Antituberculous drug susceptibility testing of Mycobacterium bovis BCG strain Montréal

Testing of Mycobacterium bovis BCG strain Montréal for susceptibility to four primary antituberculous drugs (isoniazid, ethambutol, streptomycin, and rifampin) and to one secondary drug (p-aminosalicylic acid) showed the strain to be susceptible to all five substances. Mycobacterium bovis strains ATCC 35735, which is isoniazid sensitive, and ATCC 35747, which is isoniazid resistant, were included in the test; with the exception of their respective susceptibility to isoniazid, both were inhibited by the other four drugs.     

Anti-tuberculosis drug resistance and therapeutic dead end

The irrational use of antituberculous drugs led to the emergence of resistant strains of M. tuberculosis. Every year in the world, around 440 000 new tuberculosis cases are due to bacilli that are resistant to the two main antituberculous drugs, isoniazid and rifampicin (also known as multidrug resistant or MDR). Treatment of MDR tuberculosis is difficult and has been based for twenty years on the use of fluoroquinolones and injectable antibiotics such as amikacin, kanamycin and capreomycin. Consequently, strains resistant to the latter drugs, so-called extensively drug resistant strains or XDR, have recently emerged. XDR tuberculosis is very difficult to treat and the prognosis is very close to that of untreated tuberculosis with a mortality rate that can reach 50 % to 100 %. To avoid the emergence of more resistant strains that may lead to almost untreatable disease, we must focus our efforts on the right management of drug susceptible tuberculosis. Basic principles for avoiding accumulation of resistances in selected strains are outlined in the article.     

Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis

Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA) (ref. 3), whose target of action has never been identified. Although PZA is active only against Mycobacterium tuberculosis, the PZA analog 5-chloro-pyrazinamide (5-Cl-PZA) displays a broader range of anti-mycobacterial activity. We have found that the eukaryotic-like fas1 gene (encoding fatty acid synthetase I, FASI) from M. avium, M. bovis BCG or M. tuberculosis confers resistance to 5-Cl-PZA when present on multi-copy vectors in M. smegmatis. 5-Cl-PZA and PZA markedly inhibited the activity of M. tuberculosis FASI, the biosynthesis of C16 to C24/C26 fatty acids from acetyl-CoA (ref. 6). Importantly, PZA inhibited FASI in M. tuberculosis in correlation with PZA susceptibility. These results indicate that FASI is a primary target of action for PZA in M. tuberculosis. Further characterization of FASI as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis.     

Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.     

Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex

A gene conferring low-level isoniazid (INH) resistance on Mycobacterium smegmatis was isolated from a cosmid library of the genome of an INH-resistant Mycobacterium bovis strain. The gene had good homology with ahpC , the product of which is a subunit of alkyl hydroperoxide reductase, and also with a family of thiol-specific antioxidant enzymes. A mutation was found in the promoter upon comparison with the equivalent DNA sequence from the INH-sensitive parent strain. Promoter sequences from other INH-sensitive and INH-resistant M. bovis and Mycobacterium tuberculosis strains were sequenced and the mutation was found only in the INH-resistant strains. An INH-resistant M. tuberculosis strain also had an additional mutation in the promoter region. The wild-type promoter and promoters with one and two mutations were ligated into a reporter plasmid containing the lacZ gene. The presence of the first mutation resulted in a sixfold induction of β-galactosidase activity, and the presence of both mutations caused a 10-fold induction. Increased expression of AhpC may account for some of the INH resistance of strains of the M. tuberculosis complex.     

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.     

Activity of 7-methyljuglone in combination with antituberculous drugs against Mycobacterium tuberculosis

The recent increase in the incidence of tuberculosis with the emergence of multidrug-resistant (MDR) cases has lead to the search for new drugs that are effective against MDR strains of Mycobacterium tuberculosis and can augment the potential of existing drugs against tuberculosis. In the present study, we investigated the activities of a naphthoquinone, 7-methyljuglone, isolated from the roots of Euclea natalensis alone and in combination with other antituberculous drugs against extracellular and intracellular M. tuberculosis. Combinations of 7-methyljuglone with isoniazid or rifampicin resulted in a four to six-fold reduction in the minimum inhibitory concentration of each compound. Fractional inhibitory concentration (FIC) indexes obtained were 0.2 and 0.5, respectively, for rifampicin and isoniazid, suggesting a synergistic interaction between 7-methyljuglone and these anti-TB drugs. The ability of 7-methyljuglone to enhance the activity of isoniazid and rifampicin against both extracellular and intracellular organisms suggests that 7-methyljuglone may serve as a promising compound for development as an anti-tuberculous agent.     

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional L?wenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.     

Drug resistance of strains of Mycobacterium tuberculosis isolated in Brazil.

Tuberculosis remains a serious public health problem, worsened by an increased frequency of multidrug-resistant Mycobacterium tuberculosis. We report here a retrospective study of resistance to antituberculosis drugs of 170 strains of M. tuberculosis isolated from the state of Rio Grande do Sul, Brazil. The frequency of resistance to at least one drug was 34%, while 22% were resistant to more than one drug. Among the strains isolated from patients without a history of previous treatment for tuberculosis, patients with positive serology for HIV and patients with previous treatment for tuberculosis, the resistance to at least one drug was 14, 27 and 73%, respectively. Multidrug-resistant tuberculosis, defined as resistant to at least rifampicin (RMP) and isoniazid (INH), was found in the groups of patients without previous treatment, HIV co-infected and with previous treatment for tuberculosis at 10, 17 and 44%, respectively. With the purpose of evaluating whether the sensitivity test to INH and RMP would be a good marker to indicate resistance to other antituberculosis drugs, sensitivity tests were performed with four more drugs in 32 strains, initially classified as resistant to INH, RMP or both. Of 18 strains resistant to INH and RMP simultaneously, 89% showed resistance to four more drugs.     

In Vitro Chemotherapeutic Combinations Against Isoniazid-Resistant Mycobacterium tuberculosis and Mycobacterium fortuitum

It is an acceptable medical practice to use second-line antimycobacterial drugs in combination with isoniazid in treatment of isoniazid-resistant tuberculosis. Recent investigations have demonstrated the importance of determining chemotherapeutic interaction in instances of multiple antibiotic use. We studied the inhibitory effect of combinations of isoniazid with ethambutol, rifampin, ethionamide, cycloserine, viomycin, and kanamycin against three isoniazid-resistant strains of Mycobacterium tuberculosis and three strains of M. fortuitum. The isobologram technique with drug concentrations of 0.4 to 100 mug/ml was used. With the exception of single instances in which kanamycin plus isoniazid (M. tuberculosis strain 9999) and ethionamide plus isoniazid (M. fortuitum strain 2080) seemed to have a synergistic effect, neither synergy nor antagonism was noted for any of the combinations. These studies show that the combined use of isoniazid and a second line antimycobacterial agent results in vitro in indifferent inhibitory activity.     

Drug resistance and IS6110‐RFLP patterns of Mycobacterium tuberculosis in patients with recurrent tuberculosis in northern Thailand

The emergence of drug resistant Mycobacterium tuberculosis has become a global threat to tuberculosis (TB) prevention and control efforts. This study aimed to determine the drug resistance profiles and DNA fingerprints of M. tuberculosis strains isolated from patients with relapsed or retreatment pulmonary TB in Chiang Rai province in northern Thailand. Significant differences in multidrug resistance (MDR) (P = 0.025) and resistance to isoniazid (P = 0.025) and rifampin (P = 0.046) between first and second registrations of patients with retreatment TB were found. However, there were no significant differences in resistance to any drugs in patients with relapsed TB. The rate of MDR‐TB strains was 12.2% among new patients at first registration, 22.5% among patients with recurrence who had previously undergone treatment at second registration and 12.5% at third registration. Two retreatment patients whose initial treatment had failed had developed MDR‐TB with resistance to all TB drugs tested, including rifampin, isoniazid, streptomycin and ethambutol. IS6110‐RFLP analysis revealed that 66.7% (10/15 isolates) of MDR‐TB belonged to the Beijing family. In most cases, IS6110‐RFLP patterns of isolates from the same patients were identical in relapse and retreatment groups. However, some pairs of isolates from retreatment patients after treatment failure had non‐identical IS6110‐RFLP patterns. These results suggest that, after failure and default treatment, patients with retreatment tuberculosis have a significantly greater risk of MDR‐TB, isoniazid and rifampin resistance than do other patients.     

Intralaboratory comparison of drug susceptibility testing of Mycobacterium tuberculosis by BACTEC and conventional methodology

The BACTEC radiometric method of drug susceptibility testing of Mycobacterium tuberculosis is a reliable and rapid diagnostic tool in clinical mycobacteriology. However, large scale comparative studies have also shown that the level of agreement with standard methodology was less satisfactory with strains resistant to ethambutol and streptomycin than with strains resistant to rifampin and to isoniazid. Since disagreement with drug resistance strains is far more frequent than with drug susceptible strains, it was felt that only the comparison of a large number of resistant strains would be needed to further refine this new technique. The analysis of BACTEC-derived data for isoniazid and rifampin shows that the level of agreement with conventional methodology falls well within accepted limits. Statistical analysis of the radiometric versus conventional comparisons shows no significant differences between the two methods in the case of isoniazid, rifampin, and ethambutol (3 mg/L). Streptomycin and two other ethambutol concentrations tested showed lower levels of agreement and significant statistical differences with conventional methodology.     

Bacteriological conversion in twenty urinary tuberculosis patients treated with ofloxacin, rifampin and isoniazid: a 10-year follow-up study

Twenty patients with urinary tuberculosis were treated with ofloxacin (200 mg/day, 6 months), rifampin (600 mg/day, 3 months) and isoniazid (300 mg/day, 3 months) between 1989 and 1990. All patients were new cases, diagnosed by observation and/or isolation of Mycobacterium tuberculosis in one of the three morning urine samples. Bacteriological culture conversion (negativization) was assessed as a clinical guide of efficacy, comparing it, as the only parameter, against a control group (150 patients) with urinary tuberculosis who received conventional therapy. Bacteriological follow-up studies were performed in both groups monthly for 6 months, then again 6 months later and then every year for 10 years after completion of treatment. In the 20 patients, the initial culture was positive with over 100 colonies per culture (>50%); the smear was positive in 45% of the patients (most were 2+). All strains were susceptible to rifampin, isoniazid and ofloxacin. Two patients discontinued treatment. Beginning with the first month of treatment, the bacteriological conversion was 100%, 89.5% and 100% in the remaining controls. In the control group, which received conventional treatment, the conversion was: 90%, 87%, 93% and 100% in the remaining controls. Treatment with ofloxacin resulted in a bacteriological conversion similar to that following conventional treatment (p>0.05, Fisher's exact test). After 10 years of patient follow-up, we conclude that ofloxacin, in combination with rifampin and isoniazid (both for 3 months only is effective against M. tuberculosis, providing satisfactory bacteriological and clinical efficacy. Electronic Publication     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

Multidrug chemotherapy of tuberculosis in rhesus monkeys

Occurrence of tuberculosis caused by Mycobacterium bovis in a colony of rhesus monkeys allowed evaluation of a modern multidrug therapeutic regimen. Fifteen tuberculin positive rhesus monkeys with disseminated tuberculosis were evaluated for extent of disease by radiographic techniques, physical examination and laparotomy prior to treatment. Monkeys were divided into treatment groups of 3, 6 and 12 months duration and were treated once daily with isoniazid, rifampin and ethambutol. All animals survived their treatment course, had marked clinical improvement and rapid resolution of radiographically demonstrable lesions. Lesion regression evaluated by necropsy and histopathology correlated positively with length of treatment interval. Mycobacterium bovis was not isolated from any animal following treatment. Multidrug chemotherapy of tuberculosis was considered successful and practical in rhesus monkeys at the 12 month treatment interval. Chemotherapy may provide a reasonable alternative to destruction of valuable animals infected with tuberculosis.     

Genetic antagonism and hypermutability in Mycobacterium smegmatis

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.     

Molecular genotyping of Mycobacterium tuberculosis strains isolated from patients in the Samara region by the restriction DNA fragment length polymorphism

The typing of 106 M. tuberculosis (MBT) strains isolated from patients in the Samara region by the restriction DNA fragment length polymorphism (RFLP) IS6110 revealed that most of the strains (71.7%) belonged to the W family, 5 MBT strains (4.7%) belonged to the AI family, one culture was the mixture of two strains, AI and W. In addition, 24 MBT strains (22.6%) classified with other genotypes were detected. The analysis of the sensitivity of the MBT strains to rifampicin and isoniazid, with the method of absolute concentrations and by point mutations, demonstrated that 29 MBT strains (27.3%) were sensitive to rifampicin and isoniazid and 56 MBT strains (52.9%) were resistant to rifampicin and isoniazid simultaneously. Among the MBT strains of different RFLP families, strains both sensitive and resistant to these two preparations could be detected, but strains with multiple drug resistance prevailed in the W family (61.8%).     

Photochromogenic and scotochromogenic mycobacteria: their clinical significance

In the period 1973--1977, Mycobacterium tuberculosis was isolated by cultivation in 4408 cases from the clinical specimens of patients with positive X-ray findings. On the basis of atypical colony morphology or pigment formation, 263 other mycobacterial strains were identified: of these 23 were photochromogenic and belonged to Mycobacterium kansasii. The strains were cultured on several occasions from the specimens of 4 patients with broncho-pulmonary mycobacteriosis. The strains were resistant to isoniazid and streptomycin, sensitive to ethambutol and rifampicin. A total of 18 scotochromogenic isolates cultured from 14 patients with positive X-ray findings were identified as Mycobacterium aquae (M. gordonae) and its variants: strains showing slow Tween hydrolysis and 1 strain of rapid growth. In 5 cases M. tuberculosis was also obtained, indicating the presence of a mixed mycobacterial population. All scotochromogens were resistant to isoniazid and sensitive to ethambutol, with the exception of two strains sensitive to rifampicin.