Some memoranda on the diagnostic criteria of lung disease caused by Mycobacterium avium-Mycobacterium intracellulare complex.

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.

Dietary maltitol decreases the incidence of 1,2-dimethylhydrazine-induced cecum and proximal colon tumors in rats.

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.

The activity of branched-chain alpha-keto acid dehydrogenase complex in rat digestive tracts: Effects of dietary protein and exercise.

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.

Clinical significance, biochemical features, and susceptibility patterns of sporadic isolates of the Mycobacterium chelonae-like organism.

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.

Isoelectric focusing patterns of beta-lactamases in the rapidly growing mycobacteria.

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.

Comparison of prognosis of pulmonary diseases caused by Mycobacterium avium and by Mycobacterium intracellulare.

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.

Differentiation between Mycobacterium avium and Mycobacterium intracellulare by thin-layer chromatography of lipid fraction after incubation with [35S]methionine.

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 activity of antituberculosis drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare complex and Mycobacterium kansasii in different growth phases.

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.

Clinical and laboratory features of Nocardia nova.

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.

[Studies on the nontuberculous lung mycobacteriosis in Japan. (Report of the study in the year 1987 and 1988 of the Mycobacteriosis Research Group of the Japanese National Chest Hospitals)--pulmonary infection caused by Mycobacterium kansasii has begun to appear in all over Japan including north Japan Hokkaido].

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.

Chronic tenosynovitis of the hand due to Mycobacterium nonchromogenicum: use of high-performance liquid chromatography for identification of isolates.

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.

[Chemotherapeutic regimens that were considered effective to cure pulmonary infection caused by Mycobacterium avium-Mycobacterium intracellulare 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).

[Change in biochemical and biological characteristics of Mycobacterium tuberculosis strains isolated in recent years].

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.

Diagnosis of disease caused by Mycobacterium avium complex.

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.

Clinical disease, drug susceptibility, and biochemical patterns of the unnamed third biovariant complex of Mycobacterium fortuitum.

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.

[Some observations on the mechanism of bactericidal activity of Tween 80 on mycobacteria].

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.

[In vitro experiment showing that chemotherapy of tuberculosis by aminoglycoside antibiotics may influence successive chemotherapy with rifampicin].

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.

Paradoxical effect of Tween 80 between the susceptibility to rifampicin and streptomycin and the susceptibility to ethambutol and sulfadimethoxine in the Mycobacterium avium-Mycobacterium intracellulare complex.

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.

Cefotaxime-resistant Nocardia asteroides strains are isolates of the controversial species Nocardia farcinica.

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.