- Volume 41, Issue 2, 1965

By using growth and division-inhibition at 16 hr as indices of activity, eight penicillins, differing in the side chain, were tested against an Erwinia species, and compared with benzylpenicillin. DL-α-Aminobenzylpenicillin, DL-3-chloro-α-aminobenzylpenicillin, D-α-aminobenzylpenicillin, and methicillin were more toxic for growth than was benzylpenicillin. 6-Aminopenicillanic acid and cloxacillin were about equal to benzylpenicillin in toxicity; triphenylmethylpenicillin and 2-ethoxy-1-naphthylpenicillin were much less toxic. All the compounds inhibited division of the bacteria but, at 50% of normal growth, methicillin, cloxacillin and 2-ethoxy-1-naphthylpenicillin resulted in significantly longer organisms than the others. Pantoyl lactone, when present in the medium from the time of inoculation, in all cases decreased the length of the organisms, increased growth (with a highly toxic concentration of penicillin), and decreased the accumulation of keto acids and ultraviolet-absorbing materials in the medium. It was concluded that the side chain of these penicillins is not essential for the inhibition of growth and division of this Erwinia sp.; quantitative differences in activity associated with the side chain appear to result from the influence of the side chain on factors such as penetration, or strength of binding at the active site.

A simple method of detecting arabinose by paper chromatography in the cell-walls of aerobic actinomycetes is described. Mycobacteria and Nocardia species are rich in arabinose while saprophytic species of Streptomyces are deficient. Pathogenic Streptomyces species tend to fall between the two extremes but Streptomyces somaliensis is apparently nearly devoid of arabinose.

Media frequently used for the detection of urease activity were found to be unsuitable for fluorescent pseudomonads since the presence of free ammonia was found to suppress the formation of urease in growing cultures. Incubation of cultures for 40 hr at 25° in a low concentration Casitone + yeast-extract + glucose medium, followed by the additionof urea to 0.2% (w/v), resulted in the development of an alkaline shift in the medium after further incubation for a few more hours at 37°. This method gave good results in the detection of urease activity.

Tube sensitivity tests show that, of twelve penicillins, ampicillin, benzylpenicillin and 6-aminopenicillanic acid were the most active when tested against medium-sized inocula (106 organisms) of twelve strains of penicillinase-producing Klebsiella (11 Klebsiella aerogenes and 1 K. ozaenae), which had been isolated from clinical material and were resistant to both ampicillin and tetracycline. An inoculum size effect was consistently noted when ampicillin and benzylpenicillin were tested against different inocula; the effect was significantly greater with ampicillin. The following parameters were measured for the strains, using both benzylpenicillin and ampicillin: magnitude of the inoculum size effect; inherent sensitivity (sensitivity of a small inoculum); rate of penicillin destruction; ability of penicillins to pass bacterial permeability barriers. From considerations of the relationships between these values, it has been concluded that penicillinase is primarily responsible for the observed resistance of only two of the twelve strains; although penicillinase and the lack of ability of penicillins to obtain free access into the bacterial cells add to the over-all penicillin resistance of the other ten strains, the primary reason for their penicillin resistance is neither their possession of penicillinase nor their permeability barrier: such resistance presumably reflects an innate lack of sensitivity of the cell wall synthesizing complex to inhibition by penicillins.

γ-Glutamyl transfer activity was found to be widely distributed in different bacterial species. The γ-glutamyl transfer from glutathione to water and acceptors other than water was studied with cell-free preparations of Proteus morganii. In the absence of added acceptor, the γ-glutamyl residue was predominantly transferred to water; however, some transfer to the substrate, resulting in the formation of γ-glutamylglutathione, was detected. In the presence of acceptors (amino acids or peptides) all the γ-glutamyl residue was transferred to the added acceptor. The different reactionproducts were isolated and identified. Kinetics and properties of the γ-glutamyl transfer reaction were studied.

Antigenic variation was studied in a strain of Trypanosoma brucei transmitted by Glossina morsitans and G. palpalis. In goats and rabbits infected by tsetse flies, the antigenic character of the strain did not change until the 7th day of infection; thereafter new antigens developed at 2-to 3- day intervals until the infected animal died. The antigens of the T. brucei strain developed in a similar sequence in the early stages of infections induced by different tsetse flies; in later stages of the infections, the sequences in which antigens developed varied, but many of those produced in different hosts were similar. A common antigen, provisionally called the basic strain antigen, occurred in all substrains of the strain isolated during the first 7 days of infection from animals infected by different tsetse flies. This basic strain antigen was relatively stable and, when present in try-panosomes ingested by tsetse flies, it persisted throughout the period required for cyclical development and for the remainder of the life of the infective fly. It also tended to displace variant antigens when trypanosomes with such antigens multiplied in environments free from antibody. Tsetse flies which ingested trypanosomes with variant antigens transmitted trypanosomes with either the basic strain antigen only or with a mixture of the ingested variant antigen and the basic strain antigen. The basic strain antigen also developed at an early stage of infection when nonimmune animals were infected with variants of the strain transmitted by syringe. These findings are discussed in relation to the serological classification of brucei subgroup trypanosomes and the immunization of animals against trypanosomiasis.

Deficiency of meso-inositol (i-inositol, myo-inositol) caused decreased viability of Saccharomyces carlsbergensis ATCC 9080 and altered the internal morphology of the cells as seen in preparations stained with Nile Blue A, especially the nuclear membrane. Metabolic defects resulted which led to lipid accumulation in the organism and to increased concentrations of acetoin, acetaldehyde and glycerol in the culture medium. Hypotheses about the metabolic role of inositol are examined.

The membrane of Halobacterium halobium was isolated after rupturing the organisms osmotically in 0.02 M-MgCl2. Selected properties of this membrane were compared with those of membranes isolated from organisms which had been ruptured mechanically in 5 M-NaCl. The two types of preparation were indistinguishable from one another in the electron microscope, but some differences were noted between the present series of preparations and those described earlier. These differences, of which the occasional retention of the characteristic surface pattern of this organism was notable, were attributed to a slight modification of fixation conditions used in the present work. There were no major chemical differences between the two types of preparation but some minor differences in H+ titration and carbohydrate analyses possibly reflected slight structural differences between the two types of preparation. The osmotic method described here is much quicker than the mechanical one and, unlike the mechanical one, the time involved is largely independent of the quantity of organisms being used.

The best natural habitat for bacteriophages is probably a semi-solid medium containing actively dividing host bacteria. Such conditions are provided for Bacillus and Acetobacter species in rotting grass and apples, respectively. The Bacillus phages found included one with a large head and a contractile tail, and also a so-called killer particle, which had a 350 Å head and a long contractile tail. This particle had the property of killing but not multiplying within a sensitive cell. A new morphological type of virulent Bacillus phage was also isolated; its head was oblong and the tail consisted of a short needle and a plate. The one Acetobacter phage found resembled coliphage T3 but was of particular interest because of the prominence of the head capsomeres and the three-pronged tail.

Cell walls isolated from six species of Neisseria contained 17 amino acids, 6.3--8.3% mucopeptide, and 11.4--30.0% lipid. Veillonella parvula cell walls were similar, differing principally in the presence of 24.5% mucopeptide. The mucopeptide of all seven Gram-negative cocci contained glucosamine, muramic acid, glutamic acid, alanine, glycine and DL-diaminopimelic acid as principal components; the mucopeptide from V. parvula cell walls also contained an unidentified ninhydrin-reacting component. The marked similarity of the cell walls of the Gram-negative cocci and those of Gram-negative bacilli indicates a closer relationship between these two groups than between the Gram-negative and Gram-positive cocci.

The immobilization antigens of Paramecium aurelia were located in the electron microscope by the use of antibody labelled with electron-dense ferritin. On treatment of fixed whole paramecia with ferritin-labelled antibody, followed by sectioning, ferritin granules were seen on the pellicle and cilia of homologous organisms only. By counterstaining the sections with an electron-dense stain (potassium permanganate + uranyl acetate) the absorbed globulin of the antibody was revealed as a thick fuzz on pellicle and cilia. Antigen was present over the entire surface of the organism with the exception of the gullet area. Transformation from one antigenic type to another, by change of temperature, showed the ‘new’ antigen to appear initially on the pellicle and subsequently on cilia. The ferritin labelling, with counterstaining, proved to be very sensitive; ferritin granules and globulin fuzz denoting ‘new’ antigen appearance were detected at isolated sites on the pellicle and cilia after only 1 hr of growth at the higher temperature. The number of these sites then increased, until the granules and fuzz covered the entire surface of first the pellicle and subsequently cilia. Attempts to study the internal antigens with ferritin-labelled antibody were not successful.

The classification of strains similar to Mycoplasma strain PG 27 of ‘Campo’ as Mycoplasma hominis Type 2 should be withdrawn. These strains have now been identified as M. arthritidis.

Progressive alteration of the incubation temperature of Aerobacter aerogenes cultures, growing in a chemostat at a fixed dilution rate, caused a progressive change in bacterial concentration and in bacterial RNA, carbohydrate and protein contents; the DNA content did not vary significantly. The change in bacterial RNA concentration was associated with a variation in bacterial ribosome content, similar to that found in organisms grown at a fixed temperature but different dilution rates. Changes in bacterial yield also resulted from alterations in the pH value of the culture, but corresponding changes in bacterial RNA, carbohydrate and protein contents were small.

Young non-germinating sporangia of Phytophthora erythroseptica contain nuclei and cytoplasmic organelles, similar in appearance to those in other fungi, and a conspicuous central and other vacuoles. The sporangial wall consists of an outer homogeneous layer and an inner vesicular layer, prominent in the apical region. Peripheral cytoplasmic vacuoles are frequent, particularly where the vesicular layer is well developed. In old sporangia, which lose the ability to germinate indirectly (i.e. to produce zoospores), there is no vesicular layer and there is a change in the nature of the storage material of the vacuoles. When young sporangia are stimulated to indirect germination by low-temperature treatment the central storage vacuole disappears and there is a marked elongation of mitochondrial cristae, consistent with an increased rate of respiration. The sporangial wall becomes three-layered with the development of an inner homogeneous layer which separates the plasma membrane from an increasingly prominent vesicular layer. In the region of the papilla the vesicular layer shows a striking increase in thickness; it is suggested that this layer and the inner homogeneous layer play an important part in zoospore discharge.

Absorption and centrifugation studies established that the haemagglutinin specifically associated with suckling mouse tissue infected with Coxsackie A7 virus is particulate but distinct from the infectious particle. The haemagglutinin resembles other enteroviral haemagglutinins in being inactivated by p-chloromercuribenzoic acid. This property, together with other physical and chemical properties, suggests that the haemagglutinin is a protein. There is a serological relationship between the haemagglutinin and other non-haemagglutinating, serologically reactive substances present in tissues infected with Coxsackie A7 virus.