An investigation of the distribution of bacteriophage susceptibilities was carried out by using 99 strains of M. tuberculosis isolated from patients with pulmonary tuberculosis in Japan, 99 strains obtained from Netherland and 54 strains obtained from Ceylon.
Phage susceptibilities were tested by spotting phage suspensions of routine test dilution (RTD) and 10×RTD on the lawn of mycobacteria. The name of the phages employed and the number of plaque forming unit being contained in RTD of each phage were shown in Table 1.
All of the 11 mycobacteriophages (DS6A, AG1, BK1, BG1, GS4E, PH, Clark, Legendre, Sedge, DNA 1118, and D34) were employed in the case of Japanese and Netherland strains. The results obtained were as follows (Table 2): All strains except 3 were susceptible to DS6A and AG1. These 3 exceptional strains were resistant to all phages even by the spotting of 10×RTD. Number of strains susceptible to D34 was 4 in Japanese strains and 2 in Netherland strains. Susceptibility of Netherland strains to the remaining 8 phages were generally higher than that of Japanese strains (Table 3). For instance, the number of strains susceptible to all phages except D34 by RTD was 18 in Japanese strains and 34 in Netherland strains. Forty one Japanese strains and 11 Netherland strains were susceptible only to DS6A and AG1 by RTD. However, when 10×RTD phage suspensions were employed for spotting, most of them showed susceptibility to one or more phages in addition to DS6A and AG1. Especially, in the case of BK1 and BG1, marked differences in the number of strains lysed by 10×RTD and by RTD were noted (Table 3). In this case, a possibility of nonspecific growth inhibition by concentrated phage particles was excluded, since the number of plaque forming units being contained in one drop of 10×RTD ranged from about 10² to 5×10⁴. Therefore, this phenomenon indicates that there are many strains which show intermediate susceptibility against these phages.
Phage susceptibility patterns of the 54 Ceylon strains against DS6A, BK1, PH and D34 were shown in Table 4. In general, they were more susceptible to those phages than Japanese and Netherland strains: 87% were BK1 sensitive, 68% were PH sensitive, and 8% were D34 sensitive by the spotting of RTD.
Distribution of bacteriophage types of these strains was compared using the scheme proposed by Bates and Fitzhugh. The distribution of Type A, B and C in Japan andin. Netherland was almost thesame. On the contrary, significant difference was observed between Japanese and Ceylon strains in the distribution of phage types (Table 5).
Based on the results obtained, we thought that the following conditions should be taken into account in the selection of a typing phage set of M. tuberculosis: (1) select the phages which render a clear cut, easily detectablelysis; (2) select the phages which lyse similar number of strains by the use of both 10×RTD and RTD; (3) select the phages which do not lyse too manystrains: i.e. those which lyse approximately 10 to 20% of strains are preferable; (4) select the phages the lytic pattern of which overlap each other as little as possible; (5) select the phages so as to make the distribution of strains of certain phage types evenly and minimize the number of untypable strains.
It was found that the mycobacteriophages employed in this study do not necessarily satisfy the above mentioned conditions, although it was possible tosubdivide M. tuberculosis strains to a certain extent.

View full abstract

In the preceding paper, it was reported that the increase in the amount of urinary D-β-aminoisobutyrate (β-AIB) and β-alanine was resulted from the inhibition of β-AIB: pyruvate aminotransferase and β-alanine: α-ketoglutarate aminotransferase by D-cycloserine (CS) or its metabolites. A maximal concentration of CS in the liver of rats which were administered intraperitoneally at a dose of 20mg of the drug per 100g of body weight showed only a slight inhibitory effect on the enzyme reactions. The concentration of β-AIR and β-alanine in rat liver reached to maximal level 4 hours after the injection of CS when. CS was barely defectable in the liver. This suggested that the inhibition is due to a metabolite of CS rather than CS itself. Presence of the inhibitory substance was studied in the present paper.
For the isolation of the inhibitors, rats were used and male mice were used for toxicity test of the drugs. Determination method of β-AIB: pyruvate and β-alanine: α-ketoglutarate aminotransferase was reported previously. Rabbit liver was used for the study of enzymatic conjugation of β-AOAL and α-keto acids. CS was determined using a high voltage electrophoresis.
An inhibitory metabolite of CS was detected in the rat liver after CS injection in a preliminary experiment, and the same inhibitory substance was found in urine of rats which were injected CS intraperitoneally. The inhibitor was purified from the rat.urine using combination of ion-exchange chromatographies, and was identified as β-aminoxy-alanine from its behavior on paper chromatography, paper electrophoresis and ion exchange chromatography. In order to confirm the identification, the isolated compound was conjugated with pyridoxal phosphate and compared with β-aminoxyalanine-pyridoxal phosphate conjugate on the chromatographies and UV-spectrum. The results verified the above identification. The concentration of β-aminoxyalanine required for 50% inhibition of β-AIB pyruvate and β-alanine: α-ketoglutarate aminotransferases was 10^-6M while that of CS was 10^-3M. The above results support hypothesis that a metabolite of CS, β-aminoxyalanine, is the major substanceswith inhibitory effect on the aminotransferases.
During the isolation experiment of inhibitory substances derived from CS, another fraction of ion exchange chromatography different from those of CS and β-aminoxyalanine was also found to contain an inhibitory substance. The substance was acidic. When CS or β-aminoxyalanine was incubated with substrates of transamination reaction, enzymic formation of a conjugated compound of β-aminoxyalanine and keto acids was found. β-aminoxyalanine and glyoxalate was incubated with rat liver extract, and the enzyme product was purified using ion exchange chromatography to isolate a crystalline material. Elementary analysis matched that of C₅H₈N₂O₅. This was a shiff base of β-aminoxy-alanine and glyoxalate. The concentration of the base required for 50% inhibition of β-AIB: pyruvate aminotransferase was roughly 3×10^-5M.
It is known that CS has various side effects in clinical use including headache, anxiety and convulsion. Experiment for acute toxicity of β-aminoxyalanine was carried out by using mice. LD 50 was 2.6g per kg of body weight which is slightly lower than that of CS. It was interesting that convulsion was consistently observed with the lethal dose of β-aminoxyalanine, while none with CS.

View full abstract

Pulmonary tuberculosis patients without previous chemotherapy were allocated at random to the following three regimens.
I. 1g Streptomycin biweekly+0.3g INH daily+10g PAS daily (142 subjects)
II. 1g Streptomycin biweekly+0.3g INH daily+0.45g RFP daily (137 subjects)
III. 0.3g INH daily+10g PAS daily+0.45g RFP daily (137 subjects)
RFP was administered in a single dose before the breakfast. The compatibility of the three test groups was shown in fig. 1. The number of cases excluded from the study was indicated in Table 1.
The patients withdrawn before the end of 3 months were excluded from the calculation of the rate of the sputum conversion and radiographic improvement, but they were utilized for the incidence of side effects.
As to the rate of the culture conversion, Regimens II and III including RFP were definitely superior to Regimen I (standard regimen). (fig. 2 and table 2)
The negative conversion rate of the RFP regimens was higher for culture than for smear. (fig. 3 and tables 2, 3)
Radiographic changes were demonstrated in table 4.
Incidence rate of advers reactions and dropout due to them were shown in tables 5, 6, 7 and 8. Gastrointestinal disturbance was the most frequent and liver dysfunction was observed rarely.
The rate of sputum negative conversion by culture of the cases with primary drug resistance of the Regimen I was shown in table 9, and the rate was lower than that of sensitive cases.

View full abstract

Far advanced severe pulmonary tuberculosis patients were treated by the intermittent administration of rifampicin, and their sputa findings were observed.The critical concentration. of clinical rifampicin resistance and the acquirment pattern of rifampicin resistance were studied in 47 cases; and the resistance test was done by using Kirchner's semi-liquid agar media with. 10% albumin.
Following results were obtainned.
1) Tubercle bacilli became resistant to rifampicin in a short period.
If the concentration of clinical rifampicin resistance for tubercle bacilli on Kirchner's semi-liquid agar media was defined as 1mcg/ml complete resistance, the rate of cases acquired resistance to rifampicin by the intermittent administration was 11.6% at 1 month, 25.0% at 2 months, 37.2% at 3 months, 30.8% at 4 months, 42.4% at 5 months and 48.6%, at 6 months.
2) It would be safe to say that the critical concentration of clinical rifampicin resistance for tubercle bacilli is 1mcg/ml complete resistance.
3) The acquirment pattern of rifampicin, resistance of tubercle bacilli in rifampicin treated patients was mainly the multi step-pattern and in a few cases single step-pattern on Kirchner's semi-liquid agar media.

View full abstract

This study was made to clarify the treatment method for post-operative complications in pulmonary tuberculosis patients. The subjects consisted of 263 cases with post-operative complications among 8, 386 cases who had undergone the operation during the 4 years' period from 1965 to 1968 at 51 institutions belonging to the Tuberculosis Research Committee (RYOKEN) and had been followed up 4 times at the end of November next year after the operation, respectively. All of these cases were followed up at the end of March, 1971. The fistula (including empyema with fistula), empyema (without fistula) and radiological worsening were picked up as the complication in this study. Cases with these complications were divided into two groups; one was the group judged as successfully cured within the each follow-up period and the other was the group under treatment. The former consisted of 180 cases (68.6%) and the latter 83 cases (31.4%).
The kind of post-operative complications, the background factors before the occurrence of post-operative complications, the type of operation before the occurrence of post-operative complications, the methods of treatment, the surgical procedure applied for post-operative complications, the number of operations, the duration of treatment and the results of treatments in both groups were comparatively studied.
The term of success, unsuccess and death as reported in previous papers were used in this study to evaluate the results of treatment. The case of success was separated into two categories. One was success I which consisted of cases who had shown negative sputum and had been rehabilitated or expected to be rehabilitated within three months. Another was success II which consisted of cases who had shown negative sputum but was not expected to be rehabilitated within three months.
The rates of fistula, empyema and radiological worsening were 52.7%, 21.1% and 26.2% in the cured group, and 77.1%, 6.1% and 16.8% in the group under treatment, respectively. The fistula was found more and the empyema was less in the former than in the latter.
Comparing the background factors before the occurrence of complications caused by surgical procedures, the bilateral cavitary case was only 2.8% in the cured group, while 18.1% in the group under treatment. Moreover, the bilateral operation was done in only 2 cases (13.3%) among 15 cases of the group under treatment. The rate of positive sputum. (25.3%) in the group under treatment was rather lower than in the cured group (51.1%). The case with %VC less than 50% was 9.4%, 51-70 was 22.2% and more than 71 was 65.5% in the cured group, and 31.3%, 31.3% and 33.8%, respectively, in the group under treatment. This showed that the case with lower %VC was more in the latter than in the former.
Analysing the type of operations applied before the occurrence of complications in both groups, pneumonectomy was carried out at almost same rate and resections excluding pneumonectomy in the cured group (68.9%) were applied significantly more than in the group under treatment (47.1%). Thoracoplasty. in the cured group (3.9%) was significantly lower than in the group under treatment (16.9%). Cavernostomy in the cured group (0.6%) was significantly lower than in the group under treatment (7.2%).
Observing the methods of treatment for post-operative complications, the surgical treatment was more frequently applied for fistula (87.5%) and empyema (77.0%) than chemotherapy, though chemotherapy was more frequently used for radiological worsening (87.2%) than surgical treatment in the cured group. A significant difference was found in the methods of treatment for fistula and radiological worsening. Same tendency was found in the group under treatment, but there was no significant difference between the methods of treatment for different type of complications.

View full abstract