Resumo Recentemente, foi relatado que Scardovia wiggsiae está fortemente associado à formação de cáries. Este estudo teve como objetivo estabelecer um modelo in vitro de biofilme de S. wiggsiae e investigar o efeito da nicotina no crescimento de unidades formadoras de colônias (UFC) de S. wiggsiae. O biofilme de S. wiggsiae foi cultivado durante a noite usando caldo de infusão de cérebro-coração (BHI) suplementado com 5 g de extrato de levedura / L (BHI-YE). A cultura noturna foi usada como um inóculo para cultivar biofilme de S. wiggsiae em amostras padronizadas de esmalte e dentina. As amostras foram incubadas com diferentes concentrações de nicotina (0, 0,5, 1, 2, 4, 8, 16 e 32 mg/mL) por 3 dias. Os biofilmes dissociados foram diluídos, semeados em espiral em placas de ágar sangue e incubados por 24 h. UFC/mL foram quantificados usando um contador de colônias automatizado. Uma ANOVA de duas vias foi usada para comparar o efeito de diferentes concentrações de nicotina em UFCs de S. wiggsiae. Este estudo demonstrou que o biofilme de S. wiggsiae pode ser iniciado e formado in vitro. UFCs aumentadas foram observadas com 0,5-4 mg/mL e 0,5-8 mg/mL de nicotina usando substratos de esmalte e dentina, respectivamente. A concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM) de nicotina foram determinadas, respectivamente, como 16 e 32 mg/mL. S. wiggsiae formou maior biofilme no esmalte do que espécimes de dentina em resposta ao estímulo de nicotina. Este estudo demonstrou o efeito negativo do tabagismo no aumento do biofilme de S. wiggsiae. O estabelecimento do biofilme de S. wiggsiae in vitro pode permitir que os pesquisadores no futuro tenham uma melhor compreensão da patogênese da cárie e da interação bacteriana.

Abstract Recently, Scardovia wiggsiae has been reported to be strongly associated with caries formation. This study aimed to establish an in vitro model of S. wiggsiae biofilm and to investigate the effect of nicotine on S. wiggsiae colony-forming units (CFUs) growth. S. wiggsiae biofilm was grown overnight using brain-heart infusion (BHI) broth supplemented with 5 g of yeast extract/L (BHI-YE). The overnight culture was used as an inoculum to grow S. wiggsiae biofilm on standardized enamel and dentin samples. Samples were incubated with different nicotine concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL) for 3 days. The dissociated biofilms were diluted, spiral plated on blood agar plates, and incubated for 24 h. CFUs/mL were quantified using an automated colony counter. A two-way ANOVA was used to compare the effect of different nicotine concentrations on S. wiggsiae CFUs. This study demonstrated that S. wiggsiae biofilm could be initiated and formed in vitro. Increased CFUs was observed through 0.5-4 mg/mL and 0.5-8 mg/mL of nicotine using enamel and dentin substrates, respectively. 16 and 32 mg/mL of nicotine were determined as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. S. wiggsiae formed greater biofilm on enamel than dentin specimens in response to the nicotine stimulus. This study demonstrated the negative effect of smoking on increasing S. wiggsiae biofilm. Establishing S. wiggsiae biofilm in vitro may allow researchers in the future to have a better understanding of caries pathogenesis and bacterial interaction.

Goat spermatozoa were incubated in vitro in Tris-citrate buffer, pH 7.2, containing 0, 40, 80, or 160 mug of Naja haje venom/mL buffer for 4 hours. During incubation, the percentages of sperm motility were decreased, while percentages of dead spermatozoa were increased in a time and concentration-dependent manner. The effect of venom concentrations on the ultrastructure of incubated spermatozoa was examined hourly by scanning (SEM) and transmission (TEM) electron microscopy. SEM results showed plasma membrane wrinkling at heads of some spermatozoa after 2 h incubation with 40mug venom. Most spermatozoa suffered membrane wrinkling after 4 h incubation. However, incubation with 80 mug venom caused membrane fractures in most sperm heads after 1 h incubation. The extent and depth of these fractures were increased after 2-3 h incubation. After 4 h incubation, plasma membrane focal erosion of many spermatozoa heads was common. Incubation with 160 mg venom induced sperm head swollen plasma membranes after 1 h incubation. Ruptured and disintegrated membranes were seen after 2 h; lysis and removal of external surface of spermatozoa head plasma membranes were recorded after 3-4 h incubation. TEM indicated slightly swollen areas on the sperm head plasma membrane, but showed normal nuclei, acrosomes, and tail regions after 2 h incubation in 40 mug cobra venom. The swollen areas were accompanied by sperm head membrane disintegration as well as membrane irregularities and distortion of tail mitochondrial cristae after 3-4 h incubation. However, incubation with 80 mug venom showed focal areas of membrane lysis and discontinuity in the sperm heads and tails increasing with incubation time. Severe axoneme and tail longitudinal fiber degeneration and increased numbers of distorted mitochondrial cristae were also observed after 3-4 h incubation. Spermatozoa incubation with 160 mug venom increased severity of plasma membrane dissolution, disintegration, and rupture. Axoneme disorganization and tail longitudinal fiber fusion were seen after 1-2 h; complete deformation of the motility system was recorded after 3-4 h incubation. Nuclei were normal, but acrosomes were partially damaged, and distorted mitochondrial cristae were increased after 4 h incubation. The results indicated that cobra venom induced a concentration and time-dependent alterations on spermatozoa plasma membrane as well as obvious deformation on the motility system and tail mitochondria, which are responsible for sperm viability and fertilizing ability.