Ailsa S. McKee

Effects of Carbohydrate Pulses and pH on Population Shifts within Oral Microbial Communities in vitro

A mixed culture chemostat system was used to distinguish between the effects of carbohydrate availability per se and the low pH generated from carbohydrate metabolism on the proportions of bacteria within microbial communities. Nine oral bacteria were grown at pH 7 and pulsed with glucose on ten consecutive days. In one chemostat, the pH was maintained automatically at 7 throughout the experimental period, while in the other, pH control was discontinued for six hours after each pulse. Glucose pulses at neutral pH had little effect on the composition of the microflora. Only the proportions of A. viscosus and V. dispar increased; L. casei and S. mutans remained at low levels (0.2% and 1.0%, respectively). Acetate and propionate were the predominant end-products of metabolism; lactate levels were low. In contrast, when pH was allowed to fall after each glucose pulse, the composition of the microflora altered dramatically. The amounts of L. casei and S. mutans increased both as a proportion of the total count and in absolute numbers, as did V. dispar, whereas the amounts of the other Gram-negative organisms (B. intermedius, F. nucleatum, and N. subflava) and S. sanguis were considerably reduced. Lactate formed a major portion of the metabolic end-products. Successive glucose pulses resulted in both amplified changes in the microflora and a steadily greater rate and final extent of acid production. This is in agreement with the reported shifts in the oral microflora in vivo in response to frequent carbohydrate intake. Analysis of the data strongly suggests that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, is responsible for the widely reported shifts in composition and metabolism of the oral microflora in vivo.

The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50

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 (mumax) was determined at both haemin concentrations (mumax = 0.236 +/- 0.052 and 0.271 +/- 0.039 h-1, 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 (murel) of 0.25, 0.50 and 0.75 of their respective mumax. 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.

Isolation of colonial variants of Bacteroides gingivalis W50 with a reduced virulence

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 W50/BR1 produced brown colonies whereas those of the parent strain were black; in contrast, variant W50/BE1 did not show signs of pigmentation until incubation had continued for 21 days. In subsequent studies in the chemostat, variant W50/BE1 bred true even after prolonged growth whereas other colony forms appeared after incubation of variant W50/BR1 for 14 days. The relatedness of W50/BR1 and W50/BE1 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 greater than 5 x 10(8) cfu whereas W50/BR1 was much less virulent (2 out of 10 mice killed and higher infective doses needed for higher mortality rates) and W50/BE1 was avirulent at all infective doses tested.

Prevention of Population Shifts in Oral Microbial Communities in vitro by Low Fluoride Concentrations

A continuous culture system has been used to study the effects of low (sub-MIC) levels of sodium fluoride on the stability and metabolism of a defined oral microbial community. The microflora was also subjected to glucose pulses at pH 7.0, with and without subsequent pH control. At pH 7.0, a continuous supply of 1 mmol/L NaF reduced slightly the viable counts of the oral microflora, although their proportions were relatively unaffected. At pH 7.0, during glucose pulsing, 1 mmol/L NaF prevented the rise in proportions of A. viscosus and reduced the levels of B. intermedius. Glucose pulsing without pH control and in the absence of fluoride markedly inhibited the growth of many species, and L. casei, V. dispar, and S. mutans predominated in the culture. Fluoride (1 mmol/L), either pulsed with the glucose or provided continuously, reduced both the rate of change and the degree of fall in pH, and in doing so prevented the enrichment of S. mutans in the culture. Fluoride also reduced the pH-mediated inhibition of other members of the oral community, although S. sanguis was inhibited even further. Thus, even sub-MIC levels of fluoride may have a beneficial anti-bacterial effect on dental plaque by interfering with acid production. This would reduce the pH-mediated disruption to the balance of the microflora and suppress the selection of S. mutans.

The influence of growth rate and nutrient limitation on the microbial composition and biochemical properties of a mixed culture of oral bacteria grown in a chemostat.

A sample of human dental plaque was homogenized in transport fluid and inoculated simultaneously into a glucose-limited and a glucose-excess chemostat maintained at pH 7.0 and a dilution rate (D) of 0.05 h-1. In an attempt to ensure the establishment of slow-growing bacterial populations, two further inoculations of each chemostat with fresh samples of dental plaque took place before a steady-state was attained at this dilution rate. The dilution rate was increased step-wise to D = 0.6 h-1, and then returned directly to D = 0.05 h-1. Contrary to chemostat theory, microbial communities with a high species diversity were maintained under all of the experimental conditions employed, although not all of the bacterial populations present in the inocula established successfully in the chemostat. At each steady-state the bacteriological composition and biochemical properties (fermentation products, enzyme assays and acid production) of the communities of each chemostat was determined. Higher cell yields and a slightly more diverse community were obtained from the glucose-limited chemostat at all dilution rates. A complex mixture of end products of metabolism was obtained from the glucose-limited chemostat, suggesting amino acid catabolism, while lactate was the predominant acid of the glucose-excess culture. In washed-cell experiments, communities from the glucose-excess chemostat produced the lower terminal pH values following a pulse of glucose, with the lowest pH values occurring at the higher dilution rates. A film of micro-organisms, which accumulated around the neck of the chemostat, was sampled at the end of the experiment. The microbial composition of the films from each chemostat differed markedly, and both were different to the community of the bulk fluid of the respective chemostat. Spirochaetes and a population of yeasts were detected in the films from the glucose-limited and glucose-excess chemostats, respectively. No invertase or glucosyltransferase activity, and little glucoamylase-specific glycogen was detected in the communities from either chemostat, although significant endogenous activity, particularly at high dilution rates, was obtained with washed-cells from the glucose-excess chemostat. The results suggest that the chemostat could make a valuable contribution to the study of the ecology of dental plaque.

A Longitudinal Epidemiological Study on Dental Plaque and the Development of Dental Caries—Interim Results After Two Years

1. During the two-year period, caries developed at 20% of the target premolar sites. The attack rate for these surfaces was similar in the plaque panel and the other subjects in the study. 2. The microbial composition of plaque samples from caries-free sites and from carious sites before and after radiographic detection of lesions was broadly similar. 3. Numerical domination of particular sites by S mutans before detection of caries can occur, but has only been observed so far in 2 of 15 sites. 4. Pooled date from sites which have developed lesions indicate a rise in the isolation frequency and mean numbers of S. mutans after detection of caries. This trend was particularly obvious in the one subject who developed bilateral lesions by the second examination and in three of four sites where caries was detected at the fourth examination. Similar observations have been made with lactobacilli. 5. In two of 15 instances no isolations of S mutans were made from sites which developed caries. 6. To date, no single species appears to be uniquely associated with the onset of dental caries.

A Microbiological Study of Early Caries of Approximal Surfaces in Schoolchildren

A cross-sectional epidemiological study has been undertaken to relate the bacterial composition of approximal dental plaque with the earliest stages of caries development in schoolchildren. Small samples of plaque were removed from multiple sites around the contact areas of 42 premolars extracted for orthodontic reasons from 29 schoolchildren (mean age = 13.5 yr). Caries diagnosis was based on polarized light microscopy and contact microradiography of thin sections cut through the sample sites. Fifty-seven percent of sites (37l60) showed histological evidence of demineralization. Both the isolation frequency and the mean percentage viable count of mutans streptococci and Actinomyces viscosus were higher at sites with early caries, although mutans streptococci could not be detected at 37% of sites with early caries. At these latter sites, the proportions of Veillonella were markedly reduced. Lactobacilli were rarely isolated and were never recovered from caries-free surfaces. Analysis of the data shows that the relationship between plaque bacteria and enamel is neither merely passive nor indifferent, and that particular stages of lesion formation may be associated with different combinations of bacteria.

A Mixed-culture Chemostat System to Predict the Effect of Anti-microbial Agents on the Oral Flora: Preliminary Studies using Chlorhexidine

A mixed-culture chemostat system, composed of nine bacterial species representative of plaque in health and disease, has been assessed as an improved laboratory method of evaluating the likely in vivo effects of antimicrobial agents used in dentistry. The advantages of the system include reproducibility, the long-term stable cultivation of bacteria under controllable conditions, and repeated sampling, for bacteriological and biochemical studies, without disrupting the stability of the community. The effects of (i) the continuous provision of chlorhexidine (CHX) and (ii) three pulses of CHX (final concentration in both experiments = 0.24 mmol/L) on the composition of the chemostat communities were monitored. Only L. casei survived the continuous provision of CHX; the other bacteria were killed and were lost at different rates which generally corresponded to their known sensitivities to CHX. After each CHX pulse, the numbers of bacteria fell markedly. Again, L. casei was least affected, while A. viscosus, B. intermedius, and F. nucleatum were temporarily undetectable but returned to their original levels within 2-4 generation times. Counts of S. mutans were affected more by CHX than those of S. sanguis or S. mitior. The effect of successive pulses of CHX on the viability of some bacteria and on acid production (as measured by pH-fall experiments) decreased, suggesting that adaptation to CHX had occurred. The fact that the in vitro observations paralleled previous clinical findings suggests that the mixed-culture system could be used as a predictive model of the probable effect on the oral flora of new anti-microbial agents prior to expensive trials in animals or human volunteers.

Haemin-binding proteins of Porphyromonas gingivalis W50 grown in a chemostat under haemin-limitation

Porphyromonas gingivalis W50 was grown in a chemostat at pH 7.3 under haemin-limitation and haemin-excess at a constant mean doubling time of 6.9 h. Outer membranes (OM) were extracted from whole cells using EDTA and compared by SDS-PAGE. Haemin-limited cells expressed novel outer membrane proteins (OMPs) of mol. mass 115, 113 and 19 kDa when samples were solubilized at 100 degrees C. A 46 kDa OMP was observed in haemin-excess cells but not in those from haemin-limited conditions. Tetramethylbenzidine (TMBZ) staining of gels, after OM solubilization at 20 degrees C, was used to detect haemin-binding proteins (HBPs). HBPs were observed only in OM from haemin-limited cells. The major HBP (mol. mass 32.4 kDa) corresponded to a similar sized Kenacid-blue-stained protein which was not observed in haemin-excess-derived OM. Haemin-limited cells and OM displayed a ladder-like series of Kenacid-blue-stained proteins. Lighter TMBZ-stained proteins of mol. mass 51, 53, 56 and 60 kDa, with mobilities corresponding to those of silver-stained LPS components, were observed in haemin-limited OM. No soluble HBPs were detected extracellularly. The greater number of HBPs expressed by cells grown under haemin-limitation may reflect an additional cell surface receptor system for haemin acquisition under low environmental levels of this essential cofactor.

The use of defined inocula stored in liquid nitrogen for mixed culture chemostat studies

Abstract The efficacy of using inoculum of nine oral bacteria, stored in liquid nitrogen, to establish complex microbial communities in a chemostat was investigated. Only the numbers of Fusobacterium nucleatum were significantly reduced in the inoculum on storage for up to 15 weeks, although this did not affect its establishment and final proportion within community. The chemostat communities derived from the liquid nitrogen-stored inocula were similar to those obtained using conventional inoculation procedures. Where significant variations occured (i.e., in counts of Lactobacillus caser and Actinomyces viscosus , they were greater between using conventional techniques than between those using liquid nitrogen-stored inoculat. The composition of the chemostat community could be varied simply by substituting bacteria during the preparation of the inoculum. The convenience, simplicity and reproducibility of using liquid nitrogen storage of defined inocula could be of value in a range of ecological and industrial applications.