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.     

Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co(2+), Mn(2+), and Zn(2+) restored pyrazinamidase activity, only Co(2+) enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu(2+), Fe(2+), Fe(3+), and Mg(2+) did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn(2+). However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.     

Pyrazinamide is not active in vitro against Mycobacterium avium complex

Strains of M. tuberculosis that elaborate pyrazinamidase are typically susceptible in vitro and in vivo to pyrazinamide (PZA). However, we found that 33 strains of M. avium complex (MAC), all of which were pyrazinamidase-positive, were resistant in vitro to a high concentration (100 micrograms/ml) of PZA when tested at low pH in 7H12 broth by radiometric (BACTEC) method. The drug was equally ineffective against these bacteria within cultured normal human macrophages. We conclude that the pyrazinamidase test is not suitable for susceptibility studies against M. avium complex. On the basis of our in vitro studies, which support earlier data, we believe that PZA is not appropriate for therapy of MAC disease.     

结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的影响

目的研究结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的机制和作用靶点。方法将饥饿3、5和10d的结核分枝杆菌及未经过营养饥饿的结核分枝杆菌分别加入100μg／ml吡嗪酰胺和脂肪酸、苯甲酸和水杨酸进行处理，观察营养饥饿处理过程对于吡嗪酰胺抗结核分枝杆菌作用的影响，以及酸性物质对吡嗪酰胺抗菌活性的影响。同时用流式细胞仪检测结核分枝杆菌膜电位和平均荧光强度，以探讨吡嗪酰胺的作用机制。结果未经过营养饥饿的结核分枝杆菌和饥饿3、5和10d的结核分枝杆菌经吡嗪酰胺处理后，菌落形成单位（CFU）分别减少23．08％、37．75％、82．32％和81．03％；营养饥饿5d后，结核分枝杆菌的膜电位明显降低，经吡嗪酰胺处理后的膜电位则大幅降低，加入能量物质葡萄糖后，因吡嗪酰胺作用而降低的膜电位可得到恢复。脂肪酸、苯甲酸和水杨酸对于正常生长的和营养饥饿后的结核分枝杆菌均有促进吡嗪酰胺抗菌效果的作用，对营养饥饿后的结核分枝杆菌作用更明显。结论饥饿状态和弱酸能增强吡嗪酰胺对结核分枝杆菌的抗菌作用，吡嗪酰胺可能是通过于扰结核分枝杆菌的膜电位及其能量代谢而产生作用。     

Inhibition of M. tuberculosis in vitro in monocytes and in mice by aminomethylene pyrazinamide analogs

Pyrazinamide is unusual among anti-tuberculous agents in its ability to promote a durable cure and shorten the duration of therapy. Yet the basis for this effect is not well understood. A particularly effective strategy for the development of new drugs can be to synthetically manipulate the well-established structures to improve either the spectrum of activity or some pharmacological properties. Similar to previously described aminomethylene amides such as morphazinamide, it was found that novel aminomethylene amides can have in vitro activity at higher than the very acidic pH conditions where pyrazinamide is inactive as well as retaining activity against pyrazinamide-resistant M. tuberculosis. These new compounds have shown an improved anti-tuberculous activity in infected human macrophages relative to pyrazinamide. Compound 1, in combination with rifamycin, was especially effective in both infected human macrophages and in a murine model of infection. The activity of these analogs against pyrazinamide-resistant strains suggests that the development of second generation pyrazinamide analogs may be especially fruitful.     

Pyrazinamide is not active against Mycobacterium tuberculosis residing in cultured human monocyte-derived macrophages.

SETTING: Mycobacteriology Laboratory, National Jewish Medical and Research Center, Denver, Colorado. OBJECTIVE: To evaluate the antimicrobial activity of pyrazinamide against Mycobacterium tuberculosis in cultured human monocyte-derived normal and activated macrophages. DESIGN: Monocytes separated from human blood were incubated in plastic plates for seven days to mature into macrophage monolayers. After activation with TNF-alpha or IFN-gamma or without prior treatment, the macrophages were infected with M. tuberculosis. Various concentrations of pyrazinamide (PZA), morphazinamide (MZA) or isoniazid (INH) were added the next day, and the viable counts of the intracellular bacteria were determined at days 0, 4, and 8. RESULTS: No inhibitory activity of PZA at any concentration was detected, while clear dose-dependent bacteriostatic and bactericidal activities were demonstrated by MZA and INH in the same experimental model. CONCLUSIONS: PZA has neither bacteriostatic nor bactericidal activity against M. tuberculosis persisting or multiplying in cultured monocyte-derived human macrophages, and it might be that the well-known effectiveness of this drug in tuberculosis patients is not related to its supposed activity against intracellular bacterial subpopulations.     

Pyrazinamide sterilizing activity in vitro against semidormant Mycobacterium tuberculosis bacterial populations.

Previously, we reported that pyrazinamide has very poor bactericidal activity against M. tuberculosis growing in broth at pH 5.6. In the present study, cultivation at pH 4.8 to 5.0 in 7H12 broth prevented an increase in the number of viable bacteria, but the cultures remained metabolically active. The presence of 50 micrograms/ml pyrazinamide in semidormant cultures led to a sharp decline in the number of viable bacteria, by more than 1,000-fold. This unfavorable environment probably made the bacilli especially vulnerable to pyrazinamide, whose mode of action remains unclear. To distinguish this effect of pyrazinamide on the semidormant bacteria from its mostly bacteriostatic activity against actively multiplying bacteria, we suggest interpreting the in vitro effect as "sterilizing."     

Pharmacokinetics of pyrazinamide in children suffering from pulmonary tuberculosis.

SETTING: The Paediatric and Clinical Pharmacology unit of Maulana Azad Medical College and Associated Lok Nayak Hospital, New Delhi, India. OBJECTIVE: The pharmacokinetics of the anti-tuberculosis drug pyrazinamide was evaluated in 10 children aged 6 to 12 years suffering from pulmonary tuberculosis. METHODS: Serial blood samples were collected at 0, 1, 2, 4, 6, 12 and 24 hours after administration of pyrazinamide in a dose of 35 mg/kg. Serum pyrazinamide levels were analysed by spectrophotometry. RESULTS: The serum concentrations of pyrazinamide were above the minimum inhibitory concentration of 20 microg/ml of pyrazinamide for Mycobacterium tuberculosis up to 6 hours after drug administration in all the patients, and up to 12 hours in six patients. The mean peak serum concentration of pyrazinamide was 41.2+/-11.8 microg/ml, and this was attained in (Tmax) 2.9+/-1.7 hours. The elimination half life was 10.9+/-4.5 hours, the volume of distribution 16.1+/-10.9 litres and clearance 20.2+/-16.3 ml/minute. The corresponding mean residence time was 19.9+/-14.6 hours. CONCLUSION: The serum pyrazinamide concentrations achieved with a dose of 35 mg/kg were above the minimum inhibitory concentration of pyrazinamide for M. tuberculosis for over 6 hours after drug administration. It appears that the absorption and the clearance of pyrazinamide is slower, the elimination half life longer and the volume of distribution higher in children compared with the reported values in the adult population.     

High initial drug resistance in pulmonary tuberculosis in Ghana

Between July 1985 and March 1987, initial sensitivity to anti-tuberculosis drugs was studied in patients presenting at the Chest Clinic of Agogo Hospital in the forest area of Ghana. Culture and sensitivity test results were obtained in 99 out of 123 consecutive patients with pulmonary tuberculosis who claim not to have received previous treatment. Isoniazid resistance was alarmingly high in the isolates of M. tuberculosis: 21 out of 57 (37%), and thiacetazone resistance was very high in the M. africanum isolates: 20 out of 42 (47%). Overall resistance was high: 27% to isoniazid, 23% to streptomycin, 29% to thiacetazone, 16% to both streptomycin and isoniazid, and 5% to all of these three drugs. Only 45% of the isolates were sensitive to all three drugs. Primary drug resistance to rifampicin, pyrazinamide or ethambutol was not observed. Besides the standard treatment of isoniazid, streptomycin and thiacetazone, rifampicin and pyrazinamide were usually added for the first two months of treatment. Of 13 patients who received standard treatment only, 4 of the 5 patients with resistant organisms who could be followed up failed to respond, whereas there were no failures to respond in the 5 corresponding patients with initially sensitive organisms; 3 patients could not be assessed because they defaulted.     

吡嗪酰胺酶活性与耐多药肺结核近期临床疗效的相关性研究

目的 研究吡嗪酰胺酶活性与耐多药肺结核(multidrug-resistant pulmonary tuberculosis,MDR-PTB)近期疗效的相关性,为MDR-PTB患者化学药物治疗的选择提供指导。 方法 采用前瞻性队列研究的方法,将广州市胸科医院2018年7月至2019年12月收治的85例MDR-PTB患者作为研究对象,均接受世界卫生组织推荐的6Am-Mfx-PZA-Pto-Cs/18Mfx-PZA-Pto-Cs(Am:阿米卡星;Mfx:莫西沙星;PZA:吡嗪酰胺;Pto:丙硫异烟胺;Cs:环丝氨酸)治疗方案。85例患者中,剔除9例丢失、中断治疗、失访患者,共76例患者纳入最终研究。以Wayne方法检测吡嗪酰胺酶的活性,以BACTEC MGIT 960方法检测吡嗪酰胺药物敏感性;比较吡嗪酰胺酶阳性患者与阴性患者治疗2、4、6个月末痰菌阴转、病灶吸收和空洞闭合情况。 结果 76例患者中,吡嗪酰胺酶阳性32例(42.1%),吡嗪酰胺酶阴性44例(57.9%);吡嗪酰胺表型耐药36例(47.4%),吡嗪酰胺表型敏感40例(52.6%)。吡嗪酰胺药物敏感性检测与吡嗪酰胺酶活性检测的一致性较高(Kappa=0.687,P=0.000)。吡嗪酰胺酶阳性患者治疗4个月末病灶吸收率(59.4%,19/32)及空洞闭合有效率(65.5%,19/29)均高于吡嗪酰胺酶阴性患者[分别为36.4%(16/44)和36.8%(14/38)],差异均有统计学意义(χ²=3.949,P=0.047;χ²=5.411,P=0.020)。吡嗪酰胺酶阳性患者治疗6个月后的空洞闭合有效率(86.2%,25/29)高于吡嗪酰胺酶阴性患者(63.2%,24/38),差异有统计学意义(χ²=4.447,P=0.035)。 结论 MDR-PTB患者的疗效与吡嗪酰胺酶活性有关,吡嗪酰胺酶阳性患者应用含吡嗪酰胺的MDR-PTB标准方案治疗后,病灶吸收及空洞闭合的效果优于吡嗪酰胺酶阴性患者。     

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.     

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.     

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.     

Clinical and laboratory studies in patients with pulmonary tuberculosis treated with pyrazinamide

Different combinations of tuberculostatic drugs containing pyrazinamide were prescribed to 144 patients with pulmonary tuberculosis. In 3 months of treatment, bacterial excretion was observed in 89.7% of newly diagnosed patients and in 59% of cases with chronic forms of pulmonary tuberculosis. Side effects due to pyrazinamide were registered in 16.8% of the patients. In 2-3 months of therapy there were no differences traced in the condition of the liver, lipoid peroxidation or antioxidative activity as well as in a number of immunologic indices both in subjects receiving different combinations of antituberculous drugs, containing pyrazinamide, and in the control group of patients untreated with the drug.     

Pancreatic mass caused by Mycobacterium tuberculosis with reduced drug sensitivity.

Pancreatic tuberculosis is a rare condition which should be considered in patients with a pancreatic mass, particularly if the patient is young, not jaundiced, from an area of high TB prevalence with a normal ERCP. We report a case of pancreatic tuberculosis due to Mycobacterium tuberculosis with reduced sensitivity to rifampicin and isoniazid, that was treated with rifabutin, ciprofloxacin, ethambutol and pyrazinamide following clinical failure of first-line therapy. The case presented illustrates the importance of obtaining material for culture and sensitivity testing in cases of suspected TB.     

Pyrazinamide use as a method of estimating under-reporting of tuberculosis.

OBJECTIVE: To develop a method of validating the notification of active tuberculosis by physicians in the Netherlands. METHOD: The chemotherapeutic agent pyrazinamide was used as a marker for the occurrence of tuberculosis. On the basis of defined daily doses (DDD) of pyrazinamide dispensed to out-patients, an estimate was made of the number of patients with tuberculosis in the Netherlands in the period 1994-1998. DDD is a technical unit of measurement and does not necessarily reflect the recommended or actual dose used. Usually it is based on the average dosage per day for the main indication in adults with normal organ function. The Dutch Drug Information Project (GIP) of the Health Care Insurance Board (CVZ) provided the DDD data. Based on the notification of tuberculosis patients to the Netherlands Tuberculosis Register (NTR) we calculated how much pyrazinamide (measured in DDDs) these patients would have used depending on their body weight. RESULTS: The number of DDDs prescribed according to the GIP pharmacy records differed by only 8% from the number of DDDs calculated on the basis of notification to the NTR; 6889 patients should have been registered instead of 6349. CONCLUSION: The close correlation between the use of pyrazinamide as measured by the GIP and NTR provides strong evidence that in the Netherlands tuberculosis is reported in conformity with the guidelines for notifiable diseases. The method was simple to apply and may deserve follow-up in other countries.     

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;     

Is pyrazinamide bactericidal against Mycobacterium tuberculosis?

Bactericidal activity of pyrazinamide (PZA) was tested at pH 5.6 in 7H12 broth against drug-susceptible M. tuberculosis strains. The highest tested concentrations of PZA, 500 and 1,000 micrograms/ml, killed no more than 76% of the bacterial population. These concentrations are more than 32 times greater than the minimal inhibitory concentration (MIC) and the achievable in vivo concentrations. Despite high clinical efficacy of PZA and its so-called sterilizing activity in mouse experiments, this drug is much less bactericidal in vitro than any other known antituberculosis drug.     

Treatment of isoniazid-resistant tuberculosis with isoniazid, rifampin, ethambutol, and pyrazinamide for 6 months.

SETTING: In 1992 the Seattle-King County Department of Public Health Tuberculosis Clinic began to treat patients with isoniazid-resistant tuberculosis with a regimen of isoniazid, rifampin, pyrazinamide, and ethambutol daily for 6 months. OBJECTIVE: To conduct a review of clinical and bacteriological outcomes of treatment for patients who received the four-drug, 6-month regimen for isoniazid-resistant tuberculosis. DESIGN: A retrospective review of medical records of TB cases meeting the study criteria, a Mycobacterium tuberculosis isolate resistant to isoniazid, and intent to treat with a 6-month course of isoniazid, rifampin, pyrazinamide, and ethambutol. RESULTS: Through December 1999, 44 consecutive patients with isoniazid-resistant, rifampin-susceptible tuberculosis were started on the four-drug, 6-month daily regimen. Among 42 patients followed until completion of therapy, three required changes in the regimen due to side effects. There was one case of drug-induced hepatotoxicity. Among 39 patients with pulmonary involvement, 37 converted sputum cultures from positive to negative within 2 months of starting treatment. There were no treatment failures. On passive follow-up of at least 2 years on all patients, two patients relapsed. The single patient with bacteriological relapse did not develop further drug resistance. CONCLUSION: The regimen of isoniazid, rifampin, pyrazinamide, and ethambutol given daily for 6 months produced successful outcomes when used in a public health tuberculosis clinic as routine therapy for isoniazid-resistant tuberculosis.     

Bactericidal activity of streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide alone and in combination against Mycobacterium Tuberculosis.

Log-phase cultures of Mycobacterium tuberculosis in Tween-albumin medium were exposed to streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide in concentrations in the range likely to be present in serum during treatment of patients. The bactericidal activity of the drugs was measured as the decrease in viable counts at 4 and 7 days. The activity of single drugs was highest for streptomycin and next highest for rifampin and isoniazid, but ethambutol only started to kill after 4 days. When exposed to 2 drugs, bactericidal synergism was found with streptomycin/isoniazid and isoniazid/ethambutol; additivity, with streptomycin/rifampin; indifference, with isoniazid rifampin and streptomycin/ethambutol; and antagonism, with rifampin/ethambutol and isoniazid/pyrazinamide. When cultures were exposed to the 3 drugs, isoniazid, rifampin, and ethambutol, marked antagonism was found between isoniazid and rifampin, whereas the addition of isoniazid or an increase in its concentration increased the bactericidal activity.