- Volume 21, Issue 1, 1986

The fungicidal activity of murine broncho-alveolar macrophages (BAM) against the yeast form of a virulent strain of Blastomyces dermatitidis was studied in the small-volume wells of a Terasaki plate. In a 4-h fungicidal assay, significant killing (16−33%) of the fungus by unstimulated BAM was demonstrated with BAM from normal mice and from mice rendered immune to lethal infection with B. dermatitidis. No significant differences between the activity of BAM from these two sources could be identified. Addition of 10% autologous normal or immune serum did not augment the macrophage fungicidal activity. Simultaneous experiments with peritoneal macrophages (PM) also gave reproducible killing of the yeast form in the wells of the Terasaki plate, but in the larger wells of microtitration plates, PM showed no significant fungicidal activity. On the other hand, BAM had similar fungicidal activity against B. dermatitidis in Terasaki and microtitration-plate wells. The modest fungicidal activity of BAM from immune mice against B. dermatitidis suggests that the resistance of immune mice to respiratory challenge is likely to be based on some augmentation of this first line of defence.

Lincomycin has a differential effect on exoprotein production by Vibrio cholerae. The production of some proteins, such as cholera toxin and deoxyribonuclease, is stimulated by low concentrations of the drug while production of other proteins, such as protease and alkaline phosphatase, is unaffected. Possible mechanisms of the lincomycin effect are discussed.

Six strains of Salmonella typhimurium (TML, W118, LT7, SL1027, M206 and Thax-1) of known virulence and ability to induce fluid secretion when inoculated into the rabbit ileum were examined for enterotoxin production. Enterotoxic activity, assayed in the rabbit ileal-loop test, was detected in polymyxin-B extracts from all strains (with the possible exception of Thax-1) cultured for 6 h in casamino acid-yeast extract medium. The extracts were inactive in tissue-culture assays with CHO, Y-1 adrenal and Vero cells, and in the infant mouse assay for enterotoxin. There was no correlation between enterotoxigenicity in vitro and the ability of whole organisms to induce fluid secretion in vivo. The significance of these results in relation to salmonellosis is discussed.

Haemophilus influenzae type b (HIB) and Escherichia coli J5 (J5) lipopolysaccharides (LPS) were examined to explore the basis of previously observed cross-protection. HIB-LPS and J5-LPS contained ketodeoxyoctonate, glucose, glucoheptose and glucosamine as common carbohydrate moieties, and laurate, myristate, β-hydroxymyristate and palmitate as common fatty acids, although in different ratios. J5-LPS was five times more lethal than HIB-LPS for chick embryos. Weak serological cross-reactivity was observed by haemagglutination and two-dimensional immunoelectrophoresis. No significant cross-reactivity was demonstrated by enzyme-linked immunosorbent or toxicity-neutralisation assays. The cross-reactivity observed between HIB-LPS and J5-LPS was probably due to common components in the core glycolipid.

Cross-reactivity in the delayed hypersensitivity response to mycobacteria of different Runyon groups was tested in Swiss white mice immunised with live mycobacteria. All the strains tested gave cross-reactions and, generally, slow growers gave stronger cross-reactions with other slow growers than with rapid growers and vice versa. Sonicates of cross-reacting mycobacteria were also tested for their ability to generate activated macrophages in mice immunised with Mycobacterium avium. All the mycobacterial sonicates generated activated macrophages but a sonicate of Salmonella typhi did not. The sonicate of M. tuberculosis H37Rv also generated activated macrophages, which indicates that there might be protective cross-reactions between M. tuberculosis and atypical mycobacteria.

The characteristics of seven strains of Streptobacillus moniliformis, including four isolates from a recent outbreak of Haverhill fever, are reported. Acid production from carbohydrates was uniform apart from variable reactions with mannose and salicin. Enzymatic reactions determined by the API ZYM system and fatty-acid profiles were generally consistent and may be of value in the rapid identification of S. moniliformis. Penicillin was the most active of the antibiotics tested in vitro, which supports its use as the drug of choice in the treatment of Haverhill fever.

The virulence of Bacteroides fragilis and B. vulgatus for mice was compared in a skin-infection model. These strains were also tested for pathogenic synergy in mixed infections with Escherichia coli. Strains of B. fragilis were generally more virulent than strains of B. vulgatus and, with one exception, the effect of Bacteroides strains in mixed infections merely reflected their inherent virulence.

Susceptibilities to β-lactam antibiotics and β-lactamase content of two groups of Bacteroides strains were compared. Type cultures produced low levels of β-lactamase and were susceptible to cefoxitin, latamoxef, imipenem and the combination of benzylpenicillin and clavulanic acid. Other Bacteroides strains that produced higher levels of β-lactamase were generally less susceptible to these antibiotics; this resistance was more closely related to enzyme type than to the amount of enzyme present.

The β-lactamases produced by the test strains fell into three broad groups on the basis of antibiotic degradation and inhibitor profiles: (i) those that inactivated benzylpenicillin, but not cefoxitin, latamoxef or imipenem, and were susceptible to inhibition by β-lactamase inhibitors; (ii) those that hydrolysed benzylpenicillin, cefoxitin and latamoxef, but not imipenem, and which were less susceptible to inhibition by β-lactamase inhibitors; (iii) an enzyme that inactivated all the antibiotics and was not inhibited by β-lactamase inhibitors.

A primary culture system for the cells of mouse renal-tubular epithelium was established and used to observe the adhesion of leptospires. Virulent strains of serovars copenhageni and ballum attached themselves to epithelial cells within 3 h of infection whereas an avirulent variant of serovar copenhageni did not adhere to epithelial cells at all within the experimental period of 24 h. The saprophytic Leptospira biflexa serovar patoc became attached non-specifically to inert glass surfaces as well as to the cells. The adhesion of leptospires to epithelial cells was not inhibited by homologous antibody.

Four pairs of M + SOR + and M − SOR − variants of group-A type-49 streptococci were compared as receptor strains in transduction of a streptomycin-resistance marker. The yield of transductants was 5−9-fold greater with the M − SOR − variants than with the corresponding M + SOR + variants. Treatment of M + SOR + variants of type-49 streptococci with trypsin enhanced the rate of transduction by 16−35-fold whereas trypsin treatment of corresponding M − SOR − variants resulted in minimal enhancement (5-fold or less). With trypsin treatment the numbers of transductants were approximately equal in pairs of M + SOR + and M − SOR − variants.

Enhanced transduction (10−26-fold) of streptomycin resistance was obtained by trypsin treatment of another seven M + SOR + type-49 strains, of diverse phage subtypes and from various geographical locations. A wide range of enhancement (5−46-fold) was found in eight of nine M + strains of group-A type-6 streptococci. With trypsin treatment, three of 10 transducible group-G strains showed enhanced transduction (10−13-fold) of a plasmid containing a determinant for erythromycin resistance.

Transductional enhancement is proteolytic in nature, being enhanced by trypsin, chymotrypsin, papain, pronase and streptococcal proteinase. Although interference with phage adsorption by surface proteins would appear to be the most obvious explanation for these findings, further studies are required to define more clearly the mechanism of trypsin enhancement.

Cells of group-B streptococci harvested in the late exponential phase of growth and suspended in starch-glucose phosphate-buffered saline extractor solution were observed to form and release pigment into solution. Filtrates of these solutions were analysed spectrophotometrically and two varieties of pigment were detected. Pigment, when freshly produced or in the presence of starch, had four absorption peaks at 520, 485, 455 and 435 nm. If albumin was substituted for starch in the extractor solution or if the starch-pigment complex was disrupted by treatment with amylase or by boiling, the four-peak pigment rapidly and irreversibly degraded to a second type with a single absorption band at 415 nm. The pigments formed by washed cell suspensions had absorption spectra identical to those produced by pigment formed during growth in Todd-Hewitt Broth. The formation and release of soluble pigment appeared to be an active metabolic process; a carrier molecule and an energy source were both required. Pigment yields were increased when the pH of the extractor solution was in the range 7.0−7.4 and when Mg2+, but not other divalent cations, was present. No differences in yield or type of pigment were observed when pigment was formed in anaerobic conditions. These findings support an earlier observation that group-B streptococcal pigment resembles a β carotenoid. There is some added support for the suggestion that haemolysin and pigment production by these organisms are closely linked characteristics.

A new and simple method of serotyping campylobacters has been developed which utilises co-agglutination to detect the presence of heat-stable antigens. Campylobacters are heated at 75°C for 30 min to destroy antigenic protein and allowed to react on a glass slide with staphylococci coated with antibody. Of 74 isolates, 67 gave the same result by co-agglutination and the previously described passive haemagglutination method. The co-agglutination technique may be used as a rapid screening test before serotyping by passive haemagglutination.

Mouse monoclonal antibodies raised by immunisation with a protective antigen extract from Pseudomonas aeruginosa serotype 1 varied in immunoglobulin isotype, in passive protective properties against infection by homologous P. aeruginosa serotype 1, and in cross-reactions in ELISA against antigen preparations from 15 other P. aeruginosa serotypes. All monoclonal antibodies with specificity in ELISA for the immunising antigen gave some degree of protection to mice against lethal infection by the homologous P. aeruginosa serotype. The IgG antibodies were more protective than the IgM antibodies.