RESUMO
SETTING: Mexico City, Mexico. OBJECTIVE: To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). DESIGN: Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. RESULTS: We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. CONCLUSION: ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.
Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina A/sangue , Isocitrato Liase/imunologia , Tuberculose Pulmonar/diagnóstico , alfa-Cristalinas/imunologia , Adulto , Idoso , Antígenos de Bactérias/sangue , Estudos de Casos e Controles , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , México , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Tuberculose Pulmonar/sangue , Adulto JovemRESUMO
Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection.
Assuntos
Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Animais , Animais Selvagens/microbiologia , Argentina , Brasil , Búfalos/microbiologia , Gatos/microbiologia , Bovinos/microbiologia , Humanos , México , Tipagem Molecular/veterinária , Sus scrofa/microbiologia , Suínos/microbiologia , Tuberculose/veterinária , VenezuelaRESUMO
This study examined the frequency of occurrence of non-tuberculous mycobacteria (NTM) in potable water samples from a main trauma hospital in Mexico City. Sixty-nine potable water samples were collected, 23 from each source: cistern, kitchen tap and bathroom showers. Of the 69 samples, 36 harboured NTM species. Twenty-nine of the 36 isolates were Mycobacterium mucogenicum, two Mycobacterium rhodesiae, one Mycobacterium peregrinum, one Mycobacterium fortuitum and three were Mycobacterium spp. Hospital potable water harbouring NTM represents a potential source for nosocomial infections, therefore we suggest that hospital potable water microbiological guidelines should include testing for NTM species.
Assuntos
Água Potável/microbiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Hospitais , Humanos , México , Infecções por Mycobacterium não Tuberculosas/prevenção & controleRESUMO
Recently, the incidence of human mycobacterial infections due to species other than M. tuberculosis has increased worldwide. Since disease control depends on appropriate antimicrobial therapy, the precise identification of these species of clinical importance has become a major public health concern. Identification of mycobacteria has been hampered because of the lack of specific, rapid, and inexpensive methods. Therefore, we aimed at designing and validating a bacterial lysate-based polymerase chain reaction identification scheme. This scheme can classify clinical isolates into: (1) the genus Mycobacterium, (2) the M. tuberculosis complex, (3) the nontuberculous mycobacteria, and (4) the species M. avium, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum and M. bovis of clinical importance, and M. gordonae, the most commonly encountered nonpathogenic species in clinical laboratories. By using M. fortuitum and M. avium lysates as models, the method sensitivity was determined to be 372 pg of DNA. In a blind parallel comparison between our approach and conventional biochemical tests, both assays correctly categorized 75 patient's mycobacterial isolates. However, our approach only required 4-9 h for categorization compared with at least 15 days by conventional tests. Furthermore, our methodology could also detect M. fortuitum and M. avium from liquid cultures, after only 2 and 6 days, respectively, of incubation. Our new identification scheme is therefore sensitive, specific, rapid, and economic. Additionally, it can help to provide proper treatment to patients, to control these diseases, and to improve our knowledge of the epidemiology of mycobacteriosis, all urgently needed, particularly in developing countries.
Assuntos
Bactérias/genética , Extratos Celulares/genética , Mycobacterium/patogenicidade , Sensibilidade e Especificidade , Técnicas de Tipagem Bacteriana , Humanos , Mycobacterium/classificação , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium/fisiopatologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Especificidade da EspécieRESUMO
Bovine tuberculosis is a zoonotic disease that not only causes huge economic losses but also poses an important risk for human infection. The definitive identification of a clinical isolate relies on time-consuming, highly specialized and laborious biochemical tests. We have developed a method for the rapid and reliable identification of Mycobacterium bovis and for its simultaneous differentiation from other members of the M. tuberculosis complex. Furthermore, the technique also allowed us to distinguish M. tuberculosis complex members from other Mycobacterial species. The method comprises both a single PCR and a multiplex-PCR and can be confidently applied to samples of both veterinary and human origin.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Mycobacterium bovis/classificação , Reação em Cadeia da Polimerase/métodos , Tuberculose Bovina/diagnóstico , Tuberculose/diagnóstico , Animais , Bovinos , Primers do DNA , DNA Bacteriano/análise , HumanosRESUMO
All mycobacteria studied to date have an rRNA operon, designated rrnA, located downstream from a single copy of the murA gene, which encodes an enzyme (EC 2.5.1.7) important for peptidoglycan synthesis. The rrnA operon has a promoter, P1(A), located within the coding region of murA, near the 3' end. Samples of RNA were isolated from Mycobacterium tuberculosis at different stages of the growth cycle and from Mycobacterium smegmatis grown under different conditions. RNase protection assays were used to investigate transcripts of both murA and rrnA. Transcription of murA was found to continue into the 16S rRNA gene, as if murA and rrnA form a hybrid (protein coding-rRNA coding) operon. During the growth of M. tuberculosis, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of M. smegmatis RNA revealed that the level of murA RNA depended on the growth rate and that the patterns of expression during the growth cycle were different for murA and rrnA. M. smegmatis has a second rRNA operon, rrnB, located downstream from a single copy of the tyrS gene, encoding tyrosyl-tRNA synthetase. Transcription of tyrS was found to continue into the 16S rRNA gene rrnB. The hybrid tyrS-rrnB operon contributed 0.2 to 0.6% to rrnB transcripts. The pattern of tyrS expression during the growth cycle matched the pattern of rrnB expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis.