer and developed an activated effector phenotype in the lung, even in the absence of infectious challenge. The induction of colitis and systemic cytokine response induced by the transfer and subsequent activation of CD4(+) T cells from naïve mice or CD62L(hi)CD4(+) T cells from BCG-vaccinated mice, into immunodeficient recipients, may heighten their ability to protect against mycobacterial challenge. This raises doubts about the validity of this model to study CD4(+) T cell-mediated protection against tuberculosis. PMID:22738879

232

Antibody Responses to Mycobacterial Antigens in Childhood Tuberculosis: Challenges and Potential Diagnostic Value.  

The identification of easily detectable biomarkers for active tuberculosis (TB) is a global health priority. Such biomarkers would be of particular value in childhood TB, which poses greater diagnostic challenges than adult TB. Serum antibodies can be detected by simple formats that provide extremely rapid results. However, attempts to develop accurate serodiagnostic tests for TB have been unsuccessful. Whereas antibody responses to mycobacterial antigens in adult TB have been studied extensively and reviewed, the same cannot be said for serologic data in pediatric populations. Here we appraise studies on serological responses in childhood TB and discuss findings and limitations in the context of the developing immune system, the age range, and the spectrum of TB manifestations. We found that the antibody responses to mycobacterial antigens in childhood TB can vary widely with sensitivities and specificities ranging from 14% to 85% and from 86% to 100%, respectively. We conclude that the limitations in serodiagnostic studies of childhood TB are manifold thereby restricting the interpretation of currently available data. Concerns about the methodology used in published studies suggest that conclusions about the eventual value of serodiagnosis cannot be made at this time. However, the available data suggest a potential adjunctive value for serology in the diagnosis of childhood TB. Despite the difficulties noted in this field there is optimism that the application of novel antigens and the integration of those factors which contribute to the serological responses in childhood TB can lead to useful future diagnostics. PMID:23100476

233

Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations.  

The viability of BCG vaccine has traditionally been monitored using a colony-forming unit (CFU) assay. Despite its widespread use, results from the CFU assay can be highly variable because of the characteristic clumping of mycobacteria, their requirement for complex growth media, and the three week incubation period needed to cultivate slow-growing mycobacteria. In this study, we evaluated whether an ATP luminescence assay (which measures intracellular ATP content) could be used to rapidly estimate the viability of lyophilized and/or frozen preparations of six different BCG vaccine preparations - Danish, Tokyo, Russia, Brazil, Tice, and Pasteur - and two live attenuated mycobacterial vaccine candidates - a ?lysA?panCD M. tuberculosis strain and a ?mmaA4 BCG vaccine mutant. For every vaccine tested, a significant correlation was observed between intracellular ATP concentrations and the number of viable attenuated bacilli. However, the extractable intracellular ATP levels detected per cell among the different live vaccines varied suggesting that validated ATP luminescence assays with specific appropriate standards must be developed for each individual live attenuated vaccine preparation. Overall, these data indicate that the ATP luminescence assay is a rapid, sensitive, and reliable alternative method for quantifying the viability of varying live attenuated mycobacterial vaccine preparations. PMID:22652432

234

Migratory sub-populations of afferent lymphatic dendritic cells differ in their interactions with Mycobacterium bovis Bacille Calmette Guerin.  

Understanding how pathogens or vaccine antigens are targeted to dendritic cell (DC) subsets is important for disease pathogenesis studies and vaccine design. We characterised the sub-populations of migrating bovine DC with functional and phenotypic diversity present in pseudoafferent lymph draining the skin. These skin draining DC exist as a series of maturation dependent subsets with differential capacities for antigen uptake and cytokine expression, and include both Langerhans' cells (LC) and dermal derived cells. Furthermore, Mycobacterium bovis Bacille Calmette Guerin, a vaccine which is administered by the intradermal route, was only taken up by a small number of the migrating DC, which were SIRP?(+) and expressed the mannose receptor and CD1b. This was evident following in vitro infection and also in vivo following inoculation of green fluorescent BCG over the lymphatic cannulation site. Only the SIRP?(+) DC were able to present antigen to T cells isolated from BCG vaccinated calves. Furthermore, presentation of BCG antigens by DC to T lymphocytes was ineffective compared to mycobacterial proteins. However, mycobacterial antigen 85 was delivered more effectively to DC via an adenoviral vector and the magnitude of the subsequent antigen-specific T cell response was significantly increased. This study further extends our understanding of the biology of migrating DC, identifies potential explanations for the modest success of BCG vaccination and demonstrates that targeted delivery of antigens via adenoviruses to DC can improve antigen presentation. PMID:22281103

235

Comparative severity of experimentally induced mycobacteriosis in striped bass Morone saxatilis and hybrid tilapia Oreochromis spp.  

Twenty striped bass Morone saxatilis and 20 hybrid tilapia Oreochromis niloticus x O. mossambicus x O. aureus each received a single intramuscular injection of 1.6 x 10(6) colony forming units per gram body weight of Mycobacterium marinum. Striped bass manifested significantly greater clinical and microscopic disease compared to tilapia. Whereas all the striped bass had died or were clinically ill by Day 8 post-infection, there was no apparent disruption of normal behaviour, physical appearance, or growth in any of the sacrificed or surviving tilapia. Histologically, granulomas in striped bass were generally larger and less discrete, with a higher proportion of heavily vacuolated macrophages, and large cores of necrotic cells. Visceral granulomas in tilapia were smaller, with a higher proportion of epithelioid macrophages, more pigment-containing cells, more peripheral lymphocytes, and virtually no central necrosis. Visceral granulomas were 18-fold more numerous in striped bass than in tilapia. Based upon histomorphometric data, mean proportions of acid-fast bacteria within pronephros granulomas were 4-fold greater in striped bass than tilapia, and striped bass granulomas averaged more than twice as large as tilapia granulomas. In the anterior kidney of striped bass, a positive correlation existed between mean mycobacterial proportions and mean necrosis scores. In tilapia, mean mycobacterial proportions correlated negatively with mean granuloma numbers, whereas there was no correlation between these parameters in striped bass. Results suggest that intrinsic functional differences in the immunologic systems of striped bass and hybrid tilapia may contribute to inter-species variation in mycobacteriosis susceptibility. PMID:10686670

236

Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP  

Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria.

237

Drug-sensitive tuberculosis, multidrug-resistant tuberculosis, and nontuberculous mycobacterial pulmonary disease in nonAIDS adults: comparisons of thin-section CT findings  

The aim of this work was to compare thin-section CT (TSCT) findings of drug-sensitive (DS) tuberculosis (TB), multidrug-resistant (MDR) TB, and nontuberculous mycobacterial (NTM) pulmonary disease in nonAIDS adults. During 2003, 216 (113 DS TB, 35 MDR TB, and 68 NTM) patients with smear-positive sputum for acid-fast bacilli (AFB), and who were subsequently confirmed to have mycobacterial pulmonary disease, underwent thoracic TSCT. The frequency of lung lesion patterns on TSCT and patients' demographic data were compared. The commonest TSCT findings were tree-in-bud opacities and nodules. On a per-person basis, significant differences were found in the frequency of multiple cavities and bronchiectasis (P<0.001, chi-square test and multiple logistic regression analysis). Multiple cavities were more frequent in MDR TB than in the other two groups and extensive bronchiectasis in NTM disease (multiple logistic regression analysis). Patients with MDR TB were younger than those with DS TB or NTM disease (P<0.001, multiple logistic regression analysis). Previous tuberculosis treatment history was significantly more frequent in patients with MDR TB or NTM disease (P<0.001, chi-square test and multiple logistic regression analysis). In patients with positive sputum AFB, multiple cavities, young age, and previous tuberculosis treatment history imply MDR TB, whereas extensive bronchiectasis, old age, and previous tuberculosis treatment history NTM disease. (orig.)

238

Tratamiento antirretroviral en pacientes con sida y micobacteriosis/ Anti-retroviral treatment in patients with AIDS and mycobacterial diseases  

Abstract in spanish La tuberculosis y otras micobacteriosis constituyen asociaciones o coinfecciones frecuentes en pacientes con sida y se asocian con una elevada mortalidad. En esta revisión se actualizan los tratamientos de las principales enfermedades micobacterianas asociadas al sida (tuberculosis y micobacteriosis por Mycobacterium avium), con especial énfasis en las interacciones farmacológicas entre antimicobacterianos, principalmente rifampicina y claritromicina, y fármacos antir (more) retrovirales. Se analizan los esquemas de tratamiento, su duración, la quimioprofilaxis primaria y secundaria y el momento óptimo de iniciación del tratamiento antirretroviral. Finalmente se describe el síndrome inflamatorio de reconstitución inmune y su tratamiento. Abstract in english Tuberculosis and other mycobacterial diseases are frequent coinfections in AIDS patients with an increased related mortality. In this review we have updated the treatment of the main mycobacterial diseases (tuberculosis and Mycobacterium avium disease), under the scope of pharmacological interactions between antimycobacterial drugs, specially rifampicin and clarithromycin, and anti-retroviral drugs. Antimycobacterial treatment schemes, their duration, primary and secondar (more) y chemoprophylaxis and the optimal time to start the anti-retroviral therapy are analized. Finally, the immnune reconstitution inflammatory syndrome and its treatment are discussed.

239

Increased expression of fas ligand in human tuberculosis and leprosy lesions: a potential novel mechanism of immune evasion in mycobacterial infection.  

To study the location and mechanism of apoptosis within the human tuberculosis (TB) and leprosy lesions, parallel sections were analyzed for mycobacterial antigens (M.Ag), Fas ligand (FasL), Fas, CD68 and Mac387 by immunohistochemistry, and apoptotic cells by the terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick end labelling method. Cutaneous leishmaniasis and foreign body granulomas were analyzed for comparison. The heavily infected macrophages in multibacillary TB and leprosy granulomas very strongly expressed FasL, indicating that a mycobacterial infection can induce an increased expression of FasL in a population of infected macrophages, which may protect them from the attack of Fas-expressing lymphocytes. However, macrophages with high levels of leishmania amastigotes did not selectively express FasL, suggesting that this phenomenon is specific for the mycobacteria. Interestingly, in the well-formed TB granulomas, 84% of the multinucleated giant cells strongly expressed FasL. The expression of Fas was weak (34%) or absent. A higher number (33%) of epithelioid cells expressed FasL than Fas (23%). Lymphocytes were scanty among the epithelioid cells. The frequency of apoptotic cells was higher in the epithelioid cells (0.25%) than the mononuclear cells in the mantle zone (0.14%). Thus, the epithelioid cells and the multinucleated giant cells by virtue of the increased expression of FasL may make these granulomas an immune privileged site for mycobacteria. PMID:11902340

240

A novel mechanism of growth phase-dependent tolerance to isoniazid in mycobacteria.  

Tuberculosis remains one of the most deadly infectious diseases worldwide and is a leading public health problem. Although isoniazid (INH) is a key drug for the treatment of tuberculosis, tolerance to INH necessitates prolonged treatment, which is a concern for effective tuberculosis chemotherapy. INH is a prodrug that is activated by the mycobacterial enzyme, KatG. Here, we show that mycobacterial DNA-binding protein 1 (MDP1), which is a histone-like protein conserved in mycobacteria, negatively regulates katG transcription and leads to phenotypic tolerance to INH in mycobacteria. Mycobacterium smegmatis deficient for MDP1 exhibited increased expression of KatG and showed enhanced INH activation compared with the wild-type strain. Expression of MDP1 was increased in the stationary phase and conferred growth phase-dependent tolerance to INH in M. smegmatis. Regulation of KatG expression is conserved between M. smegmatis and Mycobacterium tuberculosis complex. Artificial reduction of MDP1 in Mycobacterium bovis BCG was shown to lead to increased KatG expression and susceptibility to INH. These data suggest a mechanism by which phenotypic tolerance to INH is acquired in mycobacteria. PMID:22648414

 
 
 
 
241

Structural and functional characterization of mycobactericidal ubiquitin-derived peptides in model and bacterial membranes.  

The mycobactericidal properties of macrophages include the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal Ubiquitin-derived peptides (Ub-peptides). To better understand interactions of ubiquitin-derived peptides with mycobacteria, we further characterized the structure and function of the bactericidal Ub-peptide Ub2. We found that Ub2 adopts a ?-sheet conformation in the context of sodium dodecyl sulfate (SDS) micelles and phospholipid (POPC:POPG, 1:1) vesicles that was dependent upon the primary sequence of the peptide. Point mutations in Ub2 that reduced the net charge of the peptide decreased Ub2 bactericidal activity. We investigated Ub-peptide function in the context of model membranes and intact bacteria. Differential scanning calorimetry analysis demonstrated that Ub2 inserts into and perturbs model phospholipid vesicles. In addition, we demonstrate that Ub2 disrupts the integrity of the mycobacterial membrane, equilibrates the transmembrane potential and localizes within both the mycobacterial membrane and cytoplasm of treated bacteria. Finally, we identified additional bactericidal Ub-peptides and characterized their activity and structure. This study provides new insight into the mycobactericidal mechanisms of Ub-peptides. PMID:23173767

242

Human T cell responses induced by vaccination with Mycobacterium bovis bacillus Calmette-Guérin  

Many aspects of the widely used bacillus Calmette-Guérin (BCG) vaccine against tuberculosis are still the subject of controversy. There is a huge variation in efficacy from one clinical trial to another and no relationship between vaccine-induced skin test conversion and subsequent protection. We have studied in vitro cell-mediated immune responses primed by BCG vaccination in 22 healthy Danish donors with different levels of in vitro purified protein derivative (PPD) reactivity before vaccination. The study demonstrated a markedly different development of reactivity to mycobacterial Ags depending on the prevaccination sensitivity to PPD. Previously sensitized donors mounted a potent and highly accelerated recall response within the first week of BCG vaccination. Nonsensitized donors, in contrast, exhibited a gradually increasing responsiveness to mycobacterial Ags, reaching maximal levels between day 56 and 365 postvaccination. The recognition of different classes of Ags were induced in a stepwise manner: culture filtrate Ags were recognized 1 wk postvaccination followed by cell wall, membrane, and the cytosolic Ag fraction. The T cell response primed by BCG vaccination was characterized as a CD4 response with a Th1-like cytokine pattern and substantial levels of Ag-specific cytotoxicity. The specificity of the T cell response generated was broad and directed to a range of culture filtrate Ag fractions. The study shows that BCG vaccination of previously nonsensitized donors can provide important data on potentially protective immune responses in humans and suggest a careful evaluation of prevaccination sensitivity when investigating vaccine-induced immunity.

243

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)  

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities. ?? 2009 Blackwell Publishing Ltd.

244

Mycobacterial transcriptional signals: requirements for recognition by RNA polymerase and optimal transcriptional activity  

Majority of the promoter elements of mycobacteria do not function well in other eubacterial systems and analysis of their sequences has established the presence of only single conserved sequence located at the ?10 position. Additional sequences for the appropriate functioning of these promoters have been proposed but not characterized, probably due to the absence of sufficient number of strong mycobacterial promoters. In the current study, we have isolated functional promoter-like sequences of mycobacteria from the pool of random DNA sequences. Based on the promoter activity in Mycobacterium smegmatis and score assigned by neural network promoter prediction program, we selected one of these promoter sequences, namely A37 for characterization in order to understand the structure of housekeeping promoters of mycobacteria. A37–RNAP complexes were subjected to DNase I footprinting and subsequent mutagenesis. Our results demonstrate that in addition to ?10 sequences, DNA sequence at ?35 site can also influence the activity of mycobacterial promoters by modulating the promoter recognition by RNA polymerase and subsequent formation of open complex. We also provide evidence that despite exhibiting similarities in ?10 and ?35 sequences, promoter regions of mycobacteria and Escherichia coli differ from each other due to differences in their requirement of spacer sequences between the two positions.

245

DNA polymerase III ? subunit from Mycobacterium tuberculosis H37Rv: Homology modeling and molecular docking of its inhibitor.  

The alpha subunit of Mycobacterial DNA polymerase III holo enzyme catalyzes the polymerization of both DNA strands. The present investigation reports three dimensional (3-D) structure model of DNA polymerase III ? subunit of Mycobacterium tuberculosis H37Rv (MtbDnaE1) generated using homology modeling with the backbone structure of DNA polymerase III ? of Thermus aquaticus as a template. The model was evaluated at various structure verification servers, which assess the stereo chemical parameters of the residues in the model, as well as structural and functional domains. Comparative analysis of MtbDnaE1 structure reveals the structure of its catalytic domain to be unrelated to that of the human. Successful docking of known inhibitor of bacterial DNA polymerases, 251D onto the modeled MtbDnaE1 was also performed. Therefore, the structure model of MtbDnaE1, a potential anti-mycobacterial target, opens a new avenue for structure-based drug designing against the pathogen. ABBREVIATIONS: aa - amino acid(s), PolIII? - DNA polymerase III alpha subunit, Taq Pol III? - Pol III? of Thermus aquaticus, MtbDnaE1 - PolIII? of Mycobacterium tuberculosis. PMID:21544168

246

Usefulness of multiplex PCR in the diagnosis of genital tuberculosis in females with infertility.  

The purpose of this investigation was to evaluate the usefulness of multiplex polymerase chain reaction (m-PCR) in detecting uterine tuberculosis in women with infertility. In a prospective study, endometrial curetting from 620 females with infertility were investigated using laparoscopy, hysteroscopy, histopathology, smear microscopy, mycobacterial culture in BACTEC MGIT™ 960, and in-house m-PCR. The mean age of the women was 29.75?±?4.66 years. The majority (596) sought medical attention for infertility; of them, 455 (76.34 %) presented with primary and 141 (23.65 %) with secondary infertility. A total of 158 (25.48 %) women were diagnosed as having uterine tuberculosis by at least one of the diagnostic methods. Among them, laparoscopy was positive in 46 (29.11 %), hysteroscopy in 77 (48.73 %), histopathology in only 8 (5.06 %), smear for acid fast bacilli in 4 (2.53 %), and liquid culture in 24 (15.18 %) patients. The in-house m-PCR was positive in 135 (85.44 %) women. Of these, 129 (95.55 %) samples were positive for Mycobacterium tuberculosis, while 6 (4.44 %) were positive for non-tuberculous mycobacterial DNA. Of the 129?M. tuberculosis PCR-positive women, 112 received anti-tubercular treatment and 23 of these conceived and fell pregnant after the completion of treatment. For the diagnosis of uterine tuberculosis, m-PCR was found to be the most efficient diagnostic tool compared to the other methods. PMID:23052988

247

Impact of ozone depletion on immune function  

Depletion of stratospheric ozone is expected to lead to an increase in the amount of UV-B radiation present in sunlight. In addition to its well known ability to cause skin cancer, UV-B radiation has been shown to alter the immune system. The immune system is the body's primary defense mechanism against infectious diseases and protects against the development of certain types of cancer. Any impairment of immune function may jeopardize health by increasing susceptibility to infectious diseases, increasing the severity of infections, or delaying recovery for infections. In addition, impaired immune function can increase the incidence of certain cancers, particularly cancers of the skin. Research carried out with laboratory animals over the past 15 years has demonstrated that exposure of the skin to UV-B radiation can suppress certain types of immune responses. These include rejection of UV-induced skin cancers and melanomas, contact allergy reactions to chemicals, delayed-type hypersensitivity responses to microbial and other antigens, and phagocytosis and elimination of certain bacteria from lymphoid tissues. Recent studies with mycobacterial infection of mice demonstrated that exposure to UV-B radiation decreased the delayed hypersensitivity response to mycobacterial antigens and increased the severity of infection. In humans, UV-B radiation has also been shown to impair the contact allergy response. These studies demonstrate that UV radiation can decrease immune responses in humans and laboratory and raise the possibility that increased exposure to UV-B radiation could adversely affect human health by increasing the incidence or severity of certain infectious diseases.

248

Malaria exacerbates experimental mycobacterial infection in vitro and in vivo  

Tuberculosis (Mtb) and malaria are among the most important infectious causes of morbidity and mortality worldwide, causing an estimated 1.5 million and 1 million deaths every year, respectively. Here we demonstrate a biological interaction between malaria and mycobacteria in vitro and in vivo. Murine macrophages co-incubated with Plasmodium falciparum parasitized erythrocytes demonstrated impaired control of intracellular Mtb replication, and reduced production of reactive nitrogen species in response to mycobacteria. Infection of C57BL/6 mice with Plasmodium species exacerbated the course of acute mycobacterial infection (57% increase in peak splenic CFU, p = 0.043 for difference over time course of infection), induced disruption of the structural integrity of established granulomas, and...

249

Study of binary systems of ?-cyclodextrin with a highly potential anti-mycobacterial drug  

The present research paper is dedicated to the obtaining and physicochemical characterization of a highly potential anti-mycobacterial drug candidate with ?-cyclodextrin (?CD). The active substance is a 1,3,4-oxadiazole derivative, 2-phenyl-5-{[(2-phenyl-1,3-dioxolan-2-yl)methyl]sulfanyl}-1,3,4-oxadiaz ole, further named DIOX. DIOX??CD binary systems were obtained as a physical mixture and a lyophilized product with molar ratio between the main components equal to 1:1 and 1:2. The obtained systems were submitted to physicochemical characterization applying the following instrumental methods: infrared spectrometry, differential scanning calorimetry, and X-ray crystallographic analysis. Besides, a molecular modeling analysis has been performed. The research data suggested certain intermolecu...

250

A Mass Lesion of the Wrist: A Rare Manifestation of Tuberculosis  

The worldwide reemergence of tuberculosis is significant. In particular, the incidence of extrapulmonary tuberculosis is increasing. But tuberculous tenosynovitis is rare and may be overlooked as a cause of chronic tenosynovitis. Here, we present a case of a 24 year-old man with a mass lesion on the flexor side of the right wrist. Laboratory findings were generally negative, except for the acceleration of the erythrocyte sedimentation rate, and the tuberculosis skin test was strongly positive. Magnetic resonance imaging (MRI) of the mass lesion of the wrist revealed tenosynovitis. We performed open biopsy and mycobacterial cultures. Thus, we diagnosed the patient with tuberculous tenosynovitis. Tuberculous tenosynovitis is uncommon but should be kept in mind in cases of chronic tenosynovitis.   

251

Effect of antimicrobial peptides on ATPase activity and proton pumping in plasma membrane vesicles obtained from mycobacteria  

The potential usefulness of antimicrobial peptides (AMPs) as antimycobacterial compounds has not been extensively explored. Although a myriad of studies on AMPs from different sources have been done, some of its mechanisms of action are still unknown. Maganins are of particular interest since they do not lyse non-dividing mammalian cells. In this work, AMPs with well-recognized activity against bacteria were synthesized, characterized, purified and their antimycobacterial activity and influence on ATPase activity in mycobacterial plasma membrane vesicles were assessed. Using bioinformatics tools, a magainin-I analog peptide (MIAP) with improved antimicrobial activity was designed. The influence of MIAP on proton (H^+) pumping mediated by F1F0-ATPase in plasma membrane vesicles obtained fro...

252

Use of a Mycobacterial Cell Wall Extract (MCWE) in Susceptible Mares to Clear Experimentally Induced Endometritis With Streptococcus zooepidemicus  

The ability of an immunomodulator, mycobacterial cell wall extract (MCWE), to clear uterine infection in susceptible mares after an experimental challenge with Streptococcus zooepidemicus was evaluated. Thirty mares susceptible to endometritis, based on the presence of uterine fluid during both diestrus and estrus, were selected from a herd of 896 and inoculated with a live culture of 5 x 106 CFU of S. zooepidemicus on day 1 of estrus. Twenty-four hours later, mares were evaluated by ultrasonography, bacteriology, exfoliative cytology, and uterine biopsy to confirm infection. Forty-eight hours after inoculation, and on confirmation of uterine infection, mares were randomly assigned to one of four unbalanced experimental treatments to receive 1500 mg MCWE IU (n = 10) or IV (n = 10), or plac...

253

Dissecting memory T cell responses to TB: Concerns using adoptive transfer into immunodeficient mice  

Several studies have used adoptive transfer of purified T cell subsets into immunodeficient mice to determine the subset of T cells responsible for mediating protection against Mycobacterium tuberculosis. These studies suggested that CD62L^h^i memory CD4^+ T cells from BCG-vaccinated mice are key for protection against tuberculosis. Importantly, we observed that transfer of naive CD4^+ T cells into Rag1-/- recipients protected against a mycobacterial challenge as well as transfer of BCG-experienced CD4^+ T cells. We found that transfer of total CD4^+ T cells from naive mice or enriched CD62L^h^iCD4^+ T cells from BCG-vaccinated mice into Rag1-/- recipients induced severe colitis by 3 weeks post cell transfer, whereas transfer of CD62L^l^oCD4^+ T cells from BCG-vaccinated mice did not. Naiv...

254

Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.  

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species. PMID:21510779

255

Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures  

Ziehl?Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein?Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.8 and 100%, respectively). The overall percentages of mycolic acid detection ...

256

Polymerase chain reaction compared to other laboratory findings and to clinical evaluation in the diagnosis of cutaneous tuberculosis and atypical mycobacteria skin infection  

Abstract Background Cutaneous tuberculosis has re-emerged in the last 15 years together with the higher incidence of pulmonary tuberculosis and multidrug resistance. The choice for a single diagnostic tool among the many available today is a challenge. Our objective was to compare polymerase chain reaction (PCR) with other exams in the diagnosis of cutaneous tuberculosis and atypical mycobacteria skin infection. Methods PCR and a set of five different exams were performed in 32 patients (34 samples of paraffin-embedded tissue) evaluated for 3 years in a university hospital, considering the response to mycobacterial infection treatment as a positive case. Results PCR was the most sensitive (88%) and specific (83%) exam. Culture, immunohistochemistry and acid-fast bacilli were not in agreeme...

257

Diagnostic value of an enzyme-linked immunospot assay for interferon-g in cutaneous tuberculosis  

The aim of this study was to evaluate the diagnostic performance of an enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB; Oxford Immunotec, Oxford, UK) for interferon-g in patients with suspected cutaneous tuberculosis (TB). From March 2007 to June 2010, a total of 45 patients with suspected cutaneous TB were enrolled. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for ELISPOT assay. Ten subjects (22.2%) had culture-confirmed TB, 2 (4.4%) subjects had probable TB, and the remaining 33 (73.3%) subjects did not have TB. Twenty-one patients with mycobacterial infection had available biopsy or surgical specimens for histopathologic examination and 16 (76.2%) specimens had pathologic features (granulomatous ...

258

Application of the QuantiFERON®-TB Gold test in Erythema Induratum.  

Erythema induratum (EI) was originally described by Ernest Bazin in 1861 as nodular lesions on the legs in association with tuberculosis (TB). A 33-year-old woman was referred to our hospital with tender ulcerative nodules on her legs. Skin biopsies revealed lobular inflammation of the subcutaneous fat with vasculitis. Although conservative treatment was given, the lesions did not improve. Several months later as the lesions persisted, we suspected a TB infection and additional tests were performed. The chest roentgenogram results were normal. The skin biopsies were negative for acid-fast bacilli (AFB). Mycobacterial cultures of the blood and skin were negative. However, the QuantiFERON®-TB Gold test was positive, which led to a diagnosis of latent TB infection being made. The patient was treated with multidrug antituberculosis therapy, which successfully resolved her skin lesions. Therefore, reported herein is the application of the QuantiFERON®-TB Gold test for the confirmation of latent TB infection in EI patients. PMID:22920648

259

Ability of Cricetomys rats to detect Mycobacterium tuberculosis and discriminate it from other microorganisms.  

Trained African giant pouched rats (Cricetomys gambianus) have potential for diagnosis of tuberculosis (TB). These rats target volatile compounds of Mycobacterium tuberculosis (Mtb) that cause TB. Mtb and nontuberculous mycobacteria (NTM) species are related to Nocardia and Rhodococcus spp., which are also acid-fast bacilli and can be misdiagnosed as Mtb in smear microscopy. Diagnostic performance of C. gambianus on in vitro-cultured mycobacterial and related pulmonary microbes is unknown. This study reports on the response of TB detection rats to cultures of reference Mtb, clinical Mtb, NTM, Nocardia; Rhodococcus; Streptomyces; Bacillus; and yeasts. Trained rats significantly discriminated Mtb from other microbes (p positive sputum better than spiked sputum. Although further studies on volatiles from detectable growth phases of Mtb are vital for identification of Mtb-specific volatiles detected by rats, our study underline the potential of C. gambianus for TB diagnosis. PMID:22197664

260

Tubercle bacilli rely on a type VII army for pathogenicity  

Mycobacteria, such as the major human pathogen Mycobacterium tuberculosis, have a highly unusual and characteristic diderm cell envelope that protects them against harmful conditions. Protein secretion across this hydrophobic barrier requires specialized secretion systems. Recently, a type VII secretion (T7S) pathway has been identified that fulfills this function. Pathogenic mycobacteria have up to five different T7S systems, some of which play a crucial role in virulence. The interactions between secreted substrates and host molecules are only starting to become clear and will help in furthering our understanding of the persistence of these enigmatic pathogens. In this review, we discuss current knowledge on the role of T7S systems in mycobacterial virulence.

 
 
 
 
261

Temporal patterns of genetic and phenotypic variation in the epidemiologically important drone fly, Eristalis tenax  

Eristalis tenax L. (Diptera: Syrphidae) is commonly known as the drone fly (adult) or rat-tailed maggot (immature). Both adults and immature stages are identified as potential mechanical vectors of mycobacterial pathogens, and early-stage maggots cause accidental myiasis. We compared four samples from Mount Fruka Gora, Serbia, with the aim of obtaining insights into the temporal variations and sexual dimorphism in the species. This integrative approach was based on allozyme loci, morphometric wing parameters (shape and size) and abdominal colour patterns. Consistent sexual dimorphism was observed, indicating that male specimens had lighter abdomens and smaller and narrower wings than females. The distribution of genetic diversity at polymorphic loci indicated genetic divergence among colle...

262

Diacyltrehalose of Mycobacterium tuberculosis inhibits lipopolysaccharide- and mycobacteria-induced proinflammatory cytokine production in human monocytic cells.  

The lipids located in the outer layer of Mycobacterium tuberculosis, which include sulfolipid, phthiocerol dimycocerosate (PDIM), diacyltrehalose, and polyacyltrehalose, may play a role in host-pathogen interactions. These lipids were purified using thin-layer chromatography, and their ability to induce proinflammatory cytokines in human monocytes and in a human acute monocytic leukemia cell line (THP-1) was examined. None of the lipids tested induced significant interleukin (IL)-12p40 or tumor necrosis factor (TNF)-alpha production in monocytic cells. Diacyltrehalose significantly inhibited lipopolysaccharide- and M. tuberculosis-induced IL-12p40, TNF-alpha, and IL-6 productions in human monocytes, whereas other lipids had no effect. However, diacyltrehalose was unable to inhibit peptidoglycan-induced IL-12p40 production. These results suggest that diacyltrehalose is a mycobacterial factor capable of modulating host immune responses. PMID:17156119

263

Relationship between clinical efficacy for pulmonary MAC and drug-sensitivity test for isolated MAC in a recent 6-year period  

There are a few recent reports about the relationship between the clinical effect and drug-sensitivity test. We investigated the relationship between the clinical efficacy of treatment for pulmonary Mycobacterium avium complex (MAC) and drug-sensitivity test for isolated MAC by comparison between data from 2005 to 2007 and from 2008 to 2010. We studied 60 patients who satisfied diagnostic criteria of nontuberculous mycobacterial infection established by the American Thoracic Society in 2007 and who received combination therapy using rifampicin (RFP), ethambutol (EB), streptomycin (SM), and clarithromycin (CAM). Average CAM dosage was increased from the early (517?mg/day) to the later (800?mg/day) period. Sputum conversion rate increased from 63% in the early period to 83% in the later peri...

264

Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State, Nigeria  

This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n=64% or 79% of clustered isolates, 2-32 isolates/cluster), with patients in the age group range 25-34years being significantly associated with shared-type pattern SIT61 (p=0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages...

265

High prevalence of drug-resistant tuberculosis and other mycobacteria among HIV-infected patients in Brazil: a systematic review  

Abstract in english There is a little-noticed trend involving human immunodeficiency virus (HIV)-infected patients suspected of having tuberculosis: the triple-treatment regimen recommended in Brazil for years has been potentially ineffective in over 30% of the cases. This proportion may be attributable to drug resistance (to at least 1 drug) and/or to infection with non-tuberculous mycobacteria. This evidence was not disclosed in official statistics, but arose from a systematic review of a (more) few regional studies in which the diagnosis was reliably confirmed by mycobacterial culture. This paper clarifies that there has long been ample evidence for the potential benefits of a four-drug regimen for co-infected patients in Brazil and it reinforces the need for determining the species and drug susceptibility in all positive cultures from HIV-positive patients.

266

Different Mycobacterium avium subsp. paratuberculosis MIRU-VNTR patterns coexist within cattle herds  

A better understanding of the biodiversity of Mycobacterium avium subsp. paratuberculosis (MAP) offers more insight in the epidemiology of paratuberculosis and therefore may contribute to the control of the disease. The aim of this study was to investigate the genetic diversity in bovine MAP isolates using PCR-based methods detecting genetic elements called Variable-Number Tandem Repeats (VNTRs) and Mycobacterial Interspersed Repetitive Units (MIRUs) to determine if multiple MAP strains can coexist on farms with endemic MAP infection. For 52 temporal isolates originating from infected cattle from 32 commercial dairy herds with known trading history, MIRU-VNTR analysis was applied at 10 loci of which six showed variation. Within the group of 52 isolates, 17 different MIRU-VNTR patterns were...

267

Phyllocladane diterpenes from Anisomeles heyneana.  

New phyllocladane diterpene, phyllocladan-16?,17-dihydroxy-19-oic acid (1), together with known phyllocladane diterpene, phyllocladan-16?,19-diol (2), cembrane diterpene ovatodiolide (3), sitosteryl-3-O-?-d-glucoside (4), and verbascoside (5), were isolated from aerial parts of Anisomeles heyneana. The structure of compound 1 was elucidated by 1D and 2D NMR analyses which included HSQC, HMBC, and nuclear overhauser effect spectroscopy (NOESY) experiments as well as X-ray crystallography. This is the first report of phyllocladane diterpenes from genus Anisomeles. Compounds 1, 3, 4, and 5 were evaluated for inhibition of Mycobacterium tuberculosis and 3 was found to exhibit anti-mycobacterial activity with IC(90) 6.53 ?g/ml. Compounds 1, 3, and 5, at 100 ?g/ml, were also evaluated for inhibition of Thp-1 cell lines, and compounds 1 and 3 showed 59.02% and 96.4% inhibitions, respectively. PMID:23157282

268

Favorable infertility outcomes following anti-tubercular treatment prescribed on the sole basis of a positive polymerase chain reaction test for endometrial tuberculosis  

BACKGROUND The endometrial tuberculosis (TB) PCR test is now commonly employed for the diagnosis of female genital TB, a common cause of infertility in India. Although treatment in the absence of demonstrable tubal damage may be of doubtful benefit to fertility, the presence of mycobacterial DNA demonstrated by a positive PCR indicates infection by tubercle bacilli causing sub-clinical or latent disease potentially responsible for future clinical manifestations. This study was undertaken to assess the outcome of infertility management following early anti-tubercular treatment (ATT) based only on a positive endometrial TB-PCR test. METHODS This was an intervention study conducted at an IVF center in northern India in 443 infertile women of whom 169 (38.15%) were found to have positiv...

269

Pulmonary Mycobacterium parascrofulaceum Infection as an Immune Reconstitution Inflammatory Syndrome in an AIDS Patient  

Nontuberculous mycobacterial (NTM) infection in HIV (human immunodeficiency virus)-infected patients who have started highly active antiretroviral therapy (HAART) is well known to be one scenario of immune reconstitution inflammatory syndrome (IRIS). We encountered the first case in Japan of an HIV-infected patient with pulmonary Mycobacterium parascrofulaceum infection as IRIS. A 34 year-old man with acquired immunodeficiency syndrome (AIDS) was receiving highly active antiretroviral therapy. Lymphadenopathy was observed at the left pulmonary hilum. IRIS was suspected and thoracoscopic surgery was performed to diagnose the cause of lymphadenopathy. Granulomas were observed histologically, and M. parascrofulaceum was cultured. This organism was susceptible to Clarithromycin, rifampicin and levofloxacin. After the operation and without treatment, recurrence of M. parascrofulaceum infection was not observed. M. parascrofulaceum was isolated from several clinical specimens for the first time in 2004. To date, only five cases have been reported.   

270

BCG-infected adherent mononuclear cells release cytokines that regulate group 1 CD1 molecule expression  

Increasing evidence is now available showing that CD1-restricted T cell responses against non-peptide mycobacterial antigens could play a role in the immune resistance against tuberculosis. BCG, widely used in anti-tubercular vaccination, shares various constituents with Mycobacterium tuberculosis, but does not provide full protection. In the present study we have investigated the pattern of group 1 CD1 molecule expression in adherent mononuclear cells (AMNC) of human peripheral blood, infected in vitro with BCG. Shortly after exposure to BCG, both BCG-positive and BCG-negative AMNC showed a moderate CD1 expression elicited by BCG-induced release of GM-CSF presumably acting through an autocrine and a paracrine mechanism. This was demonstrated using two-color flow cytometry with green fluor...

271

Hallmarks of mycolic acid biosynthesis: A comparative genomics study  

Mycolic acids, which render unique qualities to mycobacteria, are known to be important for mycobacterial growth, survival, and pathogenicity. It is of interest to understand the evolutionary origins of the mycolic acid pathway (MAP), as well as the common minimum principles critical for generating the capability of mycolic acid biosynthesis. The recent curation of a comprehensive model of the MAP in Mycobacterium tuberculosis and the availability of a large number of genome sequences make it feasible to carry out detailed sequence and phylogenetic analyses, to address these questions. A comprehensive phylogenetic pathway profile analysis was carried out for 318 fully sequenced bacterial genomes, for each of the proteins present in the MAP. The organisms were clustered on the basis of co-o...

272

Osteopontin immunoreactivity in the ileum and ileocecal lymph node of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis  

Osteopontin (Opn), a highly acidic glycoprotein, promotes cellular adhesion and recruitment and has been shown to be upregulated in the granulomas of mycobacterial infections. Johnes disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is associated with granulomatous enteritis. The objective of this experiment was to identify Opn in the ileum and ileocecal lymph node (ICN) of dairy cows naturally infected with MAP and to compare the frequency and intensity of staining between noninfected healthy controls, subclinical and clinical cows. Sections from these three groups of animals were selected from a tissue archive. Immunohistochemical analysis was used to determine the location and expression of Opn. The frequency and intensity of staining was also reported. Confirmation ...

273

The contribution of a murine CNS-TB model for the understanding of the host-pathogen interactions in the formation of granulomas  

Central nervous system (CNS) tuberculosis (TB) is the most severe form of TB, characterized morphologically by brain granulomas and tuberculous meningitis (TBM). Experimental strategies for the study of the host-pathogen interaction through the analysis of granulomas and its intrinsic molecular mechanisms could provide new insights into the neuropathology of TB. To verify whether cerebellar mycobacterial infection induces the main features of the disease in human CNS and better understand the physiological mechanisms underlying the disease, we injected bacillus Calmette-Guerin (BCG) into the mouse cerebellum. BCG-induced CNS-TB is characterized by the formation of granulomas and TBM, a build up of bacterial loads in these lesions, and microglial recruitment into the lesion sites. In additi...

274

Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir  

Consumption has been a scourge of mankind since ancient times. This illness has charged a high price to human lives. Many efforts have been made to defeat Mycobacterium tuberculosis (Mt). The M. tuberculosis purine nucleoside phosphorylase (MtPNP) is considered an interesting target to pursuit new potential inhibitors, inasmuch it belongs to the purine salvage pathway and its activity might be involved in the mycobacterial latency process. Here we present the MtPNP crystallographic structure associated with acyclovir and phosphate (MtPNP:ACY:PO"4) at 2.10 A resolution. Molecular dynamics simulations were carried out in order to dissect MtPNP:ACY:PO"4 structural features, and the influence of the ligand in the binding pocket stability. Our results revealed that the ligand leads to active si...

275

Non-tuberculous slow-growing mycobacterial pulmonary infections in non-HIV-infected patients in south London  

Background: UK data on slow-growing non-tuberculous mycobacterial (NTM) pulmonary infections are sparse and there is little consensus on optimal treatment regimens. Methods: This was a retrospective study of NTM pulmonary infections in a London teaching hospital. Inclusion criteria were culture of slow-growing mycobacteria between 2000 and 2007, age > 18 y, HIV-negative, and meeting American Thoracic Society criteria. Results: Fifty-seven patients were included; 68% were males and the median age was 61 y. Predisposing factors were smoking (70%), alcohol abuse (28%), and chronic obstructive pulmonary disease (37%). Cavitation (56%) and infiltrates (42%) were common radiological findings. The predominant organism was Mycobacterium kansasii (70%). Ninety-three percent of patients with M. kans...

276

Conformational changes in 2-trans-enoyl-ACP (CoA) reductase (InhA) from M. tuberculosis induced by an inorganic complex: a molecular dynamics simulation study  

InhA, the NADH-dependent 2-trans-enoyl-ACP reductase enzyme from Mycobacterium tuberculosis (MTB), is involved in the biosynthesis of mycolic acids, the hallmark of mycobacterial cell wall. InhA has been shown to be the primary target of isoniazid (INH), one of the oldest synthetic antitubercular drugs. INH is a prodrug which is biologically activated by the MTB catalase-peroxidase KatG enzyme. The activation reaction promotes the formation of an isonicotinyl-NAD adduct which inhibits the InhA enzyme, resulting in reduction of mycolic acid biosynthesis. As a result of rational drug design efforts to design alternative drugs capable of inhibiting MTB?s InhA, the inorganic complex pentacyano(isoniazid)ferrate(II) (PIF) was developed. PIF inhibited both wild-type and INH-resistant Ile21Val mu...

277

Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides  

InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis, is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of M. tuberculosis. We report here the discovery, through high-throughput screening, of a series of arylamides as a novel class of potent InhA inhibitors. These direct InhA inhibitors require no mycobacterial enzymatic activation and thus circumvent the resistance mechanism to antitubercular prodrugs such as INH and ETA that is most commonly observed in drug-resistant clinical isolates. The crystal structure of InhA complexed with one representative inhibitor reveals the binding mode of the inhibitor within the InhA active site. Further optimization through a microtiter synthesis strategy followed by in situ activity screeni...

278

Tuberculosis drug-resistance in Lisbon, Portugal: a 6-year overview  

Abstract Multidrug-resistance and extensive drug-resistance pose a serious threat to tuberculosis management in Portugal. The country has high TB incidence rates in comparison with other European Union countries, with the Lisbon Health Region being one of the most affected. In the present study we have analysed a convenience sample of 3025 Mycobacterium tuberculosis clinical isolates, recovered over a 6-year period (2001-2006) in the Lisbon Health Region, regarding drug-resistance both to first-line and second-line drugs. Moreover, 100 of these isolates were also genotyped by 12-loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeats (MIRU-VNTR) analysis. We have compared each year and observed the existence of 22 different resistance profiles, with MDR-TB rates...

279

A Tale of Two Protease Complexes (417th Brookhaven Lecture)  

Proteins that cleave other proteins using a molecule of water, protease complexes are exquisite macromolecular machines involved in a multitude of physiological and cellular reactions. We have been studying two recently identified protease complexes using electron cryo-microscopy and X-ray crystallography. The first is a proteasome that resides in the cytoplasm of the Mycobacterial tuberculosis and is required for Tb resistance to destruction by human macrophages. The second is a gamma-secretase complex embedded in the cellular membrane of human neurons. Gamma-secretase produces Alzheimer's disease-causing A-beta peptides. Our structural studies shed light into the inner workings of these multi-protein assemblies, and they reveal a surprisingly common strategy for controlled proteolysis employed by the two drastically different machines. Further research will facilitate rational design of drugs for treating Tb infection and Alzheimer's disease.

280

Acute Pneumonia and Empyema Caused by Mycobacterium Intracellulare  

Nontuberculous mycobacterial infection is rarely accompanied by pleural involvement. We report a very rare case of M. intracellulare pulmonary disease with pleural empyema. A 56-year-old man was admitted to our hospital because of fever, purulent sputum and pleuritic chest pain. A chest radiograph and CT revealed pneumonic consolidation in the left lower lobe and loculated hydropneumothorax. The sputum smear was positive for acid fast bacilli. The aspirated pleural fluid was grossly purulent and the smear of the pleural effusion was also positive for acid fast bacilli. M. intracellulare was identified by culture and PCR from sputum and pleural fluid specimens. The patient improved with percutaneous tube drainage of the purulent effusion and antibiotic treatment including clarithromycin, rifampicin, ethambutol and streptomycin.   

 
 
 
 
281

Infección bacteriémica por Nocardia otitidiscaviarum: revisión a propósito de un caso/ Bacteremic infection due to Nocardia Otitidiscaviarum: case report and review  

Abstract in spanish Presentamos un caso de neumonía bacteremica causada por Nocardia otitidiscaviarum en un paciente con bronconeumopatia crónica obstructiva corticodependiente. Los cultivos de sangre y esputo en medios para micobacterias resultaron positivos y la identificación se realizó mediante la secuenciación de 16S rDNA. En el presente artículo se realiza una revisión de conjunto sobre el agente etiológico y se practica el estudio de susceptibilidad de la cepa mediante la técnica de E-Test. Abstract in english We present a case of bacteremic pneumonia caused by Nocardia otitidiscaviarum in a corticodependent COPD. Blood and sputum cultures on Mycobacterial media were positives and identification was done using 16S rDNA sequencing. In this article we review the most relevant comunications about Nocardia spp infection and study the strain susceptibility using E-test.

282

Identification of rat urinary metabolites of rifabutin using LC-MS^n and LC-HR-MS  

Rifabutin, an anti-mycobacterial agent, is reported to be extensively metabolized in vivo into more than 20 biotransformation products, with similar profile both in humans and rats. Among the metabolites formed, only seven have been characterized, the remaining are unknown. Hence, the purpose of the present study was to fill this gap by using modern in silico tools combined with advanced liquid chromatography-mass spectrometry (LC-MS) techniques. Initially a comprehensive mass fragmentation pattern for rifabutin was established using Frontier(TM) 5.1 software coupled with the data collected from multiple-stage MS (MS^n), high resolution MS (HR-MS) and hydrogen/deuterium exchange MS (HDE-MS) experiments. The metabolites were then predicted in silico by using different software like MetaSite...

283

Epidemiologic surveillance to detect false-positive Mycobacterium tuberculosis cultures  

This study was aimed to investigate the ability of potential indices from epidemiologic surveillance to detect false-positive cultures of Mycobacterium tuberculosis (MTB). All clinical specimens for mycobacterial culture from April 1 to August 31, 2010, were reviewed. Single-positive cultures without relevant clinical and pathologic information were categorized as suspected false-positive cultures. Genotyping methods were used to confirm false-positive cultures. The performance of epidemiologic surveillance indices to detect potential false-positive cultures was evaluated. A total of 14,462 specimens were sent to the laboratory and 214 batches were processed in 107 work days (average 67.6 specimens per batch, ranging from 21 to 130 specimens per batch). Seventy-one single-positive cultures...

284

Ag85BESAT-6 adjuvanted with IC31 promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in naive human volunteers  

Though widely used, the BCG vaccine has had little apparent effect on rates of adult pulmonary tuberculosis. Moreover, the risk of disseminated BCG disease in immunocompromised individuals means that improved TB vaccines ideally need to be able to efficiently prime mycobacterially-naive individuals as well as boost individuals previously vaccinated with BCG. Protective immunity against Mycobacterium tuberculosis is thought to depend on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-g) production. In the present study, we monitored safety and IFN-g responses in healthy TB-naive humans receiving an entirely novel vaccine, composed of the fusion protein Ag85B-ESAT-6, administered at 0 and 2 months either as recombinant protein alone or combined wi...

285

Ag85B-ESAT-6 adjuvanted with IC31 promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in volunteers with previous BCG vaccination or tuberculosis infection  

New TB vaccines are urgently needed because of the apparent lack of effect of the BCG vaccine on rates of adult contagious pulmonary tuberculosis and the risk of disseminated BCG disease in immunocompromised individuals. Since BCG appears to protect children, the primary target for vaccine development is a booster vaccine for adults but such vaccines ideally need to be able to efficiently prime mycobacterially naive individuals as well as boost individuals previously vaccinated with BCG and those latently infected with TB. Protective immunity against Mycobacterium tuberculosis depends mainly on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-g) production. In the present study, we monitored safety and IFN-g responses in healthy BCG-vaccinated an...

286

Mycobacterium ulcerans infection in North Queensland: the -Daintree ulcer-  

Abstract Background:- As the third most common mycobacterial infection in the world after tuberculosis and leprosy, Mycobacterium ulcerans is a major health and development problem that has become the focus of a World Health Organisation (WHO) initiative seeking to reduce the burden of this disease. The Daintree River catchment in north Queensland is an endemic focus for Mycobacterium ulcerans infection, known locally as the -Daintree Ulcer-. The aim of this study is to analyse the changing pattern of the disease over the last 44 years in the region. Methods:- The study is a descriptive review of all human cases where infection had been acquired in an endemic region of far north Queensland. Demographic information, lesion characteristics, management and outcomes were recorded in a database...

287

Buruli ulcer and current situation in Japan: A new emerging cutaneous Mycobacterium infection  

Abstract Buruli ulcer (BU) is a new emerging disease and the third most common chronic mycobacterial infection in humans, caused by Mycobacterium ulcerans. Approximately 5000 cases are reported annually from at least 33 countries around the globe, but more from the tropical nations. A total of 32 cases have been reported from Japan sporadically since 1980. None of the cases were related to international travel. Of the total reported, M.ulcerans ssp. shinshuense, a subspecies speculated to be domestic to Japan or in Asia, has been isolated from 23 cases. The mode of transmission and its incubation period remain unclear, despite several proposed hypotheses, including several vectors and cutaneous wound as port of entry for the pathogen. M.ulcerans invades the skin, subcutaneous tissue, fasci...

288

Extensive cortical involvement in leptomeningeal carcinomatosis.  

We present a 77-year-old previously well patient with facial asymmetry and progressive weakness of the lower extremities. An initial MRI revealed slight contrast enhancement of the meninges. Three consecutive cerebrospinal fluid examinations demonstrated low glucose concentration, marked elevation of total protein and moderate pleocytosis. No tumor cells, fungi, acid-fast bacilli or mycobacterial DNA were found. The patient's level of consciousness deteriorated dramatically, and follow-up MRI showed widespread extensive cortical hyperintensities. The lesions showed restricted diffusion on diffusion-weighted images as well as low values on the corresponding apparent diffusion coefficient maps, the changes consistent with diffuse cytotoxic edema. Neuropathological examination findings were of leptomeningeal carcinomatosis (LMC) with diffuse continuous infiltration of the cerebral cortex, cerebellum and spinal cord. The autopsy revealed a subcentimetre adenocarcinoma of the lung. To our knowledge, this is the first report demonstrating extensive cortical involvement in adenocarcinomatous LMC. PMID:22989786

289

Rifampicin and Parkinson's disease.  

Rifampicin is a macrocyclic antibiotic used extensively for the treatment of Mycobacterium tuberculosis and other mycobacterial infections. Recently, it was discovered that rifampicin exhibits neuroprotective effects. It has been shown to protect PC12 cells against MPP(+)-induced apoptosis and inhibit the expression of ?-synuclein multimers. In in vitro studies, rifampicin pretreatment protects PC12 cells against rotenone-induced cell death. Qualitative and quantitative analyses uncover that rifampicin significantly suppresses rotenone-induced apoptosis by ameliorating mitochondrial oxidative stress. It reduces microglial inflammation and improves neuron survival. Our results indicate that rifampicin is cytoprotective under a variety of experimental conditions, and suggest that it may be useful in PD therapeutics. It is the aim of this paper to review the experimental neuroprotection data reported using rifampicin with a focus on the molecular and cellular mechanisms of cytoprotective effect in in vitro models of PD. PMID:22821065

290

Diagnostic significance of humoral immune responses to recombinant antigens of Mycobacterium tuberculosis in patients with pleural tuberculosis  

Abstract Mycobacterium tuberculosis bacilli are seldom demonstrated in tuberculous pleural effusion (TPE) by conventional bacteriological methods. In this study, an indirect enzyme linked immunosorbent assay (ELISA) was developed to detect IgG against four mycobacterial recombinant antigens (ESAT 6, PlcA, HspX and Tb8.4) in 69 pleural fluids of patients with TPE and 71 patients with malignant pleural effusion. To increase the sensitivity of the assay, a multi antigen cocktail containing all the above antigens were also used. IgG positivity in ELISA for PlcA, HspX, Tb8.4, ESAT 6 antigens and multi antigen complex were 49.3, 60.8, 49.3, 53.6 and 75.4% respectively. Each one of the above four antigens and their multi antigen cocktail were highly specific in distinguishing tuberculous and mali...

291

Emphysematous cystitis in a patient presenting with paradoxical arterial embolism and intestinal mycobacteriosis without evidence of diabetes  

We describe the case of a 72-year-old woman who displayed massive multiple intramural gas collections of the bladder wall as an incidental finding on CT. The patient presented with critical ischemia of the left leg caused by paradoxical arterial embolism, raised corpuscular sedimentation rate, anemia by gastrointestinal blood loss, hypoproteinemia, diarrhea, malabsorption, and exudative enteropathia caused by mycobacterial ileocolitis. The patient had no dysuria and there was no evidence of diabetes. The intramural gas collections of the bladder wall, as shown by CT, were compatible with emphysematous cystitis. Urine samples proved infection by a multi-resistant strain of E. coli. Emphysematous cystitis is a rare form of bladder infection that can be diagnosed by plain-film radiograms or CT. (orig.)

292

Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB  

The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

293

New types of immunotherapy in children.  

Injection immunotherapy has been shown to be particularly beneficial in treating allergic rhinitis, mild to moderate asthma, and anaphylaxis caused by bee and wasp venom. It also produces a long-term, antigen-specific, protective immune effect and is the only treatment that offers the possibility of reducing the risk of asthma development in children with allergic rhinitis. Nonetheless, the potentially severe side effects associated with this form of immunotherapy limit its widespread use. Diverse preparations are being developed to increase its safety and improve its efficacy. These include alternative routes of administration, particularly the sublingual route; use of novel adjuvants, such as CpG oligonucleotides and mycobacterial vaccines; and other approaches, such as peptide immunotherapy, recombinant allergens, DNA vaccination, and combined therapy. Some of these immunotherapy forms have been evaluated in children. PMID:18940138

294

Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases.  

A new series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. The best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the ?-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents. PMID:23095026

295

Reactividad serológica y celular frente a proteínas micobacterianas en la enfermedad de Hansen/ Serological and cellular reactivity to mycobacterial proteins in Hansen´s disease  

Abstract in spanish Se diseñó un estudio para evaluar la reactividad inmunológica frente a diferentes preparaciones proteicas micobacterianas utilizando pruebas serológicas y de inmunidad celular. Para el estudio fueron incluídos pacientes con manifestaciones clínicas de lepra predominantemente de la forma multibacilar. Todos los pacientes fueron adultos con edad comprendida entre 20 y 39 años. El 58% correspondía a la forma clínica de Lepra Lepromatosa (LL) n= 81, el 29% a la forma (more) Borderline Lepromatosa (BL) n=41 y 10% a Borderline Borderline (BB) n=14. Solo el 3% fueron pacientes Borderline Tuberculoide (BT): 74% masculino y 26% femenino. El fenómeno reaccional más frecuente fue del tipo eritema nodoso leproso (ENL). Las proteínas micobacterianas ensayadas fueron: antígenos proteicos crudos totales de Mycobacterium leprae (MlSA), Mycobacterium bovis (MbSA y MbSA de excreción), antígeno proteico de excreción parcialmente purificado con una movilidad relativa de 30 kDa (Ml 30) y proteínas recombinantes de Mycobacterium (Mt70, Mb 65, Ml 36, 28, 18 y 10 kDa) encontrandose que las proteínas recombinantes (Ml10 kDa, Ml 36 kDa) a mayor carga bacilar presentaban una mayor reactividad serológica estadísticamente significativa (p= 0,0051 y 0,050 respectivamente). La proteína de 30 kDa fue predominantemente reconocida por anticuerpos de los pacientes multibacilares. Los resultados demuestran que el promedio de los valores de anticuerpos en pacientes no reaccionales fueron superiores en presencia de proteínas completas (MbSA y MbSA de exc) en comparación con el grupo de pacientes que presentaron fenómenos reaccionales (p=0,000567 y 0,000061 respectivamente) Este mismo comportamiento se observó frente a las proteínas micobacterianas individuales (30 kDa, 10 kDa y 36 kDa). La respuesta proliferativa de los linfocitos T en los pacientes multibacilares reaccionales y no reaccionales frente a las proteínas micobacterianas (MlSA, Ml 10 kDa, MbSA, MbSA de excreción) fue negativa en ambos grupos. Abstract in english The study was designed for evaluating immunological reactivity to various mycobacterial protein preparations using serological and cell-mediated immunological tests in patients with clinical leprosy signs, predominantly, with the multibacillary forms. All patients were adults with ages between 20 and 30 years. Fifty eight (n= 81) percent corresponded to Lepromatous Leprosy (LL), 29% (n= 41) to Borderline Lepromatous Leprosy (BL) and 10% (n=41) to Borderline Borderline Lep (more) rosy (BB); only 3% were Borderline Tuberculoid (BT) patients: 74% males and 26% females. The most frequent reactional phenomenon was of the Erythema Nodosum (ENL) type. The mycobacterial proteins tested were: total crude Mycobacterium leprae antigens (MISA); Mycobacterium bovis (MbSA and excretion MbSA); partially purified excretion protein antigen, with a 30kDa relative movility (Ml30); and recombinant M. leprae proteins (Mt70, Mb 65, Ml 36, 28, 18 and 10 kDa). Two of the recombinant proteins (Ml10 and Ml 36 kDa) presented a statiscally significant higher serological reactivity, directly related with a larger bacillary load (p= 0.0051 and 0.050 respectively). The 30 kDa protein was predominantly recognized by antibodies from multibacillary patients. Results show that mean antibody values were higher in non reactional patients when tested against complete proteins (MbSA and ex MbSA) when compared with the group of patients who presented reactional phenomena (p= 0.000567 and 0.000061, respectively). Comparing reactional with non reactional patients, it was seen that mean antibody values against complete proteins (MbSA and ex MbSA) were higher in non reactional individuals (p= 0.000567 and 0.000061, respectively). This same behavior occurred towards individual mycobacterial proteins (30, 10 and 36 kDa). The T lymphocyte prolypherative response in reactional and non reactional patients towards mycobacterial proteins (MlSA, Ml 10 kDa, MbSA, ex MbSA) was negative.

296

Analysis of the dormancy-inducible narK2 promoter in Mycobacterium bovis BCG.  

Upon depletion of oxygen, the obligate aerobe mycobacteria switch from growth to a state of non-replicating persistence or dormancy. Here, we report the first functional analysis of a dormancy-dependent mycobacterial promoter in Mycobacterium bovis BCG. Promoter probing using a 'lacZ reporter detected a dormancy-inducible promoter activity upstream of the coding sequence for the putative nitrite extrusion protein NarK2. Primer extension analysis mapped a transcriptional start point 47 bp upstream of the narK2 start codon. Deletion analysis revealed that the sequence -222 to -133 bp upstream from the transcriptional start point was required for basal and dormancy-inducible reporter expression. The sequence +1 to +47 downstream of the transcriptional start point had a strong inhibitory effect on the level of dormancy-induced beta-galactosidase activity. The identification of apparent activating and inhibiting regions suggests that the narK2 promoter is at least under dual control. PMID:10913697

297

Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages.  

Human alveolar macrophages (AMphi) undergo apoptosis following infection with Mycobacterium tuberculosis in vitro. Apoptosis of cells infected with intracellular pathogens may benefit the host by eliminating a supportive environment for bacterial growth. The present study compared AMphi apoptosis following infection by M. tuberculosis complex strains of differing virulence and by Mycobacterium kansasii. Avirulent or attenuated bacilli (M. tuberculosis H37Ra, Mycobacterium bovis bacillus Calmette-Guérin, and M. kansasii) induced significantly more AMphi apoptosis than virulent strains (M. tuberculosis H37Rv, Erdman, M. tuberculosis clinical isolate BMC 96.1, and M. bovis wild type). Increased apoptosis was not due to greater intracellular bacterial replication because virulent strains grew more rapidly in AMphi than attenuated strains despite causing less apoptosis. These findings suggest the existence of mycobacterial virulence determinants that modulate the apoptotic response of AMphi to intracellular infection and support the hypothesis that macrophage apoptosis contributes to innate host defense in tuberculosis. PMID:10657653

298

Immunochromatographic assays for detection of Mycobacterium tuberculosis: what is the perfect time to test?  

In this study, we aimed to correlate the analytical performance of SD BIOLINE TB Ag MPT64 Rapid Test kit (MPT64 assay) with the mycobacterial growth unit (GU) reported by the BACTEC MGIT 960 (MGIT 960) instrument. A total of 394 culture isolates reported positive by MGIT 960 were processed daily (until 'day 4') with the MPT64 assay until a positive MPT64 result was obtained and their GU values were noted daily before MPT64 testing. Based on this correlation of MPT64 positivity and corresponding GU values, a GU cut-off was determined. In the validation phase, with the experimentally determined GU cut-off value, 99.1% (576/581) of culture isolates were correctly identified as MTB within 2 days from instrument positivity. All results were available using a single-MPT64 assay strip, making the...

299

Comparative bioactivation of the novel anti-tuberculosis agent PA-824 in Mycobacteria and a subcellular fraction of human liver  

BACKGROUND AND PURPOSE PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F420)-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA-824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M.-tuberculosis and Mycobacterium smegmatis. EXPERIMENTAL APPROACH PA-824 (250-M) was incubated with the 9000g supernatant (S9) of human liver homogenates, purified Ddn, M.-tuberculosis and M.-...

300

Purification, crystallization and preliminary X-ray analysis of SGR6054, a Streptomyces homologue of the mycobacterial integration host factor mIHF  

The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b=69.35, c = 77.71-, =96.63, and diffracted X-rays to 2.22...

 
 
 
 
301

Translating basic science insight into public health action for multidrug- and extensively drug-resistant tuberculosis.  

Multidrug (MDR)- and extensively drug-resistant (XDR) tuberculosis (TB) impose a heavy toll of human suffering and social costs. Controlling drug-resistant TB is a complex global public health challenge. Basic science advances including elucidation of the genetic basis of resistance have enabled development of new assays that are transforming the diagnosis of MDR-TB. Molecular epidemiological approaches have provided new insights into the natural history of TB with important implications for drug resistance. In the future, progress in understanding Mycobacterium tuberculosis strain-specific human immune responses, integration of systems biology approaches with traditional epidemiology and insight into the biology of mycobacterial persistence have potential to be translated into new tools for diagnosis and treatment of MDR- and XDR-TB. We review recent basic sciences developments that have contributed or may contribute to improved public health response. PMID:22458269

302

TB, HIV-associated TB and multidrug-resistant TB on Thailand's border with Myanmar, 2006-2007  

Summary Objective To measure the burden and improve management of tuberculosis (TB), HIV-associated TB and MDR TB in Tak Province, Thailand, which borders Myanmar. Methods From September 2006 to August 2007, we collected uniform data about TB cases and enhanced human immunodeficiency virus (HIV) counselling and testing. We provided mycobacterial culture and drug-susceptibility testing in public or non-governmental organization facilities. Patients were classified by nationality and, for non-Thais, by migration status. Results Of 1662 TB cases in the 12-month period, 1087 (65%) occurred in non-Thais. Of non-Thais, 415 (38%) lived in Myanmar but crossed the border for healthcare. HIV infection was diagnosed in 18% of Thais compared with 12% of non-Thais (P < 0.01); HIV status was unknown for...

303

Immune reconstitution inflammatory syndrome in HIV-infected patients receiving antiretroviral therapy : pathogenesis, clinical manifestations and management  

The use of antiretroviral therapy (ART) to treat HIV infection, by restoring CD4+ cell count and immune function, is associated with significant reductions in morbidity and mortality. Soon after ART initiation, there is a rapid phase of restoration of pathogen-specific immunity. In certain patients, this results in inflammatory responses that may result in clinical deterioration known as 'the immune reconstitution inflammatory syndrome' (IRIS). IRIS may be targeted at viable infective antigens, dead or dying infective antigens, host antigens, tumour antigens and other antigens, giving rise to a heterogeneous range of clinical manifestations. The commonest forms of IRIS are associated with mycobacterial infections, fungi and herpes viruses. In most patients, ART should be continued and treatment for the associated condition optimized, and there is anecdotal evidence for the use of corticosteroids in patients who are severely affected. In this review, we discuss research relating to pathogenesis, the range of clinical manifestations, treatment options and prevention issues.

304

How environmental mycobacteria may predetermine the protective efficacy of BCG  

A proposal is made that there are 2 mechanisms of cell mediated response to mycobacteria, both of which produce positive tuberculin tests and that one of them is more protective against mycobacterial infection than is the other. These are referred to respectively as the Listeria-type and the Koch-type of responses. Contact with environmental mycobacteria will induce one or other of these types of response and BCG vaccination will enhance it. Thus in those places where the environmental species prime for the Listeria-type of response subsequent BCG vaccination will afford good protection from both tuberculosis and leprosy. Where the Koch-type of response frequently results from environmental contact BCG will be ineffective. Evidence if presented that a large contact with Mycobacterium scrofulaceum is prejudicial to at least one marker of BCG efficacy in Burma.

305

Iron transport in Mycobacterium smegmatis: uptake of iron from ferric citrate  

In mycobacterial growth medium 40 to 400 ..mu..M citrate was required to solubilize 2 ..mu..M /sup 55/Fe. This solubilized /sup 55/Fe was taken up into both iron-deficient and iron-sufficient washed cell suspensions of Mycobacterium smegmatis and Mycobacterium bovis BCG. Although the /sup 55/Fe was taken up into the cell, the citrate was not. The uptake system with M. smegmatis was not inhibited by electron transport inhibitors, uncouplers of oxidative phosphorylation, or thiol reagents and was saturable with iron at approximately 35 ..mu..M. The system was independent of the iron transport systems already known to exist in M. smegmatis: i.e., the two exochelin routes of assimilation as well as the mycobactin-salicylate system. It was not induced by the presence of 400 ..mu..M citrate in the growth medium, nor did the presence of citrate in the medium affect the production of either exochelin or mycobactin.

306

Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations  

The viability of BCG vaccine has traditionally been monitored using a colony-forming unit (CFU) assay. Despite its widespread use, results from the CFU assay can be highly variable because of the characteristic clumping of mycobacteria, their requirement for complex growth media, and the three week incubation period needed to cultivate slow-growing mycobacteria. In this study, we evaluated whether an ATP luminescence assay (which measures intracellular ATP content) could be used to rapidly estimate the viability of lyophilized and/or frozen preparations of six different BCG vaccine preparations - Danish, Tokyo, Russia, Brazil, Tice, and Pasteur - and two live attenuated mycobacterial vaccine candidates - a DlysADpanCD M. tuberculosis strain and a DmmaA4 BCG vaccine mutant. For every vaccin...

307

P012 Control of mycobacterial infection in ''humanized'' TNF knock-in mice  

Introduction: TNF is associated with the development of human immunopathologies and involved in host defense. TNF blockade to treat autoimmune inflammatory diseases revealed a crucial and non-redundant role of TNF for the control to tuberculosis infection. Tuberculosis is a chronic infection and sustained TNF and Th1 immune responses are crucial to control infection. To test and select specific human TNF inhibitors requires a special animal model. Therefore a mouse was developed where murine TNF was replaced by human TNF (human TNF knock-in) and we investigated host resistance to mycobacterial infection. Methods: Human TNF KI mice (humanized TNF mice) have been developed (Kruglov et al., in preparation) and studied for their capacity to generate protective cell-mediated immune responses an...

308

Marrow granulomas in coal workers' pneumoconiosis. A histologic study with elemental analysis  

The differential diagnosis of bone marrow granulomas is lengthy but has not previously included coal workers' pneumoconiosis. This report describes the first case in which noncaseating epithelioid granulomas containing anthracotic pigment and birefringent silica containing crystals were discovered in the marrow of a patient with progressive massive fibrosis of coal workers' pneumoconiosis. The silicotic nature of the crystals was confirmed using energy dispersive x-ray microanalysis. There was no evidence of a mycobacterial, fungal, or other etiology for the granulomas. This case demonstrates that coal workers' pneumoconiosis should be added to the differential diagnosis of bone marrow granulomas and that marrow examination may be a source of tissue for documentation of specific occupational exposures.

309

A life-threatening central nervous system-tuberculosis inflammatory reaction nonresponsive to corticosteroids and successfully controlled by infliximab in a young patient with a variant of juvenile idiopathic arthritis.  

We report a young patient with a variant of juvenile idiopathic arthritis, who, after 4 years of infliximab treatment, developed miliary tuberculosis (TB) with central nervous system involvement (meningitis and multiple tuberculomas). After anti-TB treatment, clinical and radiologic responses were observed, but severe cerebrospinal fluid and brain inflammatory reaction, nonresponsive to corticosteroids, persisted. It was considered a life-threatening paradoxical reaction based on initial cerebrospinal fluid isolation of Mycobacterium tuberculosis fully sensitive to primary anti-TB drugs. After 4 months in the hospital, infliximab was administered considering that infliximab is a potent tumor necrosis factor ? inhibiting agent that participates in the formation and preservation of granulomas and may help to modulate the exaggerated cell-mediated immune response against mycobacterial antigens. Clinical complications associated to brain inflammation resolved, and after 3 years of follow-up, the patient remains self-sufficient without neurologic sequels. PMID:22647865

310

Impaired health-related quality of life in pulmonary nontuberculous mycobacterial disease  

SummaryIntroductionThe impact of pulmonary nontuberculous mycobacterial (pNTM) disease on health-related quality of life (HRQL) has not been quantified. MethodsWe performed a prospective observational study of HRQL in 51 patients with pNTM disease. One generic (Short-form 36, version 2 -SF-36), and one pulmonary disease-specific instrument (St. George’s Respiratory Questionnaire - SGRQ) were administered to each subject. ResultsFifty-one patients with pNTM disease from one ambulatory clinic were enrolled. The mean (sd) age was 67 (10) years and 80% (41/51) were female. The most common causative NTM was MAC in 84% (43/51) followed by Mycobacterium abscessus in 8% (4/51). Radiographic disease type was nodular bronchiectasis in 71% (36/51) and fibrocavitary in 22% (11/51). For SF-36, m...

311

1,4-Diarylpiperazines and analogs as anti-tubercular agents: Synthesis and biological evaluation  

Despite progress in modern chemotherapy to combat tuberculosis, the causative pathogen Mycobacterium tuberculosis (M.tb.) is far from eradicated. Bacillary resistance to anti-mycobacterial agents, bacillary persistence and human immunodeficiency virus (HIV) co-infection hamper current drug treatment to completely cure the infection, generating a constant demand for novel drug candidates to tackle these problems. A small library of novel heterocyclic compounds was screened in a rapid luminometric in vitro assay against the laboratory M.tb. strain H37Rv. A group of amidines was found to have the highest potency and was further evaluated for acute toxicity against C3A hepatocytes. Next, the most promising compounds were evaluated for activity against a multi-drug resistant clinical isolate. T...

312

Pathological Features of Mycobacterium kansasii Infection in Black Bearded Sakis (Chiropotes satanas)  

Two black bearded sakis (Chiropotes satanas), kept in the same cage in a zoological park, developed multifocal subcutaneous nodular lesions and were diagnosed as having mycobacterial infection by microscopical examination of tissues and 16S rRNA polymerase chain reaction (PCR) and subsequent sequencing of amplicons. One animal died despite both being treated with prolonged antimicrobial therapy. This animal had disseminated disease with lesions in the liver, spleen, lymph nodes and brain. The lesions were granulomatous in nature, but organisms were not identified by acid-fast staining other than on an impression smear of one of the skin nodules. The granulomatous lesions lacked epithelioid macrophages, multinucleated giant cells and fibrous encapsulation. Mycobacterium kansasii was identif...

313

NOD2 triggers an interleukin-32???dependent human dendritic cell program in leprosy  

It is unclear whether the ability of the innate immune system to recognize distinct ligands from a single microbial pathogen via multiple pattern recognition receptors (PRRs) triggers common pathways or differentially triggers specific host responses. In the human mycobacterial infection leprosy, we found that activation of monocytes via nucleotide-binding oligomerization domain-containing protein 2 (NOD2) by its ligand muramyl dipeptide, as compared to activation via heterodimeric Toll-like receptor 2 and Toll-like receptor 1 (TLR2/1) by triacylated lipopeptide, preferentially induced differentiation into dendritic cells (DCs), which was dependent on a previously unknown interleukin-32 (IL-32)-dependent mechanism. Notably, IL-32 was sufficient to induce monocytes to rapidly differentiate ...

314

An integrated surrogate model for screening of drugs against Mycobacterium tuberculosis  

Objectives The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts the host cell environment for its active and dormant life cycle. It is evident that the lack of appropriate high-throughput screening of inhibitors within host cells is an impediment for the early stages of anti-tubercular drug discovery. We aimed to develop an integrated surrogate model that enhances the screening of large inhibitor libraries. Methods Different mycobacterial species were compared for their growth, drug susceptibility and intracellular uptake. A 6-well plate solid agar-based spot culture growth inhibition (SPOTi) assay was developed into a higher throughput format. The uptake and intracellular survival of Mycobacterium aurum within mouse macrophage cells (RAW 264.7) were ...

315

Photodynamic therapy as an adjunct to systemic treatment in a case with unilateral presumed vascularized choroidal tuberculous granuloma.  

Intraocular tuberculosis cannot be diagnosed easily in some clinical circumstances. A 29-year-old otherwise healthy woman who was initially misdiagnosed and treated empirically with systemic steroids and sulfamethoxazole-trimethoprim for toxoplasmosis was referred to us for severe visual loss. We instituted quadruple antituberculosis treatment upon chest medicine consultation as all attempts, including consecutive intravitreal antibiotic injections, could not prevent the enlargement of lesion. Systemic antituberculosis treatment halted the fulminant course but the granuloma became vascularized. Because photodynamic therapy (PDT) has very recently been shown to reduce viable mycobacterial cells in animal experiments we performed PDT, and the vascularized tuberculous granuloma was successfully treated. PDT may have an antimycobacterial effect besides its well-known antiangiogenic effect. PMID:18438611

316

Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis  

Surfactant protein D (SP-D), a lectin that recognizes carbohydrates via its C-type carbohydrate recognition domains (CRDs), regulates Mycobacterium tuberculosis (M.tb)-macrophage interactions via recognition of M.tb mannosylated cell wall components. SP-D binds to, agglutinates, and reduces phagocytosis and intracellular growth of M.tb. Species-specific variations in the CRD amino acid sequence contribute to carbohydrate recognition preferences and have been exploited to enhance the antimicrobial properties of SP-D in vitro. Here, we characterized the binding interaction between several wild-type and mutant SP-D neck + CRD trimeric subunits (NCRDs) and pathogenic and nonpathogenic mycobacterial species. Specific amino acid substitutions (i.e., the 343-amino-acid position) that flank the ca...

317

Construction and evaluation of a multistage Mycobacterium tuberculosis subunit vaccine candidate Mtb10.4-HspX  

To search for more effective vaccines to enhance the immunogenicity and protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin (BCG) and to control or even eradicate Mycobacterium tuberculosis (M. tuberculosis) in all stages of infection including the persister bacteria, antigens of Mtb10.4 (Rv0288) expressed in replicating bacilli and HspX (also called Acr, Hsp16.3, Rv2031c) highly expressed in dormant bacilli were fused together to construct a multistage fusion protein Mtb10.4-HspX (MH for short) without affinity tag with potential advantage for clinical use. The human T-cell responses to MH were evaluated for its immunogenicity. Furthermore, MH was emulsified in an adjuvant composed of N,N'-dimethyl-N,N'-dioctadecylammonium bromide (DDA) and mycobacterial cord factor trehalo...

318

[Differential diagnosis of rheumatoid granuloma].  

Rheumatoid granuloma (RG) is histomorphologically defined as a subcutaneous palisading granuloma with central fibrinoid necrosis. Clinically, it presents as a nodule typically localized at pressure points near the joints. From the rheumatic pathological point of view, the main diagnostic challenge is the differentiation of RG from granuloma anulare, especially if clinical information on the site of removal, known diseases, duration of illness, medication and existing American College of Rheumatology (ACR) criteria are missing. Other granulomatous lesions, such as mycobacterial infections, foreign body granulomas, necrobiosis lipoidica or sarcoidosis, can be differentiated from RG by histopathological criteria or by additional examinations such as pathogen specification or PCR. An immunohistochemical marker for the differential diagnosis of granulomas is not yet available. Diagnosis is based on conventional H-E staining, alcian blue-PAS staining, polarizing analysis or PCR. In the following article, the most important granulomatous entities in the differential diagnosis of RG are introduced and the main diagnostic characteristics are discussed. PMID:17043771

319

Producción in vitro de interferón gamma en ratones vacunados con ´Mycobacterium habana´ frente a antígenos de Mycobacterium tuberculosis/ In vitro interferon gamma production in mice vaccinated with ´Mycobacterium habana´ against Mycobacterium tuberculosis antigens  

Abstract in spanish Introducción: el desarrollo de nuevas vacunas antituberculosas requiere de la caracterización de la respuesta de inmunidad celular, inducida por el nuevo candidato vacunal frente a los antígenos principales de Mycobacterium tuberculosis. Objetivo: determinar el potencial inmunogénico de ´Mycobacterium habana´ TMC-5135, cuando se usa como vacuna subcutánea en ratones Balb/c. Métodos: en este estudio se inocularon subcutáneamente ratones Balb/c con la cepa viva ´M (more) ycobacterium habana´ TMC-5135 y se determinó la producción in vitro de IFN gamma en cultivos celulares de pulmón, bazo y ganglios inguinales estimulados con antígenos solubles totales y el antígeno 85b. Como grupo control se vacunaron ratones con BCG subcepa Phipps. Resultados: particularmente en los ganglios linfáticos inguinales, ambos antígenos indujeron mayor producción de IFN gamma en los ratones vacunados con ´Mycobacterium habana´que con BCG. Conclusiones: los resultados justifican la realización de nuevas investigaciones usando ´Mycobacterium habana´ TMC-5135 como candidato vacunal para prevenir la tuberculosis. Abstract in english Introduction: development of new antituberculosis vaccines requires the characterization of the cell-mediated immune responses induced by mycobacterial antigens. Objective: to determine the immunogenic potential of ´Mycobacterium habana´ TMC-5135 when using subcutaneous vaccine in Balb/c mice. Methods: in this study, Balb/c mice were inoculated subcutaneously with live ´Mycobacterium habana´ TMC-5135. The production of IFN gamma in cell suspensions obtained from the l (more) ungs, the spleen and the lymph nodes after stimulation with mycobacterial antigens Ag85b or culture filtrate antigens (CFA) was recorded. Results: the production of IFN gamma after stimulation with CFA and Ag85b was higher in mice vaccinated with ´M. habana´ than in animals immunized with BCG. Conclusions: these results encourage new research on ´M. habana´ as vaccinal candidate against tuberculosis.

320

A microfluidic system for long-term time-lapse microscopy studies of mycobacteria.  

Phenotypic heterogeneity in bacterial populations is thought to contribute to a number of important phenomena including sporulation and persistence. The latter has clinical implications in many diseases such as tuberculosis, where persistence of Mycobacterium tuberculosis within the human host is believed to be the root cause of latent tuberculosis and the ability of a minority population of cells to survive antibiotic exposure, despite being genetically identical to the bulk population that are killed. However, phenotypic variations caused by non-genetic mechanisms are difficult to study because of the transient nature of the persistent state and thereby the requirement to observe individual cells in real-time. Recently, microfluidics, combined with time-lapse microscopy, has become a powerful tool for studying population heterogeneity in bacteria. However, growth and replication of mycobacterial cells provide particular problems for the development of microfluidic systems due to their tendency to grow in three dimensions. We here describe a novel microfluidic device for the observation of growth and antibiotic killing in individual mycobacterial cells. We constructed a microfluidic device suitable for studying single cell behavior in mycobacteria. The growth of single cells of Mycobacterium smegmatis expressing green fluorescent protein was monitored using a confocal laser scanning microscope. Within the device M. smegmatis cells were tightly confined within a hydrogel matrix thus promoting planar growth. Cell growth and killing was observed in the device with dead cells highlighted by uptake of propidium iodide. Conclusions/Significance. We demonstrate that our device allows real-time analysis and long-term culture of single cells of mycobacteria, and is able to support the study of cell death during the application of antibiotics. The device will allow observation of individual cells' cell genealogy to be determined and direct observation of rare states, such as persistence. PMID:22954584

 
 
 
 
321

Global tuberculosis control: WHO report.  

Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM) and trehalose monomycolate (TMM), the apolar phthiocerol dimycocersates (PDIMs), triacyl glycerol (TAG), pentacyl trehalose (PAT), phenolic glycolipid (PGL), and mono-mycolyl glycerol (MMG). Polar lipids identified included glucose monomycolate (GMM), diphosphatidyl glycerol (DPG), phenylethanolamine (PE) and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs). These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1?, TNF? and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen. PMID:19388905

322

A novel interaction linking the FAS-II and phthiocerol dimycocerosate (PDIM) biosynthetic pathways.  

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall. PMID:18703500

323

A Novel Interaction Linking the FAS-II and Phthiocerol Dimycocerosate (PDIM) Biosynthetic Pathways*S?  

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II ?-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.

324

BCG Vaccination Enhances Resistance to M. Tuberculosis Infection in Guinea Pigs Fed a Low Casein Diet  

In order to examine the relationship between malnutrition and tuberculosis development in vivo, a malnourished guinea pig model fed with a low casein (5%) diet was developed. After being fed with the low casein diet, the guinea pigs were infected with Mycobacterium (M.) tuberculosis Kurono strain by aerosol infection, and seven weeks later were subjected to histopathologic examination, colony-forming unit (CFU) assay, fluorescence-activated cell sorter (FACS) analysis and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for interferon (IFN)-?, tumor necrosis factor (TNF)-?, interleukin (IL)-12 and inducible nitric oxide synthase (iNOS) mRNA. Another group of guinea pigs were vaccinated subcutaneously with 106 CFU BCG Tokyo for three weeks and then similarly infected by aerosol. Eighty-eight % (7/8) of the malnourished guinea pigs succumbed to mycobacterial infection within 85 days after infection, while the malnourished guinea pigs vaccinated with BCG Tokyo survived. CFU assay showed that lung and splenic CFUs were higher in the low casein diet-fed groups than in the control diet (20% casein)-fed groups, although both groups had significantly lower CFUs after vaccination with BCG Tokyo (p < 0.01). Examination of lung histopathology revealed that pulmonary granulomas were large and disorganized in the groups fed the low casein diet. The number of visible lesions on the surfaces of the fixed lungs in guinea pigs fed control diet + BCG and low casein diet + BCG was low significantly. Pan T-, CD4-, CD8- and Mac antigen-positive cells were also recognized in the infected lung tissues of low casein-fed guinea pigs and Pan T-, CD4- and Mac antigen-positive cells increased after vaccination with BCG Tokyo. Expression of IFN-?, TNF-?, IL-12 and iNOS mRNA was also recognized in the infected lung tissues of low casein-fed guinea pigs and IFN-? and TNF-? mRNA expression was enhanced with BCG vaccination. These results indicate that malnutrition exacerbates mycobacterial infection and that malnourished infected hosts may be protected by BCG vaccination.   

325

Quantiferon TB Gold and tuberculin skin tests for the detection of latent tuberculosis infection in patients treated with tumour necrosis factor alpha blocking agents.  

OBJECTIVES: The risk of activation of latent tuberculosis infection (LTBI) is increased in patients treated with anti-TNF-? drugs. Tuberculin skin test (TST) and Quantiferon-TB Gold test (QFT) are used to detect LTBI before and during anti-TNF-? treatment. We describe here a relation of these tests at various timepoints and also longitudinal QFT data. METHODS: Study group consisted of 305 patients with several rheumatic inflammatory diseases treated and/or scheduled for anti-TNF-? drugs. The QFT was performed in 303 patients during therapy and in 177 patients also during screening. The TST was used in 284 patients. Both tests simultaneously were utilised in 360 instances. RESULTS: Twenty-two patients were QFT positive; 3.9% before and 5.9% during anti-TNF-? treatment. Two patients who became QFT positive developed active tuberculosis. The TST was positive in 42% and 38% of patients before and during treatment, respectively. There was poor agreement between the two tests. Patients on glucocorticoids had a negative TST more frequently. The IFN-? response to mycobacterial antigens significantly increased after application of tuberculin, but never reached the positive threshold. There was a significant increase in mitogen-induced IFN-? production after initiation of anti-TNF-? therapy. CONCLUSIONS: Poor correlation between the QFT and TST renders the TST non-specific for LTBI. QFT is more specific to detect LTBI and conversion to a positive result may predict active TB. An increase in IFN-? production in response to mycobacterial antigens is seen when the TST is performed before the QFT. Mitogen-induced IFN-? production increases after initiation of anti-TNF-? therapy. PMID:23101473

326

Stem cell therapies for spinal cord injury  

Background Mortality in the first months of antiretroviral therapy (ART) is a significant clinical problem in sub-Saharan Africa. To date, no post-mortem study has investigated the causes of mortality in these patients. Methods HIV-positive adults who died as in-patients at a Johannesburg academic hospital underwent chart-review and ultrasound-guided needle autopsy for histological and microbiological examination of lung, liver, spleen, kidney, bone marrow, lymph node, skin and cerebrospinal fluid. A clinico-pathologic committee considered all available data and adjudicated immediate and contributing causes of death. Results Thirty-nine adults were enrolled: 14 pre-ART, 15 early-ART (7–90 days), and 10 late-ART (>90 days). Needle sampling yielded adequate specimen in 100% of kidney, skin, heart and cerebrospinal fluid samples, 97% of livers and lungs, 92% of bone marrows, 87% of spleens and 68% of lymph nodes. Mycobacterial infections were implicated in 69% of deaths (26 of 27 of these due to M. tuberculosis), bacterial infections in 33%, fungal infections in 21%, neoplasm in 26%, and non-infectious organ failure in 26%. Immune reconstitution inflammatory syndrome (IRIS) was implicated in 73% of early-ART deaths. Post-mortem investigations revealed previously undiagnosed causes of death in 49% of cases. Multiple pathologies were common with 62% of subjects with mycobacterial infection also having at least one other infectious or neoplastic cause of death. Conclusions Needle biopsy was efficient and yielded excellent pathology. The large majority of deaths in all three groups were caused by M. tuberculosis suggesting an urgent need for improved diagnosis and expedited treatment prior to and throughout the course of antiretroviral therapy. Complex, unrecognized co-morbidities pose an additional challenge. PMID:21909829

327

Genetics Home Reference: Xeroderma pigmentosum  

... cases. Read more about the ERCC2 , ERCC3 , POLH , XPA , and XPC genes. See a list of genes ... Gene Tests: ERCC5-Related Xeroderma Pigmentosum Gene Tests: XPA-Related Xeroderma Pigmentosum Gene Tests: XPC-Related Xeroderma ...

328

Ulcera lingual como signo único de infección recurrente por micobacteria en un paciente con VIH/SIDA/ Lingual ulcer as the only sign of recurrent mycobacterial infection in an HIV/AIDS-infected patient  

Abstract in spanish Se describe un paciente con VIH/SIDA en el que se identificó una infección por micobacteria en la mucosa bucal, probablemente tuberculosis, en un centro de referencia para VIH/SIDA de la Ciudad de México. El propósito del presente informe es describir los hallazgos clínicos e histológicos en un paciente con VIH/SIDA, quien después de haber sido tratado exitosamente para tuberculosis ganglionar 4 años antes, presentó una úlcera lingual como único signo que sugir (more) ió recurrencia de infección por micobacteria, probablemente tuberculosis. Hombre de 39 años de edad, atendido desde 1991 en el Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán", por el diagnóstico de infección con VIH. En 1999, el paciente presentó tuberculosis ganglionar, recibiendo tratamiento antifímico con involución de las adenopatías y desaparición de los síntomas sistémicos. En mayo del 2003 acudió a consulta por presentar una úlcera superficial en lengua, dolorosa, de 4 meses de evolución, de 0.7 cm. de diámetro, bien circunscrita, crateriforme, con bordes ligeramente elevados, irregulares e indurados. El estudio histopatológico mostró inflamación granulomatosa crónica con células gigantes multinucleadas sugestivas de infección por micobacteria, lo cual hizo pensar en recurrencia de tuberculosis, por lo que se indicó rifampicina, pirazinamida, etambutol y estreptomicina. En junio del 2003 el paciente inició TARAA, que incluyó dos ITRAN y un ITRNN. La lesión lingual evolucionó favorablemente, con cicatrización parcial a la primera semana y remisión total a los 45 días del inicio del tratamiento antifímico; a los 7 meses de seguimiento permanece sin lesión. El presente caso tiene la particularidad de que la úlcera lingual fue la única manifestación de infección por micobacteria, sugestiva de tuberculosis, en un paciente con VIH/SIDA, que pudo ocurrir como resultado de la recurrencia del episodio previo de TB ganglionar. Abstract in english The report describes an HIV/AIDS patient seen at a referral center in Mexico City, in whom a mycobacterial infection in the oral mucosa, probably tuberculosis (TB) was identified. The purpose is to describe the clinical and histological findings in an HIV-infected patient, who after being treated successfully for tuberculous lymphangitis 4 years ago, presented with a lingual ulcer as the only suggestive sign of recurrence of mycobacterial infection, probably M. tuberculos (more) is. A 39-year-old man seen inthe HIV clinic of the Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán" in Mexico City since 1991 for HIV infection. In 1999 the patient developed tuberculous lymphangitis; he was managed with a 4-drug regimen for 12 months, with improvement of local and systemic symptoms. In May of 2003, the patient presented a painful superficial lingual ulcer, 0.7 cm in diameter, well circumscribed, crateriform with slightly elevated, irregular and indurated borders, of 4 months duration. The histopathological examination showed chronic granulomatous inflammation with giant multinucleated cells, suggestive of mycobacterial infection, and recurrence of TB was considered. Rifampin, isoniazide, pyrazinamide, ethambutol and streptomycin were administered. The lingual lesion improved with partial healing at the first week and total remission at 45 days after the beginning of the antituberculous treatment. In June, 2003, the patient began highly active antiretroviral therapy (HAART) that included two NRTIs and one NNRTI. At 7 months of follow-up, the patient remains free of lingual lesions. The particularity of the present case is that the lingual ulcer was the only sign of infection by mycobacteria, suggestive of TB, in an HIV/AIDS patient that probably represented a recurrence of a previous episode.

329

Tuberculosis precolombina en el noroeste argentino (NOA): El cementerio de Rincón Chico 21 (RCH 21), Santa María, Catamarca/ Precolumbian tuberculosis in Northwest Argentina: Rincón Chico 21 cemetery (rch21), Santa María, Catamarca  

Abstract in spanish La excavación sistemática de sitios de enterratorio colectivo posibilita la recuperación de conjuntos esqueléticos en los cuales algunos individuos exhiben evidencia ósea indicadora de condiciones patológicas de origen infeccioso. La presencia y distribución de los marcadores óseos pueden reflejar la forma en que los individuos de una población respondieron o reaccionaron ante la ocurrencia de enfermedades y condiciones de morbilidad, como las ocasionadas por inf (more) ecciones micobacterianas. Presentamos los resultados de una investigación puesta en marcha para aportar al conocimiento de las condiciones paleopatológicas que pudieron haberse desarrollado entre los pueblos aborígenes del NOA. Las patologías observadas en individuos del cementerio de Rincón Chico 21 (RCh21) fueron registradas, descriptas, analizadas, discutidas e interpretadas como ocasionadas por infecciones micobacterianas (Complejo Mycobacterium tuberculosis). En el análisis crítico y la discusión, relacionados con la dinámica de las interacciones bioculturales, se considera información de asociaciones contextuales y cronológicas. Concluimos que una enfermedad de tipo tuberculosis estuvo presente entre las poblaciones aborígenes prehistóricas del NOA. En RCh21, el hallazgo de seis individuos con patologías sobre un total de 70 excavados soporta la existencia de esta enfermedad en el valle santamariano, temporalmente relacionada con momentos finales de Desarrollos Regionales y también con una creciente influencia imperial expansiva incaica. Abstract in english Systematic excavations of collective burial sites makes possible the recovery of sets of skeletons in which some skeletons show bony evidence of infectious pathological conditions. The presence and distribution of bone markers may reflect the way in which individuals responded to diseases and states of morbidity, such as those caused by mycobacterial infection. In this paper, the results of ongoing research on pathological conditions in prehistoric peoples of Northwest Ar (more) gentina are presented. Bone tissue pathologies observed in individuals from Rincón Chico 21 cemetery were recorded, described, analyzed, discussed and, finally, interpreted as caused by mycobacterial infections (Mycobacterium tuberculosis Complex). In the critical analysis and discussion of the dynamics of biocultural interactions information related to contextual associations and chronology was taken into account. It was concluded that a TB like disease was present in prehistoric populations from Northwest Argentina. At Rincón Chico 21, six individuals out of seventy so far excavated provided evidence of the existence of the disease in the Santa María Valley between the end of the Late Ceramic Period and the onset of the expansion of the Inca Empire.

330

Diagnóstico bacteriológico de la tuberculosis en Argentina: resultados de una encuesta nacional/ Bacteriological diagnosis of tuberculosis in Argentina: results of a national survey  

Abstract in spanish Con el objetivo de evaluar recursos, prácticas y medidas de bioseguridad utilizados en el diagnóstico bacteriológico de tuberculosis y determinar la utilidad y alcance de las técnicas empleadas, se llevó adelante una encuesta transversal a laboratorios del sector público de Argentina utilizando un cuestionario estructurado. El análisis de los resultados mostró que la densidad de centros de microscopía y cultivo se encuentra al nivel o encima de lo recomendado por (more) Organización Mundial de la Salud. El programa de control de calidad de microscopía cubre mayoritariamente los laboratorios con alta carga de trabajo. El promedio de baciloscopías/paciente fue bajo (1,6). El 25% de los laboratorios de cultivo empleaban protección respiratoria inadecuada. El aporte del cultivo a la confirmación de casos pulmonares en las jurisdicciones estuvo asociado a la proporción de muestras cultivadas. Globalmente, el porcentaje de pacientes pulmonares baciloscopía-negativa que fue confirmado bacteriológicamente fue de 18.9%; mientras que la may-oría de los casos extrapulmonares se confirmó sólo por cultivo (71,3%). Es prioritario aún el incremento del número de esputos estudiados por baciloscopía y cultivo, así como aumentar la cobertura de programa de control de calidad de microscopía; es fundamental proveer al personal de laboratorio de adecuada protección respiratoria y evaluación médica regular. Abstract in english In order to assess laboratory resources, practices, and biosafety measures during mycobacterial testing and determine the usefulness and scope of mycobacterial techniques, a cross-sectional survey of public laboratories was conducted in Argentina using a structured questionnaire. Sputum smear analysis showed that both smear and culture testing centers are being provided at or above the WHO recommended density for such facilities. The microscopy quality assessment program (more) covered most high-demand laboratories. Mean number of sputum smears per patient was low (1.6). The use of inadequate personal respiratory protection was identified in 25% of culture laboratories. Jurisdictions that cultured a higher proportion of their smear-tested sputa identified a higher proportion of smear-negative cases. The percentage of smear negatives among all bacteriologically confirmed pulmonary cases was 18.9%, while most extrapulmonary cases were confirmed by culture (71.3%). In conclusion, increasing the number of sputa studied by microscopy and culture (while expanding the coverage of the quality assessment program) is a priority; adequate respiratory protection and regular medical evaluation of laboratory staff is still needed in some laboratories.

331

Improving tuberculosis control through the partnership between university and the health system/ Qualificar o controle da tuberculose mediante a parceria entre a Universidade e o Sistema de Saúde  

Abstract in portuguese INTRODUÇÃO: O controle da tuberculose (TB) está relacionado com a disponibilidade de métodos de diagnóstico microbiológico qualificados, no entanto a microscopia com a sua limitada sensibilidade é o único método disponível em muitos locais.O objetivo deste estudo foi avaliar a introdução da cultura, teste de sensibilidade aos antimicrobianos (TSA) e genotipagem na rotina de um Programa Municipal de Controle da Tuberculose. MÉTODOS:A baciloscopia direta do esc (more) arro e cultura em Ogawa-Kudoh foram realizadas em 1.636 amostras de 787 pacientes. O TSA das culturas positivas foi realizado pelo método de microdiluição e a genotipagem por Mycobacterial Interspersed Repetitive Units - Variable Number Tandem Repeat (MIRU-VNTR). RESULTADOS:Foram identificados 91 pacientes com TB, com a cultura aumentando em 32% a detecção de casos em comparação com a microscopia; a resistência adquirida foi de 3,3% e a genotipagem mostrou alta diversidade genética. CONCLUSÕES: O cultivo em Ogawa-Kudoh contribuiu significativamente para o aumento na detecção de casos e é adequado para ser implementado em locais com poucos recursos. A taxa de resistência adquirida foi menor do que a relatada em recente inquérito nacional. A alta diversidade genética está possivelmente relacionada à alevada prevalência de TB na população, detecção precoce e tratamento adequado dos pacientes.A interação entre a pesquisa e serviço de saúde pública é importante para reorientar a prática, transferir tecnologia e melhorar o controle da TB. Abstract in english INTRODUCTION: Tuberculosis (TB) control is linked to the availability of qualified methods for microbiological diagnostics; however, microscopy with limited sensitivity is the only method available in many locations. The objective of this study was to evaluate the introduction of culture, drug susceptibility testing (DST), and genotyping in the routine of a Municipal Program of Tuberculosis Control. METHODS: Direct microscopy of sputum and culture in Ogawa-Kudoh were perf (more) ormed on 1,636 samples from 787 patients. DST of positive cultures was performed by resazurin microtiter assay and genotyping by mycobacterial interspersed repetitive units-variable number tandem repeat. RESULTS: A total 91 patients with TB were identified. The culture increased case detection by 32% compared with the microscopy; acquired resistance was 3.3% and the genotyping showed high genetic diversity. CONCLUSIONS: Ogawa-Kudoh contributed significantly to the increase in case detection and is suitable for implementation in poor-resource locations. The acquired resistance rate was lower than that reported in a recent Brazilian survey. The high genetic diversity is possibly related to the high TB prevalence in the population, as well as to early detection and suitable treatment of patients. The interaction between research and health care is important for reorienting the practice, transferring technology, and improving TB control.

332

Micobacteriose por Mycobacterium marinum em "linguado" Paralichthys orbignyanus e em "barber goby" Elacatinus figaro: diagnóstico histopatológico e imuno-histoquímico/ Micobacteriosis by Mycobacterium marinum in Brazilian flounder Paralichthys orbignyanus and barber goby Elacatinus figaro: histopathological and imunohistochemical diagnosis  

Abstract in portuguese As micobacterioses afetam várias espécies de peixes, tanto comerciais como ornamentais. É uma enfermidade sistêmica com formação de granulomas, conhecida como micobacteriose de peixes. O desenvolvimento da aquicultura tem aumentado o número de casos com essa enfermidade que muitas vezes é de difícil diagnóstico. Neste trabalho apresentamos dois casos de micobacterioses em Paralichthys orbignyanus e Elacatinus figaro provenientes do Laboratório de Piscicultura E (more) stuarina e Marinha da Universidade Federal do Rio Grande (FURG). As micobactérias de peixes possuem menos ácidos graxos que as de mamíferos, por isso foi utilizado o protocolo de coloração de Fite-Faraco, uma vez que o protocolo de coloração clássico de Ziehl Neelsen pode resultar em falsos negativos. Para determinar o tipo de micobactéria foi utilizada a imuno-histoquímica com a qual se pode diagnosticar a presença de Mycobacterium marinum. A enfermidade pode ser transmitida para o homem e com esse trabalho ressalta-se a necessidade de fazer o diagnóstico correto e implantar medidas de proteção para as pessoas que estão em contato com os peixes infectados. Abstract in english Mycobacteriosis affects various species of fish, including those cultured with commercial purposes. It is a chronic systemic with granuloma forming disease known as fish mycobacteriosis. In the last few years the interest of fish culture has been increasing due to the reduction of the natural fish stocks. However the intensification of fish culture has collaborated to the development of diseases such as mycobacteriosis, causing losses due to the chronic characteristics an (more) d difficult diagnosis. This study is to report two cases of mycobacteriosis observed at "Laboratório de Piscicultura Estuarina e Marinha of the Universidade Federal do Rio Grande (FURG)", one for Brazilian flounder Paralichthys orbignyanus and another for barber goby Elacatinus figaro. The Fite-Faraco staining protocol was used. Membranes of mycobacterial cells contain a waxy substance composed of mycolic acids. These are ß-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species. The fish mycobacterium is much less acid and alcohol fast than the tubercle bacillus and the classic Ziehl-Neelsen Staining Protocol may give false results. The presence of mycobacterial antigen lesions was studied by immunohistological methods using monoclonal antibodies to Mycobacterium marinum. Humans may be infected by these bacteria and develop cutaneous granuloma. In this paper the necessity is stressed for a correct diagnosis of the disease, contributing to the prevention of its establishment in aquatic facilities, and thus, preserving cultured fish as well as human beings from infection.

333

Enfermedades micobacterianas diseminadas en pacientes con VIH/SIDA. Evaluación de los hemocultivos por método rápido/ Disseminated mycobacterial infections in patients with HIV/AIDS. Evaluation of blood cultures  

Abstract in spanish Mil cuarenta hemocultivos correspondientes a 451 enfermos uruguayos con SIDA y diagnóstico clínico de micobacteriosis diseminada fueron evaluados entre 1999 y 2003. Las muestras fueron procesadas en el Centro de Referencia Nacional para Micobacterias (Montevideo, Uruguay), utilizando el sistema de hemocultivos automatizado para micobacterias MB - BacT (BioMérieux). Se detectaron 45 muestras positivas (4,3%) correspondientes a 26 enfermos (promedio 2,3 muestras por paci (more) ente). En 10/26 casos se identificó M. avium complex (MAC) y en 13/26 el germen aislado fue M. tuberculosis. El tiempo medio de incubación fue de 12,4 días (intervalo 6-19 días) para MAC y de 22,6 días (intervalo 7-35 días) para M. tuberculosis. El hemocultivo ha demostrado ser la mejor muestra para la confirmación bacteriológica de las enfermedades micobacterianas diseminadas cuando se estudian por lo menos 2 muestras por paciente. La frecuencia de aislamientos de M. tuberculosis y MAC aislados en pacientes con SIDA en Uruguay, corresponde a la de un país con una moderada prevalencia de tuberculosis. Abstract in english One thousand-forty blood cultures corresponding to 451 Uruguayan patients with AIDS and clinic diagnosis of disseminated mycobacterial infection were evaluated between 1999 and 2003. Samples were processed in the NationalReferenceCenter for Mycobacteria (Montevideo, Uruguay), using the automated blood culture system for mycobacteria MB -BacT (BioMérieux). Forty-five positive samples were detected (4.3%) corresponding to 26 patients with AIDS (average 2.3 samples per pati (more) ent). In 10/26 patients M. avium complex (MAC) was identified and in 13/26 the isolated germ was M. tuberculosis. The average time of incubation was of 12.4 days (range 6-19 days) for MAC and of 22.6 days (range 7-35 days) for M. tuberculosis. Blood culture has demonstrated to be the best sample for the bacteriological confirmation of the disseminated mycobacterial infections when at least 2 samples by patient are studied. The frequency of isolates of M. tuberculosis and MAC in AIDS patients is according with a moderate prevalence of tuberculosis in Uruguay.

334

Rapid mycobacterial liquid culture-screening method for Mycobacterium avium complex based on secreted antigen-capture enzyme-linked immunosorbent assay.  

Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended. PMID:19261776

335

Identificación molecular de micobacterias del complejo tuberculoso: Estandarización de un método de pcr basado en secuencias conservadas de la región espaciadora 16s-23s del operón de adn ribosomal/ Molecular identification of micobacteria in the tuberculous complex: standarization of a pcr method based on conserved sequences of the 16s-23s spacing region in the ribosomal dna operon  

Abstract in spanish Con la finalidad de mejorar el diagnóstico de la tuberculosis, y basados en el estudio de la secuencia de la región espaciadora 16S-23S del operón de ADN ribosomal de treinta especies de micobacterias, se diseñó un método de identificación de micobacterias pertenecientes al complejo tuberculoso utilizando la Reacción en Cadena de la Polimerasa (PCR) en combinación con enzimas de restricción. Para ello se diseñaron cuatro primers (TTS, SS2, S1A, S1B) y se emplea (more) ron las enzimas NheI y MscI. Se estandarizó la técnica de PCR y digestión enzimática, utilizando seis aislados micobacterianos (M. tuberculosis, M. kansasii, M. fortuitum, M. smegmatis, M. marinum y M. terrae), lográndose la amplificación con los primers diseñados y digestión enzimática para cada una de las micobacterias. Podemos concluir que la PCR, en combinación con el uso de enzimas de restricción, constituye un método rápido, sencillo, sensible y específico para la identificación de especies micobacterianas pertenecientes al complejo tuberculoso (M. tuberculosis, M. africanum y M. bovis). Sin embargo, es importante tener en cuenta algunas recomendaciones que reducirán los problemas de especificidad y falsos positivos obtenidos durante el estudio. Una de ellas sería la utilización del fragmento TTS-SS2 en el PCR sólo como control de la presencia e integridad del ADN bacteriano, y el PCR en combinación con la digestión con NheI solamente en el fragmento TTS-S1B. Abstract in english In order to improve the diagnosis of tuberculosis, a methodology to identify mycobacteria belonging to the tuberculosis complex has been designed, based in sequence comparison of the 16S-23S region of the ribosomal DNA operon from 30 mycobacterial species. The method uses a combination of Polymerase Chain Reaction (PCR) with four primers (TTS, SS2, S1A and S1B), and digestion by restriction enzymes NheI and MscI. The technic was standarized using six mycobacterial isolate (more) s (M. tuberculosis, M. kansasii, M. fortuitum, M. smegmatis, M. marinum, M. terrae), obtaining amplification and digestion in all cases. We conclude that the combination of PCR and digestion with restriction enzymes, represent a fast, simple, sensitive and specific method for identification of mycobacteria belonging to the tuberculosis complex (M. tuberculosis, M. bovis and M. africanum). However it is important to consider few recommendations to help decrease the unspecific amplification problems that appear during the study. One of the most important is the use of TTS-SS2 amplicon only as control for the presence and integrity of the bacterial DNA, and the combination of PCR and Nhe1 digestion only in the TTS-S1B fragment.

336

Severe febrile illness in adult hospital admissions in Tanzania: a prospective study in an area of high malaria transmission.  

Severe febrile illness is a major cause of adult hospital admission in Africa. Studies of non-malarial fever come largely from children or from high HIV prevalence settings. This prospective study of adult admissions with severe febrile illness in a malaria-endemic area with moderate/low HIV prevalence investigated admission diagnosis as well as final diagnosis based on results of investigations. Severe malaria was the admission diagnosis in 148/198 (74.7%) cases. Plasmodium falciparum was identified in 38/188 (20.2%) admissions and 26/198 (13.1%) were bacteraemic, with 13/25 (52%) prescribed empirical antibiotics. HIV was equally common among those with (16/37; 43.2%) and without P. falciparum (50/138; 36.2%) (p=0.44). In 6/22 (27.3%) deaths, blood cultures were positive for a pathogen, with Streptococcus pneumoniae, Escherichia coli and non-Typhi Salmonella predominating. Chest radiography was suspicious for bacterial/mycobacterial disease in 5/22 additional deaths. Systemic inflammatory response syndrome criteria were more sensitive but less specific than WHO severe malaria criteria for predicting mortality. Malaria is overdiagnosed in adults with severe febrile illness and was not associated with mortality in the absence of co-infection in this high-incidence setting. Adults with severe febrile illness should be tested for malaria and HIV using rapid, sensitive tests. Early antibiotic use should be promoted. Improved diagnostics for invasive bacterial disease are needed. PMID:23022040

337

A case of Mycobacterium goodii prosthetic valve endocarditis in a non-immunocompromised patient: use of 16S rDNA analysis for rapid diagnosis.  

ABSTRACT: BACKGROUND: Mycobacterium goodii is a rare cause of significant infection. M. goodii has mainly been associated with lymphadenitis, cellulitis, osteomyelitis, and wound infection. CASE PRESENTATION: A case of a 76-year-old Caucasian female is presented. The patient developed a prosthetic valve endocarditis caused by M. goodii. She had also suffered from severe neurological symptoms related to a septic emboli that could be demonstrated as an ischemic lesion found on CT of the brain. Transesophageal echocardiography verified a large vegetation attached to the prosthetic valve. Commonly used blood culture bottles showed growth of the bacteria after 3 days. CONCLUSIONS: Although M. goodii is rarely involved in these kinds of severe infections, rapidly growing mycobacteria should be recognized during conventional bacterial investigations and identified by molecular tools such as analysis of 16S rDNA. Species identification of nontuberculous mycobacteria is demanding and is preferably done in collaboration with a mycobacterial laboratory. An early diagnosis provides the opportunity for adequate treatment. In the present case, prolonged antimicrobial treatment and surgery with replacement of the prosthetic valve was successful. PMID:23151090

338

Rapid presumptive identification of the Mycobacterium tuberculosis-bovis complex by radiometric determination of heat stable urease  

Simple and rapid Bactec methodologies for the determination of neat (unaltered) and heat stable urease activity of mycobacteria are presented. Clinical isolates (63) and stock cultures (32)--consisting of: M. tuberculosis (19), M. bovis (5), M. kansasii (15), M. marinum (4), M. simiae (3), M. scrofulaceum (16), M. gordonae (6), M. szulgai (6), M. flavescens (1), M. gastri (1), M. intracellulare (6), M. fortuitum-chelonei complex (12), and M. smegmatis (1)--were tested for neat urease activity by Bactec radiometry. Mycobacterial isolates (50-100 mg wet weight) were incubated at 35 degrees C for 30 minutes with microCi14C-urea. Urease-positive mycobacteria gave Bactec growth index (GI) values greater than 100 units, whereas urease-negative species gave values less than 10 GI units. Eighty-three isolates possessing neat urease activity were heated at 80 degrees C for 30 minutes followed by incubation at 35 degrees C for 30 minutes with 1 microCi14C-urea. Mycobacterium tuberculosis-bovis complex demonstrated heat-stable urease activity (GI more than 130 units) and could be distinguished from mycobacteria other than tuberculosis (MOTT), which gave GI values equal to or less than 40 units.

339

Immunogenicity of bacillus Calmette-Guérin (BCG) in bovine neonates under traditional farming in central Ethiopia.  

Vaccination is an alternative method of controlling bovine tuberculosis (BTB) particularly in developing countries where the test and slaughter control method is not acceptable socially and economically. The objective of this study was to evaluate the immunogenicity of bacillus Calmette-Guerin (BCG) vaccination in bovine neonates. Twelve BTB free bovine neonates (six vaccinated with 0.5 mL of 2.4 x 10(6) CFU of BCG and six control) with age less than one month were used for this study. Interferon gamma (IFN-gamma) and antibody responses to mycobacterial antigens were determined at 0, 1, 3, 7, and 13 weeks of post-vaccination. The mean IFN-gamma response to bovine purified protein derivative, PPD in vaccinated group (Mean +/- SEM, 0.541 +/- 0.216) was greater than the mean IFN-gamma response to bovine PPD in non-vaccinated group (Mean +/- SEM, 0.253 +/- 0.101). Within the vaccinated group, the mean IFN-gamma response was greater in cross breed (Mean +/- SEM, 0.779 +/- 0.458) than in zebu breeds (0.303 +/- 0.178). No detectable antibody was observed in both vaccinated and non-vaccinated groups for 13 weeks post vaccination. A sharp rise in IFN-gamma response to bovine PPD was observed between at week 3, and then from week 3 to 7 post-vaccination, there was rapid falling of IFN-gamma response after which the response remained more or less constant in the consecutive weeks. This preliminary study showed the immunogenicity of BCG in bovine neonates under traditional cattle farming in Ethiopia. PMID:20391027

340

Galactose-derived phosphonate analogues as potential inhibitors of phosphatidylinositol biosynthesis in mycobacteria.  

Galactose-based phosphonate analogues of myo-inositol-1-phosphate and phosphatidylinositol have been synthesized from methyl beta-d-galactopyranoside. Michaelis-Arbuzov reaction of isopropyl diphenyl phosphite or triisopropyl phosphite with a 6-iodo-3,4-isopropylidene galactoside afforded the corresponding phosphonates. Deprotection of the diphenyl phosphonate afforded methyl beta-d-galactoside 6-phosphonate, an analogue of myo-inositol-1-phosphate. The diisopropyl esters of the diisopropyl phosphonate were selectively deprotected and the corresponding anion was coupled with 1,2-dipalmitoyl-sn-glycerol using dicyclohexylcarbodiimide. Deprotection afforded a methyl beta-d-galactoside-derived analogue of phosphatidylinositol. The galactose-derived analogues of phosphatidylinositol and myo-inositol-1-phosphate were not substrates for mycobacterial mannosyltransferases (at concentrations up to 1 mM) involved in phosphatidylinositol mannoside biosynthesis in a cell-free extract of Mycobacterium smegmatis. The galactose-derived phosphonate analogue of phosphatidylinositol was shown to be an inhibitor at 0.01 mM of PimA mannosyltransferase involved in the synthesis of phosphatidylinositol mannoside from phosphatidylinositol, and a weaker inhibitor of the next mannosyltransferase(s), which catalyzes the mannosylation of phosphatidylinositol mannoside. PMID:17340011

 
 
 
 
341

Mycobacterium paratuberculosis CobT activates dendritic cells via engagement of toll-like receptor 4 resulting in Th1 cell expansion.  

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease in animals and MAP involvement in human Crohn's disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-?B. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-? and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate MAP CobT contributes to T helper (Th) 1-polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4+/CD8+CD44highCD62Llow memory T cells in the mesenteric lymph node (MLN) of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP. PMID:23019321

342

Epidemiologic surveillance to detect false-positive Mycobacterium tuberculosis cultures.  

This study was aimed to investigate the ability of potential indices from epidemiologic surveillance to detect false-positive cultures of Mycobacterium tuberculosis (MTB). All clinical specimens for mycobacterial culture from April 1 to August 31, 2010, were reviewed. Single-positive cultures without relevant clinical and pathologic information were categorized as suspected false-positive cultures. Genotyping methods were used to confirm false-positive cultures. The performance of epidemiologic surveillance indices to detect potential false-positive cultures was evaluated. A total of 14,462 specimens were sent to the laboratory and 214 batches were processed in 107 work days (average 67.6 specimens per batch, ranging from 21 to 130 specimens per batch). Seventy-one single-positive cultures were identified, among which 5 cultures of multidrug-resistant MTB in 1 batch were false-positive, confirmed by genotyping methods. Epidemiologic surveillance with statistical process control charts for single-positive cultures per day showed good performance in epidemiologic surveillance. The false-positive rate was 38.5% in the 13 potential false-positive cultures according to the statistical process control chart for single-positive cultures per day. Although the incidence of tuberculous disease is high in Taiwan, clustering of multidrug-resistant MTB in 1 batch or clustering of single-positive cultures still suggested the occurrence of false-positive MTB cultures. Therefore, epidemiologic surveillance for the clustering of single-positive cultures with the statistical process control chart could be used to monitor the occurrence of false-positive results. PMID:22705229

343

Obtención y caracterización parcial de un extracto lipídico de la membrana externa de Mycobacterium smegmatis/ Obtainment and partial characterization of a lipidic fragment of the outer membrane of Mycobacterium smegmatis  

Abstract in spanish En la actualidad, los antígenos lipídicos de las micobacterias constituyen blancos atractivos para el desarrollo de nuevas formulaciones vacunales contra la tuberculosis. En nuestro trabajo se realizó la caracterización parcial de un extracto lipídico de pared celular de Mycobacterium smegmatis mediante cromatografía de capa delgada y Dot blot frente a gammaglobulina humana. Se identificó, fundamentalmente, la presencia de fosfolípidos y ácidos micólicos en el e (more) xtracto lipídico y se observó un elevado reconocimiento de los mismos por la gammaglobulina humana, lo cual indica la importancia de continuar los estudios de inmunoprotección empleando antígenos lipídicos de micobacterias. Abstract in english Currently, lipid antigens of mycobacteria are attractive targets for the development of new tuberculosis vaccinal formulations. A lipid extract of Mycobacterium smegmatis cell wall was characterized using a Thin Layer Chromatography and Dot blot with human gammaglobulin. Mainly we identified the presence of phospholipids and micolic acids in the lipid extract showing a high recognition by human gammaglobulin. These results indicate the relevance of continuing immunoprotection studies with mycobacterial lipid antigens.

344

Quantitative T-cell interferon-gamma responses to Mycobacterium tuberculosis-specific antigens in active and latent tuberculosis.  

The objective was to compare the quantitative T-cell responses measured by the commercial interferon-gamma (IFNgamma) release assays (IGRAs) in active and latent tuberculosis (TB) states. T-cell responses of culture-proven TB cases were compared with those of contacts with positive IGRA results and tuberculin skin tests >or= 15 mm. T-SPOT.TB results in 270 active TB cases and 183 community contacts showed the median spot-forming cells (SFCs) above negative control/2.5 x 10(5) peripheral blood mononuclear cells to be 27 (-1 to 203) vs 10 (-2 to 174) in response to ESAT-6 (p Gold In-tube in 270 cases and 142 contacts in congregate settings was 2.3 IU/ml (-0.58 to 31.44) vs 1.7 IU/ml (0.35 to 26.51, p = 0.98). Quantitative T-cell responses as measured by the T-SPOT.TB may indicate mycobacterial burden and disease activity, but cannot be used to discriminate active from latent TB. PMID:19020909

345

Temporal patterns of genetic and phenotypic variation in the epidemiologically important drone fly, Eristalis tenax.  

Eristalis tenax L. (Diptera: Syrphidae) is commonly known as the drone fly (adult) or rat-tailed maggot (immature). Both adults and immature stages are identified as potential mechanical vectors of mycobacterial pathogens, and early-stage maggots cause accidental myiasis. We compared four samples from Mount Fruška Gora, Serbia, with the aim of obtaining insights into the temporal variations and sexual dimorphism in the species. This integrative approach was based on allozyme loci, morphometric wing parameters (shape and size) and abdominal colour patterns. Consistent sexual dimorphism was observed, indicating that male specimens had lighter abdomens and smaller and narrower wings than females. The distribution of genetic diversity at polymorphic loci indicated genetic divergence among collection dates. Landmark-based geometric morphometrics revealed, contrary to the lack of divergence in wing size, significant wing shape variation throughout the year. In addition, temporal changes in the frequencies of the abdominal patterns observed are likely to relate to the biology of the species and ecological factors in the locality. Hence, the present study expands our knowledge of the genetic diversity and phenotypic plasticity of E. tenax. The quantification of such variability represents a step towards the evaluation of the adaptive potential of this species of medical and epidemiological importance. PMID:21414022

346

Morbidity and Mortality Weekly Report. Volume 61, Number 33  

The "Morbidity and Mortality Weekly Report" ("MMWR") Series is prepared by the Centers for Disease Control and Prevention (CDC). Data presented by the Notifiable Disease Data Team and 122 Cities Mortality Data Team in the weekly "MMWR" are provisional, based on weekly reports to CDC by state health departments. This issue of "Morbidity and Mortality Weekly Report" includes the following reports: (1) Lead Poisoning in Pregnant Women Who Used Ayurvedic Medications from India--New York City, 2011-2012 (Paromita Hore, Munerah Ahmed, Jacqueline Ehrlich, Celia Ng, Lourdes Steffen, Slavenka Sedlar, Phyllis Curry-Johnson, Nathan Graber, Deborah Nagin, Nancy Clark, Robert Saper, and Marissa Scalia Sucosky); (2) Vaccination Coverage Among Children in Kindergarten--United States, 2011-12 School Year (Stacie M. Greby, Karen G. Wooten, Cynthia L. Knighton, Bob Avey, and Shannon Stokley); and (3) Tattoo-Associated Nontuberculous Mycobacterial Skin Infections--Multiple States, 2011-2012 (Brenden Bedard, Byron Kennedy, Vincent Escuyer, Kara Mitchell, Jeffrey S. Duchin, Paul Pottinger, Stanley Hurst, Ken Sharp, Timothy Wickham, Sarah Jackson, Wendy Bamberg, Pamela LeBlanc, Linda M. Katz, Taranisia MacCannell, Judith Noble-Wang, Heather O'Connell, Alexander Kallen, Bette Jensen, Duc B. Nguyen, and Michael H. Kinzer). QuickStats are also included. Individual reports contain figures, tables and references.

347

A new Mycobacterium tuberculosis smooth colony reduces growth inside human macrophages and represses PDIM Operon gene expression. Does an heterogeneous population exist in intracellular mycobacteria?  

Mycobacterium tuberculosis (MTB) colony morphology was associated to the pathogen's virulence. We isolated a new MTB H37Rv smooth colony, which only appeared following human macrophages (MDM) infection. The new phenotype was Alcohol-Acid resistant, but devoid of a covering capsule and biofilm defective. We ascertained that there were no deletions in the Rv0096-Rv0101 PDIM Operon, but that its expression was repressed as compared to MTB wild type (wt). Its lipid composition displayed lower PDIM components and higher TAG as compared to wt. In MDM it induced the sigma factors sigB, sigI and sigL expression vs. synthetic medium culture, while it repressed other six sigma factors. It also induced, significantly more than wt, mprA, a mycobacterial persistence regulator. It was phagocytosed more than wt by MDM, where it grew significantly less, but persisted therein till 14 days infection. It induced significantly less IFN-?, IL-12 and IL-27 transcription than wt in infected MDM, while it increased the transcription of inducible NOS. It resided in mature, LAMP-3 positive phagolysosomes, where it never formed cords. This apparently "weaker" colony might represent an adaptive intracellular phenotype, whose infection may be less productive, but probably better equipped for a long lasting persistence in mildly activated host cells. PMID:22771837

348

Fluorescent Phospholipid Analogs as Microscopic Probes for Detection of the Mycolic Acid-Containing Layer in Corynebacterium glutamicum: Detecting Alterations in the Mycolic Acid-Containing Layer Following Ethambutol Treatment  

Corynebacterium glutamicum belongs to the mycolic acid-containing actinomycetes, which also include Mycobacterium, Nocardia, and Rhodococcus. The cells of this group possess a cell wall with a thick outer layer composed primarily of mycolic acid, which functions as a permeability barrier. To investigate the mechanism of mycolic acid-containing layer (mycolate layer) formation, we have developed a fluorescence microscopic technique detecting the mycolate layer in situ. The staining specificity of fluorescence-labeled phospholipid analogs was determined by simultaneous staining with the hydrophobic fluorescent dye Nile Red and peptidoglycan-staining fluorescence-conjugated vancomycin. We found that fluorescence-labeled phospholipid analogs preferentially stain the mycolate layer. Using this technique, we observed the effect of the anti-mycobacterial drug ethambutol on C. glutamicum mycolate-layer formation. Ethambutol interfered specifically with mycolate-layer formation on the division planes and cell poles, while the side-wall mycolate layer was not severely affected. This indicates that mycolate-layer formation occurs mainly on division planes and cell poles in C. glutamicum, where the peptidoglycan layer is actively synthesized.   

349

Epidemiology and Visual Outcomes in Patients With Infectious Scleritis.  

PURPOSE:: To describe the epidemiology of patients with infectious scleritis and identify factors associated with poor visual prognosis. METHODS:: Retrospective review of inciting factors, causative organisms, and visual outcomes of patients with infectious scleritis. RESULTS:: Fifty-five patients (56 eyes) with confirmed infectious scleritis were included. The median time from inciting event to scleritis symptoms was 1.9 months. Eyes with a history of pterygium surgery had a longer time from surgery to development of scleritis (median 49 months, range 0-183) compared to those with a history of glaucoma, cataract, and retina surgery (median 1.0-1.6 months; P = 0.001). Fungal, nocardial, and mycobacterial infections (median 17-45 days) had a longer interval between symptoms and diagnosis than eyes with non-acid-fast gram-positive and gram-negative bacteria (median 7 days; P = 0.04). Patients were followed for a median of 11.1 months (0.5-47 months). Approximately 50% of eyes lost functional vision (worse than 20/200). Presenting VA of worse than 20/200 and concomitant keratitis or endophthalmitis were associated with poorer VA outcomes. CONCLUSIONS:: Infectious scleritis can occur days to years after ocular surgery, with infection occurring after a longer interval in eyes with a history of pterygium surgery. Approximately 50% of eyes lost functional VA after infection with poor presenting VA being the strongest predictor for subsequent severe vision loss. PMID:22902495

350

Associations of Mycobacterium tuberculosis genotypes with different ethnic and migratory populations in Taiwan.  

The distribution of human Mycobacterium tuberculosis (MTB) genotypes is reportedly associated with geography, ethnicity and population migrations. Three groups of 208 patients with tuberculosis in Taiwan were sampled to test this observation: (1) 41 aborigines of Austronesian ethnicity, who have been inhabiting in Taiwan for more than 500 years; (2) 58 veterans of Han Chinese origin, who moved as the first generation from Mainland China to Taiwan 55-60 years ago; and (3) 109 patients representing the general Taiwanese population of Han Chinese whose ancestors migrated to Taiwan around 200-400 years ago. A total of 208 MTB isolates, one per patient, were analyzed by spoligotyping and mycobacterial interspersed repetitive unit (MIRU) typing. Beijing ancient strains and Haarlem strains predominated among aborigines, while Beijing modern strains were common among veterans and the general population. All Beijing strains were further analyzed by typing the NTF loci and RD deletion. Results suggest a chronological trend among Beijing isolates from the three groups: isolates from the aborigines had signatures compatible with ancient lineages, and those from veterans and the general population were more contemporary. Our data indicate that the distribution of MTB genotypes/strains in Taiwan is associated with different populations whose migratory activities occurred between 55 and 500 years ago. These results suggest that transmission of MTB may have been relatively restricted to close contacts. PMID:18378194

351

Differential recruitment and activation of natural killer cell sub-populations by Mycobacterium bovis-infected dendritic cells.  

Through complex interplay with antigen presenting cells, subsets of NK cells play an important role in shaping adaptive immune responses. Bovine tuberculosis, caused by Mycobacterium bovis, is increasing in incidence and detailed knowledge of host-pathogen interactions in the natural host is essential to facilitate disease control. We investigated the interactions of NK-cell subpopulations and M. bovis-infected dendritic cells (DCs) to determine early innate mechanisms in the response to infection. A subpopulation of NK cells (NKp46(+) CD2(-) ) selectively expressing lymphoid homing and inflammatory chemokine receptors were induced to migrate towards M. bovis-infected DCs. This migration was associated with increased expression of chemokines CCL3, 4, 5, 20 and CXCL8 by M. bovis-infected DCs. Activation of NKp46(+) CD2(-) NK cells and secretion of IFN-? was observed, a response reliant on localised IL-12 release and direct cellular interaction. In a reciprocal manner, NKp46(+) CD2(-) cells induced an increase in the intensity of cell surface MHC class II expression on DCs. In contrast, NKp46(+) CD2(+) NK cells were unable to secrete IFN-? and did not reciprocally affect DCs. This study provides novel evidence to demonstrate distinct effector responses between bovine NK-cell subsets during mycobacterial infection. PMID:23124835

352

Anti-Epidermal Growth Factor Receptor (EGFR) Antibodies Overcome Resistance of Ovarian Cancer Cells to Targeted Therapy and Natural Cytotoxicity  

The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.

353

Characterization of tannase protein sequences of bacteria and fungi: an in silico study.  

The tannase protein sequences of 149 bacteria and 36 fungi were retrieved from NCBI database. Among them only 77 bacterial and 31 fungal tannase sequences were taken which have different amino acid compositions. These sequences were analysed for different physical and chemical properties, superfamily search, multiple sequence alignment, phylogenetic tree construction and motif finding to find out the functional motif and the evolutionary relationship among them. The superfamily search for these tannase exposed the occurrence of proline iminopeptidase-like, biotin biosynthesis protein BioH, O-acetyltransferase, carboxylesterase/thioesterase 1, carbon-carbon bond hydrolase, haloperoxidase, prolyl oligopeptidase, C-terminal domain and mycobacterial antigens families and alpha/beta hydrolase superfamily. Some bacterial and fungal sequence showed similarity with different families individually. The multiple sequence alignment of these tannase protein sequences showed conserved regions at different stretches with maximum homology from amino acid residues 389-469 and 482-523 which could be used for designing degenerate primers or probes specific for tannase producing bacterial and fungal species. Phylogenetic tree showed two different clusters; one has only bacteria and another have both fungi and bacteria showing some relationship between these different genera. Although in second cluster near about all fungal species were found together in a corner which indicates the sequence level similarity among fungal genera. The distributions of fourteen motifs analysis revealed Motif 1 with a signature amino acid sequence of 29 amino acids, i.e. GCSTGGREALKQAQRWPHDYDGIIANNPA, was uniformly observed in 83.3 % of studied tannase sequences representing its participation with the structure and enzymatic function. PMID:22460647

354

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae  

Abstract in english Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary t (more) o establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

355

Health assessment of free-ranging and captive Matschie's tree kangaroos (Dendrolagus matschiei) in Papua New Guinea.  

Medical evaluations were performed on free-ranging and captive Matschie's tree kangaroos (Dendrolagus matschiei) in Papua New Guinea. The health assessment included physical examination, morphometrics, cloacal swab; and blood, hair, and feces collection. Radio-collars were placed on free-ranging tree kangaroos to determine home range and forest habitat use. The free-ranging tree kangaroos were lightly anesthetized with tiletamine/zolazepam for the data collection. A total of nine free-ranging and seven captive tree kangaroos were evaluated; medical samples were collected from six and five animals, respectively. Results of physical examination, anesthetic monitoring, serum vitamin, mineral, trace nutrient, and electrolytes, whole blood heavy metal analysis, mycobacterial screening, and fecal examinations are presented. Free-ranging tree kangaroos had significantly lower values for beta carotene, copper, selenium, molybdenum, lead, and arsenic and significantly higher values for vitamin E than captive individuals. Cloacal swabs were all negative for Mycobacterium avium via polymerase chain reaction. Some free-ranging and captive individuals had positive coprologic exams revealing Eimeria spp. oocysts and strongyle spp. type ova. These are the first medical and anesthetic data published on Matschie's tree kangaroos from Papua New Guinea. PMID:22448504

356

Synthesis, biological activity, and evaluation of the mode of action of novel antitubercular benzofurobenzopyrans substituted on A ring.  

The 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy derivatives of the antimycobacterial 3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran were synthesized by condensation of the diazonium salts of 2-chloroanilines (13-17) with 1,4-benzoquinone (18), reduction of the intermediate phenylbenzoquinones 19-22 to dihydroxybiphenyls, cyclisation to halo-2-hydroxydibenzofurans 24-27, and construction of the pyran ring by thermal rearrangement of the corresponding dimethylpropargyl ethers 35-38. Palladium catalyzed nucleophilic aromatic substitution permitted conversion of the halo to the corresponding hydroxy derivatives which were methylated to methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran. All compounds substituted on the A ring were found more potent than the reference compound 1 against Mycobacterium bovis BCG and the virulent strain Mycobacterium tuberculosis H37Rv. The effect of the most active derivatives on mycolate synthesis was explored in order to confirm the preliminary hypothesis of an effect on mycobacterial cell wall biosynthesis. The linear 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran (46) exhibiting a good antimycobacterial activity and devoid of cytotoxicity appeared to be the most promising compound. PMID:20961671

357

Re-emergence of the progenitors of a multidrug-resistant outbreak strain of Mycobacterium tuberculosis among the post-outbreak case patients.  

BACKGROUND. The progenitors of multidrug-resistant tuberculosis (MDR-TB) outbreak strains might evolve into new outbreak strains. We hypothesized that these strains could re-emerge among post-outbreak patients with TB and must thus be tracked. METHODS. To identify the progenitors of the outbreak strain, we first determined the precise IS6110 genomic insertion locations of a Haarlem3 strain that caused a severe MDR-TB outbreak in Tunisia. Next, we searched by polymerase chain reaction for these outbreak-specific IS6110 transposition sites in all the Haarlem3 post-outbreak isolates recovered in the epidemic region. RESULTS. By analyzing the distribution of the outbreak-specific IS6110 transposition sites, we were able to trap, among isolates recovered from post-outbreak new patients, drug-susceptible and drug-resistant isolates that are likely to represent the very close progenitors of the outbreak strain. Mycobacterial interspersed repetitive units-variable number of tandem repeats typing and sequential accumulation of rifampicin resistance-associated rpoB mutations further confirmed the identity of the outbreak's progenitor strains. CONCLUSIONS. The data of the current study show that the progenitors of an MDR-TB outbreak strain could re-emerge among post-outbreak TB cases. We provide an IS6110 insertion site-based approach to better trace back the events preceding the emergence of MDR-TB outbreaks, irrespective of the availability of a pre-outbreak strain collection. PMID:20021247

358

Upregulation of IL-17A, CXCL9 and CXCL10 in Early-Stage Granulomas Induced by Mycobacterium bovis in Cattle.  

To gain further insight into the immunopathogenesis of bovine tuberculosis (bTB), the cytokine and chemokine expression of cattle experimentally infected with Mycobacterium bovis was analysed in TB granulomas, using immunohistochemistry (IHC) and laser capture microdissection (LCM) followed by qPCR. Immunohistochemistry was conducted for cell types using labelling for CD68, CD3, CD4, CD8, WC1 and CD79a and for the cytokines IFN-?, TNF-? and TGF-? as well as inducible form of nitric oxide synthase (iNOS). qPCR was conducted for mRNA expression of IFN-?, TNF-?, TGF-?, IL-17A, IL-22, IL-2, granzyme A and the chemokines CXCL9 and CXCL10. Early stages of granuloma were primarily comprised of epithelioid M?s expressing high levels of IFN-? and iNOS, with significantly upregulated expression of CXCL9 and CXCL10 when compared with control tissue. These chemokines displayed a trend of decreasing mRNA expression as lesion progressed, suggesting a higher level of importance during the early stages of the immune response to mycobacterial infection. IL-22 levels showed a strong trend of decrease through granuloma development, and IL-17A was shown to be upregulated, supporting its investigation as a potential biomarker of bTB. The use of LCM and qPCR may prove especially useful for the study of IL-17A as previous attempts to analyse its expression using IHC and in situ hybridization proved unsuccessful. PMID:22909117

359

Ag85B-ESAT-6 adjuvanted with IC31 promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in naïve human volunteers.  

Though widely used, the BCG vaccine has had little apparent effect on rates of adult pulmonary tuberculosis. Moreover, the risk of disseminated BCG disease in immunocompromised individuals means that improved TB vaccines ideally need to be able to efficiently prime mycobacterially-naïve individuals as well as boost individuals previously vaccinated with BCG. Protective immunity against Mycobacterium tuberculosis is thought to depend on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-gamma) production. In the present study, we monitored safety and IFN-gamma responses in healthy TB-naïve humans receiving an entirely novel vaccine, composed of the fusion protein Ag85B-ESAT-6, administered at 0 and 2 months either as recombinant protein alone or combined with two concentrations of the novel adjuvant IC31. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against H1 and both the Ag85B and the ESAT-6 components. These strong responses persisted through 2.5 years of follow-up, indicating the induction of a substantial memory response in the vaccine recipients. PMID:20226890

360

[A new anti-mycobacterial agent, rifabutin].  

This is a review of non-clinical and clinical study results of rifabutin (Mycobutin, RBT) which was approved as a new anti-mycobacterial agent 38 years after rifampicin (RFP) was approved in Japan. The anti-bacterial actions of RBT were similar to those of RFP, but its potency was stronger (4 to 32 times in MIC90). RBT showed excellent penetration in cells (9 times in neutrophil, 15 times in monocyte, against plasma concentration) and in tissues (5 to 10 times in pulmonary tissue). Clinical efficacy of RBT (150 mg, as well as 300 mg daily) was comparable to that of RFP 600 mg daily, in the treatment of newly diagnosed tuberculosis, drug-resistant tuberculosis, and the NTM diseases. In addition, RBT 300 mg showed significant prophylactic effect on the development of disseminated MAC infection in HIV positive subjects. Most of the adverse events of RBT were the same as those of RFP, including drug-drug interactions related to the induction of CYP3A4. The concomitant use of RBT (over 450 mg) with clarithromycin induces uveitis, which warrants special attention. It is expected that the efficacy and safety of RBT in Japanese subjects will be evaluated in Japan through the accumulation of clinical experience. PMID:21061564

 
 
 
 
361

Development of an ultrasensitive polymerase chain reaction-amplified immunoassay based on mycobacterial RD antigens: implications for the serodiagnosis of tuberculosis.  

Immuno-polymerase chain reaction (I-PCR) combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification power of PCR. The present study was designed to detect antibodies to Mycobacterium tuberculosis complex-specific region of difference (RD) antigens, i.e., early secretory antigenic target-6, culture filtrate protein-10, culture filtrate protein-21, and mycobacterial protein from species tuberculosis-64, as well as antigens in pulmonary tuberculosis patients by I-PCR assay. We could detect ESAT-6 and other RD antigens up to 0.1 fg by I-PCR assay, thus resulting in 10(7) times higher sensitivity than that observed with ELISA. With paired sample analysis based on the detection of antibodies in serum and antigens in sputum of the same individual, the sensitivity of RD multi-antigen cocktail-based I-PCR assay was 72% in smear-negative cases and 91% in smear-positive cases of pulmonary tuberculosis with high specificity values. In extrapulmonary tuberculosis patients, higher sensitivity was observed by detecting cocktail of antigens by I-PCR assay as compared to sensitivity earlier observed in the same samples by ELISA. PMID:22248737

362

High Content Screening Identifies Decaprenyl-Phosphoribose 2? Epimerase as a Target for Intracellular Antimycobacterial Inhibitors  

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2? epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

363

High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.  

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials. PMID:19876393

364

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India  

Abstract in english The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was (more) composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

365

Antiretroviral agents restore Mycobacterium-specific T-cell immune responses and facilitate controlling a fatal tuberculosis-like disease in Macaques coinfected with simian immunodeficiency virus and Mycobacterium bovis BCG.  

The contribution of immune reconstitution following antiretroviral treatment to the prevention or treatment of human immunodeficiency virus-related primary or reactivation tuberculosis remains unknown. Macaque models of simian immunodeficiency virus-Mycobacterium bovis BCG (SIV/BCG) coinfection were employed to determine the extent to which anti-Mycobacterium tuberculosis immunity can be restored by antiretroviral therapy. Both SIV-infected macaques with active BCG reinfection and naive animals with simultaneous SIV/BCG coinfection were evaluated. The suppression of SIV replication by antiretroviral treatment resulted in control of the active BCG infection and blocked development of the fatal SIV-related tuberculosis-like disease. The resolution of this disease coincided with the restoration of BCG purified protein derivative (PPD)-specific T-cell immune responses. In contrast, macaques similarly coinfected with SIV/BCG but not receiving antiretroviral therapy had depressed PPD-specific primary and memory T-cell immune responses and died from tuberculosis-like disease. These results provide in vivo evidence that the restoration of anti-mycobacterial immunity by antiretroviral agents can improve the clinical outcome of an AIDS virus-related tuberculosis-like disease. PMID:11507214

366

Cooking and drying as effective mechanisms in limiting the zoonotic effect of Mycobacterium bovis in beef.  

For this study 48 non-infected muscle, lymphatic and visceral bovine tissue samples were collected from an approved red meat abattoir and spiked with 8 x 10(7) cfu/ml of M. bovis. The different spiked samples were subjected to cooking and drying (drying through the process of biltong-making) processes in a controlled laboratory environment. Mycobacterial isolates confirmed as M. bovis by means of polymerase chain reaction (PCR) were observed in 17 of a total of 576 samples that were exposed to the secondary processing method of cooking. The study showed that not only can M. bovis survive the cooking process but the survival of the bacterium will be determined by its unique adaptive changes to the surrounding composition of the environment. The results for the samples exposed to the drying process (n = 96) did not show any growth, suggesting that the process of biltong production as used in this study is likely to render infected meat safe for human consumption. PMID:20169745

367

Langhans giant cells from M. tuberculosis-induced human granulomas cannot mediate mycobacterial uptake.  

Tuberculosis is characterized by a tight interplay between Mycobacterium tuberculosis (M. tb) and host cells within granulomas. These cellular aggregates restrain M. tb spreading but do not kill all bacilli, which persist for years. A more detailed investigation of the interaction between M. tb and granuloma cells is needed to improve our understanding of this persistence and to explain the physiopathology of tuberculosis. In the present study, a recently developed in vitro human model of tuberculous granulomas has been used to analyse the modulation of granuloma cell differentiation by M. tb, in comparison to poorly virulent mycobacteria, which do not persist. It is reported that whilst all mycobacteria species induce granuloma formation, only M. tb triggers the differentiation of granuloma macrophages into very large multinucleated giant cells (MGCs) that are unable to mediate any bacterial uptake. This loss of function is not due to cell quiescence, as MGCs still display NADPH oxidase activity, but it correlates with decreased expression of phagocytosis receptors. This phenomenon is specific for the virulent species of M. tuberculosis complex, as poorly virulent species only induce the formation of small multinucleated cells (MCs) with conserved mycobacterial uptake ability, which never reach the MGC differentiation stage. The phenotype of MGCs thus strongly resembles mature dendritic cells with a loss of microbial uptake ability, despite conserved antigen presentation. In M. tb-induced granulomas, MGCs thus seem to be devoted to the destruction of bacilli that have been ingested in previous differentiation stages, ie in macrophages and MCs. PMID:17115379

368

Low incidence of colonization and no cases of disseminated Mycobacterium avium complex infection (DMAC) in Brazilian AIDS patients in the HAART era  

Abstract in english OBJECTIVE: Evaluate the incidence of mycobacterial disease and the colonization of the respiratory and gastrointestinal tracts by Mycobacterium avium complex (MAC) bacteria in AIDS patients. METHODS: Inclusion criteria: HIV-positive individuals with at least one CD4+ count (more) cidence was calculated using Poisson regression. Survival was estimated by the Kaplan Meier method and the Cox proportional hazard model. RESULTS: We followed-up 79 patients during a median period of 428 days. Blood cultures (n = 742) were negative for all mycobacteria. Positive cultures (25 samples) were obtained from non-sterile sites: Stools (19/703 specimens = 2.7%) and sputum (14/742 specimens = 1.9%). MAC was isolated in 7/703 stool samples (1%) and 1/32 sputum specimens (0.1%). The incidence of patient colonization with MAC was 0.09 /year (CI=0.05 - 0.18). CD4 counts in patients colonized with MAC were below 100 cells/mm³ in only 2 out of 8 cases. Restoration of CD4+ counts >100 cells/mm³ (HR = 0.18; CI = 0.05 - 0.70) predicted a lower risk of death (P

369

The interplay of multiple feedback loops with post-translational kinetics results in bistability of mycobacterial stress response  

Bacterial persistence is the phenomenon in which a genetically identical fraction of a bacterial population can survive exposure to stress by reduction or cessation of growth. Persistence in mycobacteria has been recently linked to a stress-response network, consisting of the MprA/MprB two-component system and alternative sigma factor ?E. This network contains multiple positive transcriptional feedback loops which may give rise to bistability, making it a good candidate for controlling the mycobacterial persistence switch. To analyze the possibility of bistability, we develop a method that involves decoupling of the network into transcriptional and post-translational interaction modules. As a result we reduce the dimensionality of the dynamical system and independently analyze input-output relations in the two modules to formulate a necessary condition for bistability in terms of their logarithmic gains. We show that neither the positive autoregulation in the MprA/MprB network nor the ?E-mediated transcriptional feedback is sufficient to induce bistability in a biochemically realistic parameter range. Nonetheless, inclusion of the post-translational regulation of ?E by RseA increases the effective cooperativity of the system, resulting in bistability that is robust to parameter variation. We predict that overexpression or deletion of RseA, the key element controlling the ultrasensitive response, can eliminate bistability.

370

Production of volatile organic compounds by mycobacteria.  

The need for improved rapid diagnostic tests for tuberculosis disease has prompted interest in the volatile organic compounds (VOCs) emitted by Mycobacterium tuberculosis complex bacteria. We have investigated VOCs emitted by Mycobacterium bovis BCG grown on Lowenstein-Jensen media using selected ion flow tube mass spectrometry and thermal desorption-gas chromatography-mass spectrometry. Compounds observed included dimethyl sulphide, 3-methyl-1-butanol, 2-methyl-1-propanol, butanone, 2-methyl-1-butanol, methyl 2-methylbutanoate, 2-phenylethanol and hydrogen sulphide. Changes in levels of acetaldehyde, methanol and ammonia were also observed. The compounds identified are not unique to M. bovis BCG, and further studies are needed to validate their diagnostic value. Investigations using an ultra-rapid gas chromatograph with a surface acoustic wave sensor (zNose) demonstrated the presence of 2-phenylethanol (PEA) in the headspace of cultures of M. bovis BCG and Mycobacterium smegmatis, when grown on Lowenstein-Jensen supplemented with glycerol. PEA is a reversible inhibitor of DNA synthesis. It is used during selective isolation of gram-positive bacteria and may also be used to inhibit mycobacterial growth. PEA production was observed to be dependent on growth of mycobacteria. Further study is required to elucidate the metabolic pathways involved and assess whether this compound is produced during in vivo growth of mycobacteria. PMID:22224870

371

Bone and joint tuberculosis.  

INTRODUCTION: Bone and joint tuberculosis has increased in the past two decades in relation with AIDS epidemics. MATERIAL AND METHODS: A literature review of bone and joint tuberculosis, focusing on Pott's disease. RESULTS: Bone and joint TB comprises a group of serious infectious diseases whose incidence has increased in the past two decades, especially in underdeveloped countries, in part due to the AIDS epidemic. Tuberculous spinal infections should be suspected in patients with an insidious, progressive history of back pain and in individuals from an endemic area, especially when the thoracic vertebrae are affected and a pattern of bone destruction with relative disc preservation and paravertebral and epidural soft tissue masses are observed. Atypical tuberculous osteoarticular manifestations involving the extraspinal skeleton, a prosthetic joint, or the trochanteric area, and nontuberculous mycobacterial infections should be considered in favorable epidemiological contexts. Surgery combined with prolonged specific antituberculous chemotherapy is mainly indicated in patients with neurological manifestations or deformities, and provides satisfactory results in most cases. CONCLUSIONS: Spinal tuberculosis is still a relative common extra spinal manifestation of spinal tuberculosis that requires a high degree of suspicion in order to avoid neurological complications and need of surgery. PMID:22711012

372

Investigating the effectiveness of St John's wort herb as an antimicrobial agent against mycobacteria.  

A persistent need exists for effective treatment agents for mycobacterial infections. This research investigated the effectiveness of the Hypericum perforatum herb (commonly known as St John's wort; SJW) in its growth inhibition of mycobacteria. A SJW extract was effective at inhibiting five nonpathogenic Mycobacterium isolates and Bacillus subtilis, but not Escherichia coli. Quantitative studies of concentration sensitivity to the SJW extract were performed with minimal bactericidal concentrations (MBC) ranging from 0.33 to 2.66?mg extract/mL. The SJW compounds hyperforin (Hfn), hypericin (Hpn), and pseudohypericin (Phn) were quantified in the extract using HPLC. The SJW extract solution of 133?mg extract/mL used in this study contained 2.3?mg Hfn/mL, 0.8?mg Hpn/mL, and 2.1?mg Phn/mL. Purified Hfn, Hpn, and Phn were tested for inhibitory activity against Mycobacterium JLS (M. JLS) at similar concentrations used in the crude extract. While Hfn was inhibitory at 46?µg/mL, none of the purified SJW constituents were bactericidal at concentrations corresponding to SJW treatments. Scanning electron microscopy (SEM) analysis of SJW-treated M. JLS cells showed changes in cell surface morphology. Copyright © 2012 John Wiley & Sons, Ltd. PMID:22294548

373

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes  

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

374

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques  

Abstract in english Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and e (more) thionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100%, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis.

375

Polymerase chain reaction as a useful and simple tool for rapid diagnosis of tuberculous meningitis in a Brazilian tertiary care hospital  

Abstract in english Meningitis is a severe and potentially fatal form of tuberculosis. The diagnostic workup involves detection of acid-fast bacilli (AFB) in the cerebrospinal fluid (CSF) by microscopy or culture, however, the difficulty in detecting the organism poses a challenge to diagnosis. The use of the polymerase chain reaction (PCR) in the diagnostic approach to Mycobacterium tuberculosis (MTB) meningitis has been reported as a fast and accurate method, with several commercial kits a (more) vailable. As an alternative, some institutions have been developing inexpensive in house assays. In our institution, we use an in house PCR for tuberculosis. We analyzed the performance of our PCR for the diagnosis of MTB meningitis in 148 consecutive patients, using MTB culture as gold standard. The sensitivity and specificity of CSF PCR for the diagnosis of MTB meningitis was 50% and 98.6% respectively with a concordance with CSF mycobacterial culture of 96% (Kappa=0.52). In contrast to CSF cultures for MTB, our PCR test is a fast, simple and inexpensive tool to diagnose tuberculous meningitis with a performance similar to that obtained with the available commercial kits.

376

Enzyme-linked immunosorbent assay for the detection of antibodies against Mycobacterium tuberculosis in bronchial washings and serum.  

This prospective study evaluates an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against Mycobacterium tuberculosis when applied to serum and bronchial washings (BW) of patients requiring bronchoscopy for definitive diagnosis. The ELISA employed an adsorbed fraction of mycobacterial sonicates as antigen. Of the 57 patients tested, 12 had tuberculosis (TB), 13 had malignant disease, 14 acute pneumonia or chronic destructive pneumonia, 13 had no demonstrable disease, and five had miscellaneous diseases. The mean optical density (OD) at 492 nm for a 1 in 40 dilution of serum of patients with TB was 0.338 +/- 0.218 and for control subjects was 0.162 +/- 0.098 (p = 0.0323). Values for neat BW were 0.317 +/- 0.122 for TB patients and 0.191 +/- 0.140 for control subjects (p = 0.0084). Using stepwise discriminant analysis, the ELISA on serum gave a sensitivity of 70.0 percent and specificity of 86.4 percent and 83.3 percent correct classification. When applied to BW, there was a 72.7 percent sensitivity, a 82.2 percent specificity, and patients were correctly classified in 80.4 percent of cases. It is concluded that the assay is a useful and reliable adjunct to conventional tests for tuberculosis. PMID:3127124

377

Pulmonary eosinophilia.  

Pulmonary eosinophilia comprises a heterogeneous group of diseases defined by eosinophilia in pulmonary infiltrates (bronchoalveolar lavage fluid) or in tissue (lung biopsy specimens). Although the inflammatory infiltrate is composed of macrophages, lymphocytes, neutrophils and eosinophils, eosinophilia is an important marker for the diagnosis and treatment. Clinical and radiological presentations can include simple pulmonary eosinophilia, chronic eosinophilic pneumonia, acute eosinophilic pneumonia, allergic bronchopulmonary aspergillosis and pulmonary eosinophilia associated with a systemic disease, such as in Churg-Strauss syndrome and hypereosinophilic syndrome. Asthma is frequently concomitant and can be a prerequisite, as in allergic bronchopulmonary aspergillosis and Churg-Strauss syndrome. In diseases with systemic involvement, the skin, the heart and the nervous system are the most affected organs. The radiological presentation can be typical, or at least suggestive, of one of three types of pulmonary eosinophilia: chronic eosinophilic pneumonia, acute eosinophilic pneumonia and allergic bronchopulmonary aspergillosis. The etiology of pulmonary eosinophilia can be either primary (idiopathic) or secondary, due to known causes, such as drugs, parasites, fungal infection, mycobacterial infection, irradiation and toxins. Pulmonary eosinophilia can be also associated with diffuse lung diseases, connective tissue diseases and neoplasia. PMID:19618037

378

Pulmonary eosinophilic infiltrates.  

Pulmonary eosinophilic infiltrates include an heterogeneous group of disorders characterized by the presence of eosinophils in the lungs as detected by bronchoalveolar lavage or tissue biopsy, with or without blood eosinophilia. The disease can be idiopathic (simple pulmonary eosinophilia, acute and chronic eosinophilic pneumonia, hypereosinophilic syndrome), secondary (to drugs, parasites, fungal and mycobacterial infection, irradiation, toxic products) or associated with diffuse lung diseases (connective tissue diseases and some neoplasms). Pathologists faced with eosinophils in the lungs (either on cytology or biopsy) should keep in mind several possibilities, although a diagnosis of certainty is rarely based on morphology alone. Correlation with laboratory tests, imaging studies and clinical presentation has a key role, even if some pulmonary eosinophilic diseases are sufficiently characteristic on clinico-radiologic ground to not require a biopsy (e.g. some drug reactions, parasitic infections, idiopathic hypereosinophilic syndrome, allergic bronchopulmonary aspergillosis). Nevertheless, pathologists can play a central role because they can be the first to note eosinophils in the lungs of a very sick patient. Knowledge of histologic features and a striking collaboration with other physicians are necessary to achieve correct diagnosis and to establish adequate treatments. PMID:21428117

379

[Eosinophilic diseases of the lungs].  

Pulmonary eosinophilias belong to a heterogenous group of the diseases characterized by pulmonary shadows related to pulmonary tissue and/or peripheral blood eosinophilia. Although the inflammatory infiltrate consists of macrophages, lymphocytes, neutrophils and eosinophils, a significant marker for the diagnosis and treatment is eosinophilia. By etiology eosinophilic diseases of the lungs fall into primary or idiopathic (common pulmonary eosinophilia, chronic eosinophilic pneumonia, hypereosinophilic syndrome), secondary or of known origin (allergic bronchopulmonary aspergillesis, bronchocentric granulematosis, parasitic invasions, drug-induced reactions, fungal and mycobacterial infection, pulmonary diseases caused by radiation or toxins). Pulmonary eosinophilia can be also associated with systemic diseases (Churg-Strauss syndrome) and tumors. Clinicoroentgenological picture of different eosinophilic diseases of the lungs is almost the same. Verification of the diagnosis is based on the presence of bronchial asthma and extrapulmonary manifestations, the level of eosinophilia in the blood, bronchoalveolar lavage and total IgE, histological and chest CT findings. This article presents modern classification, clinicoroentgenological and histological characteristics of different, primarily idiopathic, eosinophilic diseases of the lungs. PMID:22708427

380

Evaluation of rapid techniques for the detection of mycobacteria in sputum with scanty bacilli or clinically evident, smear negative cases of pulmonary and extra-pulmonary tuberculosis  

Abstract in english The objective of the current study was to compare two rapid methods, the BBL Mycobacteria Growth Indicator Tube (MGIT TM) and Biotec FASTPlaque TB TM (FPTB) assays, with the conventional Löwenstein-Jensen (LJ) media assay to diagnose mycobacterial infections from paucibacillary clinical specimens. For evaluation of the clinical utility of the BBL MGIT TM and FPTB assays, respiratory tract specimens (n = 208), with scanty bacilli or clinically evident, smear negative case (more) s and non-respiratory tract specimens (n = 119) were analyzed and the performance of each assay was compared with LJ media. MGIT and FPTB demonstrated a greater sensitivity (95.92% and 87.68%), specificity (94.59% and 98.78%), positive predictive value (94.91% and 99.16%) and negative predictive value (96.56% and 90.92%), respectively, compared to LJ culture for both respiratory tract and non-respiratory tract specimens. However, the FPTB assay was unable to detect nontuberculous mycobacteria and few Mycobacterium tuberculosis complex cases from paucibacillary clinical specimens. It is likely that the analytical sensitivity of FPTB is moderately low and may not be useful for the direct detection of tuberculosis in paucibacillary specimens. The current study concluded that MGIT was a dependable, highly efficient system for recovery of M. tuberculosis complexes and nontuberculous mycobacteria from both respiratory and non-respiratory tract specimens in combination with LJ media.

 
 
 
 
381

Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths  

Infection and transmission of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb) and extensively drug-resistant M. tuberculosis (XDR-Mtb) is a serious health problem. We analyzed a total of 1,110 Mtb isolates in Osaka Prefecture and neighboring areas from April 2000 to March 2009. A total of 89 MDR-Mtb were identified, 36 (48.5%) of which were determined to be XDR-Mtb. Among the 89 MDR-Mtb isolates, 24 (27.0%) phylogenetically distributed into six clusters based on mycobacterial interspersed repetitive units-various number of tandem repeats (MIRU-VNTR) typing. Among these six clusters, the MIRU-VNTR patterns of four (OM-V02, OM-V03, OM-V04, and OM-V06) were only found for MDR-Mtb. Further analysis revealed that all isolates belonging to OM-V02 and OM-V03, and two isolates from OM-V04 were clonal. Importantly such genotypes were not observed for drug-sensitive isolates. These suggest that few but transmissible clones can transmit after acquiring multidrug resistance and colonize even in a country with a developed, well-organized healthcare system. PMID:19165319

382

Linfadenitis supurada por BCG: Reporte de un caso/ Supurative lymphadenitis caused by BCG: A case report  

Abstract in spanish La infección micobacteriana diseminada después de la vacunación por bacilo Calmette-Guerin (BCG) es un trastorno muy raro que ocurre principalmente en pacientes con inmunodeficiencia. Se describe un caso donde se observó una reacción adversa inducida por la vacunación con BCG expresada por una linfadenitis regional supurada y abscedada, la cual se manifestó en una paciente al mes de nacida. A los 5 meses de nacida presentó fiebre, vómitos y diarreas, y exantema s (more) úbito a los 11 meses. El curso clínico y la evaluación inmunológica sugiere una inmunodeficiencia congénita Abstract in english The disseminated mycobacterial infection that appears after the BCG vaccination is a very rare disorder that occurs mainly in patients with immunodeficiency. A case is described in which an adverse reaction induced by the BCG vaccine was expressed by suppurative and abscessed regional lymphadenitis that manifested a month after the baby's birth. She had fever, vomits, and diarreas when she was 5 months old, and exanthema subitum at 11 months. The clinical course and immunological evaluation suggest a congenital immunodeficiency

383

Pulmonary Tuberculosis in Spontaneously Diabetic Goto Kakizaki Rats  

As a clinical association is thought to exist between diabetes and tuberculosis, this study was set up to examine whether GK/Jcl diabetic rats are more susceptible to Mycobacterium tuberculosis infection than non-diabetic rats. GK/Jcl diabetic rats were infected aerially with M. tuberculosis and their capacity to control mycobacterial growth, granuloma formation, cytokine secretion by alveolar macrophages and nitric oxide (NO) production was examined. The rats developed large granulomas but not necrotic lesions in their lungs, liver or spleen. This is consistent with a significant increase in number of colony-forming units of M. tuberculosis in the lungs (p<0.01). Expression levels of interferon-?, tumor necrosis factor (TNF)-? and interleukin (IL)-12 mRNA were lower in GK/Jcl diabetic rats than those in control Wistar rats. Alveolar macrophages from GK/Jcl rats secreted less TNF-? and IL-12, and produced less NO compared with those from Wistar rats. No significant difference was observed between phagocytosis of tubercle bacilli by alveolar macrophages from GK/Jcl or Wistar rats. These data show that there is a close association between experimental tuberculosis and diabetes in animals, and that alveolar macrophages from GK/Jcl diabetic rats are not fully activated by M. tuberculosis infection.   

384

Rapid Detection of Mycobacterium tuberculosis in Clinical Samples by Polymerase Chain Reaction  

A polymerase chain reaction for the rapid and specific detection of Mycobacterium tuberculosis has been used and evaluated for clinical applicability. Two oligonucleotide primers derived from the nucleotide sequence of a immunogenic protein MPB 64 amplified DNA from M. tuberculosis and M. bovis. No amplification was observed from any of ten different mycobacterial strains. A total of 126 clinical samples were amplified and tested by both dot blot hybridization and restriction enzyme analysis. M. tuberculosis was detected by PCR in 38 smear and 42 culture positive cases. An additional 16 culture negative specimens were PCR positive yielding an overall M. tuberculosis positivity rate of 46.0% (58/126) compared to 33.3% (42/126) by culture. The superior sensitivity of PCR to culture was more evident in non-pulmonary cases where PCR picked up 10 cases in addition to 6 culture positives out of 46 specimens. On the other hand, out of 80 pulmonary specimens only 6 cases in addition to 36 culture positives were picked up by PCR. The specificity of PCR was confirmed with dot blot hybridization and restriction enzyme analysis. This study corroborates that PCR offers a more sensitive and rapid alternative for the detection of M. tuberculosis to culture, and that it can be used in uncultured clinical specicimens.   

385

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG  

Abstract in english Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG e (more) xpression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ?leuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

386

Clinical and high-resolution computed tomographic findings in five patients with pulmonary tuberculosis who developed respiratory failure following chemotherapy  

AIM: The purpose of this study was to describe the clinical and high-resolution computed tomographic (HRCT) findings in patients with pulmonary tuberculosis who developed respiratory failure after starting chemotherapy. MATERIALS AND METHODS: The clinical records, chest radiographs, and HRCT findings in five patients with non-miliary pulmonary tuberculosis who developed respiratory failure after starting chemotherapy were reviewed. RESULTS: Chest radiographs taken early in the course of acute respiratory failure showed progression of the original lesions with (n = 4) or without (n = 1) new areas of opacity away from the site of the original lesions. HRCT demonstrated widespread ground-glass attenuation with a reticular pattern as well as segmental or lobar consolidation with cavitation and nodules, consistent with active tuberculous foci in all five cases. Prominent interlobular septal thickening was seen in two cases. Four of the five patients had received corticosteroids. Of these five, two died and three recovered with continued corticosteroid therapy. Transbronchial biopsy in three cases showed evidence of acute alveolar damage. CONCLUSION: In selected patients with tuberculosis who develop respiratory failure following the initiation of antituberculous therapy, HRCT may be a helpful adjunct to clinical evaluation in differentiating hypersensitivity reactions (presumed to be due to the release of mycobacterial antigens) from other pulmonary complications. Akira, M. and Sakatani, M. (2001)

387

Nontuberculous Mycobacterial (NTM) Disease in Immunocompetent Patients: Expanding Image Findings on Chest CT  

The aim of this study was to evaluate the chest CT features of nontuberculous mycobacterial (NTM) disease regardless of the specific organisms. This study included 74 consecutive patients (35 men, 39 women; mean age, 63 years; age range, 25-89 years) who were diagnosed with NTM disease according to the American Thoracic Society Guidelines (1997 and 2007) between January 2005 and July 2007. Chest CT images were randomly reviewed by two radiologists with consensus. The most common organism associated with NTM disease is M. avium-intracellulare complex (87.8%), followed by M. abscesses, M. kansasii, and M. chelonae. The most common chest CT finding was a nodular bronchiectatic lesion (n = 35, 46.7%), followed by a cavitary lesion of the upper lobe (n = 21, 28.0%), combined lesions of two prior subtypes (n = 6, 8.0%), consolidative lesion (s) (n = 5, 6.7%), a bronchogenic spreading pulmonary tuberculosis-like lesion (n = 5, 6.7%), a cavitary mass lesion with small satellite nodules (n = 2, 2.7%), and a miliary nodular lesion (n = 1, 1.3%). More than 5 segments were involved in 60 cases (81.1%). The nodular bronchiectatic lesion or cavitary lesion of upper lobe presents with multi-segmental involvement and the occurrence of combined consolidation is indicative of NTM disease

388

Genotypes of Mycobacterium avium subsp. paratuberculosis from South American countries determined by two methods based on genomic repetitive sequences.  

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of Johne's disease or paratuberculosis of ruminants and has been associated with Crohn's disease in humans. In this study, the genotypes of MAP obtained so far in South American countries using a combination of the subtyping methods Mycobacterial Interspersed Repeats Units-Variable Number of Tandem Repeats (MIRU-VNTR) and Multilocus Short Sequence Repeats (MLSSR) were analyzed. Through this analysis, seven different MIRU-VNTR genotypes and seven MLSSR genotypes have been detected. If both methods were combined, nine different genotypes were found. Results revealed the predominance of MIRU-VNTR genotype 1 (INMV 1) and MLSSR genotype A (7 g-10 g-4ggt) among MAP isolates from different host species in South America. These predominant MAP genotypes are also commonly detected in Europe and the United States. This predominance could be the result of higher animal infection ability or better culturability on solid media used for isolation. Further studies on molecular epidemiology of MAP must be carried out in South America to increase our knowledge of the global distribution of MAP. PMID:22297421

389

Unraveling the structure of membrane proteins in situ by transfer function corrected cryo-electron tomography.  

Cryo-electron tomography in combination with subtomogram averaging allows to investigate the structure of protein assemblies in their natural environment in a close to live state. To make full use of the structural information contained in tomograms it is necessary to analyze the contrast transfer function (CTF) of projections and to restore the phases of higher spatial frequencies. CTF correction is however hampered by the difficulty of determining the actual defocus values from tilt series data, which is due to the low signal-to-noise ratio of electron micrographs. In this study, an extended acquisition scheme is introduced that enables an independent CTF determination. Two high-dose images are recorded along the tilt axis on both sides of each projection, which allow an accurate determination of the defocus values of these images. These values are used to calculate the CTF for each image of the tilt series. We applied this scheme to the mycobacterial outer membrane protein MspA reconstituted in lipid vesicles and tested several variants of CTF estimation in combination with subtomogram averaging and correction of the modulation transfer function (MTF). The 3D electron density map of MspA was compared with a structure previously determined by X-ray crystallography. We were able to demonstrate that structural information up to a resolution of 16.8Å can be recovered using our CTF correction approach, whereas the uncorrected 3D map had a resolution of only 26.2Å. PMID:23000705

390

Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides.  

A family of naturally occurring mycobacterial phosphatidylinositol (PI) and its dimannosides (PIM(2), AcPIM(2), and Ac(2)PIM(2)) that all possess the predominant natural 19:0/16:0 phosphatidyl acylation pattern were prepared to study their mass spectral fragmentations. Among these, the first synthesis of a fully lipidated PIM (i.e., (16:0,18:0)(19:0/16:0)-PIM(2)) was achieved from (±)-1,2:4,5-diisopropylidene-D-myo-inositol in 16 steps in 3% overall yield. A key feature of the strategy was extending the utility of the p-(3,4-dimethoxyphenyl)benzyl protecting group for its use at the O-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies were performed on the synthetic PIMs and compared to those reported for natural PIMs identified from a lipid extract of M. bovis BCG. These analyses confirm that fragmentation patterns can be used to identify the structures of specific PIMs from the cell wall lipid extract. PMID:22845613

391

[Atypical tuberculous osteomyelitis of the humeral shaft caused by Mycobacterium avium].  

A 41-year-old man underwent skeletal scintigraphy due to chronic pain in the left foot and polyarthralgia. He was taking medication for gout and had previously had sarcoidosis, for which he had received corticosteroids and other therapy that was discontinued 4 years ago. Scintigraphy revealed a mass in the shaft of the left humerus that, according to biopsy, was an asymptomatic osteomyelitis caused by Mycobacterium avium. The shaft of the left humerus is an uncommon site for tuberculous osteomyelitis. A viable fistula remained after the biopsy that persisted despite pharmacologic treatment with ethambutol, rifabutin and clarithromycin. Four months later, sequestrectomy was performed with insertion of gentamicin-impregnated beads, which resulted in rapid resolution. The foot pain resolved spontaneously. The incidence of bone tuberculosis has increased over the last 2 decades. The most commonly affected sites are the spine and large joints. Infection with M. avium is sometimes involved. Because of the increasing incidence it is important to include mycobacterial infections in the differential diagnosis of focal bone lesions, especially when standard cultures are initially negative. PMID:16924946

392

Mycobacterium tuberculosis H37Rv is more effective compared to vaccine strains in modulating neutrophil functions: an in vitro study.  

Neutrophils are the primary cells contributing to initial defense against mycobacteria. Yet, little is known about the potential of various mycobacterial strains to stimulate neutrophils. This study was focused to compare the differential capacity of vaccine strains, Mycobacterium bovis bacillus Calmette-Guerin (BCG) and Mycobacterium indicus pranii (Mw), and laboratory strain H37Rv to activate and enhance neutrophil functions. The expression of phenotypic markers like Fc? receptor, toll-like receptor (TLR), and chemokine receptor; secretion of pro-inflammatory cytokines; and the rate of apoptosis were studied in infected neutrophils. Increased expression of CD32, CD64, TLR4, and CXCR3; increased TNF-? secretion; and downregulation of early apoptosis were observed in H37Rv-infected neutrophils. Among the vaccine strains, BCG increased the expression of only CD32 on neutrophils, while Mw was comparatively ineffective. To understand the paracrine role of neutrophils, the supernatants from infected neutrophils were used to stimulate monocytes and T helper cells. The secretory molecules from all infected neutrophils increased the expression of CCR5 on monocytes, whereas only H37Rv-infected supernatant increased the expression of CCR7 on monocytes and CD69 on T cells. Thus, H37Rv was more effective in activating neutrophils and in turn stimulating monocytes and T cells. By comparison, vaccine strains were less effective in modulating neutrophil functions. PMID:22900703

393

Nocardiasis pulmonar en un paciente asmático  

Abstract in spanish El asma bronquial es una enfermedad altamente prevalente en nuestro medio. Con mucha frecuencia estos pacientes presentan crisis severas de broncoespasmo que ameritan tratamientos farmacológicos agresivos, dentro de los que está el uso de esteroides con dosis altas y por tiempo prolongado, lo que puede producir efectos secundarios indeseables. Dentro de estos se encuentra la alteración de los mecanismos de defensa específicos, principalmente los dependientes de las c? (more) ?lulas T. Esto lleva la aparición de infecciones oportunistas por gérmenes diversos, entre los cuales están las nocardias. Se presenta el caso de un paciente asmático adulto que recibía altas dosis de esteroides y en el que se demostró una neumonía por Nocardia así como linfopenia reversible de CD4+ / CD8+. Se analizan los hallazgos y se hace una revisión del tema. Abstract in english Bronchial asthma is very common in our country. Often these patients need agresive therapy with glucocorticoids, but this approach has some side effects , mainly those on the immune system. Thus, opportunistic infections may appear with high mortality rates. These infections are mainly mycobacterial and fungal in origen. Nocardial infections are rare in these patients. We discuss here a patient with a severe asthmatic crisis who received high steroid doses and developed nocardial pneumonia and pulmonary abscesses.

394

Mycobacterium chelonae osteoarthritis in a Kemp's ridley sea turtle (Lepidochelys kempii).  

A stranded Kemp's ridley sea turtle (Lepidochelys kempii) was rescued and treated at the National Aquarium in Baltimore (Maryland, USA) for inappetence and epidermal appendicular and plastral lesions. After 4 mo of care, the turtle developed a swollen left elbow joint. Within 1 mo of initial swelling, osteolytic lesions developed in the proximal radius and ulna. The elbow joint was surgically debrided, flushed, and cultured. The incision dehisced 10 days after surgery. Mycobacterium chelonae was cultured from the left elbow joint and from a skin nodule of the dorsum of the right front flipper. The turtle was euthanized due to apparent systemic infection with M. chelonae. Mycobacterium chelonae was isolated from cultures taken at necropsy of the lung, liver, spleen, kidney, and pericardium. Osteoarthritic infections with M. chelonae have not been reported in reptiles. Additionally, primary osteoarthritic diseases of synovial joints are uncommon in reptilian species. Due to the paucity of reports of mycobacterial diseases in sea turtles, the continued documentation of these cases will increase knowledge and understanding in caring for these endangered animals. PMID:14567241

395

Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution  

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

396

HDV  

Background The current vaccine against tuberculosis (TB), BCG, has failed to control TB worldwide and the protective efficacy is moreover limited to 10–15 years. A vaccine that could efficiently boost a BCG-induced immune response and thus prolong protective immunity would therefore have a significant impact on the global TB-burden. Methods/Findings In the present study we show that the fusion protein HyVac4 (H4), consisting of the mycobacterial antigens Ag85B and TB10.4, given in the adjuvant IC31® or DDA/MPL effectively boosted and prolonged immunity induced by BCG, leading to improved protection against infection with virulent M. tuberculosis (M.tb). Increased protection correlated with an increased percentage of TB10.4 specific IFN?/TNF?/IL-2 or TNF?/IL-2 producing CD4 T cells at the site of infection. Moreover, this vaccine strategy did not compromise the use of ESAT-6 as an accurate correlate of disease development/vaccine efficacy. Indeed both CD4 and CD8 ESAT-6 specific T cells showed significant correlation with bacterial levels. Conclusions/Significance H4-IC31® can efficiently boost BCG-primed immunity leading to an increased protective anti-M.tb immune response dominated by IFN?/TNF?/IL-2 or TNF?/IL2 producing CD4 T cells. H4 in the CD4 T cell inducing adjuvant IC31® is presently in clinical trials. PMID:16903220

397

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil  

Abstract in english We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were ide (more) ntified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

398

Genetics Home Reference: Epidermolysis bullosa with pyloric atresia  

... caused by mutations in the ITGA6 , ITGB4 , and PLEC genes. These genes provide instructions for making proteins ... of EB-PA result from mutations in the PLEC gene. This gene provides instructions for making a ...

399

Scientists Link Gene to Lower Risk of Lung Cancer  

... JavaScript. Scientists Link Gene to Lower Risk of Lung Cancer Inflammation may be a disease factor, researcher says (* ... 2012 Related MedlinePlus Pages Genes and Gene Therapy Lung Cancer MONDAY, Oct. 8 (HealthDay News) -- A gene variant ...

400

earth's early biosphere - Tags « NASA Astrobiology  

Nov 19, 2012... system: celestial mechanics. cells chaos charon chelation chemcam chemical ... (asteroids) dynamo e. coli ea early animals early atmosphere early ... gcm gene detection gene duplication gene expression gene fusions ...

 
 
 
 
401

Gene Therapy for Diseases and Genetic Disorders  

... outcome by gene therapy might be achievable with immune-suppression or alternative gene delivery strategies currently being tested ... dystrophy, among others. Gene Therapy for Acquired Diseases Cancer Multiple gene therapy strategies have been developed to ...

402

GenePRIMP: A GENE PRediction IMprovement Pipeline for Prokaryotic genomes  

We present 'gene prediction improvement pipeline' (GenePRIMP; http://geneprimp.jgi-psf.org/), a computational process that performs evidence-based evaluation of gene models in prokaryotic genomes and reports anomalies including inconsistent start sites, missed genes and split genes. We found that manual curation of gene models using the anomaly reports generated by GenePRIMP improved their quality, and demonstrate the applicability of GenePRIMP in improving finishing quality and comparing different genome-sequencing and annotation technologies.

403

Cellular processing and nuclear targeting of non-viral gene delivery systems  

Gene therapy utilizes genetic material in order to cure patients either by DNA vaccines or by replacement of a defective gene with a normal one. For successful gene therapy certain elements are required: gene delivery systems with low toxicity and immunity, with efficient gene transfer and high gene...

404

RAPID SURVEY FOR PRESENCE OF A BLAST RESISTANCE GENE PI-TA IN RICE CULTIVARS USING THE DOMINANT DNA MARKERS DERIVED FROM PORTIONS OF THE PI-TA GENE  

The Pi-ta gene in rice confers resistance to strains of the blast pathogen Magnaporthe grisea (Herbert) Borr. (anamorph Pyricularia oryza Cav.) containing the corresponding avirulence gene AVR-Pita in a gene-for-gene fashion. The Pi-ta gene is a typical nucleotide-binding site type resistance gene....

405

A determination of the frequency of gene conversion in unmanipulated mouse sperm.  

Gene conversion, sometimes also called micro gene conversion or gene conversion-like events, has been proposed to act on a number of genes in higher eukaryotes, such as gamma-globin, beta-tubulin, major urinary protein, and amyloid A genes. In the immune system, immunoglobulin genes and major histoc...

406

The complex relationship of gene duplication and essentiality  

In yeast and worm, duplicate genes overlap in function so that deleting one of a pair from the genome is less likely to be lethal than deleting a singleton gene. By contrast, previous analyses showed that mouse duplicate genes were as essential as singletons. We show that the relationship between gene duplication and essentiality is complex in multicellular organisms, with developmental genes and genes that were duplicated by whole genome duplication being more essential than other duplicated genes.

407

Gene Amplification and Its Application in Cell and Tissue Engineering  

Gene amplification means the repeated replication of a certain gene without a proportional increase in the copy number of other genes and is a widespread phenomenon in eukaryotes. It is an important developmental and evolutionary process in many organisms. This article focuses on mammalian gene amplification and its application in cell and tissue engineering. The dhfr gene amplification in Chinese hamster ovary (CHO) cells, the gene amplification mechanism, the selection protocol and the application of gene amplification were reviewed.   

408

Human hemoglobin genetics  

This book contains the following 10 chapters: Introduction; The Human Hemoglobins; The Human Globin Genes; Hemoglobin Synthesis and Globin Gene Expression; The Globin Gene Mutations - A. Mechanisms and Classification; The Globin Gene Mutations - B. Their Phenotypes and Clinical Expression; The Genetics of the Human Globin Gene Loci: Formal Genetics and Gene Linkage; The Geographic Distribution of Globin Gene Variation; Labortory Identification, Screening, Education, and Counseling for Abnormal Hemoglobins and Thalassemias; and Approaches to the Treatment of the Hemoglobin Disorders.

409

High diversity of polyketide synthase genes and the melanin biosynthesis gene cluster in Penicillium marneffei  

Despite the unique phenotypic properties and clinical importance of Penicillium marneffei, the polyketide synthase genes in its genome have never been characterized. Twenty-three putative polyketide synthase genes and two putative polyketide synthase nonribosomal peptide-synthase hybrid genes were identified in the P. marneffei genome, a diversity much higher than found in other pathogenic thermal dimorphic fungi, such as Histoplasma capsulatum (one polyketide synthase gene) and Coccidioides immitis (10 polyketide synthase genes). These genes were evenly distributed on the phylogenetic tree with polyketide synthase genes of Aspergillus and other fungi, indicating that the high diversity was not a result of lineage-specific gene expansion through recent gene duplication. The melanin-biosynt...

410

Immunoglobulin genes  

This book reports on the structure, function, and expression of the genes encoding antibodies in normal and neoplastic cells. Topics covered are: B Cells; Organization and rearrangement of immunoglobin genes; Immunoglobin genes in disease; Immunoglobin gene expression; and Immunoglobin-related genes.

411

Ren-1 and Ren-2 loci are expressed in mouse kidney.  

Inbred strains of mice can be categorized into two groups based on the absence or presence of a duplicated copy of the renin structural gene; one-gene strains carry a single renin gene (Ren-1), whereas two-gene strains carry two renin genes (Ren-1 and Ren-2). To investigate the contribution that eac...

412

Wide distribution of homologs of Escherichia coli Kdp K+-ATPase among gram-negative bacteria.  

We used Southern blotting to screen a variety of bacterial genes for homology to the kdp genes of Escherichia coli, genes that encode an ATP-driven K+ transport system. We found that most enterobacteria have sequences homologous to those of the three kdp structural genes and the kdpD regulatory gene...

413

Gene 5.5 protein of bacteriophage T7 inhibits the nucleoid protein H-NS of Escherichia coli.  

Gene 5.5 of coliphage T7 is one of the most highly expressed genes during T7 infection. Gene 5.5 protein, purified from cells overexpressing the cloned gene, purifies with the nucleoid protein H-NS of Escherichia coli during three chromatographic steps. A fusion protein of gene 5.5 protein and malto...

414

Genetically modified yeast species and fermentation processes using genetically modified yeast  

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications', include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

415

Construction of a Binary Vector for Knockout and Expression Analysis of Rice Blast Fungus Genes  

We developed an efficient method to analyze gene function and expression of the rice blast fungus. We constructed a GATEWAY binary vector, which generates a gene-targeted disruptant carrying a green fluorescent protein gene under the native promoter of the target gene. Using this method, the knockout efficiency and expression patterns of two hypothetical genes were determined.   

416