Frequency and implications of pyrazinamide resistance in managing previously treated tuberculosis patients.

OBJECTIVE: To determine the extent of pyrazinamide (PZA) resistance in isolates from previously treated patients from the Western Cape, South Africa. DESIGN: Drug-resistant isolates, isolates resistant to one or more drugs other than PZA (PZA resistance is not routinely determined) (n = 127), and drug-susceptible (n = 47) clinical isolates of Mycobacterium tuberculosis from previously treated patients from the Western Cape were phenotypically (BACTEC MGIT 960) and genotypically (pncA gene sequencing) analysed for PZA resistance. RESULTS: MGIT analysis found that 68 of the 127 drug-resistant isolates were PZA-resistant. Nearly all (63/68) PZA-resistant isolates had diverse nucleotide changes scattered throughout the pncA gene, and five PZA-resistant isolates had no pncA mutations. Of the 47 phenotypically susceptible isolates, 46 were susceptible to PZA, while one isolate was PZA-monoresistant (OR = 53.0, 95% CI = 7.1-396.5). A pncA polymorphism (Thr114Met) that did not confer PZA resistance was also identified. PZA resistance was strongly associated with multidrug-resistant tuberculosis (MDR-TB). CONCLUSION: An alarmingly high proportion of South African drug-resistant M. tuberculosis isolates are PZA-resistant, indicating that PZA should not be relied upon in managing patients with MDR-TB in the Western Cape. A method for the rapid detection of PZA resistance would be beneficial in managing patients with suspected drug resistance.     

Characterisation of the pncA gene in Mycobacterium tuberculosis isolates from Gauteng, South Africa.

SETTING: The use of pyrazinamide (PZA) is important for the treatment of Mycobacterium tuberculosis as it is bactericidal to semi-dormant mycobacteria that are not affected by other drugs. The incidence of resistance to PZA and other drugs used in the treatment of M. tuberculosis is increasing in South Africa. OBJECTIVE: To characterise the pncA gene of M. tuberculosis isolates from Gauteng, South Africa, and to develop a rapid diagnostic method. DESIGN: The pncA gene and the putative regulatory gene were characterised by sequence analysis in a total of six PZA susceptible and 15 resistant isolates. The association with classical PZA susceptibility testing and PZase activity was determined. RESULTS: All PZA-resistant isolates were PZase negative as well as resistant to at least one other anti-tuberculosis drugs. Mutations were identified throughout the length of the pncA gene in 10/15 PZA-resistant isolates. Five lacked PZase activity, but the wild type pncA sequence was present. In all six PZase-positive strains, a PZA-susceptible pattern was obtained on BACTEC and the wild type pncA sequence was present. CONCLUSION: Sequencing is an effective means to identify mutations in the pncA gene in M. tuberculosis and therefore resistance to PZA. The fact that some PZA-resistant M. tuberculosis isolates lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance exist. In PZase negative strains with no genetic changes which are resistant to 100 microg/ml and susceptible to 300 microg/ml, 300 microg/ml may be a more reliable breakpoint.     

Discrepant results between pyrazinamide susceptibility testing by the reference BACTEC 460TB method and pncA DNA sequencing in patients infected with multidrug-resistant W-Beijing Mycobacterium tuberculosis strains

BACKGROUND: Mycobacterium tuberculosis strains belonging to the W-Beijing family have received broad clinical and public health attention because of their rapid worldwide spread and their frequent association with outbreaks, multidrug resistance, and treatment failures and relapses. METHODS: The present study examined a large number of multidrug-resistant strain-W isolates (isolates of 29 patients) by susceptibility testing for pyrazinamide (PZA) using the reference BACTEC 460TB method (Becton Dickinson Diagnostic Instrument Systems; Sparks, MD) and also by DNA sequencing of the pncA gene. RESULTS: We found that despite of the presence of a strain W-specific Thr47Ala in the pncA gene, all strains showed susceptibility to PZA in the reference BACTEC 460TB system due to their higher minimum inhibitory concentrations (relative to BACTEC 460TB PZA-susceptible strains). CONCLUSIONS: Our results suggest that the current radiometric reference method cannot reproducibly detect PZA resistance in patients infected with W-Beijing strains. Therefore, PZA susceptibility testing should instead be based on analysis of the pncA gene for resistance-associated mutations.     

Characterization of gyrA and gyrB mutations and fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates from Hubei Province, China

ObjectiveThe study aimed to investigate gyrA and gyrB mutations in Mycobacterium tuberculosis (MTB) clinical strains from 93 patients with pulmonary tuberculosis in Hubei Province, China, and analyze the association between mutation patterns of the genes and ofloxacin resistance level.ResultsAmong 93 MTB clinical isolates, 61 were ofloxacin-resistant by the proportion method, and 32 were ofloxacin-susceptible MDR-TB. No mutation in the gyrB gene was found in any MTB strains. In the 61 ofloxacin-resistant isolates, 54 mutations were observed in the gyrA gene. Only one mutation in the gyrA gene was found in ofloxacinsusceptible MDR-TB isolates. In this study, the mutation patterns of gyrA involved seven patterns of single codon mutation (A90V, S91P, S91T, D94N, D94Y, D94G or D94A) and two patterns of double codons mutation (S91P &; D94H, S91P &; D94A). The ofloxacin minimal inhibitory concentrations (MICs) of three patterns of single codon mutations in the gyrA gene (codons 94, 90 and 91) showed a statistically significant difference (p < 0.0001).ConclusionsThe gyrA mutations at codons 90, 91 and 94 constitute the primary mechanism of fluoroquinolone resistance in MTB, and mutations at codon 91 in the gyrA gene may be associated with low-level resistance to ofloxacin.     

Pyrazinoic acid efflux rate in Mycobacterium tuberculosis is a better proxy of pyrazinamide resistance

Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood. The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M.?tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated. The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M.?tuberculosis strains fell within a specific range, and M.?tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for M.?smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux rate was found to be 900 fold higher than the average efflux rate for M.?tuberculosis strains. No significant variability was observed in the pyrazinamide flux rate. The pyrazinoic acid efflux rate explained 61% of the variability in Bactec pyrazinamide susceptibility, 24% of Wayne activity, and 51% of the Bactec 460TB growth index. In contrast, pyrazinamidase activity accounted for only 27% of the Bactec pyrazinamide susceptibility. This finding suggests that mechanisms other than pncA mutations (reduction of pyrazinamidase activity) are also implicated in pyrazinamide resistance, and that pyrazinoic acid efflux rate acts as a better proxy for pyrazinamide resistance than the presence of pncA mutations. This is relevant to the design of molecular diagnostics for pyrazinamide susceptibility, which currently rely on pncA gene mutation detection.     

The paradox of pyrazinamide: an update on the molecular mechanisms of pyrazinamide resistance in Mycobacteria

Pyrazinamide (PZA) is an important front line anti-tuberculosis drug because of its sterilizing activity against semi-dormant tubercle bacilli. In spite of its remarkable role in shortening the treatment duration from 9 months to 6 months when used in combination with Rifampicin and Isoniazid, PZA remains a difficult paradox because of its incompletely understood mode of action and mechanism of resistance. PZA is a nicotinamide analog prodrug which is converted into the active bactericidal form pyrazinoic acid by the bacterial enzyme pyrazinamidase (PZase). PZA does not appear to have a specific cellular target and instead, exerts its bactericidal effect by disrupting the membrane energetics and acidification of cytoplasm. Majority (72-97%) of PZA-resistant isolates of M. tuberculosis exhibit mutations in their pncA gene or upstream area leading to loss of PZase activity. A wide diversity of pncA mutations scattered along the entire length of pncA gene is unique to PZA resistance. However, PZA resistant isolates with normal PZase activity and wild type pncA sequences have also been reported in several studies which indicate that alternate mechanisms of PZA resistance exist. Investigations into these mechanisms would be useful in developing alternative diagnostic/therapeutic measures. This review presents the update of various mechanisms of PZA resistance in different mycobacteria with special emphasis on mode of action of PZA and mechanisms of resistance in Mycobacterium tuberculosis.     

吡嗪酰胺耐药性结核分枝杆菌的噬菌体检测技术研究

目的 建立噬菌体生物扩增法（PhaB）快速测定吡嗪酰胺耐药性，并探讨其在结核分枝杆菌吡嗪酰胺耐药性测定中的应用价值。方法 应用建立的PhaB测定108株结核分枝杆菌临床分离株的吡嗪酰胺耐药性，并与绝对浓度法的药敏结果进行比较，对不符合的菌株进行最低抑菌浓度（MIC）测定和序列测定分析。结果 PhaB检测吡嗪酰胺耐药性的最佳测定条件为pH值5．5、药物浓度200μg／ml、37℃作用48h。用绝对浓度法检测108株结核分枝杆菌临床分离株，其中吡嗪酰胺敏感33株、耐药75株；用PhaB检测该108株结核分枝杆菌临床分离株，若以噬菌斑减少95％为判断标准。则吡嗪酰胺敏感32株，耐药76株。2种方法检测均为敏感的为28株，均为耐药的为71株，符合率为91．7％；2种方法检测结果不符的为9株，其中5株的PhaB结果与MIC结果相符，4株的结果不符，测序结果表明9株中有7株的PhaB结果与测序结果相符。如以绝对浓度法药敏结果为判断标准，PhaB检测吡嗪酰胺耐药性的敏感性为94．7％，特异性为84．8％，阳性预测值为93．4％，阴性预测值为87．5％，准确性为91．7％。结论 PhaB测定吡嗪酰胺耐药性简便、快速，3d即可获得药敏结果，可作为吡嗪酰胺耐药性的快速筛选方法。     

结核分支杆菌耐吡嗪酰胺分离株pncA基因突变的研究

目的 了解我国结核分支杆菌耐吡嗪酰胺（ＰＺＡ）分离株ｐｎｃＡ基因突变情况,研究其与耐ＰＺＡ之间的关系。方法 通过聚合酶链反应（ＰＣＲ）－单链构象多态性（ＳＳＣＰ）和ＰＣＲ－循环测序法（ＡＳ）分析７４株结核分支杆菌临床分离株的ｐｎｃＡ基因。以结核分支杆菌Ｈ３７ＲＶ标准株为对照。结果３２株药物敏感株ｐｎｃＡ基因ＳＳＣＰ分析未发现异常。２０株耐非ＰＺＡ药物的分离株中,１６株ｐｎｃＡ基因ＳＳＣＰ泳动正常;     

Mutations in the gyrA and gyrB genes of fluoroquinolone-resistant Mycobacterium tuberculosis from TB patients in Thailand

Among fluoroquinolone-resistant Mycobacterium tuberculosis (FQr-MTB) isolates, mutation at positions 90, 91, and 94 in gyrA gene and at positions 495, 516, and 533 in gyrB gene have been frequently reported. In this study, 35 isolates of FQr-MTB were collected from Siriraj Hospital and Chest Disease Institute. The quinolone-resistance-determining regions (QRDR) of gyrA and gyrB genes in all 35 FQr-MTB isolates and from the H37Ra MTB strain were amplified using polymerase chain reaction (PCR). DNA-sequencing and single-strand conformation polymorphism (SSCP) were further utilized for characterization of the mutations in the QRDR of gyrA and gyrB genes and mutation screening, respectively. From DNA-sequencing, 21 of 35 (60%) exhibited single-point mutations in different positions, at Ala90Val, Ser91Pro, and Asp94(Gly/Ala/His/Asn); and one novel mutation position at Gly88Cys in the gyrA gene and Asp495Asn in the gyrB gene. These positions were previously frequently reported to be responsible for FQr-MTB. The other 14 FQr-MTB isolates (40%) had no mutation. This study is the first report of mutation occurring only in the QRDR of the gyrB gene, without prior mutation in the gyrA QRDR among FQr-MTB isolates. By SSCP analysis for screening of the mutant FQr-MTB, the SSCP patterns of mutated FQr-MTB isolates were clearly differentiated from the SSCP patterns of FQs-MTB.     

Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru.

SETTING: Twenty-nine epidemiological unrelated and mostly multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains from Peruvian patients. OBJECTIVE: To investigate the molecular genetics of MDR-TB strains recovered in a Latin American country. DESIGN: Antimicrobial agent susceptibility testing, major genetic group designation, IS6110 fingerprinting, spoligotyping, and automated deoxyribonucleic acid sequencing of regions of the katG, rpoB, embB, gyrA, and pncA genes with mutations commonly associated with drug resistance. RESULTS: Nineteen isolates were found to be multidrug-resistant by susceptibility testing. IS6110 typing showed that virtually all isolates were unique and therefore had independently acquired drug resistance. Seventy-nine percent of isoniazid-resistant strains had a Ser315Thr amino acid change in KatG. Ninety-five percent of rifampin-resistant isolates had amino acid replacements in the rifampin-resistance determining region of RpoB. Six of 11 ethambutol-resistant strains had EmbB alterations. Eleven pyrazinamide-resistant strains had distinct mutations in pncA. CONCLUSION: Virtually all organisms evolved drug resistance independently. The types of drug resistance-associated mutations identified were very similar to changes occurring in isolates from other areas of the world. Nucleotide sequence-based strategies for rapid detection of drug resistance-conferring mutants will be applicable to organisms recovered in Peru, and potentially other areas of Latin America.     

Emergence and properties of fluoroquinolone resistant Salmonella enterica serovar Typhi strains isolated from Nepal in 2002 and 2003

A total of 171 Salmonella enterica serovar Typhi strains isolated from Nepal, mostly from patients with typhoid fever in 2002-2003, were tested for antimicrobial susceptibility by disk diffusion assay. Selected S. enterica serovar Typhi isolates were tested for MICs by E-test for ceftriaxone, ciprofloxacin and ofloxacin. Mutations of DNA gyrase gyrA and gyrB and topoisomerase IV parC and parE were identified by sequencing of PCR amplicons. By disk diffusion assay, 75/171 S. enterica serovar Typhi isolates were resistant to nalidixic acid, ampicillin, choramphenicol, streptomycin, tetracycline, sulfisoxazole, and trimethroprim/sulfamethoxazoles. Multiple drug resistance to the 7 antimicrobials was most predominant among S. enterica serovar Typhi isolates in this study. Resistance to nalidixic acid was detected in 76/111 and 56/60 of total isolates collected in 2002 and 2003, respectively. Nalidixic acid-resistant isolates in 2002 and 2003 showed MIC range for ciprofloxacin of 0.125-0.250 mg/l. Nalidixic acid-resistant isolates contained point mutations in gyrA and parC but not gyrB and parE. The gyrA mutation of nalidixic acid-resistant isolates obtained in 2002 and 2003 had amino acid substitution at position 83 of Serine-->Tyrosine and Serine-->Phenylalanine, respectively. Two different mutations of gyrA were detected among nalidixic acid-resistant isolates. Thus it is necessary to monitor mutation in DNA topoisomerase associated with increases in quinolones resistance.     

Genetic diversity and evidence for acquired antimicrobial resistance in Mycobacterium tuberculosis at a large hospital in South India.

OBJECTIVE: To assess genetic diversity and drug resistance of Mycobacterium tuberculosis isolates collected at Christian Medical College Hospital (CMCH), Vellore, India, between July 1995 and May 1996. MATERIALS AND METHODS: Isolates were subjected to IS6110-based restriction fragment length polymorphism (RFLP) analysis and tested for resistance to isoniazid, rifampin, ethambutol, streptomycin, and pyrazinamide, and DNA from selected strains was sequenced in regions associated with drug resistance. RESULTS: One hundred and one M. tuberculosis isolates were collected from 87 patients with pulmonary tuberculosis. Charts of 69 patients were reviewed for history of tuberculosis illness and treatment. DNA from 29 strains was sequenced in katG, rpoB, and gyrA, and sometimes pncA regions. Analysis by RFLP revealed a high degree of genetic diversity, with no identifiable clusters of infection. Of the strains tested, 51% were resistant to at least one antibiotic, and 43% were resistant to more than one drug. There was a high rate of resistance observed in patients whose charts indicated a history of improperly administered tuberculosis treatment, whereas little drug resistance was observed in patients never previously treated for tuberculosis. Sequencing of genes associated with drug resistance revealed several previously unreported mutations in resistant strains. CONCLUSIONS: This analysis suggests that the cases of tuberculosis in the sample are largely reactivation of long-standing infections and that the drug resistance among patients in CMCH is largely acquired or secondary rather than attributable to the spread of drug-resistant strains.     

Prevalence of katG Ser315 substitution and rpoB mutations in isoniazid-resistant Mycobacterium tuberculosis isolates from Brazil.

SETTING: Four hundred and sixty-eight isoniazid (INH) resistant Mycobacterium tuberculosis isolates recovered from a selected Brazilian population. OBJECTIVE: To check for susceptibility to other chemotherapeutic drugs used in TB treatment, and to ascertain mutations involved in INH and rifampicin (RMP) resistance. DESIGN: Antimicrobial susceptibility to RMP, streptomycin and ethambutol (EMB) was evaluated by the resistance ratio method and pyrazinamide (PZA) by activity assay. Single strand conformation polymorphism (SSCP) and sequence analysis were performed in samples from this panel to confirm mutations in codon 315 of the katG and in a 69-bp region of the rpoB gene. RESULTS: Combined resistance to INH+RMP, INH+ PZA, INH+EMB, and INH+RMP+PZA was shown in respectively 272 (58.1%), 126 (26.9%), 47 (10%), 116 (24.8%) isolates. No katG mutation was found in 19 (39.6%) of 48 strains tested. Ser315Thr substitution was found in 29 (60.4%). All RMP-resistant strains tested (n = 25) showed rpoB mutations. S531L substitution was found in 15 (60%). CONCLUSION: INH-resistant strains isolated from selected Brazilian populations frequently show resistance to other first-line anti-tuberculosis drugs. rpoB mutation was responsible for RMP resistance in all strains. Among INHr strains, katG mutations were shown in only 60.4%. Genetic approaches targeting the rpoB gene but not the katG gene have a high sensitivity to detect resistance among Brazilian M. tuberculosis strains.     

江西省结核分枝杆菌耐二线抗结核药的分子学特征

目的 初步明确江西省耐二线抗结核药相关基因突变的特征,评价等位基因特异性多重PCR（multiplex allele-specific polymerase chain reaction,MAS-PCR）检测二线抗结核药耐药性的可行性。 方法 采用DNA测序方法和MAS-PCR技术,对江西省52株耐二线抗结核药的结核分枝杆菌进行耐药相关基因突变位点检测。 结果DNA测序分析：39株耐氧氟沙星（Ofx）菌株中,32株存在gyrA基因错义突变,2株发生gyrB基因错义突变。29株耐卡那霉素（Km）或卷曲霉素（Cm）菌株中,22株为rrs1401位点A→G突变。52株耐二线抗结核药的结核分枝杆菌,40株为北京基因型（76.92%,40/52）,其中17株北京基因型菌株发生gyrA-GAC94GGC突变（42.50%,17/40）,19株北京基因型菌株发生rrs-A1401G突变（47.50%,19/40）。北京基因型与基因突变类型gyrA-GAC94GGC和rrs-A1401G无明显相关性（χ²=1.16、1.92,P值均＞0.05）。MAS-PCR检测Ofx耐药株的敏感度为61.54%（24/39）,检测Km或Cm耐药株的敏感度为79.31%（23/29）。 结论 gyrA基因94位密码子突变是江西省结核分枝杆菌Ofx耐药的主要机制; rrs-A1401G突变则是Km或Cm耐药的主要原因。MAS-PCR方法对于快速检测二线抗结核药的耐药性有一定的临床应用价值。     

深圳地区结核分枝杆菌耐药及广泛耐药分离株的分子特征

目的 研究2007-2008年深圳地区MTB耐药和广泛耐药分离株的分子特征.方法 参照世界卫生组织和同际防痨与肺部疾病联盟的标准,使用改良罗氏药敏培养基,用1%比例法药敏试验,筛选出针对异烟肼、利福平、链霉素、氧氟沙星和卡那霉素5种药物耐药或敏感的临床分离株,通过PCR扩增筛选菌株的rpoB、katG、rpsL、rrs~((1))、gyrA/B和rrs~((2))基因的相关序列,运用DNAStar和Blastn进行序列分析.结果 筛选出实验菌株123株,其中耐药株73株,全敏感株50株.异烟肼耐药株katG基因突变率为44/52,突变位点全部为S315T或S315N.利福平耐药株rpoB基因突变率为44/47,突变位点主要集中在S531L(30/44)、H526D(9/44)和H526R(1/44).链霉素耐药株以rpsl基因突变为主,突变位点为K43R(19/28)和KS8Q(6/28);rrs(1)基因突变较少见,仪有491C→T(2/28)和513A→C(1/28),2个基因突变率合计为28/41.氧氟沙星耐药株突变率为11/11,以gyrA基因突变为主,突变位点包括D94A(2/11)、S91P(4/11)和A90V(3/11),3个突变位点总突变率为9/11;     

结核分枝杆菌DNA促旋酶基因突变与耐氟喹诺酮类药物的相关性研究

目的 分析MDR TB结核分枝杆菌临床分离株的DNA促旋酶（gyr）基因突变特点,初步探究MDR-TB对氟喹诺酮类药物耐药与gyr基因突变的相关性.方法 采用最低抑菌浓度（MIC）法筛选MDR-TB,对17株临床MDRTB菌株应用DNA直接测序法检测gyr基因的突变情况,分析细菌基因突变与耐药产生的相关性.结果 Mtb 1株标准菌株（H37Rv）未见gyr A亚单位基因（gyrA）和gyr B亚单位基因（gyrB）基因突变,所有17株临床菌株gyrA均存在95位AGC→ACC.12株耐环丙沙星和左氧氟沙星菌株中,10株gyrA产生了错义突变,突变率为83.3％;突变位点位于89位、90位、91位和94位;1株发生了gyrB突变,突变位点位于500位.耐莫西沙星的9株耐药株中,8株gyrA发生突变,占88.9％.结论 gyrA基因突变是Mtb对氟喹诺酮类药物产生耐药的机制之一;gyrA的错义突变主要发生在第89位、90位、91位、94位密码子上.     

Absence of the genetic marker IS6110 from a strain of Mycobacterium tuberculosis isolated in Ontario

A 35-year-old female patient from Waterloo, Ontario was diagnosed with pulmonary tuberculosis in June 1995. Records indicated that the patient had emigrated from Laos circa 1990. A culture grown from a bronchoalveolar lavage specimen was identified as Mycobacterium tuberculosis by standard biochemical methods. Drug-susceptibility testing indicated the strain was resistant to pyrazinamide (PZA), and a mutation was detected within pncA, a gene associated with PZA resistance. Sequence data from the 16S rRNA gene and the 16S/23S rRNA gene spacer confirmed that the strain was a member of the M tuberculosis complex, and analysis of the mpcA and pncA genes supported the identification of the strain as M tuberculosis rather than Mycobacterium bovis. However, the insertion element IS6110, which is used for epidemiological tracing of M tuberculosis, was not detected in this strain by either restriction fragment length polymorphism analysis or by polymerase chain reaction. Two other genetic markers associated with the M tuberculosis complex, IS1081 and the direct repeat element, were present. The arrival of immigrants with tuberculosis from southeast Asia, where most strains of M tuberculosis lacking IS6110 have been traced, has important implications for epidemiological studies of tuberculosis in North America.     

pncA gene mutations in clinical isolates of tubercle bacillus by polymerase chain reaction-direct sequencing method: in relationship to pyrazinamide resistance]

We screened clinical isolates of tubercle bacillus for mutations in the pncA gene, which encodes pyrazinamidase (PZase), by polymerase chain reaction (PCR)-direct sequencing method. Sixty-eight strains of tubercle bacillus were isolated from 32 patients with pulmonary tuberculosis. The patients were treated with antituberculous agents including pyrazinamide (PZA) for 2 months. Thirty-two of the 68 strains were isolated from sputum samples collected from the patients before treatment; 29 strains and 7 strains were collected after 1 month and 2 months of treatment, respectively. The pncA genes in these strains, were assessed for mutations by direct sequencing of PCR products using an automated sequencer. Similarly, we examined two clinical isolates (ka567 and minami22) of tubercle bacillus, determined to be deficient in PZase activity by the Wayne method. A PZA-sensitive strain (H37Rv, ATCC27294), and a PZA-resistant strain (H37Rv-PZA-R, ATCC35828) were used as negative and positive controls for mutations in the pncA gene, respectively. None of the 68 strains demonstrated any mutations in the pncA gene; however, the 2 PZase-deficient strains had missense mutations in the pncA gene resulting in an amino acid substitution from His82 to Arg in clone ka567, and from Ala171 to Val in clone minami22.     

结核分枝杆菌对吡嗪酰胺耐药的相关基因及耐药机制研究进展

吡嗪酰胺是结核病最重要的一线治疗药物之一,但结核分枝杆菌对其耐药性在逐年增长,目前对于结核分枝杆菌吡嗪酰胺的耐药基因的分子机制尚无定论。作者对结核分枝杆菌吡嗪酰胺的相关耐药基因pncA、rpsA、panD、fadD2及FAS-I的研究进展进行综述。     

耐多药结核分枝杆菌对喹诺酮类药物的耐药性与gyr基因突变的初步研究

目的分析耐多药结核分枝杆菌（Multiple drug-resistant tuberculosis,MDR-TB）临床分离株的gyr基因突变特点,探讨MDR-TB对喹诺酮类药物耐药产生与gyr基因突变的关系。方法采用比例法对耐多药结核临床分离菌株进行氧氟沙星的药物敏感性检测,应用DNA直接测序法检测MDR-TB的gyr基因突变情况。结果 125株MDR-TB临床分离株中,50株对喹诺酮类耐药,耐药率为40%。50株耐药菌株中,40株gyr基因发生突变：其中39株gyrA基因突变,突变率为78%,突变位点包括90,91和94位氨基酸;5株gyrB基因突变,其中4株合并gyrA基因突变,gyrB基因突变位点为500,506,534和539位氨基酸。结论 MDR-TB中的喹诺酮类药物耐药态势比较严峻,其对喹诺酮类药物耐药机制主要与gyrA基因突变有关。