Evandro Watanabe

Effect of Three Methods for Cleaning Dentures on Biofilms Formed In Vitro on Acrylic Resin

PURPOSE The aim of this study was to evaluate the effect of three denture hygiene methods against different microbial biofilms formed on acrylic resin specimens. MATERIALS AND METHODS The set (sterile stainless steel basket and specimens) was contaminated (37 degrees C for 48 hours) by a microbial inoculum with 10(6) colony-forming units (CFU)/ml (standard strains: Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Candida albicans, Pseudomonas aeruginosa, and Enterococcus faecalis; field strains: S. mutans, C. albicans, C. glabrata, and C. tropicalis). After inoculation, specimens were cleansed by the following methods: (1) chemical: immersion in an alkaline peroxide solution (Bonyplus tablets) for 5 minutes; (2) mechanical: brushing with a dentifrice for removable prostheses (Dentu Creme) for 20 seconds; and (3) a combination of chemical and mechanical methods. Specimens were applied onto a Petri plate with appropriate culture medium for 10 minutes. Afterward, the specimens were removed and the plates incubated at 37 degrees C for 48 hours. RESULTS Chemical, mechanical, and combination methods showed no significant difference in the reduction of CFU for S. aureus, S. mutans (ATCC and field strain), and P. aeruginosa. Mechanical and combination methods were similar and more effective than the chemical method for E. faecalis, C. albicans (ATCC and field strain), and C. glabrata. The combination method was better than the chemical method for E. coli and C. tropicalis, and the mechanical method showed intermediate results. CONCLUSION The three denture hygiene methods showed different effects depending on the type of microbial biofilms formed on acrylic base resin specimens.

Antimicrobial activity of flavonoids and steroids isolated from two Chromolaena species

The crude extracts (dichloromethanic and ethanolic) and some compounds (8 flavonoids and 5 steroids) isolated from Chromolaena squalida (leaves and stems) and Chromolaena hirsuta (leaves and flowers) have been evaluated against 22 strains of microorganisms including bacteria (Gram-positive and Gram-negative) and yeasts. All crude extracts, flavonoids and steroids evaluated have been shown actives, mainly against Gram-positive bacteria.

Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis

Bovine mastitis represents the main form of occurrence of protothecosis in animals. The detection of mastitis caused by Prototheca sp. indicates a serious problem which can affect an entire herd. The purpose of this study is to explain some aspects of the epidemiology of mastitis due to Prototheca zopfii with the evaluation of the presence of these microorganisms in samples collected from potential sources in the dairy herd. This study was performed during a Prototheca zopfii outbreak of clinical bovine mastitis in the State of São Paulo, Brazil. The following samples were aseptically collected for microbiological examination: milk (n = 211); rectal swabs (from 15 calves and 2 lactating cows); swabs from teat cup rubbers during milking (n = 2); water (n = 6); soil (n = 6). Prototheca zopfii was isolated from 77 (36.49%) of the 211 milk samples; 11 calves and 2 cows showed Prototheca zopfii in faecal samples; both swabs collected from the teat cup rubbers showed viable forms of Prototheca zopfii; this microorganism was also isolated from 2 water samples, and 1 soil sample collected from the dry cow pasture. Prototheca zopfii seemed to be widespread throughout the dairy herd environment where this outbreak of bovine mastitis occurred.

Bovine mastitis due to algae of the genus Prototheca

Protothecosis was described in many animals, with bovine mastitis being the main form. The increasing number of isolations of Prototheca spp. from bovine mastitis cases indicates the need of a detailed evaluation of this problem. Besides this, these algae do not respond to treatment with the antimicrobians most frequently applied, leading to elimination of the affected animals, as the best method to control the disease. In two dairy farms in the State of São Paulo, Brazil, a total of 155 lactating cows and 52 dry cows were examined. Milk samples were aseptically collected from lactating cows with clinical or subclinical mastitis. From dry cows, secretion samples from all quarters were collected. All samples were then taken to microbiological exams. At dairy farm 1, Prototheca spp. was isolated from 14.95% milk samples from lactating cows, (all of them cases of subclinical mastitis), and from 8.06% samples from dry cows. At dairy farm 2, Prototheca spp. was isolated from 5.1% milk samples and there were ten cases of clinical mastitis due to this agent. Although clinical mastitis has been considered the main form of occurrence of this pathology until now, it is important to consider Prototheca spp. as subclinical mastitis pathogen. Prototheca zopfii was the main specie isolated. In this study a high rate of intramammarian infections, as clinical or subclinical mastitis during lactation as well as in dry period, was observed, high lighting the importance of suitable diagnosis, so that control and preventive measures can be implemented to avoid dissemination of the agent.

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk.

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.

Determination of the maximum inhibitory dilution of cetylpyridinium chloride-based mouthwashes against staphylococcus aureus: an in vitro study

The aim of this in vitro study was to determine the maximum inhibitory dilution (MID) of four cetylpyridinium chloride (CPC)-based mouthwashes: CPC+Propolis, CPC+Malva, CPC+Eucaliptol+Juá+Romã+Propolis (Natural Honey®) and CPC (Cepacol®), against 28 Staphylococcus aureus field strains, using the agar dilution method. Decimal dilutions ranging from 1/10 to 1/ 655,360 were prepared and added to Mueller Hinton Agar. Strains were inoculated using Steers multipoint inoculator. The inocula were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. The dishes were incubated at 37°C for 24 h. For readings, the MID was considered as the maximum dilution of mouthwash still capable of inhibiting microbial growth. The obtained data showed that CPC+Propolis had antimicrobial activity against 27 strains at 1/320 dilution and against all 28 strains at 1/160 dilution, CPC+Malva inhibited the growth of all 28 strains at 1/320 dilution, CPC+Eucaliptol+Juá+Romã+Propolis inhibited the growth of 2 strains at 1/640 dilution and all 28 strains at 1/320 dilution, and Cepacol® showed antimicrobial activity against 3 strains at 1/320 dilution and against all 28 strains at 1/160 dilution. Data were submitted to Kruskal-Wallis test, showing that the MID of Cepacol® was lower than that determined for the other products (p<0.05). In conclusion, CPC-mouthwashes showed antimicrobial activity against S. aureus and the addition of other substances to CPC improved its antimicrobial effect.

Mucoadhesive System Formed by Liquid Crystals for Buccal Administration of Poly(Hexamethylene Biguanide) Hydrochloride

Antimicrobial approaches are valuable in controlling the development of buccal diseases, but some antibacterial agents have a short duration of activity. Therefore, the development of prolonged delivery systems would be advantageous. Liquid crystalline systems comprising monoolein (GMO)/water have been considered to be a potential vehicle to deliver drugs to the buccal mucosa because of the phase properties that allow for controlled drug release as well as its mucoadhesive properties. Therefore, the aim of this study was to develop a GMO/water system for the slow release of poly(hexamethylene biguanide) hydrochloride (PHMB) on the buccal mucosa and test the properties of this system with regard to swelling, release profile, antimicrobial activity, and strength of mucoadhesion, with the overall goal of treating buccal infections. The tested systems were capable of modulating drug release, which is controlled by diffusion of the drug throughout the system. Furthermore, PHMB appeared to improve the mucoadhesive properties of the system and may synergistically act with the drug to promote antimicrobial activity against S. mutas and C. albicans, indicating that liquid crystals may be suitable for the administration of PHMB on the buccal mucosa. Therefore, this system could be proposed as a novel system for mucoadhesive drug delivery.

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.

Clinical and antimicrobial efficacy of NitrAdineTM-based disinfecting cleaning tablets in complete denture wearers

Objective This study evaluated the efficacy of NitrAdineTM-based disinfecting cleaning tablets for complete denture, in terms of denture biofilm removal and antimicrobial action. Material and methods Forty complete denture wearers (14 men and 26 women) with a mean age of 62.3±9.0 years were randomly assigned to two groups and were instructed to clean their dentures according to two methods: brushing (control) - 3 times a day with denture brush and tap water following meals; brushing and immersion (experimental) - brushing the denture 3 times a day with denture brush and tap water following meals and immersion of the denture in NitrAdineTM-based denture tablets (Medical InterporousTM). Each method was used for 21 days. Denture biofilm was disclosed by a 1% neutral red solution and quantified by means of digital photos taken from the internal surface before and after the use of the product. Microbiological assessment was conducted to quantify Candida sp. Results An independent t-test revealed a significant lower biofilm percentage for the experimental group (4.7, 95% CI 2.4 to 7.9) in comparison with the control group (mean 37.5, 95% CI 28.2 to 48.1) (t38=7.996, p<0.001). A significant reduction of yeast colony forming units could be found after treatment with Medical InterporousTM denture tablets as compared to the control group (Mann-Whitney test, Z=1.90; p<0.05). Conclusion The present findings suggest that NitrAdineTM-based disinfecting cleaning tablets are efficient in removal of denture biofilm. In addition, a clear antimicrobial action was demonstrated. Therefore, they should be recommended as a routine denture maintenance method for the prevention of the development of microbial biofilm-induced denture stomatitis.