Criteria for the assessment of adverse effects of chemotherapy on the oral microflora.

Any assessment of an antimicrobial agent for the prevention of dental caries must include a consideration of its possible adverse effects on the oral flora. It must include an examination of the resistance developed by the target pathogens and either developed by, or inherently present in, microorganisms that are known to cause opportunistic infections (yeasts, enteric organisms, Pseudomonas, clostridia, and Staphylococcus aureus). Streptococcus pyogenes should also be included. Insofar as possible, these assessments should be done in preliminary experiments, both in vitro and clinical, rather than in caries trials, so that the large numbers of subjects will not be submitted to unnecessary exposure to the formulation. It must be recognized that some combinations of agent, vehicle, and frequency are more prone than others to cause adverse effects on the oral flora, and will also influence the length of the test period that should be utilized. The microbial assessments should be done on subjects prior to use of the agent, at appropriate frequencies during therapy, and approximately 2 months after the cessation of therapy. Currently, requirements to assess the state of gingival or periodontal health by microbiological examination seem unjustified, because of the relative lack of strong evidence for their diagnostic or predictive value (Ranney, 1989) and because of the ease with which direct measurements of clinical signs and symptoms can be made. Consequently, only the latter should be required.

Biotin-containing protein as a cause of false positive clones in gene probing with streptavidin/biotin.

Using a streptavidin/biotin labeling technique, we unintentionally cloned a gene encoding a biotin carboxyl carrier protein, a subunit of biotin-dependent enzymes, from a genomic library of Streptococcus mutans strain UT-041. In colony lifts, the clone reacted positively to the streptavidin-containing detection system but could not be detected in Southern blot analysis. The amino acid sequence of the gene product, deduced from its nucleotide sequence, demonstrated all the features common to biotin carboxyl carrier proteins from other bacteria, indicating that the biotin carboxyl carrier protein in the clone had produced a "false-positive" (DNA probe-independent) reaction by binding to the streptovidin. To circumvent this problem with the detection system in gene probing in the future, we recommend that all positive clones be screened by direct incubation with streptavidin-alkaline phosphatase (SA-AP) in the absence of biotin-labeled probe DNA. Clones binding to SA-AP would be considered false positives.

The effect of chlorhexidine varnish treatment on salivary mutans streptococcal levels in child orthodontic patients.

A chlorhexidine dental varnish was applied to the teeth of 26 children, ten to 17 years of age, in an attempt to limit the increase in colonization by mutans streptococci that normally accompanies the placement of fixed orthodontic appliances and to assess the acceptance of the application procedure. Despite the insertion of the appliances in the month following the varnish application, the numbers of detectable salivary mutans streptococci in the children were found to remain significantly lower than baseline values for seven months (p less than 0.01). Among the 26 children, 16 exhibited high counts (greater than 2.5 x 10(5) cfu/mL saliva) at baseline, but none exhibited such counts until three months post-treatment, when one child did. By seven months, eight children had high counts. No significant difference in effectiveness was observed between varnish formulations containing 10% or 20% chlorhexidine acetate, or between children of different ages or past caries experience. The lack of drop-outs and the results of a questionnaire indicated that acceptance of the treatment by the children was excellent. The study indicates that chlorhexidine varnish therapy was acceptable to the children and was effective in suppressing oral mutans streptococcal levels for long periods, even when used prior to the placement of fixed orthodontic appliances.

Clinical trial in adults of an antimicrobial varnish for reducing mutans streptococci.

An abbreviated treatment with a chlorhexidine-containing varnish was compared with a similar treatment with a placebo varnish and with a prophylaxis alone for its effects on the numbers of detectable salivary mutants streptococci in 51 adults. The varnishes, applied once weekly for four weeks, were held in place with a covering layer of either of two polyurethane sealants (FluorProtector or Adhesit). On the first appointment, the varnish-sealant combination was applied to all tooth surfaces, but on succeeding appointments only the occlusal and approximal surfaces were covered. The chlorhexidine varnish, covered with either sealant, reduced the salivary mutans streptococci by an average of 3 logs (99.9%) in all of the 20 subjects treated, and below detectable levels for at least four weeks in nine of them. In the groups receiving placebo varnish-sealant combinations, the mean log number of mutans streptococci of the subjects was reduced only by approximately 0.5 log (32%), and none of the subjects experienced loss of their detectable mutans streptococci for four weeks, although one subject did so for three weeks. No significant difference between the effects of the two polyurethane sealants was observed. Treatment with a single prophylaxis had no effect on mutans streptococcus levels. Subjects treated with chlorhexidine varnish also experienced an increase in S. sanguis and a small decrease in yeasts. Loss of detectable mutans streptococci did not cause changes in the numbers of other micro-organisms examined, beyond those observed with chlorhexidine varnish treatment alone.

Changes in strains of mutans streptococci induced by treatment with chlorhexidine varnish.

Isolates of mutans streptococci were obtained from the dental plaque of ten subjects before and after the subjects had been free of detectable mutans streptococci for a mean period of 14.6 weeks (range, from two to 36 weeks). The mutans streptococci had been rendered undetectable by chlorhexidine varnish treatment. Examination of the restriction endonuclease analysis (REA) patterns of the isolates revealed that all ten subjects had one strain (REA type) after re-appearance of the mutans streptococci that was identical to one that had been present before the varnish treatment. In six of the ten subjects, only one strain was detected both before and after treatment. Each of the other four subjects appeared to gain a new strain after treatment; one of the four appeared to lose one strain, and another, four strains. The ability of strains to persist after the period of undetectability seemed unrelated to their resistance to chlorhexidine or to their ability to exhibit insoluble glucan-mediated adhesion. In the subjects harboring multiple REA types, one-seventh of the tooth surfaces sampled harbored two strains simultaneously, suggesting an inability of either strain to exclude the other aggressively. Overall, the study indicated that every subject receiving chlorhexidine varnish therapy had a primary strain of mutans streptococcus that re-emerged after treatment. In contrast, secondary strains were highly susceptible to being lost or gained.

An investigation into the use of restriction endonuclease analysis for the study of transmission of mutans streptococci.

Restriction endonuclease analysis (REA) was performed on the total cellular DNA from each of 396 strains of mutans streptococci (1) to determine its potential usefulness for the study of transmission of the organism and (2) to document the proportions and variety of strains harbored by members of a small group of families. The DNA was digested with restriction enzyme EcoRI and/or HindIII, electrophoresed on agarose gels, and the resulting patterns compared. The strains examined included fresh isolates from 58 subjects, including 19 strains from each member of five families. The sensitivity and reproducibility of REA patterns from the mutans streptococci seemed ideal for studies of their epidemiology and transmission. The pattern of each isolate from humans was unique, except for isolates from the same individual or from the same family. REA types from subjects from different families were always heterogeneous. A high frequency of multiple REA types (up to 5) was observed in many subjects. While evidence for intra-familial transmission was obtained, including transmission between spouses, there was also strong evidence of frequent sources of infection outside of the family. Mutations of strains to streptomycin resistance or to lactate dehydrogenase deficiency caused no detectable change in the REA patterns. The lack of plasmids in any of the 57 fresh isolates that were examined for them suggested that they may have contributed little to the heterogeneity of the patterns seen.

Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation was slightly but not significantly (P greater than or equal to 0.2) less. Multiple oral or fecal samples plated on 2,3,5-triphenyltetrazolium indicator medium revealed no evidence of back mutation from Ldh- to Ldh+ in vivo. Both Ldh+ strain 041 and Ldh- strain 044 demonstrated bacteriocinlike activity in vitro against a number of human strains of mutans streptococci representing serotype a (S. cricetus) and serotypes c and e (S. mutans). Serotypes b (S. rattus) and f (S. mutans) and strains of S. mitior, S. sanguis, and S. salivarius were not inhibited. Thus, Ldh mutant strain 044 possesses a number of desirable traits that suggest it should be investigated further as a possible effector strain for replacement therapy of dental caries. These traits include its stability and low cariogenicity in the sensitive gnotobiotic rat caries model, its bacteriocinlike activity against certain other cariogenic S. mutans (but not against more inocuous indigenous oral streptococci), and the fact that it is a member of the most prevalent human serotype of cariogenic streptococci.

A preliminary report of long-term elimination of detectable mutans streptococci in man.

Application of an antimicrobial varnish to the teeth of 33 adult volunteers resulted in the elimination of detectable mutans streptococci from the saliva of 21 of them for a mean period of 34.6 weeks (range, 4 to 89 weeks) without additional treatment. The mean number of applications of varnish required for elimination was 3.14 (range, 1 to 5). Extensive examination of 10 subjects made free of mutans streptococci on the basis of saliva examination revealed no detectable mutans streptococci in their dental plaque. In 14 of the subjects in whom mutans streptococci were eliminated, they subsequently re-appeared after a mean period of 22.7 weeks (range, 4 to 71 weeks). Four out of the five recurrences that were treated were eliminated with only one additional varnish application. The treatment failed to provide long-term elimination of detectable mutans streptococci in 12 of the 33 treated subjects. No serious adverse reactions were observed in any of the treated subjects. The results indicate that it is possible to eliminate mutans streptococci from man in a safe and effective manner.

Effects of cheese extract and its fractions on enamel demineralization in vitro and in vivo in humans.

In order to isolate and identify the most active anti-cariogenic components(s) of aqueous cheese extract (CE), we separated it into low (LMW) (MW less than 500), medium (MMW) (500 less than MW less than 10,000), and high (HMW) (MW greater than 10,000) molecular weight fractions by means of the Amicon ultrafiltration system. These fractions were then tested in vitro with a bacterial system containing S. mutans, adapted from that of Turtola (1977). The LMW fraction reduced the demineralization caused by the fermentation of sucrose by 96% (p less than 0.001) as compared with the water control; this was not significantly different from a 50% concentration of the CE. The MMW and HMW fractions reduced demineralization by 36 and 42%, respectively. The concentrations of acid-soluble calcium and phosphorus in CE, LMW, MMW, and HMW were 1509 and 462, 991 and 310, 231 and 7, and 162 and 3 micrograms/mL, respectively. A solution containing the same levels of calcium and phosphorus as CE was somewhat more effective in reducing demineralization in vitro than was CE itself (p less than 0.01). In vivo, the addition of these same calcium and phosphorus levels to a 10% sucrose solution reduced its cariogenicity by 67% (p less than 0.001), as judged by the intra-oral cariogenicity test (ICT). Plaque calcium and phosphorus concentrations were significantly higher in the ICT plaque samples subjected to the sucrose-Ca,P solution (p less than 0.01) than in the sucrose control. The resting pH, minimum pH, and shape of the pH curves produced by the sucrose control and sucrose-Ca,P were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of water-soluble components of cheese on experimental caries in humans.

The effect of water-soluble components of extra-old Cheddar cheese on experimental caries was tested by means of the seven-day intraoral cariogenicity test (ICT). Two bovine enamel blocks were placed in each buccal flange of the dental appliances of five volunteers. One side of each appliance (experimental) was dipped in a 25% water extract of the cheese for five min, while the other side (control) was dipped in de-ionized water. Immediately thereafter, the appliance was returned to the subject's mouth, and two 60-second rinses with 10% sucrose were performed. These procedures were repeated six times per day. The cheese-extract dippings reduced the cariogenicity of the sucrose by an average of 55.7% (p less than 0.01), as assessed by enamel microhardness. Neither the mean resting pH nor the mean minimum pH in response to sucrose was significantly different between the experimental and control sides. The concentration of calcium was significantly higher in plaque from the experimental side (32.44 micrograms/mg) as compared with the control side (19.36 micrograms/mg, p less than 0.01). The concentration of plaque phosphorus was higher on the experimental side (12.90 micrograms/mg) than on the control side (9.61 micrograms/mg); however, the difference was not statistically significant. These results show that cheese has one or more water-soluble components which reduce experimental caries in human subjects.

Development of sustained-release antimicrobial dental varnishes effective against Streptococcus mutans in vitro.

Two antimicrobial varnishes were developed to be applied to the teeth for the eradication of Streptococcus mutans infections. One of them contained chlorhexidine acetate as the antimicrobial agent and the other, erythromycin base. Both varnishes contained Sumatra benzoin. Each of the two antimicrobial agents was shown to be highly effective against S. mutans and to be compatible with the benzoin vehicle. When applied to extracted teeth, both varnishes dried to form a tough, adherent, and colorless transparent layer. Dried samples of the varnishes, when suspended in frequently-changed buffer, released their microbial agents at low but bactericidal levels for at least 12 days. After the first day, drug release from the chlorhexidine varnish showed approximately zero-order kinetics, while the erythromycin varnish showed a combination of zero-order and first-order kinetics.

Serotype c Streptococcus mutans mutatable to lactate dehydrogenase deficiency.

Three lactate-dehydrogenase-deficient mutants of serotype c S. mutans were made by using, as parents, two serotype c strains that produced unusually large amounts of ethanol, acetic acid, and acetoin, and very little lactic acid, when grown in broth containing a limiting amount of glucose. The mutants, obtained with N-methyl-N'-nitro-N-nitrosoguanidine, were stable during 12 weeks of daily subculture in broth. Crude cell-free extracts of the mutants had less than 1% of the LDH-specific activity of their parent strains. The serotype c mutants resembled serotype g mutants in having molar growth yields at least as high as those of their parents. However, in contrast to the g mutants, the c mutants produced cell crops (cell mass per ml medium) that were as high as those of their parent strains.

Lactate dehydrogenase-deficient mutants of serotype g Streptococcus mutans.

Three LDH-deficient mutants of a serotype g strain of Streptococcus mutans were produced essentially as described by Hillman (1978): They were produced using ethylmethane sulfonate, and isolated using triphenyltetrazolium-glucose agar. Similar attempts to obtain mutants from a serotype c strain were unsuccessful. The g mutant did not revert during 12 weeks of daily transfer in broth, and, when grown in glucose-containing broth, they reached pH values of 4.9 to 5.0, compared with 4.4 for the parent strain. The efficiency of conversion of glucose to cell mass (Yg) was at least as great with the mutants as with the parent strain. The LDH activities of the mutants were less than 1% of that of the parent strain. Like Hillman's (1978) mutants, ethanol, acetic acid, and acetoin were the major products resulting from the metabolism of glucose. Although at pH 7.0 the mutants grew more slowly than did the parent, at pH 5 and pH 6 one of the mutants grew as rapidly as did the parent. The stability, serotype, and ability of these mutants to grow at low pH suggest their potential usefulness for replacement therapy.

In vitro effects of low concentrations of penicillin and sulfadiazine on Streptococcus mutans.

Investigations were conducted to determine the in vitro effects of low levels of penicillin and sulfadiazine on the growth, plaque formation, and agglutination of Streptococcus mutans and on the synthesis and activity of enzymes synthesizing extracellular polymers. The concentrations tested were equivalent to those expected in the saliva of subjects receiving oral therapy with the agents. Penicillin at 0.5 ng/ml and sulfadiazine at 1 mug/ml substantially inhibited in vitro plaque formation. At these concentrations, sulfadiazine but not penicillin also inhibited growth of the organism. Neither antimicrobial agent affected the agglutination of S. mutans with dextran or the synthesis or activity of enzymes synthesizing extracellular polymers. The effect of sulfadiazine on plaque formation was attributed, at least in part, to the inhibitory action of that agent on S. mutans growth.

Effect of long-term therapies with penicillin and sulfadiazine on Streptococcus mutans and lactobaccilli in dental plaque.

Plaque samples were obtained from 13 children receiving long-term therapy with benzathine penicillin for the prevention of rheumatic fever recurrences, 31 children receiving oral sulfadiazine for the same purpose, and 29 untreated siblings. The therapies were found to have no effect upon the proportions of Streptococcus mutans or lactobacilli in dental plaque, upon the percentage of children harboring the organisms, nor upon the susceptibility of the organisms to penicillin and sulfadiazine. Of the S. mutans strains tested, 97% had a minimal inhibitory concentration of penicillin G of less than 48 ng/ml and, of the lactobacillus strains tested, 96.8% had a minimal inhibitory concentration of less than 1,600 ng/ml. All strains of both organisms were profoundly resistant to sulfadiazine.