- Volume 22, Issue 2, 1986

The growth in liquid media of Legionella pneumophila serogroups 1–6 was monitored turbidimetrically and factors affecting growth rate were studied. The presence of inhibitors, use of detoxifying agents and the method of broth preparation each had significant effects on cultivation. Cysteine was essential for growth; the optimal concentration was 100 μg/ml, but supplemental iron had no demonstrable effect.

New serotypes were sought among 165 clinical isolates of group-B streptococci that were untypable by antisera for the conventional types Ia, Ib, II and III. The strains were tested for sialic acid, an integral component of the group-B streptococcal type-polysaccharides; trypsin-treated bacteria were tested by slideagglutination with the sialic-acid-specific lectin from the snail Cepaea hortensis. Sialic acid was detected in 96 of the strains; in 95 of these, new type antigens were identified serologically (type IV, 52; provisional type V, 34; candidate type NT6, seven; provisional type V and candidate type NT6, one; candidate type 7271, one); the remaining strain was found to possess a small amount of Ia antigen. Sialic acid was not detected in 69 strains, and none of these possessed a polysaccharide type-antigen. Chemical measurement of the sialic-acid content of cultures by Aminoff’s method gave results in conformity with the lectin-agglutination test and the presence of polysaccharide type-antigens.

A strain of Staphylococcus aureus was constructed with which to compare transfer of resistance plasmids by the mechanisms of phage-mediated conjugation and conjugation. Transfer by each mechanism could be distinguished by the patterns of resistances transferred. Conjugation was favoured on dry absorbent surfaces, e.g., human skin, tissue and surgical gauze, whereas phage-mediated conjugation was favoured in fluids, e.g., milk and urine. The degree of hydration of the mating cells is postulated as one factor determining whether plasmids are transferred by phagemediated conjugation or conjugation. Preliminary evidence indicates that topical creams and ointments affect the conjugative transfer of plasmids.

The antibacterial properties of aflatoxin B1 have been evaluated against antibiotic-resistant clinical isolates of Escherichia coli and Staphylococcus aureus. The inhibition of growth ranged from 11.5 to 60.0% and 4.5 to 18.5% in the strains of S. aureus and E. coli, depending on the extent of drug resistance. Aflatoxin-B1 binding varied with toxin concentration, the presence of surfactants (Tween-80 or EDTA) as well as with the antibiotic-resistance pattern; binding was maximal in antibioticsensitive strains and least in the most resistant strains. Binding of aflatoxin B1, correlated with growth inhibition. Aflatoxin B1 also caused leakage of cell contents and decrease in inulin uptake, effects which were also concentration dependent.

Plasmids isolated from two different clinical isolates of Salmonella typhimurium, both resistant to the antibiotics ampicillin, tetracycline, streptomycin and chloramphenicol, were used to transform Escherichia coli. Segregation of antibiotic-resistance determinants occurred in both cases. Analysis of plasmids from one set of segregants by DNA-DNA hybridisation indicated that the segregation was due to precise deletions in the transforming plasmid.

A series of 178 strains of Escherichia coli, highly resistant to trimethoprim, isolated from hospital patients and patients in the community between 1979 and 1983, was examined for the presence of Tn7 on a plasmid or on the chromosome only, by transposition to RP4 and restriction endonuclease digestion with Hind III. Of the isolates, 57% carried Tn7. Comparison of hospital isolates from 1979 to 1980 and 1982 showed that although the proportions that carried Tn7 were similar (63% and 57%) there had been a significant change in the genetic location of the transposon. The proportion of plasmid-mediated Tn7 had fallen from 62% to 30% with a corresponding rise in Tn7 located exclusively on the chromosome from 38% to 70%. This change may be the result of continuing transposition of Tn7 from plasmids to the bacterial chromosome followed by plasmid loss. The consequent reduction in the mobility of trimethoprim-resistance genes may in turn lead to changes in the incidence of resistance.

Blind loops prepared in the small intestines of fasted, MgSO4-treated rats were shown to provide a simple, consistent and inexpensive means of studying mucosal colonisation by Vibrio cholerae serogroup O1. When c. 2000 cfu were injected, the number of mucosa-associated V. cholerae in each loop increased by c. 5-6 orders of magnitude in 10-14 h, without enterotoxin-induced fluid production. Scanning electronmicroscopy and culture suggested that most surface-associated organisms were present in the adherent surface mucus. V. cholerae strains varied in terms of surface-colonising capacity.

Immunisation with V. cholerae given intra-intestinally greatly reduced the rate of increase and final number of mucosa-associated vibrios within the 14-h period after challenge. The method could be used to compare the immunity induced by various immunising regimens. Immunity was sometimes accompanied by intestinal mucusborne antibody against V. cholerae lipopolysaccharide but was sometimes demonstrated in the absence of such antibody or of mucus-borne antibody to heat-sensitive surface protein.

The effect of human serum on Escherichia coli was studied with serumsensitive and serum-resistant strains. The bactericidal effect of human serum on serumsensitive strains of E. coli depended on the activation of the classical complement pathway. The role of activation of the alternative pathway was less important. After incubation in sub-bactericidal concentrations of serum these strains were also easily phagocytosed by polymorphonuclear leukocytes (PMNL). Strains of E. coli of certain O-types required not only an intact classical pathway but also the presence of specific antibodies for effective killing by serum and effective phagocytosis by PMNL, despite rapid activation of complement and rapid deposition of C3 on the bacterial surface in the absence of antibody. Capsulate strains O1K1 and O78K80 resisted the bactericidal effect of serum even in the presence of specific antibodies; phagocytosis by PMNL only occurred after opsonisation with specific antibodies.

One hundred and forty seven isolates of Serratia marcescens were collected from diverse clinical and environmental sources in south-east Texas. Natural isolates were compared with hospital strains for the occurrence of 12 potential virulence determinants. Their overall frequency was as follows: haemolytic activity 48%; lecithinase 95%; lipase 95%; motility 99%; pigmentation 24%; plasmid carriage 46%; proteolytic activity 98%; siderophore activity 99%; urease activity 5%; mannosesensitive haemagglutination 96%; mannose-resistant haemagglutination 61%; and mannose-resistant type-K haemagglutination (MR/K-HA) 68%. Clinical strains demonstrated a significantly higher occurrence of MR/K-HA (p<0.001) and nonpigmentation (p<0.01) than environmental isolates.

Interactions between representative strains of four predominant resident bacteria of the human colon, Escherichia coli, Enterococcus faecalis, Bacteroides ovatus, and Fusobacterium varium, and strains of seven enteropathogens, Yersinia enterocolitica, Shigella flexneri, Salmonella typhimurium, Vibrio parahaemolyticus, V. cholerae serogroup non O1, Staphylococcus aureus, and Clostridium perfringens, were examined in studies with an anaerobic continuous flow culture system and medium resembling the content of the mouse caecum (MCM). Potent unilateral antagonism attributable to synergic activities of the resident bacteria against the enteropathogens was evident.

The four resident bacteria persisted at levels of c. 106 cfu/ml or more in single and in any mixed cultures of the resident species. The seven enteropathogens also persisted in single cultures. In contrast, Y. enterocolitica was excluded in several days in mixed cultures with each of the four resident bacteria. Sh. flexneri and Staph. aureus were excluded in the presence of E. coli alone. C. perfringens, V. parahaemolyticus and V. cholerae serogroup non O1 were excluded in the presence of E. coli with B. ovatus and, in some cases, with additional species. S. typhimurium was the most resistant; only c. 102-fold reduction of the population level was observed in mixed culture with all four of the resident species. When the amounts of some components in the medium, such as peptone and yeast extract, were increased, C. perfringens grew and persisted even in the presence of the four resident bacteria. Sh. flexneri, in contrast decreased steadily, even in enriched media.

An agglutination assay for detecting intermicrobial adherence between the cells of Candida albicans and various oral bacteria is described. Strains of Streptococcus sanguis, S. salivarius, S. mutans, S. mitis, Fusobacterium nucleatum and Actinomyces viscosus all coagglutinated with C. albicans. No interaction could be demonstrated between the cells of Bacteroides melaninogenicus and those of C. albicans. Preliminary investigations of these interactions suggest that binding of F. nucleatum and A. viscosus to C. albicans is mediated by bacterial proteins, possibly lectins. Other mechanisms must account for the binding of oral streptococci to C. albicans. The possible implications of these findings in relation to oral mucosal colonisation and oral candidal clearance are discussed.

The presence of capsulate Bacteroides spp. and anaerobic gram-positive cocci was investigated in pus specimens from 182 children with chronic orofacial infections or abscesses and in pharyngeal swabs from 26 children without inflammation. Of 216 Bacteroides spp. and anaerobic cocci isolated from clinical infections, 170 (79%) were capsulate, compared with 34 (35%) of 96 isolates from normal pharyngeal flora (p<0.001). The commonest organisms found to be capsulate more often from infected patients than from controls belonged to the B. melaninogenicus group. The possible evolution of encapsulation in these organisms and their importance in mixed orofacial infections are discussed.

Routine procedures used to isolate Haemophilus ducreyi in a busy laboratory are reported. Identification was based on colony morphology and nutritional and biochemical properties of 120 fresh isolates of H. ducreyi. These isolates grew very well on Gonococcal Agar and Mueller-Hinton Agar incubated at 34°C in candle extinction jars containing moistened filter paper. Colonies varied in size, giving a polymorphic appearance. They were smooth, dome-shaped, and buff-yellow to grey in colour, and measured 2 mm in diameter. They could be pushed intact across the agar surface. By microscopic examination of gram-stained smears the isolates were gram-negative coccobacilli arranged in short chains, clumps or whorls and occasionally in typical “rail track” arrangements. Individual bacteria showed bipolar staining. Colonies autoagglutinated in saline. All strains were catalase-negative and did not produce indole or H2S. They were oxidase- and β-lactamase positive and required X but not V factor for growth. Now that reliable techniques have been developed and characteristics established it is possible for most clinical laboratories to isolate and identify this organism from most patients with chancroid.