- Volume 4, Issue 2, 1971

We detected subtle differences between the phagocytic activity of cells from pyridoxine-deficient and normal guinea-pigs by means of a suitable combination of parameters at two different cell-to-bacterium ratios. This difference was modest in amount, but statistically highly significant. The best parameter for discrimination was the average number of ingested bacteria per cell and the best indicator cell was the polymorphonuclear leucocyte.

Increasing the load of bacteria increased the difference in phagocytic activity between pyridoxine-deficient and control animals. These results could be achieved only by increasing the accuracy of estimates of the cell-to-bacterium ratio ten-fold above those obtained by conventional counting methods, and by eliminating the variability due to conducting experiments on different days with different bacterial suspensions.

The name Bacteroides corrodens has been applied to certain Gram-negative facultatively anaerobic organisms that are oxidase-positive, catalase-negative, indole-negative, nitrate-reducing, lysine decarboxylase-positive, non-fermentative, urease-negative, gelatinase-negative, casein hydrolysis-negative, and non-motile. During 2 yr, we have encountered no strict anaerobes with similar characteristics, but anaerobic biotypes may exist. The facultative strains require haemin for aerobic growth from small inocula, and may appear to be anaerobic if the haemin concentration in the medium is less than 5 μg per ml. Variants not requiring haemin occur. Growth is favoured by 0.005 per cent, cystine. The organisms are antigenically related, but strains may vary in the proportions of their different antigens. The G+C content is 57–58 per cent.

Four anaerobic organisms, tolerant of up to 1 per cent, oxygen and having certain superficial similarities to the facultative strains, were investigated. The organisms were oxidase-positive (with tetramethy1-p-phenylenediamine), catalase-negative, non-fermentative, urease-positive, gelatinase-positive (Frazier method), and able to hydrolyse casein. The G+C content was 28.0–29.7 per cent. In electron micrographs, the spreading strains showed multiple polar processes, but no flagella.

Agar-pitting (corroding) colonies are a striking feature of the facultative and the anaerobic organisms, but both may give non-pitting variants. The taxonomic status of the anaerobes requires further investigation.

The name Bacteroides corrodens has probably been applied by different workers to organisms that are genotypically widely dissimilar.

Several extracts prepared by the action of surface active agents on live cells of the rough Br. abortus strain 45/20 were found to protect mice and guinea-pigs against challenge with virulent Br. abortus. Incorporation in oily emulsions made little difference to the degree of immunity elicited.

An extract prepared by treatment of living cells of strain 45/20 with sodium dodecy1 sulphate followed by ethanol precipitation induced substantial immunity in cattle, despite the fact that no adjuvant was used. The extract was devoid of the smooth O somatic antigen and probably free from lipopoly-saccharide. The high protein and carbohydrate content suggested that the immunogen could be a glycoprotein.

Attempts were made to induce a carrier state in Swiss mice by intranasal inoculation of Staphylococcus aureus. A high mortality rate (90 per cent.) was observed when organisms were instilled intranasally, Aerosol infection also produced a high mortality (50 per cent.) and a rather low over-all carrier rate (30 per cent). When the organism was directly implanted with mild trauma (multiple puncture) on the nasal mucosa the mortality rate was reduced to 20 per cent. and nearly half of the initial animal population became nasal carriers of Staph. aureus. A significant number (54.8 per cent.) of 685 mice treated by the multiple puncture method developed a delayed type of hypersensitivity to the test staphylococcus and the sensitisation effect was most evident among the carrier animals.

Dissemination of Staphylococcus aureus from the site of carriage into the bloodstream and its localisation at a traumatised portion of a bone were studied in four categories of mice, viz., carrier hypersensitive, carrier nonhypersensitive, non-carrier hypersensitive, and non-carrier non-hypersensitive. The provocations used were a pyrogenic stimulus, produced by intraperitoneal injection of 0.1 ml of modified Haffkine plague vaccine, and controlled trauma to the right hind leg, either alone or in combination. It was observed that transient bacteriaemia occurred during periods of induced fever or as a sequel to trauma. It is of interest that 27 per cent. of the hypersensitive carrier mice as opposed to 2 per cent. of the non-hypersensitive carriers showed positive blood culture. Among the non-carrier mice, positive blood culture was obtained from 2.5 per cent. of the hypersensitive animals only. Significantly, localisation of Staph. aureus at the injured bone was detectable in hypersensitive mice only.

Antisera were prepared against the type-1 fimbriae of three strains of Salmonella paratyphi B and against the type-2 fimbriae of two strains of S. paratyphi B and one strain of S. pullorum. The anti-type-1 and anti-type-2 sera agglutinated fimbriate-phase bacteria possessing either type-1 or type-2 fimbriae. Neither antiserum agglutinated non-fimbriate-phase bacteria of the same strains. Mirror absorption tests were performed and in each case the fimbrial agglutinin content of the serum of either type was absorbed by bacteria with fimbriae of the other type. The results showed that the type-1 and type-2 fimbriae of S. paratyphi B contain the same fimbrial antigens. It is suggested that the type-2 fimbriae of S. paratyphi B are mutational CRM forms of the type-1 fimbriae, because they are antigenically similar to type-1 fimbriae though they lack the “ functional” adhesive properties of the latter type. A mutational origin of type-2 from type-1 bacteria is suggested by the observation that some strains of S. paratyphi B with type-2 fimbriae gave rise to mutants with type-1 fimbriae both spontaneously and after treatment with mutagens. The absence of a distinctive antigen corresponding to the haemagglutinin in the type-1 fimbriae suggests that the haemagglutinin occupies only a small proportion of the surface of the fimbriae, possibly only on their tips.

The type-2 fimbriae of S. pullorum cross-reacted in agglutination and agglutinin-absorption tests with both the type-1 and the type-2 fimbriae of S. paratyphi B. No type-1 fimbriae have been found in any strain of S. pullorum, and it is thought that the type-2 strains have arisen by mutation from a parental S. pullorurn-like organism with type-1 fimbriae that is now extinct.

Synergism has been demonstrated between selected pairs of β-lactam antibiotics against four representative strains of β-lactamase-producing Gramnegative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella aerogenes and an R-factor-bearing E. coli strain). A pour-plate double-diffusion technique with subinhibitory concentrations of antibiotics was used. Synergism has been proven by demonstrating a bactericidal effect, and by the isobologram technique. Seven out of 14 highly resistant strains of Gram-negative bacteria proved to be more sensitive to certain combinations of β-lactam antibiotics than to any of these antibiotics alone, tested at 1 mg per ml (a concentration attainable in the urine during therapy). Some aspects of the possible clinical use of combined therapy with two β-lactam antibiotics are discussed.

An indirect fluorescent antibody staining technique was adapted to study the population of potentially pathogenic E. coli serotypes relative to the total coliform population in faeces from calves. Preliminary experiments were carried out with pig faeces in which the haemolytic property of a pathogenic serotype was utilised to facilitate comparison of the results with those obtained from plate counts.

In a natural outbreak of colibacillosis in calves, diarrhoea in all except one case was associated with an increase in the proportion of pathogenic serotypes to at least 50 per cent. of the total coliform population. In some other cases, a similar increase occurred in the absence of clinical disease. The significance of these results is discussed in relation to the practical value of the method as a diagnostic procedure and as a means of studying the influence of environmental predisposing factors on the E. coli population of the gastro-intestinal tract.

A chloroform-soluble, extracellular product of Pseudomonas aeruginosa was obtained from sloppy-agar cultures. Pyrogallol 1-monomethyl ether, 1-methoxyphenazine, 1-hydroxyphenazine and pyocyanin were chemically synthesised and also tested for an inhibitory effect on mitochondrial respiration. It was found that the Ps. aeruginosa substance and 1-hydroxyphenazine inhibited the uptake of oxygen by mouse liver mitochondria utilising sodium succinate.

In a Ps. aeruginosa infection this substance would be released into the bloodstream, accumulate in the liver and cause cytolysis, thus contributing to the complex mechanism of Ps. aeruginosa pathogenicity.

Since 1-hydroxyphenazine derived from Pseudomonas aeruginosa or chemically synthesised inhibited the uptake of oxygen by mouse liver mitochondria utilising sodium succinate, attempts were made to determine the site of activity in the electron transport chain. It was found that inhibition occurred at a site corresponding to ubiquinone (coenzyme Q) or Co Q-cytochrome b; the 1-hydroxyphenazine might act as an “ electron shunt” by accepting electrons and transporting them to a “ dead end ”.

The difficulties encountered in the surface growth of Clostridium oedematiens (CI.novyi) of types B and D were investigated. Best results were obtained when nutrient agar was enriched with human blood (33 per cent.) and supplemented with (i) iron filings sprinkled on the surface of a seeded plate, or (ii) a development of Moore’s cysteine-dithiothreitol system incorporated in the medium. The growth-enhancing effects of these two systems were not identical; different degrees of enhancement were obtained with the two systems when test cultures of different ages were used.

The results of quantitative studies with these culture procedures indicate that, although spores of CI. oedematiens of types A, B, C and D may be significantly viable particles involved in the growth of inocula in pour plates seeded from old cultures in fluid media, large proportions of the surface viable counts obtained from young cultures of type-D strains are regularly derived from vegetative cells. Viable counts obtained with young cultures of type D, the most demanding strains, on solid media greatly exceeded the maximum estimates for the spore content of the inocula used. There is clear evidence that a very large proportion of type-D spores does not proceed to germination or successful outgrowth under the test conditions.

In the course of this study, amendments to the recommended anaerobic jar procedure were developed and a standardised method is described.

The MICs of fusidic acid and two sulphonamides for nine strains of Nocardia asteroides, two strains of N. caviae and one strain of N. blackwellii were studied by means of a plate-dilution technique. The median value for the MIC of fusidic acid, 2.5 fig per ml, compared very favourably with those of the sulphonamides, and fusidic acid may well have a part to play in the treatment of nocardiosis.

We would like to acknowledge the generous supply of fusidic acid by Leo Laboratories Limited, sulphadiazine by May & Baker Limited and sulphamethoxazole by the Wellcome Foundation. We are also very grateful to those mentioned in the text who supplied us with strains of Nocardia. Finally we wish to thank Dr J. C. J. Ives for helpful criticisms and advice.

The in-vitro sensitivity of 11 strains of Flavobacterium meningosepticum to 20 antimicrobial agents was studied. They were resistant to 14 antibiotics at present in clinical use, as well as to nalidixic acid. On the other hand, they were sensitive to erythromycin, novobiocin and rifampicin. Sensitivity was also found to sulphamethoxazole and trimethoprim and to a combination of these two drugs, which acted in synergy.