Dissociation in Pseudomonas aeruginosa should be taken into consideration in establishing a typing method of the bacterium. The results of a study on the dissociation of the bacterium are reported in this paper. Nine strains of Pseudomonas aeruginosa were used for the purpose of isolating their dissociants. Of them, five had been developed from a single cell. Two kinds of colonial dissociants were isolated on an agar plate from each of the nine strains; that is a large, flat, and moist colony and a small, round, and convex colony. The former was designated as type la and the latter, as type sm. Investigation was carried out to find any differences between the two types, la and sm, in frequency of appearance under cultural conditions, cell morphology revealed by electron microscopy, antigenic structure, and sensitivities to bacteriophages, pyocines, and antibiotics. 1) In broth type sm formed a pellicle which was not dispersed on shaking the tube. Electron microscopic examination showed that some of the cells of type sm were linked to one another by a string-like material. On the other hand, type la formed a homogeneous turbid culture in broth. It rarely formed a pellicle, which was easily dispersed on shaking the tube. Such string-like material as present in the cells of type sm was not observed in those of type la by electron microscopy. 2) There could not be found any difference between the two types, la and sm, in serological specificity on the thermostable O antigen in the agglutinin-absorption test. 3) Bacteriophage-sensitivity tests were performed on the dissociants derived from each of the five strains, using eight kinds of temperate phage preparations. As a result, remarkable difference were found in this sensitivity between the two types derived from each of three strains out of the five. 4) Pyocine-sensitivity tests were performed on the dissociants of types sm and la derived from each of the seven strains, using fourteen kinds of pyocine preparations. There was a difference in this sensitivity between the two types derived from each of three strains out of the seven. 5) Antibiotic-sensitivity tests were performed on the dissociants derived from each of the five strains. The results indicated no major difference between the dissociants of the given strains. 6) The frequency of appearance of the different type arising from each of the two types, sm and la, derived from each of the five strains was investigated. Three different conditions of cultivation were employed: nutrient broth at 37C overnight; nutrient broth under shaking at 37C for 20 hours; and nutrient agar slant, first at 37C overnight and then left at 4C for fifteen days. The frequency of appearance of the different type that had arisen from each of the two types was 0.1% or less in most cases and 10% or more in a few cases. From the results described above, it was concluded that the bacteriophage typing and the pyocine typing should be applied carefully to the characterization of Pseudomonas aeruginosa, since there were remarkable differences among the dissociants in sensitivity to phages and pyocines. The serological typing using thermostable O antigen is considered to be a most reliable method for the characterization of Pseudomonas aeruginosa, if it is performed with the cautions mentioned in the text, because there was found no qualitative differences in thermostable antigen between type sm and la.
The colicine type and drug-resistance pattern of R factor were investigated by using 514 strains of Shigella sonnei originated from 51 dysenteric patients in Gunma Prefecture over a 5-year period of 1958 to 1960, 1962, and 1966. The results obtained by Abbott and Shannon's method are summarized as follows. The strains isolated from 1958 to 1960 and in 1962 were divided into two colicine types, 6 and 12. Then 472 strains were isolated from 31 outbreaks in 1966 and divided into seven types: 6 (41.9%), 14 (32.3%), 12 (9.7%), 9A (6.5%), 4A (3.2%), 13A (3.2%), and 0 (3.2%), the last of which produced no colicine. This survey has disclosed that colicine type 14 has increased in frequency of isolation. It is well known that changes in colicine type are caused by superinfection of the col+ strain with other transmissible col factor. From these facts and from the epidemiological results, the increase in colicine type 14 may be accounted for by superinfection of col I to Shigella sonnei colicine type 6. The drug-resistance patterns of R factors of the Shigella strains isolated in 1966 were TC. CM. SM. SA in 81.8%, CM. SM. SA in 4.6%, and TC in 13.6%. It was found from these surveys that there was a close relationship between colicine type 14 and the quadruple (TC. CM. SM. SA) resistance pattern of R factors. In addition to these surveys, colicine typing was performed on 221 Escherichia coli strains by Abbott and Shannon's method. The results indicated that there were ten colicine types: 1A, 2, 3A, 4, 6, 7, 9, 11, 13, and 13A.
The virulence for mice was determined in the unique staphylococcal strains, the E46 and the E97, which are coagulase-negative but deoxyribonuclease-positive, and which had been described in the preceding part of the present series of papers. It was compared between these strains and two typical strains of each of Staphylococcus aureus and S. epidermidis, namely, the E111 and B40 strains for the formerand the E241 and B217 for the latter. The four strains were also mentioned in the same preceding paper. The results obtained are as follows. 1) The unique strains, especially the E46, exhibited a much higher lethal effect on mice by intraperitoneal inoculation than any other strain tested. 2) The unique strains, when inoculated subcutaneously were also proved to possess the same abscess-forming ability as the S. aureus strains. The S. epidermidis strains, however, had no such ability. 3) The lethal effect of the unique strains on mice by the intravenous route seemed to be rather weak. 4) No mice were killed by intraperitoneal administration of the culture filtrate of any unique strain or any other strain. 5) The distribution of intraperitoneally inoculated organisms in the blood, visceral organs, and ascites itself was parallel in grade with the virulence of the strain of those organisms. The unique strains, especially the E46, multiplied more vigorously than the E111 strain. In the case of the E241 strain, inoculated organisms were excluded rapidly, without manifesting any recognizable multiplication. 6) Histopathological examination revealed that the severity of lesions produced in various organs was also parallel with the virulence of the strain of the inoculated organisms. Thus, these unique strains would be available for various experiments on staphylococcal infection of mice, since they possess a high virulence and, in addition, a high sensitivity to many antibiotics, as described in the preceding paper.
Each two species of the lactic acid bacteria of intestinal origin, Streptococcus faecalis, Lactobacillus acidophilus, and Lactobacillus bifidus, were mixed and cultured in vitro to pursue the occurrence of any interaction between the two. The growth rate of Sc. faecalis was not affected by L. acidophilus, but that of L. acidophilus was considerably repressed, although the same maximum bacterial number was reached eventually in the mixed culture as in the culture of L. acidophilus alone. The growth rate of L. acidophilus was not affected by L. bifidus, but that of L. bifidus was repressed by the same dose as, or a smaller dose of inoculum than that of L. acidophilus. Compatible growth occurred only when the doses of simultaneous inocuration of L. bifidus and L. acidophilus were at the rate of 104:103. When L. bifidus and Sc. faecalis were cultured together, growth of Sc. faecalis was generally rich, but that of L. bifidus was poor. Compatible growth was observed only when L. bifidus and Sc. faecalis were inoculated at the rate of 106:101, or when Sc. faecalis was inoculated about 15 hours after inoculation of L. bifidus. It was, however, worthy to note that a medium where Escherichia coli had been pre-cultured was found to allow a better growth of the two species tested than a original medium even if the two species were cultured in the same dose of inoculum and incubated for the same period.
Deformity of feet, such as shortening of fingers and absorption of phalanges, was observed in some mice of the dd strain which had been inoculated subcutaneously with Mycobacterium leprae into the foot-pad. In order to clarify the causation of this deformity, some species of Mycobacterium were injected into the food-pad of mice. As a result, the injection of a mixture of M. fortuitum and adjuvant induced the deformity, which gave rise to abscess formation and absorption of phalangeal bones, but that of a suspension of M. fortuitum in saline solution caused no deformity in mice. That deformity, however, was different from that observed in the deformed feet of mice inoculated with M. leprae. This particular finding would be used for the differentiation of M. fortuitum and M. runyonii from any other species of Mycobacterium. No viable bacteria were seen in mice 6 weeks after the subcutaneous injection with a suspension of M. fortuitum in saline solution into the foot-pad, but killed bacteria were observed 2 years after the inoculation. Whether an organism was living or dead in the tissue of the foot-pad was determined by examining the form and length of the organism. The gathering and elongation of bacteria in living state could be observed in mice 20 weeks after the inoculation into the foot-pad. Bacterial counting is only one important method for the detection of M. leprae trasmitted into the foot-pad of the mouse, because it is impossible to culture M. leprae in any medium. Therefore, the method developed in the present investigation for the differentiation of living bacteria from dead ones will be useful for the experiment on inoculation of mice with M. leprae into the foot-pad.
In a series of their previous papers, the authors reported that 2-Mercaptoethanol (2ME)-sensitive antibody against Japanese encephalitis virus had been detected from pigs during the first two to four weeks of antibody response and that, thereafter, the antibody had become resistant to 2ME treatment. Further studies were carried out to characterize the swine antibodies obtained at various stages of antibody response by means of the gel filtration technique using Sephadex G-200. Most of the antibody of initial response was found to be contained in the 19S fraction and to be 2ME-sensitive. From serum samples of a pig which had previonsly been reported to have produced 2ME-sensitive antibody at the third oversummering, the antibody was detected in the 19S fraction. Possibility of reinfection by Japanese encephalitis virus in antibody-bearing pigs and human beings was suggested in relation to antibody response.