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Recent molecular studies have shown Mycobacterium porcinum, recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776(T)) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776(T). Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, D-mannitol, i-myo-inositol, and catalase at 68 degrees C. They were negative for L-rhamnose and D-glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776(T) gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant D-sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing.

The simultaneous appearance of new cavitary lesions in the lungs and two or more isolations of Mycobacterium avium-Mycobacterium intracellulare complex (MAC) by daily or monthly sputum examination in the initial few days or the initial 6 months shows the presence of lung disease caused by MAC. Three isolations of MAC by three to six daily or monthly sputum examination confirms the occurrence of lung disease caused by MAC. These criteria and their basis were published by the present author in 1974 and 1978. The criteria are almost the same as those published by the American Thoracic Society in 1997.

Maltitol is fermented in the colon due to only partial hydrolysis in the small intestine. In the present study, we examined effects of dietary maltitol on dimethylhydrazine-induced intestinal tumor in rats. In experiment 1, rats were fed a fiber-free diet or diets supplemented with 1 or 5 g/100 g maltitol for 27 wk. Each group of rats was injected with dimethylhydrazine or vehicle alone for the first 14 wk of the experimental period. Maltitol supplementation at 1 g/100 g of the diet significantly reduced tumor incidence in the cecum and the 5% supplement reduced tumor incidence in both the cecum and proximal colon in dimethylhydrazine-treated rats. In experiment 2, we investigated the effect of the 1 g/100 g maltitol diet on the short chain fatty acid concentrations in cecal contents of placebo and dimethylhydrazine-treated rats. Intake of the 1 g/100 g maltitol diet doubled (P < 0.05) the concentration of butyrate but did not affect acetate or propionate in the cecal contents. These results suggest that dietary maltitol has a protective effect against dimethylhydrazine-induced tumors in rat cecum and proximal colon and that butyrate produced by bacterial fermentation of maltitol in the cecum may be involved in the protection.

Branched-chain alpha-keto acid dehydrogenase complex is the rate limiting enzyme in catabolism of branched-chain amino acids. In this study, we examined effects of dietary protein and exercise on the complex activity in digestive tracts (stomach, small intestine and colon) of rats. Rats were fed a high (30%) or low (8%) protein diet for 3 weeks and a half of rats in each diet group was exercised by 85 min running just prior to sacrifice on the final day of the experiment. Total and actual (active form) activities of the complex were markedly high in stomach compared to other two tissues and the actual activity in stomach was significantly elevated by exercise only in rats fed the high protein diet. Both total and actual activities in colon were only a few percentage of those in stomach, and those in small intestine was further less. These results suggest that rat stomach is the tissue active in catabolism of branched-chain amino acids, which is promoted by combination of high protein diet and exercise.

Mycobacterium chelonae-like organisms are nonpigmented rapidly growing mycobacteria whose clinical significance is unknown. We evaluated 87 sporadic isolates encountered in a clinical laboratory. Most isolates (62%) were respiratory; only 2 of 54 (4%) (both from patients with AIDS) were clinically significant. Among 33 nonrespiratory isolates, 20 of 33 (or 61%) were clinically significant. Clinical diseases included posttraumatic wound infections and catheter-related sepsis. Routine biochemical features included growth inhibition by 5% NaCl (100%), a smooth colony morphology (94%), positive 3-day arylsulfatase reaction (84%), no color or a light tan color on iron uptake (100%), and variable nitrate reduction (45%). Additional characteristics that helped to separate this group from M. chelonae and Mycobacterium abscessus were susceptibility to cephalothin (90%) and ciprofloxacin (100%), utilization of mannitol (94%) and citrate (83%) as carbon sources, and unique patterns of mycolic acid esters by high-performance liquid chromatography. This group was quite drug susceptible, with 100% of isolates inhibited by amikacin, imipenem, cefoxitin, cefmetazole, and the newer quinolones ciprofloxacin and ofloxacin. Three examples of this group, including a proposed type strain, have been deposited in the American Type Culture Collection.

beta-lactamases from 259 strains of rapidly growing mycobacteria that included the third biovariant complex of Mycobacterium fortuitum, M. peregrinum, M. abscessus, M. chelonae, the M. chelonae-like organisms (MCLO), and M. smegmatis were analyzed by isoelectric focusing (IEF). All isolates produced acidic beta-lactamases with major band isoelectric points (pIs) between 4.4 and 6.0. Each of the 6 taxonomic groups exhibited 1 or 2 characteristic beta-lactamase IEF patterns. Heterogeneity among IEF patterns was evident in 5 of the 6 groups, however, and was greatest among the third biovariant complex of M. fortuitum. beta-lactamase patterns correlated with previously identified taxonomic subgroups of M. smegmatis and the third biovariant complex of M. fortuitum. beta-lactamase IEF analysis of MCLO strains isolated from two outbreaks demonstrated its possible usefulness for epidemiologic evaluation.

A total of 55 patients with pulmonary disease caused by Mycobacterium avium and by Mycobacterium intracellulare were compared for their prognosis. Causative mycobacteria were identified by the DNA probes (Gen-Probe) method. Of the 55 patients, 28 had pulmonary disease caused by M avium and the remaining 27, by M intracellulare. Of the former group, four patients had progressive disease, and three of them died during the observation period. In contrast, only one of the latter group had progressive disease. On the other hand, only one of the former group was cured. In contrast, six of the latter group were cured, showing the closure of cavity and the disappearance of mycobacteria from the sputum. The prognosis of pulmonary disease caused by M avium appears to be worse than that caused by M intracellulare.

Mycobacterium avium and Mycobacterium intracellulare are differentiated from each other by thin-layer chromatography of lipid fraction extracted after incubation with [35S]methionine. The former contained a petroleum ether-soluble sulfolipid and the latter did not.

Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium--M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M. tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.

Recent studies have shown that Nocardia asteroides isolates have five major antibiotic resistance patterns; one of these patterns identifies isolates of Nocardia farcinica. In the current study, we investigated a second pattern characterized by susceptibility to ampicillin and erythromycin. This pattern was seen in 17% of 223 clinical isolates identified by standard techniques as N. asteroides and associated with diseases typical for nocardiae. Biochemically, isolates with this drug pattern were relatively homogeneous and identical to the type strain and previous descriptions of Nocardia nova. The strains studied were unique among nocardiae in having both alpha- and beta-esterase activity (85 and 95%, respectively). However, the arylsulfatase activity at 14 days (75%) and antimicrobial susceptibility patterns, including susceptibility to erythromycin (100%), were the only routinely available methods that would separate N. nova strains from other members of N. asteroides. N. asteroides should be considered a complex because current clinical identification schemes include isolates of N. farcinica and N. nova and may well include additional species. This is the first detailed description of N. nova as a pathogen in humans.

Unlabelled: In this study, the Mycobacteriosis Research Group of the Japanese National Chest Hospitals (MRG) presents the reports of study years 1987 and 1988. As reported previously**, pulmonary infection caused by Mycobacterium kansasii occurred principally in South-West Japan (prefectures South-West of Tokyo) and did not appear in North Japan. However, this disease appeared in 1987 and 1988 in Hokkaido (Sapporo Hospital). Accordingly, we may say the disease occurs all over Japan. This is a noteworthy finding newly recognized in the study years. The prevalence rate of nontuberculous lung mycobacteriosis was determined as 2.92 or 2.78 in 1987 and as 2.02 or 1.91 in 1988 per 100,000 population per year. The estimated rates based on the ratio of nontuberculous lung mycobacteriosis against active lung tuberculosis and based on the ratio of nontuberculous lung mycobacteriosis against culture-positive lung tuberculosis well agreed with each other., Comment: In this country, chest physicians customarily report their cases of nontuberculous mycobacteriosis including lung tuberculosis, because the payment of treatment for patients with tuberculosis is free. Because of this custom, tuberculosis statistics surely contain cases of nontuberculous mycobacteriosis. Caution about this has been paid in calculating the prevalence rate. From the study year 1987, the MRG chairman moved from Michio Tsukamura, The National Chubu Hospital, to Nobuhiko Kita, The National Kinki Chuo Hospital.

Six cases of chronic tenosynovitis of the hand due to the Mycobacterium terrae complex were identified. All isolates from the six cases were identified as Mycobacterium nonchromogenicum by high-performance liquid chromatography and by testing for susceptibility to ofloxacin and to 5% NaCl. Ethambutol, sulfonamides (or trimethoprim-sulfamethoxazole), erythromycin, and streptomycin are the drugs most active against isolates of the M. terrae complex, and therapy with some combination of these agents plus surgical debridement offers the best current treatment of this disease. This study supports the contention arising from previous case reports of pulmonary disease that M. nonchromogenicum is the pathogenic member of the M. terrae complex.

During the period of 24 years from 1965 to 1988, we treated a total of 181 patients who had pulmonary infection caused by Mycobacterium avium--Mycobacterium intracellulare complex (MAI complex). Of these 181, 34 (19%) were cured showing sputum conversion and disappearance of cavity or marked reduction of cavity in the size to 1/2 or less or change of the cavity to thin-walled one. In these patients, negative culture continued at least for one year by monthly sputum examination. The most frequently used regimen in these patients was RFP + INH + SM, and the secondly RFP + INH + EVM, and thereafter multiple drug regimens including RFP + INH. The most frequently used drugs were RFP, INH, EVM, SM and EB. Based on the above results, we recommend the regimen RFP + INH + EVM + EB or RFP + INH + SM + EB, to which, if possible, were added a combination of MC + SX + KT. (As to abbreviations, refer to Table 3).

One hundred strains of Mycobacterium tuberculosis isolated in 1988-89 from patients who were newly hospitalized in the National Chubu Hospital were studied on their biochemical and biological characteristics and compared with the strains isolated previously. Recently isolated strains showed frequently a much stronger arylsulfatase activity, grew at a higher rate at 42 degrees C, and showed a little stronger niacin production.

Isolation of Mycobacterium avium complex from sputum specimens in association with the appearance of a new cavitary (or infiltrative) lesion was studied in 299 patients from whom the organism was isolated one or more times. Of the patients studied, 114 showed only single isolation. Of these 114, only two patients (2 percent) had association with appearance of a cavitary lesion. Of 29 patients who showed two isolations, 26 (90 percent) had the association. Of 40 patients who showed three isolations, 39 (98 percent) had the association. All 116 patients who showed four or more isolations had the association with appearance of a cavitary lesion. Accordingly, of a total of 185 patients who showed two or more isolations, 181 (98 percent) had the association. Of these 181, 176 (97 percent) showed two or more isolations in the sputum examinations made in the initial three days. Therefore, the sputum examination in the first three days after onset of disease is most important for the diagnosis of disease caused by Mycobacterium avium complex. Since the probability that casual isolation of the organism occurs twice is extremely low, we can make the diagnosis of pulmonary infection caused by this organism by evidence of two or more isolations of the organism in the first few days after the onset of disease, which is associated with appearance of a new cavitary (or infiltrative) lesion. Moreover, theoretical consideration made in this study has led us to conclude that patients who have had a single isolation of the organism together with a new cavitary lesion should be regarded as having an infection.

Previous studies of Mycobacterium fortuitum identified isolates that did not fit its two recognized biovariants. Eighty-five clinical isolates of this group, the "third biovariant complex", were evaluated. They represented 16% of 410 isolates of M. fortuitum submitted to a Texas laboratory and 22% of 45 isolates in Queensland, Australia. Most infections (76%) involved skin, soft tissue, or bone and occurred after metal puncture wounds or open fractures. Isolates differed from biovar fortuitum in resistance to pipemidic acid and use of mannitol and inositol as carbon sources. Two subgroups were present, and examples were deposited in the American Type Culture Collection. Isolates were resistant to doxycycline and one-third were resistant to cefoxitin. All were susceptible to amikacin, ciprofloxacin, sulfamethoxazole, and imipenem. Surgical debridement combined with drug therapy based on in vitro susceptibilities resulted in cures of cutaneous disease or osteomyelitis. DNA homology studies are needed to determine the taxonomic status of these organisms.

In 0.1% Tween 80 aqueous solution or in 0.1% Tween 80-containing saline (0.9% NaCl aqueous solution), Mycobacterium smegmatis strain Jucho bacteria were alive for 3 days, whereas, in 0.1% Tween 80-containing phosphate buffer solution (pH 7.1), the bacteria died rapidly. Since it was shown previously that M. smegmatis is one of the mycobacteria most susceptible to Tween 80 (Tsukamura, M.: Kekkaku 63: 695-699, 1988), the 0.1% Tween 80 aqueous solution or 0.1% Tween 80-containing saline may be used for suspending mycobacteria in order to prevent the clumping. Incorporation of 32P-ortho-phosphate to the nucleic acid fraction was most markedly inhibited by the presence of 0.5% Tween 80 in 0.067 M phosphate buffer solution (pH 7.1). The finding shows that Tween 80 inhibits directly or indirectly the synthesis of nucleic acids. Tween 80 was not bactericidal in an aqueous solution but acted bactericidally in the co-existence of phosphate, and such bactericidal activity was completely diminished by adding an ammoniacal nitrogen compound (Tsukamura, M.: Medicine and Biology (Tokyo) 96: 159-161, 1978). These findings suggest that Tween 80 does not simply act as a detergent but interferes with some metabolic way in the synthesis of nucleic acids.

When Mycobacterium tuberculosis strain H37Rv was cultivated in Ogawa egg medium containing 5 micrograms/ml streptomycin and/or 10 micrograms/ml kanamycin, which were considered as subinhibitory, it was observed that growing bacterial population contained several times more rifampicin-resistant mutants than did the parent strain. The ratio of isoniazid-resistant mutants did not change by the above treatment. The finding suggests that, even without the use of rifampicin, the bacterial population of patients becomes more resistant to rifampicin by chemotherapy in the past with streptomycin or kanamycin. Furthermore, it was shown that the ratio of streptomycin-resistant mutants increased by pre-treatment with kanamycin and the ratio of kanamycin-resistant mutants by pre-treatment with streptomycin. Parent, susceptible bacterial population develops 4R-phenotype mutants which are resistant to four drugs, high concentrations of kanamycin, lividomycin and paraomomycin and a low concentration of capreomycin, whereas streptomycin-resistant mutant population does not develop the 4R mutants but develops only mutants with the KR phenotype which is almost mono-resistant to kanamycin.

The susceptibility to rifampicin and streptomycin of the Mycobacterium avium-Mycobacterium intracellulare complex was augmented by the addition of Tween 80 into 7H10 agar medium with OADC, whereas the susceptibility to ethambutol and sulfadimethoxine was either not changed or reduced by the addition of Tween 80. In 7H10 agar medium without OADC, however, susceptibilities to both rifampicin and sulfadimethoxine were reduced by the addition of Tween 80 to the medium. A number of hypotheses are made to explain these phenomena.

The resistance development pattern of Mycobacterium smegmatis strain 17023 (Jucho) to streptomycin and kanamycin was studied. The medium used was Ogawa egg medium, and the level of resistance was determined for each clone derived from single colony by the 'actual count' method. Hence, the resistance level was estimated as the highest concentration of drugs, in which small inocula consisting of 20 to 100 colony-forming units could grow after seven days incubation. Only one type of resistance mutants resistant to more than 1,000 micrograms/ml streptomycin was isolated and these mutants were also resistant to 8 micrograms/ml kanamycin. On the other hand, only one type of kanamycin-resistant mutants resistant to 8 micrograms/ml kanamycin was isolated and these mutants were also resistant to more than 1,000 micrograms/ml streptomycin. Accordingly, there was a complete cross-resistance relationship between streptomycin and kanamycin resistances. Therefore, there existed only one phenotype, which is simultaneously resistant to streptomycin and kanamycin. The mutants occurred at a rate of about 2 x 10(-8) per viable bacterial population of the parent strain. Streptomycin-dependent mutants occurred at a rate of about 2 x 10(-9). The number of resistant phenotypes to streptomycin and kanamycin was only one in M. smegmatis, while it was five in M. tuberculosis and 2 or 3 in M. avium complex (Tsukamura, M. and Mizuno, S.: J. Gen. Microbiol. 88: 269-274, 1975; Tsukamura, M.: Kekkaku 62: 445-458, 1987). The simplicity of the resistance system of M. smegmatis suggests that this organism is evolutionally primitive in the world of mycobacteria.

A recent study of Nocardia asteroides revealed that 95% of clinical strains had one of five antibiotic resistance patterns. We found the pattern of resistance to cefotaxime and cefamandole in 19% of 200 clinical N. asteroides isolates. Isolates with this drug resistance pattern were from numerous geographic sources and were associated with significant clinical disease (56% of patients had disseminated infections). Phenotypic studies revealed that these isolates were relatively homogeneous and matched previous descriptions and reference strains of the controversial species N. farcinica. Growth at 45 degrees C, acid production from rhamnose, ability to utilize acetamide as a nitrogen and carbon source, and resistance to tobramycin and cefamandole were features of N. farcinica that could be tested in the clinical laboratory and allowed their distinction from N. asteroides. The serious nature of disease due to N. farcinica and its resistance to the newer cephalosporins suggest a clinical need for laboratory identification of this species. (Current tests used in clinical laboratories do not distinguish N. farcinica from N. asteroides.) This is the first recognition that N. farcinica has a specific drug resistance pattern and confirms the previously described concept that drug resistance patterns of N. asteroides may be associated with specific taxonomic groups.

Bactericidal activity of ofloxacin against Mycobacterium kansasii was observed in in-vitro experiment. Tested bacteria were suspended to a concentration of one mg wet weight per ml in 10 ml of Dubos liquid medium containing no drug or containing 1 or 3 micrograms/ml ofloxacin and incubated at 37 degrees C. The number of colony-forming units contained in a 0.02 ml-sample of the Dubos liquid medium was counted after incubation for 0, 1, 3 and 7 days. The number of colonies was counted in Ogawa egg medium, to which was inoculated a 0.02 ml-sample of dilutions of the medium by a spiral loop that can deliver a 0.02 ml-sample by one inoculation. The bactericidal activity of ofloxacin against M. kansasii appeared after incubation of 3 days or later. This characteristic was different from the bactericidal activity against Mycobacterium tuberculosis, which appeared after incubation of 24 hours.

Isoniazid inhibited the growth of Mycobacterium avium-Mycobacterium intracellulare strains at concentrations of 0.1-25 micrograms/ml and was even bactericidal for several strains. The bactericidal activity was observed in relatively susceptible strains. The susceptibility did not correlate with colonial morphology but correlated with species identification. Mycobacterium avium strains were more resistant to isoniazid than Mycobacterium intracellulare strains.

Bacteriostatic and bactericidal activities of rifampicin, ethambutol, enviomycin and streptomycin on Mycobacterium avium-Mycobacterium intracellulare complex (MAI complex) strains were determined. The susceptibility testings were made in Ogawa egg medium, and minimal inhibitory concentrations (MICs) were determined as the lowest concentration of drugs, in which the growth of 20 to 100 colony-forming units was completely inhibited. The MICs correspond to the MICs that can inhibit the growth of 95 to 99% of bacterial population. Bactericidal activity was determined in a modified Dubos liquid medium (1.3 g of Dubos TB Broth Base were dissolved in 180 ml of distilled water and this solution was supplemented by 20 ml of bovine serum). A one ml-sample of bacterial suspensions (10 mg wet weight per ml) was added to 9 ml of the Dubos liquid medium, and the medium was incubated at 37 degrees C for 0, 1, 3 and 7 days under shaking condition (56 strokes per minute; 8 cm amplitude). The bactericidal activity was measured as the number of colony-forming units contained in a 0.02 ml-sample of the medium. The bactericidal activities of rifampicin and ethambutol were weak or absent even in strains 13008 and 13016, which were very susceptible to all four drugs. However, the bactericidal activities of streptomycin and enviomycin could be observed in these strains.(ABSTRACT TRUNCATED AT 250 WORDS)

The determination of the susceptibilities to antituberculosis agents in Mycobacterium avium-Mycobacterium intracellulare complex (MAI complex) is influenced by the size of inoculation used in the determination. Such influences differ greatly according to the drugs. By increasing the number of colony-forming units (CFU) of the inoculation to 10(6)-fold, minimal inhibitory concentrations (MICs) of rifampicin and cycloserine have increased only 2-to 3-fold. However, MICs of ethionamide, streptomycin, enviomycin and ethambutol have increased 4-to 9-fold. In contrast, MICs of isoniazid and sulfadimethoxine have increased 30-to 35-fold. Since the determination of MICs of isoniazid and sulfadimethoxine are so greatly influenced by the size of inoculation, the determinations of MICs of these two drugs should be carried out by use of the "actual count" method (6, 7, 9) or the proportion method (8). In the actual count method, the MIC is determined by inoculating 20-100 CFU. The determination of isoniazid susceptibility of M. tuberculosis (H37Rv) was influenced only slightly by the size of inoculation, but that of M. bovis (BCG) was influenced greatly as occurred in the determination of MAI complex strains.

Treatment of nontuberculous mycobacterial infection should be carried out by the chemotherapeutic regimens most suitable for each species. We performed in-vitro susceptibility tests for various species and determined the probability in which the mycobacteria of each species are inhibited by blood concentrations attainable by the dosages usually used. From such determinations, the following regimens have been recommended: 1) M. avium-M. intracellulare complex, Rifampicin + Enviomycin + Ethambutol; 2) M. kansasii, Ofloxacin + Enviomycin + Rifampicin; 3) M. szulgai, Enviomycin - Ethambutol + Isoniazid; 4) M. fortuitum, Ofloxacin. The effectiveness of the treatment of the infection caused by M. avium complex is less than 15% even using the above regimen, while it is high in the treatment of infections caused by M. kansasii and M. szulgai. There are no effective regimens for the treatment of infection caused by M. chelonae or M. simiae. The above recommendations are useful in practice, because, at present, reliable susceptibility testing of nontuberculous mycobacteria is not yet done commonly.

In-vitro combined activity of antituberculosis drugs on Mycobacterium avium complex strain 13034 (serotype 18) was observed. A twofold dilution series of each drug was combined with 17 additional drugs and the control without drug. Each medium was inoculated by a 0.02 ml-sample of four different bacterial suspensions, original suspension (10 mg wet weight per ml) and its 10(-5), 10(-6) and 10(-7) suspensions, and incubated at 37 degrees C for 28 days. The inoculation was made by a spiral loop that can deliver a 0.02 ml-sample by one inoculation. Since a total of media reached 6480 (1620 combinations x 4 different suspensions), only one strain was investigated in this study. Inoculation size used for determining minimal inhibitory concentrations was adjusted to 20 to 80 colony-forming units per medium. This was obtained by inoculating 10(-6) suspensions. The diameter of colonies was measured on media that were inoculated with 10(-7) suspensions. Since 96% confidence limits of determining minimal inhibitory concentrations are between 1/4 to fourfold, decrease or increase of the minimal inhibitory concentration equal to or above this level was regarded as significant. Such significant change of the minimal inhibitory concentrations occurred in combinations of drugs is shown in Table 2. As shown in tables, no antagonism was observed. The combinations, in which significant decrease was seen, contained ethambutol, sulfadimethoxine and ethionamide. It was shown that these concentrations of the drugs cause marked delay of growth. In control medium, the diameter of colonies was 5-6 mm, whereas in media containing one of these drugs, it was 1 mm.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study, it was investigated whether or not Mycobacterium avium complex strains were inhibited by such low concentrations of isoniazid and ethionamide as possible in blood. The inhibitory concentrations were determined by the "Actual Count" method (Tsukamura, M.: Japan J. Tuberc. 12: 46-54, 1964), in which the inhibitory concentration was measured as a concentration that can inhibit the growth of 'countable' colony-forming units. The ratio of strains which were inhibited by such low concentrations as 0.2 microgram/ml isoniazid or 5 micrograms/ml ethionamide was 14%. The result suggests that there are some strains in which isoniazid or ethionamide are clinically effective, though their prevalence was low (14%).

Although mycobacteria grow in Dubos liquid medium showing an arithmetic linear growth, the initial few days of growth were found to correspond to an 'induction' period. In this period, rapid increase of the amount of growth occurred, whereas increase of the number of colony-forming units remained at a low level. This finding shows that the rapid increase of the amount of growth is accompanied by rapid death of multiplied bacteria. In a successive period, which was considered to correspond to the logarithmic growth phase, a 1:1 correspondence existed between the amount of growth and the number of colony-forming units. The induction period is not considered to be a lag phase, in which the bacteria grow slowly, but a period of unbalanced relationship between the growth and the viability. Even when we inoculated different sizes of bacteria, the amounts of growth became similar in both inoculations after several days of incubation. However, the number of colony-forming units remained always smaller in the use of small inocula than in the use of large inocula. In the use of small inocula, much more rapid increase of the amount of growth occurred. However, this rapid increase gave rise to rapid death of bacteria.

The bactericidal activity of antituberculosis drugs against two strains of M. avium complex 13008 (serotype 20) and 13016 (serotype 4), was observed using modified Dubos liquid medium (1.3 g of Dubos Broth Base (Difco) in 180 mil of distilled water plus 20 ml of bovine serum). The strains were most susceptible to antituberculosis drugs among M. avium complex strains (Table). The test strains were cultivated in Ogawa egg medium at 37 degrees C for 10 days, and growing colonies were homogenized by shaking with glass beads for 10 minutes and suspended in the modified Dubos liquid medium to a concentration of 2 mg wet weight per ml. The media containing drug and containing no drug were incubated at 37 degrees C for 24 hours under shaking (8 cm-moving range and 56 strokes per minute). After incubation, the medium was diluted by a 0.1% Tween 80 aqueous solution to 10(-7), and each 0.02 ml-sample of 10(-4) to 10(-7) suspensions was inoculated onto Ogawa egg medium. The Ogawa egg medium slants were incubated at 37 degrees C for 28 days, and the number of colonies was counted. The bactericidal activity was determined as a ratio of the number of colony-forming units in 10 ml-aliquot of drug-containing Dubos liquid medium against the number of colony-forming units in the control Dubos liquid medium. The results are shown in Figures 1 and 2. Under the condition tested, rifampicin, isoniazid, ethambutol, ethionamide and cycloserine did not exhibit any bactericidal activity, and only streptomycin and enviomycin exhibited bactericidal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The growth of the type strain of Mycobacterium aurum was compared between flask and tube, both of which contained the same concentration of bacteria in synthetic media and had the same air-contact dimension but had different dimensions of the glass wall of vessels contacting the medium. Much better growth occurred in the flask, which had a smaller dimension of the glass wall contacting the medium, than in the tube, which had a larger dimension. The finding shows that the growth of bacteria is influenced by the dimension of the glass wall of vessels. It was considered that a high chance of the attack shock for bacteria given by the Brownian movement lowers the multiplication of bacteria.

Tests reported to be useful for the differentiation of species or groups of mycobacteria are reviewed. Some systems of identification of these organisms are composed of relatively small or large numbers of tests. These systems remain at present "national" identification systems; no "international" system agreed on by scientists worldwide exists. Such an international system is desirable.

Mycobacterium avium complex strains previously not exposed to any antituberculosis agents could be divided into two groups according to their susceptibility to rifampicin and ansamycin; one group susceptible to 80 micrograms/ml rifampicin and to 1.25 micrograms/ml ansamycin, and another resistant to these concentrations. In each group, the ratio of the minimal inhibitory concentration of ansamycin against that of rifampicin was greatly different depending on the strain. This naturally occurring resistance to rifampicin and ansamycin was frequently correlated to naturally occurring resistance to ethambutol, kanamycin, enviomycin, kitasamycin, and minocycline, but not correlated to that to isoniazid and sulfadimethoxine. Ansamycin was more active than rifampicin against M. bovis, M. kansasii, M. marinum, M. xenopi, and M. haemophilum.

Out of 97 strains of M. tuberculosis isolated from the sputum of the same number of patients, 11 were shown to consist of a mixed population of M. tuberculosis and a strain of the M. avium-M. intracellulare (MAI) complex. The most effective method for isolating strains of the MAI complex was to incubate the culture first at 42 degrees C for 3 weeks and then at 45 degrees C for 3 weeks. By this method, the growth of M. tuberculosis was suppressed, while the contaminant mycobacteria formed colonies.

We studied the usefulness of antibody estimation by the enzyme-linked immunosorbent assay (ELISA) technique in the differential diagnosis of pulmonary mycobacterial diseases. Sera from patients with active pulmonary diseases caused by Mycobacterium tuberculosis (n = 42) or Mycobacterium avium-intracellulare complex (MAI) (n = 26) and from healthy controls (n = 75) were assayed for IgG and IgA antibody titers to antigen of M. tuberculosis and MAI. The IgG and IgA antibody titers to M. tuberculosis antigen were significantly higher in patients with mycobacterial diseases than in healthy controls, however, there was no significant difference between patients with pulmonary tuberculosis and those with pulmonary diseases due to M. avium-intracellulare complex (MAID). The IgG and IgA antibody titers to MAI antigen in MAID patients were significantly higher than in healthy controls and in patients with pulmonary tuberculosis. ELISA for IgG antibody against M. tuberculosis antigen had a sensitivity of 54.8% and a specificity of 73.1% in the differential diagnosis of pulmonary tuberculosis. The estimation of the IgA antibody against MAI antigen using ELISA is more useful for the differential diagnosis of MAID because of its sensitivity (69.2%) and specificity (90.5%).