Limitations of the use of the mtp40 fragment as a marker of differentiation between Mycobacterium tuberculosis and M. bovis

UNIFESP-EPM Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, EPM, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

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

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

Natural Occurrence of Horizontal Transfer of Mycobacterium avium-Specific Insertion Sequence IS1245 to Mycobacterium kansasii

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. the insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.Foundation for Research Support of the State of São PauloConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilInst Adolfo Lutz Registro, Setor Micobacterias, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilFoundation for Research Support of the State of São Paulo: FAPESP-06/01533-9Web of Scienc

An experimental model of mycobacterial infection under corneal flaps

In order to develop a new experimental animal model of infection with Mycobacterium chelonae in keratomileusis, we conducted a double-blind prospective study on 24 adult male New Zealand rabbits. One eye of each rabbit was submitted to automatic lamellar keratotomy with the automatic corneal shaper under general anesthesia. Eyes were immunosuppressed by a single local injection of methyl prednisolone. Twelve animals were inoculated into the keratomileusis interface with 1 µl of 10(6) heat-inactivated bacteria (heat-inactivated inoculum controls) and 12 with 1 µl of 10(6) live bacteria. Trimethoprim drops (0.1%, w/v) were used as prophylaxis for the surgical procedure every 4 h (50 µl, qid). Animals were examined by 2 observers under a slit lamp on the 1st, 3rd, 5th, 7th, 11th, 16th, and 23rd postoperative days. Slit lamp photographs were taken to document clinical signs. Animals were sacrificed when corneal disease was detected and corneal samples were taken for microbiological analysis. Eleven of 12 experimental rabbits developed corneal disease, and M. chelonae could be isolated from nine rabbits. Eleven of the 12 controls receiving a heat-inactivated inoculum did not develop corneal disease. M. chelonae was not isolated from any of the control rabbits receiving a heat-inactivated inoculum, or from the healthy cornea of control rabbits. Corneal infection by M. chelonae was successfully induced in rabbits submitted to keratomileusis. To our knowledge, this is the first animal model of M. chelonae infection following corneal flaps for refractive surgery to be described in the literature and can be used for the analysis of therapeutic responses.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de OftalmologiaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, EPM, Depto. de OftalmologiaUNIFESP, EPM, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Profiling Mycobacterium ulcerans with hsp65

Univ Fed Sao Paulo, Escola Paulista Med, Dept Microbiol, BR-04023062 Sao Paulo, BrazilInst Trop Med, B-2000 Antwerp, BelgiumUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol, BR-04023062 Sao Paulo, BrazilWeb of Scienc

Mycobacterium bovis: polymerase chain reaction identification in bovine Lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.ICB Departamento de Bioquímica e Imunologia Laboratório de Biologia Molecular de Produtos NaturaisUniversidade Federal de Minas Gerais ICB Escola de VeterináriaUniversidade Federal de Minas Gerais ICB Departamento de FarmacologiaEscola Paulista de Medicina Departamento de Microbiologia e ParasitologiaLaboratório Central do Estado do Espírito SantoInstituto Biológico de São PauloCentro de Investigación en Ciencias Veterinarias Instituto de BiotecnologiaUNIFESP, EPM, Depto. de Microbiologia e ParasitologiaSciEL

Cytotoxic T cells and mycobacteria

How the immune system kills Mycobacterium tuberculosis is still a puzzle. the classical picture of killing due to phagocytosis by activated macrophages may be only partly correct. Based on recent evidence, we express here the view that cytotoxic T lymphocytes also make an important contribution and suggest that DNA vaccines might be a good way to enhance this. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Univ São Paulo, Sch Med Ribeirao Preto, Dept Biochem & Immunol, BR-14049900 Ribeirao Preto, SP, BrazilUniv São Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Clin Anal Bromatol & Toxicol, BR-14049 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol & Immunol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol & Immunol, São Paulo, BrazilWeb of Scienc

The Detection and Sequencing of a Broad-Host-Range Conjugative IncP-1 beta Plasmid in an Epidemic Strain of Mycobacterium abscessus subsp bolletii

Background: An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004-2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE) patterns differentiated by the presence of a similar to 50 kb band. the nature of this band was investigated.Methodology/Principal Findings: Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,264-bp circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1 beta and is highly related to BRA100, pJP4, pAKD33 and pB10. the presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. the plasmid was visualized as a similar to 50-kb band using PFGE and Southern blot hybridization in 12 isolates. the remaining 25 isolates did not exhibit any evidence of this plasmid. the plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc(2)155 could not be demonstrated.Conclusions/Significance: the occurrence of a broad-host-range IncP-1 beta plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundao de Amparo a Pesquisa do Estado de São PauloUniversidade Federal de São Paulo, Disciplina Microbiol, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFed Univ Para, Inst Ciencias Biol, Lab Polimorfismo DNA, BR-66059 Belem, Para, BrazilInst Evandro Chagas, Secao Bacteriol & Micol, Ananindeua, PA, BrazilFed Univ Para, Inst Estudos Costeiros, Braganca, PA, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Disciplina Microbiol, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFAPESP: FAPESP - 06/01533-9Fundao de Amparo a Pesquisa do Estado de São Paulo: FAPESP - 8/01451-8Web of Scienc