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D Alland

Publications and source records attributed to D Alland.

14 recordsLinked to original sources
PubMed2000

Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis.

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed2000

Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing.

Past genotypic studies of Mycobacterium tuberculosis may have incorrectly estimated the importance of specific drug resistance mutations due to a number of sampling biases including an overrepresentation of multidrug-resistant (MDR) isolates. An accurate assessment of resistance mutations is crucial for understanding basic resistance mechanisms and designing genotypic drug resistance assays. We developed a rapid closed-tube PCR assay using fluorogenic reporter molecules called molecular beacons to detect reportedly common M. tuberculosis mutations associated with resistance to isoniazid and rifampin. The assay was used in a comparative genotypic investigation of two different study populations to determine whether these known mutations account for most cases of clinical drug resistance. We analyzed samples from a reference laboratory in Madrid, Spain, which receives an overrepresentation of MDR isolates similar to prior studies and from a community medical center in New York where almost all of the resistant isolates and an equal number of susceptible controls were available. The ability of the molecular beacon assay to predict resistance to isoniazid and rifampin was also assessed. The overall sensitivity and specificity of the assay for isoniazid resistance were 85 and 100%, respectively, and those for rifampin resistance were 98 and 100%, respectively. Rifampin resistance mutations were detected equally well in isolates from both study populations; however, isoniazid resistance mutations were detected in 94% of the isolates from Madrid but in only 76% of the isolates from New York (P = 0.02). In New York, isoniazid resistance mutations were significantly more common in the MDR isolates (94%) than in single-drug-resistant isolates (44%; P < 0.001). No association between previously described mutations in the kasA gene and isoniazid resistance was found. The first mutations that cause isoniazid resistance may often occur in sequences that have not been commonly associated with isoniazid resistance, possibly in other as yet uncharacterized genes. The molecular beacon assay was simple, rapid, and highly sensitive for the detection of rifampin-resistant M. tuberculosis isolates and for the detection of isoniazid resistance in MDR isolates.

Base Sequence↗
PubMed2000

Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition.

The cell wall provides an attractive target for antibiotics against Mycobacterium tuberculosis. Agents such as isoniazid and ethambutol that work by inhibiting cell wall biosynthesis are among the most highly effective antibiotics against this pathogen. Although considerable progress has been made identifying the targets for cell wall active antibiotics, little is known about the intracellular mechanisms that are activated as a consequence of cell wall injury. These mechanisms are likely to have an important role in growth regulation and in the induction of cell death by antibiotics. We previously discovered three isoniazid-induced genes (iniB, iniA, and iniC) organized in tandem on the M. tuberculosis genome. Here, we investigate the unique features of the putative iniBAC promoter. This promoter was specifically induced by a broad range of inhibitors of cell wall biosynthesis but was not inducible by other conditions that are toxic to mycobacteria via other mechanisms. Induction required inhibitory concentrations of antibiotics and could be detected only in actively growing cells. Analysis of the iniBAC promoter sequence revealed both a regulatory element upstream and a potential repressor binding region downstream of the transcriptional start site. The induction phenotype and structure of the iniBAC promoter suggest that a complex intracellular response occurs when cell wall biosynthesis is inhibited in M. tuberculosis and other mycobacteria.

Amino Acid Sequence↗
PubMed2000

Molecular determinants of drug resistance in tuberculosis.

Rapid detection of drug-resistant tuberculosis (TB) has become increasingly important in the era of pandemic human immunodeficiency virus infection and antibiotic resistance. The identification of the molecular correlates of antibiotic resistance in Mycobacterium tuberculosis have engendered the development of DNA-based assays for the identification of drug-resistant TB. This review summarizes the recent discoveries concerning resistance to isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin and the quinolones.

Antitubercular Agents↗
PubMed1999

Molecular epidemiologic evaluation of transmissibility and virulence of Mycobacterium tuberculosis.

Discovery of genotypic markers associated with increased transmissibility in Mycobacterium tuberculosis would represent an important step in advancing mycobacterial virulence studies. M. tuberculosis strains may be classified into one of three genotypes on the basis of the presence of specific nucleotide substitutions in codon 463 of the katG gene (katG-463) and codon 95 of the gyrA gene (gyrA-95). It has previously been reported that two of these three genotypes are associated with increased IS6110-based clustering, a potential proxy of virulence. We designed a case-control analysis of U.S.-born patients with tuberculosis in San Francisco, Calif., between 1991 and 1997 to investigate associations between katG-463 and gyrA-95 genotypes and epidemiologically determined measures of strain-specific infectivity and pathogenicity and IS6110-based clustering status. We used a new class of molecular probes called molecular beacons to genotype the isolates rapidly. Infectivity was defined as the propensity of isolates to cause tuberculin skin test conversions among named contacts, and pathogenicity was defined as their propensity to cause active disease among named contacts. The molecular beacon assay was a simple and reproducible method for the detection of known single nucleotide polymorphisms in large numbers of clinical M. tuberculosis isolates. The results showed that no genotype of the katG-463- and gyrA-95-based classification system was associated with increased infectivity and pathogenicity or with increased IS6110-based clustering in San Francisco during the study period. We speculate that molecular epidemiologic studies investigating clinically relevant outcomes may contribute to the knowledge of the significance of laboratory-derived virulence factors in the propagation of tuberculosis in human communities.

AIDS-Related Opportunistic Infections↗
PubMed1998

Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): the effect of isoniazid on gene expression in Mycobacterium tuberculosis.

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency. Because of the absence of poly(A)+ mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the in vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid. The DECAL method offers a powerful new tool for the study of differential gene expression.

Base Sequence↗
PubMed1998

Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis.

We developed a new approach to DNA sequence analysis that uses fluorogenic reporter molecules--molecular beacons--and demonstrated their ability to discriminate alleles in real-time PCR assays of genomic DNA. A set of overlapping molecular beacons was used to analyze an 81-bp region of the Mycobacterium tuberculosis rpoB gene for mutations that confer resistance to the antibiotic rifampin. In a blinded study of 52 rifampin-resistant and 23 rifampin-susceptible clinical isolates, this method correctly detected mutations in all of the resistant strains and in none of the susceptible strains. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. This approach can be used to analyze any DNA sequence of moderate length with single base pair accuracy.

Antibiotics, Antitubercular↗
PubMed1998

Simultaneous genotyping and species identification using hybridization pattern recognition analysis of generic Mycobacterium DNA arrays.

High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 16S genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure.

Alleles↗
PubMed1997

A city-wide outbreak of a multiple-drug-resistant strain of Mycobacterium tuberculosis in New York.

SETTING: Incident patients with active tuberculosis (TB) resistant to two or more drugs in New York City hospitals in 1992. OBJECTIVE: To examine the New York-wide distribution of Public Health Research Institute (PHRI) strain W of Mycobacterium tuberculosis, an extremely drug-resistant strain identified by a 17-band Southern hybridization pattern using IS6110, during the peak tuberculosis year of 1992. We also compared strain W with other strains frequently observed in New York. DESIGN: Blinded retrospective study of stored M. tuberculosis cultures by restriction fragment length polymorphism (RFLP) DNA fingerprinting, and chart review. RESULTS: We found 112 cultures with the strain W fingerprint and 8 variants in 21 hospitals among incident patients hospitalized in 1992. Almost all isolates were resistant to four first-line drugs and kanamycin. This single strain made up at least 22% of New York City multiple-drug-resistant (MDR) TB in 1992, far more than any other strain. Almost all W-strain cases were acquired immune deficiency syndrome (AIDS) patients. The cluster is the most drug-resistant cluster identified in New York and the largest IS6110 fingerprint cluster identified anywhere to date. CONCLUSION: Because recommended four-drug therapy will not sterilise this very resistant strain, there was a city-wide nosocomial outbreak of W-strain TB in the early 1990s among New York AIDS patients. Other frequently seen strains were either also very resistant, or, surprisingly, pansusceptible. Individual MDR strains can be spread widely in situations where AIDS and TB are both common.

Adult↗
PubMed1996

A multi-institutional outbreak of highly drug-resistant tuberculosis: epidemiology and clinical outcomes.

OBJECTIVE: To investigate a multi-institutional outbreak of highly resistant tuberculosis and evaluate patient outcome. DESIGN: Epidemiologic investigation of every tuberculosis case reported in New York City. SETTING: Patients cared for at all public and nonpublic institutions from January 1, 1990, to August 1, 1993 (43 months). PATIENTS: We reviewed medical and public health records and conducted clinical, epidemiologic, drug susceptibility, and restriction fragment length polymorphism (RFLP) analyses. A case was defined as tuberculosis in a patient with an isolate resistant to isoniazid, rifampin, ethambutol hydrochloride, and streptomycin (and rifabutin, if sensitivity testing included it), and, if RFLP testing was done, a pattern identical to or closely related to strain W. MAIN OUTCOME MEASURES: Patient survival and the conversion of sputum cultures from positive to negative. RESULTS: Of the 357 patients who met the case definition, 267 had identical or nearly identical RFLP patterns; isolates from the other 90 patients were not available for RFLP testing. Among these 267 patients, 86% were human immunodeficiency virus (HIV)-infected, 7% were HIV-negative, and 7% had unknown HIV status. All-cause mortality was 83%. Epidemiologic linkages were identified for 70% of patients, of whom 96% likely had nosocomially acquired disease at 11 hospitals. Survival was prolonged among patients who received medications to which their isolate was susceptible, especially capreomycin sulfate, and among patients with a CD4+ T-lymphocyte count greater than 0.200 x 10(9)/L (200/microL). Treatment with isoniazid and a fluoroquinolone antibiotic was also independently associated with longer survival. CONCLUSIONS: This outbreak accounted for nearly one fourth of the cases of multidrug-resistant tuberculosis in the United States during a 43-month period. Most patients had nosocomially acquired disease, were infected with HIV, and unless promptly and appropriately treated, died rapidly. With appropriate directly observed treatment, especially combinations including an injectable medication, even severely immunocompromised patients had culture conversion and prolonged, tuberculosis-free survival.

Adolescent↗
PubMed1995

Improved outcomes for patients with multidrug-resistant tuberculosis.

We conducted a retrospective study of patients with culture-confirmed multidrug-resistant tuberculosis (MDR-TB) at Bronx-Lebanon Hospital Center (South Bronx, NY) to determine what factors affected clinical and microbiological responses and survival. For the 38 patients with MDR-TB, reporting of first-line drug susceptibilities was relatively rapid (median time, 30 days). Thirty-four patients (89%) were infected with human immunodeficiency virus (HIV), and initial and overall response rates were 59% and 50%, respectively; the median survival was 315 days; and 50% of these patients died of tuberculosis. Bivariate analysis revealed that the following factors had a positive impact on response and survival: receiving > or = 2 consecutive weeks of appropriate therapy with at least two drugs to which the isolate was susceptible in vitro; starting appropriate therapy within 4 weeks of the diagnosis; and having tuberculosis that was limited to the lungs. Multivariate analysis revealed that the only variable associated with response was receipt of appropriate therapy for > or = 2 consecutive weeks. In contrast to findings in the published literature, our results indicate the outcome of MDR-TB can be improved, particularly for severely immunosuppressed HIV-infected patients. Rapid reporting of susceptibilities and prompt initiation and continuation of appropriate antituberculous therapy improved response and survival.

AIDS-Related Opportunistic Infections↗
PubMed1994

Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods.

BACKGROUND: The incidence of tuberculosis and drug resistance is increasing in the United States, but it is not clear how much of the increase is due to reactivation of latent infection and how much to recent transmission. METHODS: We performed DNA fingerprinting using restriction-fragment-length polymorphism (RFLP) analysis of at least one isolate from every patient with confirmed tuberculosis at a major hospital in the Bronx, New York, from December 1, 1989, through December 31, 1992. Medical records and census-tract data were reviewed for relevant clinical, social, and demographic data. RESULTS: Of 130 patients with tuberculosis, 104 adults (80 percent) had complete medical records and isolates whose DNA fingerprints could be evaluated. Isolates from 65 patients (62.5 percent) had unique RFLP patterns, whereas isolates from 39 patients (37.5 percent) had RFLP patterns that were identical to those of an isolate from at least 1 other study patient; the isolates in the latter group were classified into 12 clusters. Patients whose isolates were included in one of the clusters were inferred to have recently transmitted disease. Independent risk factors for having a clustered isolate included seropositivity for the human immunodeficiency virus (HIV) (odds ratio for Hispanic patients, 4.31; P = 0.02; for non-Hispanic patients, 3.12; P = 0.07), Hispanic ethnicity combined with HIV seronegativity (odds ratio, 5.13; P = 0.05), infection with drug-resistant tuberculosis (odds ratio, 4.52; P = 0.005), and younger age (odds ratio, 1.59; P = 0.02). Residence in sections of the Bronx with a median household income below $20,000 was also associated with having a clustered isolate (odds ratio, 3.22; P = 0.04). CONCLUSIONS: In the inner-city community we studied, recently transmitted tuberculosis accounts for approximately 40 percent of the incident cases and almost two thirds of drug-resistant cases. Recent transmission of tuberculosis, and not only reactivation of latent disease, contributes substantially to the increase in tuberculosis.

AIDS-Related Opportunistic Infections↗
PubMed1992

A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein.

Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.

Amino Acid Sequence↗