RESUMO
The Mycobacterium tuberculosis PE_PGRS multigene family is thought to be involved in antigenic variation, which can be generated by differential regulation of expression and a high frequency of genetic polymorphism. PE_PGRS16 and PE_PGRS26 are inversely regulated during persistent M. tuberculosis infection, suggesting that differential regulation of the expression of these two PE_PGRS genes may have a role in latency. To understand how genetic diversity, in addition to differential regulation, contributes to antigenic variability, we investigated the sequence variations in the PE_PGRS16 and PE_PGRS26 genes among 200 clinical M. tuberculosis strains, in comparison to the sequenced laboratory strain H37Rv, using PCR and DNA sequencing. Among the 200 strains, 102 (51%) and 100 (50%) had sequence variations within the PE_PGRS16 gene and the PE_PGRS26 gene, respectively. In-frame insertions and deletions, frameshifts, and SNPs were observed in both the PE_PGRS16 gene and the PE_PGRS26 gene. However, the frequency of frameshifts and in-frame deletions differed between the two PE_PGRS genes. Examining the profile of the PE_PGRS16, PE_PGRS26, and the previously investigated PE_PGRS33 amino acid sequences for each of the 200 strains, 72 different profiles were observed with frequencies ranging from 0.5% to 13%. In conclusion, a remarkable level of genetic diversity exists in the PE_PGRS16 and PE_PGRS26 genes of M. tuberculosis clinical strains. The significant sequence variations in the two PE_PGRS genes observed in this study could impact the function of these two PE_PGRS proteins and be associated with differences in the ability of the tubercle bacilli to remain persistent within the host.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Mycobacterium tuberculosis/genética , Polimorfismo Genético/genética , Tuberculose Pulmonar/microbiologia , Proteínas de Bactérias/química , Feminino , Deleção de Genes , Variação Genética/genética , Humanos , Masculino , Análise de Sequência de DNARESUMO
There is evidence that some members of the Mycobacterium tuberculosis PE PGRS gene subfamily, including PE PGRS33, may have a specific function in M. tuberculosis persistence. The impact of naturally-occurring PE PGRS33 genetic variations on the virulence and transmissibility of clinical M. tuberculosis isolates is not known. We used PCR and DNA sequencing to identify genetic variations in the PE PGRS33 gene in comparison with the sequenced laboratory strain, H37Rv, among 649 isolates from a population-based sample. The PE PGRS33 alleles were placed into two groups, based on the effect of the sequence variations on the PE PGRS33 protein, and their associations with clinical and epidemiological characteristics were assessed using multivariate logistic regression to control for potential confounding of host-related factors. Of the 639 isolates for which sequence data were obtained, 139 (21.8%) had PE PGRS33 alleles that would result in a significant change to the PE PGRS33 protein due to large insertions/deletions or frameshift mutations. These isolates were significantly associated with clustering based on genotype and absence of cavitations in the lungs, compared to isolates having PE PGRS33 alleles that would result in no or minimal change to the PE PGRS33 protein. The association of significant changes to PE PGRS33 with clinical and epidemiological characteristics suggests that PE PGRS33 may have an important role in M. tuberculosis persistence.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Genes Bacterianos/genética , Proteínas de Membrana/genética , Mycobacterium tuberculosis/genética , Tuberculose/genética , Arkansas/epidemiologia , Análise por Conglomerados , Feminino , Mutação da Fase de Leitura , Variação Genética , Humanos , Masculino , Mycobacterium tuberculosis/patogenicidade , Mapeamento de Peptídeos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Tuberculose/epidemiologiaRESUMO
BACKGROUND: As tuberculosis incidence declines in the United States, a new tool for TB control efforts is Mycobacterium tuberculosis genotyping. Colorado, Iowa, Montana, New Hampshire, West Virginia, and Wisconsin began routine genotyping of all culture-confirmed TB cases in October 2000. METHODS: M. tuberculosis isolates from cases reported October 2000 through December 2003 were genotyped by spoligotyping, mycobacterial interspersed repetitive units, and IS6110-based restriction fragment length polymorphism methods. Genotyping results were linked to demographic variables from national surveillance records. Patients who were in genotype clusters were interviewed and their records reviewed to determine possible transmission links among clustered patients. Final analysis was completed during April 2004 through June 2005. RESULTS: Of 971 reported TB cases, 774 (80%) were culture-confirmed, of which 728 (94%) were genotyped. Most genotyped isolates (634 [87%]) were unique. Within 36 clusters linking 94 individuals, four clusters involved both U.S.- and foreign-born individuals. For eight clusters, genotyping results led to the discovery of previously unsuspected transmission. Transmission links between individuals were established in 21 (58%) of the 36 clusters. CONCLUSIONS: In these six low-incidence states, most isolates had unique genotypes, suggesting that most cases arose from activation of latent infection. Few TB clusters involved the foreign-born. For 58% of genotype clusters, epidemiologic investigation ascertained that clustering represented recent M. tuberculosis transmission.
Assuntos
Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Análise por Conglomerados , Colorado/epidemiologia , Genótipo , Humanos , Incidência , Iowa/epidemiologia , Montana/epidemiologia , Mycobacterium tuberculosis/isolamento & purificação , New Hampshire/epidemiologia , Polimorfismo de Fragmento de Restrição , Vigilância da População , Medição de Risco , Fatores de Risco , Tuberculose/epidemiologia , Tuberculose/prevenção & controle , Tuberculose/transmissão , West Virginia/epidemiologia , Wisconsin/epidemiologiaRESUMO
We performed a prospective, community-based evaluation of molecular typing of Mycobacterium tuberculosis isolates as a method for tuberculosis (TB) control. We performed restriction fragment-length polymorphism (RFLP) analysis of the insertion sequences IS6110 and pTBN12 for isolates recovered from 61 of 62 patients with culture-positive TB in St. Louis during 12 months. Twenty-four (39%) of the 61 patients were infected with an isolate with an RFLP pattern that was shared with >/=1 other isolate, and 11 (46%) also had epidemiologic links with patients in their cluster of cases. One case each of laboratory cross-contamination and occupational transmission were discovered. The patients in clusters were more likely to be younger, black, United States-born, to have substance abuse problems, and to live in poorer areas. A predictive algorithm for molecular identification of clusters had a sensitivity and a specificity of 75%. This study allowed the TB-control program in St. Louis to be redirected toward the affected subpopulations.
Assuntos
Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Estudos Prospectivos , Sensibilidade e Especificidade , Tuberculose/epidemiologia , Tuberculose/prevenção & controle , Tuberculose/transmissãoRESUMO
The proportion of extrapulmonary tuberculosis cases in the United States has increased from 16% of tuberculosis cases, in 1991, to 20%, in 2001. To determine associations between the demographic, clinical, and life style characteristics of patients with tuberculosis and the occurrence of extrapulmonary tuberculosis, a retrospective case-control study was conducted. This study included 705 patients with tuberculosis, representing 98% of the culture-proven cases of tuberculosis in Arkansas from 1 January 1996 through 31 December 2000. A comparison between 85 patients with extrapulmonary tuberculosis (case patients) and 620 patients with pulmonary tuberculosis (control patients) showed women (OR, 1.98; 95% CI, 1.25-3.13), non-Hispanic blacks (OR, 2.38; 95% CI, 1.42-3.97), and HIV-positive persons (OR, 4.93; 95% CI, 1.95-12.46) to have a significantly higher risk for extrapulmonary tuberculosis than men, non-Hispanic whites, and HIV-negative persons. This study expands the knowledge base regarding the epidemiology of extrapulmonary tuberculosis and enhances our understanding of the relative contribution of host-related factors to the pathogenesis of tuberculosis.
Assuntos
Tuberculose/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Fatores Etários , Idoso , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Infecções por HIV/complicações , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Fatores Sexuais , Tuberculose/etnologia , Tuberculose/fisiopatologiaRESUMO
OBJECTIVES: To determine the reason for an increase in tuberculin skin test (TST) conversion in employees in a nursing home and to determine the source case responsible for spread of tuberculosis (TB) in two nursing homes and a hospital in a rural part of Arkansas using molecular and traditional epidemiological methods. DESIGN: TB contact investigation of residents and employees of two nursing homes and a hospital. SETTING: Two nursing homes and a hospital in rural part of Arkansas. PARTICIPANTS: One hundred fifty-seven employees and 117 residents of two nursing homes and 211 employees of a hospital in rural part of Arkansas. MEASUREMENTS: Tuberculin skin test. RESULTS: Analysis of room and work assignments of residents and employees who converted their TSTs in Nursing Home A showed that residents and employees in the same wing as the suspect source case were significantly more likely to have converted their TST than residents and employees in other wings (P = .01). A nurse from the local hospital where the suspected source case had been sent developed a tuberculous cervical abscess, and one employee in Nursing Home A developed pulmonary TB. A visitor to Nursing Home A was diagnosed with culture-positive pulmonary TB 2 years later. Genotyping of the Mycobacterium tuberculosis isolates from the four secondary cases showed identical patterns. CONCLUSION: Molecular and traditional epidemiological studies revealed an outbreak of TB that began in a nursing home and spread to a second nursing home, a local hospital, and the community.
Assuntos
Surtos de Doenças , Casas de Saúde , Doenças Profissionais/epidemiologia , Tuberculose/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Arkansas/epidemiologia , Distribuição de Qui-Quadrado , Diagnóstico Diferencial , Feminino , Genótipo , Hospitais Comunitários , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Doenças Profissionais/diagnóstico , Teste Tuberculínico , Tuberculose/diagnóstico , Tuberculose/transmissãoRESUMO
Thirty-seven multidrug-resistant and 13 pan-susceptible isolates of Mycobacterium tuberculosis were analysed for the diversity of genotypes associated with known drug-resistance mechanisms. The isolates were obtained from patients attending a university tuberculosis clinic in Monterrey, Mexico. A total of 25 IS6110-RFLP patterns were obtained from the multidrug-resistant tuberculosis (MDR-TB) isolates. Approximately 65% of the MDR-TB isolates were attributed to secondary resistance. Different drug-susceptibility patterns were seen with the clustered isolates. The percentage of isolates resistant to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STR) was 100, 97.3, 48.7 and 67.6, respectively. The most common resistance-associated polymorphisms for the four drugs were as follows: INH, Ser315Thr (67.6%) in katG; RIF, Ser450Leu (41.7%) in rpoB; EMB, Met306Ile/Val/Leu (66.7%) in embB; and STR, Lys43Arg (24%) in rpsL. Drug-resistance-associated mutations were similar to changes occurring in isolates from other areas of the world, but unique, previously unreported, mutations in katG (n=5), rpoB (n=1) and rrs (n=3) were also identified.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Genótipo , Humanos , México/epidemiologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Campylobacter spp. are a major contaminant of poultry. Eating undercooked chicken and handling raw poultry have been identified as risk factors for campylobacteriosis in humans. Previous studies have found Campylobacter spp. on 90% of poultry carcasses. In the present study, pulsed-field gel electrophoresis (PFGE) was used to assess the genetic diversity of strains on retail poultry carcasses. PFGE patterns of isolates from campylobacteriosis cases were compared to those from the poultry isolates. Over a 1-year study period (March 2000 through February 2001), whole fresh young chickens (n = 72) were obtained from three retail outlets in an urban community in the south-central United States. Campylobacter spp. were isolated from 82% of these carcasses. Strains (n = 70) were defined on the basis of their PFGE pattern. Sixty-seven percent of the carcasses from which Campylobacter spp. were isolated were contaminated with more than one PFGE-distinguishable strain. During the 1-year study period, most of the PFGE patterns (59%) were limited to isolates obtained from a single carcass. Forty-one percent of the PFGE-distinguishable strains were recovered from more than one carcass. Ninety-seven percent of the carcasses contaminated with the same strain were purchased at the same time from the same store. To examine the degree of genetic stability, four strains were followed in vitro over an estimated 1,000 doublings. The PFGE pattern of one of these isolates underwent minor changes during in vitro growth. The data indicate extensive variability in the PFGE patterns of Campylobacter spp. isolated from humans and from poultry carcasses. In spite of difficulties caused by such diversity and the fact that some carcasses are contaminated with more than one strain, the pattern variation provides a useful method for linking a particular strain to its source.
Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter/isolamento & purificação , Galinhas/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Campylobacter/classificação , Campylobacter/genética , Eletroforese em Gel de Campo Pulsado , Contaminação de Alimentos , Microbiologia de Alimentos , Variação Genética , Humanos , Fatores de RiscoAssuntos
Epidemiologia Molecular , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Controle de Doenças Transmissíveis/métodos , Impressões Digitais de DNA , Farmacorresistência Bacteriana , Técnicas Genéticas , Genoma Bacteriano , Genótipo , Humanos , Polimorfismo de Fragmento de Restrição , Tuberculose/prevenção & controle , Tuberculose/transmissãoRESUMO
It was reported previously that the major fraction of the recent decrease of tuberculosis incident cases in Arkansas had been due to a decrease in the reactivated infections. Preventing transmission of Mycobacterium tuberculosis is the key to a continued decline in tuberculosis cases. In this study, we integrated epidemiological data analysis and comparative genomics to identify host and microbial factors important to tuberculosis transmission. A significantly higher proportion of cases in large clusters (containing >10 cases) were non-Hispanic black, homeless, less than 65 years old, male sex, smear-positive sputum, excessive use of alcohol, and HIV sero-positive, compared to cases in small clusters (containing 2-5 cases) diagnosed within one year. However, being non-Hispanic black and homeless within the past year were the only two host characteristics that were identified as independent risk factors for being in large clusters. This finding suggests that social behavioral factors have a more important role in transmission of tuberculosis than does the infectiousness of the source. Comparing the genomic content of one of the large cluster strains to that of a non-clustered strain from the same community identified 25 genes that differed between the two strains, potentially contributing to the observed differences in transmission.
Assuntos
Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/transmissão , Negro ou Afro-Americano/estatística & dados numéricos , Idoso , Arkansas/epidemiologia , Análise por Conglomerados , Feminino , Variação Genética , Genótipo , Soropositividade para HIV/epidemiologia , Hispânico ou Latino/estatística & dados numéricos , Pessoas Mal Alojadas/estatística & dados numéricos , Humanos , Comunicação Interdisciplinar , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Mycobacterium tuberculosis/classificação , Fatores de Risco , Escarro/microbiologia , Tuberculose/epidemiologiaRESUMO
Mycobacterium tuberculosis lipases, a diverse class of enzymes involved in lipid metabolism, may have an important role in tuberculosis (TB) pathogenesis. We explored the association of large sequence polymorphism (LSP) in one of the M. tuberculosis lipase-encoding genes, lipR (Rv3084), with patient characteristics using a population-based sample of clinical isolates to elucidate the potential role of lipR in TB pathogenesis. LSP in lipR was found in 104 (15.6%) of 665 isolates, of which 96% belonged to principal genetic group 3. When linkage by molecular type and epidemiologic evidence were compared, molecularly clustered cases infected with a lipR LSP isolate were more often epidemiologically linked than clustered cases infected with a lipR wild-type isolate. Further epidemiologic and functional studies are necessary to determine if the association between this lipR LSP and recent transmission we identified in this population reflects a functional role of lipR in TB transmission and pathogenesis or other unidentified mechanisms.
Assuntos
Proteínas de Bactérias/genética , Esterases/genética , Hidrolases/genética , Mycobacterium tuberculosis/patogenicidade , Tuberculose/transmissão , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Arkansas/epidemiologia , Criança , Pré-Escolar , Mapeamento Cromossômico , Análise por Conglomerados , Feminino , Genes Bacterianos , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Polimorfismo Genético , Tuberculose/epidemiologia , Tuberculose/microbiologia , Virulência/genética , Adulto JovemRESUMO
Incident cases of tuberculosis may result from a recently acquired Mycobacterium tuberculosis infection or from the reactivation of a latent infection acquired in the remote past. The authors used molecular fingerprinting data to estimate the relative contributions of recent and remotely acquired infection to the yearly incidence of tuberculosis in Arkansas, a state with a largely rural population where the incidence of tuberculosis declined from 7.9 cases per 100,000 population to 4.7 cases per 100,000 between 1997 and 2003. The authors used a time-restricted definition of clustering in addition to the standard definition in order to increase the specificity of the clustering measure for recent transmission. The greatest overall declines were seen in non-Hispanic Blacks (from 13.8 cases per 100,000 in 1997 to 6.5 cases per 100,000 in 2003) and persons aged 65 years or more (from 19.9 cases per 100,000 in 1997 to 8.5 cases per 100,000 in 2003). In both groups, the incidence of nonclustered cases declined more dramatically than the incidence of clustered cases. This suggests that the decline in rates resulted primarily from declining rates of disease due to reactivation of past infections. Declines in the overall incidence of tuberculosis in a population may not necessarily result from declines in active transmission.
Assuntos
Tuberculose/epidemiologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Arkansas/epidemiologia , Distribuição de Qui-Quadrado , Análise por Conglomerados , Impressões Digitais de DNA , Feminino , Genótipo , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Distribuição de Poisson , Polimorfismo de Fragmento de Restrição , Vigilância da População , Fatores de Risco , População Rural , Tuberculose/etnologiaRESUMO
Previous studies have suggested that isolates of Mycobacterium tuberculosis responsible for tuberculosis outbreaks grow more rapidly within human mononuclear phagocytes than do other isolates. Clinical scenarios suggesting virulence of specific M. tuberculosis isolates are readily identified. Determination of appropriate "control" isolates for these studies is more problematic, but equally important for validating these assays and, ultimately, for identifying biologic differences between M. tuberculosis strains that contribute to virulence. We utilized the database from a study of Ugandan tuberculosis patients and their household (HH) contacts to identify M. tuberculosis isolates transmitted within HH and nontransmitted control isolates. Isolate pairs were evaluated from matched HH in each of three clinical scenarios: (i) coprevalent disease and no disease, (ii) incident disease and no disease, and (iii) M. tuberculosis infection (purified protein derivative [PPD] positive) and no infection (PPD negative). Intracellular growth of paired organisms was determined in a blinded fashion using two models of intracellular infection in which we have previously demonstrated correlation between intracellular growth and strain virulence, primary human monocytes (MN) and THP-1 human macrophage-like cells. In both models, transmitted isolates from coprevalent disease HH displayed more rapid growth than nontransmitted control isolates. In the THP-1 model, this was also true of transmitted isolates from HH with incident disease and their controls. Differences in production of tumor necrosis factor alpha and interleukin-10 by matched isolates showed correlation with growth patterns in the THP-1 cells but not in MN. Paired isolates characterized in this manner may be of particular interest for further investigations of the virulence of M. tuberculosis.
Assuntos
Mycobacterium tuberculosis/patogenicidade , Fagócitos/imunologia , Tuberculose/epidemiologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Características da Família , Humanos , Monócitos/imunologia , Monócitos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/isolamento & purificação , Fagócitos/microbiologia , Polimorfismo de Fragmento de Restrição , Tuberculose/imunologia , Tuberculose/transmissão , Uganda/epidemiologia , VirulênciaRESUMO
We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by GST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.
Assuntos
Evolução Molecular , Genes Bacterianos , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único , Algoritmos , Técnicas de Tipagem Bacteriana/métodos , Geografia , Humanos , Repetições Minissatélites , Família Multigênica , Especificidade da Espécie , Tuberculose/microbiologiaRESUMO
The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.
Assuntos
Antituberculosos/farmacologia , Evolução Biológica , Genes Bacterianos , Isoniazida/farmacologia , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/genética , Alelos , Antibióticos Antituberculose/farmacologia , Análise Mutacional de DNA , DNA Bacteriano/genética , DNA Intergênico/genética , Etambutol/farmacologia , Deleção de Genes , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/isolamento & purificação , Fases de Leitura Aberta , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Rifampina/farmacologia , Análise de Sequência de DNA , Estreptomicina/farmacologiaRESUMO
Mycobacterium tuberculosis strains associated with IS6110 restriction fragment-length polymorphism (RFLP) pattern clusters and strains demonstrating unique IS6110 RFLP patterns were investigated in interferon- gamma -activated THP-1 cells by measurement of binding, intracellular growth rate, and cytokine production. Binding was the same for all strains; however, strains from clusters grew significantly more rapidly than did unique strains. Maximal concentration of tumor necrosis factor (TNF)-alpha was detected at 2 days after infection, with unique strains eliciting significantly greater amounts than did strains from clusters. Interleukin (IL)-10 levels peaked at 1 day after infection with strains from clusters, whereas they peaked at 5 days after infection with unique strains. Rapid growth demonstrated by strains from clusters was highly correlated with rapid production of IL-10 and suppression of TNF-alpha in THP-1 cells during the early stages of infection. Characterization of this phenotype will further advance the investigation of virulence factors in M. tuberculosis.
Assuntos
Citocinas/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/transmissão , Linhagem Celular , Elementos de DNA Transponíveis/genética , Humanos , Interleucina-10/metabolismo , Macrófagos/imunologia , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Oligonucleotídeos/análise , Polimorfismo de Fragmento de Restrição , Tuberculose/epidemiologia , Tuberculose/microbiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PE_PGRS 33, one of about 60 PE_PGRS genes in the Mycobacterium tuberculosis genome, encodes a surface-expressed protein that may be involved in the antigenic variation of M. tuberculosis strains and evasion of the host immune system. While genetic differences between the PE_PGRS 33 genes of H37Rv and CDC 1551 have been noted, genetic variation in this gene among clinical isolates has not been evaluated. In order to gain a better understanding of the genetic basis for the role of PE_PGRS in antigenic variation and evasion of the host immune system, we investigated the genetic diversity of the PE_PGRS 33 gene among 123 clinical M. tuberculosis isolates from a population-based study, using PCR and DNA sequencing. The 123 isolates belonged to principal genetic groups 1, 2, and 3 and had IS 6110 copy numbers ranging from 1 to 22. Eighty-four (68.3%) of the 123 isolates were found to have at least one sequence variation in the PE_PGRS 33 gene, relative to that of H37Rv. Twenty-five different sequence variations were observed and included three insertions (ranging from 9 to 87 bp), nine deletions (ranging from 1 to 273 bp), one insertion-and-deletion event, and 12 single-nucleotide polymorphisms (six synonymous and six nonsynonymous). Analysis of the relationships among the different PE_PGRS 33 gene sequence variations suggests that polymorphisms in the gene are shifting along evolutionary lineages. The observed genetic diversity of the PE_PGRS 33 gene supports its role in antigenic variation and can serve as a basis for future investigations of the function of the PE_PGRS 33 gene among clinical isolates.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Variação Genética , Proteínas de Membrana/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Elementos de DNA Transponíveis/genética , DNA Bacteriano/análise , Deleção de Genes , Humanos , Proteínas de Membrana/química , Mutagênese Insercional , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
To identify Mycobacterium tuberculosis virulence factors, we integrated comparative genomics and epidemiologic data analysis to investigate the relationship between certain genomic insertions and deletions in the phospholipase-C gene D (plcD) with the clinical presentation of tuberculosis (TB). Four hundred ninety-six well-characterized M. tuberculosis clinical isolates were studied. Approximately 30% (147) of the isolates had an interruption of the plcD gene. Patients infected with the plcD mutant were twice as likely to have extrathoracic disease as those infected by a strain without an interruption (adjusted odds ratio, 2.19; 95% confidence interval, 1.27, 3.76). When we limited the analysis to the 275 isolates with distinct DNA fingerprint patterns, we observed the same association (adjusted odds ratio, 2.74; 95% confidence interval, 1.35, 5.56). Furthermore, the magnitude of the association appeared to differ with the type of extrathoracic TB. Our findings suggest that the plcD gene of M. tuberculosis is potentially involved in the pathogenesis of TB, and the clinical presentation of the disease may be influenced by the genetic variability of the plcD region.
Assuntos
Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Fosfolipases Tipo C/genética , Adulto , Idoso , Southern Blotting , Impressões Digitais de DNA , Progressão da Doença , Feminino , Genes Bacterianos/genética , Marcadores Genéticos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Análise de Sequência de DNA , Índice de Gravidade de Doença , Tuberculose/fisiopatologia , Fosfolipases Tipo C/metabolismoRESUMO
Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.
Assuntos
Antituberculosos/farmacologia , Etambutol/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Sequência de Bases , Análise por Conglomerados , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Genes Bacterianos/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Família Multigênica , Análise Multivariada , Mutação/genética , Mutação/fisiologia , FilogeniaRESUMO
In the meroistic insect ovary, the oocyte synthesizes little if any RNA. Most of the RNA which accumulates in the oocyte is synthesized by trophocytes. In the polytrophic meroistic ovary each oocyte is associated with a cyst containing 1,3,7 or 15 trophocytes. The trophocytes are derived from the same cell as the oocyte. The trophocyte cysts and the oocytes of the giant silkworm moth,Antheraea pernyi (Lepidoptera: Saturniidae), are large enough to enable their isolation by microdissection. The nucleus of each trophocyte is highly polyploid, containing hundreds of nucleoli. In order to determine whether DNA coding for rRNA (rDNA) is amplified in trophocytes ofA. pernyi, the percentage of the genome hybridizing with rRNA in somatic tissues was compared to that percentage in gametogenic tissues. RNA-DNA hybridization analysis indicates that approximately the same proportion (0.018%) of the DNA extracted from male and female gemetogenic tissues (testis, isolated trophocytes, and isolated oocytes) and somatic tissues (brain, Malpighian tubules) hybridizes with rRNA. The fact that DNA hybridizing with rRNA comprises the same proportion of the total DNA extracted from trophocytes, spermatogenic cells, and male and female somatic cells indicates that rDNA is not amplified in the trophocytes ofA. pernyi. In the polytrophic ovary, polyploidization of the entire trophocyte genome rather than amplification of a small part of it accounts for the increase of rDNA available for transcription.