Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste.

AIM: To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. DESIGN: Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. RESULTS: Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P <0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). CONCLUSION: Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

Evaluation of home disinfection protocols for acrylic baseplates of removable orthodontic appliances: A randomized clinical investigation.

INTRODUCTION: In this randomized clinical trial, we investigated, using the microbial culture technique and scanning electron microscopy, the contamination of acrylic baseplates of removable orthodontic appliances by mutans streptococci (MS) and evaluated the efficacy of different home disinfection protocols with a 0.12% chlorhexidine gluconate spray (Periogard, Colgate-Palmolive, São Bernardo do Campo, Brazil). METHODS: Fifteen dental students were randomly enrolled in a 3-stage changeover system with a 1-week interval between each stage. The acrylic baseplates were worn full time except at meals to simulate the routine use of removable appliances under clinical conditions. Three 1-week home disinfection protocols were tested in all stages by a different group of students: protocol I, toothbrushing + baseplate brushing + sterile tap water spraying once a day; protocol II, toothbrushing + baseplate brushing + Periogard spraying on the seventh day after appliance placement; and protocol III, toothbrushing + baseplate brushing + Periogard spraying on the fourth and seventh days after appliance placement. After the first week, the volunteers received new baseplates, toothbrushes, and dentifrices, and the regimens were repeated 2 more times. At the end of each week, the baseplates had a randomized disinfection protocol and were sent for microbiologic analysis. A scanning electron microscope was used to examine 3 acrylic baseplates representing each home protocol. The Friedman test (α = 0.05) compared the home protocols for the formation of MS colonies or biofilms on the acrylic surfaces. RESULTS: MS colonies or biofilms were found on all acrylic baseplates after protocol I. Protocols II and III reduced significantly (P <0.05) the number of MS colonies and biofilms on the acrylic surfaces. No significant difference (P >0.05) was observed between protocols II and III. The scanning electron microscope analysis confirmed the results of the microbiologic cultures. CONCLUSIONS: Disinfection of baseplates of removable orthodontic appliances by using 0.12% chlorhexidine spray once or twice a week reduced the contamination by MS on the acrylic surface in vivo.

Efficacy of microwaves and chlorhexidine on the disinfection of pacifiers and toothbrushes: an in vitro study.

PURPOSE: The purpose of this study was to evaluate, in vitro, the contamination of toothbrushes and pacifiers by Streptococcus mutans, and the efficacy of microwave and chlorhexidine for their disinfection. METHODS: Sixty pacifiers and 60 toothbrushes were contaminated with S mutans and then divided into groups according to the disinfection protocol: Group 1-chlorhexidine solution; Group 2-microwave sterilization; and Group 3-sterile tap water. The devices were evaluated microbiologically as to the formation of S mutans colonies/biofilms and were examined by scanning electron microscopy. The results were submitted for statistical analysis by Friedman's test at a 5% significance level. RESULTS: The results of both types of evaluation showed a large number of S mutans colonies/biofilms after spraying with sterile tap water, and chlorhexidine spraying and microwaving were effective in eliminate colonies/biofilms. Groups 1 and 2 were statistically similar to each other (P>.05) and differed significantly from Group 3 (P<.05). CONCLUSIONS: The 0.12% chlorhexidine solution spray and 7 minutes of microwave irradiation were effective for disinfection of pacifiers and toothbrushes.

Toothbrush contamination by Candida spp. and efficacy of mouthrinse spray for their disinfection.

The aim of the study was to evaluate toothbrush contamination in vivo by Candida spp. and the efficacy of Periogard and Neem Sattiva, in spray, in the disinfection of these toothbrushes. This study was performed in three phases in which mouthrinses and sterile distilled water (control group) were sprayed six times on toothbrush bristles used by 61 university students. Toothbrushes were then submitted to microbiological processing for the isolation and identification of Candida species. Fifty-nine students completed the three phases of this study, and 22 (37.3%) control group toothbrushes presented growth of Candida species. Periogard and Neem Sattiva eliminated growth of Candida spp. in 48.1 and 7.4% of toothbrushes, respectively. Contamination by Candida spp. was observed on various toothbrushes of the control group. Periogard was more efficacious than Neem Sattiva in eliminating growth of Candida spp. on the toothbrush bristles.

Antibacterial and antifungal properties of crude extracts and isolated compounds from Lychnophora markgravii.

Antimicrobial activity of dichloromethane and ethanol extracts and five compounds: pinostrobin (I), pinocembrin (II), tectochrysin (III), galangin 3-methyl ether (IV) and tiliroside (V) isolated from Lychnophora markgravii aerial parts against fifteen microorganisms was determined. The structures of these compounds were elucidated based on ESI-MS and NMR spectroscopic data. Both extracts showed antimicrobial activity against several tested microorganisms. Pinostrobin, tectochrysin and galangin 3-methyl ether showed the strongest antibacterial and antifungal effects.

Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments.

AIMS: To evaluate the checkerboard DNA-DNA hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. MATERIALS AND METHODS: Nine plastic abutments cast in a Ni-Cr alloy and nine pre-machined Co-Cr alloy abutments with plastic sleeves cast in Ni-Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 microl of a solution containing 10(8) cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant-abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA-DNA hybridization method. RESULTS: DNA-DNA hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P<0.05). CONCLUSION: Bacterial leakage through the implant-abutment interface does not significantly differ when cast or pre-machined abutments are used. The checkerboard DNA-DNA hybridization technique is suitable for the evaluation of the internal contamination of dental implants although further studies are necessary to validate the use of computational methods for the improvement of the test accuracy.

Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota.

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

Bacterial culture and DNA Checkerboard for the detection of internal contamination in dental implants.

PURPOSE: The aim of this in vitro study was to evaluate the bacterial leakage along the implant-abutment interface by the conventional bacterial culture and DNA Checkerboard hybridization method. MATERIALS AND METHODS: Twenty Branemark-compatible implants with a 3.75-mm diameter and external hexagonal platform were randomly placed in two groups of ten implant-abutment assemblies each. One group was used to analyze bacterial counts by DNA Checkerboard hybridization and the other by a conventional bacterial culture. Suspensions of Fusobacterium nucleatum (3 microl) were injected into the grooved internal cylinders of each implant assembly, and the abutment was connected by a 32 Ncm torque. The combined implant-abutments were individually placed in tubes containing the CaSaB culture medium and incubated in a bacteriological constant temperature oven for 14 days. The samples were observed daily as to the presence of turbidity, and after the designated time the microorganisms were collected from the implant interiors and analyzed by the two methods. RESULTS: After 14 days, six implant-abutment assemblies showed turbidity. Both methods indicated reduced microorganism counts in samples from the interior of the implant-abutment assemblies after incubation in the culture medium; however, the number of counts of F. nucleatum was higher by the DNA Checkerboard method when compared to the group analyzed by conventional bacterial cultures (p < 0.05). CONCLUSION: The DNA Checkerboard method was shown to be more sensitive than conventional cultures in the detection of microorganisms.

In-vivo evaluation of the contamination of Super Slick elastomeric rings by Streptococcus mutans in orthodontic patients.

INTRODUCTION: We investigated in vivo the contamination by Streptococcus mutans of Super Slick elastomeric rings (TP Orthodontics, LaPorte, Ind), manufactured with Metafasix technology (TP Orthodontics), using microbial culture and scanning electron microscopy (SEM). METHODS: Twenty patients undergoing fixed orthodontic appliance therapy were selected. Super Slick elastomeric rings (n = 160) were tied to brackets on the right maxillary premolars or molars and left mandibular premolars or molars. Conventional elastomeric rings (n = 160) were tied to brackets on the contralateral premolars or molars with the same split-mouth design. After a 15-day intraoral period, 75 elastomeric rings of each type were retrieved, submitted to microbiologic processing, and cultured in bacitracin sucrose broth-selective enrichment broth culture media. The number of S mutans colonies or biofilms on the surface of the electrometric rings was counted by using a stereomicroscope. Data were analyzed statistically with the Wilcoxon nonparametric test at the 5% significance level. Four representative rings of each type were chosen for SEM analysis. RESULTS: Statistical analysis by the Wilcoxon nonparametric test showed that the Super Slick elastomeric rings had statistically significant greater S mutans contamination than the conventional elastomeric rings (P <.0001). No formation of S mutans colonies or biofilms was observed in the elastomeric rings removed directly from their original packages. SEM micrographs showed fissures on the surface of Super Slick elastomeric rings. No fissures were found on conventional elastomeric rings. When the microbiologic culture was positive, S mutans bacterial biofilm was observed on both types of ligatures. CONCLUSIONS: There was no clinical evidence that Super Slick elastomeric rings are effective in reducing bacterial biofilm formation on their surfaces, and a recommendation for their use in orthodontic therapy for that purpose is not justifiable.

Antibacterial activity of four mouthrinses containing triclosan against salivary Staphylococcus aureus.

The maximum inhibitory dilution (MID) of triclosan-based mouthwashes against 28 Staphylococcus aureus strains was evaluated. Dilutions ranging from 1/10 to 1/655,360 were prepared. Strains were inoculated using a Steers multipoint inoculator. The MID was considered as the maximum dilution capable of inhibiting microorganism growth. The mouthwashes presented different MIDs.

In-vivo evaluation of the bacterial contamination and disinfection of acrylic baseplates of removable orthodontic appliances.

INTRODUCTION: This randomized clinical trial assessed, by using microbial culture and scanning electron microscopy (SEM), the contamination by mutans streptococci (MS) colonies/biofilms on acrylic baseplates of removable orthodontic appliances and evaluated the efficacy of antimicrobial sprays (Periogard [Colgate-Palmolive Ind. Brasileira, Osasco, SP, Brazil], Cepacol [Merrell Lepetit Farmacêutica e Industrial Ltda, Santo Amaro, SP, Brazil], and sterile tap water [control]) on their disinfection. METHODS: Seventeen children were randomly enrolled in a 3-stage changeover system with a 1-week interval between each stage. All solutions were used in all stages by a different group of children. The acrylic baseplates were worn full time except at meals. At the end of each week of the trial, the baseplates were submitted to a randomized disinfection protocol and were sent for microbiologic analysis. New baseplates were constructed, and the same sequence of procedures was repeated 2 more times. Acrylic baseplates representing each test solution were examined by SEM. The Friedman test assessed differences at the 5% significance level among the solutions for MS biofilm formation on acrylic surface. RESULTS: Cepacol and Periogard reduced the formation of MS colonies/biofilms, and both solutions differed statistically from sterile tap water (P <.001). However, Periogard was significantly more effective against MS than Cepacol (P <.001). When MS colonies/biofilms were detected on acrylic surfaces under stereomicroscopy, this was confirmed with SEM. CONCLUSIONS: Acrylic baseplates of removable orthodontic appliances worn by children were contaminated by MS colonies/biofilms in all cases after 1 week. Although Cepacol had better results than sterile tap water, spraying with Periogard showed significantly greater efficacy in reducing MS colonies/biofilms on acrylic surfaces.

Residual antibacterial activity of chlorhexidine digluconate and camphorated p-monochlorophenol in calcium hydroxide-based root canal dressings.

The purpose of this study was to evaluate the residual antibacterial activity of several calcium hydroxide [Ca(OH)2]-based pastes, placed in root canals of dogs' teeth with induced chronic periapical lesions. Root canals were instrumented with the ProFile rotary system and filled with 4 pastes: G1 (n=16): Ca(OH)2 paste + anesthetic solution; G2 (n=20): Calen paste + camphorated p-monochlorophenol (CMCP); G3 (n=18): Calen; and G4 (n=18): Ca(OH)2 paste + 2% chlorhexidine digluconate. After 21 days, the pastes were removed with size 60 K-files and placed on Petri plates with agar inoculated with Micrococcus luteus ATCC 9341. Pastes that were not placed into root canals served as control. After pre-diffusion, incubation and optimization, the inhibition zones of bacterial growth were measured and analyzed by Mann-Whitney U test at 5% significance level. All pastes showed residual antibacterial activity. The control samples had larger halos (p<0.05). The mean residual antibacterial activity halos in G1, G2, G3 and G4 were 7.6; 10.4; 17.7 and 21.4 mm, respectively. The zones of bacterial growth of G4 were significantly larger than those of G1 and G2 (p<0.05). In conclusion, regardless of the vehicle and antiseptic, all Ca(OH)2-based pastes showed different degrees of measurable residual antibacterial activity. Furthermore, unlike CMCP, chlorhexidine increased significantly the antibacterial activity of Ca(OH)2.

Evaluation of the contamination and disinfection methods of toothbrushes used by 24- to 48-month-old children.

PURPOSE: The purpose of this study was to evaluate: (1) in vivo the contamination by mutans streptococci (MS) of toothbrushes after use on 52 children (24-48 months old) by a single dentist; (2) in vivo the efficacy of 3 solutions (Periogard, Brushtox, and a Cosmocil CQa and Myacide pharma BPa-based experimental solution) in the disinfection of these toothbrushes through a randomized clinical trial; and (3) in vitro the antimicrobial activity of the solutions by the agar diffusion test using 15 microbial strains. METHODS: In the in vivo trial, children were randomly assigned to 1 of 4 groups (N=13) and a 4-stage changeover system was used with a 1-week interval between each stage. Solutions were used by a different group of children in each stage. Children were submitted to a 1-minute brushing (without toothpaste) performed by a single professional, followed by random spraying of the test solutions and microbiological analysis. RESULTS: Brushtox, Periogard, and the experimental solution reduced/prevented the formation of MS colonies/biofilms on the toothbrush bristles compared to the control (sterile tap water; P<.001). Periogard and the experimental solution showed significantly greater reduction of colonies/biofilms compared to Brushtox (P<.01). In the in vitro experiment, Periogard exhibited the greatest inhibition halo average, followed by the experimental solution, Brushtox, and sterile tap water (P<.05). CONCLUSIONS: After a single brushing, severe contamination by mutans streptococci colonies/biofilms was observed on all toothbrushes sprayed with sterile tap water (control). Although Brushtox presented better results than sterile tap water, Periogard and the experimental solution showed greater efficacy against formation of MS colonies/ biofilms on the toothbrush bristles and exhibited larger microbial growth inhibition halos.

Bacterial profile in primary teeth with necrotic pulp and periapical lesions.

The objective of this study was to evaluate the bacterial profile in root canals of human primary teeth with necrotic pulp and periapical lesions using bacterial culture. A total of 20 primary teeth with necrotic pulp and radiographically visible radiolucent areas in the region of the bone furcation and/or the periapical region were selected. After crown access, 4 sterile absorbent paper points were introduced sequentially into the root canal for collection of material. After 30 s, the paper points were removed and placed in a test tube containing reduced transport fluid (RTF) and were sent for microbiological evaluation. Anaerobic microorganisms were found in 100% of the samples, black-pigmented bacilli in 30%, aerobic microorganisms in 60%, streptococci in 85%, gram-negative aerobic rods in 15% and staphylococci were not quantified. Mutans streptococci were found in 6 root canals (30%), 5 canals with Streptococcus mutans and 1 canal with Streptococcus mutans and Streptococcus sobrinus. It was concluded that in root canals of human primary teeth with necrotic pulp and periapical lesions, the infection is polymicrobial with predominance of anaerobic microorganisms.

Antimicrobial spray for toothbrush disinfection: an in vivo evaluation.

OBJECTIVE: The aim of this study was to evaluate the efficacy of a spray containing an antimicrobial solution for toothbrush disinfection. METHODS AND MATERIALS: Three different solutions were sprayed on toothbrush bristles among 30 adults after they had brushed: (1) basic formulation (base) plus chlorhexidine; (2) base only, and (3) sterile tap water (control). Each solution was tested for 1 week. After that, the toothbrushes were collected and sonicated in Letheen Broth, diluted in 10-fold series, and plated on selective and nonselective media for detection of anaerobes, aerobes, streptococci, and gram-negative bacilli. After incubation, the colonies of those microorganisms were counted. Presence of mutans streptococci on the bristles was also confirmed. RESULTS: Spray 1 produced a significant reduction in the microbial contamination of toothbrushes for all the microorganisms, spray 2 provided some reduction of contaminants, and spray 3 demonstrated the least antimicrobial effect. CONCLUSION: The antimicrobial spray with chlorhexidine proved to be an effective and practical means for toothbrush disinfection.

The antimicrobial activity of Aspergillus fumigatus is enhanced by a pool of bacteria.

In a screening program for new antibiotic producers, a strain of Aspergillus fumigatus was isolated from Brazilian soil samples. A pool of autoclaved bacteria was added to part of the fungus culture on the second day of fermentation to increase antibiotic production. The chloroform extract from the culture broth to which the pool of autoclaved bacteria was added showed an increase of 55%, 63% and more than 100% in activity against Staphylococcus aureus, Candida albicans and Micrococcus luteus, respectively. Also, the HPLC chromatographic profiles of the chloroform extracts from both culture conditions were different. Two active compounds were isolated from the broth of the culture grown in the presence of pooled bacteria and were identified as 3,4-dimethoxyphenol and 1,3,5-trimethoxybenzene.

Antimicrobial activity of extracts and some compounds from Calea platylepis.

The antimicrobial activity of dichloromethane extracts (leaves, flowers and underground parts) and some compounds isolated from Calea platylepis were evaluated by the well diffusion method.

Reduction of salivary S. aureus and mutans group streptococci by a preprocedural chlorhexidine rinse and maximal inhibitory dilutions of chlorhexidine and cetylpyridinium.

OBJECTIVES: Staphylococcus aureus and mutans group streptococci can cause, among many other diseases, infective endocarditis and postoperative infections. The reduction of the number of these microorganisms in the oral cavity prior to surgical procedures has been related to a decreased incidence of such occurrences. The aim of this study was to evaluate the effect of a single preprocedural rinse with 0.12% chlorhexidine solution (Periogard) on the salivary counts of S aureus and mutans group streptococci and determine maximal inhibitory dilutions (MID) of this and 0.05% cetylpyridinium chloride solution (Cepacol). METHOD AND MATERIALS: Saliva was collected from 60 patients before and after 30-second mouthrinses with chlorhexidine and cultured in appropriate media. The number of microorganisms was calculated based on the colony-forming units (CFUs). For the in vitro MID determination, 25 strains of S aureus were seeded in the media containing one of the sequential dilutions of both antiseptics. RESULTS: S aureus, Streptococcus mutans, and Streptococcus sobrinus were initially isolated from 45%, 63%, and 28% of the patients, respectively. After rinsing with chlorhexidine, the reductions in the CFUs were above 99% for all the studied microorganisms. In the MID determination, all isolates were inhibited with 1/20 and 1/80 dilutions of cetylpyridinium and chlorhexidine, respectively. Dose-response curves were obtained for both antiseptics. CONCLUSION: Single preprocedural chlorhexidine mouthrinse is effective in reducing salivary microorganisms to levels currently considered safe to perform invasive procedures, and it is still effective in a 1:80 dilution.

Effect of triclosan dentifrice on toothbrush contamination.

PURPOSE: The objective of this study was to evaluate by culture and scanning electron microscopy (SEM) the contamination of toothbrushes of 30 children (5-7 years old) by mutans streptococci (MS) when dentifrices with or without triclosan are used. METHODS: The clinical procedures were divided into 3 phases at 1-week intervals. In phase 1 (group I), the children brushed their teeth without dentifrice for 4 minutes; phase 2 (group II) brushed with fluoridated dentifrice (Tandy); phase 3 (group III) brushed with dentifrice containing triclosan (Colgate Total). The toothbrushes were then submitted to microbiological processing for the counting of colony-forming units (CFUs) of MS adhered to the bristles. Four toothbrushes from each group were analyzed by SEM. RESULTS: MS were present on 93% of group I toothbrush bristles and on 77% of group II toothbrush bristles. Only 40% of group III toothbrushes were contaminated with MS. When there was a positive microbiological culture, the formation of cariogenic bacterial biofilm adhered to the bristles of all groups was identified by SEM. CONCLUSIONS: Toothbrush bristles were contaminated by MS after only one use. A dentifrice containing triclosan significantly reduced bacterial contamination of these toothbrushes.

Microbial contamination in dental unit waterlines.

The quality of water in a dental unit is of considerable importance because patients and dental staff are regularly exposed to water and aerosol generated from the dental unit. The aim of this study was to evaluate the occurrence of microbial contamination in dental unit waterlines. Water samples were collected aseptically from the waterlines (reservoir, triple-syringe, high-speed) of 15 dental units. After serial dilution to 1:10(6) in APHA, the samples were seeded by the pour-plate technique and cultured in plate count agar (Difco) for 48 h at 32 degrees C. Analysis was based on the number of colony forming units (CFU). The Wilcoxon non-parametric test indicated that the levels of water contamination were highest in the triple-syringe (13 of 15) and in the high-speed (11 of 15); both levels were higher than those of the water reservoir. There was no significant statistical difference between the level of contamination in the triple-syringe and the high-speed as determined by the Mann-Whitney test [p(H0) = 40.98%; Z = - 0.2281]. Because biofilm forms on solid surfaces constantly bathed by liquid where microorganisms are present, these results indicate that the water in the dental unit may be contaminated by biofilm that forms in these tubules.