Biomedical subjects

D H Fine

Publications and source records attributed to D H Fine.

At least 19 recordsLinked to original sources

PubMed2006

Genetic analysis of the requirement for flp-2, tadV, and rcpB in Actinobacillus actinomycetemcomitans biofilm formation.

The tad locus of Actinobacillus actinomycetemcomitans encodes a molecular transport system required for tenacious, nonspecific adherence to surfaces and formation of extremely strong biofilms. This locus is dedicated to the biogenesis of Flp pili, which are required for colonization and virulence. We have previously shown that 11 of the 14 tad locus genes are required for adherence and Flp pilus production. Here, we present genetic and phylogenetic analyses of flp-2, tadV, and rcpB genes in biofilm formation. We show that tadV, predicted to encode prepilin peptidase, is required for adherence. In contrast, targeted insertional inactivation of flp-2, a gene closely related to the prepillin gene flp-1, did not abrogate biofilm formation. Expression studies did not detect Flp2-T7 protein under standard laboratory conditions. We present phylogenetic data showing that there is no significant evidence for natural selection in the available flp-2 sequences from A. actinomycetemcomitans, suggesting that flp-2 does not play a significant role in the biology of this organism. Mutants with insertions at the 3' end of rcpB formed biofilms equivalent to wild-type A. actinomycetemcomitans. Surprisingly, 5' end chromosomal insertion mutants in rcpB were obtained only when a wild-type copy of the rcpB gene was provided in trans or when the Tad secretion system was inactivated. Together, our results strongly suggest that A. actinomycetemcomitans rcpB is essential in the context of a functional tad locus. These data show three different phenotypes for the three genes.

Aggregatibacter actinomycetemcomitans↗

PubMed2005

A non-conservative, coding single-nucleotide polymorphism in the N-terminal region of lactoferrin is associated with aggressive periodontitis in an African-American, but not a Caucasian population.

Lactoferrin is an antimicrobial protein which plays an important role in regulating bacteria that are associated with aggressive periodontitis. Lactoferrin kills directly (via its strongly cationic N-terminal region) and indirectly, through sequestering the iron that bacteria require for growth. As aggressive periodontitis has a strong heritable component, we hypothesized that genetic variation within the lactoferrin gene may play a role in susceptibility to this condition. We have identified and examined a novel, functional, single-point A/G nucleotide mutation causing a threonine/alanine substitution at position 11 (T11A) of the secreted lactoferrin protein. In a pilot case-controlled study of aggressive periodontitis, analysis of 46 African-American patients and 78 controls showed that patients were twice as likely to express the G nucleotide (alanine) allele over controls (60.3 vs 30.4%; P=0.0007, odds ratio=2.564, 95% CI=1.475-4.459). A Caucasian population of 77 patients and 131 controls showed no such association (P=0.5201, odds ratio=0.862, 95% CI=0.548-1.356). The data presented provide a new insight into the genetic susceptibility to aggressive periodontitis.

Black or African American↗

PubMed2005

In vivo antimicrobial effectiveness of an essential oil-containing mouth rinse 12 h after a single use and 14 days' use.

OBJECTIVES: Two studies were conducted to determine the antimicrobial effect of rinsing with an essential oil-containing mouth rinse 12 h after a single rinse and 12 h after 2 weeks of twice daily rinsing, during the daytime and overnight. MATERIALS AND METHODS: These studies utilized a randomized, double-blind, controlled crossover design. Following baseline sampling of bacteria from supragingival plaque and the dorsum of the tongue, subjects began twice-daily rinsing with either an essential oil mouth rinse containing 0.09% zinc chloride (Tartar Control Listerine Antiseptic) or a negative control rinse. Bacterial sampling was repeated 12 h after the first rinse, and again 12 h after the final rinse 14 days later. The sampling schedule was adjusted according to whether the study was investigating daytime or overnight activity. Samples were plated on Schaedlers medium (total anaerobes), Schaedlers Nalidixic/Vancomycin medium (Gram-negative anaerobes), and OOPS medium (volatile sulphur compound (VSC)-producing organisms). Inter-group log10 transformed colony-forming units/ml counts from samples of supragingival plaque and tongue swabs on each of the three media were compared by analysis of covariance. RESULTS: The mean bacterial counts in subjects using the essential oil mouth rinse were significantly lower (p< or =0.005) than mean counts in subjects using the control rinse in all the comparisons, i.e., tongue and supragingival plaque samples on each of three media at two sampling periods in the daytime and overnight study, respectively. Mean bacterial count percent reductions for plaque samples ranged from 56.3 to 95.3; percent reductions for tongue samples ranged from 61.1 to 96.1. There was a trend to higher reductions after 14 days' rinsing than after the initial rinse. CONCLUSION: Rinsing with the essential oil mouth rinse can have long-lasting effects in reducing anaerobic bacteria overall as well as Gram-negative anaerobes and VSC-producing bacteria. The significant reductions in numbers of these bacteria produced by the essential oil mouth rinse, both in plaque and on the dorsum of the tongue, can play a key role in explaining the essential oil mouth rinse's effectiveness in reducing supragingival plaque and gingivitis as well as its effectiveness in controlling intrinsic oral malodor over prolonged periods.

Adult↗

PubMed2004

Antimicrobial effects of a new therapeutic liquid dentifrice formulation on oral bacteria including odorigenic species.

The control of oral malodor is well-recognized in efforts to improve oral health. Antimicrobial formulations can mitigate oral malodor, however, procedures to assess effects on oral bacteria including those implicated in halitosis are unavailable. This investigation examined the antimicrobial effects of a new liquid triclosan/copolymer dentifrice (test) formulation that demonstrated significant inhibition of oral malodor in previous organoleptic clinical studies. Procedures compared antimicrobial effects of the test and control formulations on a range of oral micro-organisms including members implicated in halitosis, substantive antimicrobial effects of formulations with hydroxyapatite as a surrogate for human teeth and ex vivo effects on oral bacteria from human volunteers. With Actinomyces viscosus, as a model system, the test formulation demonstrated a dose-dependent effect. At these concentrations the test formulation provided significant antimicrobial effects on 13 strains of oral bacteria including those implicated in bad breath at selected posttreatment time points. Treatment of hydroxyapatite by the test dentifrice resulted in a significant and substantive antimicrobial effect vs. controls. Oral bacteria from subjects treated ex vivo with the test dentifrice resulted in significant reductions in cultivable oral bacteria and odorigenic bacteria producing hydrogen sulfide. In summary, microbiological methods adapted to study odorigenic bacteria demonstrate the significant antimicrobial effects of the test (triclosan/copolymer) dentifrice with laboratory and clinical strains of oral bacteria implicated in bad breath.

Actinomyces viscosus↗

PubMed2002

Sequence diversity in the major fimbrial subunit gene (flp-1) of Actinobacillus actinomycetemcomitans.

Cells of the periodontal pathogen Actinobacillus actinomycetemcomitans exhibit tight adherence to surfaces such as glass, plastic and hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize teeth and other surfaces. Tight adherence is mediated by long fibrils of bundled pili (fimbriae) that form on the surface of the cell. The flp-1 gene encodes the major pilin protein component of A. actinomycetemcomitans fimbriae. In this study we compared flp-1 DNA sequences from 43 strains of A. actinomycetemcomitans isolated in Europe, Japan and the United States and identified seven distinct flp-1 allelic classes. DNA and predicted protein sequences were almost completely conserved within each flp-1 class but were highly divergent between classes. Most amino acid substitutions occurred in the C-terminus of the pilin protein, a region that has been shown to be important for the bundling and adhesive properties of the pili. flp-1 classes correlated with serotypes and 16S rRNA genotypes in most strains. At least five strains showed evidence of horizontal transfer of flp-1 between strains of different serotypes and 16S rRNA genotypes. Four of the seven flp-1 classes were present in geographically diverse isolates. Strains representing all seven flp-1 classes, but not a strain carrying a transposon insertion in flp-1, bound avidly to polystyrene in an in vitro adherence assay. Strains representing six of the seven flp-1 classes were isolated from localized juvenile periodontitis patients, suggesting that phylogenetically diverse strains carry pathogenic potential. Our findings provide a framework for future biochemical, immunological and genetic studies of A. actinomycetemcomitans fimbriae.

Actinobacillus Infections↗

PubMed2002

Lactoferrin iron levels affect attachment of Actinobacillus actinomycetemcomitans to buccal epithelial cells.

BACKGROUND: Prior reports have suggested that the iron-binding protein lactoferrin (LF) may either kill Actinobacillus actinomycetemcomitans (Aa) or interfere with its binding to host cells. Other studies have indicated that the degree of iron saturation of LF might play a role in these interactions. However, these studies utilized strains that had lost critical attachment characteristics found in well-preserved clinical isolates of Aa. The purpose of this work was to study the effect of LF iron levels on survival and attachment of well-preserved clinical isolates of Aa. METHODS: LF containing 0%, 30%, and 100% iron saturation was tested for its ability to kill clinical isolates of Aa and to inhibit their binding to buccal epithelial cells (BECs). RESULTS: Neither iron-free LF (apo-LF) nor iron-saturated LF killed Aa clinical isolates. Increasing the iron saturation of LF resulted in an increased inhibition of Aa binding to BECs (P < or =0.005). This effect was consistent for the 3 clinical isolates tested. Pretreatment of Aa with iron-saturated LF reduced binding to BECs by 58%, 61.8%, and 64.2%, respectively, for each of the 3 clinical strains tested (P < or =0.005). Pretreatment of Aa strains with apo-LF, iron alone, or bovine serum albumin had no effect on binding. Pretreatment of BECs with LF (either apo-LF or iron-containing LF) had no influence on Aa binding. CONCLUSIONS: These results indicate that reduction in binding of Aa to epithelial cells is maximized by pretreatment of Aa cells with iron-saturated lactoferrin. These in vitro results suggest that patients with lactoferrin containing lowered levels of iron would be more susceptible to Aa colonization.

Adolescent↗

PubMed2002

Lactoferrin iron levels are reduced in saliva of patients with localized aggressive periodontitis.

BACKGROUND: Actinobacillus actinomycetemcomitans (Aa) is associated with localized aggressive periodontal disease in juveniles (LAgP). Lactoferrin (LF) is an iron-binding salivary protein that has been shown to kill Aa in its iron-free form (apo) and reduce binding to host cells in its iron-saturated form (halo). However, recent in vitro studies show that LF does not kill clinical isolates of Aa, and LF with reduced levels of bound iron does not interfere with its attachment. These findings suggest that colonization of Aa may occur more readily in an environment containing LF with low iron levels. The purpose of this study was to examine the relationship of LF iron levels in saliva of LAgP patients as compared to their age-, gender-, and race-matched controls. METHODS: Whole and parotid saliva was collected from LAgP patients and matched controls. Micrograms of LF/mg of protein as well as nanograms of iron/micrograms of LF were determined. Iron binding was determined in parotid saliva by addition of nonlabeled and 59Fe labeled iron. RESULTS: LAgP patients' whole saliva had higher LF levels than controls, but their LF contained less iron (P < or =0.005). No iron was found in LF from parotid saliva in either group. When iron was added to parotid saliva, the LAgP saliva bound 20 to 30 times less iron than controls (P< or =0.001). Finally, LF was identified as the major iron-binding protein in parotid saliva by 59Fe autoradiography and Western blotting. CONCLUSIONS: This study shows that the level of bound iron in LF is significantly reduced in LAgP patients compared to controls. These data suggest that LF from LAgP patients has a reduced capacity to bind iron and that LF iron levels may play an important role in Aa-induced LAgP.

Adolescent↗

PubMed2001

Colonization and persistence of rough and smooth colony variants of Actinobacillus actinomycetemcomitans in the mouths of rats.

Fresh isolates of Actinobacillus actinomycetemcomitans (Aa) bind avidly to surfaces in vitro, but existing in vivo studies of the adherence of Aa are limited. This study had two goals: (1) to compare the oral colonization of two isogenic strains of Aa-CU1010, a clinical isolate that expresses the adherent phenotype, and CU1012, a minimally adherent laboratory variant-and (2) to check for phenotypic reversion of these strains in a clinical setting. Rifampicin-resistant strains, developed for tracking in Sprague-Dawley rats, were tested in vitro to determine their stability and binding. In study 1, after antibiotic suppression, six rats (group I) received CU1010 in their feed. The eight rats in group II received CU1012 in their feed and four were supplemented by oral swabbing and four by gastric gavage. Group III consisted of three sham-inoculated controls. All rats were inoculated for 4 days. Microbiological data were collected at 1, 4 and 8 weeks after inoculation. Supporting data were supplied by antibody titres and clinical measures of alveolar bone loss. Study 2 consisted of six rats in each of three groups as above, but tagged strains of Aa were delivered by food alone. At all time-points in both studies, Aa was absent before inoculation and controls had no Aa or antibody to Aa. In study 1, all six rats in group I yielded positive cultures for Aa at 8 weeks. In group II, five of eight had positive cultures for Aa at 1 week, two of eight at 4 weeks and none had Aa at 8 weeks (P < or =0.001). All six rats in group I had serum anti-Aa titres compared to group II, where titres were seen in four of eight rats (P < or =0.015). In vitro data paralleled those found in vivo. No phenotypic reversion of either strain was seen in vivo. In study 2, four of six rats in group I showed Aa and had titres to Aa, while no other animals showed Aa at any time. The model provides convincing evidence that, unlike laboratory variants, clinical isolates colonize, persist and integrate into an already established, albeit reduced, econiche.

Actinobacillus Infections↗

PubMed2001

Genes for tight adherence of Actinobacillus actinomycetemcomitans: from plaque to plague to pond scum.

The Gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans forms an extremely tenacious biofilm on solid surfaces such as glass, plastic and hydroxyapatite. This characteristic is likely to be important for colonization of the oral cavity and initiation of a potentially devastating form of periodontal disease. Genetic analysis has revealed a cluster of tad genes responsible for tight adherence to surfaces. Evidence indicates that the tad genes are part of a locus encoding a novel secretion system for the assembly and release of long, bundled Flp pili. Remarkably similar tad loci appear in the genomes of a wide variety of Gram-negative and Gram-positive bacteria, including many significant pathogens, and in Archaea. We propose that the tad loci are important for microbial colonization in a variety of environmental niches.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Comparative antimicrobial activities of antiseptic mouthrinses against isogenic planktonic and biofilm forms of Actinobacillus actinomycetemcomitans.

BACKGROUND: Bacteria contained in biofilms have been shown to have a decreased susceptibility to antimicrobial agents compared to those in planktonic form. Thus, in vitro biofilm models have been developed for screening oral antimicrobial formulations in an effort to produce findings more predictive of clinical activity. This study compared the antimicrobial activity of three mouthrinse formulations when tested against isogenic strains of Actinobacillus actinomycetemcomitans (Aa), one of which was a clinical isolate which forms tenacious biofilms in vitro and the other of which was a spontaneous variant which always grows planktonically. METHOD: Biofilm-forming Aa strains CU1000 and NJ4300, obtained as clinical isolates, and their respective spontaneous planktonic variants, CU1060 and NJ4350, were grown under standard laboratory conditions and exposed for 15 s to either a negative control (phosphate buffered saline [PBS]), an essential-oil containing mouthrinse (Listerine Antiseptic [LA]), an amine fluoride/stannous fluoride-containing mouthrinse (Meridol [M]), or a triclosan and PVM/MA copolymer-containing mouthrinse (Plax [P]). The cells were then washed, serially diluted, plated, and incubated for enumeration of viable bacteria. Colony-forming units (CFU)/ml were log10 transformed and the mouthrinse groups were compared to the PBS group using analysis of variance. RESULTS: All 3 mouthrinses produced statisically significant 99.99% reductions (p< or =0.0001) in both planktonic strains compared to the PBS control. Effects on the biofilm forms of the organisms were more variable. Exposure to LA produced statistically significant (p< or =0.0001) reductions in strains CU1000 and NJ4300 of 98.20% and 96.47%, respectively, compared to PBS. M and P produced much smaller reductions which were not statistically significant. CONCLUSIONS: The results of this study, in which antimicrobial mouthrinses were tested against biofilm-forming and planktonic strains of the same organism, provide a clear demonstration of the resistance to antimicrobial agents conferred by biofilm formation and provide additional support for employing tests using biofilms to more accurately assess the relative activities of antiplaque agents in vitro.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non-specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small ( approximately 6.5 kDa), fibril-associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High-resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7-TAG epitope fused to its C-terminus was expressed in the wild-type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7-TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903phikan to isolate insertion mutations in the flp-1 gene (formerly designated flp). Mutants with insertions early in flp-1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp-1 in trans, thus providing the first evidence that flp-1 is required for fibril formation and tight, non-specific adherence. One mutant was found to have an insertion near the 3' end of flp-1 that results in the expression of a truncated and altered C-terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild-type bacteria. These findings and related observations with the Flp1-T7-TAG protein indicate that the C-terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp-1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp-like genes, including the open reading frame (flp-2, previously designated orfA) immediately downstream of flp-1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

Amino Acid Sequence↗

PubMed2001

Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f.

The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

Actinobacillus Infections↗

PubMed2001

Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase.

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.

Acid Anhydride Hydrolases↗

PubMed2000

Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels.

BACKGROUND: Clinical studies in which antimicrobial mouthrinses were shown to have significant antiplaque activity most frequently have used gingivitis as the clinically relevant endpoint. However, there is evidence to suggest that mouthrinses containing active agents effective against Streptococcus mutans, such as chlorhexidine, may also have a role in inhibiting dental caries. This clinical study was conducted to determine the effect of 2x-daily rinsing with an essential oil-containing antiseptic mouthrinse (Listerine Antiseptic) on levels of recoverable S. mutans and total streptococci in supragingival interproximal plaque and in saliva. Additionally, a follow-up in vitro study is reported which determined whether a differential susceptibility to the antiseptic mouthrinse exists among different strains of streptococci. METHOD: Following baseline saliva and plaque sampling for quantification of recoverable S. mutans and total streptococci, 29 qualifying subjects were randomly assigned either the essential oil mouthrinse or a sterile water control. They rinsed with 20 ml for 30s 2 x daily for 11 days and once on the 12th day, in addition to their usual oral hygiene procedures. On day 12, saliva and plaque samples were again collected and microbiological quantification performed. The procedures were repeated with the alternate rinse after a 1-week washout period. RESULTS: The essential oil mouthrinse produced respective reductions of 69.9% and 75.4% in total recoverable streptococci and in S. mutans in plaque, and corresponding reductions of 50.8% and 39.2% in saliva. The in vitro study revealed that streptococci from the mutans group were more susceptible to the bactericidal activity of the essential oil mouthrinse than streptococci from the mitis group. CONCLUSIONS: As antimicrobial mouthrinses are most frequently recommended to patients whose mechanical oral hygiene procedures are not adequate for the control of supragingival plaque and gingivitis, this study provides an additional rationale for the inclusion of the essential-oil mouthrinse as an adjunct to daily oral hygiene procedures.

Adult↗

PubMed2000

Secretion of RTX leukotoxin by Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxin of A. actinomycetemcomitans is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of A. actinomycetemcomitans. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of A. actinomycetemcomitans, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin. Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A. actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that A. actinomycetemcomitans has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea.

The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903phikan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad(-)). Unlike wild-type cells, Tad(-) mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad-like genes have a significant role in microbial colonization.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Proximal caries in juvenile periodontitis patients.

BACKGROUND: Caries is recognized as the prevalent proximal dental disease in adolescents, while proximal bone loss is minimal to non-existent in this population. Adolescents demonstrating an inverse disease pattern, i.e., minimal caries and active periodontitis, could provide powerful clues with regard to both diseases. However, data are inconsistent. This study was designed to clarify this relationship by comparing proximal caries prevalence in a juvenile periodontitis (JP) group to a matched non-periodontally diseased control group. METHODS: Two groups (cases [JPs] and control patients [CPs]) were matched for age, sex, and race and evaluated for decayed, missing, filled teeth and surfaces (DMFS) by radiographic analysis. Statistical analysis was performed by ANOVA and Student t test. The study consisted of four phases. Phase I was based on data from a previous study that failed to include race in the analysis. Thus, the original 23 JP patients (mostly African-Americans from New York City) were rematched for race as well as sex and age with CPs from Newark, NJ. The effect of water fluoridation (found in NYC) was evaluated in Phase II by matching the 23 original CPs (mostly Caucasian from NYC) with 23 CPs from NJ. Since differences were seen, we rematched our original JPs from NYC with a new set of race-matched CPs from NYC (Phase III). Finally, 13 JP patients from the University of Medicine and Dentistry of New Jersey (UMDNJ) were matched with CPs from NJ (Phase IV). RESULTS: Phase I and III indicated that JP patients had significantly less proximal caries than their matched CPs (P < or =0.05). Phase II confirmed the role of fluoride in caries reduction. Phase IV (NJ sample) supported our previous data and suggested that JP patients had less proximal caries than CPs (P < or =0.05). CONCLUSIONS: JP patients had significantly less proximal caries than their matched CPs when groups were balanced and radiographic evaluations were performed. In-depth studies of JP patients could provide important clues about both caries and periodontal disease etiology and pathogenesis.

Adolescent↗

PubMed2000

A model for clinical evaluation of anti-microbial effects of agents on plaque colonization.

PURPOSE: To evaluate a new improved method of microbial analysis in a cross-over clinical study by investigating the efficacy of a single application of an essential oil-containing (EOC) dentifrice as compared to its vehicle control (VC) over a 6-hr period. MATERIALS AND METHODS: Acrylic stents with retention areas for 7, 3 mm x 3 mm hydroxyapatite (HA) squares were fabricated for 12 subjects. 30 min following stent placement, one HA square was removed and sampled for viable microflora. After stent replacement, subjects were assigned either the EOC dentifrice or its VC and brushed under supervision for 1 min. Stents remained in place for the next 6 hrs. A HA square was removed at hourly intervals for 6 hrs following brushing. The microflora was analyzed for total anaerobes on Schaedler's media, for total gram-negative anaerobes on Schaedler-NV selective media, and for total Fusobacterium species on CVE selective media. Plates were incubated anaerobically at 37 degrees C for 2-5 days. Colony forming units were calculated. For each time point, pairwise t-tests were performed using the adjusted means and the pooled error term from the analysis of variance (ANOVA). RESULTS: Treatment with the EOC dentifrice resulted in a statistically reduced plaque growth. Differences were seen as reductions in: (1) total gram-negative anaerobes seen from 1-6 hrs (P < or = 0.011), (2) total anaerobic bacteria which achieved significance at 3 hrs and continued through 6 hrs (P < or = 0.005), and (3) Fusobacterium species as seen from 4-6 hrs (P < or = 0.002).

Adult↗