- Volume 8, Issue 2, 1975

Heated saline extracts of 89 strains, and (1) supernates of phenol-water extracts (L1 fractions), (2) purified lipopolysaccharide, (3) trichloracetic-acid (TCA) extracts, and (4) sodium-hydroxide extracts of 23 strains representing all Pseudomonas aeruginosa O antigens were subjected to electrophoresis. Precipitation lines obtained with homologous and heterologous antisera were evaluated by electrodensitometric measurement. The characteristics of the immunoelectrophoretic groups established were as follows. Group I: two lines running at different rates towards the anode; three subgroups on the basis of the behaviour of alkali-treated antigens. Group II: triple line at the starting well, alkali sensitive. Group III: triple line at the starting well, alkali resistant; two subgroups according to reactivity or non-reactivity of L1 fractions. Group IV: triple line on the cathode side, alkali resistant, L1 fraction non-reactive. Group V: single line on the anode side, alkali sensitive, L1 fraction and TCA extract non-reactive. O antigens identified by agglutination corresponded closely with the immunoelectrophoretic pattern: strains with identical O antigens or sharing major somatic components fell, with one exception, into the same immunoelectrophoretic group.

A glycoprotein fraction (fraction VII) suitable for use as a substrate in assays of microbial neuraminidase was prepared from pooled human plasma. It is pasteurised during preparation to eliminate the risk of transmission of serum hepatitis. This results in polymerisation of some of the αl-acid glyco-protein, but fraction VII is shown to be an excellent substrate for the neura-minidase of Clostridium welchii (C. perfringens). A sensitive assay procedure is described. A number of factors may interfere with the measurement of NANA released by the action of microbial neuraminidase and procedures are described for evaluation of this problem. Fraction VII is easy to prepare, cheap and available in standard form in large amounts (inquiries should be addressed to J. K. S.); it is recommended for routine use as a convenient substrate for neuraminidase assays.

The production of neuraminidase by a classical strain of Clostridium welchii (C. perfringens) type A was studied. Good yields were produced in 5% Proteose Peptone-water medium (PPW5); the enzyme was essentially extracellular but some further neuraminidase could be released by ultrasonic disintegration of the cells. This also released N-acyl neuraminic acid-aldolase (NAN-aldolase) and the degree to which this interferes with the assay for neuraminidase was evaluated.

Forty-one British reference food-poisoning strains of C. welchii type A were examined for extracellular neuraminidase production in PPW5. Twelve of 17 strains that produce so-called heat-sensitive spores were neuraminidase positive whereas 20 of 24 strains that are non-haemolytic and produce very heat-resistant spores were neuraminidase negative. Variation was found in the ability to produce neuraminidase among strains of a single Hobbs’ serotype; four Hobbs’ type-13 strains produced neuraminidase but a fifth did not. Disruption of the cells of a Hobbs’ type-2 strain that did not produce any extra-cellular neuraminidase released NAN-aldolase but there was no evidence of cell-associated neuraminidase. British food-poisoning strains of C. welchii type A thus include some that are clearly neuraminidase positive and some that still cannot be shown to produce neuraminidase. There is no correlation between lack of neuraminidase production and the ability to cause food poisoning, although the majority of non-haemolytic heat-resistant strains do not produce neuraminidase. It remains possible that neuraminidase may play a part in C. welchii gas gangrene; it is suggested that the ability to define neuraminidase-negative strains may now be of value in investigating this possibility.

Staphylococcus aureus strains were treated with ethylmethane sulphonate with the object of obtaining mutants that differed from the wild type in one of the properties that might contribute to virulence.

Mutants with isolated loss or deficient production of α-haemolysin, coagulase, leucocidin and staphylokinase were obtained. Changes in more than one property were often observed. The loss of δ-lysin production was always coupled with an appreciable reduction in P-V leucocidin production. With that exception, systematised combined loss of a restricted number of staphylococcal characteristics was not observed. Mutants that had lost all characteristics of S. aureus were phage-resistant. Isolated loss of staphylokinase production and deficient P-V leucocidin production were found to be due to the independent loss of two prophages.

The contribution of a number of extracellular products of Staphylococcus aureus to the virulence of the organism for mice was studied by comparing a wild-type strain with various mutants derived from it in three virulence tests: (1) subcutaneous and (2) intravenous challenge in normal mice, and (3) subcutaneous challenge in mice after total body X-irradiation.

Mutants with lower production or non-production of coagulase, staphylokinase and leucocidin were just as virulent for mice as the wild type in all three tests. Unlike the wild type, mutants with low production or non-production of α-lysin never gave necrosis after subcutaneous injection in normal mice. One mutant with loss of δ-lysin and unaltered α-lysin production gave necrosis only when injected in high doses. Dermonecrosis seems to be caused by a combination of α- and δ-lysin.

Intravenous injection of each of the two types of mutant in normal mice gave a lower mortality rate than that obtained with the wild type. Mutants with deficient α-lysin production, but not δ-lysin-deficient mutants, multiplied more slowly in the kidneys than the wild type under these conditions. α-Lysin appears to have a growth-enhancing effect for the organism in vivo, but δ-lysin does not.

Differences in virulence between the wild type and mutants could not be demonstrated in irradiated mice.

In tests with broth-culture products of Clostridium oedematiens, none of 15 type-B strains showed β-toxin lecithinase activities exceeding 20 egg-units per ml, whereas 12 of 13 type-D strains consistently produced much greater amounts of the lecithinase.

The types also differed in their susceptibility to lysis by mitomycin C (MC). Of 13 type-D strains tested, 12 were sensitive to MC at a concentration of 1 or 2 μg per ml, whilst 14 of 15 type-B strains were insensitive. Phage-like particles were observed in the MC-lysates of some type-D strains. No type-specific differences in the production of indole or the fermentation of maltose were demonstrated.

The taxonomic relationships between Clostridium bifermentans and C. sordellii were reinvestigated by numerical taxonomy, studies of DNA-DNA homology and DNA duplex thermal stability, and by analysis of cell-wall sugar components. Although the results indicate that both species may be grouped into one geno-species, C. sordellii strains could be differentiated from C. bifermentans strains on the basis of a few phenetic criteria that include the inability to ferment mannose and sorbitol, the absence of mannose in the cell wall, the production of urease, the absence of arginine deaminase activity, and susceptibility to inhibition of growth by mannose.

The degree of correspondence between urinary and faecal Escherichia coli O-groups has been assessed in non-pregnant women with symptomatic urinary-tract infection or asymptomatic bacteriuria.

In 20 of 26 patients with symptomatic urinary tract infection E. coli of the same O-group as that of the urinary infecting strain was also present in the patient’s faecal flora whereas such a correspondence was found in the faecal flora of only five of 25 patients with asymptomatic bacteriuria. This finding indicates that the majority of episodes of symptomatic urinary tract infection in non-pregnant women are not preceded by a significant period of asymptomatic bacteriuria.

E. coli O6 showed correspondence between urinary and faecal isolates more frequently than did other O-groups, but it had a relatively low prevalence in the faecal flora of patients with urinary-tract infection caused by E. coli of other O-groups. This finding lends support to previous suggestions that E. coli O6 may be especially pathogenic for the urinary tract.

Chromatographically separated antigens of Mycobacterium leprae were tested for their ability to elicit skin reactions in guinea-pigs sensitised with homologous and heterologous mycobacteria. Of the three antigen-positive fractions obtained, one showed specific activity and the other two cross-reactivity, as indicated by studies of hypersensitivity and passive cutaneous anaphylasix.

The fraction exhibiting specificity contained only one antigen, which was protein in nature, whereas the other two fractions contained more than one antigen and possessed both protein and polysaccharide constituents. Because the single-antigen-containing fraction showed both positive skin and PCA reactivity, the suggestion is made that this fraction may contain either an antigen with two determinants or may contain two antigens that are not easily distinguishable by immunodiffusion methods.

Perforated plastic chambers implanted subcutaneously in guinea-pigs and rabbits became encapsulated and filled with sterile transudate. When these chambers in guinea-pigs were inoculated with various strains of Neisseria gonorrhoeae, persistent infections were achieved without the use of anti-inflammation agents and in the presence of a substantial predominantly polymorphonuclear inflammatory response. Two strains with small colonies similar to types 1 and 2, and one strain with large colonies similar to type 4 of Kellog et al., (1963 and 1968), showed differences in infectivity comparable with those that might be expected in man, and passage through guinea-pig chambers increased this infectivity. Rabbit chambers could not be infected without the use of an anti-inflammation drug (betamethasone), and differences in infectivity between strains were not as clear cut. The growth of N. gonorrhoeae in chambers in the guinea-pig provides a convenient model system for studying some aspects of the pathogenicity of this organism.

A strain of Staphylococcus aureus (no. FAR4) has been isolated at intervals, for 32 months, from the sputum of a patient with cystic fibrosis of the lung. Changes in the properties of isolates of this strain over the first 18 months have been reported previously (Lacey et al., 1973 and 1974). During the last 14 months (May 1973 to July 1974), further evolution has occurred to produce a total of 31 distinct phenotypes. Recent changes are as follows.

1. The ability of isolates to produce penicillinase in vitro was closely correlated with flucloxacillin therapy. Inactivation of flucloxacillin by penicillinase was demonstrated by diffusion testing (but not MIC determination) in vitro and may have occurred to a significant extent in vivo.

2. Lincomycin-resistant mutants slowly disappeared from the sputum after the termination of clindamycin therapy.

3. All of the recent isolates were resistant to erythromycin, possibly because of the linkage of the genes coding for erythromycin resistance with those coding for the production of δ-haemolysin; δ-haemolysin may be an important “virulence factor”.

The morphology of 10 strains of T-mycoplasma was studied in wet preparations of viable cells by darkfield, phase-contrast and interference microscopy, and in fixed preparations by various techniques of electron microscopy. Mycoplasma-like artefacts in the horse-serum component of the medium were eliminated by filtration. All 10 strains were similar. Individual cells were spherical, 0.25-1.0 μm in size, with a bounding trilaminar membrane, 10 nm thick and containing 7.5-1 2.5-nm particles, and a layer of pilus-like projections, 5–8 nm long, on the outer surface. A possible capsular matrix was observed only by the pseudoreplica technique. The cells contained 12–15-nm ribosomes, nuclear fibroids 7.5–9 nm wide, and vacuoles. During replication, the cell elongated slightly and the ribsomes migrated to the ends of the cell leaving a ribosome-free area into which the bounding membrane invaginated to form a bud. The bud eventually separated by completion of the process of invagination; a cross-septum did not form. Usually only a single bud developed but sometimes two appeared simultaneously.

Shanson’s medium, a MacConkey-type agar containing added inhibitors, was compared with deoxycholate-citrate agar for the examination of faeces. The isolation rate of salmonellae on the two media was similar, but Shanson’s medium was the more selective; this meant that the amount of work required to produce a result was reduced.

The induction of L-forms of Haemophilus influenzae by penicillin, amoxycillin and glycine has been studied in vitro on a nutrient-agar medium. The minimal inducing concentrations of the antibiotics were generally the same as their minimal inhibitory concentrations, but the addition of a sub-inducing concentration of glycine lowered the minimal inducing concentration of penicillin.

Preliminary observations have shown that L-forms are induced by penicillin or amoxycillin on a medium in which mucoid sputum forms the sole source of nutrients, and that they remain viable for at least 48 h in the absence of added osmotic stabiliser. The minimal inducing concentration on “sputum agar” is within the range of concentrations measured in sputum from patients receiving amoxycillin therapy.

The implications of these observations in relation to bactericidal therapy of haemophilus infections of the respiratory tract are discussed.

Optimal conditions for the NBT-reduction test have been sought. Increasing heparin concentrations up to 100 units per ml and a delay in performance of the test, especially when blood specimens are kept at room temperature, resulted in higher values for the NBT index, which then sometimes exceeded the upper limit of normal in healthy people and in uninfected patients. The effect of pH, composition of the buffer, and dye concentration was also investigated. Phosphate-buffered saline pH 7.2 containing 0.1% NBT dye, without glucose, gave the most reliable results.

In endotoxin-stimulated NBT tests, the following procedure is recommended: incubation of 0.1 ml whole blood with lyophilised endotoxin 20 μg per ml, for 15 min. in a 37°C water bath, followed by the standard test with a 0.2% NBT solution. By this technique, the leucocyte reaction to various types of lipopolysaccharides was of the same order of magnitude. Drug therapy having an effect on blood components lowered this reaction, whatever the source of endotoxin used as stimulant.

The importance of NBT-reduction tests is discussed. Standard conditions of test performance are strictly requisite if comparable results are to be obtained and if data not corresponding with the apparent clinical and other laboratory findings are to be evaluated correctly. The stimulated NBT test, performed in parallel with the standard test, is useful in the interpretation of abnormal results and in the detection of factors with a temporary or permanent effect on the phagocytic activity of PMN leucocytes.