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PubMed1998

Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis.

The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 micrograms/ml.

Amidohydrolases↗
PubMed2000

Mycobacterium smegmatis has two pyrazinamidase enzymes, PncA and pzaA.

The Mycobacterium smegmatis pncA gene, encoding nicotinamidase/pyrazinamidase, was identified. While it was similar to counterparts from other mycobacteria, the M. smegmatis PncA had little homology to the other M. smegmatis pyrazinamidase/nicotinamidase, encoded by the pzaA gene. Transformation of Mycobacterium bovis strain BCG with M. smegmatis pncA or pzaA conferred susceptibility to pyrazinamide.

Amidohydrolases↗
PubMed2006

Detection of multidrug resistance in Mycobacterium tuberculosis.

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.

Antitubercular Agents↗
PubMed1990

Modified method for testing the quality of albumin-containing enrichments used in growth media for mycobacteria.

Many commercially available media for cultivation of mycobacteria have failed to support the growth of these organisms. This is especially true of media prepared with albumin-containing enrichments. Earlier, we developed a method for rapid identification of good albumin enrichments for agar-based media used to test the susceptibility of tubercle bacilli to pyrazinamide. The method was modified to make testing of the acceptability of albumin enrichments for primary isolation media for mycobacteria possible. We describe here a simple turbidimetric test using a specific Bacillus subtilis strain to assay quickly (24 h) different lots of albumin-containing enrichments that may be used in the preparation of growth media for mycobacteria.

Albumins↗
PubMed1997

Rapid differentiation of bovine and human tubercle bacilli based on a characteristic mutation in the bovine pyrazinamidase gene.

Bovine tuberculosis (TB) caused by Mycobacterium bovis is an important veterinary disease that can also afflict humans. Although M. bovis shares an almost identical genome with M. tuberculosis, subtle differences in host specificity and several biochemical parameters can be used to distinguish the two closely related species. The current method for distinguishing M. bovis from M. tuberculosis relies on tedious testing of biochemical parameters, including natural resistance to pyrazinamide and defective pyrazinamidase (PZase) activity of M. bovis strains. In this study, we report the development of a rapid PCR-single-strand conformation polymorphism (SSCP) assay to differentiate M. bovis from M. tuberculosis strains, based on the detection of a single characteristic point mutation in the PZase gene (pncA) of M. bovis. Eighty-seven of 89 M. bovis strains could be distinguished from M. tuberculosis strains. Surprisingly, two animal isolates which were initially identified as M. bovis were shown to be M. africanum because they had a wild-type pncA sequence with positive PZase. These two M. africanum strains contain multiple (three and six) copies of insertion sequence IS6110, a feature they have in common with M. tuberculosis. The implication of this finding for the taxonomy of M. tuberculosis complex is discussed in relation to host preference and epidemiology. The development of a rapid PCR-SSCP test for distinguishing M. bovis from M. tuberculosis will be useful for monitoring the spread of bovine TB to humans in areas where bovine TB is endemic and for directing the treatment of human TB caused by M. bovis.

Amidohydrolases↗
PubMed1997

Genetic characterization of multidrug-resistant Mycobacterium bovis strains from a hospital outbreak involving human immunodeficiency virus-positive patients.

Nineteen multidrug-resistant (MDR) Mycobacterium complex strains isolated in a nosocomial outbreak were characterized at the molecular level. The strains were microbiologically characterized as Mycobacterium bovis. The mpt40 sequence was not present in chromosomal DNA from these strains, supporting the fact that they were M. bovis. All of the isolates were resistant to isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, para-aminosalicylic acid, clarithromycin, cycloserine, ethionamide, ofloxacin, capreomycin, and amikacin. By performing the standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP) analysis, we were able to differentiate two groups (groups A and B) containing two (16 isolates) and three (3 isolates) IS6110 copies, respectively. These strains were typed by spoligotyping, developed to distinguish M. bovis strains and also to distinguish them from M. tuberculosis strains (J. Kamerbeek et al., J. Clin. Microbiol. 35:907-914, 1997). All the strains were confirmed to be M. bovis. In addition, spoligotyping showed a difference in only 1 of 43 spacers between RFLP groups A and B. The rpo beta region of several strains representative of each identified group was cloned and sequenced, and identical mutations (Ser-531 to Leu) responsible for the rifampin resistance phenotype were found. To our knowledge, this is the first characterization at the molecular level of an MDR M. bovis strain responsible for a nosocomial outbreak.

AIDS-Related Opportunistic Infections↗
PubMed2000

Differentiation of clinical Mycobacterium tuberculosis complex isolates by gyrB DNA sequence polymorphism analysis.

The discriminatory power of gyrB DNA sequence polymorphisms for differentiation of the species of the Mycobacterium tuberculosis complex (MTBC) was evaluated by sequencing and restriction fragment length polymorphism (RFLP) analysis of a 1,020-bp fragment amplified from clinical isolates of M. tuberculosis, Mycobacterium bovis (pyrazinamide [PZA] resistant as well as PZA susceptible), Mycobacterium africanum subtypes I and II, and Mycobacterium microti types vole and llama. We found sequence polymorphisms in four regions described previously and at one additional position. These differences in the gyrB sequences allow an accurate discrimination of M. bovis, M. microti, and M. africanum subtype I. The PZA-susceptible subtypes of M. bovis shared the M. bovis-specific substitution at position 756 with the PZA-resistant strains, but can be unambiguously differentiated by a characteristic substitution at position 1311. As a drawback, M. tuberculosis and M. africanum subtype II showed an identical gyrB sequence that facilitates discrimination from the other species, but not from each other. A PCR-RFLP technique applying three restriction enzymes could be shown to be a rapid and easy-to-perform tool for the differentiation of the members of the MTBC. Based on these results, we present a clear diagnostic algorithm for the differentiation of species of the MTBC.

Animals↗
PubMed2002

Identification of the causative organism of tuberculous lymphadenitis in ethiopia by PCR.

UNLABELLED: Tuberculous lymphadenitis (TBLN) is a common form of extrapulmonary tuberculosis with multiple differential diagnoses. Demonstration of the etiologic agent by smear microscopy or culture of fine needle aspirate (FNA) specimens is often unsuccessful. FNA specimens from 40 patients presenting at a rural health center in South Ethiopia and diagnosed as positive for TBLN on the basis of clinical and cytological criteria were analyzed for mycobacterial DNA by PCR. Thirty (75%) had cervical lymphadenitis and 11 (27.5%) were seropositive for human immunodeficiency virus (HIV). Three primer sets were initially used to identify the causative agent at the genus (antigen 85 complex), complex (IS6110 insertion sequence), and species (pncA gene and allelic variation) levels. Among the forty TBLN cases, 35 (87.5%) were positive by PCR at the genus and complex levels. Based on PCR for detection of allelic variation at position 169, 24 (68.6%) of the 35 were positive for Mycobacterium tuberculosis and 6 (17.1%) were positive for M. bovis. These six were positive in additional PCR assays using the JB21-JB22 primer set, which is highly specific for M. bovis. Five (14.1%) showed amplification for both M. tuberculosis and M. bovis with the allele-specific primer set. Cooccurrence of pyrazinamide (PZA)-sensitive and -resistant M. tuberculosis in those five cases was indicated, since all were negative in assays with the JB21-JB22 primer set. This feature was seen in 3 of 11 HIV-positive and 2 of 29 HIV-negative individuals (P < 0.001). CONCLUSION: among 35 PCR-positive cases of TBLN from southern Ethiopia, 29 (82.9%) were caused by M. tuberculosis and six (17.1%) were caused by M. bovis.

Biopsy, Needle↗
PubMed2002

Temperature-mediated heteroduplex analysis performed by using denaturing high-performance liquid chromatography to identify sequence polymorphisms in Mycobacterium tuberculosis complex organisms.

PCR products containing sequence polymorphisms were prepared from six mycobacterial genes, denatured, mixed with reference PCR products, and reannealed; the mixtures were then examined with a denaturing high-performance liquid chromatography system (WAVE) equipped with a temperature-controlled alkalated polystyrene divinyl benzene column. Mismatching of bases in heteroduplexes of the PCR products causes elution patterns of the DNA from the column to be altered. The six mycobacterial genes studied were oxyR, in which a specific polymorphism (G(1031)A) is found only in certain species of the Mycobacterium tuberculosis complex, and five genes in which mutations associated with antituberculosis drug resistance have been found. The resistance genes (with affected drug and PCR product sizes given parenthetically) were rpoB (rifampin; 258 bp), katG (isoniazid; 205 bp), pncA (pyrazinamide; 579 bp); rpsL (streptomycin; 196 bp), and embB (ethambutol; 185 bp). Elution patterns of heteroduplexes of all 20 polymorphisms studied shifted detectably at column temperatures ranging from 65.3 to 68 degrees C and elution times of 3.5 to 6 min. These results show that temperature-mediated heteroduplex analysis is a potentially useful genotypic screen for mutations associated with antituberculosis drug resistance and for the G(1031)A polymorphism in oxyR. The method may allow users to detect novel as well as heterogeneous mutations without using expensive kits or detection labels.

Antitubercular Agents↗
PubMed2002

Characterization of Mycobacterium tuberculosis complex isolates from the cerebrospinal fluid of meningitis patients at six fever hospitals in Egypt.

Mycobacterium tuberculosis complex isolates from cerebrospinal fluid of 67 meningitis patients were obtained from six fever hospitals in Egypt. One M. bovis and 66 M. tuberculosis isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis of oxyR. Among the M. tuberculosis isolates, 53 unique strain types (with 3 to 16 copies of IS6110) were found by RFLP analyses. Nine clusters (eight with two isolates each and one with six isolates) were also found. Thirty-six spoligotypes, including at least 10 that have been previously reported from other countries, were also observed. Forty-one (62.1%) of the isolates were in spoligotype clusters, and 22 (33%) of the isolates were in RFLP clusters. Fifty-one of the isolates were susceptible in vitro to all of the antituberculosis drugs tested, 11 were monoresistant to capreomycin, rifampin, isoniazid (INH), pyrazinamide, or streptomycin (STR), 4 were resistant to STR and INH, and 1 was resistant to STR, INH, and ethambutol.

Bacterial Proteins↗
PubMed2004

Genomic characterization of an endemic Mycobacterium tuberculosis strain: evolutionary and epidemiologic implications.

In a study of 302 Mycobacterium tuberculosis clinical isolates from the low-incidence Canadian-born population of Quebec, we characterized a large endemic strain family by using genomic deletions. The DS6(Quebec) deleted region (11.4 kb) defined a strain family of 143 isolates encompassing two subgroups: one characterized by pyrazinamide (PZA) susceptibility and the other marked by a PZA-monoresistant phenotype. A second deletion (8 bp) in the pncA gene was shared by all 76 isolates with the PZA resistance phenotype, whereas a third DRv0961 deletion (970 bp) defined a further subset of 15 isolates. From their deletion profiles, we derived a most parsimonious evolutionary scenario and compared multiple standard genotyping modalities (using IS6110 restriction fragment length polymorphism [RFLP], spoligotyping, and mycobacterial interspersed repetitive units [MIRU]) across the deletion-based subgroups. The use of a single genotyping modality yielded an unexpectedly high proportion of clustered isolates for a high IS6110 copy strain (27% by IS6110 RFLP, 61% by MIRU, and 77% by spoligotyping). By combining all three modalities, only 14% were genotypically clustered overall, a result more congruent with the epidemiologic profile of reactivation tuberculosis, as suggested by the older age (mean age, 60 years), rural setting, and low proportion of epidemiologic links. These results provide insight into the evolution of genotypes in endemic strains and the potential for false clustering in molecular epidemiologic studies.

Biological Evolution↗
PubMed2006

Evaluation of MGIT 960-based antimicrobial testing and determination of critical concentrations of first- and second-line antimicrobial drugs with drug-resistant clinical strains of Mycobacterium tuberculosis.

The objectives of this study were to (i) compare agreement of the MGIT 960 system for first-line drugs with a methodology (the resistance ratio method [RRM]) that had been used in clinical trials, relating drug susceptibility to clinical outcome; (ii) compare the performance of the MGIT 960, RRM, and microtiter plate assay (MPA) methodologies for second-line drug testing; and (iii) define critical concentrations for ciprofloxacin and moxifloxacin for liquid-culture-based testing. The large collection of clinical isolates of Mycobacterium tuberculosis (n = 247) used included 176 (71%) multidrug-resistant isolates. The results for MGIT 960 and the RRM for rifampin and isoniazid (n = 200) were in excellent (99 to 100%) agreement for all strains. For streptomycin, 97% of the results at the critical concentration and 92% at high concentration, and for pyrazinamide 92% of results overall, were concordant, but for ethambutol, fewer than 85% (65% for the critical concentration and 84% for the high concentration) of the MGIT-based results were concordant with those for the RRM. The MGIT 960, RRM, and MPA assays (n = 133) correlated well for most second-line drugs tested. For susceptibility to ofloxacin, the MGIT 960 and MPA results were in full agreement. The amikacin and rifabutin results obtained by MGIT 960 agreed with the RRM results in 131 (99%) cases, and for capreomycin, they agreed for 129 of 133 isolates tested (97%). For prothionamide testing, only a limited number of drug-resistant isolates were available for testing and drawing definitive conclusions. We propose critical concentrations of 1.0 microg/ml and 0.125 microg/ml for ciprofloxacin and moxifloxacin, respectively, for liquid-culture-based testing.

Antitubercular Agents↗
PubMed1989

Management and outcome of chemotherapy for childhood tuberculosis. Medical Research Council Tuberculosis and Chest Diseases Unit.

The management and outcome of chemotherapy is reported for 393 children with tuberculosis notified in 1983 (191 (49%) of white, 155 (39%) of Indian, Pakistani, or Bangladeshi, and 47 (12%) of other ethnic origins). Most (313) had respiratory disease, 65 had extrathoracic lymph node disease, and 15 had both. Only 15 (4%) of the 390 children for whom information was available did not complete chemotherapy, 10 because of default. All except 23 (6%) of the children known to have completed chemotherapy received isoniazid and rifampicin, 194 (52%) without additional drugs, 126 (34%) with ethambutol, eight (2%) with pyrazinamide, and seven (2%) with both drugs in the initial phase. The median duration of treatment was nine months. At the time they were last seen, all except six of the 375 children who completed chemotherapy were classified by the clinician as cured either on the primary course of chemotherapy (348, 93%) or after modification for failure or relapse (11.3%), or toxicity (10.3%). The remaining children were still on treatment for relapse (n = 2) or had defaulted from follow up (n = 4).

Adolescent↗
PubMed1972

Adverse reactions to daily and intermittent rifampicin regimens for pulmonary tuberculosis in Hong Kong.

This paper reports the nature, incidence, and severity of adverse reactions to regimens of rifampicin and ethambutol given once weekly, twice weekly, or daily and to a standard reserve regimen in a total of 330 Chinese failure patients who completed at least six months' chemotherapy in a therapeutic comparison in Hong Kong.The adverse reactions which occurred on the regimens of intermittent rifampicin were termed cutaneous, abdominal, "flu", and respiratory; in addition, purpura and abnormal liver function tests were encountered. There was an association of adverse reactions with the interval between doses and with the dose size of rifampicin, the highest incidence occurring with once-weekly rifampicin in high dosage. A procedure was developed for managing adverse reactions to intermittent rifampicin. Of 202 patients treated with intermittent rifampicin 60 developed adverse reactions, but in only 7 (3%) was it necessary to terminate the drug, though a further 10 (5%) were changed to daily rifampicin. On daily rifampicin, generalized hypersensitivity, cutaneous reactions, (one with purpura), and impaired liver function were encountered. Adverse reactions on the standard ethionamide, pyrazinamide, and cycloserine regimen were frequent and some were serious.

Alanine Transaminase↗
PubMed1996

Mycobacterium kansasii: its presentation, treatment and outcome in HIV infected patients.

AIM: To report the clinical significance and treatment of Mycobacterium kansasii infection in the context of HIV disease. DESIGN/METHODS: Retrospective case review of all isolates of M kansasii until June 1994. RESULTS: Ten cases of M kansasii were isolated. All but one patient with this infection had clinical symptoms compatible with generalised infection. The majority had chest infections with the organism isolated on induced sputum but not routine sputum. All isolates were sensitive to ethambutol and nine of 10 to rifampicin. All isolates were resistant to isoniazid and pyrazinamide. CONCLUSION: M kansasii is a pathogen in HIV infected patients and should be treated when isolated. Treatment should be with rifampicin and ethambutol but not isoniazid, as has been recommended previously.

AIDS-Related Opportunistic Infections↗
PubMed1982

Bacteriological survey of tuberculous lymphadenitis in South-east England: 1973-80.

During the eight years 1973-80 the Public Health Laboratory Service Regional Centre for Tuberculosis Bacteriology received cultures of mycobacteria from 2339 patients with tuberculous lymphadenitis. Of these, 2272 were M tuberculosis (2207 human and 65 bovine strains) and 67 were other mycobacterial species, usually M avium and its intracellulare variant. Disease due to the human strains of M tuberculosis occurred most often in young women of Asian ethnic origin. Many bovine strains isolated from Asian patients differ from the classical bovine type in being sensitive to pyrazinamide: the origin of these strains, whether from other people or from cattle, is unknown. Lymphadenitis due to bovine strains tended to occur in an older age group than the human strains and probably include relatively more cases of reactivation diseases. Infection caused by the other mycobacterial species occurred mainly in young children of European origin. Tuberculosis, therefore, remains an important cause of lymphadenopathy in Britain.

Adolescent↗
PubMed1986

Renal hypouricaemia in a patient with 48,XXYY syndrome.

Studies on hypouricaemia observed in a patient with 48,XXYY syndrome revealed an abnormality in renal urate handling. His renal urate clearance was abnormally increased. Inosine administration and provocative tests using probenecid and pyrazinamide identified an isolated renal tubular abnormality with increased urate secretion. Since the serum urate in his brother with a normal sex chromosome constitution was also low, the association of renal hypouricaemia and 48,XXYY syndrome in this patient is probably coincidental. Although the brother was not investigated, these siblings may be a previously unreported case of familial hypouricaemia due to isolated renal hypersecretion.

Adult↗
PubMed1990

Pulmonary infiltrates and fever induced by isoniazid.

An 88 year old woman with bacteriologically confirmed pulmonary tuberculosis was first treated with isoniazid, rifampicin and pyrazinamide. Two weeks later she developed pulmonary infiltrates with fever. A drug-induced reaction was suspected but the reaction recurred three times until isoniazid was identified as the cause. The reaction became worse each time, finally being nearly fatal.

Aged↗