Drug resistance of 3, 000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in α cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM. SA) and R (TC. CM. SM. SA) factors was most frequent. It was found that 25 R (SM. SA) factors isolated from strains in hetero-R had the genetic determinant iR^-, while most of the R (TC. CM. SM. SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM. SA) and R (TC. CM. SM. SA) derived from the same host cells, were brought together in host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.

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Two types of slowly growing, nonphotochromogenic mycobacteria, Mycobacterium terrae and Mycobacterium novum, were isolated from soil by mouse body passage method. The former was presented previously by the present author as α new species. Its characteristics are better clarified in this paper based on the data of 93 strains. Mycobacterium novurn is a slowly growing nonphotochromogen. It grows at 10 to 14 days on egg media and does not grow on Sauton agar. It grows on Ogawa egg medium con-taining either 0.2% (w/v) sodium p-aminosalicylate, 0.1% (w/v) sodium salicylate or 0.25mg/ml NH₂OH•HCl. It is differentiated from M. tuberculosis, M. bovis and M. microti by these characters. It grows at 28C and 37C, but does not grow at 45C. Other characteristics are: nitrate not reduced; negative two week arylsulphatase; negative niacin test; and no amidase demonstrated. None of the carbohydrates and nitrogen compounds tested could be utilized as the sole source of carbon or of nitrogen in synthetic agar medium. It survived in mouse organs for three to four weeks. It was noticed that these mycobacteria occur very commonly in soil.

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Five R factors capable of conferring resistance to ampicillin, together with resistance to 4 other drugs, tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA), were found in Escherichia coli strains isolated from clinical sources. Ampicillin resistance was due to synthesis of penicillinase in the host bacterium which harbored the R factor. The penicillinases produced by three R factors were similar each other in their substrate specificity and were also similar to the enzyme reported by previous workers. The penicillinases produced by 2 other R factors were found to be greatly different from penicillinases reported thus far. The latter were shown to hydrolyse ampicillin and cloxacillin efficiently, but penicillin-G and 6-aminopenicillanic acid to a lesser extent.

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On the basis of resistance patterns to penicillins, strains of Staphyloroccns aureus highly resistant to penicillin-G which were isolated from clinical sources, could be classified into 2 groups, A-1 and A-2. Group A-2 is minor group which accounts for about 10% of these strains and exhibits lower degree of resistance to penicillin-G and phenethicillin than that exhibited by group A-1 strains. However, there was no marked difference between both groups in inducible production of penicillinase. The genetic determinants for penicillinase synthesis in strains of both groups were transduced into
recipient and the characteristics of the determinants were compared with each other under identical conditions. The results suggested that the genetic determinants for penicillinase synthesis in groups A-1 and A-2 strains differed from each other in both the structural gene for penicillinase production and the regulatory gene for the formation of the repressor.

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As an approach to clarify biochemically the mechanism of intracellular killing of bacteria by phagocytic cells, the effect of several metabolic inhibitors on the bactericidal activity of mouse peritoneal macrophages (MP) on ingested Salmonella enteritidis and Escherichia coli was examined. 5×10^-4M iodoacetate and 10^-2M NaF added to the medium interfered significantly with the bactericidal activity of the MP, whereas sodium malonate, KCN, dinitrophenol, and anaerobiosis did not. Thus a close relation between the glycolytic pathway of MP and their bactericidal activity on ingested bacteria was demonstrated. However, oxamic acid and exclusion of external glucose, both of which significantly reduced lactate production by MP, did not affect the bactericidal activity of such cells. The lack of influence of puromycin on the bactericidal activity of MP excluded the possibility that de novo synthesis of lysosomal enzymes and bactericidal substances by the cells is necessary for accomplishment of the intracellular killing of bacteria. NaF inhibited significantly acid phosphatase and esterase activities, and iodoacetate slightly inhibited uricase activity, while sodium malonate, KCN and dinitrophenol did not exhibit any inhibitory effect on the activities of all the lysosomal enzymes examined. From these findings, the mechanism of the intracellular killing of the ingested bacteria by MP was discussed.

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Twenty-five strains of RNA phages were tested for their filtration and elution patterns (F-E patterns) by a millipore filtration method. These strains, including MS2, f2 and R17, were separated in three groups in this aspect. We named these groups as group I, II and III. According to this grouping, MS2, f2 and R17 belonged to group I and Qβ belonged to group III. Furthermore, it was shown that group III was further divided in two sub-groups (IIIa and IIIb) by this method. Grouping based on the F-E patterns was in extremely good accordance with the grouping based on the serological properties. This grouping was also supported by the results of chemical and physical analyses of these RNA phages, that is, RNA phages which belonged to the same group had several common properties. Basic information on the millipore filtration method was also presented in this paper.

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A statistical method is proposed for recognition of a bacterial species or for differentiation of two groups of bacterial strains. Comparison between two groups is done by the "t"-test. When the mean S-values (similarity value) of two groups are A and B, and the mean S-value for all possible combinations between strains of both groups is S, a condition necessary for defining the two groups as different species is to demonstrate the existence of equations A>S and B>S. Unless this condition is fulfilled, the two groups should be considered unseparable. The condition necessary for recognition of two groups as one species is to demonstrate the existence of equations A=S and B=S. A few examples of the test were shown using the mycobacteria, and it was suggested that this statistical method is useful in the recognition of a species.

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When either colistin at 1, 000μg/ml or sulfisomezole at 125μg/ml was used separately, growth of a strain of Proteus mirabilis was not inhibited. However, when 1μg/ml of colistin and 25μg/ml of sulfisomezole were used together in agar media, growth was inhibited. The synergistic action of colistin and sulfisomezole was also demonstrated in broth culture, when a smaller inoculum such as 10⁶ cells/ml was used. The lethal and lytic effect of this synergism parallels the characteristic effect of colistin towards colistin-sensitive gram-negative organisms. When the mode of this synergistic action was analyzed by adding each compound in sequence to a growing culture of Proteus, it was found that growth of organism for about 4 generations in the presence of sulfisomezole was a prerequisite for revealing the lethal and lytic effects of colistin. In cultures where these two compounds were present at the beginning of incubation, the synergistic effect was abolished by the addition of p-aminobenzoic acid (PABA) at an early stage of incubation, but not at a late stage. Methionine, serine, and betaine, when used together, had the same effect as PABA. An insufficiency of the three compounds induced by sulfisomezole, was considered to afford the receptor site of colistin to Proteus.

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