J. de Graaff

Differences in virulence within the species Bacteroides gingivalis

In order to gain insight into the relative importance of several virulence factors of Bacteroides gingivalis, 8 strains with a varying virulence were studied. The virulence of B. gingivalis was determined in a mouse model. Strains HG 66, HG 76 and HG 184 were very virulent causing phlegmonos abscesses with lesions and necrosis. The strains HG 405 and HG 462 caused phlegmonous abscesses with pus. Strains HG 91, HG 94 and HG 185 were less virulent and induced gravity abscesses. In vitro strains HG 66, HG 76 and HG 184 induced low amounts of chemiluminescence by polymorphonuclear leucocytes. All other strains including HG 405 and HG 462 caused a relatively high chemiluminescence. Most strains displayed a high sensitivity to the bactericidal activity of fresh serum except for the highly virulent strains HG 66, HG 76 and HG 184. No differences in extracellular proteolytic activity on Azocoll, production of volatile fatty acids and ammonia were found between the B. gingivalis strains studied. In conclusion, differences in virulence were shown within the species B. gingivalis; the relative importance of several virulence factors was investigated.

Short-chain fatty acids produced by anaerobic bacteria alter the physiological responses of human neutrophils to chemotactic peptide.

The effect of some short-chain fatty acids (SCFA) produced by anaerobic bacteria, namely acetic, propionic, isobutyric, butyric, isovaleric and succinic acids, on production of light and release of lysozyme by human neutrophils exposed to chemotactic peptide fMet-Leu-Phe was investigated. A short period of incubation of neutrophils with SCFA led to marked inhibition of both granulocytic chemiluminescence and degranulation (P less than 0.001). Ultrastructural studies of neutrophils, incubated with concentrations of SCFA inhibiting the chemotactic response, chemiluminescence and release of lysozyme (30 mmol/l), effected alterations in cellular morphology with formation of protrusions of varying shape. The data reported indicate that SCFA might be regarded as important pathogenicity factors. The observed effect on neutrophils could also partially explain the ability of anaerobes to inhibit their own phagocytosis and killing as well as that of the aerobic species present in mixed infections.

Bacterial Involvement in Denture-induced Stomatitis

Denture and mucosal plaque samples were collected from eight full-denture wearers of whom four suffered from denture-induced stomatitis (DIS). Cultures were made, and a proportional identification to species level was carried out of bacteria and yeasts. An inventory was made of the predominant flora. Results showed that the predominant microflora of both groups, both on dentures and the denture-bearing mucosa, consisted mainly of Gram-positive bacteria. Differences in the proportions of cocci were found between the predominant bacterial flora on the dentures. In the control group, 69% of the denture flora consisted of cocci, while on the dentures of the DIS group a mean of 33% cocci was found. Neither group of palates revealed any differences in the proportions of cocci. On these locations a mean of 69% was found. The plaque of dentures and the palates of the healthy group showed means of 35% and 31%, respectively, of obligate anaerobic bacteria, while in the DIS group these percentages were 56% and 43%, respectively. No obligately aerobic bacteria were found. Candida species was detected in both groups, both on dentures and palates in very low numbers (DIS group, median palates 0.02%, median dentures 0.25%). The predominant organisms were Streptococcus species, of which S. salivarius was mostly present on the palates of both groups. Other species which were regularly found at different locations were Veillonella parvula and species of Lactobacillus, Bacteroides, and Actinomyces.

Butyrate: a cytotoxin for Vero cells produced by Bacteroides gingivalis and Bacteroides asaccharolyticus.

Culture filtrates of B. gingivalis and B. asaccharolyticus are cytotoxic for Vero cells. It is shown that the cytotoxic effect is due to the butyrate concentrations present in the culture filtrates of these strains. This cytotoxic effect proved to be reversible. Strains of the B. melaninogenicus subspecies intermedius and melaninogenicus did not produce butyrate and did not show cytotoxic activity towards Vero Cells.The significance of the production of toxic concentrations of butyrate for the etiology of especially periodontal diseases is discussed.

Enzymatic characterization of oral and non-oral black-pigmented Bacteroides species

The purpose of this study was to investigate the production of various enzymes by oral and non-oral black-pigmented Bacteroides species using chromogenic substrates. The 19 substrates present in the API ZYM system did not differentiate between B. melaninogenicus, B. denticola, B. loescheii and B. levii. The asaccharolytic black-pigmented Bacteroides species showed each species specific enzyme activity, however, differences were based on one enzyme only as far as B. asaccharolyticus and B. endodontalis are concerned. An extended number of 40 chromogenic substrates were tested in order to find more species specific enzyme. With a set of 20 substrates it appeared to be possible to discriminate between all species tested. The possibility to use enzymes for the identification of black-pigmented Bacteroides is discussed.

Effects of Ammonia and Volatile Fatty Acids Produced by Oral Bacteria on Tissue Culture Cells

Culture filtrates of several bacterial species isolated from the oral cavity were tested for their effects on two types of tissue culture cells: Vero cells, the continuous cell line of African green monkey kidney cells; and chondrocytes, isolated from 15-day-old chick embryo tibiae. Only a limited number of bacterial species — i.e., the asaccharolytic black-pigmented Bacteroides species and Fusobacterium species - affected the two cell types. The effect on Vero cells, detected by the rounding of the cells, correlated with the butyric acid concentration in the bacterial supernatant, which confirms previous findings. A small enhancement of this effect was found with propionic acid and ammonium ions. The same strains which affected Vero cells also affected chondrocytes, detected by a vacuolization of the cells. However, volatile fatty acids on their own had no visible effect on these cells. Instead, ammonium ion in the culture filtrate, when present in concentrations of 20 to 60 mmollL, proved to be responsible for vacuolization of the chondrocytes. The volatile fatty acids (butyric and propionic) had a limited additive effect. No effects were visible with cell extracts of the bacteria.Culture filtrates of several bacterial species isolated from the oral cavity were tested for their effects on two types of tissue culture cells: Vero cells, the continuous cell line of African green monkey kidney cells; and chondrocytes, isolated from 15-day-old chick embryo tibiae. Only a limited number of bacterial species — i.e., the asaccharolytic black-pigmented Bacteroides species and Fusobacterium species - affected the two cell types. The effect on Vero cells, detected by the rounding of the cells, correlated with the butyric acid concentration in the bacterial supernatant, which confirms previous findings. A small enhancement of this effect was found with propionic acid and ammonium ions. The same strains which affected Vero cells also affected chondrocytes, detected by a vacuolization of the cells. However, volatile fatty acids on their own had no visible effect on these cells. Instead, ammonium ion in the culture filtrate, when present in concentrations of 20 to 60 mmollL, proved to be responsi...

Pathogenic synergy: mixed infections in the oral cavity

In almost all infections in the oral cavity, mixed populations of bacteria are present. However, recent evidence points to a certain specificity in these infections:Streptococcus mutans is related to caries and black-pigmentedBacteroides species are suspected pathogens in periodontal disease. Periodontal diseases, endodontic infections and submucous abscesses in the oral cavity are probably mixed infections in which anaerobic bacteria together with facultatives or other anaerobes are present. In experimental mixed anaerobic infections black-pigmentedBacteroides strains have been shown to play a key role. Little is known about the pathogenic synergy between the bacteria involved in mixed infections. Important mechanisms could be nutritional interrelationships and interactions with the host defense. Within the group of black-pigmentedBacteroides B. gingivalis seems to be the most virulent species. These bacteria possess a great number of virulence factors, which might be important in the pathogenesis of oral infections.

Hemolysin plasmid coding for the virulence of a nephropathogenic Escherichia coli strain.

The nephropathogenic Escherichia coli strain P673 was shown to harbor two plasmids with molecular sizes of 70 and 41 megadaltons, respectively. The 70-megadalton plasmid, pCW1, coded for tetracycline resistance, whereas hemolysin production was coded by the 41-megadalton plasmid, pCW2. Plasmid pCW1 proved to be self-transmissible, in contrast to pCW2. Transfer of the hemolysin character was associated with the appearance of a 110-megadalton plasmid, pCW3. The incompatibility of pCW3 with both native plasmids and restriction enzyme analysis led to the conclusion that pCW3 is a cointegrate of pCW1 and pCW2, pCW2, carrying the hemolytic determinant, is involved in the nephropathogenic character of strain P673, because (i) elimination of pCW2 from P673 was associated with a loss of virulence and (ii) the nephropathogenicity of the avirulent mutant could be restored by reintroduction of pCW2 DNA as part of a cointegrate structure.

Age at menarche and menopause of uterine cancer patients

An investigation was undertaken of the ages at menarche and at menopause of cervical and endometrial cancer patients for the years 1950-55 and 1960-65. Analysis of the ages at menarche in relation to the year of birth did not show a difference between the uterine cancer groups, whereas the age at menopause did show such a difference. The menopause occurred later in the endometrial than in the cervical cancer group. There was an earlier mean age at menarche and a later mean age at menopause per decade. Therefore, the menopause seems a constitutional factor involved in the development of endometrial cancer and perhaps also cervical cancer.

Cross-inhibition Between Black-pigmented Bacteroides Species

Cross-inhibition within the group of black-pigmented Bacteroides, including both oral and non-oral strains, was studied by means of a membrane filter technique. It was found that B. gingivalis possessed the most extended inhibitory capacity among all species tested. B. gingivalis showed inhibitory activity against B. intermedius, B. endodontalis, B. loescheii, and B. melaninogenicus. B. endodontalis was active against some B. intermedius strains. Among the saccharolytic species, some B . melaninogenicus strains were inhibitory for some B. endodontalis strains, some B. gingivalis strains, and some B. intermedius strains. These inhibitory activities observed in vitro may play a role in the colonization of the periodontal pocket.