- Volume 20, Issue 3, 1985

Existing models of Candida albicans infection are semi-quantitative and do not allow continuous observations to be made on individual animals. We have used the inflammatory response in the footpad as an indirect measure of the number of yeast cells in a localised lesion. C. albicans infection of the footpad has been used in series of experiments in which changes in yeast-cell numbers in the local lesion have been compared with the degree of footpad oedema. Studies in animals treated with cyclophosphamide or amphotericin have confirmed that paw oedema parallels yeast-cell numbers in the local lesion. This quantitative approach will be helpful in the study of localised infection with C. albicans and other fungi and in the evaluation of antifungal agents.

Oral candidal infection and the carriage of oral bacteria in rats has been studied in animals on a high carbohydrate diet and treated with tetracycline. Candidal infection was not significantly enhanced by carbohydrate alone but was promoted by tetracycline; carbohydrate plus tetracycline was no more effective than tetracycline alone. Carriage of lactobacilli was enhanced by carbohydrate but streptococcal carriage was depressed; there was no effect on the number of rats carrying enterobacteria. Administration of tetracycline reduced the carriage of all three groups of bacteria but the isolation rate for enterobacteria increased towards the end of the experiment, becoming nearly the same as at the start. The prevalence of C. albicans did not vary with these changes in bacterial populations.

Mice were given, intraperitoneally, inocula of a cell-wall preparation and fractions thereof from Fonsecaea pedrosoi, F. compactum, Cladosporium carrioni and Phialophora verrucosum. Large doses of cell-wall preparation, with or without trypsin treatment, produced a pronounced loss of body weight, a granulomatous reaction and, sometimes, death. After extraction of the cell wall preparation with IN NaOH, three fractions were obtained: an alkali-insoluble fraction 1; an alkali-soluble acid-insoluble fraction 2; and an alkali- and acid-soluble fraction 3. Intravenous administration showed that only fraction 1 induced a granulomatous reaction and death in mice.

A disk-diffusion method for identification of yeasts was developed that depended on their different but distinct susceptibilities to the following chemicals: janus green, ethidium bromide, 2, 3, 5-triphenyltetrazolium chloride, brilliant green, cycloheximide and rhodamine 6G. For 594 of 623 routinely isolated yeasts, the disk-diffusion and the commercial API 20C auxanogram tests gave the same identification, an agreement of 95·3%. Only 8 of 1052 isolates from clinical specimens were not identified by the disk-diffusion method. The method is simple, inexpensive and technically straightforward and for most isolates gives an identification in 24 h.

The encapsulated strain M of Staphylococcus aureus and the encapsulated strain 76 of S. simulans survived incubation with bovine neutrophils in media containing heated bovine serum 0·5% v/v or milk whey 10% v/v. With two unencapsulated strains of S. aureus, M12 and M60, <5% of the original inoculum survived. When the concentrations of bovine serum and milk whey were increased to 2% and 50% respectively, survival of the encapsulated strains was similar to that of the unencapsulated strains. The ability of skimmed milk 50% v/v to opsonise strain M was found in milk from 95% of 62 cows.

The individual values for percentage intracellular survival of strains of S. aureus phagocytosed by bovine neutrophils in the presence of bovine serum 2% v/v or milk whey 50% v/v were 0·48, 0·30 and 0·24 for strains M60, M and M12 respectively. Intracellular survival of strain M60 was significantly greater than that of strains M12 and M. The encapsulated coagulase-negative strain 76 (S. simulans) was not susceptible to lysis by lysostaphin, and it was not possible to destroy extracellular staphylococci to measure intracellular survival. It is concluded that the capsule of S. aureus strain M does not contribute to intracellular survival.

Three hundred and seventy-one penicillin-resistant Staphylococcus aureus strains of phage type 95 isolated in the years 1977–1983 were investigated. They had characteristic resistance patterns to cadmium (Cd), arsenate (As) and mercury (Hg). Most were susceptible to all three heavy metals; 25 strains were resistant to As only and one to Cd and As. The susceptible strains had a uniform medium level of penicillinase production, whereas the As-resistant strains produced large amounts of penicillinase. In most of the strains, penicillin resistance was located on a very unstable penicillinase plasmid.

The combinaton of rare properties found in Danish type-95 strains seems to point to the spread of one or two clones. Co-reactions with other phage groups or complexes and results of lysogenisation experiments suggest that the Danish type-95 strains are derived from strains of the 52, 52A, 80, 81 complex.

Escherichia coli strains isolated from patients with urinary infections were tested for their ability to adhere to human uro-epithelial cells. In any single experiment, the numbers of bacteria adhering to individual uro-epithelial cells showed great variations; some cells had hundreds of bacteria adhering to them whereas other cells had few or none. This non-Normal distribution of bacterial attachment must be taken into account when carrying out statistical analyses of the results. The wide discrepancies reported in the literature regarding bacterial adhesion to uro-epithelial cells must, in part, be related to the type of statistical analysis used. In many cases, a Normal rather than a non-Normal distribution has been assumed. We found that even when all variables were kept constant, the experiment was still not reproducible. Therefore the technique shows a high degree of both inter- and intra-experimental error. Adhesion depended on such factors as the type of fimbriae produced by the bacteria, differing viability of uro-epithelial cells and varying pH of the medium used for a particular experiment. It is concluded that the results of in-vitro experiments demonstrating adhesion of E. coli to uro-epithelial cells are difficult to relate to bacterial adhesion in vivo but better results could be obtained if more attention were paid to standardisation of methods and their analysis.

Strains of Escherichia coli producing type-1 fimbriae, associated with mannose-sensitive haemagglutinin (MSHA), or three antigenically different kinds of ‘MRE’ fimbriae, associated with mannose-resistant and eluting haemagglutinins (MREHAs), adhered poorly to HEp2 epithelial cells in an in-vitro adhesion model previously used to demonstrate the importance of motility and type-1 fimbriae for the attachment of strains of Salmonella typhimurium to HEp2 cells. Strains of E. coli producing narrow-spectrum MREHA, agglutinating human erythrocytes only of 14 red-cell species tested, adhered well to HEp2 cells, particularly so when bacteria produced MSHA (and type-1 fimbriae) along with the narrow-spectrum ‘man-only’ MREHA. These findings are discussed with regard to recent observations suggesting that narrow-spectrum ‘man-only’ MREHA in E. coli may be associated with fine, fibrillar appendages 2-nm wide.

The pathogenic potential of black-pigmented Bacteroides strains was studied in an animal model in which the effect on bone tissue could be determined. Bacteria suspended in agar were injected subcutaneously in the left hind paw of a mouse. After 3–5 days, B. gingivalis strain HG 66 had caused a massive infiltration with polymorphonuclear cells, destruction of the periosteum on the metatarsals and bone resorption by osteoclasts. After 7 days only a few osteoclasts remained and reactive bone formation was observed. In a comparative study with strains of several black-pigmented Bacteroides species, differences in bone resorbing potential were seen. B. gingivalis strains caused severe inflammation which resulted in bone resorption. Strains of B. asaccharolyticus, B. endodontalis, B. intermedius, B. melaninogenicus and B. loeschei caused less inflammation and less bone resorption. Killed bacteria or agar alone caused a relatively mild inflammation and no bone resorption.

Enterotoxigenic strains of Clostridium perfringens have recently been implicated in some cases of antibiotic-associated diarrhoea. We present here the results of an epidemiological study of this disease. Five cases of diarrhoea caused by C. perfringens serotype 41 occurred during a 9-week period, and then during a 6-week period there were three cases due to serotype 27 and two due to serotype 24; in all but one case two geriatric wards were involved. In total there were 16 cases in 22 months. All cases were identified by the detection of C. perfringens enterotoxin in the faeces. The mean number of C. perfringens in these cases was 108·8 cfu/g of faeces. Of 37 patients who had negative test results for C. perfringens enterotoxin, 18 had positive cultures for C. perfringens, with mean faecal counts of 105.3 cfu/g, and nine of these patients had diarrhoea. Thirteen different serotypes were isolated from these 18 patients, including type 41 from seven patients and type 27 from one. Hand carriage of the offending serotype was demonstrated in three of four infected patients, none of four controls and two of 14 ward staff. C. perfringens of serotypes causing disease was isolated from 59% of environmental areas where there was active disease, 27% of areas where there had been disease which had since resolved and 9% of areas where there was no history of disease. The findings imply that cross infection may occur.

The results were compared of submitting simple swabs, swabs in Stuart’s Transport Medium (STM) and swabs in Robertson’s cooked-meat broth (RCMB), from 100 potentially or definitely infected sites in patients undergoing general surgery. Significantly more positive bacterial cultures were obtained from swabs sent in RCMB (65), than from swabs sent either in STM (39) or as simple swabs (32). The isolation of potentially significant organisms from only the RCMB series could influence clinical management. The conventional reluctance of bacteriologists to accept evidence obtained from RCMB cultures seeded directly in the ward or at operation is challenged.

The envelope phenotypes and coagglutination (CoA) W serogroups of 301 unselected clinical isolates of Neisseria gonorrhoeae were studied. Of the 287 isolates from infections acquired in the Edinburgh area, 252 (88%) were of the wild-type phenotype, 17 (6%) were of Env phenotype with increased permeability of the cell envelope, and 18 (6%) were of Mtr phenotype with reduced permeability of the cell envelope. Mtr strains were isolated significantly more often from homosexual men than from heterosexual men and women (p>0·001). Of the isolates from homosexual men there were considerably fewer Mtr phenotypes among rectal isolates than among urethral and throat isolates. All isolates from homosexual men, 57% from heterosexual men and 52% from women were of CoA serogroup WII (including WII/WIII). Although the Mtr phenotype was strongly associated with serogroup-WII isolates from homosexual men, there was no such correlation between the Mtr phenotype and serogroup-WII isolates from heterosexual patients. It is suggested that hydrophobic compounds in the rectal environment are not the major factor in selecting serogroup-WII strains of Mtr phenotype associated with homosexually acquired infection. The hypothesis that the Mtr phenotype and serogroup WII are selected independently as a result of a more general selective pressure such as antibiotic usage, common to all infected sites, is discussed.

Conventional chemotherapy of tuberculosis and leprosy requires rifampicin to be administered orally. The long period of treatment and adverse side effects of the drug lead to poor compliance. To overcome this, subdermal implants incorporating rifampicin in pure and micro-encapsulated forms with biodegradable material were used as a new drug delivery system in experimental tuberculosis of guinea pigs. Two experiments were performed with 45-mg and 100-mg drug implants. Progress of infection was followed at intervals by studying necropsy scores and weights of the organs of predilection and levels of the drug in the blood were determined. There was a constant and sustained release of the drug in therapeutic concentrations for 30 and 50 days until the implants were completely assimilated without causing any damage to the implant site. The importance of multiple implants at long intervals is discussed.

An animal model was used to establish the risk of transmitting a virus infection by subcutaneous jet injection. Virus transmission was studied with mice chronically infected with LDH virus. The virus infection was transmitted by subcutaneous jet injection in 16 cases out of 49. Other routes of cross-infection were ruled out. Before using the jet injector as a harmless instrument for mass subcutaneous injection, further experiments on the risks of virus transmission should be performed.

The nuclease-neutralisation test was evaluated as a means of identifying nuclease-positive staphylococci isolated from different animals. The test identified 510 of 520 strains (98%) of staphylococci characterised by biochemical testing.

Edited by the Steering Committee of the Anaerobe Discussion Group: M. J. Hill, S. P. Borriello, J. M. Hardie, M. J. Hudson, R. J. Lyons and S. Tobaqchali. 1984. Martinus Nijhoff, Dordrecht, The Netherlands. Pp. xi and 287.