- Volume 15, Issue 2, 1982

Strains of thermophilic campylobacters of human origin were examined for bacteriocine activity and for susceptibility to R-type pyocines of Pseudomonas aeruginosa. None of 50 strains inhibited the growth of any other strain, but 13 of 80 strains (16%) were sensitive to R-type pyocines. Absorption of one of the partially purified pyocine preparations with a sensitive strain resulted in the removal of pyocine activity and a decrease in viable count of the organism by 85%.

Culture filtrates of all eight strains of Bacteroides gingivalis and all five strains of B. asaccharolyticus were toxic for Vero cells. Cytotoxicity was in general greater with material from cultures of B. gingivalis than from B. asaccharolyticus but none of the culture filtrates from eight strains of B. melaninogenicus showed activity in this test. The toxic material was released during prolonged incubation and more detailed study of preparations from one strain indicated that it had a molecular weight of less than 3500 and was heat stable.

CBA/N mice, which could not produce antibodies against lipopoly-saccharide (LPS) from either Escherichia coli or Leptospira interrogans serovar pomona, produced levels of agglutinating antibodies against leptospires similar to those produced by immunologically normal CBA mice. CBA/N mice were thus resistant to acute leptospiral infection and CBA/N immune serum passively protected immunosuppressed mice from infection. The results suggest that antibodies against LPS are not important in protection against experimental leptospiral infection in mice.

The thiosulphate: cyanide sulphurtransferase (rhodanese) test of Vandenbergh, Bawdon and Berk (1979) has been simplified and 2469 strains from a wide variety of sources representing different biochemical, serological or phage-pattern entities were tested. The percentages of rhodanese-producing strains were: Escherichia coli 98%, Shigella flexneri serovars 1–5%, X and Y 0%, other shigellae 73–100%, Yersinia spp. 0%, Salmonella subgenera I–IV 0%, Citrobacter freundi 16%, Klebsiella 37%, Enterobacter 4%, Hafnia alvei 61%, Proteus spp. 0%, Pseudomonas spp. 98–100%. Rhodanese production by S. flexneri serovar 6 supports the view that this group of bacteria should be removed from S. flexneri and placed in another species of Shigella.

The in-vitro bactericidal effect of serum for Klebsiella rhinoscleromatis was tested. Experiments with C2-deficient and hypogammaglobulinaemic human sera suggested that killing depended on activation of the classical complement pathway, although the alternative pathway probably amplified the effect. Serum from a patient with active rhinoscleroma, and another cured of the disease, showed normal killing.

During pneumococcal infection of mice, nucleic-acid products, including deoxynucleotides, may be released into the serum from cellular disintegration in at least three organs, the lungs, spleen and liver. The serum, after sterilisation to remove contaminating pneumococci, stimulated multiplication of virulent but not avirulent pneumococci in vitro. It also stimulated growth of virulent pneumococci in serum from uninfected animals and could be replaced, at least in part, by certain nucleic-acid degradation products at concentrations found in infected serum. The effects of the serum were lost after dialysis or dilution.

Antiserum specific for salmonella O7 antigen was raised by immunisation of rabbits with an artificial conjugate consisting of oligosaccharide and bovine serum albumin (Os-BSA). The oligosaccharide was a pentasaccharide isolated after cleavage of the O antigen polysaccharide chain of Salmonella thompson (O antigen 6, 7) with endo-glycanase from bacteriophage 14. The usefulness of the S. thompson Os-BSA antiserum for rapid and accurate identification of isolates of Salmonella of serogroup C1 (O6, 7) was shown by indirect immunofluorescence tests in which 77 strains of Salmonella of serogroup C1 were correctly identified from among 848 intestinal strains investigated. The finding that three strains of Escherichia coli and most strains of Candida were also positive in immunofluorescence tests with this antiserum is readily explained by the known structural similarities among the antigenic determinants of E. coli, Candida and Salmonella of serogroup C1. The specificity of the antiserum for the O7 antigen determinant was further demonstrated in enzyme-linked immunosorbent assay tests and in co-agglutination tests with staphylococci sensitised with S. thompson Os-BSA antiserum.

Material from 18 lungs positive for Legionella pneumophila and 21 strains of legionellae grown on bacteriological media or in the yolk sac of fertile hen’s eggs were examined by direct negative stain and immunoferritin electronmicroscopy. The 18 lung samples and all preparations of cultivated organisms showed the presence of bacteria with the typical morphology of L. pneumophila. By immunoferritin electronmicroscopy, with specific antisera, it was possible to serogroup the organisms. The immunoferritin method in reverse made it possible to detect and titrate antibody in sera from patients. Direct and immunoferritin electronmicroscopy were as sensitive as immunofluores-cent antibody tests for detecting the antigens and antibodies of Legionnaires’ disease. Additional advantages of the ultrastructural technique are discussed.

The succession of bacterial populations in the large bowel of seven breast-fed and seven formula-fed infants was examined during the first year of life. The composition of the intestinal microflora varied according to the infant’s diet. During the first week of life breast-fed and formula-fed infants were colonised by enterobacteria and enterococci followed by bifidobacteria, Bacteroides spp., clostridia and anaerobic streptococci. From week 4 until solid foods were given, breast-fed babies had a simple flora consisting of bifidobacteria and relatively few enterobacteria and enterococci. Formula-fed babies during the corresponding period were more often colonised by other anaerobes in addition to bifidobacteria and had higher counts of facultatively anaerobic bacteria. The introduction of solid food to the breast-fed infants caused a major disturbance in the microbial ecology of the large bowel as counts of enterobacteria and enterococci rose sharply and colonisation by Bacteroides spp., clostridia and anaerobic streptococci occurred. This was not observed when formula-fed infants began to take solids; instead, counts of facultative anaerobes remained high while colonisation by anaerobes other than bifidobacteria continued. At 12 months, the anaerobic bacterial populations of the large bowel of breast-fed and formula-fed infants were beginning to resemble those of adults in number and composition and there was a corresponding decrease in the number of facultative anaerobes. These changes are discussed in relation to changes in susceptibility to gastro-intestinal infection.

Filtrates of Treponema hyodysenteriae grown in rabbit serum broth supplemented with sodium ribonucleate (Na-RNA) 1.0% contained a haemolysin for sheep, rabbit, and pig erythrocytes. Haemolysin was not detected in the absence of Na-RNA. Haemolytic titres were highest when viable counts were maximal at the end of the logarithmic growth phase. Haemolysin could not be demonstrated in the spirochaete-free supernates of either centrifuged washings from blood-agar cultures, or centrifuged frozen-and-thawed cultures in semi-solid blood agar. In contrast, washed spirochaetes lysed sheep erythrocytes suspended in phosphate-buffered saline (PBS) or PBS-cysteine, provided that bovine serum albumin (BSA) fraction V was present. Haemolysin was produced aerobically as well as anaerobically. Ultrasonically disrupted spirochaetes did not produce haemolysin.

After incubation for 30 min at 37°C, haemolysin was present in supernate or filtrate from centrifuged suspensions of resting cells of T. hyodysenteriae in PBS containing glucose or maltose, cysteine hydrochloride, Mg2+, and Na-RNA or ribonucleic acid (RNA)-core. By analogy with its function as a non-specific carrier molecule for the oxygen-stable streptococcal haemolysin streptolysin S, ribonucleic acid can be regarded as a carrier substance for the treponemal haemolysin. RNA-core was a more efficient carrier than Na-RNA; BSA fraction V and Tween 80 were less efficient than Na-RNA. Haemolysin was not produced in the absence of a carrier. Haemolysin production occurred at temperatures up to 42°C, but was reduced at 18°C and below. The haemolysin was inactivated slowly at 37°C and 42°C, and more rapidly at 60°C. Titres also decreased slightly after 18 h at 4°C.

Haemolysin was produced in only small amounts by resting cells of a weakly haemolytic porcine-intestinal spirochaete, and only during a restricted period in the logarithmic phase. A prozone was observed when this haemolysin was titrated.

The demonstration that the treponemal haemolysin can be produced from resting cells will facilitate its purification and thus expedite studies on its interaction with other types of mammalian cell.

Effector responses of cell-mediated immunity were studied in mice immediately and 3 months after immunisation with different kinds of salmonella vaccine. Whereas immunisation with live Salmonella enteritidis generated delayed hypersensitivity and activated macrophages, immunisation with formalised vaccine generated neither. Although activated macrophages had declined considerably by 3 months after immunisation, the delayed-hypersensitivity response had persisted. It was possible to generate activated macrophages in these mice by stimulation with sonicate antigens of the immunising strain or Salmonella paratyphi C. Delayed-hypersensitivity responses were elicited in mice, immunised with live S. enteritidis, by sonicate antigens of the homologous strain, S. gallinarum or S. paratyphi C.

EDTA-released outer-membrane antigen complexes were prepared from 172 laboratory and reference strains and 43 fresh clinical or faecal isolates representing 20 species or subspecies of Bacteroides, together with 13 species of other genera. These antigens were titrated against antisera to whole, live cells of 21 species or subspecies of Bacteroidaceae in an indirect enzyme-linked immunosorbent assay (ELISA). The presence of species-specific antigens was investigated and cross reactions between species were noted. Results showed that a high proportion of the species possess species-specific antigens with little significant cross reactivity.

We believe that the ELISA system described here detects antigens that represent the whole cell surface of bacteroides organisms. We have exploited the sensitivity of the system and its quantitative potential to define approaches for those wishing to use serological approaches for either the identification of Bacteroides species or for the titration of serum antibodies in patients with a possible bacteroides infection.

A two-tier system for biotyping Escherichia coli gave a fine and reliable differentiation of strains and is capable of future extension by the addition of new types and new tests. Strains were allocated to a primary biotype (1-16) by their reactions in four primary tests in culture media containing raffinose, sorbose, ornithine or dulcitol. Subtypes were distinguished within the primary biotypes by reactions in six secondary tests for rhamnose fermentation, lysine decarboxylation, aesculin hydrolysis, motility, type-1 fimbriation and prototrophy. Full biotypes were designated by letters indicating subtype reactions appended to primary type numbers.

A series of 599 strains (1242 cultures) of E. coli from diverse sources was classified into 16 primary biotypes and into 213 full biotypes. The biotype characters of a strain were generally stable during its spread in the natural environment and in non-selective media used for storage.