ABSTRACT
ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.
RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.
Subject(s)
Humans , Nontuberculous Mycobacteria/isolation & purification , Mycobacterium avium/genetics , Mycobacterium Infections, Nontuberculous/diagnosis , Bacterial Proteins/genetics , Genetic Variation , Brazil , DNA-Directed RNA Polymerases/genetics , Bacterial Typing Techniques , Chaperonin 60/genetics , Mycobacterium avium/isolation & purification , Mycobacterium Infections, Nontuberculous/microbiologyABSTRACT
IS901 RFLP analysis of 36 Mycobacterium avium subsp. avium (MAA) isolates from 15 pheasants (Phasianus colchicus) and two goshawks (Accipiter gentilis) from four pheasant farms was performed. Using this method, six different IS901 RFLP types (E, F, G, M, Q, and V) were identified. The distribution of IS901 RFLP profiles was tightly linked to individual flocks. Matching IS901 RFLP profiles observed in the present study indicate MAA transmission between pheasants and goshawks in the same locality. In two flocks, different pheasants within a flock as well as in various organs of five individual pheasants were found to have two distinct IS901 RFLP profiles.
Subject(s)
Animals , Bone Marrow/microbiology , Galliformes , Intestines/microbiology , Liver/microbiology , Mycobacterium avium/genetics , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , Poultry Diseases/microbiology , Spleen/microbiology , Tuberculosis, Avian/microbiologyABSTRACT
In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.
Subject(s)
Animals , Humans , Mice , Adhesins, Bacterial/genetics , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/therapeutic use , Cell Proliferation , Cytokines/metabolism , Dendritic Cells/cytology , Disease Models, Animal , Gene Expression Regulation , Glycogen Synthase Kinase 3/metabolism , Mice, Inbred C57BL , Mycobacterium avium/genetics , Paratuberculosis/metabolism , Protein Binding , Signal Transduction , T-Lymphocytes, Cytotoxic/metabolism , Thymoma/genetics , Toll-Like Receptor 4/agonistsABSTRACT
BACKGROUND & OBJECTIVE: We report a new polymerase chain reaction (PCR) - restriction fragment length polymorphism (RFLP) assay using mycobacterial groES as a target to identify Mycobacterium avium and M. intracellulare in clinical samples. METHODS: The assay was standardized using M. avium and M. intracellulare standard strains obtained from ATCC and was tested with 45 M. avium-M. intracellulare complex (MAC) clinical isolates (Of which 31 were from HIV(+) individuals). The standard and clinical strains were typed with HPLC based mycolic acid fingerprinting. RESULTS: Three polymorphisms (BamHI, BstNI and HgaI) were identified for inter-species differentiation among standard strains; of which, only HgaI was found to be useful in clinical isolates. Of the 45 isolates, 25 were M. avium and 20 were M. intracelluare. MAC isolates, which could not be differentiated by HPLC analysis, were also typed by this method. INTERPRETATION & CONCLUSION: The use of mycobacterial groES as a PCR-RFLP target for M. avium and M. intracellulare is a simple and rapid method that can complement HPLC in their differentiation.
Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Base Sequence , DNA Primers/genetics , DNA, Bacterial/genetics , Genes, Bacterial , Chaperonin 10/genetics , Heat-Shock Proteins/genetics , Humans , Mycobacterium avium/genetics , Mycobacterium avium Complex/genetics , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Species SpecificityABSTRACT
Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.
Subject(s)
Animals , Humans , Acquired Immunodeficiency Syndrome/microbiology , Bacterial Typing Techniques/methods , Genetic Variation , Mycobacterium avium/genetics , Mycobacterium avium/isolation & purification , Brazil , Chickens/microbiology , Food Microbiology , Genotype , Mycobacterium avium/classification , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Swine/microbiology , Vegetables/microbiologyABSTRACT
The Mycobacterium avium complex (MAC), especially M. avium, is an important opportunistic pathogen of AIDS patients in the United States. In Puerto Rico, the incidence of infections caused by MAC has not been determined. This is due, in part, to the difficulties associated to the microbiological identification of the microorganisms. In this work, a commercially available kit (AccuProbe, Gen-Probe, Inc., San Diego, CA) utilizing a DNA probe complementary to rRNA of M. avium and M. intracellulare was used to identify seventeen MAC strains and one unknown atypical mycobacterium recovered in culture in Puerto Rico from clinical samples. The results obtained revealed that M. avium was the predominant species recovered (83 per cent of isolates tested). Only two cultures were identified as M. intracellulare. The unknown culture, which did not react with either probe, turned out to be M. gordonae. The probe tests not only are simple to perform, but provide cultural identification results in as little as two hours. This study, the first one of its kind in Puerto Rico, demonstrates that the nucleic acid probes for the cultural identification of M. avium and M. intracellulare offer the potential of providing a prompt diagnosis and much needed data on the epidemiology of MAC infections in Puerto Rico