- Volume 22, Issue 3, 1986

Glutaraldehyde-inactivated cells and cell-wall fractions of Candida albicans were studied for their capacity to induce or inhibit the in-vitro proliferation of human peripheral blood mononuclear cells (PBMC), as measured by 3H-thymidine incorporation. Both the intact cells (CA) and a phosphorylated gluco-mannanprotein complex of the cell wall (GMP), in μg doses, were strong inducers of PBMC proliferation, with a peak of activity at 6-9 days of culture and varying with the PBMC donor. A significant but much lower proliferation was observed on exposure of PBMC to a low-protein (<3% by weight) mannan component (M) of the cell wall. Both a hot-alkali extracted mannan-protein complex (M-alk), comparable to GMP in crude chemical composition, and an alkali-insoluble cell-wall glucan (GG) were inactive. None of the Candida fractions induced a lymphoproliferation of umbilical cord blood cells and all fractions, except GG, were equally effective in binding human anti-Candida antibodies as shown by a sensitive ELISA-inhibition assay. Moreover, a monoclonal antibody against the class II determinant of the HLA complex inhibited PBMC proliferation irrespective of the Candida antigen used. Taken together, the data shows that in inducing lymphoproliferation, Candida fractions act as specific antigens rather than as non-specific mitogens. Use of intact Candida cells and chemically-defined cell-wall components appears preferable to use of undefined antigenic mixtures as stimulators of PBMC proliferation.

The effect of CO2 on the growth of 31 strains of Candida albicans was studied in serum and in a defined medium containing urea, ammonium chloride, asparagine, glutamine or acetamide as the nitrogen source. CO2 10% enhanced the mycelial growth of all strains when the medium contained an appropriate constituent to mediate its effect. The effect of CO2 was most clearly demonstrated at 30°C when it induced a characteristic growth form consisting of a single swollen blastospore giving rise to a long, unbranched mycelial tube with few secondary blastospores; in atmospheric concentrations of CO2 only blastospore growth occurred. Growth in the blastospore form was more rapid in CO2 10% than in air. Bicarbonate ions had no effect on mycelium formation. The result suggest that the induction of germ-tubes and mycelial growth is essentially a physical phenomenon caused by the intracellular accumulation of CO2 in limited nutrient conditions, a view consistent with other reported laboratory and clinical findings.

Methicillin-resistant strains of Staphylococcus aureus isolated at the Royal Prince Alfred Hospital since 1965 were differentiated by phage-typing and by their lysogenic status. Most of these strains were isolated during two periods, 1965-72 and 1976-85. Nearly all of the strains isolated in the first period had one of four phagetyping patterns. Strains with each typing pattern carried two prophages; these eight phages were all different, as characterised by serological grouping and lytic spectrum. Lysogenisation of the non-lysogenic strain 1489 with each of these phages narrowed its phage-typing pattern; the typing pattern of the double lysogens was generally similar to and occasionally identical with that of the host strain that had yielded the pair of phages. In the second period, strains with one of five other phage-typing patterns predominated. Representatives of each of these carried the lysogenic phage C. The first methicillin-resistant strain carrying this phage had been isolated in 1974. The current methicillin-resistant S. aureus strains thus appear to form a distinct group that can be differentiated from those seen in earlier years.

Carbenicillin resistance in strains of Pseudomonas aeruginosa isolated in Britain is mediated more frequently by “intrinsic factors” than by β-lactamase production. Intrinsically carbenicillin-resistant isolates almost invariably were more resistant to azlocillin, cefoperazone, cefotaxime, ceftazidime, chloramphenicol, tetracycline and nalidixic acid than were carbenicillin-susceptible strains. This crossresistance to different classes of antimicrobials suggested an impermeability-based mechanism of resistance, perhaps involving the outer membrane. The structure and composition of the outer membrane of the pseudomonas cell also influences the Oserotype specificity and the susceptibility to many bacteriophages. We therefore examined these properties for possible relationships to antibiotic resistance. Carbenicillin-resistant (122) and -sensitive (127) P. aeruginosa strains from 24 hospitals were compared. Serotype O:1, O:3, O:6, O:10 and O:11 strains predominated in both groups. Non-typable and polyagglutinating strains were infrequent in both groups. With one possible exception, none of 18 bacteriophages showed a significant preference for carbenicillin-resistant or -sensitive strains. Variation between strains was observed in the electrophoretic profile of LPS and this could be related in part to serotype, but not to antibiotic resistance. Our results contrast with those of earlier small-scale studies which have claimed relationships between surface properties and antibiogram in P. aeruginosa, and suggest that interpretation of the minor changes in LPS sometimes observed in association with the development of antibiotic resistance in vitro requires caution.

The effect of Bacteroides asaccharolyticus on the opsonisation and phagocytosis of gram-positive and gram-negative bacteria by human polymorphonuclear leukocytes (PMNL) was investigated. Uptake of most isolates of staphylococci, streptococci and clostridia by PMNL after opsonisation with serum treated with B. asaccharolyticus was largely unimpaired. The same treatment of serum before opsonisation of isolates of Pseudomonas, Enterobacter, Klebsiella and Gardnerella resulted in the uptake by PMNL varying with individual isolates; a large reduction occurred with some and none with others. Treatment of serum with B. fragilis lipopolysaccharide before opsonisation of Proteus mirabilis produced a marked reduction in uptake, whereas treatment with B. fragilis capsular polysaccharide had little effect.

Several strains of Escherichia coli were markedly sensitised to killing at pH 2.5 or 3.5 when the ColV, I-K94 virulence plasmid was introduced into them. For strain 1829, the effect on acid sensitivity was due to the presence of plasmid in the previously resistant strain rather than to its introduction into an acid-sensitive variant already in the population. Acid sensitivity was also conferred by the ColV-K30 and ColB-K98 plasmids and the resistance plasmid R124-F2; other plasmids tested had no marked effect. Studies of ColV+ strains carrying mutant plasmids indicated that it was the presence of ColV-encoded transfer components that made ColV, I-K94+ strains acidsensitive. Organisms in the exponential phase of growth were more sensitive to acid than were those from stationary phase cultures and this difference was more marked for ColV, I-K94+ strains than for Col− ones. Moreover, ColV+ strains exposed to conditions of low pH for short periods subsequently grew less well than the Col− parent and appeared to be sensitised by the damage to the effects of H2O2. These results indicate that some ColV+ strains may be more sensitive to gastric acid and to phagocytic acidity than are Col− strains. ColV, I-K94+ strains grew as well as Col− ones in broth or urine at pH 4.5-6.0 which suggests that the presence of the plasmid would not be detrimental to bacterial growth in the urinary tract. The presence of transfer components in the outer membrane of ColV, I-K94+ bacteria may destabilise the lipopolysaccharide layer allowing increased penetration of hydrogen ions.

An indirect haemagglutination (IHA) test was performed with chick red blood cells (RBC) on sera from 26 confirmed cases of echinococcosis and 45 control sera. The results were compared with those obtained in an IHA test with sheep RBC on the same batches of sera; both tests were equally sensitive. The chick cells settled quickly and the results could be determined within 30-45 min. Heterophilic antigen was not a problem. This study also showed that chick cells stabilised by the doublealdehyde method, could be sensitised with the antigen and then stored at 4°C for up to 31 days before use in the IHA test without loss of sensitivity. The use of sensitised double-aldehyde stabilised (DAS) chick cells in IHA tests provides a rapid diagnostic test in echinococcosis.

A modification of the indirect haemagglutination test is described for the serodiagnosis of echinococcosis. In the modified test the Cowan I strain of Staphylococcus aureus, which contains protein A, was used to enhance haemagglutination of sensitised red cells. The test was performed in parallel with the indirect haemagglutination test on 31 sera from surgically-confirmed cases of hydatid disease and on 45 sera from healthy blood donors. Use of S. aureus protein A enhanced the sensitivity of the test and greatly increased the titres obtained with most of the sera. None of the sera from healthy blood donors exhibited a titre > 64, whereas all but two of the sera from cases of hydatid disease exhibited titres > 128. The immunoassay is simple, inexpensive and requires little technical skill. It has the potential for wide application in the serodiagnosis of echinococcosis.

Cytotoxic effects of bacteria found in dental plaque are usually attributed to lipopolysaccharides (LPS) or ill-defined toxins. Many bacteria implicated in periodontal disease produce surface exopolymers (capsules) recently shown to stimulate bone resorption. Capsular material and LPS extracted from Actinobacillus (Haemophilus) actinomycetemcomitans were purified and examined for their effects on cultures of human gingival fibroblasts. DNA and collagen synthesis were significantly inhibited by capsular material (0.1-50 μg/ml). LPS caused only modest inhibition of DNA synthesis at 10 and 50 μg/ml, and had no effect on collagen synthesis. Release of lactate dehydrogenase from fibroblasts was not increased by LPS nor by capsular material, showing that the inhibitory effects were not due to cell death. Capsular material, but not LPS, caused a pronounced increase in cell size; a doubling of the nuclear area occurred within 72 h exposure. These results indicate that the capsule of A. actinomycetemcomitans may play an active role in the tissue destruction characterising inflammatory periodontal disease.

Spleen cells from mice immunised with group-A type-M15 streptococci and boosted with purified IgG Fc-receptor (FcR) from this type were fused with Sp 2/0 mouse myeloma cells. The resulting hybridomas were screened by ELISA for antibody production. Two IgM-secreting cell lines were selected. The monoclonal antibodies and ascites fluids inhibited the binding of 125I-labelled human IgG and IgG Fc-fragments to group-A type-M15 streptococci. The monoclonal antibodies also displaced purified FcR towards the anode in electrophoresis. They opsonised group-A type M-15 streptococci for phagocytosis by human granulocytes in the presence of fresh human serum. It was concluded that FcR is important for group-A streptococcal virulence.

Some 10% of group-A streptococci have inhibitory activity against all nine strains (eight of them streptococci) in a set of indicators in an inhibitor-production typing (P-typing) scheme. This activity was associated with the concurrent synthesis of cell-associated proteinase by the streptococcal strain. Inhibitor production was prevented either by incubation of the test strain in conditions inimical to proeteinase production, e.g., at low temperature and alkaline pH, or by addition to the medium of substances, such as glucose, iodoacetic acid, lincomycin, Congo red or trypan blue, that had an anti-proteinase effect. Inhibitory activity was not detectable in liquid cultures, but freeze-thaw extracts of cultures of group-A streptococcus strain A1013 on Gibco Columbia Agar Base (Gibco Diagnostics, Madison, WI, USA) had some inhibitory activity. The inhibitor was concentrated and partially purified, and the active agent was shown to be a high-mol.-wt cationic protein which was bactericidal for various bacteria in the logarithmic growth phase, including the homologous producer strain.

Organ culture of rat trachea was used as an experimental model to examine the ability of lipo-oligosaccharide from Haemophilus influenzae to damage respiratory tract mucosal tissue. Lipo-oligosaccharide from two strains of H. influenzae produced a significant decrease in the ciliary activity of tracheal rings observed over a 3-5 day period. No loss of ciliary activity was observed with the lipid-free moiety of the lipooligosaccharide.

An IgA monoclonal antibody (MUM/F1-1/copenhageni) was produced from a mouse immunised with Leptospira interrogans serovar copenhageni. The antibody showed partial serogroup specificity by agglutination and by reaction in enzyme immunoassay, and opsonised homologous leptospires for phagocytosis by cultured mouse macrophages. Immunodiffusion and Western-blotting experiments indicated that MUM/F1-1/copenhageni reacted with a carbohydrate determinant in the leptospiral lipopolysaccharide. Daily administration of purified MUM/F1-1/copenhageni IgA before and after challenge with 2 x 108 virulent homologous leptospires passively protected newborn guinea pigs against lethal leptospirosis.