Emergence of antibiotic resistant Streptococcus sanguis in dental plaque of children after frequent antibiotic therapy.

PURPOSE: In the pediatric population, several different antibiotic regimens are currently recommended for the treatment of otitis media. This study investigated whether therapy for otitis media was associated with the emergence of antibiotic-resistant oral bacteria. METHODS: Streptococcus sanguis (S. sanguis) was isolated from supragingival dental plaque of children after a recent course of antibiotic. The isolated strains were tested for resistance to penicillin, amoxicillin, trimethoprim-sulfamethoxazole, and erythromycin and compared to isolated strains from age- and sex-matched control subjects, who had received no antibiotics within two years before sampling. RESULTS: While control subjects harbored no resistant strains of S. sanguis, about 60% of children who had received antibiotics harbored S. sanguis which were resistant to at least one of the tested antibiotics. Nearly half of these strains were resistant to two or more antibiotics. Resistance to penicillin and amoxicillin decreased with the age of the child and with the length of time since exposure to the antibiotic. However, resistance to trimethoprim-sulfamethoxazole or erythromycin showed no relationship to the age of the child or the length of time since exposure to the antibiotic. CONCLUSION: The data show that children who had been treated for otitis media with common antibiotic protocols do harbor antibiotic-resistant oral streptococci which may complicate prophylactic and therapeutic regimens for bacterial endocarditis.

Investigation of the role of human breast milk in caries development.

PURPOSE: The objective of this study was to determine the caries-related risk associated with human breast milk (HBM). METHODS: First, the plaque pH of 18 children (12-24 months) was monitored before and after a five-minute feeding with HBM to determine the pH drop and minimum pH obtained. Second, Streptococcus sobrinus 6715 was cultured for 3 hr in HBM, and the increase in the number of colony forming units (cfus) and the culture pH was measured. Third, HBM was incubated for 24 hr with powdered enamel to determine the solubility of mineral in the absence of bacteria. Fourth, HBM was mixed with acid to determine the buffering capabilities. Finally, enamel windows were created on extracted premolar crowns (N = 33) that were colonized with Mutans Streptococci and incubated with HBM. Caries was assessed visually and radiographically for 12 weeks. RESULTS: One- and two-way ANOVAs of these five assays demonstrated that HBM did not cause a significant drop in plaque pH when compared to rinsing with water; HBM supported moderate bacterial growth; calcium and phosphate were actually deposited onto enamel powder after incubation with HBM; the buffer capacity of HBM was very poor; and HBM alone did not cause enamel decalcification even after 12 weeks exposure. However, when supplemented with 10% sucrose, HBM caused dentinal caries in 3.2 weeks. CONCLUSION: It is concluded that human breast milk is not cariogenic.

Estimation of the caries-related risk associated with infant formulas.

PURPOSE: Baby bottle tooth decay (BBTD) affects 6% of children under three years of age and is associated with inappropriate bottle use. The objective of this study was to estimate the caries-related risk associated with 26 infant formulas and whole milk. METHODS: First, the plaque pH of adult volunteers was monitored before and after an oral rinse with infant formula to determine the minimum pH obtained in response to each formula. Second, Streptococcus sobrinus 6715 was cultured in each infant formula, and the increase in the number of colony forming units was measured. Third, each infant formula was incubated with powdered enamel and the solubility of enamel mineral was calculated in the absence of bacteria. Fourth, each formula was mixed with standardized concentrations of acid to determine the buffering capabilities. Finally, enamel windows were created on extracted permanent molars and exfoliated primary incisor crowns that were then colonized with mutans streptococci and incubated with infant formula. Caries was assessed visually and radiographically for 18 weeks. The length of time required for the development of enamel caries, dentinal caries and pulpal involvement was recorded. RESULTS: One-way or two-way ANOVA of these five assays demonstrated that 1. Plaque pH varied in response to oral rinsing with infant formula and most formulas did have the ability to reduce the pH significantly below the pH obtained after rinsing with water 2. Some infant formulas supported significant bacterial growth 3. Enamel mineral was dissolved by incubation with certain infant formula 4. The buffer capacity varied among the infant formulas tested 5. The length of time required for caries to reach dentin or pulp differed for the formulas, with some formulas causing dentinal caries by 3.4 weeks and pulpal involvement by 7.2 weeks.

A survey of the American Academy of Pediatric Dentistry membership: infant oral health care.

The AAPD has long held that preventive oral health care instituted during the first year of life results in long-term enhanced oral health status. A 33-item questionnaire addressing membership attitudes regarding infant oral health care was mailed to 1500 active AAPD members in the spring of 1996. The 913 (60.9%) responses were received, and descriptive statistics were obtained. Results suggest that the respondents were representative of the demographics of the AAPD membership. While 72.6% of the respondents agreed with the AAPD policy, only 46.6% practiced the AAPD policy of performing the first oral evaluation at age 12 months or younger. Agreement with the policy and the age at which infant evaluations were recommended were dependent upon the age of the respondent. Younger practitioners were significantly more likely to agree with the policy (85%) and perform evaluations according to the guidelines (66%). The Academy must provide better communication to the established membership regarding the rationale for early visits and how to perform infant evaluation. Nearly 20% of respondents reported that they did not perform infant evaluations, mainly because 1) existing conditions, and not age, should be the reason for seeing these patients (78.4%), and 2) parents don't see the value (64.0%). A variety of responses were given when asked how the AAPD could assist these persons in providing this service to their patients. The most common suggestions were: 1. Educate pediatricians/primary care providers about the value of early dental evaluations, 2. Offer guidelines or a protocol for incorporation of infant evaluations into an office routine, 3. Prepare materials and/or brochures for education of parents, and 4. Organize a public relations promotion demonstrating the value of early examinations.

Primary incisor decay before age 4 as a risk factor for future dental caries.

The purpose of this investigation was to determine whether early childhood caries (ECC) is a risk factor for future dental caries. One hundred fifteen dental charts of children younger than 4 years of age when initially treated were reviewed and abstracted for primary incisor caries and age at the initial examination, gender, recall dental visits, sealants, and age at the last dental examination. In addition, the number of carious, extracted, and restored teeth (cert/CERT: primary/secondary) at the last examination was determined. Children with ECC at their initial examination (N = 58) had a 93.0% cert rate, a 67.2% CERT rate and a 60.3% CERT in first molars rate by their last dental examination. Non-ECC children at their initial examination (n = 57) had less than half the rate of each cert/CERT parameter (43.9%, 22.8%, and 26.3%, respectively) at their last dental visit. The odds ratios for each cert/CERT parameter posed by ECC status were 17.3 for cert, 7.0 for CERT, and 4.3 for CERT in first molars. When these odds were adjusted for other study parameters by a forward step-wise logistic regression analysis, ECC status continued to be a risk factor for each cert/CERT parameter. We conclude that 1) early childhood caries is a risk factor for future caries, 2) increased age is a risk factor for CERT, and 3) recalls and sealants are protective factors.

Evaluation of plaque pH changes following oral rinse with eight infant formulas.

Inappropriate feeding habits have been identified as major factors associated with the development of baby bottle tooth decay or nursing caries. An in vivo/in vitro combination technique was developed to investigated the plaque pH changes associated with rinsing with eight different infant formulas. These eight formulas represented four categories: 1) formulas with iron, 2) formulas with low iron, 3) soy formulas 4) and protein hydrolyzate formulas (from the manufacturers Mead Johnson Nutritionals and Ross Laboratories). All formulas had the ability to reduce the pH significantly below the pre-rinse plaque pH. Furthermore, the average minimum pH for formulas from the two manufacturers did not differ within each formula category except for the soy-based formulas, where, rinsing with Isomil produced a significantly lower plaque pH than ProSobee. These results suggest that infant formulas are acidogenic and therefore may play a significant role in the development of baby bottle tooth decay.

Altered expression of the platelet aggregation-associated protein from Streptococcus sanguis after growth in the presence of collagen.

Certain strains of Streptococcus sanguis adhere selectively to human platelets (Adh+) and, in plasma, induce them to aggregate into in vitro thrombi (Agg+). The induction of aggregation is mediated by the platelet aggregation-associated protein (PAAP) expressed on the cell surface of the streptococcus. In endocarditis, expression of PAAP may be regulated by association with host proteins on damaged heart valves. To begin to test this hypothesis, three strains of S. sanguis were each cultured in the presence or absence of collagens (types I to X), laminin, or PAAP-derived peptide preparations. After harvesting and washing, the platelet-interactive phenotype of strains 133-79 (Adh+ Agg+), L74 (Adh+ Agg-), and 10556 (Adh- Agg-) was unchanged. The cells from each culture were then digested mildly with trypsin to isolate PAAP. PAAP isolated from strain 133-79 (Adh+ Agg+) grown in the absence of added collagen, other proteins, or peptides inhibited platelet aggregation in response to untreated cells of S. sanguis. Platelet aggregation was induced immediately, however, by PAAP from strain 133-79 isolated after growth in the presence of 300 nM type I collagen, while lower concentrations yielded protein fragments that potentiated the response to intact cells. Aggregation-inducing PAAP could be removed by anti-PAAP (PGEQGPK) immunoaffinity chromatography, but only inhibitory activity could be recovered. The agonist effect of PAAP was not associated with collagen itself, since the PAAP preparations did not contain detectable amounts of hydroxyproline. PAAP antigens isolated from cells grown in the presence and absence of collagen had similar apparent molecular weights, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. When electrophoresis was performed under nondenaturing conditions, however, PAAP isolated from cells grown in type I collagen migrated more slowly. Strain L74 grown with type I collagen yielded tryptic fragments of proteins that inhibited aggregation significantly better than control peptides (no collagen in the medium). Strain 10556 was apparently unaffected by growth in type I collagen. The effect of type I collagen was somewhat unique. Growth in the presence of collagen types II to VI (300 nM) yielded protein fragments that potentiated without inducing platelet aggregation, while other collagens, laminin, and PAAP-derived peptides did not affect platelet aggregation. These results suggest that growth in the presence of type I collagen and, perhaps, collagens II to VI alters the expression and conformation of PAAP in certain strains of S. sanguis.

Evidence for the covalent linkage of carbohydrate polymers to a glycoprotein from Streptococcus sanguis.

The platelet aggregation-associated protein (PAAP) from Streptococcus sanguis contains 39% carbohydrate in rhamnose-rich polysaccharides. Synthesized by cultured protoplasts, these polysaccharides were shown to be covalently linked to this cell wall protein using specific inhibitors of glycosylation, glycosidase treatment, amino acid and carbohydrate analyses of prepared minimal glycopeptides and isolated protein, and NMR spectroscopy. To our knowledge, this is the first direct proof of an N-asparaginyl linkage of carbohydrate to a eubacterial protein.

The Streptococcus sanguis platelet aggregation-associated protein. Identification and characterization of the minimal platelet-interactive domain.

Streptococcus sanguis expresses a cell wall-bound protein that induces the activation and aggregation of platelets. This platelet aggregation-associated protein (PAAP) contains a collagen-like, platelet-interactive domain within a 23-kDa protein fragment. To isolate the minimal platelet-interactive domain, p23 PAAP was digested with collagenase, and the digest chromatographed to isolate fractions with activity inhibitory to S. sanguis-induced platelet aggregation. The active fraction was then digested with cyanogen bromide, the product chromatographed, and a smaller inhibitory peptide isolated. Finally, this fraction was digested with endoproteinase Lys-C, and the digest fractionated. After each step, inhibitory activity resolved into single chromatographic peaks of 13 kDa (p13 PAAP), 2.7 kDa (p2.7 PAAP), and a minimal 7-mer peptide, respectively. These PAAP fragments showed similar ID50 (19-28 nM), suggesting that each contained a single copy of the platelet-interactive domain. The minimal 7-mer peptide was purified by immunoaffinity chromatography and reverse phase high pressure liquid chromatography. The primary structure was determined to be Pro-Gly-Glu-Gln-Gly-Pro-Lys. This sequence conforms to the predicted structural motif of the platelet-interactive domains of types I and III collagen. This 7-mer peptide is therefore the platelet-interactive domain of the PAAP from S. sanguis. Its structure explains the molecular basis for immunological cross-reactivity and functional similarity to the platelet-interactive domains of collagens.

The platelet interactivity phenotype of Streptococcus sanguis influences the course of experimental endocarditis.

A strain of Streptococcus sanguis that induced rabbit platelets to aggregate in vitro (Agg+ phenotype) was hypothesized to be a more virulent pathogen than an Agg- strain in experimental endocarditis in rabbits. A left ventricular catheter was implanted, and then an Agg+ or Agg- strain was inoculated intravenously. Vegetations formed on the aortic semilunar valves but were unaffected by the duration of implantation of the catheter. Vegetations enlarged by accumulating platelets and their mass increased directly with the duration of endocarditis. Inoculation of the Agg+ strain consistently caused endocarditis with significantly larger vegetations, a more severe clinical course (including febrile episodes, hematological changes, and signs of myocardial ischemia), more gross lesions in major organs, and greater mortality than inoculation with the Agg- strain, saline, or the Agg+ strain pretreated with monospecific rabbit immunoglobulin G or Fab fragments against its platelet aggregation-associated protein (PAAP; class II). In experimental endocarditis, PAAP expressed by Agg+ S. sanguis appeared to be an important virulence factor.

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.

Platelet-interactive products of Streptococcus sanguis protoplasts.

To isolate a more native, platelet-interactive macromolecule (class II antigen) of Streptococcus sanguis, cultured protoplasts were used as a source. Protoplasts were optimally prepared from fresh washed cells by digestion with 80 U of mutanolysin per ml for 75 min at 37 degrees C while osmotically stabilized in 26% (wt/vol) raffinose. Osmotically stabilized forms were surrounded by a 9-nm bilaminar membrane, as shown by transmission electron microscopy. Protoplasts were cultured in chemically defined synthetic medium and osmotically stabilized by ammonium chloride. Spent culture media were harvested daily for 7 days. Each day, soluble proteins were isolated from media, preincubated with platelet-rich plasma, and tested for inhibition of platelet aggregation induced by S. sanguis cells. Products released from S. sanguis protoplasts and reactive with an anti-class II antigen immunoaffinity matrix were able to inhibit S. sanguis-induced platelet aggregation. As resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, anti-class II-reactive protoplast products included silver-stained bands of 67, 79, 115, 216, and 248 kDa. The 115-kDa protein fraction was isolated by gel filtration and ion-exchange chromatography. This form of the class II antigen contained N-formylmethionine at its amino terminus. Rhamnose constituted 18.2% of the total residual dry weight and nearly half of its carbohydrate content. Diester phosphorus constituted 1% of this fraction. After trypsinization of the protoplast products from either preparation, a 65-kDa protein fragment was recovered. This protoplast protein fragment and the S. sanguis cell-derived 65-kDa class II antigen, previously implicated in the induction of platelet aggregation, were shown to be functionally and immunologically identical.

Purification and partial characterization of a 65-kDa platelet aggregation-associated protein antigen from the surface of Streptococcus sanguis.

Cells of Streptococcus sanguis express a collagen-like immunodeterminant (class II antigen) on their cell walls that induces aggregation of platelets in plasma. These platelet aggregation-associated proteins (PAAPs) are recovered in cell-free preparations obtained from cells of S. sanguis after 5 min of sonic or limited trypsin treatment. Pretreatment of platelet-rich plasma with these soluble preparations selectively inhibits platelet aggregation in response to S. sanguis cells. A PAAP antigen was isolated and purified from minimal tryptic digests of S. sanguis cells using (i) immunoaffinity chromatography or (ii) gel filtration and ion-exchange chromatography. A monospecific rabbit antiserum was prepared against PAAP (from procedure ii) and used to verify identity with PAAP fragments in different preparations. Criteria of purity included single precipitins in immunoelectrophoresis and crossed immunoelectrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblot, and COOH (Lys)- and NH2 (Pro)-terminal analyses. The 65-kDa (p65) antigen isolated by immunoaffinity chromatography had 50-fold greater specific inhibitory activity in S. sanguis-induced PRP aggregation than the original tryptic digest and about 1.4 times that recovered by sequential column chromatography. Amino acids of the p65 PAAP fragment constituted 89.5% of the total dry weight, with glycine, lysine, and glutamic acid predominant. Lesser amounts of proline were also noted. Monosaccharides, including glucose and galactose, comprised 4.0% of the total. A platelet interactive determinant of p65 was localized to a 23-kDa tryptic fragment after further trypsin treatment. Amino acids of this 23-kDa fragment constituted 99.8% of the total dry weight. In their native state on the cell wall of platelet-interactive strains of S. sanguis, platelet aggregation-associated proteins are probably assembled on fibrils as polyvalent agonists.

Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis.

Certain strains of Streptococcus sanguis adhere (Adh+) selectively to human platelets and, in plasma, induce them to aggregate (Agg+) into in vitro thrombi. In this study, we examined 18 recent endocarditis and dental plaque isolates of microorganisms that were biotyped as S. sanguis for coexpression of platelet interactivity phenotypes with another possible virulence factor in bacterial endocarditis, dextran synthesis. Detectable production of extracellular glucosyltransferase ranged from 0.2 to 66 mU/mg of culture fluid for 10 representative strains tested. Production of extracellular or cell-associated glucosyltransferase, fructosyltransferase, and soluble or insoluble dextrans was not necessarily coexpressed with platelet interactivity phenotypes, since the levels of production of soluble and insoluble dextrans varied among representative Adh+ Agg+ and Adh- Agg- strains. Analysis of a second panel of 38 fresh dental plaque isolates showed that S. sanguis distributes in a reproducible manner into the possible phenotype groups. Strains with different platelet interactivity phenotypes were distinguished with a panel of four murine monoclonal antibodies (MAbs) raised against Adh+ Agg+ strain 133-79 and screened to rule out artifactual reactions with antigenic components in culture media. The MAbs reacted selectively with Adh+ Agg+ strains in a direct-binding, whole-cell, enzyme-linked immunosorbent assay and also inhibited their interactions with platelets. Analysis of minimal tryptic digests of many strains, including variants that failed to bind the MAbs, suggested that some noninteractivity phenotypes possess cryptic surface determinants. Since the ability to adhere to platelets and induce them to aggregate is relatively stable, these traits may be useful in a phenotyping scheme for these Lancefield nontypeable streptococci.

A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation.

The basis of similarities in the mechanism of human platelet aggregation induced by soluble collagen and the dental plaque bacterium Streptococcus sanguis was analyzed. Structural and functional comparisons were made by using molecular probes, including rabbit antibody fractions reactive with components on S. sanguis and a synthetic, collagen-like octapeptide mimicking segments from cyanogen bromide fragments 6 and 4 of types I and III collagen, respectively. When platelets were pretreated with tryptic peptides or class II antigen of S. sanguis or with the synthetic, collagen-like octapeptide, the onset of aggregation in response to S. sanguis and collagen was prolonged. When compared to other peptides of similar size and charge, the collagen-like peptide's action towards platelets was shown to be selective. Indeed, absorption of antiserum to S. sanguis cells with particulate type I collagen removed specificities directed at a single S. sanguis antigen. These observations suggested that a common platelet-interactive immunodeterminant on soluble types I and III collagens, particulate type I collagen, and S. sanguis cells was present. Selective inhibition by antibody was used to show structural similarities between the S. sanguis surface proteins and collagen. When either agonist was pretreated with anti-S. sanguis IgG or Fab fragments, the lag time to onset of platelet aggregation was increased. Greater increases in the lag time to aggregation was seen when S. sanguis cells or collagen were pretreated with anti-S. sanguis IgG or Fab fragments made relatively specific for the class II antigen. Neutralization of the platelet-interactive action of the octapeptide by anti-S. sanguis antibody fractions showed that the immunodeterminant common to S. sanguis and collagen triggered platelets in plasma to aggregate. Although the anti-S. sanguis antibodies could inhibit fibrillogenesis, this action was apparently independent of interactions with platelets. In contrast, S. sanguis could bind or adhere to platelets by different determinants. Our data suggest that platelets have at least two distinct sites that bind collagen or S. sanguis. One of these may be a common site for collagen and S. sanguis agonists.

Reduced absorption of 45calcium from isolated duodenal segments in vivo in juvenile but not adult X-linked hypophosphatemic mice.

In juvenile X-linked hypophosphatemic (Hyp) mice, whole body calcium balances are significantly lower than in genetically normal mice. This is associated with low duodenal vitamin D-dependent calcium-binding protein and a failure of skeletal mineralization. To seek more specific evidence of an intestinal defect in these mice, absorption of 45Ca was measured in isolated duodenal segments in vivo in mice from 2-13 weeks of age. The duodenum was isolated by sutures and 45Ca was injected into the lumen in 150 mM NaCl and 2 mM CaCl2 at pH = 7.2. Absorption was measured by the amount of isotope remaining in the lumen and by the plasma isotope level. Hemizygous Hyp male and heterozygous Hyp female mice absorbed significantly less 45Ca at 4 and 7 weeks of age than genetically normal mice while Hyp mice at 2, 10, and 13 weeks of age were not significantly affected. At 4 and 7 weeks of age, the Hyp mice also had significantly reduced plasma radioactivity midway through the collection period as well as at the end of the period. To explore a possible mechanism for this malabsorption, 1,25(OH)2-vitamin D receptors were measured in cytosol prepared from 4-week-old normal and Hyp duodenum. There were non-significant differences between the normal and Hyp mice in both binding affinity, Kd, and the number of receptors, nmax. In conclusion, juvenile Hyp mice at 4 and 7 weeks of ages malabsorbed calcium from their duodenum. Hyp mice younger than this period were not affected nor were adult Hyp mice.(ABSTRACT TRUNCATED AT 250 WORDS)