Comparison of chairside microbiological screening systems and conventional selective media in children with and without visible dental caries.

PURPOSE: The purpose of this study was to compare the results of commercial chairside microbial tests (CT) and conventional selective media (GS, gold standards) for mutans streptococci (MS) and lactobacilli (LB) using oral specimens from young children with and without visible dental caries. METHODS: Using cotton swabs to collect oral microbial specimens from children 10 to 36 months old, microbial counts of CT and GS were compared with caries experience of the subjects. Contamination levels by non-MS or non-LB isolates on CT and GS were also determined. The CT employed were: (1) CRT bacteria for MS and LB; (2) CarioCheck Plus for MS and LB; and (3) Dentocult SM and Mucount for MS. RESULTS: All CT and GS for MS represented caries status of the participants (P<.001, Fisher exact test; P<.015 linear regression), whereas only GS for LB showed significant association with caries status (P<.001, Fisher exact test; P<.001, linear regression). Non-MS or non-LB isolates were observed on most media, and CT usually exhibited higher contaminant levels than GS. Dentocult SM and Mucount did not harbor contaminants. CONCLUSIONS: Despite contamination, CT and GS for MS and GS for LB exhibited satisfactory outcomes based on cross-sectional caries experience of infants and toddlers.

Evaluation of microbiological screening systems using dental plaque specimens from young children aged 6-36 months.

Using cotton swab specimens of dental plaque from children aged 6-36 months, four commercial chairside tests for oral bacteria were evaluated by comparison with conventional selective culture: mitis salivarius kanamycin bacitracin agar for mutans streptococci and Rogosa SL agar for lactobacilli. Representative colonies of all isolates were identified by commercial identification kits. According to qualitative evaluations, all chairside tests for mutans streptococci were effective in our population. Those for lactobacilli were not as effective, due mainly to a high recovery of yeast contaminants.

Oral malodor in children and volatile sulfur compound-producing bacteria in saliva: preliminary microbiological investigation.

PURPOSE: This study examined and compared levels of salivary bacteria which produced volatile sulfur compounds (VSC) in young children with and without oral malodor. METHODS: Clinic populations of children aged two to seven years, whose parents presented with an unsolicited major complaint of oral malodor in their child (OM+), or aged-matched controls in whom oral malodor was not detected by parents (OM-), were investigated. Saliva specimens were cultured anaerobically on media that differentiated VSC+ bacteria. These were quantified and identified. Levels in OM+ and OM- children were compared and statistically analyzed. RESULTS: OM+ children harbored significantly higher levels of VSC+ isolates in saliva than OM- children (OM+ = 44% of total viable counts, TVC; OM- = 24% of TVC; P = 0.0083). Types of recovered bacterial species did not differ in the two groups, but levels of Prevotella oralis were significantly higher in OM+ children (P = 0.0001). Veillonella species followed by P. oralis were the predominant VSC+ isolates recovered in both populations. CONCLUSIONS: VSC+ salivary bacteria may differ both in type and quantity in young children with and without parent-perceived oral malodor.

Antigen activation of THP-1 human monocytic cells after stimulation with lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

A human THP-1 monocyte cell line culture system has been utilized to evaluate the morphological changes in THP-1 cells and to measure expression of activation antigens (CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR) as evidence of maturation of THP-1 cells in response to stimulation by lipopolysaccharide (LPS) from the oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis, and granulocyte-macrophage colony-stimulating factor. THP-1 cells were stimulated with LPS (1 microgram/ml) of P. gingivalis or F. nucleatum for different time periods (1, 2, 4 and 7 d). Detection of different activation antigens on THP-1 cells was performed by indirect immunohistochemical staining followed by light microscopy. Confirmational studies were performed in parallel using indirect immunofluorescence and immunogold electron microscopy for detection of the corresponding activation antigens. Expression of different activation antigens by resting THP-1 cells revealed HLA-DR to be on 3% of the cells; CD-11b, 9%; CD-11c, 8%; CD-14, 22%; CD-35, 9% and CD-68, 7%. The CD-71 activation antigen was not expressed in untreated THP-1 cells. LPS stimulation increased expression of all activation antigens. A significant (p < 0.05) increase in expression of CD-11b, CD-11c, CD-14, CD-35, CD-68 and CD-71 was observed when GM-CSF (50 IU/ml) was supplemented during the treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis. Activation and differentiation of THP-1 cells by LPS from oral microorganisms in the presence of GM-CSF supports a role for human macrophages in acute and chronic periodontal diseases and may explain the clinically observable periodontal exacerbations in some patients after GM-CSF therapy.

Dental unit waterlines: biofilms, disinfection and recurrence.

BACKGROUND: Transmission of microbial pathogens to patients from biofilm within dental unit waterlines, or DUWLs, is a concern. To reduce the risk of toxicity to dental patients when water coolants are used, numerous chemical agents have been tested. In a series of trials, the authors investigated the recurrence of microbial growth after treating DUWLs with sodium hypochlorite (bleach), or B; glutaraldehyde, or G; or isopropanol 15.3 percent, or I. METHODS: The authors excised tubing sections from dental units in a general clinic. The tubing sections were evaluated at baseline and after overnight treatment. Effluent water samples and biofilm samples from tubing sections also were evaluated, by culture, at baseline and after treatment with the chemical agents. Biofilm within the tubing was examined by scanning electron microscopy, or SEM, and the authors identified bacterial isolates using standard techniques. The authors performed minimum inhibitory concentration tests on identified isolates pre- and posttreatment and compared the results to determine possible differences in resistance. RESULTS: In baseline evaluations, the authors determined that the effluent and biofilm matrix harbored an average of 1 x 10(5) colony-forming units, or CFU, per square centimeter and 1 x 10(4) CFU/cm2 recoverable microorganisms, respectively. A single overnight treatment of the DUWLs with B, G or I rendered effluent and biofilm samples that were free of recoverable bacteria. The number of viable bacteria in the effluent and the biofilm of B- or I-treated DUWLs returned to pretreatment levels by day six and day 15, respectively. DUWLs treated with G showed evidence of bacterial recurrence in the effluent and the biofilm to pretreatment levels by day three. The authors compared recurrence of biofilm and effluent posttreatment with untreated control tubing. The lower recurrence of viable bacteria in both biofilm and effluent samples for tubing treated with B and I was significant (P < or = .05). No evidence of resistance to the agents was noted during the study. Multiple treatments held the bacterial population to below recoverable levels but failed to remove the biofilm matrix, as evidenced by SEM. CONCLUSIONS: B, G and I eliminated recoverable bacteria after treatment and inhibited their recurrence in DUWL. Recolonization rates varied by agent. CLINICAL IMPLICATIONS: The residual effect of these agents raises concerns about the slow release of potentially toxic substances from the residual biofilm matrix. These agents reduce microorganisms in effluent water but do little to destroy the biofilm matrix in the DUWL, even with periodic treatments. Bacterial populations in the dental unit water rapidly recolonize the DUWL. Chemical agents or agents that potentially could be trapped in the matrix can represent an additional risk to the patient.

Functional changes in THP-1 human monocytic cells after stimulation with lipopolysaccharide of oral microorganisms and granulocyte macrophage colony stimulating factor.

A human THP-1 monocyte cell line culture system has been utilized to observe the effect of granulocyte macrophage colony stimulating factor (GM-CSF) supplementation with lipopolysaccharide (LPS) of oral microorganisms to stimulate monocyte/macrophage functional activity. LPS of oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis was produced by phenol-water extraction and characterized. The phagocytosis assay was performed using F1TC labeled Saccharomyces yeast particles. Phagocytic functional activity was observed in 10-11% of resting THP-1 cells. Treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis increased the phagocytic activity of THP-1 cells 2-3 fold. GM-CSF significantly increased phagocytosis either alone or when supplemented with LPS of F. nucleatum or P. gingivalis. A chemotaxis assay was performed using a 48 well chemotaxis chamber. Chemotactic functional activity of THP-1 cells was increased 2-fold after 4 days of treatment with GM-CSF. Stimulation of THP-1 cells with LPS of F. nucleatum or P. gingivalis significantly reduced the chemotactic activity indicating the maturation towards a fixed macrophage. There were functional variations (chemotaxis and phagocytosis) in THP-1 cells in response to LPS of oral microorganisms following stimulation with GM-CSF.

Granulocyte-macrophage colony-stimulating factor amplification of interleukin-1beta and tumor necrosis factor alpha production in THP-1 human monocytic cells stimulated with lipopolysaccharide of oral microorganisms.

Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1beta and TNF-alpha production following GM-CSF supplementation with lipopolysaccharide (LPS) from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. LPS of P. gingivalis or F. nucleatum was prepared by a phenol-water extraction method and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determination of total protein and endotoxin contents. Resting THP-1 cells were treated with LPS of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) by using different concentrations for various time periods. Production of IL-1beta and TNF-alpha in THP-1 cells was measured by solid-phase enzyme-linked immunosorbent assay. Reverse transcription (RT)-PCR was used to evaluate the gene expression of resting and treated THP-1 cells. IL-1beta was not detected in untreated THP-1 cells. IL-1beta production was, however, stimulated sharply at 4 h. GM-CSF amplified IL-1beta production in THP-1 cells treated with LPS from both oral anaerobes. No IL-1beta-specific mRNA transcript was detected in untreated THP-1 cells. However, IL-1beta mRNA was detected by RT-PCR 2 h after stimulation of THP-1 cells with LPS from both organisms. GM-CSF did not shorten the IL-1beta transcriptional activation time. GM-CSF plus F. nucleatum or P. gingivalis LPS activated THP-1 cells to produce a 1.6-fold increase in TNF-alpha production at 4 h over LPS stimulation alone. These investigations with the in vitro THP-1 model indicate that there may be an increase in the cellular immune response to oral endotoxin following GM-CSF therapy, as evidenced by production of the tissue-reactive cytokines IL-1beta and TNF-alpha.

Interleukin-6 production by human monocytes treated with granulocyte-macrophage colony-stimulating factor in the presence of lipopolysaccharide of oral microorganisms.

This study focused on the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide of the putative periodontal pathogens Porphyromonas gingivalis or Fusobacterium nucleatum on IL-6 production by THP-1 cells (a human monocytic cell line). Resting THP-1 cells were alternatively treated with GM-CSF (50 IU/ml) and lipopolysaccharide of P. gingivalis or F. nucleatum, in varying concentrations for varying time periods. IL-6 production in supernatant fluids of treated cells was evaluated by an enzyme-linked immunosorbent assay (ELISA) and a reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression. Untreated THP-1 cells did not produce IL-6 as determined by ELISA. RT-PCR also failed to detect IL-6 mRNA in untreated THP-1 cells, indicating that IL-6 was not constitutively produced. After stimulation of THP-1 cells with lipopolysaccharide of F. nucleatum or P. gingivalis, IL-6 was produced, peaking at 4 h (200-300 pg/ml) and thereafter sharply declining by 8 h. When GM-CSF was added together with lipopolysaccharide of P. gingivalis or F. nucleatum, there was a synergistic quantitative increase in production of IL-6 as measured by ELISA as compared with lipopolysaccharide alone. IL-6 mRNA was detected by RT-PCR, 15 min after stimulation with lipopolysaccharide of either P. gingivalis or F. nucleatum. GM-CSF supplementation with lipopolysaccharide of P. gingivalis shortened the transcription of IL-6 mRNA to 5 min, a shift which was not observed with lipopolysaccharide of F. nucleatum, possibly indicating a different mechanism of initiation of transcription. Production of IL-6 by GM-CSF-treated THP-1 cells in the presence of lipopolysaccharide of oral microorganisms may provide a model for studying the role of macrophages in acute and chronic periodontal diseases, including the clinical periodontal exacerbation as observed in chemotherapy patients receiving GM-CSF for bone marrow recovery.

Inhibition of pure cultures of oral bacteria by root canal filling materials.

This study compared the antimicrobial effectiveness of nine dental materials and a negative control agent against 21 strains or species of bacteria using an agar diffusion assay. The materials were: 1. Camphorated parachlorophenol mixed with calcium hydroxide (CPC + Ca(OH)2). 2. CPC mixed with zinc oxide (CPC + ZnO). 3. Formocresol mixed with zinc oxide and eugenol (FC + ZOE). 4. Chlorhexidine mixed with ZOE (CHX + ZOE). 5. Kri paste. 6. ZOE. 7. Zinc oxide mixed with sterile water (ZnO + H2O). 8. Calcium hydroxide mixed with sterile water (Ca(OH)2 + H2O). 9. Vitapex. 10. Vaseline (control). The test bacteria represented species commonly isolated from nonvital primary and permanent tooth root canals. The antimicrobial effectiveness of the materials was divided into five groups based on the diameters of the zones of inhibition against all test bacteria and distribution of the data. All materials except Vaseline showed antimicrobial activity against some of the 21 organisms. Generally, all materials inhibited gram-negative anaerobic bacteria more effectively than aerotolerant gram-negative or gram-positive bacteria. Materials containing CPC or FC (except Kri paste) produced strong or medium strong inhibition against most bacteria. CHX + ZOE, Kri paste, ZnO + H2O, and ZOE inhibited all or most bacteria, but to lesser extent than CPC + Ca(OH)2, CPC + ZnO, or FC + ZOE. Ca(OH)2 + H2O, Vitapex, and Vaseline generally were nonihibitory. The findings should allow a comparative evaluation of antimicrobial effectiveness to be made of materials commonly used in pulpectomy procedures with primary teeth.

Development and evaluation of a selective and differential medium for the primary isolation of Peptostreptococcus micros.

Peptostreptococcus micros, an anaerobic gram-positive coccus, has been associated with periodontal and endodontic lesions, including those refractory to treatment, as well as many human polymicrobial infections in other body locations. A selective and differential medium for the primary isolation of P. micros was developed and evaluated. Columbia CNA agar, a selective medium for gram-positive cocci, was supplemented with glutathione and lead acetate (P. micros medium: PMM). P. micros has a characteristic of rapidly utilizing the reduced form of glutathione to form hydrogen sulfide, which reacts with lead acetate producing a black precipitate in the medium. When grown on PMM, P. micros can be easily identified by its typical colonial morphology and the presence of a black precipitate directly under the colony. PMM was compared for the growth of P. micros with phenylethyl alcohol agar (PEA) and Columbia base medium (CBM) with 80 strains of P. micros and 30 strains of other gram-positive cocci. All P. micros isolates tested grew and showed the typical morphology of P. micros on PMM. Using colony counts on CBM as controls, there was an average 81.8% recovery in the number of P. micros colonies on PMM, in contrast to an average 6.1% recovery on PEA. Subgingival plaque and tongue samples from 12 adult periodontitis and 6 early-onset periodontitis patients were cultured onto PMM for the isolation of P. micros. P. micros was isolated on PMM and identified biochemically and enzymatically from both adult periodontitis and early-onset periodontitis patients with higher percentages isolated from the diseased periodontal pockets of adult periodontitis patients; furthermore, this is the first isolation of P. micros from tongue samples taken from periodontally diseased patients. This medium in cultural studies will further our understanding and assist future investigations of P. micros involved in disease processes.

In vitro inhibition of bacteria from root canals of primary teeth by various dental materials.

The primary tooth pulpectomy is a common clinical procedure. The choice of filling material is important to the success rate, but antibacterial properties of such materials against organisms known to inhabit infected primary root canals have not been well documented. This study compared the antibacterial effectiveness of 10 materials: 1. Calcium hydroxide mixed with camphorated parachlorophenol (Ca(OH)2+CPC) 2. Calcium hydroxide mixed with sterile water (Ca(OH)2+H2O) 3. Zinc oxide mixed with CPC (ZnO+CPC) 4. Zinc oxide mixed with eugenol (ZOE) 5. ZOE mixed with formocresol (ZOE+FC) 6. Zinc oxide mixed with sterile water (ZnO+H2O) 7. ZOE mixed with chlorhexidine dihydrochloride (ZOE+CHX) 8. Kri paste 9. Vitapex paste 10. Vaseline (control) These materials were compared against microbial specimens obtained from 13 infected primary teeth by using an agar diffusion assay. The results suggest that the materials could be divided into three categories. Category I, with the strongest antibacterial effect included ZnO+CPC, Ca(OH)2+CPC, and ZOE+FC. Category II, with a medium antibacterial effect included ZOE+CHX, Kri, ZOE, and ZnO+H2O. Category III, with no or minimal antibacterial effect included Vitapex, Ca(OH)2+H2O, and Vaseline. There were no significant differences within each category, but there were significant differences between the categories. The one exception was the antibacterial effect of ZOE+FC which was not significantly different from ZOE+CHX, Kri, or ZOE.