- Volume 35, Issue 6, 1991

Two monoclonal antibodies (MAbs), 3D9 with reported specificity for Candida albicans hyphae, and 3B7 with reported specificity for morphological forms of C. albicans found in vivo, were tested by indirect immunofluorescence with C. albicans cells that were grown in 12 different environments (four different culture media incubated at various temperatures) and whose cellular morphology was estimated in terms of morphology index (Mi). Both MAbs reacted strongly with cells with Mi> 3.0, i.e., with pseudohyphal and hyphal forms, but in Eagle’s medium at 26°C and in a modified Sabouraud’s broth medium at 30°C, some reactivity was also found with cells of lower Mi (i.e., yeast forms). Therefore, it was concluded that the hyphal phenotype and the epitopes reactive with the MAbs were co-expressed but that the epitopes could also be expressed independently of the hyphal phenotype. The results confirm the propensity of C. albicans for variation of its surface antigenic composition.

Fifteen siderophore producing urinary isolates of Escherichia coli were compared for aerobactin and enterochelin production in trypticase soy broth and pooled normal human urine. Significant increase in siderophore production (both phenolate and hydroxamate) was observed when organisms were grown in urine. Mouse kidney pathogenic potential of the strains grown in urine was compared with that of bacteria grown in trypticase soy broth in an ascending model of pyelonephritis in female Swiss Webster mice. Organisms grown in urine and instilled into a mouse bladder demonstrated markedly enhanced renal pathogenicity (p<0.01). Further information about the influence of urinary constituents on siderophore production could help in understanding the pathogenesis of pyelonephritis.

Entero-invasive Escherichia coli (EIEC) and shigellae were tested for contact-haemolysin (CH) with red blood cells (RBCs) of guinea-pig, rabbit, rat, mouse, monkey, man, sheep and chicken; all bacteria showed the best lysis with guinea-pig RBCs. The best culture medium for CH activity of shigellae was tryptic soy broth, and for EIEC it was casamino acid-yeast extract broth with 1 mM CaCl2. CH production by all species was best at the slightly alkaline pH which is optimal for growth; it was also dependent on the presence of a large (140-Mda) plasmid. Pre-treatment of bacteria with homologous antisera inhibited CH activity. Various treatments of bacterial cells and RBCs suggested that CH may be a protein molecule, and that a chitotriose-like moiety may serve as CH receptor. RBCs that were incubated with bacteria at 4°C, or with heat-killed bacteria at 37°C, were not lysed; also, isolated cell-surface components (lipopolysaccharide and outer-membrane protein) did not lyse RBCs. This suggests that metabolically active cells are required for CH activity. Production of CH by both EIEC and shigellae is consistent with a common mechanism for the virulence of these organisms.

Eubacterium yurii subsp. yurii is an anaerobic, gram-positive rod. On isolation E. yurii forms cellular arrangements resembling test-tube brushes (TTB). Although TTB decreased in size and number on repeated laboratory subculture in enriched media, media poor in available iron enhanced formation of these. Plasmids were not demonstrated, even after chloramphenicol enhancement. To characterise the nature and possible physiological roles of the structures of the TTB, they were examined by transmission electronmicroscopy (TEM) with thin-section, negative-staining, shadow-casting, freeze-etching and freeze-fracturing techniques, and by scanning electronmicroscopy (SEM). Previous studies by phase-contrast microscopy revealed an amorphous core, the size of which varied in direct proportion to the number of associated bacterial cells. Thin sections of the TTB showed a gram-positive cell wall with additional surface layers. Negative staining, shadow casting and freeze etching revealed a surface layer comprising subunits in tetragonal array (P4 symmetry). Shadow casting showed also that the outermost layer of the cells was composed of fibrillar structures closely associated with, but distinct from, the tetragonal layer. The fibrils extended from the cell surface in clumps or strands. The presence of these fibrils was confirmed by the freeze-fracture technique and SEM. Chemical analysis of the core material of the TTB showed it to be low in carbohydrate (0.06%) and protein (0.2%). Energy-dispersive X-ray spectrometry showed that the core was composed mostly of iron. SEM evidence suggests that the intertwining of the fibrils of several individual cells was responsible for the unique brush-like arrangements characteristic of E. yurii and may function to entrap or localise iron within the core, possibly as a mechanism of iron sequestration.

Tightly spiralled bacteria („Gastrospirillum suis”) were seen in the pyloric mucosa of the stomach of 13 (10.8%) of 120 pigs that appeared clinically healthy at slaughter and in the fundic mucosa of three (5.0%) out of 60 pigs. The spiral organism could not be cultured from any pig. Chronic gastritis was observed in the pyloric mucosa of 53 (44.2%) of 120 pigs and in the fundic mucosa of 7 (11.7%) of 60 pigs. The 13 pigs with spiral bacteria in the pyloric region comprised one animal (7.7%) with normal pyloric mucosa, two (15.4%) with „borderline gastritis”, and 10 (76.9%) with chronic gastritis–in one instance accompanied by signs of activity (numerous polymorphonuclear cells). The three pigs with spiral bacteria in the fundic mucosa comprised two animals with a normal fundic region and one with „borderline gastritis”. The presence of the spiral bacterium was significantly associated with pyloric gastritis (p = 0.013) and with numbers of lymphoid follicles (p = 0.014).

Experiments to measure the invasiveness of seven strains of Salmonella typhimurium for HEp-2 cells showed that high inocula (100 bacteria/HEp-2 cell), as used by most workers to synchronise events and to increase the number of bacteria which invade, resulted in recovery of significantly less than 1% of the original inoculum after treatment with gentamicin to kill extracellular bacteria. Also, the cell culture medium became acidic, and microscopic examination of Giemsa-stained monolayers immediately following gentamicin treatment revealed high concentrations of bacteria associated with the cells. Moreover, with bacterium-cell interaction beyond 2 h, many HEp-2 cells became rounded, especially with virulent strains W118 and TML. Thus, the biological significance of the quantitative data was uncertain. The fall in pH and the rounding of HEp-2 cells were prevented by the use of a low (1:1) bacterium: cell ratio; but the recovery of bacteria after treatment with gentamicin was still lower than expected by microscopic examination. After treatment of cells with Triton X-100 to release bacteria, many remained bound to residual cell nuclei. Additional treatment with a rubber policeman, and vigorous pipetting to disperse aggregates of bacteria and cell debris, increased the recovery to c. 10% of the initial inoculum after interaction for 2 h, and 30–80% after 4 h, depending on the strain and experimental conditions. The pattern of invasiveness, but not the absolute count, was highly reproducible on different days and in different hands. However, after interaction exceeding 2 h, the distribution of bacteria was uneven, many cells having no associated organisms, others showing microcolonies. Either this variation does not happen with high inocula, or it is occluded by the high concentration of bacteria associated with the monolayer. Uptake of bacteria depends on the batch of fetal calf serum used in the cell culture medium. The bacterial phenotype is important: bacteria in early or mid log phase entered cells more efficiently, and bacteria grown in Hartley Digest Broth were significantly better at invading HEp-2 cells than those grown in Myosate Broth. Centrifuge-assisted inoculation of HEp-2 cells with bacteria may grossly distort the results, particularly with some avirulent strains.

In studies which have involved determination of fractional inhibitory concentrations, synergy has been described between the 4-quinolones, which inhibit the A subunit of DNA gyrase, and either coumermycin or novobiocin, which inhibit the B subunit of the same enzyme. In this study, fixed concentrations of ciprofloxacin or ofloxacin were combined with varying concentrations of coumermycin or novobiocin and vice versa in nutrient broth. The bactericidal activities of the different mixtures against either Staphylococcus aureus E3T or S. warneri were determined and found to be less than those of equivalent concentrations of either 4-quinolone alone. The observation that gyrase B subunit inhibitors antagonised the bactericidal activity of 4-quinolones is in accordance with the report previously made by others that ciprofloxacin combined with coumermycin was less effective than ciprofloxacin alone in treating staphylococcal endocarditis in rats. Our results indicate that both inhibitory and bactericidal activity should be taken into account when assessing possible interactions in vivo between 4-quinolones and other antimicrobial agents.

We investigated whether production of histamine by bacteria isolated from sputum of patients with infective lung diseases could be attributed to the presence of histidine decarboxylase (HD). Twenty gram-positive and 20 gram-negative organisms were studied for their ability to decarboxylate 14C-histidine in vitro over the pH range 4.5-7.5. Of the bacteria investigated, lysates from the gram-negative species Haemophilus influenzae, H. parainfluenzae, Moraxella (Branhamella) catarrhalis and Pseudomonas aeruginosa liberated 14CO2 and histamine from 14C-histidine in the presence of the cofactor pyridoxal phosphate. In contrast, results obtained in the absence of cofactor were similar to those of negative (lysate-free) controls suggesting that the HD enzymes of these species resembled those previously described in other gram-negative bacteria. No HD activity was detected over this pH range in lysates from gram-positive species. This finding correlated with earlier observations that these gram-positive organisms did not produce histamine in vitro.

A collection of strains representing all the currently recognised species of oral or viridans streptococci (Streptococcus sanguis, S. parasanguis, S. gordonii, S. oralis, S. mitis, S. salivarius, S. vestibularis, S. anginosus, S. constellatus, S. intermedius, S. mutans and S. sobrinus) were tested for the production of a range of glycosidase activities with 4-methylumbelliferyl-linked fluorogenic substrates, and for reactions in a range of conventional fermentation and hydrolytic tests. The resulting biochemical scheme, consisting of 14 tests, enabled the differentiation of all these species and distinguished three biotypes within S. sanguis. The scheme reported here represents an improvement over currently available schemes for the identification of viridans streptococci.