WorldWideScience
 
 
1

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases.

Santos Rubens R; Sartori Alexandrina; Lima Deison S; Souza Patrícia RM; Coelho-Castelo Arlete AM; Bonato Vânia LD; Silva Célio L

2009-01-01

2

HSP65-PRA identification of non-tuberculosis mycobacteria from 4892 samples suspicious for mycobacterial infections.  

Science.gov (United States)

Various molecular methods have been used for the rapid identification of mycobacterial species. In this survey, evaluation of antibiotic resistance and PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was carried out for identification of non-tuberculosis mycobacteria (NTM) isolates from different clinical specimens. Forty-eight different mycobacterial isolates were selected and followed by the conventional and PRA of hsp65 for species identification. The antibiotic susceptibility test was carried out according to standard methods. A 439 bp PCR product of hsp65 in all selected isolates was amplified and digested with the BstEII and HaeIII restriction enzymes. The restriction fragment length polymorphism (RFLP) patterns were analyzed for species identification. Using PRA for 48 mycobacterial selected isolates, including 15 M. tuberculosis, one M. bovis and all 32 isolates of NTM, revealed 11 different species among the NTM isolates. The most frequent NTM isolates were M. kansasii, M. gordonae III, M. marinum, M. chelonae, M. scrofluaceum and M. gastri. In most cases, the PRA results were perfectly in accordance with the classical biochemical method. Combination of resistance to rifampin and isoniazid was present among M. kansasi, M. gordoniae III, M. scrofluaceum, M. chelonae, M. marinum, M. gastri, M. gordoniae II and M. trivale isolates. A high incidence of co-resistance to six, five, four and three anti-TB drugs was observed in 18.5%, 9.1%, 6.6% and 11.7% of all NTM isolates, respectively. Our results showed that PRA, in comparison with classical methods, is rapid and accurate enough for the identification of mycobacterial species from LJ medium. Additionally, we found that in Iran we have a highly diverse population of NTM isolates among patients suspected of having TB. PMID:22963505

Saifi, M; Jabbarzadeh, E; Bahrmand, A R; Karimi, A; Pourazar, S; Fateh, A; Masoumi, M; Vahidi, E

2012-09-11

3

HSP65-PRA identification of non-tuberculosis mycobacteria from 4892 samples suspicious for mycobacterial infections.  

UK PubMed Central (United Kingdom)

Various molecular methods have been used for the rapid identification of mycobacterial species. In this survey, evaluation of antibiotic resistance and PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was carried out for identification of non-tuberculosis mycobacteria (NTM) isolates from different clinical specimens. Forty-eight different mycobacterial isolates were selected and followed by the conventional and PRA of hsp65 for species identification. The antibiotic susceptibility test was carried out according to standard methods. A 439 bp PCR product of hsp65 in all selected isolates was amplified and digested with the BstEII and HaeIII restriction enzymes. The restriction fragment length polymorphism (RFLP) patterns were analyzed for species identification. Using PRA for 48 mycobacterial selected isolates, including 15 M. tuberculosis, one M. bovis and all 32 isolates of NTM, revealed 11 different species among the NTM isolates. The most frequent NTM isolates were M. kansasii, M. gordonae III, M. marinum, M. chelonae, M. scrofluaceum and M. gastri. In most cases, the PRA results were perfectly in accordance with the classical biochemical method. Combination of resistance to rifampin and isoniazid was present among M. kansasi, M. gordoniae III, M. scrofluaceum, M. chelonae, M. marinum, M. gastri, M. gordoniae II and M. trivale isolates. A high incidence of co-resistance to six, five, four and three anti-TB drugs was observed in 18.5%, 9.1%, 6.6% and 11.7% of all NTM isolates, respectively. Our results showed that PRA, in comparison with classical methods, is rapid and accurate enough for the identification of mycobacterial species from LJ medium. Additionally, we found that in Iran we have a highly diverse population of NTM isolates among patients suspected of having TB.

Saifi M; Jabbarzadeh E; Bahrmand AR; Karimi A; Pourazar S; Fateh A; Masoumi M; Vahidi E

2013-08-01

4

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

Kim, Hyun-Ju; Mun, Ho-Suk; Kim, Hong; Oh, Eun-Ju; Ha, Youngju; Bai, Gill-Han; Park, Young-Gil; Cha, Chang-Yong; Kook, Yoon-Hoh

5

The genomic heterogeneity among Mycobacterium terrae complex displayed by sequencing of 16S rRNA and hsp 65 genes.  

UK PubMed Central (United Kingdom)

The species identification within Mycobacterium terrae complex has been known to be very difficult. In this study, the genomic diversity of M. terrae complex with eighteen clinical isolates, which were initially identified as M. terrae complex by phenotypic method, was investigated, including that of three type strains (M. terrae, M. nonchromogenicum, and M. triviale ). 16S rRNA and 65-kDa heat shock protein (hsp 65) gene sequences of mycobacteria were determined and aligned with eleven other references for the comparison using similarity search against the GenBank and Ribosomal Database Project II (RDP) databases. 16S rRNA and hsp 65 genes of M. terrae complex showed genomic heterogeneity. Amongst the eighteen clinical isolates, nine were identified as M. nonchromogenicum, eight as M. terrae, one as M. mucogenicum with the molecular characteristic of rapid growth. M. nonchromogenicum could be subdivided into three subgroups, while M. terrae could be subdivided into two subgroups using a 5 bp criterion (>1% difference). Seven isolates in two subgroups of M. nonchromogenicum were Mycobacterium sp. strain MCRO 6, which was closely related to M. nonchromogenicum. The hsp 65 gene could not differentiate one M. nonchromogenicum from M. avium or one M. terrae from M. intracellulare. The nucleotide sequence analysis of 16S rRNA and hsp 65 genes was shown to be useful in identifying the M. terrae complex, but hsp 65 was less discriminating than 16S rRNA.

Lee CK; Gi HM; Cho Y; Kim YK; Lee KN; Song KJ; Song JW; Park KS; Park EM; Lee H; Bai GH

2004-01-01

6

Análise de restrição enzimática do gene hsp65 de isolados clínicos de pacientes com suspeita de tuberculose pulmonar em Teresina, Piauí/ Restriction enzyme analysis of the hsp65 gene in clinical isolates from patients suspected of having pulmonary tuberculosis in Teresina, Brazil  

Scientific Electronic Library Online (English)

Full Text Available Abstract in portuguese OBJETIVO: Identificar as espécies de micobactérias encontradas no escarro de pacientes com suspeita de tuberculose pulmonar e analisar o impacto dessas identificações na abordagem terapêutica. MÉTODOS: Foram avaliados 106 pacientes com suspeita de tuberculose pulmonar encaminhados para o serviço de pneumologia de um hospital público em Teresina, Piauí. Espécimes de escarro matinal foram avaliados quanto à presença de micobactérias por baciloscopia e cultura. (more) Foram utilizadas PCR e análise de restrição enzimática do gene hsp65 (PRA-hsp65) para a identificação das cepas de micobactérias isoladas em cultura. RESULTADOS: Foram analisadas 206 amostras de escarro. A idade dos pacientes variou de 15 a 87 anos, sendo 67% do gênero masculino. Tosse ocorreu em 100% dos casos. O padrão radiográfico predominante foi de lesão moderada, observada em 70%. A positividade no esfregaço foi de 76%, e isolamento em cultura ocorreu em 91% das culturas executadas. Testes tradicionais identificaram micobactérias não tuberculosas (MNT) em 9% dos isolados. O método PRA-hsp65 confirmou esses dados, mostrando sete padrões de bandas capazes de identificar as espécies de MNT isoladas: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5 e M. gordonae 7. Todos os pacientes com MNT tinham mais de 60 anos, e observaram-se bronquiectasias em 88% das radiografias. Houve dois casos de reinfecção, identificados inicialmente como infecção por M. abscessus e M. kansasii. CONCLUSÕES: As MNT causam infecção pulmonar em pacientes imunocompetentes, e a identificação das MNT é importante para estabelecer o diagnóstico correto e a decisão terapêutica mais adequada. O método PRA-hsp65 é útil para identificar espécies de MNT e pode ser implantado em laboratórios de biologia molecular não especializados em micobactérias. Abstract in english OBJECTIVE: To identify mycobacterial species in the sputum of patients suspected of having pulmonary tuberculosis and to determine the impact that the acquisition of this knowledge has on the therapeutic approach. METHODS: We evaluated 106 patients suspected of having pulmonary tuberculosis and referred to the pulmonology department of a public hospital in the city of Teresina, Brazil. Morning sputum specimens were evaluated for the presence of mycobacteria by sputum smea (more) r microscopy and culture. We used PCR and restriction enzyme analysis of the hsp65 gene (PRA-hsp65) to identify the strains of mycobacteria isolated in culture. RESULTS: A total of 206 sputum samples were analyzed. Patient ages ranged from 15 to 87 years, and 67% were male. There was cough in 100% of the cases. The predominant radiographic pattern was moderate disease, observed in 70%. Smear positivity was 76%, and isolation in culture occurred in 91% of the cultures. Traditional tests identified nontuberculous mycobacteria (NTM) in 9% of the isolates. The PRA-hsp65 method confirmed these data, showing seven band patterns that were able to identify the isolated species of NTM: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5; and M. gordonae 7. All of the patients with NTM were over 60 years of age, and bronchiectasis was seen in 88% of the X-rays. There were two cases of reinfection, initially attributed to M. abscessus and M. kansasii. CONCLUSIONS: In immunocompetent patients, NTM can infect the lungs. It is important to identify the specific NTM in order to establish the correct diagnosis and choose the most appropriate therapeutic regimen. The PRA-hsp65 method is useful in identifying NTM species and can be implemented in molecular biology laboratories that do not specialize in the identification of mycobacteria.

Bona, Maria das Graças Motta e; Leal, Maria José Soares; Martins, Liline Maria Soares; Silva, Raimundo Nonato da; Castro, José Adail Fonseca de; Monte, Semiramis Jamil Hadad do

2011-10-01

7

Análise de restrição enzimática do gene hsp65 de isolados clínicos de pacientes com suspeita de tuberculose pulmonar em Teresina, Piauí Restriction enzyme analysis of the hsp65 gene in clinical isolates from patients suspected of having pulmonary tuberculosis in Teresina, Brazil  

Directory of Open Access Journals (Sweden)

Full Text Available OBJETIVO: Identificar as espécies de micobactérias encontradas no escarro de pacientes com suspeita de tuberculose pulmonar e analisar o impacto dessas identificações na abordagem terapêutica. MÉTODOS: Foram avaliados 106 pacientes com suspeita de tuberculose pulmonar encaminhados para o serviço de pneumologia de um hospital público em Teresina, Piauí. Espécimes de escarro matinal foram avaliados quanto à presença de micobactérias por baciloscopia e cultura. Foram utilizadas PCR e análise de restrição enzimática do gene hsp65 (PRA-hsp65) para a identificação das cepas de micobactérias isoladas em cultura. RESULTADOS: Foram analisadas 206 amostras de escarro. A idade dos pacientes variou de 15 a 87 anos, sendo 67% do gênero masculino. Tosse ocorreu em 100% dos casos. O padrão radiográfico predominante foi de lesão moderada, observada em 70%. A positividade no esfregaço foi de 76%, e isolamento em cultura ocorreu em 91% das culturas executadas. Testes tradicionais identificaram micobactérias não tuberculosas (MNT) em 9% dos isolados. O método PRA-hsp65 confirmou esses dados, mostrando sete padrões de bandas capazes de identificar as espécies de MNT isoladas: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5 e M. gordonae 7. Todos os pacientes com MNT tinham mais de 60 anos, e observaram-se bronquiectasias em 88% das radiografias. Houve dois casos de reinfecção, identificados inicialmente como infecção por M. abscessus e M. kansasii. CONCLUSÕES: As MNT causam infecção pulmonar em pacientes imunocompetentes, e a identificação das MNT é importante para estabelecer o diagnóstico correto e a decisão terapêutica mais adequada. O método PRA-hsp65 é útil para identificar espécies de MNT e pode ser implantado em laboratórios de biologia molecular não especializados em micobactérias.OBJECTIVE: To identify mycobacterial species in the sputum of patients suspected of having pulmonary tuberculosis and to determine the impact that the acquisition of this knowledge has on the therapeutic approach. METHODS: We evaluated 106 patients suspected of having pulmonary tuberculosis and referred to the pulmonology department of a public hospital in the city of Teresina, Brazil. Morning sputum specimens were evaluated for the presence of mycobacteria by sputum smear microscopy and culture. We used PCR and restriction enzyme analysis of the hsp65 gene (PRA-hsp65) to identify the strains of mycobacteria isolated in culture. RESULTS: A total of 206 sputum samples were analyzed. Patient ages ranged from 15 to 87 years, and 67% were male. There was cough in 100% of the cases. The predominant radiographic pattern was moderate disease, observed in 70%. Smear positivity was 76%, and isolation in culture occurred in 91% of the cultures. Traditional tests identified nontuberculous mycobacteria (NTM) in 9% of the isolates. The PRA-hsp65 method confirmed these data, showing seven band patterns that were able to identify the isolated species of NTM: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5; and M. gordonae 7. All of the patients with NTM were over 60 years of age, and bronchiectasis was seen in 88% of the X-rays. There were two cases of reinfection, initially attributed to M. abscessus and M. kansasii. CONCLUSIONS: In immunocompetent patients, NTM can infect the lungs. It is important to identify the specific NTM in order to establish the correct diagnosis and choose the most appropriate therapeutic regimen. The PRA-hsp65 method is useful in identifying NTM species and can be implemented in molecular biology laboratories that do not specialize in the identification of mycobacteria.

Maria das Graças Motta e Bona; Maria José Soares Leal; Liline Maria Soares Martins; Raimundo Nonato da Silva; José Adail Fonseca de Castro; Semiramis Jamil Hadad do Monte

2011-01-01

8

Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of Mycobacterial species from clinical isolates  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. Results Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates) for the MTC and 91.4% (161 isolates) for the NTM. Among the discordant results (8.6%) for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA. Conclusions This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.

Huang Chen-Cheng; Chen Jiann-Hwa; Hu Shiau-Ting; Chiou Chien-Shun; Huang Wei-Chang; Hsu Jeng-Yuan; Lu Jang-Jih; Shen Gwan-Han

2012-01-01

9

Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of mycobacterial species from clinical isolates.  

UK PubMed Central (United Kingdom)

BACKGROUND: Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. RESULTS: Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates) for the MTC and 91.4% (161 isolates) for the NTM. Among the discordant results (8.6%) for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA. CONCLUSIONS: This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.

Huang CC; Chen JH; Hu ST; Chiou CS; Huang WC; Hsu JY; Lu JJ; Shen GH

2012-01-01

10

Pyrosequence Analysis of the hsp65 Genes of Nontuberculous Mycobacterium Communities in Unchlorinated Drinking Water in the Netherlands.  

UK PubMed Central (United Kingdom)

Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance.

van der Wielen PW; Heijnen L; van der Kooij D

2013-10-01

11

Pyrosequence Analysis of the hsp65 Genes of Nontuberculous Mycobacterium Communities in Unchlorinated Drinking Water in the Netherlands.  

Science.gov (United States)

Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance. PMID:23913420

van der Wielen, Paul W J J; Heijnen, Leo; van der Kooij, Dick

2013-08-02

12

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Coelho-Castelo AAM; Trombone AP; Rosada RS; Santos RR; Bonato VLD; Sartori A; Silva CL

2006-01-01

13

hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory PCR e análise de padrões de restrição do gene hsp65 (PRA) para identificação de micobactérias no laboratório clínico  

Directory of Open Access Journals (Sweden)

Full Text Available More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.Mais de 70 espécies de micobactérias já foram definidas e algumas delas podem causar enfermidade em humanos, especialmente em pacientes imunocomprometidos. A identificação de espécie, na maioria dos laboratórios clínicos, se baseia em características fenotípicas e testes bioquímicos e resultados definitivos só são obtidos após duas a quatro semanas. Métodos rápidos de identificação reduzem o tempo necessário para o diagnóstico e podem antecipar a instituição do tratamento específico, aumentando as chances de sucesso. A análise de padrões de restrição do gene hsp65 amplificado por PCR (PRA) foi utilizada como método rápido de identificação em 103 isolamentos clínicos. Os padrões de bandas foram interpretados por comparação com tabelas publicadas e padrões disponíveis em um site de Internet (http://www.hospvd.ch:8005). Resultados concordantes de PRA e identificação bioquímica foram obtidos em 76 de 83 isolamentos (91,5%). Os resultados de 20 isolamentos não puderam ser comparados porque a identificação fenotípica ou por PRA foi inconclusiva. Os resultados deste trabalho mostram que PRA pode ser útil para identificação de rotina de micobactérias por ser um método acurado, rápido e mais econômico do que a identificação convencional.

Carolina Feher da SILVA; Suely Yoko Mizuka UEKI; Débora de Cássia Pires GEIGER; Sylvia Cardoso LEÃO

2001-01-01

14

Report of 2 indigenous cases of leprosy from a European country: use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for identification of Mycobacterium leprae directly from a clinical sample.  

UK PubMed Central (United Kingdom)

In this article, we report on 2 indigenous cases of leprosy detected in a European country. We also report on the use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for rapid identification of Mycobacterium leprae directly from the clinical sample.

Neonakis IK; Gitti Z; Kontos F; Baritaki S; Zerva L; Krambovitis E; Spandidos DA

2009-07-01

15

Report of 2 indigenous cases of leprosy from a European country: use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for identification of Mycobacterium leprae directly from a clinical sample.  

Science.gov (United States)

In this article, we report on 2 indigenous cases of leprosy detected in a European country. We also report on the use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for rapid identification of Mycobacterium leprae directly from the clinical sample. PMID:19376672

Neonakis, Ioannis K; Gitti, Zoe; Kontos, Fanourios; Baritaki, Stavroula; Zerva, Loukia; Krambovitis, Elias; Spandidos, Demetrios A

2009-04-18

16

DETECÇÃO DO COMPLEXO Mycobacterium tuberculosis NO LEITE PELA REAÇÃO EM CADEIA DA POLIMERASE SEGUIDA DE ANÁLISE DE RESTRIÇÃO DO FRAGMENTO AMPLIFICADO (PRA) DETECTION OF Mycobacterium tuberculosis COMPLEX BY PCR-RESTRICTION FRAGMENT LENGTH POLYMORFISM ANALYSIS OF THE HSP65 GENE  

Directory of Open Access Journals (Sweden)

Full Text Available Mycobacterium bovis é membro do complexo Mycobacterium tuberculosis (MTBC), grupo este composto por espécies com grande homologia genética. É o agente etiológico da tuberculose bovina, importante zoonose transmissível ao homem, principalmente através da inalação do bacilo e/ou pelo consumo de leite e derivados não-pasteurizados provenientes de vacas tuberculosas. O objetivo deste estudo foi padronizar a identificação de micobactérias do complexo M. tuberculosis presentes no leite, por metodologia molecular. Fez-se a extração de DNA diretamente do leite contaminado e realizou-se a identificação molecular pela reação em cadeia da polimerase seguida de análise de restrição do fragmento amplificado (PRA). Utilizaram-se inhagens de referência e leite cru artificialmente contaminado com M. bovis IP. Um fragmento de 441pb do gene hsp65 foi amplificado, tratado com BstEII e HaeIII e empregou-se o perfil de restrição enzimática obtido para identificar o complexo M. tuberculosis no leite. Com a PRA foi possível detectar com especificidade e sensibilidade a presença de M. bovis em até 10 UFC/mL de leite. A metodologia padronizada poderá auxiliar os métodos microbiológicos e bioquímicos tradicionalmente usados na identificação do bacilo em alimentos suspeitos de contaminação, como, por exemplo, o leite proveniente de animais suspeitos de infecção por M. bovis.Palavras-chaves: Análise de perfil de restrição enzimática (PRA), complexo Mycobacterium tuberculosis, leite, Mycobacterium bovis, limite de detecção (PCR). Mycobacterium bovis is a member of the M. tuberculosis complex, a group composed by species with high genetic homology. The pathogen is the etiological agent of bovine tuberculosis, an important zoonosis that is mainly transmitted by inhalation of infectious droplet nuclei or by ingestion of milk and crude milk derivative products from tuberculosis cows. The definitive identification of M. bovis, up to species level, is time consuming and difficult. In this work, the objective was to standardize a polymerase chain reaction followed by an enzyme restriction analysis in order to identify the M. tuberculosis complex in milk, without a microbiological isolation step. Reference strains and raw milk seeded with M. Bovis, were used as the starting material.  A 441pb fragment of the hsp65 gene was amplified and digested by two restriction enzymes BstEII and HaeIII. The obtained profile was used to identify the M. tuberculosis complex in milk. The minimum limit of detection of M. bovis in milk was 10CFU/mL. PRA methodology proved to be a specific and sensible method. It can be used to assist the microbiological and biochemical methods commonly used to identifying the bacilli in clinical samples, as milk  Key word: Detection limit (PRA), Mycobacterium tuberculosis complex, milk Mycobacterium bovis, Restriction Enzyme Analysis (PCR),

Eduardo Eustáquio de Souza Figueiredo; Marlei Gomes da Silva; Leila de Souza Fonseca; Joab Trajano Silva; Vânia Margaret Flosi Paschoalin

2008-01-01

17

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardi (more) ovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 µg DNA-HSP65. On day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Fonseca, D.M.; Bonato, V.L.D.; Silva, C.L.; Sartori, A.

2007-11-01

18

Purification of clinical-grade recombinant HSP65-MUCI fusion protein.  

UK PubMed Central (United Kingdom)

HSP65-MUCI is a fusion protein between BCG (Bacille Calmette-Guerin)-derived HSP65 (heat-shock protein 65) and human MUCI (mucin I) VNTR (variable number of tandem repeats)-domain peptides that has shown antitumour efficacy. China's Food and Drug Administration has recently approved a Phase I clinical trial using HSP65-MUCI for the treatment of MUCI-positive breast cancer. In order to produce sufficient quantities of clinical-grade HSP65-MUCI, we established a pilot-scale purification scheme comprising two steps of column chromatography: HIC (hydrophobic-interaction chromatography) and IEX (ion-exchange chromatography). The pH values of the buffers used in homogenization and HIC were adjusted to pH 9.0 to maintain protein stability and prevent protein degradation. Using this manufacturing process, we obtained clinical-grade HSP65-MUCI with a yield of 400 mg per 70 g of wet cell pellet and >96% purity.

Cao Z; Feng Y; Wei H; Fang M; Hu X; Yu Y; Wang L; Wan M

2010-09-01

19

Influência do biofármaco DNA-hsp65 na lesão pulmonar induzida por bleomicina/ Influence of a DNA-hsp65 vaccine on bleomycin-induced lung injury  

Scientific Electronic Library Online (English)

Full Text Available Abstract in portuguese OBJETIVO: Avaliar a influência do biofármaco DNA-hsp65 em um modelo de distúrbio fibrosante pulmonar experimental. MÉTODOS: Foram estudados 120 camundongos machos C57BL/6, divididos em quatro grupos: grupo SS, animais tratados com salina (placebo) e injetados com salina intratraqueal (IT); grupo SB, tratados com salina (placebo) e injetados com bleomicina IT; grupo PB, tratados com plasmídeo, sem gene bacteriano, e injetados com bleomicina IT; e grupo BB, tratados co (more) m DNA-hsp65 e injetados com bleomicina IT. A bleomicina foi injetada 15 dias após a última imunização, e os animais sacrificados seis semanas após o uso da droga IT. O pulmão esquerdo retirado foi utilizado para análise morfológica, e o pulmão direito para dosagens de hidroxiprolina. RESULTADOS: A proporção de camundongos que apresentaram morte não-programada depois de 48 h da injeção IT foi maior no grupo SB em comparação ao grupo SS (57,7% vs. 11,1%). A área percentual média de interstício septal foi maior nos grupos SB e PB (53,1 ± 8,6% e 53,6 ± 9,3%, respectivamente) em comparação aos grupos SS e BB (32,9 ± 2,7% e 34,3 ± 6,1%, respectivamente). Os grupos SB, PB e BB mostraram aumentos nos valores médios da área de interstício septal corada por picrosirius em comparação ao grupo SS (SS: 2,0 ± 1,4%; SB: 8,2 ± 4,9%; PB: 7,2 ± 4,2%; e BB:6,6±4,1%).O conteúdo pulmonar de hidroxiprolina no grupo SS foi inferior ao dos demais grupos (SS: 104,9 ± 20,9 pg/pulmão; SB: 160,4 ±47,8 pg/pulmão; PB:170,0 ± 72,0 pg/pulmão; e BB: 162,5 ± 39,7 pg/pulmão). CONCLUSÕES: A imunização com o biofármaco DNA-hsp65 interferiu na deposição de matriz não-colágena em um modelo de lesão pulmonar induzida por bleomicina. Abstract in english OBJECTIVE: To evaluate the effects of immunization with a DNA-hsp65 vaccine in an experimental model of pulmonary fibrosis. METHODS: A total of 120 male C57BL/6 mice were distributed into four groups: SS, injected with saline (placebo) and then receiving intratracheal (IT) instillation of saline; SB, injected with saline (placebo) and then receiving IT instillation of bleomycin; PB, treated with plasmid only, without bacterial genome, and then receiving IT instillation of (more) bleomycin; and BB, treated with the vaccine and then receiving IT instillation of bleomycin. Bleomycin was instilled 15 days after the last immunization, and the animals were killed six weeks thereafter. The left and right lungs were removed, the former for morphological analysis and the latter for hydroxyproline measurements. RESULTS: The proportion of deaths within the first 48 h after the IT instillation (deaths attributed to the surgical procedure) was higher in the SB group than in the SS group (57.7% vs. 11.1%). The mean area of pulmonary interstitial septa was greater in the SB and PB groups (53.1 ± 8.6% and 53.6±9.3%, respectively) than in the SS and BB groups (32.9 ± 2.7% and 34.3 ± 6.1%, respectively). The mean area of interstitial septa stained by picrosirius was greater in the SB, PB and BB groups than in the SS group (8.2 ± 4.9%, 7.2 ± 4.2% and 6.6 ± 4.1%, respectively, vs. 2.0±1.4%). The total hydroxyproline content in the lung was significantly lower in the SS group (104.9 ± 20.9 pg/lung) than in the other groups (SB: 160.4 ± 47.8 pg/lung; PB: 170.0 ± 72.0 pg/lung; and BB: 162.5 ± 39.7 pg/lung). CONCLUSIONS: Immunization with the DNA-hsp65 vaccine reduced the deposition of noncollagen matrix in a model of bleomycin-induced lung lesion.

Padua, Adriana Ignacio de; Silva, Célio Lopes; Ramos, Simone Gusmão; Faccioli, Lúcia Helena; Martinez, José Antônio Baddini

2008-11-01

20

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.

Trombone Ana Paula F; Silva Celio L; Almeida Luciana P; Rosada Rogerio S; Lima Karla M; Oliver Constance; Jamur Maria C; Coelho-Castelo Arlete AM

2007-01-01

 
 
 
 
21

Discovery of a novel hsp65 genotype within Mycobacterium massiliense associated with the rough colony morphology.  

UK PubMed Central (United Kingdom)

So far, genetic diversity among strains within Mycobacterium massiliense has rarely been studied. To investigate the genetic diversity among M. massiliense, we conducted phylogenetic analysis based on hsp65 (603-bp) and rpoB (711-bp) sequences from 65 M. massiliense Korean isolates. We found that hsp65 sequence analysis could clearly differentiate them into two distinct genotypes, Type I and Type II, which were isolated from 35 (53.8%) and 30 patients (46.2%), respectively. The rpoB sequence analysis revealed a total of four genotypes (R-I to R-IV) within M. massiliense strains, three of which (R-I, R-II and R-III) correlated with hsp65 Type I, and other (R-IV), which correlated with Type II. Interestingly, genotyping by the hsp65 method agreed well with colony morphology. Despite some exceptions, Type I and II correlated with smooth and rough colonies, respectively. Also, both types were completely different from one another in terms of MALDI-TOF mass spectrometry profiles of whole lipid. In addition, we developed PCR-restriction analysis (PRA) based on the Hinf I digestion of 644-bp hsp65 PCR amplicons, which enables the two genotypes within M. massiliense to be easily and reliably separated. In conclusion, two distinct hsp65 genotypes exist within M. massiliense strains, which differ from one another in terms of both morphology and lipid profile. Furthermore, our data indicates that Type II is a novel M. massiliense genotype being herein presented for the first time. The disparity in clinical traits between these two hsp65 genotypes needs to be exploited in the future study.

Kim BJ; Yi SY; Shim TS; Do SY; Yu HK; Park YG; Kook YH; Kim BJ

2012-01-01

22

Discovery of a novel hsp65 genotype within Mycobacterium massiliense associated with the rough colony morphology.  

Science.gov (United States)

So far, genetic diversity among strains within Mycobacterium massiliense has rarely been studied. To investigate the genetic diversity among M. massiliense, we conducted phylogenetic analysis based on hsp65 (603-bp) and rpoB (711-bp) sequences from 65 M. massiliense Korean isolates. We found that hsp65 sequence analysis could clearly differentiate them into two distinct genotypes, Type I and Type II, which were isolated from 35 (53.8%) and 30 patients (46.2%), respectively. The rpoB sequence analysis revealed a total of four genotypes (R-I to R-IV) within M. massiliense strains, three of which (R-I, R-II and R-III) correlated with hsp65 Type I, and other (R-IV), which correlated with Type II. Interestingly, genotyping by the hsp65 method agreed well with colony morphology. Despite some exceptions, Type I and II correlated with smooth and rough colonies, respectively. Also, both types were completely different from one another in terms of MALDI-TOF mass spectrometry profiles of whole lipid. In addition, we developed PCR-restriction analysis (PRA) based on the Hinf I digestion of 644-bp hsp65 PCR amplicons, which enables the two genotypes within M. massiliense to be easily and reliably separated. In conclusion, two distinct hsp65 genotypes exist within M. massiliense strains, which differ from one another in terms of both morphology and lipid profile. Furthermore, our data indicates that Type II is a novel M. massiliense genotype being herein presented for the first time. The disparity in clinical traits between these two hsp65 genotypes needs to be exploited in the future study. PMID:22693637

Kim, Byoung-Jun; Yi, Su-Yeon; Shim, Tae-Sun; Do, Seung Yeon; Yu, Hee-Kyung; Park, Young-Gil; Kook, Yoon-Hoh; Kim, Bum-Joon

2012-06-05

23

Purification of clinical-grade recombinant HSP65-MUCI fusion protein.  

Science.gov (United States)

HSP65-MUCI is a fusion protein between BCG (Bacille Calmette-Guerin)-derived HSP65 (heat-shock protein 65) and human MUCI (mucin I) VNTR (variable number of tandem repeats)-domain peptides that has shown antitumour efficacy. China's Food and Drug Administration has recently approved a Phase I clinical trial using HSP65-MUCI for the treatment of MUCI-positive breast cancer. In order to produce sufficient quantities of clinical-grade HSP65-MUCI, we established a pilot-scale purification scheme comprising two steps of column chromatography: HIC (hydrophobic-interaction chromatography) and IEX (ion-exchange chromatography). The pH values of the buffers used in homogenization and HIC were adjusted to pH 9.0 to maintain protein stability and prevent protein degradation. Using this manufacturing process, we obtained clinical-grade HSP65-MUCI with a yield of 400 mg per 70 g of wet cell pellet and >96% purity. PMID:20704567

Cao, Zhao; Feng, Yu; Wei, Hongfei; Fang, Mingli; Hu, Xiaoping; Yu, Yongli; Wang, Liying; Wan, Min

2010-09-01

24

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-? by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma. The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.

Gonçalves Eduardo DC; Bonato Vânia; da Fonseca Denise M; Soares Edson G; Brandão Izaíra T; Soares Ana; Silva Célio L

2007-01-01

25

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strate (more) gy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-? but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E.; Ramos, S.G.; Silva, C.L.; Coelho-Castelo, A.A.M.

2012-12-01

26

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis  

Directory of Open Access Journals (Sweden)

Full Text Available In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-? but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

C.D. Rocha; A.P.F. Trombone; J.C.C. Lorenzi; L.P. Almeida; A.F. Gembre; E. Padilha; S.G. Ramos; C.L. Silva; A.A.M. Coelho-Castelo

2012-01-01

27

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally. Results We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 ?g of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-? and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 ?g). Conclusion Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.

Rosada Rogério S; Torre Lucimara; Frantz Fabiani G; Trombone Ana PF; Zárate-Bladés Carlos R; Fonseca Denise M; Souza Patrícia RM; Brandão Izaíra T; Masson Ana P; Soares Édson G; Ramos Simone G; Faccioli Lúcia H; Silva Célio L; Santana Maria HA; Coelho-Castelo Arlete AM

2008-01-01

28

Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (rpoB)  

Digital Repository Infrastructure Vision for European Research (DRIVER)

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the ? subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were direct...

Kim, Bum-Joon; Lee, Seung-Hyun; Lyu, Mi-Ae; Kim, Seo-Jeong; Bai, Gill-Han; Kim, Sang-Jae; Chae, Gue-Tae; Kim, Eui-Chong

29

Expanding the mycobacterial diversity of metalworking fluids (MWFs): evidence showing MWF colonization by Mycobacterium abscessus.  

UK PubMed Central (United Kingdom)

Nontuberculous mycobacteria (NTM) have been associated with hypersensitivity pneumonitis in machinists. Only two species of NTM, namely Mycobacterium immunogenum and Mycobacterium chelonae, have been reported thus far to have the ability to colonize contaminated metalworking fluids (MWFs). Here, we report, for the first time, the presence and characterization (phenotypic and genotypic) of a third species, Mycobacterium abscessus, colonizing these harsh alkaline machining fluids. Two Mycobacterium morphotypes, smooth (S) and rough (R), were isolated (two isolates each) from an in-use industrial MWFs. Biocide susceptibility analysis using triclosan as a model yielded the same minimal inhibitory concentration for the two morphotypes. PCR-restriction analysis-based speciation of the morphotypes confirmed their identity as M. abscessus. Genotyping based on partial DNA sequences corresponding to the variable regions of the hsp65 gene and 16S-23S rRNA operon internal transcribed spacer region and randomly amplified polymorphic DNA-PCR analysis showed that both morphotypes belong to a single genotype. In addition, we isolated and confirmed two novel mycobacterial genotypes, one each of M. immunogenum and M. chelonae from additional in-use MWF screening. Taken together, this study expands the known mycobacterial species- and strain-diversity colonizing MWF. Furthermore, the study emphasizes the need for including M. abscessus species in the existing mycobacterial screening of contaminated MWF.

Kapoor R; Yadav JS

2012-02-01

30

Immunization with a HSP65-HER2 fusion peptide selectively eliminates HER2(+) B16 melanoma cells in a xenograft tumor mouse model.  

UK PubMed Central (United Kingdom)

HER2/neu peptide-based vaccines can eliminate human tumors overexpressing the human epidermal growth factor receptor 2 (HER2/neu), but the efficacy of this therapeutic strategy is suboptimal. Heat shock proteins (HSPs) are capable of eliciting efficient cytotoxic T lymphocyte (CTL) responses by cross-presentation. To evaluate whether immunization with a HSP65-HER2 fusion peptide could selectively eliminate HER2(+) B16 melanoma cells in a xenograft tumor mouse model, a HSP65-HER2 fusion peptide was incubated with immature dendritic cells (iDCs) in vitro to determine whether loading of iDCs with HSP65-HER2 could induce the expression of the immunomodulatory cell surface molecule, CD86. In vivo mouse immunizations with HSP65-HER2 or PBS (control) were performed to determine the antitumor effects by longitudinally monitoring changes in tumor volume, weight, and incidence. The effects on percentages of HER2(+) B16 cells in tumors were assessed by confocal microscopy and flow cytometry. The results indicated that loading of iDCs with HSP65-HER2 induced the expression of CD86 in vitro, suggesting that the hybrid antigen was able to stimulate an immune response. Immunization with HSP65-HER2 had no significant influence on tumor weight or volume but significantly reduced tumor incidence (62.5 % in mice injected with 25 ?g of HSP65-HER2 vs. 100 % in PBS-injected controls; P?HSP65-HER2 immunization significantly reduced the percentages of HER2(+) B16 cells in xenografted tumors (1.86 % vs. 30.56 % in PBS-injected controls; P?=?0.01). Our findings suggest that immunization with the HSP65-HER2 fusion peptide selectively eliminates HER2(+) B16 melanoma cells in a xenograft tumor mouse model and may represent a novel and efficacious targeted therapy of HER2/neu(+) tumors.

Wang J; Wang X; Chen Y; Wan M; Xiang Z; Wu X; Wei H; Wang L; Zhang P; Wang L; Yu Y

2013-02-01

31

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection.  

UK PubMed Central (United Kingdom)

Of the hundreds of new tuberculosis (TB) vaccine candidates, some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65+CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

dos Santos SA; Zárate-Bladés CR; de Sá Galetti FC; Brandão IT; Masson AP; Soares EG; Araújo AP; Silva CL

2010-12-01

32

Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.  

Science.gov (United States)

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a "mycobacterial" gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the "non mycobacterial" factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients. PMID:19543527

Ripoll, Fabienne; Pasek, Sophie; Schenowitz, Chantal; Dossat, Carole; Barbe, Valérie; Rottman, Martin; Macheras, Edouard; Heym, Beate; Herrmann, Jean-Louis; Daffé, Mamadou; Brosch, Roland; Risler, Jean-Loup; Gaillard, Jean-Louis

2009-06-19

33

Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.  

UK PubMed Central (United Kingdom)

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a "mycobacterial" gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the "non mycobacterial" factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.

Ripoll F; Pasek S; Schenowitz C; Dossat C; Barbe V; Rottman M; Macheras E; Heym B; Herrmann JL; Daffé M; Brosch R; Risler JL; Gaillard JL

2009-01-01

34

Massive gene acquisitions in Mycobacterium indicus pranii provide a perspective on mycobacterial evolution.  

UK PubMed Central (United Kingdom)

Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria.

Saini V; Raghuvanshi S; Khurana JP; Ahmed N; Hasnain SE; Tyagi AK; Tyagi AK

2012-11-01

35

Is mycobacterial heat shock protein 16 kDa, a marker of the dormant stage of Mycobacterium tuberculosis, a sarcoid antigen?  

Science.gov (United States)

We demonstrated opposite presence of mycobacterial heat shock proteins (Mtb-hsp) 70 kDa, 65 kDa, 16 kDa in sera and lymph nodes in sarcoidosis (SA). Higher occurrence of serum Mtb-hsp70 than Mtb-hsp 65 and Mtb-hsp 16 could be caused by sequestration of Mtb-hsp 65 and Mtb-hsp 16 in circulating immune complexes (CIs). It is possible that in genetically different predisposed hosts, Mtb-hsp 16 induced by dose-dependent nitrate/nitrite (NOx) may be involved in latent tuberculosis (TB), active TB, or SA development. We evaluated Mtb-hsp 70, Mtb-hsp 65, Mtb-hsp 16 presence in precipitated CIs and serum NOx level in 20 SA patients, 19 TB patients, and 21 healthy volunteers using PEG precipitation, Western Blot, and Griess methods. We revealed higher NOx concentrations in SA and TB than in controls, but lower in SA than TB. Mtb-hsp 16, Mtb-hsp 65, and Mtb-hsp70 concentrations in precipitated CIs were higher in SA than in TB and controls. In all tested groups, Mtb-hsp 16 concentration was higher than Mtb-hsp70 and Mtb-hsp 65. We suggest that lower levels of NOx may induce a M. tuberculosis genetic dormancy program via higher Mtb-hsp 16 expression in SA. It seems that Mtb-hsp 16 may be more important than Mtb-hsp70 and Mtb-hsp 65 in CIs formation and initiate an autoimmune response in SA related to mycobacteria's stationary-phase. PMID:23079237

Dubaniewicz, Anna; Holownia, Adam; Kalinowski, Leszek; Wybieralska, Monika; Dobrucki, Iwona T; Singh, Mahavir

2012-10-16

36

[A comparative study of the gene chip technology and acid-fast staining or mycobacterial culture to diagnose].  

UK PubMed Central (United Kingdom)

OBJECTIVE: To investigate the clinical value of mycobacterial DNA microarray technology for diagnosis of childhood tuberculosis. METHODS: 120 clinical specimens were collected from hospitalized child patients. Acid-fast staining, mycobacterial culture and DNA microarray assays were performed using these clinical specimens. The results of DNA microarray assays were compared with the results of acid-fast staining and mycobacterial culture. RESULTS: The sensitivity of DNA microarray assays for specimens from children with tuberculosis was 24.3% (17/70), of acid-fast staining 17.1% (12/70), of mycobacterial culture 20.0% (14/70), and the specificity of the three methods was all 100.0% (50/50). The difference between results of DNA microarray assays and that of acid-fast staining or mycobacterial culture was not significant. CONCLUSION: DNA microarray assay has reference value for the diagnosis of childhood tuberculosis. It provides a new way for the diagnosis of childhood tuberculosis.

Deng JJ; Xiao GG; Wan CM; Zeng CL; Cao L; Shu M; Mu DZ; Zhou W

2013-05-01

37

Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis.  

UK PubMed Central (United Kingdom)

Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the ? subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other than rpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the M. leprae or M. tuberculosis rpoB gene with or without mutation and disrupted their own rpoB genes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected in M. leprae isolates or rifampin-resistant M. tuberculosis isolates are not involved in rifampin resistance.

Nakata N; Kai M; Makino M

2012-04-01

38

A novel homozygous p.R1105X mutation of the AP4E1 gene in twins with hereditary spastic paraplegia and mycobacterial disease.  

UK PubMed Central (United Kingdom)

We report identical twins with intellectual disability, progressive spastic paraplegia and short stature, born to a consanguineous family. Intriguingly, both children presented with lymphadenitis caused by the live Bacillus Calmette-Guérin (BCG) vaccine. Two syndromes - hereditary spastic paraplegia (HSP) and mycobacterial disease - thus occurred simultaneously. Whole-exome sequencing (WES) revealed a homozygous nonsense mutation (p.R1105X) of the AP4E1 gene, which was confirmed by Sanger sequencing. The p.R1105X mutation has no effect on AP4E1 mRNA levels, but results in lower levels of AP-4? protein and of the other components of the AP-4 complex, as shown by western blotting, immunoprecipitation and immunofluorescence. Thus, the C-terminal part of the AP-4? subunit plays an important role in maintaining the integrity of the AP-4 complex. No abnormalities of the IL-12/IFN-? axis or oxidative burst pathways were identified. In conclusion, we identified twins with autosomal recessive AP-4 deficiency associated with HSP and mycobacterial disease, suggesting that AP-4 may play important role in the neurological and immunological systems.

Kong XF; Bousfiha A; Rouissi A; Itan Y; Abhyankar A; Bryant V; Okada S; Ailal F; Bustamante J; Casanova JL; Hirst J; Boisson-Dupuis S

2013-01-01

39

Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies.  

UK PubMed Central (United Kingdom)

Mycobacterial speciation by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members.

Whang J; Lee BS; Choi GE; Cho SN; Kil PY; Collins MT; Shin SJ

2011-05-01

40

Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies.  

Science.gov (United States)

Mycobacterial speciation by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members. PMID:21429694

Whang, Jake; Lee, Byung Soo; Choi, Go-Eun; Cho, Sang-Nae; Kil, Park Young; Collins, Michael T; Shin, Sung Jae

2011-03-22

 
 
 
 
41

[Non tuberculous mycobacterial infections].  

UK PubMed Central (United Kingdom)

PURPOSE: Non tuberculous mycobacterial (NTM) infections, also called atypical mycobacterial infections, are caused by environmental mycobacteria and usually occur in cases of general or local immunosupression. These infections usually concern the lungs, the lymphatic system, the skin or the bones tissues. They are sometimes disseminated. In spite of new efficient antibiotics, including macrolides, therapeutic failures are common and favoured by long treatments with their potential adverse effects and drug interactions. CURRENT KNOWLEDGE AND KEY POINTS: The prevalence of atypical mycobacterial infections is increasing and is also observed in internal medicine and geriatric wards. Their clinical expression can be varied. Nowadays, these infections are more and more frequent in non-infected HIV patients, whether immunosupressed or not. Concerning other localisations of atypical mycobacterial infections, iatrogenic causes seem to be increasing and cases of nosocomial transmissions have also been described. When a NTM is found in a sample, its role in the cause of an infection must be assessed with criterias distinguishing infection from colonisation. FUTURE PROSPECTS AND PROJECTS: For those who are not locally or generally immunosupressed, it is important to search for an immunological deficiency. Indeed, patients having congenital deficiencies occurring in the interferon and interleukine pathways can develop repeated NTM infections. Therefore, for pulmonary infections in treatment failure and for disseminated infections, an adjuvant treatment by interferon gamma could be proposed. New molecules have recently been tested and can be used in some atypical mycobacterial infections.

Prendki V; Germaud P; Bemer P; Masseau A; Hamidou M

2008-05-01

42

Nontuberculous Mycobacterial Lung Disease Caused by Mycobacterium chelonae: A Case Report.  

UK PubMed Central (United Kingdom)

Mycobacterium chelonae lung disease is very rare. We report a case of lung disease caused by M. chelonae in a previously healthy woman. A 69-year-old woman was referred to our hospital because of hemoptysis. A computed tomography (CT) scan of the chest revealed bronchiolitis associated with bronchiectasis in the lingular division of the left upper lobe. Nontuberculous mycobacteria were isolated three times from sputum specimens. All isolates were identified as M. chelonae by various molecular methods that characterized rpoB and hsp65 gene sequences. Although some new lesions including bronchiolitis in the superior segment of the left lower lobe developed on the chest CT scan 35 months after diagnosis, she has been followed up without antibiotic therapy because of her mild symptoms. To the best of our knowledge, this is the first case of M. chelonae lung disease in Korea in which the etiologic organisms were confirmed using molecular techniques.

Ko Y; Kim W; Shin BS; Yoo H; Eom JS; Lee JH; Jhun BW; Kim SY; Choi GE; Shin SJ; Koh WJ

2013-04-01

43

Phenotypic heterogeneity in mycobacterial stringent response.  

UK PubMed Central (United Kingdom)

BACKGROUND: A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity. RESULTS: In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise. CONCLUSIONS: The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

Ghosh S; Sureka K; Ghosh B; Bose I; Basu J; Kundu M

2011-01-01

44

Phenotypic heterogeneity in mycobacterial stringent response  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity. Results In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise. Conclusions The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

Ghosh Sayantari; Sureka Kamakshi; Ghosh Bhaswar; Bose Indrani; Basu Joyoti; Kundu Manikuntala

2011-01-01

45

Mycobacterial cervical lymphadenitis in childhood.  

Directory of Open Access Journals (Sweden)

Full Text Available A study of 190 children of chronic cervical lymphadenitis showed tuberculous etiology on histopathological examination in 92 (48.4%) and bacteriological evidence of mycobacterial infection (smear and/or culture) in 42 (22.1%). Of these 42, twelve (28.6%) showed histopathological diagnosis of non-specific lymphadenitis. Positive culture for mycobacteria was obtained in 40, of which 30 (75%) were typical M. tuberculosis and 10 (25%) were atypical mycobacteria. The most predominant species of typical mycobacteria was M. scrofulaceum (60%) followed by M. avium intracellulare (40%). There was no remarkable difference in the histopathological pattern of those in which M. tuberculosis was grown and those in which bacterial growth was that of atypical mycobacteria. The diagnosis of chronic cervical lymphadenitis should therefore be taken a step beyond histopathology, up to complete bacteriological examination, especially to confirm the cases of mycobacterial lymphadenitis caused by atypical mycobacteria.

Jindal N; Devi B; Aggarwal A

2003-01-01

46

Mycobacterium tuberculosis Complex and Mycobacterial Heat Shock Proteins in Lymph Node Tissue from Patients with Pulmonary Sarcoidosis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We demonstrated that not whole Mycobacterium tuberculosis but its particular antigens, hsp70Mtb, hsp65Mtb, and hsp16Mtb, are present in lymph node tissues of patients with sarcoidosis (SA). hsp16Mtb occurs in the early stage of SA, whereas hsp70Mtb occurs in stage II of SA. hsp65Mtb is highly expres...

Dubaniewicz, Anna; Dubaniewicz-Wybieralska, Miros?awa; Sternau, Adam; Zwolska, Zofia; I?ycka-?wieszewska, Ewa

47

Atypical mycobacterial infections mimicking mycetoma  

Directory of Open Access Journals (Sweden)

Full Text Available ABSTRACT: Two cases of mycobacterial infection by Mycobacterium fortuitum and Mycobacterium chelonae are described in immunecompetent individuals. In both patients the lesions clinically simulated mycetoma. One patient developed multiple discharging sinuses over the abdomen, 3 months after a penetrating injury while the second had an indurated plaque with sinuses over the left thigh without a prior history of trauma. M fortuitum complex infections should thus be considered in the differential diagnosis of mycetoma. An increased awareness is required for the diagnosis of these infections.

Handa S; Sharma VK.

1993-01-01

48

Protein Turnover in Mycobacterial Proteomics  

Directory of Open Access Journals (Sweden)

Full Text Available Understanding the biology of Mycobacterium tuberculosis is one of the primary challenges in current tuberculosis research. Investigation of mycobacterial biology using the systems biology approach has deciphered much information with regard to the bacilli and tuberculosis pathogenesis. The modulation of its environment and the ability to enter a dormant phase are the hallmarks of M. tuberculosis. Until now, proteome studies have been able to understand much about the role of various proteins, mostly in growing M. tuberculosis cells. It has been difficult to study dormant M. tuberculosis by conventional proteomic techniques with very few proteins being found to be differentially expressed. Discrepancy between proteome and transcriptome studies lead to the conclusion that a certain aspect of the mycobacterial proteome is not being explored. Analysis of protein turnover may be the answer to this dilemma. This review, while giving a gist of the proteome response of mycobacteria to various stresses, analyzes the data obtained from abundance studies versus data from protein turnover studies in M. tuberculosis. This review brings forth the point that protein turnover analysis is capable of discerning more subtle changes in protein synthesis, degradation, and secretion activities. Thus, turnover studies could be incorporated to provide a more in-depth view into the proteome, especially in dormant or persistent cells. Turnover analysis might prove helpful in drug discovery and a better understanding of the dynamic nature of the proteome of mycobacteria.

Prahlad K. Rao; Qingbo Li

2009-01-01

49

METHOD FOR PREPARING RECOMBINANT MYCOBACTERIAL POLYPEPTIDES IN YEAST AND THEIR USE IN DIAGNOSIS OF MYCOBACTERIAL RELATED DISEASES  

UK PubMed Central (United Kingdom)

The present invention relates to a method for preparing a mycobacterial polypeptide qualitatively similar to the native mycobacterial protein. The present invention also relates to the mycobacterial polypeptide obtained by the meth od of invention and its use in method and kit for the diagnosis of mycobacterial related diseases, such as tuberculosis.

BARBOUCHE MOHAMED RIDHA; LARGUECHE BEYA; FATHALLAH MOHAMED DAHMANI; DELLAGI KOUSSAY; BENABDESSELEM CHAOUKI; HO JOHN L

50

METHOD FOR PREPARING RECOMBINANT MYCOBACTERIAL POLYPEPTIDES IN YEAST AND THEIR USE IN DIAGNOSIS OF MYCOBACTERIAL RELATED DISEASES  

UK PubMed Central (United Kingdom)

The present invention relates to a method for preparing a mycobacterial polypeptide qualitatively similar to the native mycobacterial protein. The present invention also relates to the mycobacterial polypeptide obtained by the method of invention and its use in method and kit for the diagnosis of mycobacterial related diseases, such as tuberculosis.

FATHALLAH MOHAMED DAHMANI; BARBOUCHE MOHAMED RIDHA; HO JOHN L; BENABDESSELEM CHAOUKI; DELLAGI KOUSSAY; LARGUECHE BEYA

51

Physiological role and potential clinical interest of mycobacterial pigments.  

UK PubMed Central (United Kingdom)

The production of pigments by bacterial colonies has sparked interest among bacteriologists since the 19th century, whether for taxonomy or, in the case of carotenoids for their association with antibiotics resistance. Mycobacteria have gained a very special place in the bacterial world due to their clinical importance. Alone, Mycobacterium tuberculosis is responsible for about two million deaths annually worldwide making tuberculosis one of the most influential diseases in the history of mankind. Almost half of the Nontuberculous Mycobacteria species identified are associated with opportunistic infections in animals and humans. Mycobacterial pigmentary characteristics started to be documented about 80 years ago; but to date, their main use has been only for limited taxonomic and identification purposes. While mycobacterial pigments, especially carotenoids have been clearly associated with cellular photoprotection and survival, the regulation of their production and their physiological role have been largely unstudied. Recent advances in deciphering mycobacterial genomes and characterization of carotenoid synthesis genes, combined with an urgent need for innovative approaches to understand Mycobacterium tuberculosis pathogenic properties open new avenues for exciting research opportunities that might lead to new therapeutic strategies against a devastating secular disease.

Robledo JA; Murillo AM; Rouzaud F

2011-02-01

52

Mycobacterial mutants with defective control of phagosomal acidification.  

Directory of Open Access Journals (Sweden)

Full Text Available The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.

Stewart Graham R; Patel Janisha; Robertson Brian D; Rae Aaron; Young Douglas B

2005-01-01

53

Non-tuberculous mycobacterial keratitis.  

UK PubMed Central (United Kingdom)

Non-tuberculous mycobacteria are environmental, opportunistic pathogens that are increasingly being recognized as important causes of many human diseases. Among them, rapidly growing mycobacteria are the most notorious organisms causing infectious keratitis. Non-tuberculous mycobacterial (NTM) keratitis commonly occurs after trauma or refractive surgery, and can masquerade as fungal, herpetic or amoebic keratitis. Therefore, the diagnosis is often delayed. Prolonged medical treatment and judicious surgical debridement are required in order to eradicate the pathogens. Combination therapy with aminoglycosides, macrolides and fluoroquinolones improves the prognosis and decreases the occurrence of drug resistance. However, regardless of the development of new diagnostic techniques and antimicrobials, NTM keratitis remains a clinical challenge for most ophthalmologists. In this article, we provide a concise introduction to the epidemiological features and clinical characteristics of NTM keratitis, and the modern diagnostic tools used for it. We also summarize the current concepts of prevention and treatment for this potentially devastating condition.

Chu HS; Hu FR

2013-03-01

54

Rapidly growing mycobacterial bloodstream infections.  

UK PubMed Central (United Kingdom)

About 20 species of rapidly growing mycobacteria species that are capable of infecting human beings and causing bloodstream infections have been identified. Many more of these species are being discovered worldwide, especially in resource-poor settings. These microorganisms have been known to cause outbreaks and pseudo-outbreaks. Although rapidly growing mycobacteria are not highly virulent or life threatening, they have a high predisposition to create biofilms and to colonise and infect intravascular catheters. Early detection and identification of specific species can help to estimate predictable antimicrobial susceptibility patterns. However, because susceptibility data originate from developed countries, studies in resource-poor settings urgently need to be done. The best outcome of cure without recurrence depends on a combination of at least 4 weeks of treatment with two or more active antimicrobial agents, plus removal of the intravascular catheter. We review and discuss the epidemiology, pathogenesis, diagnosis, management, and outcomes of rapidly growing mycobacterial bloodstream infections.

El Helou G; Viola GM; Hachem R; Han XY; Raad II

2013-02-01

55

Drug testing in mouse models of tuberculosis and nontuberculous mycobacterial infections.  

UK PubMed Central (United Kingdom)

Mice as a species are susceptible to tuberculosis infection while mouse inbred strains present wide spectrum of susceptibility/resistance to this infection. However, non-tuberculosis Mycobacterial infections usually cannot be modeled in mice of common inbred strains. Introduction of specific properties, such as gene mutations, recombinants, targeted gene knockouts significantly extended the use of mice to mimic human Mycobacterial infections, including non-tuberculosis ones. This review describes the available mouse models of tuberculosis and non-tuberculosis infections and drug therapy in these models. Mouse models of non-tuberculosis infections are significantly less developed than tuberculosis models, hampering the development of therapies.

Nikonenko BV; Apt AS

2013-05-01

56

Association of CFTR gene variants with nontuberculous mycobacterial lung disease in a Korean population with a low prevalence of cystic fibrosis.  

UK PubMed Central (United Kingdom)

Several lines of evidence suggest that in Caucasian populations, mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are associated with susceptibility to lung disease caused by nontuberculous mycobacteria (NTM). However, there is little data available in Asian populations, in which the prevalence of CF is very low. Therefore, we investigated this potential relationship in a Korean population. Sixty patients who fulfilled the diagnostic criteria for NTM lung disease were screened for genetic alterations in the CFTR gene by whole-exon resequencing. For all identified CFTR gene variants, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) genotyping was performed. Genotype and haplotype data were compared between 360 patients with NTM lung disease and 446 healthy controls. Among 13 CFTR genetic variants that were found by whole-exon resequencing, Q1352H showed a significantly higher frequency in NTM patients than in controls, giving an odds ratio (OR) of 4.27 (95% confidence interval (CI), 1.43-12.78). A haplotype with Q1352H showed the strongest association with the disease, with an OR of 3.73 (95% CI, 1.50-9.25). Furthermore, all Q1352H alleles were associated with the V allele of the V470M variant. Our results suggest that CFTR gene variants may increase susceptibility to NTM lung disease in the Korean population. Q1352H appears to be strongly related to NTM lung disease susceptibility in the Korean population.

Jang MA; Kim SY; Jeong BH; Park HY; Jeon K; Kim JW; Ki CS; Koh WJ

2013-05-01

57

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.

Ishikawa Larissa; França Thaís; Chiuso-Minicucci Fernanda; Zorzella-Pezavento Sofia; Marra Nelson; Pereira Paulo; Silva Célio; Sartori Alexandrina

2009-01-01

58

Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth.  

UK PubMed Central (United Kingdom)

Galactofuranose (Galf) residues are fundamental components of the cell wall of mycobacteria. A key enzyme, UDP-galactopyranose mutase (UGM), that participates in Galf incorporation mediates isomerization of UDP-Galf from UDP-galactopyranose (UDP-Galp). UGM is of special interest as a therapeutic target because the gene encoding it is essential for mycobacterial viability and there is no comparable enzyme in humans. We used structure-activity relationships and molecular design to devise UGM inhibitors. From a focused library of synthetic aminothiazoles, several compounds that block the UGM from Klebsiella pneumoniae or Mycobacterium tuberculosis were identified. These inhibitors block the growth of M. smegmatis.

Dykhuizen EC; May JF; Tongpenyai A; Kiessling LL

2008-05-01

59

Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth.  

Science.gov (United States)

Galactofuranose (Galf) residues are fundamental components of the cell wall of mycobacteria. A key enzyme, UDP-galactopyranose mutase (UGM), that participates in Galf incorporation mediates isomerization of UDP-Galf from UDP-galactopyranose (UDP-Galp). UGM is of special interest as a therapeutic target because the gene encoding it is essential for mycobacterial viability and there is no comparable enzyme in humans. We used structure-activity relationships and molecular design to devise UGM inhibitors. From a focused library of synthetic aminothiazoles, several compounds that block the UGM from Klebsiella pneumoniae or Mycobacterium tuberculosis were identified. These inhibitors block the growth of M. smegmatis. PMID:18447352

Dykhuizen, Emily C; May, John F; Tongpenyai, Aimon; Kiessling, Laura L

2008-05-01

60

Serodiagnosis of environmental mycobacterial infections.  

Science.gov (United States)

To demonstrate the usefulness of enzyme-linked immunosorbent assay for serodiagnosis of mycobacterioses due to environmental mycobacteria we utilized a panel of glycolipid antigens selective for Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium scrofulaceum and Mycobacterium gordonae. The levels of circulating antibodies were determined against the environmental mycobacteria, and Mycobacterium tuberculosis in human immunodeficiency virus-negative and -positive patient sera. The method used immunomagnetic separation of the antigens, with covalent immobilization of antibodies to superparamagnetic amine and carboxyl terminated particles in solutions of the specific antigens. Enzyme-linked immunosorbent assay was performed on 195 patient sera: 34 with infections due to environmental mycobacteria, 114 with tuberculosis, 47 with other respiratory diseases. There were 46 human immunodeficiency virus-1 infected individuals. Among the 34 infections due to environmental mycobacteria, 9 patients were singularly infected with an environmental mycobacterium, and 25 co-infected with both M. tuberculosis and an environmental mycobacterium. Sensitivity, specificity and false positivity ranges were determined for each of the volunteer groups: tuberculosis positive, human immunodeficiency virus negative; tuberculosis positive, human immunodeficiency virus positive; those with infections due to individual environmental mycobacteria (such as M. scrofulaceum and M. kansasii); and those with other respiratory diseases. We demonstrate that such multiple assays, can be useful for the early diagnosis of diverse environmental mycobacterial infections to allow the start of treatment earlier than henceforth. PMID:21641939

Stavri, Henriette; Ulea, Irina; Radu, Dorel L; Branaru, Manuela Gheorghiu; Moldovan, Olga; Bogdan, Miron A; Tudose, Cornelia; Raileanu, Marinela; Duiculescu, Dan; Ene, Luminita; Olar, Viorel; Ionita, Catalin; Popa, Gabriela Loredana; Popa, Mircea I; Brennan, Patrick J

2011-05-20

 
 
 
 
61

Serodiagnosis of environmental mycobacterial infections.  

UK PubMed Central (United Kingdom)

To demonstrate the usefulness of enzyme-linked immunosorbent assay for serodiagnosis of mycobacterioses due to environmental mycobacteria we utilized a panel of glycolipid antigens selective for Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium scrofulaceum and Mycobacterium gordonae. The levels of circulating antibodies were determined against the environmental mycobacteria, and Mycobacterium tuberculosis in human immunodeficiency virus-negative and -positive patient sera. The method used immunomagnetic separation of the antigens, with covalent immobilization of antibodies to superparamagnetic amine and carboxyl terminated particles in solutions of the specific antigens. Enzyme-linked immunosorbent assay was performed on 195 patient sera: 34 with infections due to environmental mycobacteria, 114 with tuberculosis, 47 with other respiratory diseases. There were 46 human immunodeficiency virus-1 infected individuals. Among the 34 infections due to environmental mycobacteria, 9 patients were singularly infected with an environmental mycobacterium, and 25 co-infected with both M. tuberculosis and an environmental mycobacterium. Sensitivity, specificity and false positivity ranges were determined for each of the volunteer groups: tuberculosis positive, human immunodeficiency virus negative; tuberculosis positive, human immunodeficiency virus positive; those with infections due to individual environmental mycobacteria (such as M. scrofulaceum and M. kansasii); and those with other respiratory diseases. We demonstrate that such multiple assays, can be useful for the early diagnosis of diverse environmental mycobacterial infections to allow the start of treatment earlier than henceforth.

Stavri H; Ulea I; Radu DL; Branaru MG; Moldovan O; Bogdan MA; Tudose C; Raileanu M; Duiculescu D; Ene L; Olar V; Ionita C; Popa GL; Popa MI; Brennan PJ

2011-09-01

62

MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE IN EGYPTIAN CHILDREN  

Directory of Open Access Journals (Sweden)

Full Text Available Background: Tuberculosis remains a major health problem in developing countries especially with the emergence of multidrug resistant strains. Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare disorder with impaired immunity against mycobacterial pathogens. Reported MSMD etiologies highlight the crucial role of the Interferon gamma /Interleukin 12 (IFN-g/ IL-12) axis and the phagocyte respiratory burst axis.Purpose: Screen patients with possible presentations for MSMD.Methods: Patients with disseminated BCG infection following vaccination, atypical mycobacterial infections or recurrent tuberculosis infections were recruited from the Primary Immune Deficiency Clinic at Cairo University Specialized Pediatric Hospital, Egypt and immune and genetic laboratory investigations were conducted at Human Genetic of Infectious Diseases laboratory in Necker Medical School, France from 2005-2009. IFN-g level in patient’s plasma as well as mutations in the eight previously identified MSMD-causing genes were explored.Results: Nine cases from eight (unrelated) kindreds were evaluated in detail. We detected a high level of IFN-g in plasma in one patient. Through Sanger sequencing, a homozygous mutation in the IFNGR1 gene at position 485 corresponding to an amino acid change from serine to phenylalanine (S485F), was detected in this patient.Conclusion: We report the first identified cases of MSMD among Egyptian patients, including in particular a new IFNGR1 mutation underlying IFN-gR1 deficiency. The eight remaining patients need to be explored further. These findings have implications regarding the compulsory Bacillus Calmette Guerin vaccination policy in Egypt, especially given the high consanguinity rate.Keywords: Interferon gamma axis, mycobacterium tuberculosis, BCG, consanguinity

Nermeen Galal; Jeannette Boutros; Aisha Marsafy; Xiao-Fei Kong; Jacqueline Feinberg; Jean-Laurent Casanova; Stéphanie Boisson-Dupuis; Jacinta Bustamante

2012-01-01

63

Mycobacterial species as case-study of comparative genome analysis  

DEFF Research Database (Denmark)

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.

Zakham, F.; Belayachi, L.

2011-01-01

64

Mycobacterial species as case-study of comparative genome analysis.  

UK PubMed Central (United Kingdom)

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.

Zakham F; Belayachi L; Ussery D; Akrim M; Benjouad A; El Aouad R; Ennaji MM

2011-01-01

65

Receptor-mediated recognition of mycobacterial pathogens.  

Science.gov (United States)

Mycobacteria are a genus of bacteria that range from the non-pathogenic Mycobacterium smegmatis to Mycobacterium tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria primarily infect host tissues through inhalation or ingestion. They are phagocytosed by host macrophages and dendritic cells. Here, conserved pathogen-associated molecular patterns (PAMPs) on the surface of mycobacteria are recognized by phagocytic pattern recognition receptors (PRRs). Several families of PRRs have been shown to non-opsonically recognize mycobacterial PAMPs, including membrane-bound C-type lectin receptors, membrane-bound and cytosolic Toll-like receptors and cytosolic NOD-like receptors. Recently, a possible role for intracellular cytosolic PRRs in the recognition of mycobacterial pathogens has been proposed. Here, we discuss currentideas on receptor-mediated recognition of mycobacterial pathogens by macrophages and dendritic cells. PMID:23795683

Killick, Kate E; Ní Cheallaigh, Clíona; O'Farrelly, Cliona; Hokamp, Karsten; MacHugh, David E; Harris, James

2013-07-29

66

Receptor-mediated recognition of mycobacterial pathogens.  

UK PubMed Central (United Kingdom)

Mycobacteria are a genus of bacteria that range from the non-pathogenic Mycobacterium smegmatis to Mycobacterium tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria primarily infect host tissues through inhalation or ingestion. They are phagocytosed by host macrophages and dendritic cells. Here, conserved pathogen-associated molecular patterns (PAMPs) on the surface of mycobacteria are recognized by phagocytic pattern recognition receptors (PRRs). Several families of PRRs have been shown to non-opsonically recognize mycobacterial PAMPs, including membrane-bound C-type lectin receptors, membrane-bound and cytosolic Toll-like receptors and cytosolic NOD-like receptors. Recently, a possible role for intracellular cytosolic PRRs in the recognition of mycobacterial pathogens has been proposed. Here, we discuss currentideas on receptor-mediated recognition of mycobacterial pathogens by macrophages and dendritic cells.

Killick KE; Ní Cheallaigh C; O'Farrelly C; Hokamp K; MacHugh DE; Harris J

2013-09-01

67

Identification of mycobacterial lectins from genomic data.  

UK PubMed Central (United Kingdom)

Sixty-four sequences containing lectin domains with homologs of known three-dimensional structure were identified through a search of mycobacterial genomes. They appear to belong to the ?-prism II, the C-type, the Microcystis virdis (MV), and the ?-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC, and the ?-grasp domains, respectively while mycobacterial ?-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three-dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and nonpathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one or the other of the four lectin domains can be gleaned through the examination of homologs proteins, although the structure of none of them is available. Their biological role is also yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins.

Abhinav KV; Sharma A; Vijayan M

2013-04-01

68

Mycobacterial signaling through toll-like receptors  

Science.gov (United States)

Studies over the past decade have helped to decipher molecular networks dependent on Toll-like receptor (TLR) signaling, in mycobacteria-infected macrophages. Stimulation of TLRs by mycobacteria and their antigenic components rapidly induces intracellular signaling cascades involved in the activation of nuclear factor-?B and mitogen-activated protein kinase pathways, which play important roles in orchestrating proinflammatory responses and innate defense through generation of a variety of antimicrobial effector molecules. Recent studies have provided evidence that mycobacterial TLR-signaling cross talks with other intracellular antimicrobial innate pathways, the autophagy process and functional vitamin D receptor (VDR) signaling. In this article we describe recent advances in the recognition, responses, and regulation of mycobacterial signaling through TLRs.

Basu, Joyoti; Shin, Dong-Min; Jo, Eun-Kyeong

2012-01-01

69

Rapid lysis technique for mycobacterial species.  

Science.gov (United States)

The lysis of mycobacterial cells typically has been difficult and time-consuming. We report a method for the physical rupture of Mycobacterium paratuberculosis and several other members of the family Mycobacteriaceae using a Mini-Beadbeater cell disrupter (Biospec Products; Bartlesville, Okla.) and zirconium beads, a process which yields DNA and RNA of high molecular weight and in greater quantity than that obtained by rupture in a Ribi pressure cell. PMID:2447121

Hurley, S S; Splitter, G A; Welch, R A

1987-11-01

70

Rapid lysis technique for mycobacterial species.  

UK PubMed Central (United Kingdom)

The lysis of mycobacterial cells typically has been difficult and time-consuming. We report a method for the physical rupture of Mycobacterium paratuberculosis and several other members of the family Mycobacteriaceae using a Mini-Beadbeater cell disrupter (Biospec Products; Bartlesville, Okla.) and zirconium beads, a process which yields DNA and RNA of high molecular weight and in greater quantity than that obtained by rupture in a Ribi pressure cell.

Hurley SS; Splitter GA; Welch RA

1987-11-01

71

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 ?g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Lorenzi Julio CC; Trombone Ana PF; Rocha Carolina D; Almeida Luciana P; Lousada Ricardo L; Malardo Thiago; Fontoura Isabela C; Rossetti Renata AM; Gembre Ana F; Silva Aristóbolo M; Silva Celio L; Coelho-Castelo Arlete AM

2010-01-01

72

An accurate method for the qualitative detection and quantification of mycobacterial promoter activity.  

UK PubMed Central (United Kingdom)

The present study was designed to determine the half-life of gfp(m) (2+) mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFP(m) (2+) protein, and to find out whether mycobacterial promoter activity can be quantitated more accurately using the mRNA levels of the reporter gene, gfp(m) (2+), than the fluorescence intensity of the GFP(m) (2+) protein. Quantitative PCR of gfp(m) (2+) mRNA in the pulse-chased samples of the rifampicin-treated Mycobacterium smeg-matis/gfpm(2+) transformant showed the half-life of gfp(m) (2+) mRNA to be 4.081 min. The levels of the gfp(m) (2+) mRNA and the fluorescence intensity of the GFP(m) (2+) protein, which were expressed by the promoters of Mycobacterium tuberculosis cell division gene, ftsZ (MtftsZ), were determined using quantitative PCR and fluorescence spectrophotometry, respectively. The data revealed that quantification of mycobacterial promoter activity by determining the gfp(m) (2+) mRNA levels is more accurate and statistically significant than the measurement of GFP(m) (2+) fluorescence intensity, especially for weak promoters.

Mishra S; Anand D; Vijayarangan N; Ajitkumar P

2013-01-01

73

Mycobacterial infections in marrow transplant patients.  

Science.gov (United States)

Bone marrow transplant recipients undergo ablation of host immune defenses with total-body irradiation or high dose chemotherapy, or both. Over a 5.6-year period, mycobacterial infections were observed in 7 of 682 patients with leukemia who received marrow grafts. Four patients had pulmonary and three extrapulmonary infection. Granulomas were observed in the lungs of three patients, in the liver of one patient, and in the skin of one patient. Cultures revealed Mycobacterium tuberculosis in two patients, Mycobacterium fortuitum in two patients, and Mycobacterium kansasii in one patient. In the six patients treated with antimycobacterial therapy in either the pretransplant or posttransplant period, complete resolution of the infection was achieved. Pretransplant chest radiograph abnormalities suggesting mycobacterial infections should be aggressively evaluated in these immunocompromised hosts. Prophylaxis should be considered in marrow graft recipients with a well-established history of inadequately treated tuberculosis, previous Bacille Calmette-Guerin immunotherapy, known family contacts, recent skin test conversion, or past skin test positivity. PMID:6356515

Navari, R M; Sullivan, K M; Springmeyer, S C; Siegel, M S; Meyers, J D; Buckner, C D; Sanders, J E; Stewart, P S; Clift, R A; Fefer, A

1983-11-01

74

Nontuberculous mycobacterial otomastoiditis: a case report.  

UK PubMed Central (United Kingdom)

Nontuberculous mycobacterial otomastoiditis is rare and can be easily confused with various different forms of otitis media. We describe the case of a 50-year-old woman who presented with left-sided chronic otitis media that had persisted for more than 1 year. It was not eradicated by standard antimicrobial therapy and surgical debridement. After appropriate antibiotic therapy for nontuberculous mycobacteria was added to the therapeutic regimen, the patient improved significantly and the lesion had healed by 6 months. Based on our experience with this case, we conclude that early bacterial culture and staining for acid-fast bacilli in ear drainage material or granulation tissue should be performed when standard antimicrobial therapy fails to eradicate chronic otitis media of an undetermined origin that is accompanied by granulation tissue over the external auditory canal or middle ear. Polymerase chain reaction testing is also effective for rapid diagnosis. Surgical debridement and removal of the foreign body can successfully treat nontuberculous mycobacterial otomastoiditis only when effective antimicrobial therapy is also administered.

Tsai LT; Wang CY; Lin CD; Tsai MH

2013-01-01

75

Mycobacterial infections: Still a millennium bug - the imaging features of mycobacterial infections  

Energy Technology Data Exchange (ETDEWEB)

Mycobacterial infection is re-emerging as a major health care concern. Although Mycobacterium tuberculosis (MTB) is still the most important pathogen, atypical mycobacterium (AMB) infections are becoming increasingly common. We present a pictorial review of the imaging features of these infections in the chest, abdomen, brain and musculoskeletal system. Imaging similarities and differences between the normal and the immunocompromised host will be highlighted. Koh, D. M. et al. Clinical Radiology (2001)

Koh, D.M.; Bell, J.R.G.; Burkill, G.J.C.; Padley, S.P.G.; Healy, J.C

2001-07-01

76

Pulmonary Nontuberculous Mycobacterial Infection in Congenital Contractural Arachnodactyly  

Science.gov (United States)

SUMMARY Congenital contractural arachnodactyly (CCA) is caused by mutations within fibrillin-2 (FBN2), which is crucial for microfibril structure. Affected individuals may have contractures, chest wall deformities, scoliosis, abnormal ear folding and elongated limbs. We describe a novel FBN2 mutation in a woman with CCA who also has pulmonary nontuberculous mycobacterial infection. The population with pulmonary nontuberculous mycobacterial infections shares phenotypic features with CCA, such as elongated body habitus, scoliosis and pectus deformities. While it is unlikely that FBN2 defects account for susceptibility to nontuberculous mycobacterial infection in the majority of cases, the overlap between these two diseases suggests some shared pathophysiology.

Paulson, Michelle L.; Olivier, Kenneth N.; Holland, Steven M.

2013-01-01

77

Tetrahydrolipstatin inhibition, functional analyses, and three-dimensional structure of a lipase essential for mycobacterial viability.  

UK PubMed Central (United Kingdom)

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 ? resolution, revealed an ?/? 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.

Crellin PK; Vivian JP; Scoble J; Chow FM; West NP; Brammananth R; Proellocks NI; Shahine A; Le Nours J; Wilce MC; Britton WJ; Coppel RL; Rossjohn J; Beddoe T

2010-09-01

78

Tetrahydrolipstatin inhibition, functional analyses, and three-dimensional structure of a lipase essential for mycobacterial viability.  

Science.gov (United States)

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 ? resolution, revealed an ?/? 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. PMID:20656688

Crellin, Paul K; Vivian, Julian P; Scoble, Judith; Chow, Frances M; West, Nicholas P; Brammananth, Rajini; Proellocks, Nicholas I; Shahine, Adam; Le Nours, Jerome; Wilce, Matthew C J; Britton, Warwick J; Coppel, Ross L; Rossjohn, Jamie; Beddoe, Travis

2010-07-23

79

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

Science.gov (United States)

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 ? resolution, revealed an ?/? 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.

Crellin, Paul K.; Vivian, Julian P.; Scoble, Judith; Chow, Frances M.; West, Nicholas P.; Brammananth, Rajini; Proellocks, Nicholas I.; Shahine, Adam; Le Nours, Jerome; Wilce, Matthew C. J.; Britton, Warwick J.; Coppel, Ross L.; Rossjohn, Jamie; Beddoe, Travis

2010-01-01

80

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

Energy Technology Data Exchange (ETDEWEB)

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.

Crellin, Paul K.; Vivian, Julian P.; Scoble, Judith; Chow, Frances M.; West, Nicholas P.; Brammananth, Rajini; Proellocks, Nicholas I.; Shahine, Adam; Le Nours, Jerome; Wilce, Matthew C.J.; Britton, Warwick J.; Coppel, Ross L.; Rossjohn, Jamie; Beddoe, Travis (Monash); (Centenary)

2010-09-17

 
 
 
 
81

Molecular Characterization of Environmental Non-Tuberculous Mycobacteria Using PCR- RFLP Analysis of 441 Bp Heat Shock Protein 65 Fragments.  

UK PubMed Central (United Kingdom)

BACKGROUND: Non- Tuberculous Mycobacteria are environmental opportunistic pathogens that can be found in various terrestrial and aquatic habitats. There are an epidemiological links between species isolated in tap water and those isolated from patients. hsp65 gene has more variability in its sequences, compared to the some more conserved genes in NTM, for identification of mycobacteria to species level. In this study, the prevalence of NTM in Isfahan City water samples was determined using culture, biochemical tests and PCR-RFLP analyses of hsp65 gene. METHODS: Eighty-five water samples were collected and cultured. The mycobacterial isolates were identified by conventional biochemical tests. A 441 bp fragment of hsp65 genes was amplified and digested by two restriction enzymes, BstEII and HaeII. Digested products were analyzed using polyacrilamid gel electrophoresis (PAGE). RESULTS: 25.9% of the water samples contained different species of NTM. Dominant isolates were M. fortuitum (26.7%), M. chelonae like organism (13.3%) and M. mucogenicum (13.3%). Nineteen isolates of Mycobacteria were differentiated using hsp65 genes PCR-RFLP. Three isolates could not be identified at the species level because their RFLP patterns were different from other known PCR-RFLP profiles. There were different hsp65 gene PCR-RFLP profiles produced by digestion with BstEII and HaeIII. CONCLUSION: This study showed that PCR-RFLP of hsp65 gene in mycobacteria is more reliable method for identification of NTM at the specie level than conventional phenotypic methods (P<0.05). In comparing of RFLP patterns of this study to other investigation, some minor differences were negligible.

Nasr-Esfahani B; Sarikhani E; Moghim S; Faghri J; Fazeli H; Hoseini N; Rezaei-Yazdi H

2012-01-01

82

Molecular Characterization of Environmental Non-Tuberculous Mycobacteria Using PCR- RFLP Analysis of 441 Bp Heat Shock Protein 65 Fragments  

Directory of Open Access Journals (Sweden)

Full Text Available Background: Non- Tuberculous Mycobacteria are environmental opportunistic pathogens that can be found in various terrestrial and aquatic habitats. There are an epidemiological links between species isolated in tap water and those isolated from patients. hsp65 gene has more variability in its sequences, compared to the some more conserved genes in NTM, for identification of mycobacteria to species level. In this study, the prevalence of NTM in Isfahan City water samples was determined using culture, biochemical tests and PCR-RFLP analyses of hsp65 gene.Methods: Eighty-five water samples were collected and cultured. The mycobacterial isolates were identified by conventional biochemical tests. A 441 bp fragment of hsp65 genes was amplified and digested by two restriction enzymes, BstEII and HaeII. Digested products were analyzed using polyacrilamid gel electrophoresis (PAGE).Results: 25.9% of the water samples contained different species of NTM. Dominant isolates were M. fortuitum (26.7%), M. chelonae like organism (13.3%) and M. mucogenicum (13.3%). Nineteen isolates of Mycobacteria were differentiated using hsp65 genes PCR-RFLP. Three isolates could not be identified at the species level because their RFLP patterns were different from other known PCR-RFLP profiles. There were different hsp65 gene PCR-RFLP profiles produced by digestion with BstEII and HaeIII. Conclusion: This study showed that PCR-RFLP of hsp65 gene in mycobacteria is more reliable method for identification of NTM at the specie level than conventional phenotypic methods (P<0.05). In comparing of RFLP patterns of this study to other investigation, some minor differences were negligible.

B Nasr-Esfahani; E Sarikhani; S Moghim; J Faghri; H Fazeli; N Hoseini; H Rezaei-Yazdi

2012-01-01

83

Novel diagnostic algorithm using tuf gene amplification and restriction fragment length polymorphism is promising tool for identification of nontuberculous mycobacteria.  

Science.gov (United States)

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84- 58) and M. chelonae (477-84-80-58), and M. kansasii (141- 136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level. PMID:19349759

Shin, Ji-Hyun; Cho, Eun-Jin; Lee, Jung-Yeon; Yu, Jae-Yon; Kang, Yeon-Ho

2009-03-01

84

Novel diagnostic algorithm using tuf gene amplification and restriction fragment length polymorphism is promising tool for identification of nontuberculous mycobacteria.  

UK PubMed Central (United Kingdom)

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84- 58) and M. chelonae (477-84-80-58), and M. kansasii (141- 136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level.

Shin JH; Cho EJ; Lee JY; Yu JY; Kang YH

2009-03-01

85

Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages.  

Science.gov (United States)

Mycobacterial shuttle vectors contain dual origins of replication for growth in both Escherichia coli and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression in E. coli, Mycobacterium smegmatis, and M. avium at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a lacZ? promoter. This high-level fluorescent protein expression allowed easy visualization of M. smegmatis and M. avium in infected macrophages. The M. tuberculosis gene esat-6 was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression in M. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide. M. smegmatis clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in E. coli, was insufficient to suppress a strong inflammatory response elicited by M. smegmatis or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species. PMID:22820329

Eitson, Jennifer L; Medeiros, Jennifer J; Hoover, Ashley R; Srivastava, Shashikant; Roybal, Kole T; Aínsa, José A; Hansen, Eric J; Gumbo, Tawanda; van Oers, Nicolai S C

2012-07-20

86

Membrane TNF confers protection to acute mycobacterial infection  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-c...

Fremond Cecile; Allie Nasiema; Dambuza Ivy; Grivennikov Sergei I; Yeremeev Vladimir; Quesniaux Valerie FJ; Jacobs Muazzam

87

Malaria exacerbates experimental mycobacterial infection in vitro and in vivo.  

UK PubMed Central (United Kingdom)

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 caused reactivation of latent mycobacterial infection (2.6-fold increase in peak splenic CFU, p = 0.016 for difference over time course of reactivation). Malaria pigment deposition within the granulomas of co-infected mice suggested that the influx of dysfunctional hemozoin-laden monocytes into the locus of mycobacterial control may contribute to impaired containment of mycobacteria. Collectively, these results point to malaria-induced dysregulation of innate and adaptive anti-mycobacterial defences, and suggest that the interaction of these globally important pathogens may potentiate Mtb infection and transmission.

Hawkes M; Li X; Crockett M; Diassiti A; Liles WC; Liu J; Kain KC

2010-10-01

88

Role for mycobacterial infection in pathogenesis of primary biliary cirrhosis?  

Directory of Open Access Journals (Sweden)

Full Text Available Primary biliary cirrhosis (PBC) is a progressive cholestatic liver disease characterized by the immune-mediated destruction of biliary epithelial cells in small intrahepatic bile ducts. The disease is characterized by circulating antimitochondrial antibodies (AMAs) as well as disease-specific antinuclear antibodies, cholestatic liver function tests, and characteristic histological features, including granulomas. A variety of organisms are involved in granuloma formation, of which mycobacteria are the most commonly associated. This has led to the hypothesis that mycobacteria may be involved in the pathogenesis of PBC, along with other infectious agents. Additionally, AMAs are found in a subgroup of patients with mycobacterial infections, such as leprosy and pulmonary tuberculosis. Antibodies against species-specific mycobacterial proteins have been reported in patients with PBC, but it is not clear whether these antibodies are specific for the disease. In addition, data in support of the involvement of the role of molecular mimicry between mycobacterial and human mitochondrial antigens as triggers of cross-reactive immune responses leading to the loss of immunological tolerance, and the induction of pathological features have been published. Thus, antibodies against mycobacterial heat shock protein appear to cross-recognize AMA-specific autoantigens, but it is not clear whether these autoantibodies are mycobacterium-species-specific, and whether they are pathogenic or incidental. The view that mycobacteria are infectious triggers of PBC is intriguing, but the data provided so far are not conclusive.

Daniel Smyk; Eirini I Rigopoulou; Yoh Zen; Robin Daniel Abeles; Charalambos Billinis; Albert Pares; Dimitrios P Bogdanos

2012-01-01

89

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation.  

Science.gov (United States)

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH. PMID:22026918

Wang, Xu-De; Gu, Jing; Wang, Ting; Bi, Li-Jun; Zhang, Zhi-Ping; Cui, Zong-Qiang; Wei, Hong-Ping; Deng, Jiao-Yu; Zhang, Xian-En

2011-11-03

90

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation.  

UK PubMed Central (United Kingdom)

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.

Wang XD; Gu J; Wang T; Bi LJ; Zhang ZP; Cui ZQ; Wei HP; Deng JY; Zhang XE

2011-12-01

91

[Genetic susceptibility to infectious diseases: immunogenetical approaches to mycobacterial infections and subacute sclerosing panencephalitis].  

Science.gov (United States)

Genetic susceptibility to infectious diseases can be explained by nucleotide alteration (mutation, polymorphism, etc.) of genes encoding molecules involved in the entry of or the immune response to microorganisms. We have conducted studies on host genetic factors for the development of mycobacterial infections and subacute sclerosing panencephalitis (SSPE) in the past decade. First, we identified autosomal dominant IFN-gamma receptor deficiency as a predominant genetic basis of patients with bacille Calmette-Guérin osteomyelitis in Japan. Second, by gene-based association analysis of 21 candidate genes, it was suggested that genetic variants of IL-12 receptor beta1 gene (IL12RB1) confer genetic susceptibility to tuberculosis, and are associated with the progression of the disease in Japanese. Third, we demonstrated that variants of several genes encoding molecules associated with innate immunity (MxA and TLR3 genes) and acquired immunity (IL4 and programmed cell death 1 [PD1] genes) were associated with the development of SSPE. Immunogenetical approaches to infectious diseases would help us to evaluate the risk for disease development and progression, individualize prevention and treatment strategies, and create new therapies. PMID:20549906

Kusuhara, Koichi

2010-06-01

92

[Genetic susceptibility to infectious diseases: immunogenetical approaches to mycobacterial infections and subacute sclerosing panencephalitis].  

UK PubMed Central (United Kingdom)

Genetic susceptibility to infectious diseases can be explained by nucleotide alteration (mutation, polymorphism, etc.) of genes encoding molecules involved in the entry of or the immune response to microorganisms. We have conducted studies on host genetic factors for the development of mycobacterial infections and subacute sclerosing panencephalitis (SSPE) in the past decade. First, we identified autosomal dominant IFN-gamma receptor deficiency as a predominant genetic basis of patients with bacille Calmette-Guérin osteomyelitis in Japan. Second, by gene-based association analysis of 21 candidate genes, it was suggested that genetic variants of IL-12 receptor beta1 gene (IL12RB1) confer genetic susceptibility to tuberculosis, and are associated with the progression of the disease in Japanese. Third, we demonstrated that variants of several genes encoding molecules associated with innate immunity (MxA and TLR3 genes) and acquired immunity (IL4 and programmed cell death 1 [PD1] genes) were associated with the development of SSPE. Immunogenetical approaches to infectious diseases would help us to evaluate the risk for disease development and progression, individualize prevention and treatment strategies, and create new therapies.

Kusuhara K

2010-06-01

93

Relationship between mycobacterial species and their carotenoid pigments.  

UK PubMed Central (United Kingdom)

A study of the relationship between mycobacterial species and their carotenoid pigments was carried out. According to the carotenoid pigments contained, the mycobacterial species tested were divided into four groups: the first group of Mycobacterium kansasii and M. marinum, which formed principally only beta-carotene; the second group of M. gordonae, M. scrofulaceum, M. szulgai, M. xenopi, M. flavescens, M. phlei, M. rhodesiae, M. neoaurum, and M. aichiense, which formed beta-carotene and a zeaxanthin-like substance; the third group of M. aurum and M. obuense, which formed beta-carotene and an eschscholtzxanthin-like substance; and the fourth group of M. chubuense and M. tokaiense, which formed beta-carotene and zeaxanthin- and eschscholtzxanthin-like substances. The common carotenoid pigment throughout the genus Mycobacterium was beta-carotene and the hypophasic carotenoids differed according to the species.

Ichiyama S; Shimokata K; Tsukamura M

1988-01-01

94

Relationship between mycobacterial species and their carotenoid pigments.  

Science.gov (United States)

A study of the relationship between mycobacterial species and their carotenoid pigments was carried out. According to the carotenoid pigments contained, the mycobacterial species tested were divided into four groups: the first group of Mycobacterium kansasii and M. marinum, which formed principally only beta-carotene; the second group of M. gordonae, M. scrofulaceum, M. szulgai, M. xenopi, M. flavescens, M. phlei, M. rhodesiae, M. neoaurum, and M. aichiense, which formed beta-carotene and a zeaxanthin-like substance; the third group of M. aurum and M. obuense, which formed beta-carotene and an eschscholtzxanthin-like substance; and the fourth group of M. chubuense and M. tokaiense, which formed beta-carotene and zeaxanthin- and eschscholtzxanthin-like substances. The common carotenoid pigment throughout the genus Mycobacterium was beta-carotene and the hypophasic carotenoids differed according to the species. PMID:3173145

Ichiyama, S; Shimokata, K; Tsukamura, M

1988-01-01

95

DNA encoding individual mycobacterial antigens protects mice against tuberculosis  

Directory of Open Access Journals (Sweden)

Full Text Available Over the last few years, some of our experiments in which mycobacterial antigens were presented to the immune system as if they were viral antigens have had a significant impact on our understanding of protective immunity against tuberculosis. They have also markedly enhanced the prospects for new vaccines. We now know that individual mycobacterial protein antigens can confer protection equal to that from live BCG vaccine in mice. A critical determinant of the outcome of immunization appears to be the degree to which antigen-specific cytotoxic T cells are generated by the immune response. Our most recent studies indicate that DNA vaccination is an effective way to establish long-lasting cytotoxic T cell memory and protection against tuberculosis.

C.L. Silva; V.L.D. Bonato; V.M.F. Lima

1999-01-01

96

Mycobacterial pseudotumor of the plantar fascia: how common is it?  

UK PubMed Central (United Kingdom)

Mycobacterial spindle cell pseudotumor (MSCP) is an extremely rare complication of mycobacterial infections. It has been reported to occur in various sites such as skin, lymph nodes, bone marrow, lungs, and spleen. This tumor-like lesion can be confused clinically as well as radiographically with dermatofibroma, nodular fasciitis, xanthogranuloma, and Kaposi's sarcoma. While this lesion is rare and has been previously reported to occur only in superficial skin, we emphasize its consideration and inclusion in the differential diagnoses when a deep soft tissue mass is complicated by symptoms of deep tissue infection secondary to abscess formation in immunocompromised hosts. Here, we present the clinical and radiologic findings of a case of MSCP involving the deep plantar sheaths.

Sideras PA; Heiba S; Machac J; Hechtman J; Vatti S

2013-07-01

97

Mycobacterial pseudotumor of the plantar fascia: how common is it?  

Science.gov (United States)

Mycobacterial spindle cell pseudotumor (MSCP) is an extremely rare complication of mycobacterial infections. It has been reported to occur in various sites such as skin, lymph nodes, bone marrow, lungs, and spleen. This tumor-like lesion can be confused clinically as well as radiographically with dermatofibroma, nodular fasciitis, xanthogranuloma, and Kaposi's sarcoma. While this lesion is rare and has been previously reported to occur only in superficial skin, we emphasize its consideration and inclusion in the differential diagnoses when a deep soft tissue mass is complicated by symptoms of deep tissue infection secondary to abscess formation in immunocompromised hosts. Here, we present the clinical and radiologic findings of a case of MSCP involving the deep plantar sheaths. PMID:23768743

Sideras, Panagiotis A; Heiba, Sherif; Machac, Josef; Hechtman, Jaclyn; Vatti, Sridhar

2013-04-04

98

Nontuberculous Mycobacterial Infection after Fractionated CO2 Laser Resurfacing  

Science.gov (United States)

Nontuberculous mycobacteria are increasingly associated with cutaneous infections after cosmetic procedures. Fractionated CO2 resurfacing, a widely used technique for photorejuvenation, has been associated with a more favorable side effect profile than alternative procedures. We describe 2 cases of nontuberculous mycobacterial infection after treatment with a fractionated CO2 laser at a private clinic. Densely distributed erythematous papules and pustules developed within the treated area within 2 weeks of the laser procedure. Diagnosis was confirmed by histologic analysis and culture. Both infections responded to a 4-month course of a multidrug regimen. An environmental investigation of the clinic was performed, but no source of infection was found. The case isolates differed from each other and from isolates obtained from the clinic, suggesting that the infection was acquired by postprocedure exposure. Papules and pustules after fractionated CO2 resurfacing should raise the suspicion of nontuberculous mycobacterial infection.

Culton, Donna A.; Miller, Becky A.; Miller, Melissa B.; MacKuen, Courteney; Groben, Pamela; White, Becky; Cox, Gary M.; Stout, Jason E.

2013-01-01

99

Nontuberculous mycobacterial infection after fractionated CO(2) laser resurfacing.  

Science.gov (United States)

Nontuberculous mycobacteria are increasingly associated with cutaneous infections after cosmetic procedures. Fractionated CO2 resurfacing, a widely used technique for photorejuvenation, has been associated with a more favorable side effect profile than alternative procedures. We describe 2 cases of nontuberculous mycobacterial infection after treatment with a fractionated CO2 laser at a private clinic. Densely distributed erythematous papules and pustules developed within the treated area within 2 weeks of the laser procedure. Diagnosis was confirmed by histologic analysis and culture. Both infections responded to a 4-month course of a multidrug regimen. An environmental investigation of the clinic was performed, but no source of infection was found. The case isolates differed from each other and from isolates obtained from the clinic, suggesting that the infection was acquired by postprocedure exposure. Papules and pustules after fractionated CO2 resurfacing should raise the suspicion of nontuberculous mycobacterial infection. PMID:23628077

Culton, Donna A; Lachiewicz, Anne M; Miller, Becky A; Miller, Melissa B; Mackuen, Courteney; Groben, Pamela; White, Becky; Cox, Gary M; Stout, Jason E

2013-03-01

100

Nontuberculous mycobacterial infection after fractionated CO(2) laser resurfacing.  

UK PubMed Central (United Kingdom)

Nontuberculous mycobacteria are increasingly associated with cutaneous infections after cosmetic procedures. Fractionated CO2 resurfacing, a widely used technique for photorejuvenation, has been associated with a more favorable side effect profile than alternative procedures. We describe 2 cases of nontuberculous mycobacterial infection after treatment with a fractionated CO2 laser at a private clinic. Densely distributed erythematous papules and pustules developed within the treated area within 2 weeks of the laser procedure. Diagnosis was confirmed by histologic analysis and culture. Both infections responded to a 4-month course of a multidrug regimen. An environmental investigation of the clinic was performed, but no source of infection was found. The case isolates differed from each other and from isolates obtained from the clinic, suggesting that the infection was acquired by postprocedure exposure. Papules and pustules after fractionated CO2 resurfacing should raise the suspicion of nontuberculous mycobacterial infection.

Culton DA; Lachiewicz AM; Miller BA; Miller MB; Mackuen C; Groben P; White B; Cox GM; Stout JE

2013-03-01

 
 
 
 
101

Fatal atypical mycobacterial infection in a cardiac transplant recipient.  

UK PubMed Central (United Kingdom)

A 37-year-old female underwent heart transplantation for giant cell myocarditis. The patient died within three-and-a-half months of cardiac transplantation. Postmortem specimens from the heart and lung showed multiple necrotizing granulomas with numerous acid-fast bacilli. Polymerase chain reaction done on both the postmortem samples confirmed the presence of atypical mycobacterial infection. This fatal case of atypical mycobacteriosis in a cardiac transplant patient is reported for its rarity.

Ray R; Chakravorty S; Tyagi JS; Airan B; Talwar KK; Venugopal P; Chopra P

2001-01-01

102

Fatal atypical mycobacterial infection in a cardiac transplant recipient.  

Science.gov (United States)

A 37-year-old female underwent heart transplantation for giant cell myocarditis. The patient died within three-and-a-half months of cardiac transplantation. Postmortem specimens from the heart and lung showed multiple necrotizing granulomas with numerous acid-fast bacilli. Polymerase chain reaction done on both the postmortem samples confirmed the presence of atypical mycobacterial infection. This fatal case of atypical mycobacteriosis in a cardiac transplant patient is reported for its rarity. PMID:11456134

Ray, R; Chakravorty, S; Tyagi, J S; Airan, B; Talwar, K K; Venugopal, P; Chopra, P

103

Clinical and pathological characteristics of mycobacterial tenosynovitis and arthritis.  

UK PubMed Central (United Kingdom)

PURPOSE: To investigate the clinical characteristics and pathological features of patients with mycobacterial tenosynovitis and arthritis. METHODS: All patients with tenosynovitis and arthritis caused by Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) who were treated at a medical center in Taiwan from 2001 to 2010 were analyzed. RESULTS: Thirty-two patients with mycobacterial tenosynovitis and arthritis were identified. MTB was isolated exclusively from patients with arthritis of large joints (n = 11), while NTM were isolated from patients with arthritis of large joints (n = 4) and from those with tenosynovitis (n = 17). Among patients with tenosynovitis due to NTM, the most commonly found NTM were M. marinum (n = 7), M. intracellulare (n = 5), and M. abscessus (sensu stricto) (n = 2). Six of the seven patients with tenosynovitis due to M. marinum had suffered fishing-related injuries to the hands. All four patients with NTM arthritis had recurrent septic arthritis after surgery. NTM were isolated once from the debrided tissue specimens in three of these patients; the other patient died of systemic infection caused by M. intracellulare and multiple bacterial pathogens. CONCLUSION: Mycobacterial tenosynovitis should be considered in patients who present with indolent symptoms of chronic tenosynovitis. Complete clinical information, including history of trauma or joint replacement surgery and underlying systemic disease, is helpful in establishing an early diagnosis of the disease.

Hsiao CH; Cheng A; Huang YT; Liao CH; Hsueh PR

2013-04-01

104

Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium. Results We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides. We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers. Conclusion Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes.

Benaroudj Nadia; Raynal Bertrand; Miot Marika; Ortiz-Lombardia Miguel

2011-01-01

105

Production of mycobacterial cell wall glycopeptidolipids requires a member of the MbtH-like protein family  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production. Results Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility. Conclusions Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane.

Tatham Elizabeth; sundaram Chavadi Sivagami; Mohandas Poornima; Edupuganti Uthamaphani R; Angala Shiva K; Chatterjee Delphi; Quadri Luis E N

2012-01-01

106

Partial overlap of anti-mycobacterial, and anti-Saccharomyces cerevisiae mannan antibodies in Crohns disease  

Directory of Open Access Journals (Sweden)

Full Text Available AIM: To test whether humoral immune reaction against mycobacteria may play a role in anti-Saccharomyces cerevisiae antibodies (ASCA) generation in Crohn's disease (CD) and/or whether it correlates with clinical subtypes.METHODS: The dominant ASCA epitope was detected by Galanthus nivalis lectin (GNL)-binding assay. ASCA and IgG against mycobacterial lysates [M avium, M smegmatis, M chelonae, M bovis BCG, M avium ssp. paratuberculosis (MAP)] or purified lipoarabinomannans (LAM) were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice.RESULTS: GNL bound to different extents to mycobacterial lysates, abundantly to purified mannose-capped (Man) LAM from M tuberculosis, but not to uncapped LAM from M smegmatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA (r = 0.37-0.64; P = 0.003-P < 0.001). ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent cross-reactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP (P = 0.024, 0.004 and 0.045, respectively).CONCLUSION: Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.

Stefan Müller, Thomas Schaffer, Alain M Schoepfer, Annamarie Hilty, Thomas Bodmer, Frank Seibold

2008-01-01

107

Diptera as vectors of mycobacterial infections in cattle and pigs.  

Science.gov (United States)

Mycobacteria were isolated from 14 (4.5%) of 314 samples, containing 7791 adult Diptera, which were collected in the Czech Republic and Slovakia in 1997-2000. These flies were collected from three cattle herds with paratuberculosis, two pig herds with mycobacterial infections and one farm that kept both cattle and pigs and that did not have problems of mycobacterial infections. Mycobacterium intracellulare was isolated from Eristalis tenax Linnaeus (Diptera: Syrphidae) captured from a pig herd. Mycobacterium avium ssp. avium (serotype 8) was isolated from flies of the genera Drosophila Fallen (Diptera: Drosophilidae) and Musca Linnaeus (Diptera: Muscidae) originating from a pig herd. Mycobacterium spp. were isolated from Musca spp. and Mycobacterium fortuitum was isolated from dung flies of the genus Scatophaga Meigen (Diptera: Scatophagidae), Musca spp. and Stomoxys calcitrans Linnaeus (Diptera: Muscidae) captured in the same herd. Mycobacterium scrofulaceum was isolated from S. calcitrans from the farm with both cattle and pigs. Mycobacterium avium ssp. paratuberculosis was isolated from Scatophaga spp. collected from pastures grazed by one of the cattle herds and from Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) and Lucilia caesar Linnaeus (Diptera: Calliphoridae) captured in a slaughterhouse, where cattle infected with paratuberculosis were slaughtered. Mycobacterium phlei was isolated from flies of the genus Lucilia captured at a waste bin. These data indicate that mycobacteria may be spread by adult flies that have been in contact with material contaminated with these pathogens. PMID:11434556

Fischer, O; Mátlová, L; Dvorská, L; Svástová, P; Bartl, J; Melichárek, I; Weston, R T; Pavlík, I

2001-06-01

108

Diptera as vectors of mycobacterial infections in cattle and pigs.  

UK PubMed Central (United Kingdom)

Mycobacteria were isolated from 14 (4.5%) of 314 samples, containing 7791 adult Diptera, which were collected in the Czech Republic and Slovakia in 1997-2000. These flies were collected from three cattle herds with paratuberculosis, two pig herds with mycobacterial infections and one farm that kept both cattle and pigs and that did not have problems of mycobacterial infections. Mycobacterium intracellulare was isolated from Eristalis tenax Linnaeus (Diptera: Syrphidae) captured from a pig herd. Mycobacterium avium ssp. avium (serotype 8) was isolated from flies of the genera Drosophila Fallen (Diptera: Drosophilidae) and Musca Linnaeus (Diptera: Muscidae) originating from a pig herd. Mycobacterium spp. were isolated from Musca spp. and Mycobacterium fortuitum was isolated from dung flies of the genus Scatophaga Meigen (Diptera: Scatophagidae), Musca spp. and Stomoxys calcitrans Linnaeus (Diptera: Muscidae) captured in the same herd. Mycobacterium scrofulaceum was isolated from S. calcitrans from the farm with both cattle and pigs. Mycobacterium avium ssp. paratuberculosis was isolated from Scatophaga spp. collected from pastures grazed by one of the cattle herds and from Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) and Lucilia caesar Linnaeus (Diptera: Calliphoridae) captured in a slaughterhouse, where cattle infected with paratuberculosis were slaughtered. Mycobacterium phlei was isolated from flies of the genus Lucilia captured at a waste bin. These data indicate that mycobacteria may be spread by adult flies that have been in contact with material contaminated with these pathogens.

Fischer O; Mátlová L; Dvorská L; Svástová P; Bartl J; Melichárek I; Weston RT; Pavlík I

2001-06-01

109

Nontuberculous Mycobacterial Lung Disease Caused by Mycobacterium lentiflavum in a Patient with Bronchiectasis.  

UK PubMed Central (United Kingdom)

We report a rare case of lung disease caused by Mycobacterium lentiflavum in a previously healthy woman. A 54-year-old woman was referred to our hospital due to chronic cough and sputum. A computed tomography scan of the chest revealed bilateral bronchiectasis with bronchiolitis in the right middle lobe and the lingular division of the left upper lobe. Nontuberculous mycobacteria were isolated twice from three expectorated sputum specimens. All isolates were identified as M. lentiflavum by multilocus sequence analysis based on rpoB, hsp65, and 16S rRNA fragments. To the best of our knowledge, this is the first documented case of M. lentiflavum lung disease in an immunocompetent adult in Korea.

Jeong BH; Song JU; Kim W; Han SG; Ko Y; Song J; Chang B; Hong G; Kim SY; Choi GE; Shin SJ; Koh WJ

2013-04-01

110

Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.  

UK PubMed Central (United Kingdom)

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

Furuya T; Hayashi M; Kino K

2013-10-01

111

Diagnosis and management of atypical mycobacterial infection after laparoscopic surgery.  

UK PubMed Central (United Kingdom)

Atypical mycobacterial infections at the laparoscopic port site are a frequent problem encountered in patients undergoing laparoscopic surgery. In this study we concentrate on the clinical diagnosis, management and prevention of this problem. In this series we assess 19 patients presenting with port hole infections after laparoscopic surgery and were treated with a combination of oral clarithromycin and ciprofloxacin. Seven patients who had persistent nodules were given injections of amikacin directly into the infection foci along with standard oral therapy. Most of the patients treated with standard oral therapy for 28 days showed recovery. The patients with persistent nodules 4 weeks after completion of therapy were treated with injections of amikacin directly into the nodule which lead to resolution of symptoms. For prevention of infection, proper sterilization and storage of instruments is recommended. Laparoscopic port hole infections is a preventable problem and can also be treated by nonsurgical method.

Chaudhuri S; Sarkar D; Mukerji R

2010-12-01

112

New targets and inhibitors of mycobacterial sulfur metabolism.  

UK PubMed Central (United Kingdom)

The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress made in the development of inhibitors of sulfur metabolism enzymes.

Paritala H; Carroll KS

2013-04-01

113

[Atypical mycobacterial infection after kidney transplant: two clinical cases].  

UK PubMed Central (United Kingdom)

Infections are an important cause of morbidity and mortality during kidney transplant. In areas where tuberculosis is not endemic, Mycobacteria other than tuberculosis (MOOT), also known as 'atypical' Mycobacteria, are more frequently involved in mycobacterial infections than M. tuberculosis. The incidence of MOOT infection in renal transplant recipients ranges from 0.16 to 0.38 percent. This low rate of reported incidence is, however, often due to delay in diagnosis and lack of therapeutic protocols. Further difficulty is caused by the interaction of antimycobacterial drugs with the post-transplant immunosuppressive regimen, necessitating close monitoring of plasma concentrations and careful dose modification. We present two cases of Mycobacterium Chelonae infection in kidney transplant recipients which differ in both clinical presentation and pharmacological approach.

Mele A; Bilancio G; Luciani R; Bellizzi V; Palladino G

2013-01-01

114

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important ...

Gonçalves, Eduardo DC; Bonato, Vânia Luiza D; da Fonseca, Denise M; Soares, Edson G; Brandão, Izaíra T; Soares, Ana Paula M

115

Sulfatase-activated fluorophores for rapid discrimination of mycobacterial species and strains.  

Science.gov (United States)

Most current diagnostic tests for tuberculosis do not reveal the species or strain of pathogen causing pulmonary infection, which can lead to inappropriate treatment regimens and the spread of disease. Here, we report an assay for mycobacterial strain assignment based on genetically conserved mycobacterial sulfatases. We developed a sulfatase-activated probe, 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-sulfate, that detects enzyme activity in native protein gels, allowing the rapid detection of sulfatases in mycobacterial lysates. This assay revealed that mycobacterial strains have distinct sulfatase fingerprints that can be used to judge both the species and lineage. Our results demonstrate the potential of enzyme-activated probes for rapid pathogen discrimination for infectious diseases. PMID:23878250

Beatty, Kimberly E; Williams, Monique; Carlson, Brian L; Swarts, Benjamin M; Warren, Robin M; van Helden, Paul D; Bertozzi, Carolyn R

2013-07-22

116

Dendritic cell subsets in mycobacterial infection: control of bacterial growth and T cell responses.  

UK PubMed Central (United Kingdom)

Anti-mycobacterial immunity is guided by specialised antigen presenting cells known as dendritic cells, which are essential for both initiating and maintaining T cell immune responses during infection. The dendritic cell population can be divided into functionally distinct subsets that differ in their ability to present antigen and produce key TH1 cytokines, such as IL-12. This review discusses recent studies, in murine models, investigating which dendritic cell populations are important for mycobacterial control.

Prendergast KA; Kirman JR

2013-03-01

117

Antibodies to mycobacterial 65-kD heat shock protein cross-react with the main mitochondrial antigens in patients with primary biliary cirrhosis.  

UK PubMed Central (United Kingdom)

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease associated with autoimmune disorders. The aetiology is unknown, although it has been suggested that the disease may be related to infectious agents. Previous studies revealed that sera from patients with PBC react against Mycobacterium gordonae. This specific reactivity, characterized by a recognition of two membrane polypeptides of 70-65 and 55 kD, cross-react with the two major mitochondrial autoantigens of PBC. As the most immunogenic components of mycobacteria are the heat shock proteins (hsp), which have been associated with autoimmunity, this study has been undertaken to characterize whether the reacting polypeptides in PBC are hsp from M. gordonae. Cultures of M. gordonae were incubated at 37 degrees C and 46 degrees C before sonication, protein extraction and separation by SDS-PAGE. Exposure of M. gordonae to heat shock treatment resulted in membrane protein overexpression, similar to the 70-65-kD polypeptide recognized by the sera from patients with PBC. Immunoprecipitation assays with a monoclonal antibody directed against the Hsp65 kD of mycobacteria and with sera from patients with PBC revealed similar reacting profiles characterized by the precipitation of the overexpressed 65-kD polypeptide from M. gordonae. Competitive immunoblotting showed that binding of the monoclonal antibody to the Hsp65 kD protein was prevented by preincubation with sera from patients with PBC, but not with sera from healthy subjects. Furthermore, monoclonal antibody to the Hsp65 kD protein recognized the main mitochondrial autoantigens of PBC (PDH-E2 and BCKDH-E2). These data indicate the existence of cross-reacting epitopes contained on M. gordonae Hsp65 kD and the main mitochondrial antigens in patients with PBC.

Vilagut L; Parés A; Viñas O; Vila J; Jiménez de Anta MT; Rodés J

1997-08-01

118

Antibodies to mycobacterial 65-kD heat shock protein cross-react with the main mitochondrial antigens in patients with primary biliary cirrhosis.  

Science.gov (United States)

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease associated with autoimmune disorders. The aetiology is unknown, although it has been suggested that the disease may be related to infectious agents. Previous studies revealed that sera from patients with PBC react against Mycobacterium gordonae. This specific reactivity, characterized by a recognition of two membrane polypeptides of 70-65 and 55 kD, cross-react with the two major mitochondrial autoantigens of PBC. As the most immunogenic components of mycobacteria are the heat shock proteins (hsp), which have been associated with autoimmunity, this study has been undertaken to characterize whether the reacting polypeptides in PBC are hsp from M. gordonae. Cultures of M. gordonae were incubated at 37 degrees C and 46 degrees C before sonication, protein extraction and separation by SDS-PAGE. Exposure of M. gordonae to heat shock treatment resulted in membrane protein overexpression, similar to the 70-65-kD polypeptide recognized by the sera from patients with PBC. Immunoprecipitation assays with a monoclonal antibody directed against the Hsp65 kD of mycobacteria and with sera from patients with PBC revealed similar reacting profiles characterized by the precipitation of the overexpressed 65-kD polypeptide from M. gordonae. Competitive immunoblotting showed that binding of the monoclonal antibody to the Hsp65 kD protein was prevented by preincubation with sera from patients with PBC, but not with sera from healthy subjects. Furthermore, monoclonal antibody to the Hsp65 kD protein recognized the main mitochondrial autoantigens of PBC (PDH-E2 and BCKDH-E2). These data indicate the existence of cross-reacting epitopes contained on M. gordonae Hsp65 kD and the main mitochondrial antigens in patients with PBC. PMID:9279530

Vilagut, L; Parés, A; Viñas, O; Vila, J; Jiménez de Anta, M T; Rodés, J

1997-08-01

119

Imbalanced effector and regulatory cytokine responses may underlie mycobacterial immune restoration disease  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Immune restoration disease (IRD) is an adverse consequence of antiretroviral therapy, where the restored pathogen-specific response causes immunopathology. Mycobacteria are the pathogens that most frequently provoke IRD and mycobacterial IRD is a common cause of morbidity in HIV-infected patients co-infected with mycobacteria. We hypothesised that the excessive effector immune response in mycobacterial IRD reflects impaired regulation by IL-10. Results We studied two patients who experienced mycobacterial IRD during ART. One patient developed a second episode of IRD with distinct clinical characteristics. Findings were compared with patients 'at risk' of developing IRD who had uneventful immune recovery. Peripheral blood mononuclear cells (PBMC) from all subjects were stimulated with mycobacterial antigens in the form of purified protein derivative (PPD). Supernatants were assayed for IFN? and IL-10. In response to PPD, PBMC from IRD patients generated IFN? during the first IRD episode, whilst cells from non-IRD controls produced more IL-10. Conclusion We present preliminary data from two HIV-infected patients showing an imbalance between IFN? and IL-10 responses to mycobacterial antigens during mycobacterial IRD. Our findings suggest that imbalanced effector and regulatory cytokine responses should be investigated as a cause of IRD.

Lim Andrew; D'Orsogna Lloyd; Price Patricia; French Martyn A

2008-01-01

120

MycoProtease-DB: Useful resource for Mycobacterium tuberculosis complex and nontuberculous mycobacterial proteases  

Directory of Open Access Journals (Sweden)

Full Text Available MycoProtease-DB is an online MS SQL and CGI-PERL driven relational database that domiciles protease information of Mycobacterium tuberculosis (MTB) complex and Nontuberculous Mycobacteria (NTM), whose complete genome sequence is available. Our effort is to provide comprehensive information on proteases of 5 strains of Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, F11 and KZN 1435), 3 strains of Mycobacterium bovis (AF2122/97, BCG Pasteur 1173P2 and BCG Tokyo 172) and 4 strains of NTM (Mycobacterium avium 104, Mycobacterium smegmatis MC2 155, Mycobacterium avium paratuberculosis K-10 and Nocardia farcinica IFM 10152) at gene, protein and structural level. MycoProtease-DB currently hosts 1324 proteases, which include 906 proteases from MTB complex with 237distinct proteases & 418 from NTM with 404 distinct proteases. Flexible database design and easy expandability & retrieval of information are the main features of MycoProtease-DB. All the data were validated with various online resources and published literatures for reliable serving as comprehensive resources of various Mycobacterial proteases

Lingaraja Jena,; Satish Kumar &; Bhaskar Chinnaiah Harinath

2012-01-01

 
 
 
 
121

MycoProtease-DB: Useful resource for Mycobacterium tuberculosis complex and nontuberculous mycobacterial proteases.  

UK PubMed Central (United Kingdom)

UNLABELLED: MycoProtease-DB is an online MS SQL and CGI-PERL driven relational database that domiciles protease information of Mycobacterium tuberculosis (MTB) complex and Nontuberculous Mycobacteria (NTM), whose complete genome sequence is available. Our effort is to provide comprehensive information on proteases of 5 strains of Mycobacterium tuberculosis (H(37)Rv, H(37)Ra, CDC1551, F11 and KZN 1435), 3 strains of Mycobacterium bovis (AF2122/97, BCG Pasteur 1173P2 and BCG Tokyo 172) and 4 strains of NTM (Mycobacterium avium 104, Mycobacterium smegmatis MC2 155, Mycobacterium avium paratuberculosis K-10 and Nocardia farcinica IFM 10152) at gene, protein and structural level. MycoProtease-DB currently hosts 1324 proteases, which include 906 proteases from MTB complex with 237distinct proteases & 418 from NTM with 404 distinct proteases. Flexible database design and easy expandability & retrieval of information are the main features of MycoProtease-DB. All the data were validated with various online resources and published literatures for reliable serving as comprehensive resources of various Mycobacterial proteases. AVAILABILITY: The Database is publicly available at http://www.bicjbtdrc-mgims.in/MycoProtease-DB/

Jena L; Kumar S; Harinath BC

2012-01-01

122

DedA protein relates to action-mechanism of halicyclamine A, a marine spongean macrocyclic alkaloid, as an anti-dormant mycobacterial substance.  

Science.gov (United States)

A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegmatis with the genomic DNA library of M. bovis BCG, which were constructed in the multi-copy shuttle cosmid pYUB145. Sequencing analysis of the cosmids isolated from the halicyclamine A-resistant transformants revealed that the responsible gene was involved in the genome region between 2920.549 kb and 2933.210 kb. Further experiments using the transformants over-expressing individual gene contained in the responsible region were executed, and the transformant, which over-expressed BCG2664 gene assigned as dedA gene, was found to become halicyclamine A-resistant. This evidence strongly suggested that DedA protein correlates with the action-mechanism of halicyclamine A as an anti-dormant mycobacterial substance. PMID:21747743

Arai, Masayoshi; Liu, Liu; Fujimoto, Takao; Setiawan, Andi; Kobayashi, Motomasa

2011-06-08

123

DedA Protein Relates to Action-Mechanism of Halicyclamine A, a Marine Spongean Macrocyclic Alkaloid, as an Anti-dormant Mycobacterial Substance  

Directory of Open Access Journals (Sweden)

Full Text Available A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegmatis with the genomic DNA library of M. bovis BCG, which were constructed in the multi-copy shuttle cosmid pYUB145. Sequencing analysis of the cosmids isolated from the halicyclamine A-resistant transformants revealed that the responsible gene was involved in the genome region between 2920.549 kb and 2933.210 kb. Further experiments using the transformants over-expressing individual gene contained in the responsible region were executed, and the transformant, which over-expressed BCG2664 gene assigned as dedA gene, was found to become halicyclamine A-resistant. This evidence strongly suggested that DedA protein correlates with the action-mechanism of halicyclamine A as an anti-dormant mycobacterial substance.

Masayoshi Arai; Liu Liu; Takao Fujimoto; Andi Setiawan; Motomasa Kobayashi

2011-01-01

124

DedA protein relates to action-mechanism of halicyclamine A, a marine spongean macrocyclic alkaloid, as an anti-dormant mycobacterial substance.  

UK PubMed Central (United Kingdom)

A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegmatis with the genomic DNA library of M. bovis BCG, which were constructed in the multi-copy shuttle cosmid pYUB145. Sequencing analysis of the cosmids isolated from the halicyclamine A-resistant transformants revealed that the responsible gene was involved in the genome region between 2920.549 kb and 2933.210 kb. Further experiments using the transformants over-expressing individual gene contained in the responsible region were executed, and the transformant, which over-expressed BCG2664 gene assigned as dedA gene, was found to become halicyclamine A-resistant. This evidence strongly suggested that DedA protein correlates with the action-mechanism of halicyclamine A as an anti-dormant mycobacterial substance.

Arai M; Liu L; Fujimoto T; Setiawan A; Kobayashi M

2011-01-01

125

Doença pulmonar por Mycobacterium tuberculosis e micobactérias não-tuberculosas entre pacientes recém-diagnosticados como HIV positivos em Moçambique, África Mycobacterium tuberculosis and nontuberculous mycobacterial isolates among patients with recent HIV infection in Mozambique  

Directory of Open Access Journals (Sweden)

Full Text Available OBJETIVO: A micobacteriose é frequentemente diagnosticada entre pacientes infectados pelo HIV. Em Moçambique, onde apenas um pequeno número de pacientes encontra-se sob tratamento anti-retroviral, e a tuberculose tem alta prevalência, existe a necessidade de melhor caracterização destes agentes bacterianos, em nível de espécie, bem como de se caracterizar os padrões de resistência às drogas antituberculosas. MÉTODOS: Em uma coorte de 503 indivíduos HIV positivos suspeitos de tuberculose pulmonar, 320 apresentaram positividade para baciloscopia ou cultura no escarro e no lavado brônquico. RESULTADOS: Bacilos álcool-ácido resistentes foram detectados no escarro em 73% dos casos com cultura positiva. De 277 isolados em cultura, apenas 3 mostraram-se tratar de micobactérias não-tuberculosas: 2 Mycobacterium avium e uma M. simiae. Todos os isolados de M. tuberculosis inicialmente caracterizados através de reação em cadeia de polimerase (RCP) do gene hsp65 foram posteriormente caracterizados como tal através de RCP do gene gyrB. Resistência à isoniazida foi encontrada em 14% dos casos; à rifampicina em 6%; e multirresistência em 5%. Pacientes previamente tratados para tuberculose mostraram tendência a taxas maiores de resistência às drogas de primeira linha. O padrão radiológico mais freqüente encontrado foi o infiltrado intersticial (67%), seguido da presença de linfonodos mediastinais (30%), bronquiectasias (28%), padrão miliar (18%) e cavidades (12%). Os pacientes infectados por micobactérias não-tuberculosas não apresentaram manifestações clínicas distintas das apresentadas pelos outros pacientes. A mediana de linfócitos CD4 entre todos os pacientes foi de 134 células/mm³. CONCLUSÕES: Tuberculose e AIDS em Moçambique estão fortemente associadas, como é de se esperar em países com alta prevalência de tuberculose. Embora as taxas de resistência a drogas sejam altas, o esquema isoniazida-rifampicina continua sendo a escolha apropriada para o início do tratamento.OBJECTIVE: Mycobacteriosis is frequently diagnosed among HIV-infected patients. In Mozambique, where few patients are under antiretroviral therapy and the prevalence of tuberculosis is high, there is need for better characterization of mycobacteria at the species level, as well as for the identification of patterns of resistance to antituberculous drugs. METHODS: We studied a sample of 503 HIV-infected individuals suspected of having pulmonary tuberculosis. Of those 503, 320 tested positive for mycobacteria through sputum smear microscopy or culture of bronchoalveolar lavage fluid. RESULTS: Acid-fast bacilli were observed in the sputum of 73% of the individuals presenting positive cultures. Of 277 isolates tested, only 3 were nontuberculous mycobacteria: 2 were identified as Mycobacterium avium and one was identified as M. simiae. Strains initially characterized as M. tuberculosis complex through polymerase chain reaction restriction analysis (PRA) of the hsp65 gene were later confirmed as such through PRA of the gyrB gene. Among the M. tuberculosis isolates, resistance patterns were as follows: to isoniazid, 14%; to rifampin, 6%; and multidrug resistance, 5%. Previously treated cases showed significantly higher rates of resistance to first-line antituberculous drugs. The most common radiological pattern was interstitial infiltrate (in 67%), followed by mediastinal lymph node enlargement (in 30%), bronchiectasis (in 28%), miliary nodules (in 18%) and cavitation (in 12%). Patients infected with nontuberculous mycobacteria presented clinical profiles indistinguishable from those of other patients. The median CD4 lymphocyte count in this group was 134 cells/mm³. CONCLUSIONS: There is a strong association between tuberculosis and AIDS in Mozambique, as expected in a country with a high prevalence of tuberculosis. Although drug resistance rates are high, the isoniazid-rifampin regimen continues to be the appropriate choice for initial therapy.

Elizabete Abrantes Nunes; Eduardo Mello De Capitani; Elizabete Coelho; Alessandra Costa Panunto; Orvalho Augusto Joaquim; Marcelo de Carvalho Ramos

2008-01-01

126

Novel Polymorphic Region of the rpoB Gene Containing Mycobacterium Species-Specific Sequences and Its Use in Identification of Mycobacteria  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Sequence analysis of a specific region of the mycobacterium rpoB gene in 35 mycobacterial strains representing 26 different mycobacterial species of clinical importance showed that there exists a highly polymorphic region. Based on the sequences of the polymorphic region, the oligonucleotide probes ...

Lee, Hyeyoung; Bang, Hye-Eun; Bai, Gill-Han; Cho, Sang-Nae

127

Ethnicity and mycobacterial lineage as determinants of tuberculosis disease phenotype.  

UK PubMed Central (United Kingdom)

BACKGROUND: Emerging evidence suggests that Mycobacterium tuberculosis (Mtb) lineage and host ethnicity can determine tuberculosis (TB) clinical disease patterns but their relative importance and interaction are unknown. METHODS: We evaluated prospectively collected TB surveillance and Mtb strain typing data in an ethnically heterogeneous UK population. Lineage assignment was denoted using 15-loci mycobacterial interspersed repetitive units containing variable numbers of tandem repeats (MIRU-VNTR) and MIRU-VNTRplus. Geographical and ethnic associations of the six global Mtb lineages were identified and the influence of lineage and demographic factors on clinical phenotype were assessed using multivariate logistic regression. RESULTS: Data were available for 1070 individuals with active TB which was pulmonary only, extrapulmonary only and concurrent pulmonary-extrapulmonary in 52.1%, 36.9% and 11.0% respectively. The most prevalent lineages were Euro-American (43.7%), East African Indian (30.2%), Indo-Oceanic (13.6%) and East Asian (12.2%) and were geo-ethnically restricted with, for example, Indian subcontinent ethnicity inversely associated with Euro-American lineage (OR 0.23; 95% CI 0.14 to 0.39) and positively associated with the East African-Indian lineage (OR 4.04; 95% CI 2.19 to 7.45). Disease phenotype was most strongly associated with ethnicity (OR for extrathoracic disease 21.14 (95% CI 6.08 to 73.48) for Indian subcontinent and 14.05 (3.97 to 49.65) for Afro-Caribbean), after adjusting for lineage. With East Asian lineage as the reference category, the Euro-American (OR 0.54; 95% CI 0.32 to 0.91) and East-African Indian (OR 0.50; 95% CI 0.29 to 0.86) lineages were negatively associated with extrathoracic disease, compared with pulmonary disease, after adjusting for ethnicity. CONCLUSIONS: Ethnicity is a powerful determinant of clinical TB phenotype independently of mycobacterial lineage and the role of ethnicity-associated factors in pathogenesis warrants investigation.

Pareek M; Evans J; Innes J; Smith G; Hingley-Wilson S; Lougheed KE; Sridhar S; Dedicoat M; Hawkey P; Lalvani A

2013-03-01

128

Induction of Mycobacterial Resistance to Quinolone Class Antimicrobials  

Science.gov (United States)

An agar plate assay was developed for detecting the induction of drug-resistant mycobacterial mutants during exposure to inhibitors of DNA gyrase. When Mycobacterium smegmatis on drug-containing agar, resistant colonies arose over a period of 2 weeks. A recA deficiency reduced mutant recovery, consistent with involvement of the SOS response in mutant induction. The C-8-methoxy compounds gatifloxacin and moxifloxacin allowed the recovery of fewer resistant mutants than either ciprofloxacin or levofloxacin when present at the same multiple of the MIC; a quinolone-like 8-methoxy-quinazoline-2,4-dione was more effective at restricting the emergence of resistant mutants than its cognate fluoroquinolone. Thus, the structure of fluoroquinolone-like compounds affects mutant recovery. A spontaneous mutator mutant of M. smegmatis, obtained by growth in medium containing both isoniazid and rifampin, increased mutant induction during exposure to ciprofloxacin. Moreover, the mutator increased the size of spontaneous resistant mutant subpopulations, as detected by population analysis. Induction of ciprofloxacin resistance was also observed with Mycobacterium tuberculosis H37Rv. When measured with clinical isolates, no difference in mutant recovery was observed between multidrug-resistant (MDR) and pansusceptible isolates. This finding is consistent with at least some MDR isolates of M. tuberculosis lacking mutators detectable by the agar plate assay. Collectively, the data indicate that the use of fluoroquinolones against tuberculosis may induce resistance and that the choice of quinolone may be important for restricting the recovery of induced mutants.

Malik, Muhammad; Chavda, Kalyan; Zhao, Xilin; Shah, Nirali; Hussain, Syed; Kurepina, Natalia; Kreiswirth, Barry N.; Kerns, Robert J.

2012-01-01

129

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

DEFF Research Database (Denmark)

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.

Ravn, P; Pedersen, B K

1994-01-01

130

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli.  

Science.gov (United States)

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes. PMID:23892738

Furuya, Toshiki; Hayashi, Mika; Kino, Kuniki

2013-07-26

131

Identification of mycobacterial membrane proteins and their types using over-represented tripeptide compositions.  

UK PubMed Central (United Kingdom)

Mycobacterium can cause many serious diseases, such as tuberculosis and leprosy. Its membrane proteins play a critical role for multidrug-resistance and its tenacious survival ability. Knowing the types of membrane proteins will provide novel insights into understanding their functions and facilitate drug target discovery. In this study, a novel method was developed for predicting mycobacterial membrane protein and their types by using over-represented tripeptides. A total of 295 non-membrane proteins and 274 membrane proteins were collected to evaluate the performance of proposed method. The results of jackknife cross-validation test show that our method achieves an overall accuracy of 93.0% in discriminating between mycobacterial membrane proteins and mycobacterial non-membrane proteins and an overall accuracy of 93.1% in classifying mycobacterial membrane protein types. By comparing with other methods, the proposed method showed excellent predictive performance. Based on the proposed method, we built a predictor, called MycoMemSVM, which is freely available at http://lin.uestc.edu.cn/server/MycoMemSVM. It is anticipated that MycoMemSVM will become a useful tool for the annotation of mycobacterial membrane proteins and the development of anti-mycobacterium drug design.

Ding C; Yuan LF; Guo SH; Lin H; Chen W

2012-12-01

132

Potential biomarkers for identification of mycobacterial cultures by proton transfer reaction mass spectrometry analysis.  

UK PubMed Central (United Kingdom)

RATIONALE: Several mycobacterial species can produce serious infections in humans, and the treatment required depends on the infecting species. Fast identification, ideally with minimal manipulation of the infecting species, is therefore critical; here, we propose a method potentially allowing cultures to be identified by headspace analysis and use it to screen for differences between mycobacterial species based on the volatiles released during growth. METHODS: Short-chain volatile organic compound emissions from two non-tuberculosis slow growing mycobacterial species, Mycobacterium avium and Mycobacterium kansasii, and a non-pathogenic fast growing species, Mycobacterium smegmatis, in Middlebrook M7H9 culturing media were followed online with a proton transfer reaction quadrupole mass spectrometer. RESULTS: Measurable differences between the headspace of the two slow growing mycobacteria M. kansasii and M. avium were found, as well as differences with respect to the faster growing mycobacteria M. smegmatis. Three compounds, attributed to sulfur-containing volatiles--dimethyl sulfide, propanethiol and dimethyl disulfide--were found to be specific to M. avium. CONCLUSIONS: Clear differences were detected in the low molecular weight volatile emissions compounds of the mycobacterial species under study, without the need for sample manipulation. Further studies with other mycobacterial species will reveal if the differences observed are specific to the species studied here. Furthermore, the use of an ion trap as a mass analyzer with the same ionization technique, allowing molecular detection over a wider molecular range, could allow the detection of additional biomarkers thus capturing a wider molecular range.

Crespo E; de Ronde H; Kuijper S; Pol A; Kolk AH; Cristescu SM; Anthony RM; Harren FJ

2012-03-01

133

Use of the GenoType Mycobacterium CM and AS assays to analyze 76 nontuberculous mycobacterial isolates from Greece.  

Science.gov (United States)

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, additional probes should be designed and added to the strip. PMID:16757630

Gitti, Zoe; Neonakis, Ioannis; Fanti, Garyfallia; Kontos, Fanourios; Maraki, Sofia; Tselentis, Yiannis

2006-06-01

134

Use of the GenoType Mycobacterium CM and AS assays to analyze 76 nontuberculous mycobacterial isolates from Greece.  

UK PubMed Central (United Kingdom)

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, additional probes should be designed and added to the strip.

Gitti Z; Neonakis I; Fanti G; Kontos F; Maraki S; Tselentis Y

2006-06-01

135

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

Gitti, Zoe; Neonakis, Ioannis; Fanti, Garyfallia; Kontos, Fanourios; Maraki, Sofia; Tselentis, Yiannis

136

Membrane TNF confers protection to acute mycobacterial infection  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF). Methods C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice. Results While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice. Conclusion Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.

Fremond Cecile; Allie Nasiema; Dambuza Ivy; Grivennikov Sergei I; Yeremeev Vladimir; Quesniaux Valerie FJ; Jacobs Muazzam; Ryffel Bernhard

2005-01-01

137

[Pulmonary aspergillosis complicating atypical mycobacterial infection in two patients suffering from chronic obstructive pulmonary disease].  

UK PubMed Central (United Kingdom)

INTRODUCTION: Atypical mycobacteria and Aspergillus are opportunistic organisms responsible for severe pulmonary diseases whose development is encouraged by the presence of chronic obstructive pulmonary disease (COPD) and related immunosuppression. CASE REPORTS: We report the cases of two patients, both alcoholics with emphysematous COPD, who developed chronic pulmonary aspergillosis following atypical mycobacterial infection. Patient 1 developed chronic necrotising aspergillosis several months after the diagnosis of infection with Mycobacterium avium. Patient 2 developed an aspergilloma several weeks after the diagnosis of infection with Mycobacterium xenopi. The association of these two pathologies presents diagnostic and therapeutic problems that are discussed. CONCLUSION: The development of Aspergillus pulmonary disease may complicate atypical mycobacterial infections and explain a poor response to treatment. Our two case reports suggest that a systematic search should be made for pulmonary aspergillosis during the follow-up of patients with atypical mycobacterial infection.

Montaigne E; Petit FX; Gourdier AL; Urban T; Gagnadoux F

2012-01-01

138

Mycobacterial PGL virulence factor biosynthesis: mechanism and small-molecule inhibition of polyketide chain initiation  

Science.gov (United States)

Summary Phenolic glycolipids (PGLs) are polyketide-derived virulence factors produced by Mycobacterium tuberculosis, Mycobacterium leprae, and other mycobacterial pathogens. We have combined bioinformatic, genetic, biochemical, and chemical biology approaches to illuminate the mechanism of chain initiation required for assembly of the p-hydroxyphenyl-polyketide moiety of PGLs. Our studies have led to the identification of a stand-alone, didomain initiation module, FadD22, comprised of a p-hydroxybenzoic acid adenylation domain and an aroyl carrier protein domain. FadD22 forms the first acyl-S-enzyme covalent intermediate in the p-hydroxyphenyl-polyketide chain assembly line. We also used this information to develop the first small-molecule inhibitor of PGL biosynthesis. Overall, these studies provide new insights into the biosynthesis of an important group of small-molecule mycobacterial virulence factors and support the feasibility of targeting PGL biosynthesis to develop new drugs to treat mycobacterial infections.

Ferreras, Julian A.; Stirrett, Karen L.; Lu, Xuequan; Ryu, Jae-Sang; Soll, Clifford E.; Tan, Derek S.; Quadri, Luis E. N.

2008-01-01

139

Multiple mycobacterial antigens are targets of the adaptive immune response in pulmonary sarcoidosis  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Introduction Sarcoidosis is a multisystem granulomatous disease for which the association with mycobacteria continues to strengthen. It is hypothesized that a single, poorly degradable antigen is responsible for sarcoidosis pathogenesis. Several reports from independent groups support mycobacterial antigens having a role in sarcoidosis pathogenesis. To identify other microbial targets of the adaptive immune response, we tested the ability of CD4+ and CD8+ T cells to recognize multiple mycobacterial antigens. Methods Fifty-four subjects were enrolled in this study: 31 sarcoidosis patients, nine non-tuberculosis mycobacterial (NTM) infection controls, and 14 PPD- controls. Using flow cytometry, we assessed for Th1 immune responses to ESAT-6, katG, Ag85A, sodA, and HSP. Results Alveolar T-cells from twenty-two of the 31 sarcoidosis patients produced a CD4+ response to at least one of ESAT-6, katG, Ag85A, sodA, or HSP, compared to two of 14 PPD- controls (p = 0.0008) and five of nine NTM controls (p = 0.44), while eighteen of the 31 sarcoidosis subjects tested produced a CD8+ response to at least one of the mycobacterial antigens compared to two of 14 PPD- controls (p = 0.009) and three of nine NTM controls (0.26). Not only did the BAL-derived T cells respond to multiple virulence factors, but also to multiple, distinct epitopes within a given protein. The detection of proliferation upon stimulation with the mycobacterial virulence factors demonstrates that these responses are initiated by antigen specific recognition. Conclusions Together these results reveal that antigen-specific CD4+ and CD8+ T cells responses to multiple mycobacterial epitopes are present within sites of active sarcoidosis involvement, and that these antigen-specific responses are present at the time of diagnosis.

Oswald-Richter Kyra A; Beachboard Dia C; Zhan Xiaoyan; Gaskill Christa F; Abraham Susamma; Jenkins Cathy; Culver Daniel A; Drake Wonder

2010-01-01

140

A thin-layer chromatographic method for separating methyl esters of mycobacterial mycolic acids.  

Science.gov (United States)

A two-dimensional thin-layer chromatographic method was developed which allowed separation of the different mycobacterial mycolic acids as methyl esters. Dichloromethane (once) and petroleum ether:acetone (95:5, v/v twice or three times) were used as solvents. Alkaline saponification of freeze-dried cells followed by methylation of the mycolic acids using iodomethane gave satisfactory results, whereas methylation using boron trichloride-methanol complex or trans-esterification through direct acid methanolysis was found to degrade epoxy-mycolates. The chromatographic method developed here is rapid and informative, and should prove valuable in routine mycobacterial differentiation. PMID:3565012

Valero-Guillén, P; Martin-Luengo, F; Jimenez, J; Larsson, L

1986-12-01

 
 
 
 
141

A thin-layer chromatographic method for separating methyl esters of mycobacterial mycolic acids.  

UK PubMed Central (United Kingdom)

A two-dimensional thin-layer chromatographic method was developed which allowed separation of the different mycobacterial mycolic acids as methyl esters. Dichloromethane (once) and petroleum ether:acetone (95:5, v/v twice or three times) were used as solvents. Alkaline saponification of freeze-dried cells followed by methylation of the mycolic acids using iodomethane gave satisfactory results, whereas methylation using boron trichloride-methanol complex or trans-esterification through direct acid methanolysis was found to degrade epoxy-mycolates. The chromatographic method developed here is rapid and informative, and should prove valuable in routine mycobacterial differentiation.

Valero-Guillén P; Martin-Luengo F; Jimenez J; Larsson L

1986-12-01

142

Prevalence and associated risk factors of mycobacterial infections in slaughter pigs from Mubende district in Uganda.  

UK PubMed Central (United Kingdom)

To date, the public health relevance of mycobacterial infections in pigs is not well investigated despite high risk of infection. Recently, there has been a documented increase in opportunistic infections and risk of acquiring opportunistic mycobacterial infections in HIV/AIDS patients in Mubende district; unfortunately, there has been no published information on the epidemiology of mycobacterial infections in this area. This study was carried out between September 2008 and February 2009. Investigations were done to assess the prevalence and associated risk factors of mycobacterial infections in slaughtered pigs in Mubende district of Uganda. A total of 997 pigs (53.7% male and 46.3% female) from 31 different slaughterhouses were examined for the presence of lesions compatible with TB and mycobacterial infections. Pathologic tissue specimens were collected for culturing and isolation of mycobacteria. A cross-sectional technique was used based on convenient visits to slaughterhouses but random selection of individual slaughtered pigs for a detailed post-mortem inspection on a daily basis. The results reflected a 9.3% and 3.1% (95% CI) prevalence of Mycobacterium species based on necropsy examinations and culture isolation, respectively. The highest prevalence of mycobacterial infection was recorded in Buwekula County (the mixed agro-zone) whilst the lowest was in Kassanda County (pastoral zone). A multivariable logistical regression analysis identified age (P < or = 0.001) and sex (P < or = 0.05) as risk factors for mycobacterial infections in pigs. Post-estimation statistics of the regression model evaluation and validation fit it well into the data (HL, chi (2) = 5.9; P = 0.69 for necropsy, HL chi (2) = 2.9; P = 0.94 for culturing). This study documented a high prevalence of mycobacterial infections in slaughter pigs in Mubende district. The fact that pigs and human often share common housing and environment poses a high risk of zoonotic transmission. This then warrants further molecular investigation to identify the specific Mycobacterium species and their public health importance in this area.

Muwonge A; Kankya C; Godfroid J; Djonne B; Opuda-Asibo J; Biffa D; Ayanaw T; Munyeme M; Skjerve E

2010-06-01

143

Laboratory procedures for the detection and identification of cutaneous non-tuberculous mycobacterial infections.  

UK PubMed Central (United Kingdom)

There is evidence that the incidence of cutaneous non-tuberculous mycobacterial (NTM) infection is increasing worldwide. Novel culture methods and new analytical procedures have led to significant advancements in understanding the origin and progression of NTM infections. Differential identification of NTM isolates is important because culture characteristics and/or sensitivity to anti-mycobacterium drugs vary between different mycobacterial species. In this manuscript, we describe the latest diagnostic techniques for cutaneous NTM infection and show how these methodologies can be used for the diagnosis of Buruli ulcer in Japan.

Nakanaga K; Hoshino Y; Yotsu RR; Makino M; Ishii N

2013-03-01

144

Haploinsufficiency at the human IFNGR2 locus contributes to mycobacterial disease.  

UK PubMed Central (United Kingdom)

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare syndrome, the known genetic etiologies of which impair the production of, or the response to interferon-gamma (IFN-?). We report here a patient (P1) with MSMD whose cells display mildly impaired responses to IFN-?, at levels, however, similar to those from MSMD patients with autosomal recessive (AR) partial IFN-?R2 or STAT1 deficiency. Whole-exome sequencing (WES) and Sanger sequencing revealed only one candidate variation for both MSMD-causing and IFN-?-related genes. P1 carried a heterozygous frame-shift IFNGR2 mutation inherited from her father. We show that the mutant allele is intrinsically loss-of-function and not dominant-negative, suggesting haploinsufficiency at the IFNGR2 locus. We also show that Epstein-Barr virus transformed B lymphocyte cells from 10 heterozygous relatives of patients with AR complete IFN-?R2 deficiency respond poorly to IFN-?, in some cases as poorly as the cells of P1. Naive CD4(+) T cells and memory IL-4-producing T cells from these individuals also responded poorly to IFN-?, whereas monocytes and monocyte-derived macrophages (MDMs) did not. This is consistent with the lower levels of expression of IFN-?R2 in lymphoid than in myeloid cells. Overall, MSMD in this patient is probably due to autosomal dominant (AD) IFN-?R2 deficiency, resulting from haploinsufficiency, at least in lymphoid cells. The clinical penetrance of AD IFN-?R2 deficiency is incomplete, possibly due, at least partly, to the variability of cellular responses to IFN-? in these individuals.

Kong XF; Vogt G; Itan Y; Macura-Biegun A; Szaflarska A; Kowalczyk D; Chapgier A; Abhyankar A; Furthner D; Djambas Khayat C; Okada S; Bryant VL; Bogunovic D; Kreins A; Moncada-Vélez M; Migaud M; Al-Ajaji S; Al-Muhsen S; Holland SM; Abel L; Picard C; Chaussabel D; Bustamante J; Casanova JL; Boisson-Dupuis S

2013-02-01

145

Secretome differences between the taxonomically related but clinically differing mycobacterial species Mycobacterium abscessus and M. chelonae  

Directory of Open Access Journals (Sweden)

Full Text Available Rapidly growing non-tuberculous mycobacteria (NTM) are significant human pathogens which show high inter-species differences in clinical characteristics (virulence, host immune response) during infection even within a given NTM complex. Understanding the differences between the secreted proteomes of the member species for an NTM complex may reveal the basis of their differential virulence and host pathogenesis potential including host immune reactions. In this study, major secreted proteins of the two taxonomically close but clinically differing member species M. abscessus and M. chelonae of the M. chelonae-M. abscessus(MCA) complex were compared using an approach based on 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF analyses. The two secretomes showed dramatic differences. Of the 73 major secreted proteins identified, majority were expressed in a species-specific manner, including 37 in M. chelonae and 32 in M. abscessus. Interestingly, 9 of these differentially expressed proteins were orphan proteins showing homology to either hypothetical proteins or those with no defined function. The other 60 distinctly expressed proteins were homologs of those associated with various bacterial cellular functions and virulence, namely cell wall synthesis or lipid metabolism, metabolic and respiratory pathways, stress response and signal transduction, gene regulation, and immune response. This information on species-specific secreted proteins would help understand the critical virulence factors and host pathogenesis mechanisms in these mycobacterial species and provide the basis for developing better therapeutic strategies. These proteins may also serve as potential targets for species-specific diagnosis as an additional outcome. To our knowledge, this is the first attempt to characterize the secretome of M. chelonae (for which the genome sequence is not yet available) and the secretome differences between M. abscessus and M. chelonae.

Jagjit S. Yadav; Manish Gupta

2012-01-01

146

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 phosphol...

Crellin, Paul K.; Vivian, Julian P.; Scoble, Judith; Chow, Frances M.; West, Nicholas P.; Brammananth, Rajini; Proellocks, Nicholas I.

147

Biochip System for Rapid and Accurate Identification of Mycobacterial Species from Isolates and Sputum?  

Science.gov (United States)

The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.

Zhu, Lingxiang; Jiang, Guanglu; Wang, Shengfen; Wang, Can; Li, Qiang; Yu, Hao; Zhou, Yang; Zhao, Bing; Huang, Hairong; Xing, Wanli; Mitchelson, Keith; Cheng, Jing; Zhao, Yanlin; Guo, Yong

2010-01-01

148

Biochip System for Rapid and Accurate Identification of Mycobacterial Species from Isolates and Sputum?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, includ...

Zhu, Lingxiang; Jiang, Guanglu; Wang, Shengfen; Wang, Can; Li, Qiang; Yu, Hao; Zhou, Yang; Zhao, Bing; Huang, Hairong

149

Biochip system for rapid and accurate identification of mycobacterial species from isolates and sputum.  

UK PubMed Central (United Kingdom)

The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.

Zhu L; Jiang G; Wang S; Wang C; Li Q; Yu H; Zhou Y; Zhao B; Huang H; Xing W; Mitchelson K; Cheng J; Zhao Y; Guo Y

2010-10-01

150

Characterization and Comparison of Mycobacterial Antigens by Two-Dimensional Immunoelectrophoresis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Two-dimensional immunoelectrophoresis (2D-IEP), in which a complex of antigens is subjected to electrophoresis first through an agarose matrix in one direction and secondly through an antiserum-agarose matrix at right angles to the first direction, was evaluated as a tool for analysis of mycobacteri...

Roberts, Dianna B.; Wright, George L.; Affronti, Lewis F.; Reich, Melvin

151

The hydroxy-naphthoquinone lapachol arrests mycobacterial growth and immunomodulates host macrophages.  

UK PubMed Central (United Kingdom)

The present study reports the anti-mycobacterial activity of 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone (lapachol) as well as its influence on macrophage functions. Lapachol (L) did not induce apoptosis/necrosis of THP-1 macrophages at ?32 ?g/mL. Mycobacterium avium liquid growth was arrested by ?32 ?g/mL and intra-macrophage proliferation by ?16 ?g/mL lapachol. The main immuno-modulatory effects of lapachol observed were an up-regulation of interferon-?-receptor 1 (IFN-?R1) and major histocompatibility complex class II (MHCII) surface expression, and a marked inhibition of IL-10 secretion. Lapachol did not affect resting, IFN-?- or toll-like receptor 2 (TLR2)-induced levels of oxygen and nitrogen metabolism key proteins nor the TLR2-mediated secretion of TNF-?, nor induced either oxidative or endoplasmic reticulum (ER) stress. Lapachol inhibited the surface expression of the co-stimulatory molecule CD86 but not that of CD80 and CD83. The results obtained indicate that the substituted naphthoquinone lapachol exhibits an anti-mycobacterial activity that is more efficient intra- than extra-cellularly, and exerts immuno-modulatory effects some of which may enhance the capacity of the host cell to control mycobacterial growth. The immune-modulatory action of lapachol could contribute to its more efficient intra-macrophage anti-mycobacterial activity.

Oliveira RA; Azevedo-Ximenes E; Luzzati R; Garcia RC

2010-11-01

152

The hydroxy-naphthoquinone lapachol arrests mycobacterial growth and immunomodulates host macrophages.  

Science.gov (United States)

The present study reports the anti-mycobacterial activity of 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone (lapachol) as well as its influence on macrophage functions. Lapachol (L) did not induce apoptosis/necrosis of THP-1 macrophages at ?32 ?g/mL. Mycobacterium avium liquid growth was arrested by ?32 ?g/mL and intra-macrophage proliferation by ?16 ?g/mL lapachol. The main immuno-modulatory effects of lapachol observed were an up-regulation of interferon-?-receptor 1 (IFN-?R1) and major histocompatibility complex class II (MHCII) surface expression, and a marked inhibition of IL-10 secretion. Lapachol did not affect resting, IFN-?- or toll-like receptor 2 (TLR2)-induced levels of oxygen and nitrogen metabolism key proteins nor the TLR2-mediated secretion of TNF-?, nor induced either oxidative or endoplasmic reticulum (ER) stress. Lapachol inhibited the surface expression of the co-stimulatory molecule CD86 but not that of CD80 and CD83. The results obtained indicate that the substituted naphthoquinone lapachol exhibits an anti-mycobacterial activity that is more efficient intra- than extra-cellularly, and exerts immuno-modulatory effects some of which may enhance the capacity of the host cell to control mycobacterial growth. The immune-modulatory action of lapachol could contribute to its more efficient intra-macrophage anti-mycobacterial activity. PMID:20837170

Oliveira, Renato A S; Azevedo-Ximenes, Eulalia; Luzzati, Roberto; Garcia, Rodolfo C

2010-09-15

153

Molecular Evidence of Lateral Gene Transfer in rpoB Gene of Mycobacterium yongonense Strains via Multilocus Sequence Analysis  

Science.gov (United States)

Recently, a novel species, Mycobacterium yongonense (DSM 45126T), was introduced and while it is phylogenetically related to Mycobacterium intracellulare, it has a distinct RNA polymerase ?-subunit gene (rpoB) sequence that is identical to that of Mycobacterium parascrofulaceum, which is a distantly related scotochromogen, which suggests the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event. The aims of this study are to prove the presence of the LGT event in the rpoB gene of the M. yongonense strains via multilocus sequence analysis (MLSA). In order to determine the potential of an LGT event in the rpoB gene of the M. yongonense, the MLSA based on full rpoB sequences (3447 or 3450 bp) and on partial sequences of five other targets [16S rRNA (1383 or 1395 bp), hsp65 (603 bp), dnaJ (192 bp), recA (1053 bp), and sodA (501 bp)] were conducted. Incongruences between the phylogenetic analysis of the full rpoB and the five other genes in a total of three M. yongonense strains [two clinical strains (MOTT-12 and MOTT-27) and one type strain (DSM 45126T)] were observed, suggesting that rpoB gene of three M. yongonense strains may have been acquired very recently via an LGT event from M. parascrofulaceum, which is a distantly related scotochromogen.

Kim, Byoung-Jun; Hong, Seok-Hyun; Kook, Yoon-Hoh; Kim, Bum-Joon

2013-01-01

154

Molecular evidence of lateral gene transfer in rpoB gene of Mycobacterium yongonense strains via multilocus sequence analysis.  

UK PubMed Central (United Kingdom)

Recently, a novel species, Mycobacterium yongonense (DSM 45126(T)), was introduced and while it is phylogenetically related to Mycobacterium intracellulare, it has a distinct RNA polymerase ?-subunit gene (rpoB) sequence that is identical to that of Mycobacterium parascrofulaceum, which is a distantly related scotochromogen, which suggests the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event. The aims of this study are to prove the presence of the LGT event in the rpoB gene of the M. yongonense strains via multilocus sequence analysis (MLSA). In order to determine the potential of an LGT event in the rpoB gene of the M. yongonense, the MLSA based on full rpoB sequences (3447 or 3450 bp) and on partial sequences of five other targets [16S rRNA (1383 or 1395 bp), hsp65 (603 bp), dnaJ (192 bp), recA (1053 bp), and sodA (501 bp)] were conducted. Incongruences between the phylogenetic analysis of the full rpoB and the five other genes in a total of three M. yongonense strains [two clinical strains (MOTT-12 and MOTT-27) and one type strain (DSM 45126(T))] were observed, suggesting that rpoB gene of three M. yongonense strains may have been acquired very recently via an LGT event from M. parascrofulaceum, which is a distantly related scotochromogen.

Kim BJ; Hong SH; Kook YH; Kim BJ

2013-01-01

155

Anti-mycobacterial activity of root and leaf extracts of Anthocleista djalonensis (Loganiaceae) and Diospyros mespiliformis (Ebenaceae)  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We screened the aqueous and methanol leaf and root extracts of Anthocleista djalonensis, Diospyros mespiliformis, and their combinations for possible anti-mycobacterial activities using Mycobacterium smegmatis as a surrogate screen. These plants are reputed among folk practices ...

Esimone Charles; Nworu Chukwuemeka; Onuigbo Ebere; Omeje Justina; Nsirim Kelechi; Ogbu Joy; Ngwu Maria; Chah Kennedy

156

Atypical mycobacterial exit-site infection and peritonitis in peritoneal dialysis patients on prophylactic exit-site gentamicin cream.  

UK PubMed Central (United Kingdom)

We report 9 cases of exit-site infection and continuous ambulatory peritoneal dialysis peritonitis associated with atypical mycobacteria. All patients had been using topical gentamicin cream as prophylaxis for exit-site infection before the onset of these infections. Gentamicin cream is postulated to be a potential risk factor for atypical mycobacterial infection because of selective pressure on other micro-organisms. The microbiology of atypical mycobacteria and the treatment for atypical mycobacterial infections are discussed.

Lo MW; Mak SK; Wong YY; Lo KC; Chan SF; Tong GM; Lo KY; Wong PN; Tse CW; Kam KM; Wong AK

2013-05-01

157

Mycobacterial disease and impaired IFN-? immunity in humans with inherited ISG15 deficiency.  

UK PubMed Central (United Kingdom)

ISG15 is an interferon (IFN)-?/?-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes-granulocyte, in particular-reduced the production of IFN-? by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-?-inducing secreted molecule for optimal antimycobacterial immunity.

Bogunovic D; Byun M; Durfee LA; Abhyankar A; Sanal O; Mansouri D; Salem S; Radovanovic I; Grant AV; Adimi P; Mansouri N; Okada S; Bryant VL; Kong XF; Kreins A; Velez MM; Boisson B; Khalilzadeh S; Ozcelik U; Darazam IA; Schoggins JW; Rice CM; Al-Muhsen S; Behr M; Vogt G; Puel A; Bustamante J; Gros P; Huibregtse JM; Abel L; Boisson-Dupuis S; Casanova JL

2012-09-01

158

7.5-A cryo-em structure of the mycobacterial fatty acid synthase.  

UK PubMed Central (United Kingdom)

The mycobacterial fatty acid synthase (FAS) complex is a giant 2.0-MDa ?(6) homohexameric multifunctional enzyme that catalyzes synthesis of fatty acid precursors of mycolic acids, which are major components of the cell wall in Mycobacteria and play an important role in pathogenicity. Here, we present a three-dimensional reconstruction of the Mycobacterium smegmatis FAS complex at 7.5Å, highly homologous to the Mycobacterium tuberculosis multienzyme, by cryo-electron microscopy. Based on the obtained structural data, which allowed us to identify secondary-structure elements, and sequence homology with the fungal FAS, we generated an accurate architectural model of the complex. The FAS system from Mycobacteria resembles a minimized version of the fungal FAS with much larger openings in the reaction chambers. These architectural features of the mycobacterial FAS may be important for the interaction with mycolic acid processing and condensing enzymes that further modify the precursors produced by FAS and for autoactivation of the FAS complex.

Boehringer D; Ban N; Leibundgut M

2013-03-01

159

Definition and annotation of (myco)bacterial non-coding RNA.  

UK PubMed Central (United Kingdom)

RNA in bacteria may be broadly classified into coding and non-coding types. The prior, also known as messenger RNA, encode proteins as their final product. The non-coding RNA include all RNAs that are not translated into a protein. Examples of extensively studied and therefore prominent non-coding RNAs include rRNA, tRNA, tmRNA, whose designations reflect the functions performed by these RNAs. Discoveries of non-coding RNAs in mycobacteria have been reported in the recent years. At this early stage of this discipline of mycobacterial research, there is an opportunity for the scientific community to establish a consistent, systematic and objective approach to annotation of these RNAs. We are providing recommendations for this systematic annotation that we hope will be adopted by the mycobacterial research community. These may also serve as templates for annotation of non-coding RNAs in other bacteria.

Lamichhane G; Arnvig KB; McDonough KA

2013-01-01

160

Comparative genomic insights into the biosynthesis and regulation of mycobacterial siderophores.  

UK PubMed Central (United Kingdom)

Iron is essential for nearly all biological events. Siderophores are indispensable for most organisms to obtain iron from iron-limiting milieus. This holds particularly true for pathogens such as the causative agent of tuberculosis - Mycobacterium tuberculosis. The categories of mycobacterial siderophores, their biosynthesis and regulation are summarized here. The siderophore biosynthesis and regulation differences between the pathogenic and non-pathogenic mycobacteria are highlighted from comparative genomic perspective, with an aim to find clues for drug or drug target within siderophore metabolism.

Li W; He J; Xie L; Chen T; Xie J

2013-01-01

 
 
 
 
161

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence  

Directory of Open Access Journals (Sweden)

Full Text Available When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

André Alves Dias; Dominique Raze; Cristiana Soares de Lima; Maria Angela de Melo Marques; Hervé Drobecq; Anne-Sophie Debrie; Michelle Lopes Ribeiro-Guimarães; Franck Biet; Maria Cristina Vidal Pessolani

2012-01-01

162

Disease progression of Mycobacterium avium pulmonary infection and the mycobacterial variable number tandem repeat (VNTR) typing.  

UK PubMed Central (United Kingdom)

Nontuberculous mycobacteriosis may progress to fatal chronic respiratory infections. Some cases remain stable over a relatively long period of time. With no well established progression predictors yet available, we conducted a retrospective analysis of the association between mycobacterial variable numbers of tandem repeat (VNTR) and clinical progression in 37 patients who were seen at the Department of Respiratory Medicine, Tohoku University Hospital between 2005 and 2006 and from whose respiratory tract specimens M. avium was isolated and cultured. The disease type in the 15 patients who began an antimicrobial therapy within 1 year after a bacteriological diagnosis was defined as progressive, and that in the 9 patients who began an antimicrobial therapy 2 years or longer after diagnosis was defined as stable. A cluster analysis of the mycobacterial VNTR genotypes showed concentrations of the progressive-type isolates and the stable-type isolates in different clusters. Furthermore, the study demonstrated that multiple logistic regression analysis can be used to construct a model for estimating, with statistical significance, progression of nontuberculous mycobacteriosis based on the mycobacterial VNTR genotype. These results indicated that whether a nontuberculous mycobacteriosis is progressive can be estimated by the VNTR genotyping of the nontuberculous mycobacterium.

Kikuchi T

2013-01-01

163

Pulmonary non-tuberculous mycobacterial infection in congenital contractural arachnodactyly.  

Science.gov (United States)

Congenital contractural arachnodactyly (CCA) is caused by mutations within the fibrillin-2 gene (FBN2), which is crucial for microfibril structure. Affected individuals may have contractures, chest wall deformities, scoliosis, abnormal ear folding and elongated limbs. We describe a novel FBN2 mutation in a woman with CCA who also had pulmonary non-tuberculous mycobacteria (NTM) infection. The population with pulmonary NTM infections shares phenotypic features with CCA, such as elongated body habitus, scoliosis and pectus deformities. While it is unlikely that FBN2 defects account for susceptibility to NTM infection in the majority of cases, the overlap between these two diseases suggests some shared pathophysiology. PMID:22325249

Paulson, M L; Olivier, K N; Holland, S M

2012-04-01

164

Pulmonary non-tuberculous mycobacterial infection in congenital contractural arachnodactyly.  

UK PubMed Central (United Kingdom)

Congenital contractural arachnodactyly (CCA) is caused by mutations within the fibrillin-2 gene (FBN2), which is crucial for microfibril structure. Affected individuals may have contractures, chest wall deformities, scoliosis, abnormal ear folding and elongated limbs. We describe a novel FBN2 mutation in a woman with CCA who also had pulmonary non-tuberculous mycobacteria (NTM) infection. The population with pulmonary NTM infections shares phenotypic features with CCA, such as elongated body habitus, scoliosis and pectus deformities. While it is unlikely that FBN2 defects account for susceptibility to NTM infection in the majority of cases, the overlap between these two diseases suggests some shared pathophysiology.

Paulson ML; Olivier KN; Holland SM

2012-04-01

165

Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3? to 5? DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA...

Unciuleac, Mihaela-Carmen; Shuman, Stewart

166

Identification of drug susceptibility pattern and mycobacterial species in sputum smear positive pulmonary tuberculosis patients with and without HIV co-infection in north west Ethiopia  

DEFF Research Database (Denmark)

Ethiopia is among the high-burden countries of tuberculosis (TB) in the world Since mycobacterial culture and susceptibility testing are not routinely performed in Ethiopia, recent data on susceptibility patterns and the mycobacterial species cultured from sputum smear positive patients are limited.

Mekonen, Mekdem; Abate, Ebba

2010-01-01

167

Acute mycobacterial flexor tenosynovitis following accidental bacillus calmette-guerin inoculation in a health care worker: case report.  

UK PubMed Central (United Kingdom)

Solutions containing bacillus Calmette-Guérin (BCG), a live attenuated form of Mycobacterium bovis or Mycobacterium tuberculosis, commonly are injected intravesically to treat tumors of the urinary bladder. We report a case of acute mycobacterial flexor tenosynovitis in a health care worker who inadvertently inoculated her finger via needlestick while preparing BCG solution for intravesicular administration. She was treated successfully with immediate operative intervention followed by 6 months of antimycobacterial antibiotics. Of 3 previous reports of hand infections following self-inoculation with BCG solutions, this case is unique owing to rapid onset of acute mycobacterial flexor tenosynovitis and positive intraoperative mycobacterial cultures. Needlesticks with BCG-containing solutions, especially into the flexor tendon sheath, should be treated with timely surgical debridement and appropriate antimycobacterial management.

Mundinger GS; Douglas KC; Higgins JP

2013-02-01

168

Probing the interaction of the diarylquinoline TMC207 with its target mycobacterial ATP synthase.  

Science.gov (United States)

Infections with Mycobacterium tuberculosis are substantially increasing on a worldwide scale and new antibiotics are urgently needed to combat concomitantly emerging drug-resistant mycobacterial strains. The diarylquinoline TMC207 is a highly promising drug candidate for treatment of tuberculosis. This compound kills M. tuberculosis by binding to a new target, mycobacterial ATP synthase. In this study we used biochemical assays and binding studies to characterize the interaction between TMC207 and ATP synthase. We show that TMC207 acts independent of the proton motive force and does not compete with protons for a common binding site. The drug is active on mycobacterial ATP synthesis at neutral and acidic pH with no significant change in affinity between pH 5.25 and pH 7.5, indicating that the protonated form of TMC207 is the active drug entity. The interaction of TMC207 with ATP synthase can be explained by a one-site binding mechanism, the drug molecule thus binds to a defined binding site on ATP synthase. TMC207 affinity for its target decreases with increasing ionic strength, suggesting that electrostatic forces play a significant role in drug binding. Our results are consistent with previous docking studies and provide experimental support for a predicted function of TMC207 in mimicking key residues in the proton transfer chain and blocking rotary movement of subunit c during catalysis. Furthermore, the high affinity of TMC207 at low proton motive force and low pH values may in part explain the exceptional ability of this compound to efficiently kill mycobacteria in different microenvironments. PMID:21858172

Haagsma, Anna C; Podasca, Ioana; Koul, Anil; Andries, Koen; Guillemont, Jerome; Lill, Holger; Bald, Dirk

2011-08-17

169

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential di (more) agnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

Lima, Andrea Santos; Duarte, Rafael Silva; Montenegro, Lilian Maria Lapa; Schindler, Haiana Charifker

2013-07-01

170

Effect of Mycobacterial Drug Resistance Patterns on Patients’ Survival: A Cohort Study in Thailand  

Directory of Open Access Journals (Sweden)

Full Text Available Background: Drug resistance substantially increases tuberculosis (TB) mortality. This study aimed to describe the prevalence of mycobacterial drug resistance pattern and association of common resistance patterns with TB mortality in Thailand. Method: A retrospective cohort study was conducted using TB surveillance data. A total of 9,518 culture-confirmed, pulmonary TB patients registered from 1 October 2004 to 31 December 2008 from the Thailand TB Active Surveillance Network were included in this study. Patients were followed up until TB treatment completion or death. Mycobacterial drug resistance patterns were categorized as pan-susceptible, rifampicin resistance, isoniazid monoresistance, and ethambutol/streptomycin resistance. Drug susceptibility testing (DST) was determined by Mycobacterial Growth Indicator Tube (MGIT) liquid culture systems. Survival analysis was applied. Result: Isoniazid monoresistance was the most common pattern, while rifampicin resistance had the largest impact on mortality. Cox regression analysis showed a significantly higher risk of death among patients with rifampicin resistance (adjusted hazard ratio (aHR) 1.9, 95% confident interval (CI), 1.5-2.5) and isoniazid monoresistance (aHR 1.4, 95% CI 1.1-1.7) than those with pan-susceptible group after adjustment for age, nationality, human immunodeficiency virus (HIV) and antiretroviral therapy (ART) status, diabetes mellitus, cavitary disease on chest x-ray, treatment observation, and province. HIV co-infection was associated with higher mortality in patients both on ART (aHR 1.9, 95% CI 1.5-2.5) and not on ART (aHR 8.1, 95% CI 6.8-9.8). Conclusion: Rifampicin resistance and isoniazid monoresistance were associated with increased TB mortality. HIV-coinfection was associated with a higher risk of death including among those taking antiretroviral therapy.

Amornrat Anuwatnonthakate; Sara J. Whitehead; Jay K. Varma; Udomsak Silachamroon; Yuthichai Kasetjaroen; Saiyud Moolphate; Pranom Limsomboon; Jiraphun Inyaphong; Narin Suriyon; Suporn Kavinum; Navarat Chiengson; Phatchara Tunteerapat; Jaranit Kaewkungwal

2013-01-01

171

Impairment of mycobacterial but not viral immunity by a germline human STAT1 mutation.  

UK PubMed Central (United Kingdom)

Interferons (IFN) alpha/beta and gamma induce the formation of two transcriptional activators: gamma-activating factor (GAF) and interferon-stimulated gamma factor 3 (ISGF3). We report a natural heterozygous germline STAT1 mutation associated with susceptibility to mycobacterial but not viral disease. This mutation causes a loss of GAF and ISGF3 activation but is dominant for one cellular phenotype and recessive for the other. It impairs the nuclear accumulation of GAF but not of ISGF3 in heterozygous cells stimulated by IFNs. Thus, the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF.

Dupuis S; Dargemont C; Fieschi C; Thomassin N; Rosenzweig S; Harris J; Holland SM; Schreiber RD; Casanova JL

2001-07-01

172

Selectivity of TMC207 towards mycobacterial ATP synthase compared with that towards the eukaryotic homologue.  

Science.gov (United States)

The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC(50)] of >200 microM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC(50) of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target. PMID:19075053

Haagsma, Anna C; Abdillahi-Ibrahim, Rooda; Wagner, Marijke J; Krab, Klaas; Vergauwen, Karen; Guillemont, Jerome; Andries, Koen; Lill, Holger; Koul, Anil; Bald, Dirk

2008-12-15

173

Mycobacterial lesions in fish, amphibians, reptiles, rodents, lagomorphs, and ferrets with reference to animal models.  

UK PubMed Central (United Kingdom)

Mycobacteriosis is a serious disease across many animal species. Approximately more than 120 species are currently recognized in the genus Mycobacterium. This article describes the zoonotic potential of mycobacteria and mycobacteriosis in fish, amphibians, rodents, rabbits, and ferrets. It considers clinical signs; histology; molecular methods of identification, such as polymerase chain reaction and DNA sequencing; routes of infection; and disease progression. Studying the disease in animals may aid in understanding the pathogenesis of mycobacterial infections in humans and identify better therapy and preventative options such as vaccines.

Reavill DR; Schmidt RE

2012-01-01

174

Blood Neutrophil Counts in HIV-Infected Patients with Pulmonary Tuberculosis: Association with Sputum Mycobacterial Load.  

UK PubMed Central (United Kingdom)

BACKGROUND: Increasing evidence suggests that neutrophils play a role in the host response to Mycobacterium tuberculosis. We determined whether neutrophil counts in peripheral blood are associated with tuberculosis (TB) and with mycobacterial load in sputum in HIV-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: Adults enrolling in an antiretroviral treatment (ART) clinic in a Cape Town township were screened for TB regardless of symptoms. Paired sputum samples were examined using liquid culture, fluorescence microscopy, and the Xpert MTB/RIF assay. Absolute neutrophil counts (ANC) were measured in blood samples. Of 602 HIV-infected patients screened, 523 produced one or more sputum samples and had complete results available for analysis. Among these 523 patients, the median CD4 count was 169×10(9)/L (IQR, 96-232) and median ANC was 2.6×10(9)/L (IQR, 1.9-3.6). Culture-positive pulmonary tuberculosis was diagnosed in 89 patients. Patients with TB had a median ANC of 3.4×10(9)/L (IQR, 2.4-5.1) compared to 2.5×10(9)/L (IQR, 1.8-3.4) among those who were culture negative (p<0.0001). In multivariable analyses, having pulmonary TB was associated with an adjusted risk ratio (aRR) of 2.6 (95%CI, 1.5-4.5) for having an ANC level that exceeded the median value (ANC ?2.6×10(9)/L; p?=?0.0006) and an aRR of 6.8 (95%CI, 2.3-20.4) for having neutrophilia defined by a neutrophil count exceeding the upper limit of the normal range (ANC >7.5×10(9)/L; p?=?0.0005). Patients were then classified into four mutually exclusive groups with increasing sputum mycobacterial load as defined by the results of culture, Xpert MTB/RIF and sputum smear microscopy. Multivariable analyses demonstrated that increasing sputum mycobacterial load was positively associated with blood ANC ?2.6×10(9)/L and with neutrophilia. CONCLUSIONS/SIGNIFICANCE: Increased blood neutrophil counts were independently associated with pulmonary TB and sputum mycobacterial burden in this HIV-infected patient group. This observation supports the growing body of literature regarding the potential role for neutrophils in the host response to TB.

Kerkhoff AD; Wood R; Lowe DM; Vogt M; Lawn SD

2013-01-01

175

Permeabilization of the mycobacterial envelope for protein cytolocalization studies by immunofluorescence microscopy  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The establishment of the cellular localization of proteins in M. tuberculosis will provide of valuable information for the identification of new drug/vaccine/diagnostic targets. Cytolocalization by inmunofluorescence microscopy has been limited in mycobacteria because to difficulties in effectively permeabilize it. Results A treatment combining lysozyme with triton X-100 was found to be an effective permeabilization method of the mycobacterial envelope. Conclusion A rapid and simple permeabilization protocol has been successfully assessed in pure cultures of both Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. This method can be successful used in the cytolocalization of proteins by immunolabeling.

Cimino Mena; Alamo Lorenzo; Salazar Leiria

2006-01-01

176

Mycobacterium arosiense sp. nov., a slowly growing, scotochromogenic species causing osteomyelitis in an immunocompromised child  

DEFF Research Database (Denmark)

A yellow-pigmented, scotochromogenic, slowly growing mycobacterial strain, designated T1921(T), was isolated from the disseminated osteomyelitic lesions of a 7-year-old child with an underlying partial gamma interferon receptor alpha-1 deficiency. Hybridization by the line probe assay indicated the presence of a Mycobacterium species. Sequencing of the 16S rRNA gene, the internally transcribed spacer (ITS) region and the hsp65 and rpoB genes revealed that strain T1921(T) could be differentiated from all recognized species of the genus Mycobacterium. Phylogenetic analysis based on the 16S rRNA gene indicated that strain T1921(T) was related most closely to Mycobacterium intracellulare, whereas analysis based on the ITS and hsp65 and rpoB genes indicated that it was most closely related to Mycobacterium avium. Phenotypic tests were not able to differentiate strain T1921(T) from similar slowly growing mycobacteria. Strain T1921(T) is considered to represent a novel species of the genus Mycobacterium, for which the name Mycobacterium arosiense sp. nov. is proposed. The type strain is T1921(T) (=DSM 45069(T) =ATCC BAA-1401(T)) Udgivelsesdato: 2008/10

Bang, D.; Herlin, T.

2008-01-01

177

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

Scientific Electronic Library Online (English)

Full Text Available 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.

Castaño, Diana; Rojas, Mauricio

2010-06-01

178

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

Directory of Open Access Journals (Sweden)

Full Text Available 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 endosomas 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.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 nutrients 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.

Diana Castaño; Mauricio Rojas

2010-01-01

179

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

Energy Technology Data Exchange (ETDEWEB)

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.

Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

1988-08-01

180

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

International Nuclear Information System (INIS)

[en] 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

1988-01-01

 
 
 
 
181

Mycobacterial spindle cell pseudotumor of the appendix vermiformis in a patient with AIDS  

Directory of Open Access Journals (Sweden)

Full Text Available Mycobacterial pseudotumor (MP) is a rare pathologic presentation of both Mycobacterium tuberculosis and non-tuberculous mycobacterial disease, hitherto reported to occur only in immunosuppressed patients with or without human immunodeficiency virus infection. This lesion shares close pathologic resemblance to certain mesenchymal neoplasms, particularly Kaposi's sarcoma (KS), from which it must be properly differentiated due to distinct prognosis and therapy. We report a case of MP obliterating the lumen of the appendix vermiformis in a 34-year-old patient who died of complications of AIDS at our hospital in Rio de Janeiro. A total of 24 cases of MP (including our patient) have been described in the literature. MP has been found especially in lymph nodes, but extranodal lesions have been described in the skin, spleen, lung, bone marrow, brain and, in our patient, the appendix vermiformis. We offer a review of the other 23 published case reports of MP in both HIV-infected and uninfected patients and discuss the pathologic features that differentiate MP from KS.

Carlos Alberto Basílio-de-Oliveira; Walter A. Eyer-Silva; Heliomar de Azevedo Valle; Ana Lúcia Rodrigues; Ana Luisa Pinheiro Pimentel; Carlos Alberto Morais-de-Sá

2001-01-01

182

Mycobacterial spindle cell pseudotumor of the appendix vermiformis in a patient with AIDS  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english Mycobacterial pseudotumor (MP) is a rare pathologic presentation of both Mycobacterium tuberculosis and non-tuberculous mycobacterial disease, hitherto reported to occur only in immunosuppressed patients with or without human immunodeficiency virus infection. This lesion shares close pathologic resemblance to certain mesenchymal neoplasms, particularly Kaposi's sarcoma (KS), from which it must be properly differentiated due to distinct prognosis and therapy. We report a c (more) ase of MP obliterating the lumen of the appendix vermiformis in a 34-year-old patient who died of complications of AIDS at our hospital in Rio de Janeiro. A total of 24 cases of MP (including our patient) have been described in the literature. MP has been found especially in lymph nodes, but extranodal lesions have been described in the skin, spleen, lung, bone marrow, brain and, in our patient, the appendix vermiformis. We offer a review of the other 23 published case reports of MP in both HIV-infected and uninfected patients and discuss the pathologic features that differentiate MP from KS.

Basílio-de-Oliveira, Carlos Alberto; Eyer-Silva, Walter A.; Valle, Heliomar de Azevedo; Rodrigues, Ana Lúcia; Pimentel, Ana Luisa Pinheiro; Morais-de-Sá, Carlos Alberto

2001-04-01

183

Cytokines in the balance of protection and pathology during mycobacterial infections.  

UK PubMed Central (United Kingdom)

The outcome of natural infections with pathogenic mycobacteria can range from early asymptomatic clearance through latent infection to clinical disease. Different host and pathogen-specific factors have been implicated in determining the outcome of these infections; however, it is clear that the interaction of mycobacteria with the innate and acquired components of the immune system plays a central role. Specifically, the recognition of mycobacterial components by innate immune cells through different pathogen recognition receptors (PPRs) induces a cytokine response that can promote early control of the infection. In fact, in the majority of individuals that come into contact with mycobacteria, this response is enough to control the infection. Among PRRs, Toll-like receptors (TLRs), Nucleotide Oligomerization Domain (NOD)-like receptors, and C-type lectins have all been implicated in recognition of mycobacteria and in the initiation of the cytokine response. Defining the mechanisms by which distinct mycobacterial components and their receptors stimulate the immune response is an area of intense research.

Torrado E; Cooper AM

2013-01-01

184

Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage  

Science.gov (United States)

Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn2+ fingers from the mycobacterial topoI could be associated with Zn2+ export and homeostasis.

Ahmed, Wareed; Bhat, Anuradha Gopal; Leelaram, Majety Naga; Menon, Shruti; Nagaraja, Valakunja

2013-01-01

185

Epithelial G protein-coupled receptor kinases regulate the initial inflammatory response during mycobacterial infection.  

UK PubMed Central (United Kingdom)

The interaction between mycobacteria and epithelium is unexplored, but may determine the outcome of the infection. We have analyzed the role of two G protein-coupled receptors, CXCR1 and CXCR2 that are important regulators of many pulmonary diseases. We found that mycobacteria significantly increased the expression of both CXCR1 and CXCR2 on alveolar epithelial cells and both receptors were found to be important for neutrophil diapedesis across primary endothelial cells towards infected mucosa. Mycobacteria, lipoarabinomannan or 19-kDa glycolipoprotein up-regulated the inhibitory G protein-coupled receptor kinase (GRK)2, while GRK3 was less affected. Mycobacteria-induced GRK2 up-regulation decreased chemokine transcription and secretion thereby affecting the neutrophil recruitment to infected mucosa. These events were completely abolished by blocking these receptors prior to infection as the blocking increased epithelial immune responses. We have identified novel interactions occurring in the initial phase of mycobacterial infections by which mycobacterial manipulate epithelial inflammatory responses.

Håkansson G; Lutay N; Andersson M; Hallgren O; Westergren-Thorsson G; Svensson M; Godaly G

2013-07-01

186

Assay development for identifying inhibitors of the mycobacterial FadD32 activity.  

Science.gov (United States)

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of T m (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors. PMID:23364516

Galandrin, Ségolène; Guillet, Valérie; Rane, Rajendra S; Léger, Mathieu; N, Radha; Eynard, Nathalie; Das, Kaveri; Balganesh, Tanjore S; Mourey, Lionel; Daffé, Mamadou; Marrakchi, Hedia

2013-01-30

187

Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1?C expression strain  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1. Results In order to improve purification efficiency and yield of polyhistidine-tagged mycobacterial target proteins, we created a mutant version of GroEL1 by removing the coding sequence for the histidine-rich C-terminus, termed GroEL1?C. GroEL1?C, which is a functional protein, is no longer able to bind nickel affinity beads. Using a selection of challenging test proteins, we show that GroEL1?C is no longer present in protein samples purified from the groEL1?C expression strain and demonstrate the feasibility and advantages of purifying and characterising proteins produced using this strain. Conclusions This novel Mycobacterium smegmatis expression strain allows efficient expression and purification of mycobacterial proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification.

Noens Elke E; Williams Chris; Anandhakrishnan Madhankumar; Poulsen Christian; Ehebauer Matthias T; Wilmanns Matthias

2011-01-01

188

The Internal Organization of Mycobacterial Partition Assembly: Does the DNA Wrap a Protein Core?  

Science.gov (United States)

Before cell division in many bacteria, the ParBs spread on a large segment of DNA encompassing the origin-proximal parS site(s) to form the partition assembly that participates in chromosome segregation. Little is known about the structural organization of chromosomal partition assembly. We report solution X-ray and neutron scattering data characterizing the size parameters and internal organization of a nucleoprotein assembly formed by the mycobacterial chromosomal ParB and a 120-meric DNA containing a parS-encompassing region from the mycobacterial genome. The cross-sectional radii of gyration and linear mass density describing the rod-like ParB-DNA assembly were determined from solution scattering. A “DNA outside, protein inside” mode of partition assembly organization consistent with the neutron scattering hydrogen/deuterium contrast variation data is discussed. In this organization, the high scattering DNA is positioned towards the outer region of the partition assembly. The new results presented here provide a basis for understanding how ParBs organize the parS-proximal chromosome, thus setting the stage for further interactions with the DNA condensins, the origin tethering factors and the ParA.

Qian, Shuo; Dean, Rebecca; Urban, Volker S.; Chaudhuri, Barnali N.

2012-01-01

189

Identification and structural characterization of an unusual mycobacterial monomeromycolyl-diacylglycerol.  

Science.gov (United States)

Systematic thin layer chromatographic (TLC) analysis of apolar lipids in Mycobacterium kansasii revealed the presence of a previously uncharacterized novel component. The product was ubiquitously found in a panel of M. kansasii clinical isolates, as well as other pathogenic and non-pathogenic mycobacterial species. TLC analysis of [(14)C]-acetate- or [(14)C]-glycerol-labelled M. kansasii cultures tentatively assigned the novel product as an unusual triacylglycerol-related lipid. Subsequent purification, followed by structural determination using (1)H-nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ES/MS), led to the identification of this product as a monomeromycolyl-diacylglycerol (MMDAG). Treatment of M. kansasii with either isoniazid (INH), a well-known type II fatty acid synthase (FAS-II) and mycolic acid biosynthesis inhibitor, or tetrahydrolipstatin (THL), a drug approved for treating obesity, correlated with a reduced incorporation of [(14)C]-acetate into both mycolic acids and MMDAG. Addition of INH or THL to the cultures induced major morphological changes and, surprisingly, resulted in an increased number of lipid storage bodies, as determined by electron microscopy. The potent antimycobacterial activity of THL was confirmed against a variety of mycobacterial species, including INH-susceptible and -resistant Mycobacterium tuberculosis strains. Therefore, THL and other beta-lactones may be promising drugs for the development of new antitubercular therapy. PMID:16091048

Kremer, Laurent; de Chastellier, Chantal; Dobson, Gary; Gibson, Kevin J C; Bifani, Pablo; Balor, Stéphanie; Gorvel, Jean-Pierre; Locht, Camille; Minnikin, David E; Besra, Gurdyal S

2005-08-01

190

An Inducible System for the Identification of Target Genes for a Regulator in Mycobacteria  

Directory of Open Access Journals (Sweden)

Full Text Available We described principle and application of an inducible system for identification of target genes of a given mycobacterial protein with a regulatory function. This vector system, a promoter-probe vector, carries (i) promoterless lacZ as the reporter gene and (ii) the mycobacterial gene encoding the regulatory protein under the transcriptional control of the promoter of highly inducible mycobacterial gene encoding acetamidase. A promoter library of M.tuberculosis is constructed in this vector upstream of the lacZ gene. It is then possible to screen for those promoters that are responsive to the presence of the regulatory protein by inducing the expression of the regulatory gene. The presence of lacZ permits the screening of the promoters based on simple blue-white selection. This system is specifically designed for those regulatory genes of M.tuberculosis which are associated with virulence and thus are absent from M.smegmatis (the non-pathogenic, saprophytic species of mycobacteria), although in principle, modifications can be incorporated in the selection scheme to make it applicable for the identification of target promoter(s) of any regulatory gene of mycobacterial origin. This strategy will be helpful in quick identification of targets for the development of anti-tubercular drugs and in alleviating some of the stumbling blocks faced by the investigators working in the area of molecular genetics of M.tuberculosis.

Shruti Jain; Garima Khare; Pushplata Tripathi; Anil K. Tyagi

2008-01-01

191

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical character...

Unciuleac, Mihaela-Carmen; Shuman, Stewart

192

Effect of intestinal resection on serum antibodies to the mycobacterial 45/48 kilodalton doublet antigen in Crohn's disease.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Interest in the role of mycobacterial infection in Crohn's disease has been revived by the cultural detection of Mycobacterium paratuberculosis in patients with Crohn's disease. This hypothesis was examined serologically using assays with high specificity for Crohn's disease. The effect of intestina...

Kreuzpaintner, G; Das, P K; Stronkhorst, A; Slob, A W; Strohmeyer, G

193

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis? †  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the com...

Supply, Philip; Allix, Caroline; Lesjean, Sarah; Cardoso-Oelemann, Mara; Rüsch-Gerdes, Sabine; Willery, Eve; Savine, Evgueni

194

Rhomboids of Mycobacteria: characterization using an aarA mutant of Providencia stuartii and gene deletion in Mycobacterium smegmatis.  

UK PubMed Central (United Kingdom)

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.

Kateete DP; Katabazi FA; Okeng A; Okee M; Musinguzi C; Asiimwe BB; Kyobe S; Asiimwe J; Boom WH; Joloba ML

2012-01-01

195

Support Vector Machine-based method for predicting subcellular localization of mycobacterial proteins using evolutionary information and motifs  

Science.gov (United States)

Background In past number of methods have been developed for predicting subcellular location of eukaryotic, prokaryotic (Gram-negative and Gram-positive bacteria) and human proteins but no method has been developed for mycobacterial proteins which may represent repertoire of potent immunogens of this dreaded pathogen. In this study, attempt has been made to develop method for predicting subcellular location of mycobacterial proteins. Results The models were trained and tested on 852 mycobacterial proteins and evaluated using five-fold cross-validation technique. First SVM (Support Vector Machine) model was developed using amino acid composition and overall accuracy of 82.51% was achieved with average accuracy (mean of class-wise accuracy) of 68.47%. In order to utilize evolutionary information, a SVM model was developed using PSSM (Position-Specific Scoring Matrix) profiles obtained from PSI-BLAST (Position-Specific Iterated BLAST) and overall accuracy achieved was of 86.62% with average accuracy of 73.71%. In addition, HMM (Hidden Markov Model), MEME/MAST (Multiple Em for Motif Elicitation/Motif Alignment and Search Tool) and hybrid model that combined two or more models were also developed. We achieved maximum overall accuracy of 86.8% with average accuracy of 89.00% using combination of PSSM based SVM model and MEME/MAST. Performance of our method was compared with that of the existing methods developed for predicting subcellular locations of Gram-positive bacterial proteins. Conclusion A highly accurate method has been developed for predicting subcellular location of mycobacterial proteins. This method also predicts very important class of proteins that is membrane-attached proteins. This method will be useful in annotating newly sequenced or hypothetical mycobacterial proteins. Based on above study, a freely accessible web server TBpred http://www.imtech.res.in/raghava/tbpred/ has been developed.

2007-01-01

196

Greater Preexisting Interferon ? Responses to Mycobacterial Antigens and Lower Bacillary Load During HIV-Associated Tuberculosis.  

Science.gov (United States)

The role of preexisting interferon (IFN) ? responses in controlling bacillary burden in human immunodeficiency virus (HIV)-associated tuberculosis is not known. Among BCG-immunized HIV-infected adults who developed tuberculosis in a phase III trial of an investigational tuberculosis vaccine, greater baseline IFN-? responses to early secretory antigenic target 6 and Mycobacterium tuberculosis whole-cell lysate were associated with reduced bacillary burden on sputum smear grade, days to culture positivity on agar, and sputum culture grade during subsequent tuberculosis. This association was most consistent among recipients of the investigational vaccine. When HIV-associated tuberculosis develops, greater preexisting IFN-? responses to mycobacterial antigens are associated with reduced tuberculosis bacillary burden. ClinicalTrials.gov Identifier.?NCT0052195. PMID:23908490

Lahey, Timothy; Czechura, Tom; Crabtree, Scott; Arbeit, Robert D; Matee, Mecky; Horsburgh, C Robert; Mackenzie, Todd; Bakari, Muhammad; Pallangyo, Kisali; von Reyn, C Fordham

2013-08-01

197

Non-tuberculous mycobacterial infection of the parotid gland in an immunocompetent elderly patient.  

Science.gov (United States)

We experienced an extremely rare case in which combined antibacterial therapy for a non-tuberculous mycobacteria (NTM) infection of the parotid gland achieved a favourable outcome in an elderly immunocompetent patient. Although a 79-year-old man, who presented with swelling and fistula formation in the left parotid gland region, initially received combined antituberculous therapy due to a positive result of acid-fast staining, the lesion did not respond to these agents. Thereafter, since the culture examination did not detect Mycobacterium tuberculosis or NTM, we excluded tuberculosis and considered the possibility of an NTM infection caused by a rare mycobacterial species. Therefore, we switched to the clarithromycin-based antibacterial treatment for eight consecutive months without a surgical intervention, resulting in the complete disappearance of the lesion and no evidence of recurrence detected for 4 years. This conservative chemotherapy might be a feasible alternative to a surgical intervention for treatment against NTM infections of the parotid gland. PMID:24132446

Yamanaka, Toshiaki; Okamoto, Hideyuki; Hosoi, Hiroshi

2013-10-16

198

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria.  

Science.gov (United States)

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+ fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme. PMID:19564134

Leelaram, Majety Naga; Bhat, Anuradha Gopal; Suneetha, Nunna; Nagaraja, Valakunja; Manjunath, Ramanathapuram

2009-06-28

199

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria.  

UK PubMed Central (United Kingdom)

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+ fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.

Leelaram MN; Bhat AG; Suneetha N; Nagaraja V; Manjunath R

2009-07-01

200

Exposure to a cutinase-like serine esterase triggers rapid lysis of multiple mycobacterial species.  

UK PubMed Central (United Kingdom)

Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.

Yang Y; Bhatti A; Ke D; Gonzalez-Juarrero M; Lenaerts A; Kremer L; Guerardel Y; Zhang P; Ojha AK

2013-01-01

 
 
 
 
201

Selective adsorption of europium(III) and americium(III) in non-proliferative mycobacterial suspensions  

International Nuclear Information System (INIS)

[en] The absorption effect of 5% w/w non-proliferative cell suspensions of Mycobacterium smegmatis on labelled solutions of Eu3+ ions, both alone and in mixtures with Am3+, Co2+ and Cs+, was studied at pH 1.0 as a function of time and cationic concentration. For 10 ?M concentrations of Eu, Co and Cs, selective adsorption of the trivalent lanthanide and actinide ions was practically quantitative after 90 min; no significant adsorption was observed for cobalt and cesium ions. Column adsorption measurements with the mycobacterial biomass showed that desorption of the M3+ ions did not occur at less than 2M HCl and remained incomplete even at higher acidities. (author) 7 refs.; 2 figs.; 2 tabs

1989-03-16

202

Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species  

International Nuclear Information System (INIS)

Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on 14CO2 evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of 14CO2 evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or ''no viable organisms'' within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

1984-01-01

203

Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species  

Energy Technology Data Exchange (ETDEWEB)

Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on /sup 14/CO/sub 2/ evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of /sup 14/CO/sub 2/ evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or ''no viable organisms'' within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

Siddiqi, S.H.; Hwangbo, C.C.; Silcox, V.; Good, R.C.; Snider, D.E. Jr.; Middlebrook, G.

1984-10-01

204

Patients with nontuberculous mycobacterial lung disease exhibit unique body and immune phenotypes.  

UK PubMed Central (United Kingdom)

RATIONALE: Among patients with nontuberculous mycobacterial lung disease is a subset of previously healthy women with a slender body morphotype, often with scoliosis and/or pectus excavatum. We hypothesize that unidentified factors predispose these individuals to pulmonary nontuberculous mycobacterial disease. OBJECTIVES: To compare body morphotype, serum adipokine levels, and whole-blood cytokine responses of patients with pulmonary nontuberculous mycobacteria (pNTM) with contemporary control subjects who are well matched demographically. METHODS: We enrolled 103 patients with pNTM and 101 uninfected control subjects of similar demographics. Body mass index and body fat were quantified. All patients with pNTM and a subset of control subjects were evaluated for scoliosis and pectus excavatum. Serum leptin and adiponectin were measured. Specific cytokines important to host-defense against mycobacteria were measured in whole blood before and after stimulation. MEASUREMENTS AND MAIN RESULTS: Patients with pNTM and control subjects were well matched for age, gender, and race. Patients with pNTM had significantly lower body mass index and body fat and were significantly taller than control subjects. Scoliosis and pectus excavatum were significantly more prevalent in patients with pNTM. The normal relationships between the adipokines and body fat were lost in the patients with pNTM, a novel finding. IFN-? and IL-10 levels were significantly suppressed in stimulated whole blood of patients with pNTM. CONCLUSIONS: This is the first study to comprehensively compare body morphotype, adipokines, and cytokine responses between patients with NTM lung disease and demographically matched controls. Our findings suggest a novel, predisposing immunophenotype that should be mechanistically defined.

Kartalija M; Ovrutsky AR; Bryan CL; Pott GB; Fantuzzi G; Thomas J; Strand MJ; Bai X; Ramamoorthy P; Rothman MS; Nagabhushanam V; McDermott M; Levin AR; Frazer-Abel A; Giclas PC; Korner J; Iseman MD; Shapiro L; Chan ED

2013-01-01

205

Extrapulmonary mycobacterial infections in a cohort of HIV-positive patients: ultrasound experience from Vicenza, Italy.  

UK PubMed Central (United Kingdom)

PURPOSE: Extrapulmonary tuberculosis (EPTB) is frequently seen in human immunodeficiency virus (HIV)-infected individuals in Sub-Saharan Africa and recent work has shown point-of-care (POC) ultrasound to be a diagnostic aid in the resource-limited, highly endemic setting. Its role in industrialized countries, however, has rarely been studied. With international migration, EPTB is increasingly seen in European hospitals. This study reports ultrasound findings and discusses the diagnostic relevance of EPTB in an industrialized country setting. METHODS: In this retrospective study, we describe a cohort of 27 patients with a predominantly immigrant background diagnosed with HIV and EPTB in Northern Italy and evaluate the role of ultrasound in their clinical management. All inpatient files of HIV-positive individuals admitted to our hospital with culture-proven diagnosis of EPTB were reviewed, along with chest X-rays and ultrasound studies. The outcome and results of long-term follow-up were extracted. RESULTS: A total of 243 HIV-positive inpatients were identified between January 2005 and November 2011. Twenty-seven of the patients [11.1 %, 95 % confidence interval (CI) 7.4-15.7] were diagnosed with EPTB. Ultrasound showed a typical pattern of enlarged abdominal lymph nodes and focal lesions in the spleen and liver in 22 patients (81.5 %, 95 % CI 7.4-15.7) and, thus, helped to raise the suspicion of mycobacterial infection. CONCLUSION: As disseminated mycobacterial infections in HIV-positive patients can be treated effectively if diagnosed early, and typical sonographic findings are seen in the majority of these patients, we suggest that POC ultrasound should be integrated in diagnostic and screening algorithms for EPTB in developed countries.

Giordani MT; Brunetti E; Binazzi R; Benedetti P; Stecca C; Goblirsch S; Heller T

2013-04-01

206

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

UK PubMed Central (United Kingdom)

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 1.56 ?g/mL. Neamphamide B (1) was also effective to Mycobacterium bovis BCG with MIC in the ranging of 6.25-12.5 ?g/mL.

Yamano Y; Arai M; Kobayashi M

2012-07-01

207

Antigen Specificity in Experimental Bovine Tuberculosis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This report describes the kinetics of T-cell responses to a panel of mycobacterial antigens (PPD-M, PPD-A, ESAT-6, Ag85, 38kD, MPB64, MPB70, MPB83, hsp16.1, hsp65, and hsp70) following experimental infection of cattle with Mycobacterium bovis. Increased antigen-specific lymphocyte proliferation, gam...

Rhodes, S. G.; Gavier-Widen, D.; Buddle, B. M.; Whelan, A. O.; Singh, M.; Hewinson, R. G.; Vordermeier, H. M.

208

Detection of Mycobacterium bovis and Mycobacterium tuberculosis from Cattle: Possible Public Health Relevance  

DEFF Research Database (Denmark)

Mycobacterium bovis and Mycobacterium tuberculosis infect both animals and humans. The disease epidemiology by these agents differs in developed and developing countries due to the differences in the implementation of the prevention and control strategies. The present study describes the detection of M. bovis and M. tuberculosis from specimens of lungs and pulmonary lymph nodes of four cattle died in an organized herd of 183 cattle in the state of Himachal Pradesh, India, with inconclusive skin test results. Identification and distinction of these closely related mycobacterial species was done by PCR-RFLP targeting hsp65 gene followed by spacer oligonucleotide typing. Mixed infection of M. bovis and M. tuberculosis was detected in one cattle.

Thakur, Aneesh; Sharma, Mandeep

2012-01-01

209

Disseminated infection due to Mycobacterium avium subsp. avium in an Asian elephant (Elephas maximus).  

UK PubMed Central (United Kingdom)

A disseminated infection caused by Mycobacterium avium subspecies avium (MAA) was diagnosed in a 57-yr-old male Asian elephant (Elephas maximus) housed at the Seoul Zoo, Gyeonggi, Republic of Korea. An apparent granulomatous inflammation with central caseous necrosis was evident in the lung sections. To confirm mycobacterial infection, polymerase chain reaction-restriction enzyme polymorphism analysis (PCR-RFLP) of the rpoB and hsp65 genes was performed from multiple organs and cultured bacteria. The PCR-RFLP revealed a M. avium subspecies. MAA was identified by multiplex PCR for detection of IS901 and IS1311. Thus, it is believed that MAA caused the disseminated infection in this case. Although the source of infection was not determined, the elephant may have become infected through contamination of soil and feed by free-living birds infected with MAA. This is the first reported case of disseminated infection due to MAA in a captive elephant in the Republic of Korea.

Yong H; Choi GE; Lee BS; Whang J; Shin SJ

2011-12-01

210

Síndromes micobacterianos felinos y su importancia en la salud pública - Feline mycobacterial syndromes and its importance to public health  

Directory of Open Access Journals (Sweden)

Full Text Available ResumenEn felinos domésticos las especies del género Mycobacterium causan tres síndromes: tuberculosis ocasionada por el complejo M. tuberculosis, lepra felina por M. lepraemurium y micobacteriosis atípica causada por varias especies de micobacterias no tuberculosas y no lepromatosas. SummaryIn domestic felines three mycobacterial syndromes are described:tuberculoses caused by M. tuberculosis complex, feline leprosy caused by M. lepraemurium and atypical mycobacteriosis caused by non tuberculous and non lepromatous mycobacteria.

Jorge, María Cristina; Traversa, María Julia; Schettino, Daniel Mateo; Sanz, Horacio

2010-01-01

211

Síndromes micobacterianos felinos y su importancia en la salud pública - Feline mycobacterial syndromes and its importance to public health  

Directory of Open Access Journals (Sweden)

Full Text Available ResumenEn felinos domésticos las especies del género Mycobacterium causan tres síndromes: tuberculosis ocasionada por el complejo M. tuberculosis, lepra felina por M. lepraemurium y micobacteriosis atípica causada por varias especies de micobacterias no tuberculosas y no lepromatosas.AbstractIn domestic felines three mycobacterial syndromes are described: tuberculoses caused by M. tuberculosis complex, feline leprosy caused by M. lepraemurium and atypical mycobacteriosis caused by non tuberculous and non lepromatous mycobacteria.

Jorge, María Cristina; Traversa, María Julia; Schettino, Daniel Mateo; Sanz Horacio

2012-01-01

212

Haliclonacyclamines, tetracyclic alkylpiperidine alkaloids, as anti-dormant mycobacterial substances from a marine sponge of Haliclona sp.  

Science.gov (United States)

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 microg/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 microg/ml. PMID:19801875

Arai, Masayoshi; Ishida, Shunsuke; Setiawan, Andi; Kobayashi, Motomasa

2009-10-01

213

Haliclonacyclamines, tetracyclic alkylpiperidine alkaloids, as anti-dormant mycobacterial substances from a marine sponge of Haliclona sp.  

UK PubMed Central (United Kingdom)

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 microg/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 microg/ml.

Arai M; Ishida S; Setiawan A; Kobayashi M

2009-10-01

214

Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling.  

Science.gov (United States)

Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose-capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-gamma) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine production. PMID:15666089

Shabaana, A K; Kulangara, K; Semac, I; Parel, Y; Ilangumaran, S; Dharmalingam, K; Chizzolini, C; Hoessli, D C

2005-01-01

215

Increased serum anti-mycobacterial antibody titers in rheumatoid arthritis patients: Is there any specific antigenic target?  

International Nuclear Information System (INIS)

Objective was to investigate the presence of immunoreactivity against mycobacterial antigens in the sera of patients with rheumatoid arthritis (Ra) and to detect the target of the immune reaction. This study was carried out on 60 patients with RA, and 25 patients with no joint diseases in the laboratory of Clinical Microbiology Department of Ankara University Medical Faculty, Ankara, Turkey between July 2003 to January 2004. Secreted and cellular antigens of Mycobacterium tuberculosis (M. tuberculosis) H37Rv and Mycobacterium bovis (M. bovis) were isolated and purified by high performance liquid chromatography to antigenic fractions. The immunoreactivity of patient and control sera against these antigens were determined by enzyme-linked immunosorbent assay (ELISA). Immunoreactivity against mycobacterial antigens in RA patients were significantly higher than controls. Significant difference between patients and controls has been determined with M. bovis Bacillus Calmette Guerin (BCG) culture fluid and sonicate antigens, but not with M. tuberculosis H37Rv. This suggests that the antigen triggering immune response in patients with RA may belong to or mainly expressed on M. bovis BCG. The ELISA results showed significant difference between RA patients and controls with all antigenic fractions. Presence of increased immunoreactivity against mycobacterial antigens in the sera of patients with RA was detected. When statistical analysis was considered, we cannot put forward any antigenic fraction alone as the one responsible for the increased reactivity. (author)

2007-01-01

216

Anti-mycobacterial activity of root and leaf extracts of Anthocleista djalonensis (Loganiaceae) and Diospyros mespiliformis (Ebenaceae)  

Directory of Open Access Journals (Sweden)

Full Text Available We screened the aqueous and methanol leaf and root extracts of Anthocleista djalonensis, Diospyros mespiliformis, and their combinations for possible anti-mycobacterial activities using Mycobacterium smegmatis as a surrogate screen. These plants are reputed among folk practices as potent remedy in the management of tuberculosis and leprosy cases. In the sensitivity screening study, only the methanol extracts of A. djalonensis and D. mespiliformis showed anti-mycobacterial activity, while the aqueous extracts exhibited no inhibitory activity on M. smegmatis. The minimum inhibitory concentration (MIC) of the methanol leaf and root extract of A. djalonensis against M. smegmatis were 125 ?g/ml. The MIC of the methanol leaf and root extracts of D. mespiliformis is 167 and 250 ?g/ml, respectively. In the interaction studies, four out of nine decimal combinations of the two medicinal plant extracts exhibited synergism with fractional inhibitory concentration indices < 1 and a negative activity index values. The 8:2 ratio of D. mespiliformis and A. djalonensis exhibited the greatest degree of antimycobacterial synergy against M. smegmatis. The result of this study supports the claims of efficacy reported in the folk use of these plants in mycobacterial infection and the plants could therefore be investigated further and harnessed as potent antimycobacterial agents.

Esimone Charles; Nworu Chukwuemeka; Onuigbo Ebere; Omeje Justina; Nsirim Kelechi; Ogbu Joy; Ngwu Maria; Chah Kennedy

2009-01-01

217

Comparison of Three Methods for Rapid Identification of Mycobacterial Clinical Isolates to the Species Level?  

Science.gov (United States)

A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of Mycobacterium species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the Mycobacterium genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding Mycobacterium species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from M. intracellulare reference strains had a negative hybridization signal with probe c, which is specific for M. intracellulare. The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria.

Wu, Xueqiong; Zhang, Junxian; Liang, Jianqin; Lu, Yang; Li, Hongmin; Li, Chuihuan; Yue, Jun; Zhang, Lishui; Liu, Zhihui

2007-01-01

218

Comparison of three methods for rapid identification of mycobacterial clinical isolates to the species level.  

Science.gov (United States)

A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of Mycobacterium species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the Mycobacterium genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding Mycobacterium species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from M. intracellulare reference strains had a negative hybridization signal with probe c, which is specific for M. intracellulare. The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria. PMID:17360840

Wu, Xueqiong; Zhang, Junxian; Liang, Jianqin; Lu, Yang; Li, Hongmin; Li, Chuihuan; Yue, Jun; Zhang, Lishui; Liu, Zhihui

2007-03-14

219

Comparison of three methods for rapid identification of mycobacterial clinical isolates to the species level.  

UK PubMed Central (United Kingdom)

A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of Mycobacterium species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the Mycobacterium genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding Mycobacterium species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from M. intracellulare reference strains had a negative hybridization signal with probe c, which is specific for M. intracellulare. The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria.

Wu X; Zhang J; Liang J; Lu Y; Li H; Li C; Yue J; Zhang L; Liu Z

2007-06-01

220

A novel anti-mycobacterial function of mitogen-activated protein kinase phosphatase-1  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Mycobacterium tuberculosis (MTB) is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-? (TNF-?), which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin (BCG), a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-? via mitogen-activated protein kinase (MAPK) in human blood monocytes. Since MAPK phosphatase-1 (MKP-1) is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. Results Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK (p38 MAPK and ERK1/2) and TNF-? inducible by BCG. Conclusions Since TNF-? is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity.

Cheung Benny KW; Yim Howard CH; Lee Norris CM; Lau Allan SY

2009-01-01

 
 
 
 
221

The effect of endotoxin and endotoxin tolerance on inflammation induced by mycobacterial adjuvant.  

UK PubMed Central (United Kingdom)

Peptidoglycan, the substance in mycobacteria thought to be responsible for inducing adjuvant arthritis, and endotoxin (lipopolysaccharide or LPS) share many inflammatory properties. Since repeated administration of LPS produces tolerance, i.e., resistance to the toxic and inflammatory effects of LPS, we tested whether LPS and/or LPS tolerance might influence inflammation due to mycobacterial adjuvant. Male Sprague-Dawley rats were injected with Escherichia coli LPS or saline intraperitoneally and then challenged with 100 micrograms killed Mycobacteria butyricum (adjuvant) in the footpad. A single dose of 100 micrograms LPS three or 24 hours before adjuvant markedly, but transiently, reduced the local footpad swelling that begins within hours of the adjuvant injection and histologically resembles a sterile abscess. Animals that received multiple doses of LPS and were therefore tolerant or animals that received LPS 72 hours before adjuvant demonstrated adjuvant-induced footpad swelling nearly equal to controls. The anti-inflammatory effect of LPS was transient since footpad swelling in all groups was nearly comparable six days after the adjuvant injection and LPS failed to inhibit consistently the arthritis that develops two or more weeks after adjuvant injection. These studies establish that LPS can markedly inhibit the prodrome of adjuvant arthritis (footpad swelling due to M. butyricum), that inhibition of this prodrome does not prevent the subsequent development of arthritis, and that LPS tolerance diminishes this anti-inflammatory effect of LPS.

Rosenbaum JT; Mandell RB

1983-07-01

222

Utility of mycobacterial interspersed repetitive unit typing for differentiating Mycobacterium tuberculosis isolates in Wuhan, China.  

UK PubMed Central (United Kingdom)

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis, and may represent a feasible approach to study M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating M. tuberculosis strains, isolates from 105 patients in Wuhan City, China, were genotyped by this method as compared to spoligotyping. MIRU typing identified 55 types that defined 21 clusters and 34 unique isolates. The discriminatory power was high [Hunter-Gaston discriminatory index (HGDI), 0.97]. Spoligotyping showed that 86 (81.9 %) of 105 isolates belonged to the Beijing family genotype. For Beijing family and non-Beijing strains, the discriminatory power of MIRU was high (HGDI, 0.95 and 0.98, respectively). Among the alleles of the MIRU loci for the Beijing family, only locus 26 was highly discriminative, but for non-Beijing strains, loci 10, 16 and 26 were highly discriminative. MIRU typing is a simple and fast method which may be used for preliminary screening of M. tuberculosis isolates in China.

Han H; Wang F; Xiao Y; Ren Y; Chao Y; Guo A; Ye L

2007-09-01

223

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation.  

UK PubMed Central (United Kingdom)

PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation.

Williams KJ; Bennett MH; Barton GR; Jenkins VA; Robertson BD

2013-03-01

224

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells.  

Science.gov (United States)

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323-339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner. PMID:23269491

Spiering, R; van der Zee, R; Wagenaar, J; van Eden, W; Broere, F

2012-12-27

225

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells.  

UK PubMed Central (United Kingdom)

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323-339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner.

Spiering R; van der Zee R; Wagenaar J; van Eden W; Broere F

2013-07-01

226

Tattoo-associated nontuberculous mycobacterial skin infections--multiple states, 2011-2012.  

UK PubMed Central (United Kingdom)

Permanent tattoos have become increasingly common, with 21% of adults in the United States reporting having at least one tattoo. On rare occasions, outbreaks of nontuberculous mycobacterial (NTM) skin infections have been reported after tattooing. In January 2012, public health officials in New York received reports of Mycobacterium chelonae skin infections in 14 New York residents who received tattoos during September-December 2011. All infections were associated with use of the same nationally distributed, prediluted gray ink manufactured by company A. CDC disseminated an Epi-X public health alert to identify additional tattoo-associated NTM skin infections; previously identified cases were reported from three states (Washington, Iowa, and Colorado). Public health investigations by CDC, state and local health departments, and the Food and Drug Administration (FDA) found NTM contamination in tattoo inks used in two of five identified clusters. All infected persons were exposed to one of four different brands of ink. NTM contamination of inks can occur during the manufacturing process as a result of using contaminated ingredients or poor manufacturing practices, or when inks are diluted with nonsterile water by tattoo artists. No specific FDA regulatory requirement explicitly provides that tattoo inks must be sterile. However, CDC recommends that ink manufacturers ensure ink is sterile and that tattoo artists avoid contamination of ink through dilution with nonsterile water. Consumers also should be aware of the health risks associated with getting an intradermal tattoo.

2012-08-01

227

Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection.  

UK PubMed Central (United Kingdom)

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen-specific IFN-? and IL-2-expressing CD4+ and CD8+ T cells. In exosome-vaccinated mice there was a similar TH 1 immune response but a more limited TH 2 response compared to BCG-vaccinated mice. Using a low-dose M. tuberculosis mouse aerosol infection model, exosomes from CFP-treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell-free vaccine against an M. tuberculosis infection. This article is protected by copyright. All rights reserved.

Cheng Y; Schorey JS

2013-08-01

228

Cervical Nontuberculous Mycobacterial Lymphadenitis Mimicking a Thyroid Tumor and Infiltrating Deep into the Neck  

Directory of Open Access Journals (Sweden)

Full Text Available Aim: Nontuberculous Mycobacterial Lymphadenitis (NML), which occurs in 1.2 per 100,000 children, is very rare. And those which emerge at the anterior cervical portion and infiltrate deep into the neck are even more rare. Generally, this disorder is uncommon existed near the thyroid gland. We report here a case of NML mimicking a thyroid tumor and infiltrating into the deep part of the anterior neck. Case: A mass at the anterior portion of her neck was found at 5 years old. It was not mobile and palpated as an irregularly surfaced hard mass whose size was 3 cm at the anterior lower portion of her neck. Ultrasonography showed an oval mass which existed near the slightly inferior part of the right lobe of the thyroid gland. Enhanced computed tomography showed a mass near the slightly inferior part of the right lobe of the thyroid gland. The mass was resected with the platysma and the right sternohyoid muscle. In the HE staining, epithelioid cell and Langhans type giant cells surrounding coagulative necrosis lesions which seemed to be caseation necrosis existed, similarly to cervical NML. Discussion: No consensus exists for the treatment of NML, but many documents advise complete excision. When the lesion cannot be completely removed, excision as far as possible and additional antibiotics are recommended. The characteristics of imaging of NML around the thyroid gland and infiltrating deep into the anterior neck and mediastinum are discussed.

Kazuhiro Mino; Tadao Okada; Shouhei Honda; Hisayuki Miyagi; Nobuhisa Ishiguro; Kanako C. Kubota; Taketomi Akinobu

2012-01-01

229

Occurrence of Nontuberculous Mycobacterial Pulmonary Infection in an Endemic Area of Tuberculosis  

Science.gov (United States)

The majority of investigations of the epidemiology of nontuberculous mycobacteria (NTM) have focused on highly developed nations with a low prevalence of tuberculosis. In contrast, the Para state of north Brazil represents an area of high tuberculosis prevalence and increasing NTM incidence. Toward the goal of understanding the dynamics of infection by all Mycobacterium species, we report patient characteristics and the identification of NTM strains isolated from sputum samples from patients that were residents of Para, a state in the Amazon region, Northern of Brazil, over the period January 2010 through December 2011 (2 years). The 29 NTM patients comprised 13.5% of positive mycobacterial cultures over the 2-year period. A major risk factor for NTM pulmonary disease was previous tuberculosis (76%). Further, the average age of NTM patients (52 years) was significantly higher than that of tuberculosis patients (39 years) and more were female (72.4% vs. 37.4%). Unlike other Brazilian states, NTM pulmonary patients in Para were infected with a different spectrum of mycobacteria; primarily the rapidly growing Mycobacterium massiliense and Mycobacterium simiae complex.

da Costa, Ana Roberta Fusco; Falkinham, Joseph O.; Lopes, Maria Luiza; Barretto, Adriana Rodrigues; Felicio, Joao Soares; Sales, Lucia Helena Messias; Bahia, Jeann Ricardo da Costa; Conceicao, Emilyn Costa; Lima, Karla Valeria Batista

2013-01-01

230

Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.  

Science.gov (United States)

Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1) isomer and PIM(2) mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1) and PIM(2) analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1) and PIM(2) analogues. CD14 was dispensable for PIM(1) and PIM(2) analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1) and PIM(2) analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway. PMID:21949737

Court, Nathalie; Rose, Stéphanie; Bourigault, Marie-Laure; Front, Sophie; Martin, Olivier R; Dowling, Jennifer K; Kenny, Elaine F; O'Neill, Luke; Erard, François; Quesniaux, Valerie F J

2011-09-16

231

Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.  

UK PubMed Central (United Kingdom)

Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1) isomer and PIM(2) mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1) and PIM(2) analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1) and PIM(2) analogues. CD14 was dispensable for PIM(1) and PIM(2) analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1) and PIM(2) analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.

Court N; Rose S; Bourigault ML; Front S; Martin OR; Dowling JK; Kenny EF; O'Neill L; Erard F; Quesniaux VF

2011-01-01

232

HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.  

UK PubMed Central (United Kingdom)

HAMP domains, ?55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the ?-helices of HAMP domains, plays an important structural role in HAMP function.

Mondéjar LG; Lupas A; Schultz A; Schultz JE

2012-01-01

233

A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet.  

UK PubMed Central (United Kingdom)

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.

Mollenkopf HJ; Jungblut PR; Raupach B; Mattow J; Lamer S; Zimny-Arndt U; Schaible UE; Kaufmann SH

1999-08-01

234

Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy  

Directory of Open Access Journals (Sweden)

Full Text Available This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

Doreen Manuela Floss; Michael Mockey; Galliano Zanello; Damien Brosson; Marie Diogon; Roger Frutos; Timothée Bruel; Valérie Rodrigues; Edwin Garzon; Claire Chevaleyre; Mustapha Berri; Henri Salmon; Udo Conrad; Laurence Dedieu

2010-01-01

235

Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85.  

UK PubMed Central (United Kingdom)

Mycolic acids, long-chain (C70-C90) alpha-alkyl, beta-hydroxy fatty acids, are characteristic cell envelope components of mycobacteria; similar but shorter-chain substances occur in corynebacteria and related taxa. These compounds apparently play an important role in the physiology of these bacteria. The deduced N-terminal region of PS1, one of the two major secreted proteins of Corynebacterium glutamicum encoded by the csp1 gene, is similar to the antigens 85 complex of Mycobacterium tuberculosis which has been shown to be associated in vitro with a mycoloyltransferase activity onto trehalose. Overexpression of PS1 in the wild-type strain of C. glutamicum suggested the implication of the protein in the transfer of corynomycolates, evidenced by an increase esterification of the cell wall arabinogalactan with corynomycolic acid residues and an accumulation of trehalose dicorynomycolates. Overexpression of truncated forms of PS1 demonstrated that the crucial region for transfer activity of the protein involves all the region of homology with antigens 85. To establish the putative mycoloyltransferase activity of PS1, a csp1-inactivated mutant of C. glutamicum was biochemically characterized. Inactivation of the gene resulted in: (i) a 50% decrease in the cell wall corynomycolate content; (ii) the alteration of the permeability of the C. glutamicum cell envelope; (iii) the decrease of the trehalose dicorynomycolate content; (iv) the accumulation of trehalose monocorynomycolate; and (v) the appearance of a glycolipid identified as 6-corynomycoloylglucose. Complementation of the mutant by the csp1 gene fully restored the wild-type phenotype. Finally, a mycoloyltransferase assay established that PS1 possesses a trehalose mycoloyltransferase activity. To define the in vivo function of antigens 85, the csp1-inactivated mutant was complemented with the fbpA, fbpB or fbpC genes. Complementation with the different fbp genes restored the normal cell wall corynomycolate content and permeability, but did not affect either the fate of trehalose corynomycolates or the occurrence of glucose corynomycolate. Thus, PS1 is one of the enzymes that transfer corynomycoloyl residues onto both the cell wall arabinogalactan and trehalose monocorynomycolate, whereas in the whole bacterium the mycobacterial antigens 85A, 85B and 85C can transfer mycolates only onto the cell wall acceptor in C. glutamicum.

Puech V; Bayan N; Salim K; Leblon G; Daffé M

2000-03-01

236

Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85.  

Science.gov (United States)

Mycolic acids, long-chain (C70-C90) alpha-alkyl, beta-hydroxy fatty acids, are characteristic cell envelope components of mycobacteria; similar but shorter-chain substances occur in corynebacteria and related taxa. These compounds apparently play an important role in the physiology of these bacteria. The deduced N-terminal region of PS1, one of the two major secreted proteins of Corynebacterium glutamicum encoded by the csp1 gene, is similar to the antigens 85 complex of Mycobacterium tuberculosis which has been shown to be associated in vitro with a mycoloyltransferase activity onto trehalose. Overexpression of PS1 in the wild-type strain of C. glutamicum suggested the implication of the protein in the transfer of corynomycolates, evidenced by an increase esterification of the cell wall arabinogalactan with corynomycolic acid residues and an accumulation of trehalose dicorynomycolates. Overexpression of truncated forms of PS1 demonstrated that the crucial region for transfer activity of the protein involves all the region of homology with antigens 85. To establish the putative mycoloyltransferase activity of PS1, a csp1-inactivated mutant of C. glutamicum was biochemically characterized. Inactivation of the gene resulted in: (i) a 50% decrease in the cell wall corynomycolate content; (ii) the alteration of the permeability of the C. glutamicum cell envelope; (iii) the decrease of the trehalose dicorynomycolate content; (iv) the accumulation of trehalose monocorynomycolate; and (v) the appearance of a glycolipid identified as 6-corynomycoloylglucose. Complementation of the mutant by the csp1 gene fully restored the wild-type phenotype. Finally, a mycoloyltransferase assay established that PS1 possesses a trehalose mycoloyltransferase activity. To define the in vivo function of antigens 85, the csp1-inactivated mutant was complemented with the fbpA, fbpB or fbpC genes. Complementation with the different fbp genes restored the normal cell wall corynomycolate content and permeability, but did not affect either the fate of trehalose corynomycolates or the occurrence of glucose corynomycolate. Thus, PS1 is one of the enzymes that transfer corynomycoloyl residues onto both the cell wall arabinogalactan and trehalose monocorynomycolate, whereas in the whole bacterium the mycobacterial antigens 85A, 85B and 85C can transfer mycolates only onto the cell wall acceptor in C. glutamicum. PMID:10712685

Puech, V; Bayan, N; Salim, K; Leblon, G; Daffé, M

2000-03-01

237

Species Identification of Mycobacteria by PCR-Restriction Fragment Length Polymorphism of the rpoB Gene  

Digital Repository Infrastructure Vision for European Research (DRIVER)

PCR-restriction fragment length polymorphism analysis (PRA) using the novel region of the rpoB gene was developed for rapid and precise identification of mycobacteria to the species level. A total of 50 mycobacterial reference strains and 3 related bacterial strains were used to amplify the 360-bp r...

Lee, Hyeyoung; Park, Hee-Jung; Cho, Sang-Nae; Bai, Gill-Han; Kim, Sang-Jae

238

The use of a two-gene sequencing approach to accurately distinguish between the species within the Mycobacterium abscessus complex and Mycobacterium chelonae.  

UK PubMed Central (United Kingdom)

Mycobacterium abscessus [M. abscessus (sensu lato) or M. abscessus complex] comprises three closely related species: M. abscessus (sensu stricto), hereafter referred to as M. abscessus, M. bolletii and M. massiliense. We describe here an accurate and robust method for distinguishing M. chelonae from M. abscessus, M. bolletii and M. massiliense, using polymerase chain reaction (PCR) and the sequencing of house-keeping gene targets (hsp65 and rpoB). Sequencing of the sodA gene is of little additional value in discriminating between species, but M. massiliense can be rapidly identified by amplification of the truncated erm(41) gene without the need for amplicon sequencing. We have applied the method to 81 isolates from 40 patients from two hospitals, the majority of whom were cystic fibrosis (CF) patients. Of these patients, 21 had previously been identified as M. chelonae and 59 as M. abscessus complex using commercial line probe assays. We identified these as 46 M. abscessus isolates, 20 M. massiliense isolates, five M. bolletii isolates and nine M. chelonae isolates and confirmed the one M. fortuitum isolate. This is the first study that has identified the individual members of the M. abscessus complex in a UK cohort of mainly CF patients.

Blauwendraat C; Dixon GL; Hartley JC; Foweraker J; Harris KA

2012-08-01

239

CT findings of mycobacterial infection other than tuberculosis : comparison with tuberculosis  

Energy Technology Data Exchange (ETDEWEB)

To compare the CT findings of mycobacterial infection other than tuberculosis (MOTT) with those of tuberculosis (TB). The chest CT scans of 30 immunocompetent patients with culture-proven pulmonary MOTT (M:F =3D 11:19; mean age, 51.2 yrs.) and of 24 patients with active tuberculosis (M:F 12:12; mean age, 42.5 yrs.) were analyzed by two radiologists; decisions were reached by consensus. Common findings for both MOTT and TB included brochogenically-spread bronchogenic spread nodular lesion (93.3% for MOTT, 100% for TB), bronchiectasis (90%, 83.3%), bronchial wall thickening (66.7%, 54.2%), granuloma (63.3%, 75%), parenchymal scarring (53.3%, 54.2%), and mediastinal lymphadenopathy (50%, 37.5%). Less commonly observed findings were emphysema (46.7%, 29.7%), atelectasis (36.7%, 29.2%), narrowing of a major airway (23.3%, 25%), consolidation (23.3%, 29.2%)and pleural disease (16.7%, 29.2%). Except for cavity (30%, 53.3%; P less than 0.05), the frequencies of each finding were not different between the two groups. A lobe-matched frequency comparison showed that only bronchiectasis in the right middle lobe (40%, 16.7%), right lower lobe (63.3%, 33.3%) and lingula division (53.3%, 25%) was significantly more common in MOTT than in TB (p less than 0.05). The number of lobes in which bronchiectasis and bronchial wall thickening were involved was greater in MOTT (3.20) than in TB (2.04) (p=3D 0.011). Although the CT findings of MOTT and TB overlap considerably, cavities are more common in TB, while in MOTT, bronchiectasis in the lower lung zone is more common and bronchiectasis tends to be more extensive. (author)

Yoon, Chang Jin; Goo, Jin Mo; Seo, Joon Beom; Kim, Se Hyung; Im, Jung Gi [College of Medicine and the Institute of Radiation Medicine, Seoul National University, Seoul (Korea, Republic of)

2000-03-01

240

Epidemiology of Nontuberculous Mycobacterial Infections and Associated Chronic Macrolide Use among Persons with Cystic Fibrosis.  

UK PubMed Central (United Kingdom)

Rationale: Persons with cystic fibrosis (CF) are at high risk of nontuberculous mycobacterial (NTM) infection, with treatment requiring prolonged multi-drug regimens that include macrolides. While macrolides, specifically azithromycin, are used in the management of CF patients with chronic Pseudomonas, macrolide-resistant NTM infections are of growing concern. We sought to evaluate the relationship between macrolide use and NTM infections among CF patients. Objective: To evaluate the relationship between chronic macrolide use and NTM infection among CF patients included in the 2011 CF Patient Registry (CFPR). Main Results: The 2011 CFPR included 27,112 patients; 5,403 (20%) were cultured for mycobacteria in 2010-2011 and met all inclusion criteria. Of these, 191 (4%) were NTM-positive in 2011 only (incident cases), while 5,212 (96%) were NTM-negative in both 2010 and 2011 (controls). Among the cases, 122 (64%) were culture-positive for Mycobacterium avium complex (MAC) and 69 (36%) for M. abscessus. Azithromycin use in 2010 was less frequently reported among MAC cases (57%; OR=0.7, p<0.05) and M. abscessus cases (51%; OR=0.5, p<0.01) than in controls (66%). Among adolescents and adults, patients with the greatest number of years on chronic macrolides were the least likely to develop incident NTM in 2011 (p<0.01). Conclusions: Patients with incident NTM infections from either MAC or M. abscessus were less likely to have had chronic azithromycin treatment in the past year. However, because macrolide monotherapy may lead to macrolide resistance, routine screening for NTM should be considered for persons with CF.

Binder AM; Adjemian J; Olivier KN; Prevots DR

2013-08-01

 
 
 
 
241

Emergence of clinically relevant Non-Tuberculous Mycobacterial infections in Saudi Arabia.  

UK PubMed Central (United Kingdom)

BACKGROUND: Non-Tuberculous Mycobacteria (NTM) are emerging around the world due to a higher prevalence of immunosuppressive illness and therapy. Saudi Arabia is not an exception as there have been novel mycobacterial species also identified. In addition, several published case reports from different parts of the country suggest a growing pathogenic potential of NTM. As the first nationwide study, we sought to gain an insight into the species diversity of NTM clinical isolates. METHODOLOGY/PRINCIPAL FINDINGS: During June 2009-July 2010, 95 clinical isolates were collected from tuberculosis reference laboratories in major provinces within Saudi Arabia and subjected to standard line probe assay techniques to identify their species. Diagnostic guidelines of the American Thoracic Society were applied to determine the clinical relevance of respiratory isolates. Species diversity (13 species) was very high and dominated (61.0%) by rapid growing NTM. The major species obtained were Mycobacterium abscessus, M. fortuitum, M. intracellulare followed by M. kansassi, M. gordanae and M. avium. Interestingly this study reports for the first time the clinical relevance of M. celatum, M. xenopi, M. scrofulceum, M. lentiflavum, M. asiaticum and M. simiae in Saudi Arabia. Of the total, 67.1% were clinically relevant respiratory cases, 23.2% were non-respiratory cases and 9.7% were respiratory colonizers. Coexisting illness was reported in 53.7% of the studied cases. The major risk factors observed among the patients were previous history of tuberculosis, chronic obstructive pulmonary disorder and human immunodeficiency virus infection. CONCLUSION/SIGNIFICANCE: The high rates of clinically confirmed respiratory cases suggest that NTM infections are indeed a new challenge to health authorities. The current findings show an opposite picture of the Western world where M. avium complex and particularly slow growing NTM are the most predominant respiratory pathogens. The complexity of species demands an immediate strengthening of the current diagnostic facilities.

Varghese B; Memish Z; Abuljadayel N; Al-Hakeem R; Alrabiah F; Al-Hajoj SA

2013-05-01

242

Towards new antituberculotic targets: biochemical characterisation of mycobacterial RNase E/G  

Directory of Open Access Journals (Sweden)

Full Text Available The World Health Organization estimates that each year 3 million people die from tuberculosis (TB) and 8 million people become infected. No new anti-TB drugs have been introduced in the past 30 years, even though their development becomes increasingly important to face new challenges posed by multidrug-resistant and extensively drug-resistant strains and by acute infection with M. tuberculosis of HIV positive patients. Owing to its apparently important role in RNA metabolism, the RNase E/G family of endoribonucleases can be considered as a promising target for antimicrobial drugs. This consideration promted us to characterise biochemical properties of the M. tuberculosis RNase E/G homologue. To learn more about specific properties of RNase E/G homologues a M. tuberculosis RNase E/G (MycRne) was overexpressed in E. coli and purified as a 6His-tagged polypeptide. To characterise MycRne, we used in vitro cleavage assays and primer extension analysis of total RNA extracted from mycobacteria. We show that affinity purified MycRne has an endoribonucleolytic activity, which is dependent on the 5'-phosphorylation status of RNA. We could also show that RNase E/G has Mg2+ dependent activity and similar to E. coli RNase E, MycRne was able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA. Although, similar to E. coli RNase E, the mycobacterial RNase E/G homologue plays a role in rRNA processing, the substrate specificities of these enzymes show differences. This suggests that RNase E/G can be used as a promising target for antimicrobial drugs that can be optimized to specifically target pathogenic species.

Agnes Csanadi; Mirijam-Elisabeth Zeller; Andras Miczak; Thierry Rose; Thierry Bizebard; Vladimir R. Kaberdin

2008-01-01

243

Bilateral Candida and atypical mycobacterial infection after frontalis sling suspension with silicone rod to correct congenital ptosis.  

Science.gov (United States)

In this case report, the authors describe an unusual complication of a frontalis sling suspension with silicone rods. A 5-year-old girl with blepharophimosis syndrome underwent frontalis sling suspension using an open sky technique. Four weeks after surgery, she was noted to have pustules over both upper eyelids and eyebrows. Cultures from the surgical sites grew Mycobacterium chelonae and Candida parapsilosis. Intravenous antibiotics and antifungals and sling explantation were curative. One month after sling explantation, the patient maintained an adequate marginal reflex distance 1. Atypical mycobacterial and Candida infection should be considered in the differential diagnoses of postoperative infection after frontalis sling suspension with silicone rods. PMID:23381566

Davies, Brett W; Bratton, Emily M; Durairaj, Vikram D; Hink, Eric M

244

Bilateral Candida and atypical mycobacterial infection after frontalis sling suspension with silicone rod to correct congenital ptosis.  

UK PubMed Central (United Kingdom)

In this case report, the authors describe an unusual complication of a frontalis sling suspension with silicone rods. A 5-year-old girl with blepharophimosis syndrome underwent frontalis sling suspension using an open sky technique. Four weeks after surgery, she was noted to have pustules over both upper eyelids and eyebrows. Cultures from the surgical sites grew Mycobacterium chelonae and Candida parapsilosis. Intravenous antibiotics and antifungals and sling explantation were curative. One month after sling explantation, the patient maintained an adequate marginal reflex distance 1. Atypical mycobacterial and Candida infection should be considered in the differential diagnoses of postoperative infection after frontalis sling suspension with silicone rods.

Davies BW; Bratton EM; Durairaj VD; Hink EM

2013-07-01

245

Mycobacterial HSP60 and HSP70 prevents the severe form of acute DSS colitis in Balb/c mice.  

Czech Academy of Sciences Publication Activity Database

. Innsbruck : Elsevier, 2007, s. 49-49.[European Crohn´s and Colitis Organisation (ECCO) Congress. Innsbruck (AT), 01.03.2007-03.03.2007]Grant CEP: GA AV ?R IAA5020205; GA AV ?R 1QS500200572; GA ?R GD310/03/H147Grant ostatní: XE(XE) Broad Medical Research Program IBD-0159Výzkumný zám?r: CEZ:AV0Z50200510Zdroj financování: R - rámcový projekt EKKlí?ová slova: mycobacterial hsp60Kód oboru RIV: EE - Mikrobiologie, virologie

Kverka, MiloslavG; Zákostelská, Zuzana; Tlaskalová, Helena; Van der Zee, R.; Van Eden, W.

246

Prime-boost BCG vaccination with DNA vaccines based in ?-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model.  

UK PubMed Central (United Kingdom)

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0-89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that ?-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding ?-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-?) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB.

Cervantes-Villagrana AR; Hernández-Pando R; Biragyn A; Castañeda-Delgado J; Bodogai M; Martínez-Fierro M; Sada E; Trujillo V; Enciso-Moreno A; Rivas-Santiago B

2013-01-01

247

Prime-boost BCG vaccination with DNA vaccines based in ?-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model.  

Science.gov (United States)

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0-89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that ?-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding ?-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-?) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB. PMID:23196205

Cervantes-Villagrana, Alberto R; Hernández-Pando, Rogelio; Biragyn, Arya; Castañeda-Delgado, Julio; Bodogai, Monica; Martínez-Fierro, Margarita; Sada, Eduardo; Trujillo, Valentin; Enciso-Moreno, Antonio; Rivas-Santiago, Bruno

2012-11-26

248

Effect of HIV protease inhibitors and Orlistat on mycobacterial ES-31 serine protease, a potential drug target in Mycobacterium tuberculosis.  

UK PubMed Central (United Kingdom)

BACKGROUND: Mycobacterial excretory secretory-31 (SEVA TB ES-31) antigen is shown to possess protease and lipase activities. AIM: To study the effect of commonly used HIV-protease inhibitors and lipase inhibitor Orlistat if any on mycobacterial ES-31 serine protease in vitro enzyme activity and on the growth of M.tb H37Ra bacilli in axenic culture. METHODS: Effect of HIV-protease inhibitors namely Ritonavir, Lopinavir and Indinavir and Orlistat on protease activity of ES-31 was assessed using azocasein assay and on bacillary growth in axenic culture of Mycobacterium tuberculosis H37Ra. The concentration of ES-31 antigen in culture filtrate was determined by sandwich peroxidase ELISA using anti ES-31 antibody and the growth of bacilli by CFU count. RESULTS: HIV-protease inhibitors such as Ritonavir, Lopinavir and Indinavir and lipase inhibitor Orlistat inhibited serine protease activity by 41.3 - 69.7% in vitro. These inhibitors also showed decreased bacterial growth in axenic culture and further confirmed by decreased concentration of ES-31 serine protease secretion in the culture fluid. Ritonavir showed maximum inhibition of 77% on the growth of the bacilli in axenic culture while anti obesity drug Orlistat showed 61% inhibition. CONCLUSION: SEVA TB ES-31 with serine protease and lipase activities may be a potential drug target in tuberculosis management.

Majumdar A; Wankhade G; Kamble PD; Harinath BC

2011-01-01

249

Analytical performance of the Roche LightCycler® Mycobacterium Detection Kit for the diagnosis of clinically important mycobacterial species.  

UK PubMed Central (United Kingdom)

BACKGROUND: The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility. METHODOLOGY/PRINCIPAL FINDINGS: Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay's reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent. SIGNIFICANCE: The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.

Omar SV; Roth A; Ismail NA; Erasmus L; Ehlers M; Kock M; Paulse N; Said HM; Hoosen AA; Reischl U

2011-01-01

250

Coresistance to isoniazid and ethionamide maps to mycothiol biosynthetic genes in Mycobacterium bovis.  

Science.gov (United States)

A search to identify new mechanisms of isoniazid resistance in Mycobacterium bovis led to the isolation of mutants defective in mycothiol biosynthesis due to mutations in genes coding for the glycosyltransferase (mshA) or the cysteine ligase (mshC). These mutants showed low-level resistance to isoniazid but were highly resistant to ethionamide. This study further illustrates that mutations in mycothiol biosynthesis genes may contribute to isoniazid or ethionamide resistance across mycobacterial species. PMID:21709101

Vilchèze, Catherine; Av-Gay, Yossef; Barnes, S Whitney; Larsen, Michelle H; Walker, John R; Glynne, Richard J; Jacobs, William R

2011-06-27

251

Coresistance to isoniazid and ethionamide maps to mycothiol biosynthetic genes in Mycobacterium bovis.  

UK PubMed Central (United Kingdom)

A search to identify new mechanisms of isoniazid resistance in Mycobacterium bovis led to the isolation of mutants defective in mycothiol biosynthesis due to mutations in genes coding for the glycosyltransferase (mshA) or the cysteine ligase (mshC). These mutants showed low-level resistance to isoniazid but were highly resistant to ethionamide. This study further illustrates that mutations in mycothiol biosynthesis genes may contribute to isoniazid or ethionamide resistance across mycobacterial species.

Vilchèze C; Av-Gay Y; Barnes SW; Larsen MH; Walker JR; Glynne RJ; Jacobs WR Jr

2011-09-01

252

[The diagnosis and treatment of rapidly growing non-tuberculous mycobacterial keratitis].  

UK PubMed Central (United Kingdom)

OBJECTIVE: To study the clinical features, diagnosis and treatment of non-tuberculous mycobacterial keratitis (NTMK). METHODS: It was retrospective case series study. Twelve eyes in 12 patients with NTMK following corneal foreign body trauma in 2007 were studied retrospectively including the case histories, clinical findings, laboratory examinations, diagnosis, treatment and prognosis. The main laboratory examination included corneal scrapings by culturing, polymerase chain reaction (PCR) and transmission electron microscopy (TEM), corneal lesions by histopathologic examinations and TEM. The patients received local and systemic antibiotics therapy, lesion cleaning followed by cauterization with tincture of iodine (5%) and (or) keratoplasty. RESULTS: All cases had a history of corneal trauma, there was corneal metallic foreign body removal at one hospital in 11 cases, corneal reed trauma in 1 case. The characteristic signs involved grayish-blue crystalloid keratopathy, multifocal infiltrates, satellites, radical form changes in the Descemet's membrane. The results of laboratory examinations of the scrapings of the cornea infection were as follows: all cultures (12/12) were positive for rapidly growing mycobacteria, and isolates from 5 patients were all diagnosed as mycobacterium chelonae subspecies abscess; acid-fast staining revealed positive bacilli in all the 4 patients; seven of 8 patients were positive for bacterium by PCR. Transmission electron microscopy in all the 3 specimens showed many slender rod-shaped or short coarse-shaped bacteria which were phagocytized by monocytes, and some necrotic tissue. Infections in 10 eyes were resolved by combined treatment regimen including a combination of antimicrobial agents (amikacin, rifampin, gatifloxacin, ciprofloxacin, azithromycin and/or ofloxacin, etc.) and local lesion cleaning followed by cauterization with 5% tincture of iodine within 2-5 months; two cases resolved by keratoplasty which poorly responded to antibiotic therapy for 6 months. CONCLUSIONS: NTMK is a rare, recalcitrant opportunistic infection which can occur in an epidemic fashion following corneal foreign body trauma. The diagnosis of NTMK is difficult, and may easily be misdiagnosed as fungal keratitis. Acid-fast staining, TEM, especially bacterial culture can help to obtain definitive diagnosis. NTMK has a long response period to medical management. The majority of patients can be cured by local and systemic antibiotics therapy, and the recalcitrant infections could be resolved by keratoplasty.

Guan HJ; Cheng ZP; Yin L; Wu YY; Hu N; Zhang JF; Shi HH

2009-06-01

253

The mycobacterial glycolipid glucose monomycolate induces a memory T cell response comparable to a model protein antigen and no B cell response upon experimental vaccination of cattle  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Glycolipids are presented to T cells by human group 1 CD1 proteins, but are not used as subunit vaccines yet. Experimental immunizations with pure mycobacterial glucose monomycolate (GMM) and keyhole limpet haemocyanin (KLH) in cattle, a species which, unlike mice, expresses group 1 CD1, showed that...

Nguyen, Thi Kim Anh; Koets, Ad P.; Santema, Wiebren J.; van Eden, Willem; Rutten, Victor P.M.G.; Van Rhijn, Ildiko

254

Automated High-Throughput Mycobacterial Interspersed Repetitive Unit Typing of Mycobacterium tuberculosis Strains by a Combination of PCR and Nondenaturing High-Performance Liquid Chromatography  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex isolates is portable, 100% reproducible, and highly discriminatory. Nondenaturing high-performance liquid chromatography (non-dHPLC) with use of a WAVE microbial analysis...

Evans, Jason T.; Hawkey, Peter M.; Smith, E. Grace; Boese, Kerstin A.; Warren, Roderic E.; Hong, George

255

DedA Protein Relates to Action-Mechanism of Halicyclamine A, a Marine Spongean Macrocyclic Alkaloid, as an Anti-dormant Mycobacterial Substance  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegm...

Arai, Masayoshi; Liu, Liu; Fujimoto, Takao; Setiawan, Andi; Kobayashi, Motomasa

256

Bovine NK Cells Can Produce Gamma Interferon in Response to the Secreted Mycobacterial Proteins ESAT-6 and MPP14 but Not in Response to MPB70  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Bovine NK cells have recently been characterized and the present study describes the interaction between NK cells, antigen-presenting cells, and secreted mycobacterial proteins. Gamma interferon (IFN-?) production by NK cells was seen in approximately 30% of noninfected calves in response to the Myc...

Olsen, Ingrid; Boysen, Preben; Kulberg, Siri; Hope, Jayne C.; Jungersen, Gregers; Storset, Anne K.

257

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry.  

UK PubMed Central (United Kingdom)

PURPOSE: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MATERIALS AND METHODS: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. RESULTS: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. CONCLUSIONS: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

Panda A; Kurapati S; Samantaray JC; Myneedu VP; Verma A; Srinivasan A; Ahmad H; Behera D; Singh UB

2013-04-01

258

[Implementation of the technical requirements of the UNE-EN-ISO 15189 quality standard in a mycobacterial laboratory].  

Science.gov (United States)

The UNE-EN-ISO 15189:2007 standard defines the requirements for quality and competence that must be met by medical laboratories. These laboratories should use this international standard to develop their own quality management systems and to evaluate their own competencies; in turn, this standard will be used by accreditation bodies to confirm or recognize the laboratories' competence. In clinical microbiology laboratories, application of the standard implies the implementation of the technical and specific management requirements that must be met to achieve optimal quality when carrying out microbiological tests. In Spain, accreditation is granted by the Spanish Accreditation Body (Entidad Nacional de Acreditación). This review aims to discuss the practical application of the standard's technical requirements in mycobacterial laboratory. Firstly, we define the scope of accreditation. Secondly, we specify how the items of the standard on personnel management, control of equipment, environmental facilities, method validation, internal controls and customer satisfaction surveys were developed and implemented in our laboratory. PMID:23453231

Guna Serrano, M del Remedio; Ocete Mochón, M Dolores; Lahiguera, M José; Bresó, M Carmen; Gimeno Cardona, Concepción

2013-02-01

259

Anti-mycobacterial treatment reduces high plasma levels of CXC-chemokines detected in active tuberculosis by cytometric bead array  

Directory of Open Access Journals (Sweden)

Full Text Available Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometric bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

Caroline de Souza Almeida; Clarice Abramo; Caio César de Souza Alves; Luciano Mazzoccoli; Ana Paula Ferreira; Henrique Couto Teixeira

2009-01-01

260

Techniques of DNA hybridization detect small numbers of mycobacteria with no cross-hybridization with non-mycobacterial respiratory organisms  

International Nuclear Information System (INIS)

The traditional methods used in identifying mycobacteria, such as acid-fast bacillus stains and culture, are often time-consuming, insensitive, and nonspecific. As part of an ongoing program to improve diagnosis and characterization of mycobacteria, the authors have found that deoxyribonucleic acid (DNA) hybridization techniques using isotopically labeled, single-stranded, total DNA can be used to detect as little as 10(-4) micrograms of Mycobacterium tuberculosis (MTb) DNA. This amount of DNA represents approximately 2 X 10(4) genomes. They have also shown the MTb DNA is sufficiently different from the DNA of non-mycobacterial microorganisms such that cross-hybridization with MTb DNA does not occur under the hybridization conditions employed. The authors speculate that DNA hybridization techniques may allow the rapid, sensitive, and specific identification of mycobacteria

1985-01-01

 
 
 
 
261

Techniques of DNA hybridization detect small numbers of mycobacteria with no cross-hybridization with non-mycobacterial respiratory organisms  

Energy Technology Data Exchange (ETDEWEB)

The traditional methods used in identifying mycobacteria, such as acid-fast bacillus stains and culture, are often time-consuming, insensitive, and nonspecific. As part of an ongoing program to improve diagnosis and characterization of mycobacteria, the authors have found that deoxyribonucleic acid (DNA) hybridization techniques using isotopically labeled, single-stranded, total DNA can be used to detect as little as 10(-4) micrograms of Mycobacterium tuberculosis (MTb) DNA. This amount of DNA represents approximately 2 X 10(4) genomes. They have also shown the MTb DNA is sufficiently different from the DNA of non-mycobacterial microorganisms such that cross-hybridization with MTb DNA does not occur under the hybridization conditions employed. The authors speculate that DNA hybridization techniques may allow the rapid, sensitive, and specific identification of mycobacteria.

Shoemaker, S.A.; Fisher, J.H.; Scoggin, C.H.

1985-05-01

262

A hybrid soft solar cell based on the mycobacterial porin MspA linked to a sensitizer-viologen Diad.  

Science.gov (United States)

A prototype of a nano solar cell containing the mycobacterial channel protein MspA has been successfully designed. MspA, an octameric transmembrane channel protein from Mycobacterium smegmatis, is one of the most stable proteins known to date. Eight Ruthenium(II) aminophenanthroline-viologen maleimide Diads (Ru-Diads) have been successfully bound to the MspA mutant MspAA96C via cysteine-maleimide bonds. MspA is known to form double layers in which it acts as nanoscopic surfactant. The nanostructured layer that is formed by (Ru-Diad)8MspA at the TiO2 electrode is photochemically active. The resulting "protein nano solar cell" features an incident photon conversion efficiency of 1% at 400 nm. This can be regarded as a proof-of-principle that stable proteins can be successfully integrated into the design of solar cells. PMID:23611424

Perera, Ayomi S; Subbaiyan, Navaneetha K; Kalita, Mausam; Wendel, Sebastian O; Samarakoon, Thilani N; D'Souza, Francis; Bossmann, Stefan H

2013-04-30

263

A hybrid soft solar cell based on the mycobacterial porin MspA linked to a sensitizer-viologen Diad.  

UK PubMed Central (United Kingdom)

A prototype of a nano solar cell containing the mycobacterial channel protein MspA has been successfully designed. MspA, an octameric transmembrane channel protein from Mycobacterium smegmatis, is one of the most stable proteins known to date. Eight Ruthenium(II) aminophenanthroline-viologen maleimide Diads (Ru-Diads) have been successfully bound to the MspA mutant MspAA96C via cysteine-maleimide bonds. MspA is known to form double layers in which it acts as nanoscopic surfactant. The nanostructured layer that is formed by (Ru-Diad)8MspA at the TiO2 electrode is photochemically active. The resulting "protein nano solar cell" features an incident photon conversion efficiency of 1% at 400 nm. This can be regarded as a proof-of-principle that stable proteins can be successfully integrated into the design of solar cells.

Perera AS; Subbaiyan NK; Kalita M; Wendel SO; Samarakoon TN; D'Souza F; Bossmann SH

2013-05-01

264

Divergent Effects of Mycobacterial Cell Wall Glycolipids on Maturation and Function of Human Monocyte-Derived Dendritic Cells  

Science.gov (United States)

Background Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system. Principal Findings We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect. Conclusions These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.

Mazurek, Jolanta; Ignatowicz, Lech; Kallenius, Gunilla; Svenson, Stefan B.; Pawlowski, Andrzej; Hamasur, Beston

2012-01-01

265

The role of the mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG in host cell interaction  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Mycobacterium tuberculosis differs from most pathogens in its ability to multiply inside monocytes and to persist during long periods of time within granuloma in a status of latency. A class of proteins called mycobacterial histone-like proteins has been associated with regulation of replication and latency, but their precise role in the infection process has yet to be uncovered. Our study aimed at defining the impact of the histone-like protein MDP1 from M. bovis BCG (mycobacterial DNA-binding protein 1, corresponding to Rv2986c from M. tuberculosis) on early steps of infection. Results Previously, a BCG (Bacillus Calmette Guérin) strain had been generated by antisense-technique exhibiting reduced MDP1 expression. This strain was now used to analyse the impact of reduced amount of MDP1 on the interaction with human blood monocytes, macrophage lines and PBMC (peripheral blood mononuclear cells). MDP1 was revealed to be required for growth at acidic pH and for intracellular replication in human blood monocytes. Down-regulation of MDP1 resulted in reduced secretion of the cytokine IL-1? by infected human PBMC. In addition, a reduction of MDP1 expression had a major impact on the formation of fused multi-nucleated macrophages. In monocyte preparations from human blood as well as in human and mouse macrophage cell lines, both the percentage of multi-nucleated cells and the number of nuclei per cell were much enhanced when the monocytes were infected with BCG expressing less MDP1. Conclusion MDP1 from M. bovis BCG affects the growth at acidic pH and the intracellular replication in human monocytes. It furthermore affects cytokine secretion by host cells, and the formation of fused multi-nucleated macrophages. Our results suggest an important role of MDP1 in persistent infection.

Kunisch Ralph; Kamal Elisabeth; Lewin Astrid

2012-01-01

266

Cutaneous manifestations of Nocardia brasiliensis infection in Taiwan during 2002-2012-clinical studies and molecular typing of pathogen by gyrB and 16S gene sequencing.  

UK PubMed Central (United Kingdom)

To observe the clinicopathologic and resistance profiles of the Nocardia brasiliensis causing cutaneous nocardiosis in Taiwan, 12 N. brasiliensis isolates were prospectively collected from patients with cutaneous nocardiosis in a hospital during 2002-2012. Clinicopathologic data were obtained, and isolates were identified by biochemical methods and 16S rRNA sequencing. Susceptibilities to 14 antimicrobial compounds were tested. Isolates were further genotyped by sequencing of 16S rRNA, secA1, hsp65, and gyrB genes. The nodulopustular pyoderma associated with sporotrichoid spreading was the most common skin presentations caused by N. brasiliensis. All of the isolates were susceptible to amikacin, gentamicin, tobramycin, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole and resistant to kanamycin, erythromycin, and oxacillin, while susceptibilities to imipenem, vancomycin, penicillin-G, tetracycline, clindamycin, and ciprofloxacin varied among the 12 isolates. GyrB genotyping delineated the 12 isolates into 2 major groups, which was coincident with different single nucleotide substitutions at position 160 (G versus T) of 16S rRNA, different levels of imipenem minimum inhibition concentration (4-32 versus 0.25-0.75 mg/L), and prevalence of lymphadenitis (66.7 versus 16.7%). We have noted that tiny pustular lesions can be the first sign of cutaneous nocardiosis, which we believe has not been previously emphasized. No resistance to trimethoprim and sulfamethoxazole was found; therefore, sulphonamide drugs remain effective for treatment of cutaneous nocardiosis in Taiwan.

Chen KW; Lu CW; Huang TC; Lu CF; Liau YL; Lin JF; Li SY

2013-09-01

267

Cutaneous manifestations of Nocardia brasiliensis infection in Taiwan during 2002-2012-clinical studies and molecular typing of pathogen by gyrB and 16S gene sequencing.  

Science.gov (United States)

To observe the clinicopathologic and resistance profiles of the Nocardia brasiliensis causing cutaneous nocardiosis in Taiwan, 12 N. brasiliensis isolates were prospectively collected from patients with cutaneous nocardiosis in a hospital during 2002-2012. Clinicopathologic data were obtained, and isolates were identified by biochemical methods and 16S rRNA sequencing. Susceptibilities to 14 antimicrobial compounds were tested. Isolates were further genotyped by sequencing of 16S rRNA, secA1, hsp65, and gyrB genes. The nodulopustular pyoderma associated with sporotrichoid spreading was the most common skin presentations caused by N. brasiliensis. All of the isolates were susceptible to amikacin, gentamicin, tobramycin, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole and resistant to kanamycin, erythromycin, and oxacillin, while susceptibilities to imipenem, vancomycin, penicillin-G, tetracycline, clindamycin, and ciprofloxacin varied among the 12 isolates. GyrB genotyping delineated the 12 isolates into 2 major groups, which was coincident with different single nucleotide substitutions at position 160 (G versus T) of 16S rRNA, different levels of imipenem minimum inhibition concentration (4-32 versus 0.25-0.75 mg/L), and prevalence of lymphadenitis (66.7 versus 16.7%). We have noted that tiny pustular lesions can be the first sign of cutaneous nocardiosis, which we believe has not been previously emphasized. No resistance to trimethoprim and sulfamethoxazole was found; therefore, sulphonamide drugs remain effective for treatment of cutaneous nocardiosis in Taiwan. PMID:23791388

Chen, Kuo-Wei; Lu, Chun-Wei; Huang, Ting-Chi; Lu, Chin-Fang; Liau, Yea-Ling; Lin, Jeng-Fong; Li, Shu-Ying

2013-06-19

268

Successful treatment of refractory cutaneous infection caused by Mycobacterium marinum with a combined regimen containing amikacin  

Directory of Open Access Journals (Sweden)

Full Text Available Yingxue Huang,* Xiulian Xu,* Yi Liu, Kan Wu, Wei Zhang, Pai Liu, Xuesi Zeng, Jianfang Sun, Yiqun Jiang, Hongsheng WangKey Laboratory of Molecular Biology for Skin Diseases, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, China *These authors contributed equally to this workBackground: The incidence of Mycobacterium marinum infection has been increasing. First-line antituberculous drugs and other common antibiotics are effective for most cutaneous M. marinum infections; however, treatment failure still occurs in some rare cases. We report a case of a 70-year-old man with refractory cutaneous infection caused by M. marinum. Reasons for delayed diagnosis and related factors of the refractory infection are also discussed.Methods: Samples of lesional skin were inoculated on Löwenstein–Jensen medium for acid-fast bacilli. Species of mycobacterium were identified by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis. We then carried out genotyping by using mycobacterial interspersed repetitive units and sequencing of heat shock protein 65 (hsp65) and 16S rDNA genes.Results: Tissue cultures for acid-fast bacilli were positive. PCR-RFLP analysis and sequencing of hsp65 and 16S rDNA genes confirmed the isolated organisms to be M. marinum. Systemic therapy with rifampicin, clarithromycin, and amikacin empirically over 6 months led to complete resolution of skin lesions leaving only some residual scars.Conclusion: Key diagnostic elements for M. marinum infections include a high index of suspicion raised by chronic lesions, poor response to conventional treatments, and a history of fish-related exposure. Strong clinical suggestion of M. marinum infection warrants initial empirical treatment. The duration of therapy is usually several months or even longer, especially for elderly patients. Amikacin can be considered in multidrug therapy for treatment of some refractory M. marinum infections.Keywords: amikacin, clarithromycin, skin infection, Mycobacterium marinum, nontuberculous mycobacteria

Huang Y; Xu X; Liu Y; Wu K; Zhang W; Liu P; Zeng X; Sun J; Jiang Y; Wang H

2012-01-01

269

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

International Nuclear Information System (INIS)

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

2006-01-01

270

Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases.  

UK PubMed Central (United Kingdom)

dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg(2+)-coordination. Our results on transient/steady-state kinetics, ligand binding and a 1.80 A resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg(2+) accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function.

Takács E; Nagy G; Leveles I; Harmat V; Lopata A; Tóth J; Vértessy BG

2010-07-01

271

Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases  

Science.gov (United States)

dUTPases are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg2+-coordination. Our results on transient/steady-state kinetics, ligand-binding and a 1.80 Å-resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg2+ accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function.

Takacs, Eniko; Nagy, Gergely; Leveles, Ibolya; Harmat, Veronika; Lopata, Anna; Toth, Judit; Vertessy, Beata G.

2010-01-01

272

XDR TB in a case of IL12R?1 deficiency: a case report of Mendelian Susceptibility to Mycobacterial Disease from India.  

UK PubMed Central (United Kingdom)

Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a relatively new term that describes a spectrum of inherited defects in the IL-12/23 and IFN- ? pathways that result in a selective predisposition to disease caused by poorly pathogenic mycobacteria. In contrast to previous reports of patients infected with environmental mycobacteria and BCG, this manuscript elucidates the clinical course and diagnosis of MSMD in a child harboring extensively drug resistant (XDR) Mycobacterium tuberculosis.

Merchant RH; Ahmed J; Ahmad N

2013-09-01

273

Immunodiagnosis of tuberculous meningitis: rapid detection of mycobacterial antigens in cerebrospinal fluid by reverse passive hemagglutination assay and their characterization by Western blotting.  

UK PubMed Central (United Kingdom)

Tuberculous meningitis (TBM) is one of the commonest chronic infections of the central nervous system (CNS). Diagnosis of TBM has been a problem as it causes various clinical manifestations which can be confused with those of other chronic infections of the CNS such as neurocysticercosis (NCC), neurobrucellosis and cryptococcal meningitis, that are prevalent in many underdeveloped and developing countries. Differential diagnosis of TBM can be made by detecting circulating mycobacterial antigens in CSF by immunoassays. In this study, a reverse passive hemagglutination (RPHA) has been developed using rabbit antimycobacterial IgG for detection of circulating mycobacterial antigens in CSFs from chronic infections of the CNS in order to develop a rapid, simple, sensitive and cost-effective method. Circulating mycobacterial antigens were characterized by immunoblot assay. The sensitivity limit of RPHA was 400 ng ml(-1). RPHA was specific as antimycobacterial IgG did not show any reaction with porcine Cysticercus cellulosae which was used as a control antigen. RPHA could detect mycobacterial antigens in CSF at a sensitivity level of 94.11% with a specificity of 99.0%. Immunoblot analysis of RPHA positive CSFs revealed predominantly 30-32 kDa and 71 kDa antigens whilst 6, 86, 120, 96 and 110 kDa showed varied degree of reactivity. Antigens of masses 30-32 and 71 kDa were absent in culture filtrate of Mycobacterium tuberculosis H37Rv grown in Proskeur-Beck liquid medium. RPHA is a rapid, simple and sensitive immunological method with a long shelf life of 6-8 weeks if stabilized coated erythrocytes are stored at +4 degrees C. RPHA could be used as an additional immunodiagnostic tool in both differential diagnosis and prognosis of TBM. Immunoblot results indicate that 30-32 kDa and 71 kDa antigens are cell wall derived.

Katti MK

2001-07-01

274

Type I interferon suppresses type II interferon-triggered human anti-mycobacterial responses.  

Science.gov (United States)

Type I interferons (IFN-? and IFN-?) are important for protection against many viral infections, whereas type II interferon (IFN-?) is essential for host defense against some bacterial and parasitic pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-? and IFN-? gene expression programs. IFN-? and its downstream vitamin D-dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-? and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-?-induced macrophage vitamin D-dependent antimicrobial peptide response was inhibited by IFN-? and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections. PMID:23449998

Teles, Rosane M B; Graeber, Thomas G; Krutzik, Stephan R; Montoya, Dennis; Schenk, Mirjam; Lee, Delphine J; Komisopoulou, Evangelia; Kelly-Scumpia, Kindra; Chun, Rene; Iyer, Shankar S; Sarno, Euzenir N; Rea, Thomas H; Hewison, Martin; Adams, John S; Popper, Stephen J; Relman, David A; Stenger, Steffen; Bloom, Barry R; Cheng, Genhong; Modlin, Robert L

2013-02-28

275

Transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with BCG at birth  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Novel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult. Methods To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix® HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes. Results We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-?, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated. Conclusion Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.

Fletcher Helen A; Keyser Alana; Bowmaker Mark; Sayles Peter C; Kaplan Gilla; Hussey Greg; Hill Adrian VS; Hanekom Willem A

2009-01-01

276

Molecular identification of nontuberculous mycobacteria isolates in a Brazilian mycobacteria reference laboratory.  

UK PubMed Central (United Kingdom)

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.

da Costa AR; Lopes ML; Furlaneto IP; de Sousa MS; Lima KV

2010-12-01

277

MmpL11 protein transports mycolic acid-containing lipids to the mycobacterial cell wall and contributes to biofilm formation in Mycobacterium smegmatis.  

Science.gov (United States)

A growing body of evidence indicates that MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not been fully elucidated. We report the characterization of Mycobacterium smegmatis MmpL11. We showed previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to host antimicrobial peptides. We report herein the further characterization of the M. smegmatis mmpL11 mutant and identification of the MmpL11 substrates. We found that biofilm formation by the M. smegmatis mmpL11 mutant was distinct from that by wild-type M. smegmatis. Analysis of cell wall lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across pathogenic and nonpathogenic mycobacteria. Phenotypes of the M. smegmatis mmpL11 mutant are complemented by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved role in mycobacterial cell wall biogenesis. PMID:23836904

Pacheco, Sophia A; Hsu, Fong-Fu; Powers, Katelyn M; Purdy, Georgiana E

2013-07-08

278

MmpL11 protein transports mycolic acid-containing lipids to the mycobacterial cell wall and contributes to biofilm formation in Mycobacterium smegmatis.  

UK PubMed Central (United Kingdom)

A growing body of evidence indicates that MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not been fully elucidated. We report the characterization of Mycobacterium smegmatis MmpL11. We showed previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to host antimicrobial peptides. We report herein the further characterization of the M. smegmatis mmpL11 mutant and identification of the MmpL11 substrates. We found that biofilm formation by the M. smegmatis mmpL11 mutant was distinct from that by wild-type M. smegmatis. Analysis of cell wall lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across pathogenic and nonpathogenic mycobacteria. Phenotypes of the M. smegmatis mmpL11 mutant are complemented by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved role in mycobacterial cell wall biogenesis.

Pacheco SA; Hsu FF; Powers KM; Purdy GE

2013-08-01

279

Identification of non-tuberculous mycobacteria from the Central Public Health Laboratory from Mato Grosso do Sul and analysis of clinical relevance/ Identificação de micobactérias não-tuberculosas do Laboratório Central de Saúde Pública de Mato Grosso de Sul e análise de dados clínicos dos pacientes  

Scientific Electronic Library Online (English)

Full Text Available Abstract in portuguese Micobactérias não-tuberculosas isoladas no Laboratório Central de Saúde Pública de Mato Grosso do Sul em 2003 e 2004 foram identificadas usando métodos fenotípicos convencionais (TI) e PCR-Restriction Enzyme Analysis (PRA) tendo o gene hsp65 como alvo (PRA-hsp65). Em 15 dos 32 isolados analisados os resultados obtidos com ambos métodos foram concordantes, sendo 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum e 1 Nocardia bras (more) iliensis. TI de 12 isolados não foi conclusiva. Perfis não descritos de PRA-hsp65 foram observados com 11 isolados. Dados dos prontuários médicos foram avaliados para inferir a relevância clínica dos isolados. Abstract in english Non-tuberculous mycobacteria isolated at the Central Public Health Laboratory from Mato Grosso do Sul in 2003 and 2004 were identified by conventional phenotypic methods (TI) and by PCR-Restriction Enzyme Analysis (PRA) using the hsp65 gene as target (PRA-hsp65). With 15 of the 32 analysed isolates, results of both methods were concordant, being 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum and 1 Nocardia brasiliensis. TI of 12 iso (more) lates was inconclusive. Novel PRA-hsp65 patterns were observed with 11 isolates. Medical data were evaluated for inference of clinical relevance of these isolates.

Moraes, Paulo Ricardo de Souza; Chimara, Erica; Telles, Maria Alice da Silva; Ueki, Suely Yoko Misuka; Cunha, Eunice Atsuko Totumi; Honer, Michael Robin; Leão, Sylvia Cardoso

2008-06-01

280

Progressing features of atypical mycobacterial infection in the lung on conventional and high resolution CT (HRCT) images  

International Nuclear Information System (INIS)

The aim of this study was to clarify the localization of abnormalities within secondary pulmonary lobules and the changes in follow-up studies of pulmonary atypical mycobacterial infection (AMI) by conventional and high-resolution computed tomography (HRCT). Forty-six patients (16 men and 30 women; 43-84 years) with pulmonary AMI (M. intracellulare 36; M. avium 10) in the lung were examined by conventional and HRCT. In peripheral zones, all patients had the nodule located in the terminal or lobular bronchiole, and most of the patients also had nodules accompanied with a wedge-shaped or linear shadow connected with the pleura. In the follow-up scans, new centrilobular nodules appeared in other segments, and consolidation or ground-glass pattern appeared newly and was preceded by nodules. Bronchiectasis became more severe in five of 38 follow-up patients. The common HRCT findings of AMI were centrilobular, peribronchovascular nodules, bronchiectasis, consolidation, and pleural thickening/adhesion. The nodules frequently connected with the pleura. The initial and follow-up studies suggest that the disease may begin in the terminal bronchiole or as preexisting bronchiectasis and spread transbronchially along the draining bronchus or towards the pleura to produce lesions such as new nodules, cavities, consolidation, pleuritis, and bronchiectasis, or more severe bronchiectasis. (author)

2001-01-01

 
 
 
 
281

The mycobacterial cord factor adjuvant analogue trehalose-6,6'-dibehenate (TDB) activates the Nlrp3 inflammasome.  

UK PubMed Central (United Kingdom)

The success of a vaccine consists in the induction of an innate immune response and subsequent activation of the adaptive immune system. Because antigens are usually not immunogenic, the addition of adjuvants that activate innate immunity is required. The mycobacterial cord factor trehalose-6,6'-dimycolate (TDM) and its synthetic adjuvant analogue trehalose-6,6'-dibehenate (TDB) rely on the C-type lectin Mincle and the signaling molecules Syk and Card9 to trigger innate immunity. In this study, we show that stimulation of bone marrow-derived dendritic cells (BMDCs) with TDB induces Nlrp3 inflammasome-dependent IL-1? secretion. While Card9 is required for NF-?B activation by TDB, it is dispensable for TDB-induced activation of the Nlrp3 inflammasome. Additionally, efflux of intracellular potassium, lysosomal rupture, and oxygen radical (ROS) production are crucial for caspase-1 processing and IL-1? secretion by TDB. In an in vivo inflammation model, we demonstrate that the recruitment of neutrophils by TDB is significantly reduced in the Nlrp3-deficient mice compared to the wild-type mice, while the production of chemokines in vitro is not influenced by the absence of Nlrp3. These results identify the Nlrp3 inflammasome as an essential mediator for the induction of an innate immune response triggered by TDB.

Schweneker K; Gorka O; Schweneker M; Poeck H; Tschopp J; Peschel C; Ruland J; Gross O

2013-04-01

282

Application of Mycobacterial Interspersed Repetitive Unit Typing to Manitoba Tuberculosis Cases: Can Restriction Fragment Length Polymorphism Be Forgotten?  

Science.gov (United States)

Since 1993, all Mycobacterium tuberculosis isolates recovered in the province of Manitoba, Canada, have been genotyped by the standard IS6110-restriction fragment length polymorphism (RFLP) method for routine surveillance, prevention, and control purposes. To date, our laboratory has collected 1,290 isolates, from which we have identified approximately 390 unique fingerprint patterns or “types.” Although the standard method is well known for being a lengthy and labor-intensive procedure, a more efficient alternative for typing tuberculosis isolates, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method, has recently gained acceptance. Consequently, all isolates acquired in 2003 (n = 126) were typed by both methods in order to determine the utility of replacing the RFLP method with MIRU typing for all future isolates. Application of Hunter's discriminatory index to the available study population showed that the MIRU method was close in discriminatory power (D) to the RFLP method (DMIRU = 0.831 to 0.984 versus DRFLP = 0.821 to 0.997). Clustering of isolates by using MIRU data correlated with RFLP-derived clustering, lending useful information for either an investigation or confirmation of an incidence of recent transmission. In addition, it was determined that each predominant RFLP type in Manitoba had a corresponding, recognizable MIRU type. It is conceivable that in the future RFLP typing can be replaced with MIRU for real-time, ongoing tuberculosis surveillance in the province.

Blackwood, K. S.; Wolfe, J. N.; Kabani, A. M.

2004-01-01

283

Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten?  

UK PubMed Central (United Kingdom)

Since 1993, all Mycobacterium tuberculosis isolates recovered in the province of Manitoba, Canada, have been genotyped by the standard IS6110-restriction fragment length polymorphism (RFLP) method for routine surveillance, prevention, and control purposes. To date, our laboratory has collected 1,290 isolates, from which we have identified approximately 390 unique fingerprint patterns or "types." Although the standard method is well known for being a lengthy and labor-intensive procedure, a more efficient alternative for typing tuberculosis isolates, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method, has recently gained acceptance. Consequently, all isolates acquired in 2003 (n = 126) were typed by both methods in order to determine the utility of replacing the RFLP method with MIRU typing for all future isolates. Application of Hunter's discriminatory index to the available study population showed that the MIRU method was close in discriminatory power (D) to the RFLP method (D(MIRU) = 0.831 to 0.984 versus D(RFLP) = 0.821 to 0.997). Clustering of isolates by using MIRU data correlated with RFLP-derived clustering, lending useful information for either an investigation or confirmation of an incidence of recent transmission. In addition, it was determined that each predominant RFLP type in Manitoba had a corresponding, recognizable MIRU type. It is conceivable that in the future RFLP typing can be replaced with MIRU for real-time, ongoing tuberculosis surveillance in the province.

Blackwood KS; Wolfe JN; Kabani AM

2004-11-01

284

Mycobacterial species diversity at a general hospital on the island of Crete: first detection of Mycobacterium lentiflavum in Greece.  

UK PubMed Central (United Kingdom)

The objective of the present study was to investigate the diversity of mycobacterial isolates in a general hospital in Crete, Greece. 48 positive Lowenstein-Jensen cultures over a 3-y period were analysed by means of AccuProbe and GenoType assays. Non-tuberculous mycobacteria (NTM) comprised the majority of the isolates (56.3%, 27/48) vs 33.3% (16/48) of M. tuberculosis; 10.4% of the isolates could not be classified. Among NTM, M. lentiflavum was the predominant species isolated (9/27) followed by M. kansasii, M. gordonae and M. peregrinum, whereas no M. avium complex isolates were detected. This is the first detection of M. lentiflavum in Greece. The susceptibilities of the M. lentiflavum isolates to an extended panel of antibiotics were determined by the proportions method and the medical files of the 9 patients were reviewed. Three isolates were from urine, which is an unusual site. All strains exhibited multidrug resistance. The patients were adults with immunosuppression or predisposing conditions for NTM infection. Diagnosis of true infection was either not pursued or the patients died shortly after isolation.

Neonakis IK; Gitti Z; Kourbeti IS; Michelaki H; Baritaki M; Alevraki G; Papadomanolaki E; Tsafaraki E; Tsouri A; Baritaki S; Krambovitis E; Spandidos DA

2007-01-01

285

Mycobacterial species diversity at a general hospital on the island of Crete: first detection of Mycobacterium lentiflavum in Greece.  

Science.gov (United States)

The objective of the present study was to investigate the diversity of mycobacterial isolates in a general hospital in Crete, Greece. 48 positive Lowenstein-Jensen cultures over a 3-y period were analysed by means of AccuProbe and GenoType assays. Non-tuberculous mycobacteria (NTM) comprised the majority of the isolates (56.3%, 27/48) vs 33.3% (16/48) of M. tuberculosis; 10.4% of the isolates could not be classified. Among NTM, M. lentiflavum was the predominant species isolated (9/27) followed by M. kansasii, M. gordonae and M. peregrinum, whereas no M. avium complex isolates were detected. This is the first detection of M. lentiflavum in Greece. The susceptibilities of the M. lentiflavum isolates to an extended panel of antibiotics were determined by the proportions method and the medical files of the 9 patients were reviewed. Three isolates were from urine, which is an unusual site. All strains exhibited multidrug resistance. The patients were adults with immunosuppression or predisposing conditions for NTM infection. Diagnosis of true infection was either not pursued or the patients died shortly after isolation. PMID:17852893

Neonakis, Ioannis K; Gitti, Zoe; Kourbeti, Irene S; Michelaki, Helen; Baritaki, Maria; Alevraki, Georgia; Papadomanolaki, Evangelia; Tsafaraki, Ekaterini; Tsouri, Anna; Baritaki, Stavroula; Krambovitis, Elias; Spandidos, Demetrios A

2007-01-01

286

Mycobacterial and nonbacterial pulmonary complications in hospitalized patients with human immunodeficiency virus infection: A prospective, cohort study  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background A prospective observational study was done to describe nonbacterial pulmonary complications in hospitalized patients with human immunodeficiency virus (HIV) infection. Methods The study included 1,225 consecutive hospital admissions of 599 HIV-infected patients treated from April 1995 through March 1998. Data included demographics, risk factors for HIV infection, Acute Physiology and Chronic Health Evaluation (APACHE) II score, pulmonary complications, CD4+ lymphocyte count, hospital stay and case-fatality rate. Results Patient age (mean ± SD) was 38.2 ± 8.9 years, 62% were men, and 84% were African American. The median APACHE II score was 14, and median CD4+ lymphocyte count was 60/?L. Pulmonary complications were Pneumocystis carinii pneumonia (85) in 78 patients, Mycobacterium avium complex (51) in 38, Mycobacterium tuberculosis (40) in 35, Mycobacterium gordonae (11) in 11, Mycobacterium kansasii (10) in 9, Cytomegalovirus (10) in 10, Nocardia asteroides (3) in 3, fungus ball (2) in 2, respiratory syncytial virus (1), herpes simplex virus (1), Histoplasma capsulatum (1), lymphoma (3) in 3, bronchogenic carcinoma (2) in 2, and Kaposi sarcoma (1). The case-fatality rate of patients was 11% with Pneumocystis carinii pneumonia; 5%, Mycobacterium tuberculosis; 6%, Mycobacterium avium complex; and 7%, noninfectious pulmonary complications. Conclusion Most pulmonary complications in hospitalized patients with HIV are from Pneumocystis and mycobacterial infection.

Afessa Bekele

2001-01-01

287

Elisa protocol for rapid screening of potential anti-tubercular drugs based on antigenic reactivity of mycobacterial ES-31 serine protease - a drug target supported by axenic culture of Mycobacterium tuberculosis H37 Ra strain in the presence of inhibitor.  

UK PubMed Central (United Kingdom)

BACKGROUND: Mycobacterial ES-31 serine protease has been reported to be a drug target using protease and lipase inhibitors in axenic and macrophage cultures. Simple screening techniques are needed for rapid testing of anti-tubercular drugs. AIM: To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity. MATERIAL AND METHODS: Indirect ELISA for assessment of antigenic reactivity of mycobacterial ES-31 serine protease was standardized using ES-31Ag and anti-DSS-goat-serum and assessed the inhibition of the antigenic reactivity by isoniazid, an anti-tubercular drug and serine protease inhibitor and orlistat, a lipase inhibitor. RESULTS: Optimal antigenic reactivity of mycobacterial ES-31 serine protease was observed at 5 microg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4 microg/well, while orlistat showed inhibition of 60% at 0.5 microg/well. Inhibition of Mtb H,37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4 microg/well and orlistat at 0.5 microg/well were observed respectively on bacterial growth. CONCLUSION: Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs.

Hutke V; Wankhade G; Waghmare PJ; Harinath BC

2013-07-01

288

In Vitro Gene Expression Dissected: Chemostat Surgery for Mycobacterium Tuberculosis  

Directory of Open Access Journals (Sweden)

Full Text Available A unique approach, combining defined and reproducible in vitro models with DNA microarrays, has been developed to study environmental modulation of mycobacterial gene expression. The gene expression profiles of samples of Mycobacterium tuberculosis, from independent chemostat cultures grown under defined and reproducible conditions, were found to be highly correlated. This approach is now being used to study the effect of relevant stimuli, such as limited oxygen availability, on mycobacterial gene expression. A modification of the chemostat culture system, enabling largevolume controlled batch culture, has been developed to study starvation survival. Cultures of M. tuberculosis have been maintained under nutrient-starved conditions for extended periods, with 106 – 107 bacilli surviving in a culturable state after 100 days. The design of the culture system has made it possible to control the environment and collect multiple time-course samples to study patterns of gene expression. These studies demonstrate that it is possible to perform long-term studies and obtain reproducible expression data using controlled and defined in vitro models.

Brian W. James; Joanna Bacon; Tobias Hampshire; Kim Morley; Philip D. Marsh

2002-01-01

289

Synthetic UDP-galactofuranose analogs reveal critical enzyme-substrate interactions in GlfT2-catalyzed mycobacterial galactan assembly.  

UK PubMed Central (United Kingdom)

Mycobacterial cell wall galactan, composed of alternating ?-(1?5) and ?-(1?6) galactofuranosyl residues, is assembled by the action of two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2, which use UDP-galactofuranose (UDP-Galf) as the donor substrate. Kinetic analysis of synthetic UDP-Galf analogs identified critical interactions involved in donor substrate recognition by GlfT2, a processive polymerizing glycosyltransferase. Testing of methylated UDP-Galf analogs showed the donor substrate-binding pocket is sterically crowded. Evaluation of deoxy UDP-Galf analogs revealed that the C-6 hydroxyl group is not essential for substrate activity, and that interactions with the UDP-Galf C-3 hydroxyl group orient the substrate for turnover but appears to play no role in substrate recognition, making the 3-deoxy-analog a moderate competitive inhibitor of the enzyme. Moreover, the addition of a Galf residue deoxygenated at C-5 or C-6, or an l-arabinofuranose residue, to the growing galactan chain resulted in "dead end" reaction products, which no longer act as an acceptor for the enzyme. This finding shows dual recognition of both the terminal C-5 and C-6 hydroxyl groups of the acceptor substrate are required for GlfT2 activity, which is consistent with a recent model developed based upon a crystal structure of the enzyme. These observations provide insight into specific protein-carbohydrate interactions in the GlfT2 active site and may facilitate the design of future inhibitors.

Poulin MB; Zhou R; Lowary TL

2012-05-01

290

The mutation rate of mycobacterial repetitive unit loci in strains of M. tuberculosis from cynomolgus macaque infection.  

UK PubMed Central (United Kingdom)

BACKGROUND: Mycobacterial interspersed repetitive units (MIRUs) are minisatellites within the Mycobacterium tuberculosis (Mtb) genome. Copy number variation (CNV) in MIRU loci is used for epidemiological typing, making the rate of variation important for tracking the transmission of Mtb strains. In this study, we developed and assessed a whole-genome sequencing (WGS) approach to detect MIRU CNV in Mtb. We applied this methodology to a panel of Mtb strains isolated from the macaque model of tuberculosis (TB), the animal model that best mimics human disease. From these data, we have estimated the rate of MIRU variation in the host environment, providing a benchmark rate for future epidemiologic work. RESULTS: We assessed variation at the 24 MIRU loci used for typing in a set of Mtb strains isolated from infected cynomolgus macaques. We previously performed WGS of these strains and here have applied both read depth (RD) and paired-end mapping (PEM) metrics to identify putative copy number variants. To assess the relative power of these approaches, all MIRU loci were resequenced using Sanger sequencing. We detected two insertion/deletion events both of which could be identified as candidates by PEM criteria. With these data, we estimate a MIRU mutation rate of 2.70 × 10-03 (95% CI: 3.30 × 10-04- 9.80 × 10-03) per locus, per year. CONCLUSION: Our results represent the first experimental estimate of the MIRU mutation rate in Mtb. This rate is comparable to the highest previous estimates gathered from epidemiologic data and meta-analyses. Our findings allow for a more rigorous interpretation of data gathered from MIRU typing.

Ragheb MN; Ford CB; Chase MR; Lin PL; Flynn JL; Fortune SM

2013-01-01

291

Influenza A virus infection impairs mycobacteria-specific T cell responses and mycobacterial clearance in the lung during pulmonary coinfection.  

Science.gov (United States)

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-? CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria. PMID:23698750

Flórido, Manuela; Grima, Michael A; Gillis, Caitlin M; Xia, Yingju; Turner, Stephen J; Triccas, James A; Stambas, John; Britton, Warwick J

2013-05-22

292

Influenza A virus infection impairs mycobacteria-specific T cell responses and mycobacterial clearance in the lung during pulmonary coinfection.  

UK PubMed Central (United Kingdom)

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-? CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.

Flórido M; Grima MA; Gillis CM; Xia Y; Turner SJ; Triccas JA; Stambas J; Britton WJ

2013-07-01

293

Protein export by the mycobacterial SecA2 system is determined by the preprotein mature domain.  

UK PubMed Central (United Kingdom)

At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles "housekeeping" export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.

Feltcher ME; Gibbons HS; Ligon LS; Braunstein M

2013-02-01

294

Predicting results of mycobacterial culture on sputum smear reversion after anti-tuberculous treatment: a case control study  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Little is currently known regarding sputum smear reversion (acid-fast smear becomes positive again after negative conversion) during anti-tuberculous treatment. This study aimed to evaluate its occurrence in patients with pulmonary tuberculosis (TB) and identify factors predicting results of mycobacterial culture for smear-reversion of sputum samples. Methods The retrospective review was performed in a tertiary referral center and a local teaching hospital in Taiwan. From 2000 to 2007, patients with smear-positive culture-confirmed pulmonary TB experiencing smear reversion after 14 days of anti-tuberculous treatment were identified. Results The 739 patients with smear-positive pulmonary TB had 74 (10%) episodes of sputum smear reversion that grew Mycobacterium tuberculosis in 22 (30%) (Mtb group). The remaining 52 episodes of culture-negative sputum samples were classified as the non-Mtb group. The anti-tuberculous regimen was modified after confirming smear reversion in 15 (20%). Fourteen episodes in the Mtb group and 15 in the non-Mtb group occurred during hospitalization. All were admitted to the negative-pressure rooms at the time of smear reversion. Statistical analysis showed that any TB drug resistance, smear reversion within the first two months of treatment or before culture conversion, and the absence of radiographic improvement before smear reversion were associated with the Mtb group. None of the smear reversion was due to viable M. tuberculosis if none of the four factors were present. Conclusions Sputum smear reversion develops in 10% of patients with smear-positive pulmonary TB, with 30% due to viable M. tuberculosis bacilli. Isolation and regimen modification may not be necessary for all drug-susceptible patients who already have radiographic improvement and develop smear reversion after two months of treatment or after sputum culture conversion.

Shu Chin-Chung; Wang Jann-Tay; Lee Chih-Hsin; Wang Jann-Yuan; Lee Li-Na; Yu Chong-Jen

2010-01-01

295

Does neutralization of gastric aspirates from children with suspected intrathoracic tuberculosis affect mycobacterial yields on MGIT culture?  

Science.gov (United States)

The microbiological confirmation of pulmonary tuberculosis in children relies on cultures of gastric aspirate (GA) specimens. Conventionally, GAs are neutralized to improve culture yields of mycobacteria. However, there are limited data to support this practice. To study the utility of neutralization of GAs with sodium bicarbonate in children with intrathoracic tuberculosis, a total of 116 children of either sex, aged 6 months to 14 years (median age, 120 months; interquartile range [IQR], 7 to 192 months), underwent gastric aspiration on 2 consecutive days. Gastric aspirates were divided into two aliquots, and only one aliquot was neutralized with 1% sodium bicarbonate. Both aliquots were processed for smear and culture examinations. Out of the 232 gastric aspirates, 12 (5.17%) were acid-fast bacilli (AFB) smear positive. There were no differences in smear positivity rates from samples with or without neutralization. The yield of Mycobacterium tuberculosis on a Bactec MGIT 960 culture system was significantly lower in the neutralized samples (16.3% [38/232]) than in the nonneutralized samples (21.5% [50/232]) (P = 0.023). There was no significant difference between the neutralized and the nonneutralized samples in time to detection using the MGIT 960 system (average, 24.6 days; IQR, 12 to 37 days) (P = 0.9). The contamination rates were significantly higher in the neutralized samples than in the nonneutralized samples (17.2% [40/232] versus 3.9% [9/232]) (P = 0.001). The agreement for positive mycobacterial culture between the two approaches was 66.5% (P = 0.001). Hence, we recommend that gastric aspirate samples not be neutralized with sodium bicarbonate prior to culture for M. tuberculosis. PMID:23536406

Parashar, Deepak; Kabra, Sushil K; Lodha, Rakesh; Singh, Varinder; Mukherjee, Aparna; Arya, Tina; Grewal, Harleen M S; Singh, Sarman

2013-03-27

296

Does neutralization of gastric aspirates from children with suspected intrathoracic tuberculosis affect mycobacterial yields on MGIT culture?  

UK PubMed Central (United Kingdom)

The microbiological confirmation of pulmonary tuberculosis in children relies on cultures of gastric aspirate (GA) specimens. Conventionally, GAs are neutralized to improve culture yields of mycobacteria. However, there are limited data to support this practice. To study the utility of neutralization of GAs with sodium bicarbonate in children with intrathoracic tuberculosis, a total of 116 children of either sex, aged 6 months to 14 years (median age, 120 months; interquartile range [IQR], 7 to 192 months), underwent gastric aspiration on 2 consecutive days. Gastric aspirates were divided into two aliquots, and only one aliquot was neutralized with 1% sodium bicarbonate. Both aliquots were processed for smear and culture examinations. Out of the 232 gastric aspirates, 12 (5.17%) were acid-fast bacilli (AFB) smear positive. There were no differences in smear positivity rates from samples with or without neutralization. The yield of Mycobacterium tuberculosis on a Bactec MGIT 960 culture system was significantly lower in the neutralized samples (16.3% [38/232]) than in the nonneutralized samples (21.5% [50/232]) (P = 0.023). There was no significant difference between the neutralized and the nonneutralized samples in time to detection using the MGIT 960 system (average, 24.6 days; IQR, 12 to 37 days) (P = 0.9). The contamination rates were significantly higher in the neutralized samples than in the nonneutralized samples (17.2% [40/232] versus 3.9% [9/232]) (P = 0.001). The agreement for positive mycobacterial culture between the two approaches was 66.5% (P = 0.001). Hence, we recommend that gastric aspirate samples not be neutralized with sodium bicarbonate prior to culture for M. tuberculosis.

Parashar D; Kabra SK; Lodha R; Singh V; Mukherjee A; Arya T; Grewal HM; Singh S

2013-06-01

297

Diverse humoral immune responses and changes in IgG antibody levels against mycobacterial lipid antigens in active tuberculosis.  

Science.gov (United States)

Humoral immune responses of active TB patients against six mycobacterial lipid antigens [trehalose 6,6'-dimycolate (TDM) from Mycobacterium bovis BCG (TDM-T) and Mycobacterium avium complex (TDM-M), trehalose 6-monomycolate (TMM) from M. bovis BCG (TMM-T) and M. avium complex (TMM-M), triacyl (PL-2) and tetraacyl (PL-1) phosphatidylinositol dimannosides] were examined by ELISA. IgG antibodies of TB patients with active disease reacted against the six lipid antigens distinctively, but heterogeneously. If tests were combined and an overall positive was scored cumulatively when any one of the six tests was positive, a good discrimination between patient and normal subject was obtained. A positive result in any one of the six tests was obtained in 91.5% of all 924 hospitalized patients and 93.3% of 210 patients at their first visit to the outpatient clinic. The IgG antibody response differed considerably from patient to patient, and the response patterns were grouped into several types. IgG antibody levels paralleled the bacterial burden; however, the smear-negative (culture-positive) patient group also showed high positive rates and mean ELISA DeltaA values against the six lipid antigens. There were also marked differences in positive rate and mean DeltaA values between cavity-positive and -negative groups, the former being higher than the latter. After anti-TB chemotherapy was initiated, IgG antibody levels decreased dramatically, paralleling the decrease in the amount of excretion of bacteria. Since multiple-antigen ELISA using particular lipid antigens was highly sensitive, and IgG antibody levels vary greatly at different stages of the disease, this technique is applicable for early diagnosis of smear-negative (and -positive) active TB and the prognosis for completion of anti-TB chemotherapy. PMID:15942013

Fujita, Yukiko; Doi, Takeshi; Sato, Koji; Yano, Ikuya

2005-06-01

298

Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.  

Science.gov (United States)

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies. PMID:17005759

Supply, Philip; Allix, Caroline; Lesjean, Sarah; Cardoso-Oelemann, Mara; Rüsch-Gerdes, Sabine; Willery, Eve; Savine, Evgueni; de Haas, Petra; van Deutekom, Henk; Roring, Solvig; Bifani, Pablo; Kurepina, Natalia; Kreiswirth, Barry; Sola, Christophe; Rastogi, Nalin; Vatin, Vincent; Gutierrez, Maria Cristina; Fauville, Maryse; Niemann, Stefan; Skuce, Robin; Kremer, Kristin; Locht, Camille; van Soolingen, Dick

2006-09-27

299

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis? †  

Science.gov (United States)

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%—and by 23% in combination with spoligotyping—among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold—by threefold in combination with spoligotyping—under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

Supply, Philip; Allix, Caroline; Lesjean, Sarah; Cardoso-Oelemann, Mara; Rusch-Gerdes, Sabine; Willery, Eve; Savine, Evgueni; de Haas, Petra; van Deutekom, Henk; Roring, Solvig; Bifani, Pablo; Kurepina, Natalia; Kreiswirth, Barry; Sola, Christophe; Rastogi, Nalin; Vatin, Vincent; Gutierrez, Maria Cristina; Fauville, Maryse; Niemann, Stefan; Skuce, Robin; Kremer, Kristin; Locht, Camille; van Soolingen, Dick

2006-01-01

300

The DosS-DosT/DosR Mycobacterial Sensor System  

Directory of Open Access Journals (Sweden)

Full Text Available DosS/DosR is a two-component regulatory system in which DosS, a heme-containing sensor also known as DevS, under certain conditions undergoes autophosphorylation and then transfers the phosphate to DosR, a DNA-binding protein that controls the entry of Mycobacterium tuberculosis and other mycobacteria into a latent, dormant state. DosT, a second sensor closely related to DosS, is present in M. tuberculosis and participates in the control of the dormancy response mediated by DosR. The binding of phosphorylated DosR to DNA initiates the expression of approximately fifty dormancy-linked genes. DosT is accepted to be a gas sensor that is activated in the ferrous state by the absence of an oxygen ligand or by the binding of NO or CO. DosS functions in a similar fashion as a gas sensor, but contradictory evidence has led to the suggestion that it also functions as a redox state sensor. This review focuses on the structure, biophysical properties, and function of the DosS/DosT heme sensors.

Santhosh Sivaramakrishnan; Paul R. Ortiz de Montellano

2013-01-01

 
 
 
 
301

Molecular characterization of Mycobacterium tuberculosis H37Rv/Ra variants: distinguishing the mycobacterial laboratory strain.  

UK PubMed Central (United Kingdom)

The Mycobacterium tuberculosis strains H37Rv and H37Ra are the most commonly used controls for M. tuberculosis identification in the clinical and research laboratory setting. To reduce the likelihood of misidentification and possible cross-contamination with this laboratory neotype, it is important to be able to distinguish H37 from clinical isolates. To provide a reference for identifying H37, we used multiple molecular techniques to characterize H37 strains, including 18 of the most frequently used variants available through the American Type Culture Collection. Isolates were genotyped using gene probes to IS6110 and IS1085. In addition, we performed polymorphic GC-rich sequence typing (PGRS), spoligotyping, determination of variable number of tandem repeats (VNTR), and PCR amplification of the mtp40, msx4, and mpp8 polymorphic regions. Southern hybridization with IS6110 provided the most discrimination, differentiating the 18 H37 isolates into 10 discrete patterns made up of 9 H37Rv variants and 1 H37Ra variant. PGRS, IS1085, mpp8, and spoligotyping were not able to distinguish any H37 variants, while VNTR and msx4 discriminated two. Only IS6110 and spoligotyping could distinguish the H37 strain from clinical isolates. In summary, spoligotyping and IS6110 provide a rapid and accurate way to identify H37 contamination, though IS6110 can, in addition, classify many of the H37 variants that would otherwise require phenotypic segregation.

Bifani P; Moghazeh S; Shopsin B; Driscoll J; Ravikovitch A; Kreiswirth BN

2000-09-01

302

Molecular characterization of Mycobacterium tuberculosis H37Rv/Ra variants: distinguishing the mycobacterial laboratory strain.  

Science.gov (United States)

The Mycobacterium tuberculosis strains H37Rv and H37Ra are the most commonly used controls for M. tuberculosis identification in the clinical and research laboratory setting. To reduce the likelihood of misidentification and possible cross-contamination with this laboratory neotype, it is important to be able to distinguish H37 from clinical isolates. To provide a reference for identifying H37, we used multiple molecular techniques to characterize H37 strains, including 18 of the most frequently used variants available through the American Type Culture Collection. Isolates were genotyped using gene probes to IS6110 and IS1085. In addition, we performed polymorphic GC-rich sequence typing (PGRS), spoligotyping, determination of variable number of tandem repeats (VNTR), and PCR amplification of the mtp40, msx4, and mpp8 polymorphic regions. Southern hybridization with IS6110 provided the most discrimination, differentiating the 18 H37 isolates into 10 discrete patterns made up of 9 H37Rv variants and 1 H37Ra variant. PGRS, IS1085, mpp8, and spoligotyping were not able to distinguish any H37 variants, while VNTR and msx4 discriminated two. Only IS6110 and spoligotyping could distinguish the H37 strain from clinical isolates. In summary, spoligotyping and IS6110 provide a rapid and accurate way to identify H37 contamination, though IS6110 can, in addition, classify many of the H37 variants that would otherwise require phenotypic segregation. PMID:10970357

Bifani, P; Moghazeh, S; Shopsin, B; Driscoll, J; Ravikovitch, A; Kreiswirth, B N

2000-09-01

303

Molecular Characterization of Mycobacterium tuberculosis H37Rv/Ra Variants: Distinguishing the Mycobacterial Laboratory Strain†  

Science.gov (United States)

The Mycobacterium tuberculosis strains H37Rv and H37Ra are the most commonly used controls for M. tuberculosis identification in the clinical and research laboratory setting. To reduce the likelihood of misidentification and possible cross-contamination with this laboratory neotype, it is important to be able to distinguish H37 from clinical isolates. To provide a reference for identifying H37, we used multiple molecular techniques to characterize H37 strains, including 18 of the most frequently used variants available through the American Type Culture Collection. Isolates were genotyped using gene probes to IS6110 and IS1085. In addition, we performed polymorphic GC-rich sequence typing (PGRS), spoligotyping, determination of variable number of tandem repeats (VNTR), and PCR amplification of the mtp40, msx4, and mpp8 polymorphic regions. Southern hybridization with IS6110 provided the most discrimination, differentiating the 18 H37 isolates into 10 discrete patterns made up of 9 H37Rv variants and 1 H37Ra variant. PGRS, IS1085, mpp8, and spoligotyping were not able to distinguish any H37 variants, while VNTR and msx4 discriminated two. Only IS6110 and spoligotyping could distinguish the H37 strain from clinical isolates. In summary, spoligotyping and IS6110 provide a rapid and accurate way to identify H37 contamination, though IS6110 can, in addition, classify many of the H37 variants that would otherwise require phenotypic segregation.

Bifani, P.; Moghazeh, S.; Shopsin, B.; Driscoll, J.; Ravikovitch, A.; Kreiswirth, B. N.

2000-01-01

304

Greater pre-existing interferon gamma responses to mycobacterial antigens are associated with lower bacillary load during HIV-associated tuberculosis.  

UK PubMed Central (United Kingdom)

The role of pre-existing interferon gamma (IFN-?) responses in controlling bacillary burden in HIV-associated tuberculosis (TB) is not known. Among BCG-immunized HIV-infected adults who developed TB in a phase III trial of an investigational TB vaccine, greater baseline IFN-? responses to ESAT-6 and Mycobacterium tuberculosis whole cell lysate were associated with reduced bacillary burden on sputum smear grade, days to culture positivity on agar, and sputum culture grade during subsequent tuberculosis. This association was most consistent among recipients of the investigational vaccine. When HIV-associated TB develops, greater pre-existing IFN-? responses to mycobacterial antigens are associated with reduced TB bacillary burden.

Lahey T; Czechura T; Crabtree S; Arbeit RD; Matee M; Horsburgh CR; Mackenzie T; Bakari M; Pallangyo K; von Reyn CF

2013-08-01

305

Mycobacterium bourgelatii sp. nov., a rapidly growing, non-chromogenic species isolated from the lymph nodes of cattle.  

UK PubMed Central (United Kingdom)

Three independent strains of a rapidly growing, non-chromogenic Mycobacterium were isolated from lymph nodes of French cattle. Identification of the isolates was carried out using a polyphasic approach. The nearly complete SSUrRNA gene sequences (> 1,200 bp) of the MLB-A23, MLB-A30, and MLB-A84T strains were identical. A phylogenetic analysis of these unique SSUrRNA gene sequences showed that these strains were most closely related to Mycobacterium intermedium. Further phylogenetic analysis based on concatenated sequences (2,854 bp) of four housekeeping genes (hsp65, rpoB, sodA, tuf), the tmRNA and SSUrRNA genes indicated that these three strains represented a distinct species that shares a common ancestor with M. intermedium. Phylogenetic and phenotypic data strongly indicate that the MLB-A23, MLB-A30 and MLB-A84T strains belong to a new mycobacterial species for which the name Mycobacterium bourgelatii sp. nov. is proposed. The type strain is MLB-A84T (=DSM 45746T= CIP110557T).

Guérin-Faublée V; Flandrois JP; Pichat C; Boschiroli ML; Lamy B

2013-08-01

306

[Use of different PCR-based techniques integrated into a non-tuberculous identification algorithm].  

UK PubMed Central (United Kingdom)

INTRODUCTION: The aim of the present work was to demonstrate the utility of a non-tuberculous mycobacteria (NTM) identification algorithm, which integrates different PCR-based techniques and basic phenotypic features. Moreover, the algorithm for pattern restriction analysis of hsp65 (hsp65 PRA) interpretation has been updated. METHODS: The workflow chosen consisted of the identification by a DNA hybridization probe method, followed by PCR-restriction enzyme analysis of hsp65 (hsp65 PRA) in those isolates that cannot be identified by hybridization probes. If necessary, 16S rRNA gene and hsp65 gene sequencing were used for speciation. RESULTS: A total of 236 NTM were collected, in which 102 (43.2%) isolates were identified by DNA specific probes and 76 (32.2%) isolates were identified with hsp65 PRA. Partial sequencing of the 16S rRNA gene was used for species identification of the remaining 58 (24.5%) isolates. Fifty-three (22.4%) were identified using this method. Five isolates (2.1%) were submitted for partial sequencing of hsp65 gene and one isolate was identified with this method. Four strains (1.7%) could not be identified at species level. Three new PRA patterns were found. Seven isolates tested positive with the AccuProbe Mycobacterium avium complex identification test but did not test positive with the M. avium or Mycobacterium intracellulare specific probes. Five and two of these isolates were identified as M. intracellulare and Mycobacterium colombiense, respectively. CONCLUSION: This approach allowed us to identify almost all NTM isolates found in this study, including some recently described species.

Esparcia Ó; Español M; Garrigó M; Moreno C; Montemayor M; Navarro F; Coll P

2012-01-01

307

High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.  

UK PubMed Central (United Kingdom)

Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS). Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

Mestre O; Hurtado-Ortiz R; Dos Vultos T; Namouchi A; Cimino M; Pimentel M; Neyrolles O; Gicquel B

2013-01-01

308

Triclosan inhibition of mycobacterial InhA in Saccharomyces cerevisiae: yeast mitochondria as a novel platform for in vivo antimycolate assays.  

UK PubMed Central (United Kingdom)

AIMS: To demonstrate the suitability of yeast to act as a novel biotechnological platform for conducting in vivo inhibition assays using drugs with low efficacies towards their mycobacterial targets, such as occurs in the situation with triclosan and InhA. METHODS AND RESULTS: A surrogate yeast host represented by Saccharomyces cerevisiae etr1Delta cells lacking Etr1p, the 2-trans-enoyl-thioester reductase of mitochondrial type 2 fatty acid synthase (FASII), was designed to rely on the Mycobacterium tuberculosis FASII enzyme InhA. Although InhA is 10,000 times less sensitive to the antimicrobial drug triclosan than is bacterial FabI, the respiratory growth of yeast cells depending on InhA was severely affected on glycerol medium containing triclosan. CONCLUSIONS: The yeast system could detect enzyme inhibition despite the use of a drug with only low efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: Tuberculosis affects a third of the human population, and InhA is a major drug target for combating this disease. InhA is inhibited by isoniazid, but triclosan-derived compounds are presently being developed as antimycolates. A demonstration of triclosan inhibition of InhA in yeast represents a meaningful variation in studying this effect in mycobacteria, because it occurred without the potentially confusing aspects of perturbing protein-protein interactions which are presumed vital to mycobacterial FASII, inactivating other important enzymes or eliciting a dedicated transcriptional response in Myco. tuberculosis.

Gurvitz A

2010-04-01

309

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

Energy Technology Data Exchange (ETDEWEB)

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.)

Chung, Myung Jin; Lee, Kyung Soo; Kim, Tae Sung; Kim, Sung Mok [Sungkyunkwan University School of Medicine, Department of Radiology and Center for Imaging Science, Samsung Medical Center, Seoul (Korea); Koh, Won-Jung; Kwon, O Jung [Sungkyunkwan University School of Medicine, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center, Seoul (Korea); Kang, Eun Young [Korea University Guro Hospital, Department of Diagnostic Radiology, Korea University College of Medicine, Seoul (Korea); Kim, Seonwoo [Sungkyunkwan University School of Medicine, Biostatistics Unit of the Samsung Biomedical Research Institute, Samsung Medical Center, Seoul (Korea)

2006-09-15

310

[Cervical adenitis caused by Mycobacterium kansasii: advantage of the INNO-LiPA V2 test in diagnosis of nontuberculous mycobacterial diseases  

UK PubMed Central (United Kingdom)

Mycobacterium kansasii is one of the main mycobacterial species. Among the numerous subtypes, I and II subtypes are the only pathogens ones. A 56 years old woman has been under observation during her hospitalization for a cervical adenitis check-up; these one having been evolving for 2 months, without inflammatory syndrome. The failures of various antibiotics treatments lead one to withdraw some cervical ganglions whose histopathologic aspect reminds tuberculosis. These adenitis being isolated. An INH, rifampin, and ethambutol associated treatment is set up. After 2 weeks, the different cultures enable to isolated numerous photochromogenic colonies; a positive result is obtained with M. kansasii probe (Accuprobe). The identification is confirmed in 24 hours time with another molecular hybridation test (INNO-LiPA V2); the various specific probes of the different M. kansasii subtypes show it's not a I or II subtype (subtype IV by sequence analysis). This observation underlines the advantage and rapidity of the new diagnosis methods for nontuberculous mycobacterial diseases.

Salles Y; Fabre M; Guitterez MC; Chaudier B; Soler C

2007-12-01

311

Mycobacterium yongonense sp. nov., a slow-growing non-chromogenic species closely related to Mycobacterium intracellulare.  

UK PubMed Central (United Kingdom)

A slow-growing non-chromogenic mycobacterium was isolated from a patient with pulmonary disease. Phenotypically, strain 05-1390(T) was similar to Mycobacterium intracellulare ATCC 13950(T). The 16S rRNA gene sequence (1385 bp) of strain 05-1390(T) showed a high degree of similarity to those of the M. intracellulare complex, namely Mycobacterium marseillense 5351974(T) (100 %), M. intracellulare ATCC 13950(T) (99.8 %) and Mycobacterium chimaera DSM 44623(T) (99.9 %). Phylogenetic analysis based on internal transcribed spacer 1 (ITS1) and the hsp65 gene indicated that strain 05-1390(T) was closely related to M. intracellulare ATCC 13950(T), but that it was a distinct phylogenetic entity. Of particular interest, an analysis based on the rpoB gene (701 bp) showed that it is closely related to Mycobacterium parascrofulaceum ATCC BAA-614(T) (99.4 %), a scotochromogenic strain, rather than to the M. intracellulare-related strains. Unique MALDI-TOF MS profiles also supported the taxonomic status of this strain as a distinct species. These data support the conclusion that strain 05-1390(T) represents a novel mycobacterial species, for which the name Mycobacterium yongonense sp. nov. is proposed; the type strain is 05-1390(T) ( = DSM 45126(T) = KCTC 19555(T)).

Kim BJ; Math RK; Jeon CO; Yu HK; Park YG; Kook YH; Kim BJ

2013-01-01

312

Dipterinyl calcium pentahydrate inhibits intracellular mycobacterial growth in human monocytes via the C-C chemokine MIP-1? and nitric oxide.  

UK PubMed Central (United Kingdom)

Tuberculosis remains one of the top three leading causes of morbidity and mortality worldwide, complicated by the emergence of drug-resistant Mycobacterium tuberculosis strains and high rates of HIV coinfection. It is important to develop new antimycobacterial drugs and immunomodulatory therapeutics and compounds that enhance antituberculous immunity. Dipterinyl calcium pentahydrate (DCP), a calcium-complexed pterin compound, has previously been shown to inhibit human breast cancer cells and hepatitis B virus (HBV). DCP inhibitory effects were attributed to induction of apoptosis and/or increased production of interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study, we tested the ability of DCP to mediate inhibition of intracellular mycobacteria within human monocytes. DCP treatment of infected monocytes resulted in a significant reduction in viability of intracellular but not extracellular Mycobacterium bovis BCG. The antimicrobial activity of DCP was comparable to that of pyrazinamide (PZA), one of the first-line antituberculosis drugs currently used. DCP potentiated monocyte antimycobacterial activity by induction of the cysteine-cysteine (C-C) chemokine macrophage inflammatory protein 1? (MIP-1?) and inducible nitric oxide synthase 2. Addition of human anti-MIP-1? neutralizing antibody or a specific inhibitor of the l-arginase-nitric oxide pathway (N(G)-monomethyl l-arginine [l-NMMA] monoacetate) reversed the inhibitory effects of DCP on intracellular mycobacterial growth. These findings indicate that DCP induced mycobacterial killing via MIP-1?- and nitric oxide-dependent effects. Hence, DCP acts as an immunoregulatory compound enhancing the antimycobacterial activity of human monocytes.

Sakala IG; Eickhoff CS; Blazevic A; Moheno P; Silver RF; Hoft DF

2013-06-01

313

A time-to-event pharmacodynamic model describing treatment response in patients with pulmonary tuberculosis using days to positivity in automated liquid mycobacterial culture.  

UK PubMed Central (United Kingdom)

Days to positivity in automated liquid mycobacterial culture have been shown to correlate with mycobacterial load and have been proposed as a useful biomarker for treatment responses in tuberculosis. However, there is currently no quantitative method or model to analyze the change in days to positivity with time on treatment. The objectives of this study were to describe the decline in numbers of mycobacteria in sputum collected once weekly for 8 weeks from patients on treatment for tuberculosis using days to positivity in liquid culture. One hundred forty-four patients with smear-positive pulmonary tuberculosis were recruited from a tuberculosis clinic in Cape Town, South Africa. A nonlinear mixed-effects repeated-time-to-event modeling approach was used to analyze the time-to-positivity data. A biexponential model described the decline in the estimated number of bacteria in patients' sputum samples, while a logistic model with a lag time described the growth of the bacteria in liquid culture. At baseline, the estimated number of rapidly killed bacteria is typically 41 times higher than that of those that are killed slowly. The time to kill half of the rapidly killed bacteria was about 1.8 days, while it was 39 days for slowly killed bacteria. Patients with lung cavitation had higher bacterial loads than patients without lung cavitation. The model successfully described the increase in days to positivity as treatment progressed, differentiating between bacteria that are killed rapidly and those that are killed slowly. Our model can be used to analyze similar data from studies testing new drug regimens.

Chigutsa E; Patel K; Denti P; Visser M; Maartens G; Kirkpatrick CM; McIlleron H; Karlsson MO

2013-02-01

314

Mycobacterium salmoniphilum infection in burbot Lota lota.  

Science.gov (United States)

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

Zerihun, Mulualem Adam; Berg, Vidar; Lyche, Jan L; Colquhoun, Duncan J; Poppe, Trygve T

2011-05-24

315

Mycobacterium salmoniphilum infection in burbot Lota lota.  

UK PubMed Central (United Kingdom)

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.

Zerihun MA; Berg V; Lyche JL; Colquhoun DJ; Poppe TT

2011-05-01

316

Evaluation of INNO-LiPA mycobacteria v2 assay for identification of rapidly growing mycobacteria.  

UK PubMed Central (United Kingdom)

A total of 54 rapidly growing mycobacteria (RGM) isolated from patients attended in the two hospitals of Cádiz Bay (Spain) were selected during a seven-year-period (2000-2006) in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Löwenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzyme analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3%) were correctly identified by conventional techniques and 47 strains (87.0%) by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group.

García-Agudo L; Jesús I; Rodríguez-Iglesias M; García-Martos P

2011-07-01

317

Evaluation of INNO-LiPA mycobacteria v2 assay for identification of rapidly growing mycobacteria.  

Science.gov (United States)

A total of 54 rapidly growing mycobacteria (RGM) isolated from patients attended in the two hospitals of Cádiz Bay (Spain) were selected during a seven-year-period (2000-2006) in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Löwenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzyme analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3%) were correctly identified by conventional techniques and 47 strains (87.0%) by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group. PMID:24031745

García-Agudo, Lidia; Jesús, Iría; Rodríguez-Iglesias, Manuel; García-Martos, Pedro

2011-09-01

318

Evaluation of INNO-LiPA mycobacteria v2 assay for identification of rapidly growing mycobacteria  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english A total of 54 rapidly growing mycobacteria (RGM) isolated from patients attended in the two hospitals of Cádiz Bay (Spain) were selected during a seven-year-period (2000-2006) in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Löwenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzym (more) e analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3%) were correctly identified by conventional techniques and 47 strains (87.0%) by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group.

García-Agudo, Lidia; Jesús, Iría; Rodríguez-Iglesias, Manuel; García-Martos, Pedro

2011-09-01

319

Evaluation of INNO-LiPA mycobacteria v2 assay for identification of rapidly growing mycobacteria  

Directory of Open Access Journals (Sweden)

Full Text Available A total of 54 rapidly growing mycobacteria (RGM) isolated from patients attended in the two hospitals of Cádiz Bay (Spain) were selected during a seven-year-period (2000-2006) in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Löwenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzyme analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3%) were correctly identified by conventional techniques and 47 strains (87.0%) by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group.

Lidia García-Agudo; Iría Jesús; Manuel Rodríguez-Iglesias; Pedro García-Martos

2011-01-01

320

The mycobacterial binuclear iron monooxygenases require a specific chaperonin-like protein for functional expression in a heterologous host.  

Science.gov (United States)

The mimABCD gene clusters in Mycobacterium smegmatis strain mc(2) 155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the Rhodococcus-Escherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD. PMID:23171424

Furuya, Toshiki; Hayashi, Mika; Semba, Hisashi; Kino, Kuniki

2013-01-02

 
 
 
 
321

The mycobacterial binuclear iron monooxygenases require a specific chaperonin-like protein for functional expression in a heterologous host.  

UK PubMed Central (United Kingdom)

The mimABCD gene clusters in Mycobacterium smegmatis strain mc(2) 155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the Rhodococcus-Escherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.

Furuya T; Hayashi M; Semba H; Kino K

2013-02-01

322

MASSIVE PRODUCTION OF MYCOBACTERIAL GLUCOSYL-3-PHOSPHOGLYCERATE SYNTHASE AND THE USE OF THIS ENZYME AS A TARGET FOR ANTIMYCOBACTERIAL SUBSTANCES  

UK PubMed Central (United Kingdom)

The present invention describes the isolation, identification and characterization of a gene (gpgS) and of the function of the corresponding enzyme responsible for the synthesis of glucosyl-3-phosphoglycerate (GpgS), the precursor of glucosylglycerate, a component of the methylglucose polysaccharides from mycobacteria and some closely related bacteria. This gene is conserved among mycobacteria and is essential for the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis.; The invention discloses the gpgS gene, processes for production of the enzymes encoded by the genes from Mycobacterium bovis and Mycobacterium smegmatis, the properties of the enzymes, the construction of plasmids that include the genes and of Escherichia coli clones containing these plasmids and that can be used for the large scale production of the enzymes, aiming at the identification or development of specific molecules that can inhibit this enzyme involved in the metabolic pathway leading to the synthesis of the methylglucose polysaccharides from mycobacteria. Therefore the present invention is applicable to the area of the pharmaceutical industry.

SIMOES DA COSTA MILTON; DA SILVA EMPADINHAS NUNO MIGUEL

323

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as...

Onwueme, Kenolisa C.; Ferreras, Julian A.; Buglino, John; Lima, Christopher D.; Quadri, Luis E. N.

324

Studies towards the identification of mycobacterial cell wall biosynthesis inhibitors and synthetic studies of buergerinin F, buergerinin G, and feigrisolide B.  

UK PubMed Central (United Kingdom)

This dissertation consists of four sections. The first section describes the synthesis of methyl 4a-carba-D-galactofuranosides. Oligosaccharides containing furanose moieties are important constituents of the cell wall of Mycobacterium tuberculosis, the intracellular causative bacterial pathogen of tuberculosis. A polysaccharide, arabinogalactan, attached to long chain lipids called mycolic acids, in combination with glycolipids found at the periphery of the cell wall envelope, provide a very efficient hydrophobic barrier that prevents the intrusion of drugs into the microorganism. Compounds that interfere with the biosynthesis of the arabinogalactan are therefore potential anti-tuberculosis agents. All arabinose and galactose residues of the arabinogalactan are found in the furanose form. Because furanose polysaccharides are critical for the survival of mycobacteria, the enzymes responsible for the biosynthesis of mycobacterial cell wall are very promising targets for drug action. A route for the synthesis of metabolically stable carbasugar analogs of galactofuranosides, was developed from D-galactose by using ring-closing metathesis as a key transformation. The second section, which is related to the first section in that it is focused on the identification of new anti-tuberculosis agents, details the synthesis of C-glycosidic glycopeptides. A series of C-arabinosyl glycopeptides was synthesized by solid-phase peptide synthesis. The Fmoc-protected arabinofuranoside building block was prepared from D-arabinose. The key transformations were Horner-Emmons olefination and catalytic asymmetric hydrogenation of resulting enamide ester. The synthesized dipeptides will be screened for their ability to inhibit arabinosyltransferases involved in mycobacterial cell wall assembly. The third section reports the synthesis of buergerinin F and buergerinin G, recently isolated from a plant Scrophularia buergeriana. The synthesis of buergerinin F and G was achieved from thymidine. We were able to establish the structures and absolute stereochemistry of these natural products. The final section addresses studies towards the synthesis of feigrisolide B, a seven-membered lactone isolated from fungi Streptomyces griseus. The synthetic study includes coupling reaction of two key intermediates and deoxygenation protocol developed in our group. Three more steps from the prepared intermediate would provide feigrisolide B.

Han J

325

Mutation in alkylhydroperoxidase D gene dramatically decreases persistence of Mycobacterium bovis bacillus calmette-guerin in infected macrophage  

Directory of Open Access Journals (Sweden)

Full Text Available Background and Objectives: Mycobacterium tuberculosis is the leading cause of death from a single bacterial species in the world and is subjected to a highly oxidative environment in its host macrophage and consequently has evolved protective mechanisms against reactive oxygen and nitrogen intermediates. Alkyl hydroperoxidase D (AhpD) is a molecule from these mycobacterial defense systems that has a dual function. It not only works with Alkyl hydroperoxidase C (AhpC) in mycobacterial defense system against oxidative stress but also has a role in oxidation/reduction of succinyltransferase B (SucB), dihydrolipoamide dehydrogenase (LPD) and AhpC. The present study was undertaken to find out the effects of inactivation of ahpD gene in the intra-macrophage persistence of resulted BCG mutant. Materials and Methods: We did allelic exchange mutagenesis in Mycobacterium bovis BCG and evaluate the effects of this mutagenesis in intracellular persistence of wild type BCG strains and ahpD mutant ones by comparing colony forming units (CFU) in infected macrophage. Results: Our findings showed that after producing allelic exchange mutagenesis in ahpD gene of M.bovis BCG a sever decrease in the CFU?s of ahpD mutant BCG strains has been observed and intracellular persistence of ahpD mutant BCG strains decreased significantly. Conclusion: Mutagenesis in ahpD gene will cause significant decrease in intracellular survival of ahpD mutant strains than wild type M.bovis BCG strains and could leads to an inefficiency in pyruvate dehydrogenase pathway and could also impair impairs mycobacterial defense system against oxidative and nitrosative stress.

Farivar Taghi; Varnousfaderani Pouran; Borji Abasalt

2008-01-01

326

Serologic follow-up of IgG responses against recombinant mycobacterial proteins ML0405, ML2331 and LID-1 in a leprosy hyperendemic area in Venezuela  

Directory of Open Access Journals (Sweden)

Full Text Available Leprosy is a slowly evolving disease that occurs mainly in adults. In this study, the Mamaría Village, state of Portuguesa was selected because it had one of the highest prevalence rates (13.25%) of leprosy cases in 1997. Between 1998-2004, 20.2% of the 89 cases registered in this village were less than 15 years old and 61.8% were males. Pau-cibacillary (PB) lesions were the predominant clinical forms identified, although also multibacillary (MB) forms were found. Additionally, 76% of the patients were bacteriologically negative. At the time of diagnosis, 75% of the patients presented with grade 0 disabilities, 23% with grade 1 and 2% with grade 2. Serum samples were collected from 18 PB and 15 MB patients, in addition to 14 family contacts, at the beginning and end of treatment. All the groups were re-evaluated during a three-year period (2008-2011). The proteins used for evaluation were ML0405, ML2331 and LID-1. These mycobacterial proteins were highly specific for Mycobacterium leprae and the IgG responses decreased in both MB and PB patients during multidrug treatment. Our results suggest that these antigens could be used as markers for successful treatment of non-reactional lepromatous patients.

Elsa Rada; Malcolm S Duthie; Steven G Reed; Nacarid Aranzazu; Jacinto Convit

2012-01-01

327

Serologic follow-up of IgG responses against recombinant mycobacterial proteins ML0405, ML2331 and LID-1 in a leprosy hyperendemic area in Venezuela  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english Leprosy is a slowly evolving disease that occurs mainly in adults. In this study, the Mamaría Village, state of Portuguesa was selected because it had one of the highest prevalence rates (13.25%) of leprosy cases in 1997. Between 1998-2004, 20.2% of the 89 cases registered in this village were less than 15 years old and 61.8% were males. Pau-cibacillary (PB) lesions were the predominant clinical forms identified, although also multibacillary (MB) forms were found. Additi (more) onally, 76% of the patients were bacteriologically negative. At the time of diagnosis, 75% of the patients presented with grade 0 disabilities, 23% with grade 1 and 2% with grade 2. Serum samples were collected from 18 PB and 15 MB patients, in addition to 14 family contacts, at the beginning and end of treatment. All the groups were re-evaluated during a three-year period (2008-2011). The proteins used for evaluation were ML0405, ML2331 and LID-1. These mycobacterial proteins were highly specific for Mycobacterium leprae and the IgG responses decreased in both MB and PB patients during multidrug treatment. Our results suggest that these antigens could be used as markers for successful treatment of non-reactional lepromatous patients.

Rada, Elsa; Duthie, Malcolm S; Reed, Steven G; Aranzazu, Nacarid; Convit, Jacinto

2012-12-01

328

Prospective evaluation of in-house polymerase chain reaction for diagnosis of mycobacterial diseases in patients with HIV infection and lung infiltrates.  

UK PubMed Central (United Kingdom)

SETTING: Rapid diagnosis of tuberculosis (TB) in AIDS is critical for optimal treatment to reduce mycobacterial dissemination, HIV-1 replication and mortality. The inadequate sensitivity of Ziehl-Neelsen staining and its inability to distinguish atypical mycobacteria delays accurate diagnosis. OBJECTIVE: To evaluate the polymerase chain reaction (PCR) for diagnosis of TB in bronchoalveolar lavage (BAL), blood and extra-pulmonary samples from patients with AIDS and pulmonary infiltrates. DESIGN: Specimens from 103 HIV-1-infected patients were prospectively analysed using bacteriological methods and IS6110-PCR. Smear-positive samples were also tested using 16S ribosomal-DNA-PCR to identify Mycobacterium avium complex (MAC) infections. Gold standard diagnosis relied on positive cultures or treatment outcome. RESULTS: Thirty-four patients exhibited TB, one TB and MAC and four MAC. The sensitivity of IS6110-PCR was 100% in smear-positive samples, 81.8% in smear-negative BAL, 66.7% in extra-pulmonary samples and 42.9% in blood. Its specificity was 97.1% in BAL and 100% in extra-pulmonary and blood specimens. The 16S rDNA-PCR identified M. avium from all smear-positive samples that grew MAC. CONCLUSIONS: IS6110-PCR proved useful in evaluating episodes with probable clinical diagnosis of pulmonary or mixed TB and negative smears, whereas 16S rDNA-PCR would be helpful for prompt differential diagnosis of MAC in smear-positive specimens.

Schijman AG; Losso MH; Montoto M; Saez CB; Smayevsky J; Benetucci JA

2004-01-01

329

Patients with non-tuberculous mycobacterial lung disease have elevated transforming growth factor-beta following ex vivo stimulation of blood with live Mycobacterium intracellulare.  

UK PubMed Central (United Kingdom)

We previously found that a subset of patients with pulmonary non-tuberculous mycobacterial (pNTM) disease were taller, leaner, and had a higher prevalence of pectus excavatum and scoliosis than uninfected controls. Additionally, whole blood of pNTM patients stimulated ex vivo with live Mycobacterium intracellulare produced significantly less interferon-gamma (IFN?) compared to that of uninfected controls. Since IFN? production can be suppressed by transforming growth factor-beta (TGF?), an immunosuppressive cytokine, we measured basal and M. intracellulare-stimulated blood levels of TGF? in a group of 20 pNTM patients and 20 uninfected controls. In contrast to the IFN? findings, we found that stimulated blood from pNTM patients produced significantly higher levels of TGF? compared to controls. Since pNTM patients frequently possess body features that overlap with Marfan syndrome (MFS), and increased TGF? expression is important in the pathogenesis of MFS, we posit that a yet-to-be-identified syndrome related to MFS predisposes certain individuals to develop pNTM disease.

Ovrutsky AR; Merkel PA; Schonteich E; Bai X; Kinney W; Iseman MD; Kartalija M; Knight V; Chan ED

2013-09-01

330

Various stages in the life cycle of syrphid flies (Eristalis tenax; Diptera: Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds.  

UK PubMed Central (United Kingdom)

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment.

Fischer OA; Mátlová L; Dvorská L; Svástová P; Bartos M; Weston RT; Pavlík I

2006-01-01

331

Various stages in the life cycle of syrphid flies (Eristalis tenax; Diptera: Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds.  

Science.gov (United States)

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment. PMID:16821726

Fischer, O A; Mátlová, L; Dvorská, L; Svástová, P; Bartos, M; Weston, R T; Pavlík, I

2006-01-01

332

The adrenal steroid response during tuberculosis and its effects on the mycobacterial-driven IFN-gamma production of patients and their household contacts.  

Science.gov (United States)

Earlier studies revealed that patients with tuberculosis (TB) have imbalanced immunoendocrine responses and that adrenal steroids [cortisol and dehydroepiandrosterone (DHEA)] can modify their specific cell-mediated immune response. Because most household contacts (HHCs) of contagious TB patients develop a subclinical and self-controlled process (latent TB), we studied some features of their immune and endocrine responses, particularly those related to the hypothalamic-pituitary-adrenal axis. Nineteen HHCs, 24 untreated TB patients (15 moderate, 9 advanced), and 18 healthy controls of similar age were studied. Patients had increased and reduced levels of cortisol and DHEA, respectively. DHEA levels were also reduced in HHCs. Stimulation of peripheral blood mononuclear cells (PBMC) with Mycobacterium tuberculosis sonicate resulted in increased in vitro lymphoproliferation in HHCs, while advanced patients showed the lowest response. Significantly higher amounts of interferon (IFN)-gamma were detected in supernatants from stimulated PBMC of HHCs when compared to controls and TB patients. Addition of cortisol to the cultures inhibited mycobacterial antigen-driven IFN-gamma production in all groups, although HHC supernatant contained significantly higher concentrations. In contrast, addition of DHEA to cultures of cells from HHCs resulted in increased IFN-gamma levels. These results suggest the existence of a particular immunoendocrine relation assuring a preserved IFN-gamma production in healthy housemates of TB patients. PMID:19236347

Bozza, Veronica; D'Attilio, Luciano; Didoli, Griselda; Santucci, Natalia; Nannini, Luis; Bogue, Cristina; Del Rey, Adriana; Besedovsky, Hugo; Bay, Maria Luisa; Bottasso, Oscar

2009-02-01

333

The adrenal steroid response during tuberculosis and its effects on the mycobacterial-driven IFN-gamma production of patients and their household contacts.  

UK PubMed Central (United Kingdom)

Earlier studies revealed that patients with tuberculosis (TB) have imbalanced immunoendocrine responses and that adrenal steroids [cortisol and dehydroepiandrosterone (DHEA)] can modify their specific cell-mediated immune response. Because most household contacts (HHCs) of contagious TB patients develop a subclinical and self-controlled process (latent TB), we studied some features of their immune and endocrine responses, particularly those related to the hypothalamic-pituitary-adrenal axis. Nineteen HHCs, 24 untreated TB patients (15 moderate, 9 advanced), and 18 healthy controls of similar age were studied. Patients had increased and reduced levels of cortisol and DHEA, respectively. DHEA levels were also reduced in HHCs. Stimulation of peripheral blood mononuclear cells (PBMC) with Mycobacterium tuberculosis sonicate resulted in increased in vitro lymphoproliferation in HHCs, while advanced patients showed the lowest response. Significantly higher amounts of interferon (IFN)-gamma were detected in supernatants from stimulated PBMC of HHCs when compared to controls and TB patients. Addition of cortisol to the cultures inhibited mycobacterial antigen-driven IFN-gamma production in all groups, although HHC supernatant contained significantly higher concentrations. In contrast, addition of DHEA to cultures of cells from HHCs resulted in increased IFN-gamma levels. These results suggest the existence of a particular immunoendocrine relation assuring a preserved IFN-gamma production in healthy housemates of TB patients.

Bozza V; D'Attilio L; Didoli G; Santucci N; Nannini L; Bogue C; Del Rey A; Besedovsky H; Bay ML; Bottasso O

2009-02-01

334

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

Scientific Electronic Library Online (English)

Full Text Available 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.

Rada, Elsa; Aranzazu, Nacarid; Rodríguez, Vestalia; Borges, Rafael; Convit, Jacinto

2010-09-01

335

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

Science.gov (United States)

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

Mehta, Promod K; Kalra, Mamta; Khuller, Gopal Krishan; Behera, Digambar; Verma, Indu

2012-02-01

336

Mycobacterium thermoresistibile: Case report of a rarely isolated mycobacterium from Europe and review of literature  

Directory of Open Access Journals (Sweden)

Full Text Available Mycobacterium thermoresistibile is a non-tuberculous mycobacterium strongly associated with human infections. Since 1966, there have only been six reports of its isolation from clinical samples. We report on the first case from Europe and review all the previous cases. Identification was achieved with sequencing of the 16S rRNA and hsp65 genes. This study presents its phenotypic and biochemical profile, susceptibilities to selected antibiotics and hsp65 polymerase chain reaction-restriction fragment length polymorphism profile with BsteII and Hae III .

Neonakis I; Gitti Z; Kontos F; Baritaki S; Petinaki E; Baritaki M; Zerva L; Spandidos D

2009-01-01

337

Mycobacterium thermoresistibile: case report of a rarely isolated mycobacterium from Europe and review of literature.  

Science.gov (United States)

Mycobacterium thermoresistibile is a non-tuberculous mycobacterium strongly associated with human infections. Since 1966, there have only been six reports of its isolation from clinical samples. We report on the first case from Europe and review all the previous cases. Identification was achieved with sequencing of the 16S rRNA and hsp65 genes. This study presents its phenotypic and biochemical profile, susceptibilities to selected antibiotics and hsp65 polymerase chain reaction-restriction fragment length polymorphism profile with BsteII and Hae III . PMID:19584513

Neonakis, I K; Gitti, Z; Kontos, F; Baritaki, S; Petinaki, E; Baritaki, M; Zerva, L; Spandidos, D A

338

Mycobacterium thermoresistibile: case report of a rarely isolated mycobacterium from Europe and review of literature.  

UK PubMed Central (United Kingdom)

Mycobacterium thermoresistibile is a non-tuberculous mycobacterium strongly associated with human infections. Since 1966, there have only been six reports of its isolation from clinical samples. We report on the first case from Europe and review all the previous cases. Identification was achieved with sequencing of the 16S rRNA and hsp65 genes. This study presents its phenotypic and biochemical profile, susceptibilities to selected antibiotics and hsp65 polymerase chain reaction-restriction fragment length polymorphism profile with BsteII and Hae III .

Neonakis IK; Gitti Z; Kontos F; Baritaki S; Petinaki E; Baritaki M; Zerva L; Spandidos DA

2009-07-01

339

Mycobacterium arupense pulmonary infection: antibiotic resistance and restriction fragment length polymorphism analysis.  

UK PubMed Central (United Kingdom)

Mycobacterium arupense is a novel mycobacterium species. It was first identified from clinical specimens in 2006 and since then there have been only two reports of its recovery from clinical samples. In the present case M. arupense was isolated from the sputum of a 62-year-old man with a malignant mass in his left kidney, who presented with a one-month history of recurrent fever, dyspnea and haemoptysis. M. arupense was identified with sequencing of hsp65 and 16S rRNA genes. In the present study, its biochemical profile along with its resistance status and hsp65 RFLP analysis is presented.

Neonakis IK; Gitti Z; Kontos F; Baritaki S; Petinaki E; Baritaki M; Liakou V; Zerva L; Spandidos DA

2010-04-01

340

Mycobacterium arupense pulmonary infection: antibiotic resistance and restriction fragment length polymorphism analysis.  

Science.gov (United States)

Mycobacterium arupense is a novel mycobacterium species. It was first identified from clinical specimens in 2006 and since then there have been only two reports of its recovery from clinical samples. In the present case M. arupense was isolated from the sputum of a 62-year-old man with a malignant mass in his left kidney, who presented with a one-month history of recurrent fever, dyspnea and haemoptysis. M. arupense was identified with sequencing of hsp65 and 16S rRNA genes. In the present study, its biochemical profile along with its resistance status and hsp65 RFLP analysis is presented. PMID:20404471

Neonakis, I K; Gitti, Z; Kontos, F; Baritaki, S; Petinaki, E; Baritaki, M; Liakou, V; Zerva, L; Spandidos, D A

 
 
 
 
341

Mycobacterium arupense pulmonary infection: Antibiotic resistance and restriction fragment length polymorphism analysis  

Directory of Open Access Journals (Sweden)

Full Text Available Mycobacterium arupense is a novel mycobacterium species. It was first identified from clinical specimens in 2006 and since then there have been only two reports of its recovery from clinical samples. In the present case M. arupense was isolated from the sputum of a 62-year-old man with a malignant mass in his left kidney, who presented with a one-month history of recurrent fever, dyspnea and haemoptysis. M. arupense was identified with sequencing of hsp65 and 16S rRNA genes. In the present study, its biochemical profile along with its resistance status and hsp65 RFLP analysis is presented.

Neonakis I; Gitti Z; Kontos F; Baritaki S; Petinaki E; Baritaki M; Liakou V; Zerva L; Spandidos D

2010-01-01

342

Cluster J mycobacteriophages: intron splicing in capsid and tail genes.  

Science.gov (United States)

Bacteriophages isolated on Mycobacterium smegmatis mc(2)155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution. PMID:23874930

Pope, Welkin H; Jacobs-Sera, Deborah; Best, Aaron A; Broussard, Gregory W; Connerly, Pamela L; Dedrick, Rebekah M; Kremer, Timothy A; Offner, Susan; Ogiefo, Amenawon H; Pizzorno, Marie C; Rockenbach, Kate; Russell, Daniel A; Stowe, Emily L; Stukey, Joseph; Thibault, Sarah A; Conway, James F; Hendrix, Roger W; Hatfull, Graham F

2013-07-09

343

Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria.  

UK PubMed Central (United Kingdom)

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Parikh A; Kumar D; Chawla Y; Kurthkoti K; Khan S; Varshney U; Nandicoori VK

2013-03-01

344

Differential expression of inflammatory and immune response genes in mesenteric lymph nodes of Iberian red deer (Cervus elaphus hispanicus) naturally infected with Mycobacterium bovis.  

Science.gov (United States)

Little information is available about gene expression in natural mycobacterial infection of wildlife species. Iberian red deer can serve as reservoir of Mycobacterium bovis in Spain, thus increasing the risk of bovine tuberculosis (bTB) in humans and cattle. Herein, we characterized the differential expression of inflammatory and immune response genes in mesenteric lymph nodes of deer naturally infected with M. bovis using microarray hybridization. Results were validated by determination of serum protein concentrations and/or real-time RT-PCR. Of the 600 genes that were analyzed in the microarray, 17 genes displayed an expression fold change greater than 1.7 in infected or uninfected deer (P0.05). These genes included tight junction proteins, IL-11R, bactenecin, CD62L, CD74, desmoglein, IgA and IgM that constitute new findings and suggest new mechanisms by which M. bovis may modulate host inflammatory and immune responses. These results contribute to our basic understanding of the mechanisms of pathogenesis and immunity to natural mycobacterial infections and may have important implications for the control of bTB. PMID:17604102

Fernández de Mera, Isabel G; Pérez de la Lastra, José Manuel; Ayoubi, Patricia; Naranjo, Victoria; Kocan, Katherine M; Gortazar, Christian; de la Fuente, José

2007-06-14

345

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

Scientific Electronic Library Online (English)

Full Text Available 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.

Godoy, María José; Orozco, Loren; Hernández, Cohinta; DaMata, Omaira; De Waard, Jacobus; González Rico, Susana

2008-12-01

346

Characterization of mycobacteria from a major Brazilian outbreak suggests that revision of the taxonomic status of members of the Mycobacterium chelonae-M. abscessus group is needed.  

Science.gov (United States)

An outbreak of postsurgical infections caused by rapidly growing mycobacteria has been ongoing in Brazil since 2004. The degrees of similarity of the rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both the Mycobacterium massiliense and the M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting identification results. Therefore, an extensive characterization of members of the M. chelonae-M. abscessus group was carried out. The M. abscessus, M. chelonae, M. immunogenum, M. massiliense, and M. bolletii type strains and a subset of clinical isolates were analyzed by biochemical tests, high-performance liquid chromatography, drug susceptibility testing, PCR-restriction enzyme analysis of hsp65 (PRA-hsp65), rpoB, and hsp65 gene sequencing and analysis of phylogenetic trees, DNA-DNA hybridization (DDH), and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (RFLP-16S rRNA). The clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. The results of DDH also confirmed the >70% relatedness of the clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains; and indistinguishable RFLP-16S rRNA patterns were obtained. On the contrary, the separation of clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains from M. chelonae and M. immunogenum was supported by the results of PRA-hsp65, DDH, and RFLP-16S rRNA and by the rpoB and hsp65 phylogenetic trees. Taken together, these results led to the proposition that M. abscessus, M. massiliense, and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense, and these two subspecies can be distinguished by two different PRA-hsp65 patterns, which differ by a single HaeIII band, and by differences in their rpoB (3.4%) and hsp65 (1.3%) sequences. PMID:19571015

Leao, Sylvia Cardoso; Tortoli, Enrico; Viana-Niero, Cristina; Ueki, Suely Yoko Mizuka; Lima, Karla Valeria Batista; Lopes, Maria Luiza; Yubero, Jesus; Menendez, Maria Carmen; Garcia, Maria Jesus

2009-07-01

347

Yield and cost effectiveness of mycobacterial infection detection using a simple IGRA-based protocol in UK subjects with inflammatory bowel disease suitable for anti-TNF? therapy.  

UK PubMed Central (United Kingdom)

BACKGROUND AND AIMS: Testing for LTBI is recommended prior to anti-TNF? agents. This includes an assessment of TB risk factors, chest radiograph, and interferon-gamma release assay alone or with concurrent Tuberculin skin testing. Here we review our experience and cost-effectiveness of using T-SPOT.TB IGRA to detect mycobacterial infection in patients with IBD suitable for anti-TNF? therapy. METHODS: This was a single-centre, retrospective review and economic evaluation (compared to British Thoracic Society guidance) of 125 adult IBD patients (90 anti-TNF? naïve, 35 established on anti-TNF?) tested for LTBI using T-SPOT.TB IGRA. RESULTS: All subjects had normal chest radiographs and no clinical evidence for TB. 109 (87%) were BCG vaccinated. 27 (22%) of all patients tested were not using immunomodulation at the time of testing. 66 (53%) were taking thiopurines, 22 (18%)corticosteroids, and 35 (28%) anti-TNF? agents. One hundred twenty two (98%) had a negative IGRA result, two (2%) had positive results, and one (1%) had an indeterminate IGRA. A strategy using IGRA to guide TB preventative treatment produced cost savings of £10.79 per person compared to the BTS guidance. Eighty eight percent of the anti-TNF? naïve group have subsequently received treatment with either infliximab or adalimumab (median follow-up of 24 months, IQR 18-30) with no cases of TB disease occurring. CONCLUSIONS: The use of a simple screening protocol for LTBI incorporating T-SPOT.TB IGRA in place of TST in a largely BCG vaccinated population, many using immunomodulatory agents, appears to work well and is a cost-effective strategy in our IBD service.

Greveson K; Goodhand J; Capocci S; Woodward S; Murray C; Cropley I; Hamilton M; Lipman M

2013-06-01

348

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants.  

UK PubMed Central (United Kingdom)

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity.

Leelaram MN; Suneetha N; Nagaraja V; Manjunath R

2010-05-01

349

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants.  

Science.gov (United States)

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity. PMID:20302873

Leelaram, Majety Naga; Suneetha, Nunna; Nagaraja, Valakunja; Manjunath, Ramanathapuram

2010-03-17

350

Optimisation of a functional mycobacterial growth-inhibition assay to improve its suitability for infant TB vaccine studies?  

Science.gov (United States)

The development of vaccines against tuberculosis continues to be hindered by the lack of correlates of protection. Immunity to Mycobacterium tuberculosis (M.tb) infection relies predominantly on cell mediated response, which is routinely measured using a read-out of host cytokine profiles. However, to date none of the cytokine profiles have been found to predict protection. A number of functional in vitro approaches that measure growth of mycobacteria pre- and post-vaccination as a potential functional surrogate marker for vaccine take have been developed. The use of a reporter-gene tagged BCG-lux assay measuring the viability of mycobacteria in whole blood samples has previously been described by our group to assess vaccine immunogenicity. Since only very small blood samples are usually available in paediatric studies, we now report a modification of the BCG-lux assay to reduce the volume required and make it more field-friendly. Our results show that a 2-fold reduction in blood volume made no significant difference to bacterial growth ratios, used as the main read-out. These results confirm the suitability of the BCG-lux assay for functional studies of vaccine immunogenicity and immunopathogenesis in young children and could play a role in late-phase TB vaccine trials of novel candidates.

Burl, S.; Holder, B.S.; Lo, B.K.M.; Kampmann, B.

2013-01-01

351

Optimisation of a functional mycobacterial growth-inhibition assay to improve its suitability for infant TB vaccine studies.  

Science.gov (United States)

The development of vaccines against tuberculosis continues to be hindered by the lack of correlates of protection. Immunity to Mycobacterium tuberculosis (M.tb) infection relies predominantly on cell mediated response, which is routinely measured using a read-out of host cytokine profiles. However, to date none of the cytokine profiles have been found to predict protection. A number of functional in vitro approaches that measure growth of mycobacteria pre- and post-vaccination as a potential functional surrogate marker for vaccine take have been developed. The use of a reporter-gene tagged BCG-lux assay measuring the viability of mycobacteria in whole blood samples has previously been described by our group to assess vaccine immunogenicity. Since only very small blood samples are usually available in paediatric studies, we now report a modification of the BCG-lux assay to reduce the volume required and make it more field-friendly. Our results show that a 2-fold reduction in blood volume made no significant difference to bacterial growth ratios, used as the main read-out. These results confirm the suitability of the BCG-lux assay for functional studies of vaccine immunogenicity and immunopathogenesis in young children and could play a role in late-phase TB vaccine trials of novel candidates. PMID:23707325

Burl, S; Holder, B S; Lo, B K M; Kampmann, B

2013-05-23

352

The contraceptive depot medroxyprogesterone acetate impairs mycobacterial control and inhibits cytokine secretion in mice infected with Mycobacterium tuberculosis.  

Science.gov (United States)

The contraceptive depot medroxyprogesterone acetate (DMPA), with progestin as the single active compound, possesses selective glucocorticoid activity and can alter the expression of glucocorticoid receptor-regulated genes. We therefore propose that pharmacological doses of DMPA used for endocrine therapy could have significant immune modulatory effects and impact on susceptibility to, as well as clinical manifestation and outcome of, infectious diseases. We investigated the effect of contraceptive doses of DMPA in two different murine Mycobacterium tuberculosis models. Multiplex bead array analysis revealed that DMPA altered serum cytokine levels of tumor necrosis factor alpha (TNF-?), granulocyte colony-stimulating factor (G-CSF), and interleukin 10 (IL-10) in C57BL/6 mice and gamma interferon (IFN-?) in BALB/c mice. DMPA also suppressed antigen-specific production of TNF-?, G-CSF, IL-10, and IL-6 and induced the production of IP-10 in C57BL/6 mice. In BALB/c mice, DMPA altered the antigen-specific secretion of IFN-?, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and monocyte chemotactic protein 1 (MCP-1). Furthermore, we show that C57BL/6 mice treated with doses of DMPA, which result in serum concentrations similar to those observed in contraceptive users, have a significantly higher bacterial load in their lungs. Our data show for the first time that DMPA impacts tuberculosis (TB) disease severity in a mouse model and that the effects of this contraceptive are not confined to infections of the genital tract. This could have major implications for the contraceptive policies not only in developing countries like South Africa but also worldwide. PMID:23381991

Kleynhans, Léanie; Du Plessis, Nelita; Allie, Nasiema; Jacobs, Muazzam; Kidd, Martin; van Helden, Paul D; Walzl, Gerhard; Ronacher, Katharina

2013-02-04

353

The contraceptive depot medroxyprogesterone acetate impairs mycobacterial control and inhibits cytokine secretion in mice infected with Mycobacterium tuberculosis.  

UK PubMed Central (United Kingdom)

The contraceptive depot medroxyprogesterone acetate (DMPA), with progestin as the single active compound, possesses selective glucocorticoid activity and can alter the expression of glucocorticoid receptor-regulated genes. We therefore propose that pharmacological doses of DMPA used for endocrine therapy could have significant immune modulatory effects and impact on susceptibility to, as well as clinical manifestation and outcome of, infectious diseases. We investigated the effect of contraceptive doses of DMPA in two different murine Mycobacterium tuberculosis models. Multiplex bead array analysis revealed that DMPA altered serum cytokine levels of tumor necrosis factor alpha (TNF-?), granulocyte colony-stimulating factor (G-CSF), and interleukin 10 (IL-10) in C57BL/6 mice and gamma interferon (IFN-?) in BALB/c mice. DMPA also suppressed antigen-specific production of TNF-?, G-CSF, IL-10, and IL-6 and induced the production of IP-10 in C57BL/6 mice. In BALB/c mice, DMPA altered the antigen-specific secretion of IFN-?, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and monocyte chemotactic protein 1 (MCP-1). Furthermore, we show that C57BL/6 mice treated with doses of DMPA, which result in serum concentrations similar to those observed in contraceptive users, have a significantly higher bacterial load in their lungs. Our data show for the first time that DMPA impacts tuberculosis (TB) disease severity in a mouse model and that the effects of this contraceptive are not confined to infections of the genital tract. This could have major implications for the contraceptive policies not only in developing countries like South Africa but also worldwide.

Kleynhans L; Du Plessis N; Allie N; Jacobs M; Kidd M; van Helden PD; Walzl G; Ronacher K

2013-04-01

354

NUCLEOSIDE DIPHOSPHATE KINASE GENE IS EXPRESSED THROUGH MULTIPLE TRANSCRIPTS IN Mycobacterium smegmatis  

Directory of Open Access Journals (Sweden)

Full Text Available Nucleoside diphosphate kinase, NDK, plays a vital role in maintaining pools of nucleoside triphosphates and their respective deoxynucleoside triphosphates for the synthesis of RNA and DNA. Transcriptional regulation of ndk in mycoacteria remains unknown, although modulation of ndk expression under stress conditions involving DNA and, RNA synthesis arrest and cell division arrest had been studied in several bacterial systems. Therefore, in the present study, the start sites of transcription of ndk of Mycobacterium smegmatis (Msmndk) were identified and putative promoter regions were predicted. Using transcriptional fusions of the cloned putative promoter regions to mycobacterial codon-optimised reporter gene, gfpm2+, promoter activity was examined under active phase of growth, nutrient starvation and other stress conditions involving DNA replication inhibition and cell division arrest. Msmndk was found to be expressed through two transcripts, T1 and T2, arising from P1 and P2 promoters, respectively. Both the promoters belonged to C group of mycobacterial promoters, which do not possess consensus to any known canonical sigma factor recognition sequences. The levels of T2, but not of T1, were found to be low under the different stress conditions studied. The data documents modulation of ndk transcripts in mycobacteria.

Muthu Arumugam, Deepak Anand, Namperumalsamy Vijayarangan, Chandrasekaran Anbukayalvizhi, Megha Rao, Srinivasan Vijay, Haryadi Rajeswari and Parthasarathi Ajit kumar

2012-01-01

355

Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis.  

UK PubMed Central (United Kingdom)

BACKGROUND: Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. RESULTS: Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. CONCLUSIONS: We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

Movahedzadeh F; Wheeler PR; Dinadayala P; Av-Gay Y; Parish T; Daffé M; Stoker NG

2010-01-01

356

Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. Results Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. Conclusions We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

Movahedzadeh Farahnaz; Wheeler Paul R; Dinadayala Premkumar; Av-Gay Yossef; Parish Tanya; Daffé Mamadou; Stoker Neil G

2010-01-01

357

Genes and gene regulation  

Energy Technology Data Exchange (ETDEWEB)

Genetics has long been a central topic for biologists, and recent progress has captured the public imagination as well. This book addresses questions that are at the leading edge of this continually advancing discipline. In tune with the increasing emphasis on molecular biology and genetic engineering, this text emphasizes the molecular aspects of gene expression, and the evolution of gene sequence organization and control. It reviews the genetic material of viruses, bacteria, and of higher organisms. Cells and organisms are compared in terms of gene numbers, their arrangements within a cell, and the control mechanisms which regulate the activity of genes.

MacLean, N.

1988-01-01

358

Characterization of the MSMEG_2631 gene (mmp) encoding a multidrug and toxic compound extrusion (MATE) family protein in Mycobacterium smegmatis and exploration of its polyspecific nature using biolog phenotype microarray.  

UK PubMed Central (United Kingdom)

In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc(2)155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na(+)-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions.

Mishra MN; Daniels L

2013-04-01

359

Diagnosis of nontuberculous mycobacterial infections.  

UK PubMed Central (United Kingdom)

The nontuberculous mycobacteria (NTM) are typically environmental organisms residing in soil and water. Although generally of low pathogenicity to humans, NTM can cause a wide array of clinical diseases; pulmonary disease is most frequent, followed by lymphadenitis in children, skin disease by M. marinum (particularly in fish tank fanciers), and other extrapulmonary or disseminated infections in severely immunocompromised patients. Of the >140 NTM species reported in the literature, 25 species have been strongly associated with NTM diseases; the remainder are environmental organisms rarely encountered in clinical samples. Correct species identification is very important because NTM species differ in their clinical relevance. Further, NTM differ strongly in their growth rate, temperature tolerance, and drug susceptibility. The diagnosis of NTM disease is complex and requires good communication between clinicians, radiologists, and microbiologists. Isolation of M. kansasii and (in northwestern Europe) M. malmoense from pulmonary specimens usually indicates disease, whereas Mycobacterium gordonae and, to a lesser extent, M. simiae or M. chelonae are typically contaminants rather than causative agents of true disease. Mycobacterium avium complex (MAC), M. xenopi, and M. abscessus form an intermediate category between these two extremes. This review covers the clinical and laboratory diagnosis of NTM diseases and particularities for the different disease types and patient populations. Because of limited sensitivity and specificity of symptoms, radiology, and direct microscopy of clinical samples, culture remains the gold standard. Yet culture is time consuming and demands the use of multiple media types and incubation temperatures to optimize the yield. Outside of reference centers, such elaborate culture algorithms are scarce.

van Ingen J

2013-02-01