- Volume 21, Issue 2, 1986

Mixed infections with three Fusobacterium species and seven other bacterial species were studied in a subcutaneous abscess model in mice. Fifteen Fusobacterium isolates (eight F. nucleatum, four F. necrophorum, and three F. varium) and one isolate each of Bacteroides fragilis, B. asaccharolyticus, Staphylococcus aureus, Group A β-haemolytic streptococcus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were studied. Electronmicrographs showed the presence of a thin mucopolysaccharide wall before and after inoculation into mice in 12 isolates which included all of 11 Fusobacterium isolates that induced subcutaneous abscesses. After co-inoculation of Fusobacterium isolates with other species and selective therapy with antimicrobial agents, S. aureus and K. pneumoniae were found to be of equal or greater importance in abscess induction than were Fusobacterium isolates, while Fusobacterium isolates were found to be more important than Group A streptococci and E. coli. Mutual enhancement of the numbers of organisms in mixed infections was observed with Fusobacterium spp. and K. pneumoniae, P. aeruginosa or Bacteriodes spp. Suppression of Fusobacterium spp. was noticed only when they were co-inoculated with Group A streptococci. The additive or synergistic capabilities of Fusobacterium species highlighted their potential pathogenicity in infection.

A qualitative and quantitative bacteriological study was performed on pus specimens otained by needle aspiration of 50 acute dento-alveolar abscesses. Most samples contained a mixture of species (average 3.3); 20 (40%) of the abscesses contained anaerobes alone, 3 (6%) contained facultative anaerobes only and the remaining 27 (54%) contained mixtures of both types of bacteria, with anaerobes predominating. In total, 166 bacterial strains were isolated, 75% of which were strictly anaerobic; the most common species were Peptococcus spp, Bacteroides oralis and B. melaninogenicus. Among facultative anaerobes, Streptococcus milleri was particularly common. The mean concentration of bacteria in each abscess was 106.9±0.2. The mean concentration of anaerobic bacteria was 106.2±0.1 and of facultatively anaerobic bacteria 105.7±0.2.

Adhesive properties of five species of Bacteroides were compared by direct haemagglutination with erythrocytes of different origin. Only strains of Bacteroides fragilis agglutinated erythrocytes and different patterns of haemagglutination were observed. None of eight carbohydrates tested inhibited haemagglutination. The activity was destroyed by heat and by periodate treatment, but not by three proteases tested.

Twenty-seven strains of gram-negative anaerobic bacteria isolated from patients with non-gonococcal urethritis (NGU-associated strains) were compared with reference strains of Bacteroides ureolyticus and representatives of other species of Bacteroides and Fusobacterium. Conventional biochemical tests, electrophoretic (PAGE) protein profiles and DNA base compositions of 29.5±1.2 mol % G+C indicated striking similarities between the NGU-associated strains and B. ureolyticus. Three of the reference strains previously assigned to that species were atypical in some respects as was one of the strains from urethritis and one strain, NCTC 10939, was incorrectly classified as B. ureolyticus. Casein hydrolysis by B. ureolyticus was the principal test difference between the two sets of strains. DNA-DNA hybridisation results of > 72% at optimum temperature conditions provided further evidence of the close relationship between B. ureolyticus and the NGU-associated strains and we conclude that these organisms should be identified as B. ureolyticus. There was insufficient evidence for recognising subspecies or biotypes within this revised concept of B. ureolyticus although several types (PAGE-type I, 17 strains; type II, 6 strains; type III, 3 strains) were detected which might provide the basis for future studies of hospital strains. The DNA base composition and hybridisation data indicated that B. ureolyticus was not related to other species of Bacteroides or Fusobacterium.

The adherence properties of non-fimbriate entero-invasive Escherichia coli O124 in the guinea pig intestinal tract were studied. Quantitative in-vitro determinations were done by incubating radiolabelled bacteria with suspensions of viable intestinal cells released by treating loops of the guinea pig intestine with solutions containing EDTA, dithiothreitol and citrate. Non-bound bacteria were separated from the intestinal cells on a Percoll gradient. Only cells released from the colon, especially from its transverse and descending regions, avidly adhered to E. coli O124 (68−79 bacteria/cell), whereas the attachment to ileal cells was negligible. The adherence process was Ca++ and temperature-dependent, had an optimal pH of 6.2 and was inhibited by fucose, glucose or mannose. Several pretreatment studies of the bacteria or the colonic cells showed that the adherence was mediated by a carbohydrate-binding protein (adhesin or lectin) on the colonic cells and not on the bacterial surface. Results of studies of in-vitro adherence to intestinal loops and to intact intestinal surfaces correlated well with the in-vitro findings. These results indicate that the adherence of entero-invasive E. coli O124 to the gut is similar to the attachment of Shigella flexneri and is quite different from that of enterotoxigenic E. coli.

The surface hydrophobicity of Klebsiella aerogenes is influenced by the presence of capsular (K) and lipopolysaccharide (O) antigens. Loss of both K and O antigens (K−O−), but not the K antigen alone (K−O+), increased surface hydrophobicity and susceptibility to phagocytosis. Unheated serum (i.e., containing complement) increased the surface hydrophobicity and phagocytosis of the K−O+ and K−O− strains, but not of the K+O+ encapsulated parent strain. Despite the altered susceptibility to phagocytosis caused by the presence or absence of the K and O antigens, their loss did not influence sensitivity to a range of hydrophilic, hydrophobic or cationic antimicrobial agents.

The importance of K antigen of Klebsiella as a virulence factor was studied in nine pairs of K+ and K- strains, each pair isogenic apart from the presence of K antigen. Loss of K antigen by nine K+ strains resulted in the reduced virulence of their K- variants in a mouse-skin model. This reduced virulence of K- strains for mice may be explained in all strains by a higher degree of phagocytosis as measured by chemiluminescence response of human polymorphonuclear leukocytes (PMNL) and in most strains by enhanced killing by either human PMNL or human serum or both. Although the protective role of the K antigen in serum-induced killing and killing by PMNL was generally evident, our results also suggested that other virulence factors were sometimes involved.

The faecal carriage rates of different species of Proteeae were assessed in studies with 220 faecal isolates from 219 individuals of whom approximately one-third were well and the remainder had gastro-enteritis. As a result of the development of new media that allowed replacement of the phenylalanine deaminase test with the tryptophan deaminase test and made it possible to combine tests for indole and urease production and for hydrogen sulphide and ornithine decarboxylase formation in two single-tube tests, all strains were speciated with speed, economy and accuracy. Most (96%) isolates were either Proteus mirabilis (62%) or Morganella morgani (34%). The significance of these findings in relation to urinary tract infection is discussed. P. vulgaris was found in only one (0.45%) faecal specimen and this rarity of carriage in faeces is believed to be the main reason for its rare association with urinary tract infections. The frequent association of M. morgani, in the absence of other enteropathogenic bacteria, with severe gastroenteritis was noted with interest.

The recently-introduced semi-automated bacterial identification system, the Quantum II BID, is designed to identify fermentative and non-fermentative gram-negative bacilli in 4−5 h. The system was evaluated independently by the two participating laboratories. Inter-laboratory reproducibility was determined by testing 181 strains in each laboratory and found to be 97.8%. A further 893 organisms, comprising 417 fresh clinical isolates and 476 stock cultures, were then tested by the system in the two laboratories. Of the fresh clinical isolates, 95.7% yielded the same result as the comparative system used (API 20E) and 93.1% of the stock strains were assigned to their expected, previously established identity. Overall agreement between the two systems for all strains examined was 94.3%. There was no significant statistical difference between the results obtained in the two laboratories.

Inbred female C3H mice were given 2 × 107 cfu virulent Salmonella typhimurium by intraperitoneal injection. Peritoneal washings were harvested between 3 h and 72 h after infection and examined by electronmicroscopy. There was evidence of intracellular killing by polymorphs and macrophages. The degeneration of intracellular salmonellae was seen initially as enlarging central electron-lucent areas in the cytoplasm and peripheral condensation of cytoplasmic granules, followed by disruption of the bacterial envelope and disintegration of cellular structure. Alternatively, the initial injury appeared as an irregular and discontinuous bacterial envelope with compression of the bacterium and diffuse condensation of cytoplasmic granules. It was also evident that virulent salmonellae multiplied extracellularly in the peritoneal cavity of the infected mice.

The antigenic composition of an endocarditis-associated isolate of Streptococcus faecalis was studied by immunoblotting of whole cells and cell walls from sodium-dodecyl sulphate polyacrylamide gels on to nitrocellulose and detection with serum from patients and hyperimmune rabbit serum. A major envelope protein antigen of mol. wt 53 × 103 detected with patient’s serum was also present in three urinary strains of Str. faecalis and a laboratory strain of Str. faecalis ss. zymogenes but not in Staphylococcus aureus. Other common antigens of Str. faecalis were of mol. wt (103) 65, 63, 56, 49.5, 30 and 21. Two other protein antigens (43 and 37 × 103 mol. wt) reacted strongly with asparagus pea lectin-peroxidase conjugate indicating the presence of fucosyl residues. Other lectin-peroxidase conjugates were used to demonstrate the presence of various glycosyl residues on envelope proteins. Growth of Str. faecalis in serum to mimic in-vivo growth conditions in endocarditis infections dramatically altered the antigenic patterns. Only two major antigens of mol. wt (103) 56 and 53 reacted with sera from endocarditis patients. These antigens may, therefore, be of diagnostic or protective potential.

Extracellular proteins produced by Staphylococcus aureus strains were examined by Western Blot analysis with blood donor plasma as a source of antibodies. Comparison of epidemiologically related strains showed strong concordance between plot pattern and phage type.

Calcium-dependent binding of C-reactive protein (CRP) to Aspergillus fumigatus was determined by enzyme-linked immunosorbent assay. A homogenate of young hyphae was fractionated by hydrophobic interaction chromatography followed by gel filtration. High CRP-binding activity was found in a fraction of mol. wt c. 500,000 which was characterised by strong binding to the hydrophobic column. Three fractions of less conspicuous CRP-binding activity were identified (c. 500 000, 150 000 and 150 000−50 000 mol. wt respectively). In these four fractions, phosphorylcholine was detected by an anti-phosphorylcholine mouse hybridoma antibody. Some CRP-binding activity in fractions with low affinity for the hydrophobic column did not correspond closely with the presence of phosphorylcholine. It is suggested that C-reactive substance in A. fumigatus is heterogeneous. The C-reactive substances did not correspond with fractions containing major antigens (470 000 and 250 000 mol. wt respectively) which elicit a strong immune response in man.

Serologically polyagglutinating (PA) and non-typable (NT) strains of Pseudomonas aeruginosa are frequently isolated from cystic fibrosis (CF) patients, but are uncommon in other patients. From serologically typical parent strains, we isolated two variants (one PA, the other NT) which differed from the parent in bacteriophage susceptibility or in sensitivity to the bactericidal action of normal human serum. The PA and NT variants (strains 7/1 and 18S respectively) reacted with antiserum to the parent strains 7 and 18R but did not absorb homologous specific O antibody from antiserum to the parent strains. In contrast the parent strains absorbed anti-PA and anti-NT antibodies from antisera to the variant strains. The yield of lipopolysaccharide (LPS) from acetone-dried cells of the parent strain 7 was similar to that of the PA derivative; but the NT strain 18S yielded only half the LPS of its parent strain. LPS of the variant 7/1 gave a banding profile by SDS-PAGE similar to that of the parent LPS 7, but lacked high-molecular-weight components. LPS of the variant 18S appeared to be grossly different in profile from LPS 18R. Of 533 isolates of P. aeruginosa that were tested with O antisera and with antisera to the two variants, 15% were O-typable and 22% were O-non-typable; 26% reacted with anti-PA serum alone, 10% with anti-NT serum alone, and 27% were agglutinated by both sera. There was a statistically significant correlation between serum sensitivity of CF isolates and their reaction with the PA or NT antisera.

Edited by A. Mizrahi and A. L. van Wezel. 1984. Alan R. Liss, Inc., New York. Pp. xv and 361. £52.00.