Bacteroides gingivalis W50 was grown in a chemostat under steady-state conditions at pH 7.5 ± 0.2 and a constant growth rate of 6.9 h for periods of up to 6 weeks (146 bacterial generations) in a complex medium. Hemin was capable of limiting the growth of cells up to a concentration of approximately 0.5 ,ug/ml since higher concentrations of hemin did not increase cell yields; cells grew in the absence of exogenously added vitamin Kl. Only a limited number of amino acids was metabolized during growth, but because none of these was totally depleted, the limiting nutrient under hemin excess conditions was probably a peptide. A range of fermentation products was produced under all conditions of growth; higher concentrations of cytotoxic metabolites such as propionate and butyrate were formed under hemin excess conditions, although more ammonia was released under hemin limitation. When viewed by electron microscopy, cells grown under hemin limitation appeared to be either coccobacillary or short rods and possessed few fimbriae per cell, but large numbers of extracellular vesicles could be seen both surrounding the cell surface and free in the environment. In contrast, cells grown under hemin excess conditions were more commonly coccus shaped and were more heavily fimbriated but had fewer extracellular vesicles. Marked differences were found in the susceptibility of mice to infection with cells grown under different concentrations of hemin. Cells transferred to media without any added hemin were avirulent, whereas those grown under conditions of hemin limitation (0.33 and 0.40 ,ug/ml) produced a 20 and 50% mortality in mice, respectively. In contrast cells grown under hemin excess always caused 100% mortality in mice, although this virulence was dose dependent. When virulent, the bacteria caused an extensive, spreading infection with necrosis of the skin and subcutaneous tissues. Collagen disintegration was seen histologically, implying a role for collagenase production in the pathogenicity of these bacteria.
Porphyromonas gingivalis strain W50 was grown under haemin-limitation and haemin-excess conditions in a chemostat at pH 7.5. The maximum specific growth rate (,urn,,) was determined at both haemin concentrations (,urn, = 0236 f 0.052 and 0.271 +0*039 h-l, respectively). This enabled dilution rates to be adjusted so that the virulence and enzyme activity of haemin-limited and haemin-replete cells could be compared at identical relative growth rates @ , , ) of 0025,050 and 075 of their respective ,urnax. The data showed that the fastest growing cells were significantly more virulent than those grown more slowly, irrespective of haemin concentration. However, at each growth rate tested, cells grown under haemin-excess conditions were always more virulent than haemin-limited cells. Trypsin-like enzyme activity of whole cultures was also greater at each growth rate under haemin-excess conditions while, conversely, collagenolytic activity was generally higher in haemin-limited cultures. Thus, although growth rate had an effect on the virulence and enzyme activity of P. gingivalis, the availability of haemin for growth was the most significant factor.
Nine commonly isolated oral bacterial populations were inoculated into a glucose-limited and a glucose-excess (amino acid-limited) chemostat maintained at a constant pH 7.0 and a mean community generation time of 13.9 h. The bacterial populations were Streptococcus mutans ATCC 2-27351, Strep. sanguis NCTC 7865, Strep. mitior EF 186, Actinomyces viscosus WVU 627, Lactobacillus casei AC 413, Neisseria sp. A1078, Veillonella alkalescens ATCC 17745, Bacteroides intermedius T 588 and Fusobacterium nucleatum NCTC 10593. All nine populations became established in the glucose-limited chemostat although Strep. sanguis and Neisseria sp. were present only after a second and third inoculation, respectively. In contrast, even following repeated inoculations, Strep. mutans, B. intermedius and Neisseria sp. could not be maintained under glucose-excess conditions. A more extensive pattern of fermentation products and amino acid catabolism occurred under glucose-limited growth; this simultaneous utilization of mixed substrates also contributed to the higher yields (Y molar glucose) and greater species diversity of these communities. Microscopic and biochemical evidence suggested that cell-to-cell interactions and food chains were occurring among community members. To compare the reproductibility of this system, communities were established on three occasions under glucose-limitation and twice under glucose-excess conditions. The bacterial composition of the steady-state communities and their metabolic behaviour were similar when grown under identical conditions but varied in a consistent manner according to the nutrient responsible for limiting growth. Although a direct simulation of the oral cavity was not attempted, the results show that the chemostat could be used as an environmentally-related model to grow complex but reproducible communities of oral bacteria for long periods from a defined inoculum.
Summary. The spontaneous appearance of unusual colony forms was observed during prolonged growth of Bacteroides gingivalis W50 in a chemostat. Two variants were selected for further study which could be distinguished from the parent strain by the rate and intensity of pigmentation of their colonies. For example, after anaerobic incubation for 14 days, variant WSO/BRl produced brown colonies whereas those of the parent strain were black; in contrast, variant W5O/BE1 did not show signs of pigmentation until incubation had continued for 21 days. In subsequent studies in the chemostat, variant W5O/BE1 bred true even after prolonged growth whereas other colony forms appeared after incubation of variant WSO/BRl for 14 days. The relatedness of W5O/BR1 and WSO/BEl to the parent strain was confirmed by comparisons of the whole-cell fatty-acid profiles, the patterns of pre-formed enzymes and by the metabolic end products after growth. However, the variants did differ from the parent strain in their virulence in a mouse pathogenicity model. The parent strain killed all mice given infective doses > 5 x lo8 cfu whereas W5O/BRl was much less virulent (2 out of 10 mice killed and higher infective doses needed for higher mortality rates) and W5O/BE1 was avirulent at all infective doses tested.
Since the pH of the gingival crevice increases from below neutrality in health to above pH 8 in disease, we decided to investigate the effect of environmental pH on the growth anol enzyme activity of Bacteroides gingivalis W50. Cells were grown in a chemostat under hemin-excess conditions over a range of pH values; stable growth was observed only between pH 6.7 and 8.3, with the maximum yields obtaiped between pH 7.0 and 8.0. The enzyme profile of cells varied markedly with pH. Enzymes with a specificity for gingival connective tissue (collagenase, hyaluronidase) were produced optimally at or below neutral pH, whereas trypsinlike activity increased with the growth pH and was maximal at pH 8.0. Chymotrypsinlike activity was generally low, although its activity was highest at the extremes of growth pH, i.e., at pH 6.7 and 8.3. Inhibitor studies provided evidence that the breakdown of collagen involved the concerted action of both a collagenase and the trypsinlike enzyme.
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