- Volume 4, Issue 1, 1971

The possible role of EB virus in post-transfusion mononucleosis (PTM) was investigated serologically. Sera were obtained from patients before and after cardiopulmonary bypass for open-heart surgery, and tested for antibodie by immunofluorescence. No association of EB virus with cases of PTM was found; nor was there evidence of activation of infection, reinfection or super infection with EB virus resulting from the massive blood exchange.

It appears that cytomegalovirus is implicated aetiologically in most cases of PTM, while EB virus infection is rarely associated with this disease. There was no evidence of reciprocal cross-reactivity between EB virus and CMV antibodies.

Cultures of Staphylococcus aureus from 36 food-poisoning incidents in Great Britain during the period 1961–69 were examined for their ability to produce enterotoxins A, B, C and, in some tests, enterotoxin D. Enterotoxin was produced by means of a sac-culture technique and detected serologically by the slide gel double-diffusion method.

Enterotoxins A, B, C, or D were demonstrated from strains from 33 of 36 incidents (92 per cent.) and in some instances two enterotoxins were produced by one strain; enterotoxin A was the predominant type. Strains from 34 of 36 (94 per cent.) were lysed by phages of group III and in 13 of these incidents reactions were also obtained with phages of group I.

Of 36 cultures of Staph. aureus isolated from a range of routine food samples not associated with food-poisoning incidents, 13 produced enterotoxins A, B, or C.

Several strains of TRIC agent produced agglutinins for mouse and rat erythrocytes during growth in embryonated hens’ eggs. The agglutinins appeared irregularly and in a low percentage of inoculated eggs. A small inoculum dose was more likely to give a good yield, and a lethal inoculum failed to produce detectable amounts of haemagglutinin. The haemagglutinins were separable from the infectious particles and, with the exception of those associated with the T’ang strain, were unstable on storage. The agglutinin for rat erythrocytes was more often present in the allantoic fluid whilst the mouse cell agglutinin was more cell-associated. The T’ang strain also produced a haemolysin.

It has been suggested that the bactericidal antibody associated with immunity to acute primary Haemophilus influenzae infections is that produced against the capsular antigen of H. influenzae type b. In a previous study, however, the bactericidal and the type-b capsular antibodies were found to have different age-distributions. In the present studies the age-distributions of children with bactericidal antibody and of those with agglutinins against non-capsulated strains were found to be similar. Furthermore, it was possible to absorb type-b bactericidins from rabbit antisera with non-capsulated strains without removing the type-b capsular antibody. These findings, together with other evidence in the literature, suggest that the antigen responsible for the production of the bactericidal antibody is somatic and is possessed by non-capsulated strains, although attempts to produce type-b bactericidins in rabbits immunised with these non-capsulated strains were unsuccessful.

A highly toxic fraction causing dermonecrosis in guinea-pigs and a lethal effect in mice was obtained from Bordetella pertussis phase-1 organisms. Intravenous injection of this toxic fraction into mice produced a considerable reduction in spleen weight within 7 days. The histological findings indicate that B. pertussis causes an atrophy of the spleen. The term “lienotoxicity” is proposed to designate this toxic phenomenon. Whether dermonecrosis, lethality and lienotoxicity are caused by three different agents contained in the preparation studied has not been determined.

The survival of two colicine-sensitive strains of Escherichia coli when grown in mixed culture with each of three colicine-producing strains was investigated. The universal colicine indicator strain, E. coli B/TKM, was completely eliminated during mixed culture with each of the colicinogenic strains, whereas colicine-resistant mutants of it showed normal growth. The second colicine-sensitive strain, E. coli no. C600/21, was also eliminated by the colicine-B-producing strain, but not by either the colicine-E2- or E3-producing strains. The bacteria that were refractory to colicine E2 or E3 remained viable even in high concentrations of the colicine yet were unable to multiply; they regained full colicine sensitivity after growth in peptone water or when subcultured with a non-colicine producing strain of E. coli, but not when grown with a strain producing the same colicine. The refractory cells were non-colicinogenic and free colicine was present in the supernatant fluids of the mixed cultures. After treatment with trypsin to destroy the absorbed colicine the refractory cells regained their colicine sensitivity.

A naturally occurring neomycin-resistant strain of Staphylococcus aureus was found to donate its resistance, together with cross-resistance to kanamycin and paromomycin, to a variety of sensitive staphylococci. The transfer occurred spontaneously and at relatively high frequency both in broth cultures and experimentally on the skin surface of volunteers.

The mechanism of the transfer was transduction, which is believed to be mediated by an element composed of a defective prophage of serological group B and a determinant for neomycin resistance. The latter is probably located on a plasmid in the resistant cell, since strains harbouring the element tended to lose their resistance spontaneously, particularly at high temperatures. The effect of UV light on the transduction of the element was also characteristic of known plasmids.

The phage-typing patterns of staphylococci able to acquire the neomycin-resistance marker in vitro were limited but similar to those of naturally occurring resistant strains. Since neomycin resistance of the type found naturally could not be established in the laboratory, it is probable that the acquisition of neomycin resistance may have occurred in vivo by high-frequency transduction.

Sera were tested for capsular antibody and for bactericidal factor against Haemophilus influenzae type b. The age-distribution of the bactericidal factor corresponded to the known age-distribution of immunity to serious infection with H. influenzae type b, but that of the capsular antibody did not. This finding argues against the belief that the protective antibody to H. influenzae type-b infections is that produced against its capsular antigen.

Hormonal effects on the uptake of amino acids by Staphylococcus aureus have been studied in relation to the reported antibacterial action of the hormones. Progesterone at 40 µg per ml retarded the uptake of 14C-alanine and 14C-glutamic acid. Diethylstilboestrol, testosterone and oestradiol-17β exerted similar inhibitory effects, whereas 17α-hydroxyprogesterone did not. Maximum inhibition of alanine uptake occurred at a cellular density corresponding to 260 µg dry weight per ml, at a pH value between 5.7 and 7.0, and with diethylstilboestrol at a concentration of 20 µg per ml. The assimilation of alanine by Shigella flexneri and Salmonella paratyphi was not inhibited by progesterone. The data are correlated with inhibitory phenomena previously observed and indicate parameters for further research toward elucidation of the hormonal mechanism of inhibition of Staph. aureus.

Hormonal effects on substrate incorporation into various cellular fractions such as protein, nucleic acid and mucopeptide, have been investigated to elucidate the reported steroidal retardation of staphylococcal growth. Bacteria suspended in synthetic medium were exposed to 14C-compounds in the presence and absence of hormones. Progesterone at 40 μg per ml significantly altered the entrance of glutamic acid, lysine and alanine into all cellular fractions of Staphylococcus aureus. With progesterone and epiandrosterone, the critical concentrations required to reduce incorporation of 14C-alanine in staphylococci were 15 and 20 μg per ml respectively. Short-term incubation experiments with alanine revealed that the initial inhibition occurred in the entry of the 14C-label into the cold TCA fraction. In further studies it was found that progesterone significantly altered the entrance of eight substrates into cellular pools as indicated by studies with the cold TCA fractions. Thus, it appears as if the hormones directly influence the passage of nutrients into bacterial cells with subsequent effects on the incorporation of the nutrients into diverse cellular macromolecules. This may be related to the previously reported antimicrobial effects of hormones on staphylococci.

Sucrose density-gradient centrifugation was used for the study of serum IgM and IgG antibodies in rubella infection in early pregnancy. Seventeen women with proven rubella in the first trimester of pregnancy were tested and all were found to have an IgM antibody response to the virus. IgM antibody was present for 1 mth following the onset of illness but not later, and it was absent in women who did not have acute rubella. With one exception, IgM antibodies were not found in women who had recently given birth to an infant with congenital rubella.

The technique involved the collection of six fractions from the density gradient. The second fraction, which contained IgM but was free from IgG, was tested for rubella antibody by means of an HI test. Examination of this one fraction from a single serum collected within 1 mth of the onset of illness was sufficient to confirm or exclude a diagnosis of rubella.

Secondary cultures of human amnion cells developed characteristic morpho-logical changes when re-fed with media containing yeast extract. When cultures so treated were inoculated with vaccinia virus, the usual cytopathic effect failed to develop although isolated microplaques were occasionally observed after prolonged incubation. Some studies were made of the nature of the active substance and its mode of action.

Mycoplasma orale type 1 and M. hominis increased in number in the maintenance medium of cultures of L132 cells. The former mycoplasma grew slowly, but the latter grew rapidly and persisted in the cultures for more than a month. Neither mycoplasma grew in maintenance medium that had not been in contact with cells. In contrast, two T-mycoplasmas of human origin, one from the genital tract and the other from the oropharynx, grew rapidly in cultures of L132 cells and sometimes in maintenance medium from tubes without cells also, but persisted for 3–4 days only.

Even though there was growth of T-mycoplasmas in the maintenance medium of infected cell cultures, the cells contributed to the mycoplasma growth. Thus, serum-free maintenance medium alone did not support growth, but did so when used in cell cultures. Very small amounts of urea—probably derived from the cells—may have been responsible for the growth. The addition of 0.01–0.05 per cent. urea increased the number of viable T-mycoplasma organisms. In the presence or absence of additional urea, most organisms were found free in the maintenance medium overlying cell monolayers, but some were present in close association with the cells. Treatment of T-mycoplasma-infected cultures with streptomycin, which was lethal for extracellular T-mycoplasma organisms, followed by disruption of the cells, led to the release of viable organisms, indicating that some organisms were intracellular.

Minimal cytopathic changes were seen in cultures of L132, HeLa and Vero cells, particularly when 0.05 per cent. urea was added. Chronic infection could not be established in the cultures because the mycoplasmas usually died before the optimum time for cell subculture. The appearance of a T-mycoplasma inhibitor in the cultures possibly may have been partly responsible for this. The failure of T-mycoplasmas to persist in these cell cultures contrasts with their behaviour in the human host and in organ cultures.

The repeated isolation of a haemolytic species of Haemophilus requiring V factor and preferring increased CO2 tension for growth is described. Other characters helpful in establishing the identity of such strains are given and the name Haemophilus paraphrohaemolyticus is proposed.