Qualitative and quantitative molecular analysis of bacteria in root canals of primary teeth with pulp necrosis

Abstract Information about bacterial diversity, such as the number of each species in the root canals of primary teeth, contributes to improving our effective management of infections of endodontic origin in primary teeth. This study made a qualitative and quantitative assessment of the bacteria in the root canals of primary teeth with necrotic pulp, using the fluorescence in situ hybridization (FISH) technique. Thirty-one primary teeth with pulp necrosis from 31 children were evaluated using the FISH technique, to detect the presence and density of Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Streptococcus, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythia and Treponema denticola. Descriptive measures explained the data related to density, and Student's t-test assessed the differences among the densities of each bacterium, according to signs and symptoms. The bacterial density was paired and correlated. All bacteria tested were detected and identified in all the samples. The average number of bacterial individuals from each species ranged from 1.9 x 108 cells/mL (S. mutans) to 3.1 x 108 cells/mL (F. nucleatum) (p > 0.05). The sum of the mean counts of each bacterium represented almost 80% of the entire microbial community. Patients with pain had significantly more T. denticola, and those with edema showed a greater density of Streptococcus and P. nigrescens (p < 0.05). This study revealed that all 12 bacteria evaluated were found in all primary teeth with pulp necrosis. There was no predominance among the species studied; all species had a similar number of individuals.

Assessment of the nitrification process in a culture of pacific white shrimp, using artificial substrate and bacterial inoculum in a biofloc technology system (BFT) / Avaliação do processo de nitrificação no cultivo do camarão branco do pacífico Litopenaeus vannamei com uso de substrato artificial e inóculo de bactérias em sistema de bioflocos (BFT)

ABSTRACT: Litopenaeus vannamei is the most cultured marine shrimp in all types of systems including the Bioflocs Technology System (BFT). Bioflocs are formed by microorganisms, among these, autotrophic bacteria are responsible for the nitrification process. This study aimed to identify and promote the development of nitrifying bacteria by adding artificial substrates and biofloc inoculum in L. vannamei culture in a BFT system. The experiment consisted of four treatments with three replics (4x3) as follows: (1) Control: clear water in which bioflocs were formed; (2) IN (10%): clear water with biofloc inoculum (10%); (3) IB: clear water with substrate (immature "bioballs"); and (4) MB: clear water with mature "bioballs" inoculum from a recirculation system. Treatments were stocked with shrimp juveniles (4.92±0.45 g) in 12 tanks with 200 L working volume at a stocking density of 200 shrimp/m³. Shrimps were fed twice a day with a commercial feed (38% CP) following a feeding table, and daily observations intake were made over the four weeks of the experiment. Biofloc and "bioballs" samples were collected to detect the growth of the population of nitrifying and heterotrophic bacteria by FISH. There was no significant difference between treatments (P>0.05) for survival, obtaining mean values greater than 88%. The IN (10%) treatment had lower concentrations of ammonia and nitrite, and nitrate concentration increased, while MB had a higher weight and biomass final, productivity, weekly weight gain and lower conversion of apparent feed for production performance results.

RESUMO: Litopenaeus vannamei é o camarão marinho mais cultivado em todos os tipos de sistemas, incluindo o Sistema de Tecnologia de Bioflocos (BFT). Os bioflocos são formados por microorganismos, entre estes, bactérias autotróficas que são responsáveis pelo processo de nitrificação. Este estudo teve como objetivo identificar e promover o desenvolvimento de bactérias nitrificantes pela adição de substratos artificiais e inóculo de bioflocos no cultivo de L. vannamei em sistema BFT. O experimento consistiu de quatro tratamentos com três repetições (4x3), sendo: (1) Controle: água limpa, na qual foram formados os bioflocos; (2) IN (10%): água limpa com inóculo de bioflocos (10%); (3) BI: água limpa com substrato ("bioballs" imaturos); e (4) BM: água limpa com inóculo de "bioballs" maduros de um sistema de recirculação. Para tanto, os tratamentos foram estocados com juvenis de camarão (4,92±0,45 g) em 12 tanques com 200 L de volume útil com densidade de 200 camarões/m³. Os camarões foram alimentados duas vezes ao dia com ração comercial (38% PB) seguindo uma tabela de alimentação, e observações diárias foram feitas ao longo das quatro semanas de experimento. Amostras de bioflocos e "bioballs" foram coletadas para detectar o crescimento da população de bactérias nitrificantes e heterotróficas por FISH. Não houve diferença significativa entre os tratamentos (P>0,05) para sobrevivência, obtendo-se valores médios superiores a 88%. O tratamento com IN (10%) apresentou menores concentrações de amônia e nitrito, e a concentração de nitrato aumentou, enquanto o BM apresentou maior peso e biomassa final, produtividade, ganho de peso semanal e menor conversão alimentar aparente para resultados de desempenho zootécnico.

Bacteriocin-like inhibitory substance in aquaculture: a classic method of protein precipitation for a new aplicability / Substância inibidora tipo bacteriocina na aquicultura: método clássico de precipitação de proteínas para novas aplicações

Techniques to decrease losses from bacterial diseases are always important to improve the fish production. The use of antagonistic substances (bacteriocins) has been proven to be a viable option. The aim of this study was to evaluate different methods of purification for bacteriocin like inhibitory substances (BLIS). For the purification process, we isolated and used two Gram-positive bacilli that produce antagonistic substances for pathogens in aquaculture. Tests for detection of interfering factors were also performed. After the confirmation that the antagonistic action was due the BLIS activity, we carried out the purification methods. The methods tested were: cell free supernatant, acid extraction and ammonium sulfate precipitation at two concentrations (20 and 50%). Salmonella Tiphy CFP/IAL1472 and Aeromonas hydrophila (isolated in a tilapia production environment) were used as indicators of the efficiency of extracts in controlling pathogenic potentials. Ammonium sulfate precipitation at 50% was the most appropriate for purifying the antagonistic substance for both indicators. The extracts of the two isolates remained active for 22 days at 25ºC. These are promising results regarding the water and fish health without the use of antibiotics, in this manner being a safer environmental practice.

Técnicas para diminuir as perdas causadas por doenças bacterianas são importantes para melhorar continuamente a produção de pescado. O uso de substâncias antagônicas (bacteriocinas) tem-se mostrado uma opção viável. O objetivo do trabalho foi avaliar diferentes métodos de purificação de bacteriocinas como substâncias inibidoras (BLIS). Dois bacilos Gram-positivos, produtores de substâncias antagonistas para agentes patogênicos da aquicultura, foram utilizados em processos de purificação. Depois de confirmada a ação antagônica pela atividade de BLIS, os métodos de purificação foram realizados. Os métodos testados foram: células livres de sobrenadante, extração ácida e precipitação por sulfato de amônia em duas concentrações (20 e 50%). Salmonella Typhi PCP/IAL1472 e Aeromonas hydrophila (isolada de um ambiente de tilapicultura) foram utilizadas como indicadores de eficiência dos extratos. O precipitado por sulfato de amônio a 50% foi o mais adequado para purificar a substância antagonista para ambos os isolados indicadores. Os extratos dos dois isolados permaneceram ativos por 22 dias em 25ºC. Estes resultados são promissores do ponto de vista da manutenção da sanidade da água e dos peixes, sem uso de antibióticos, constituindo uma prática ambientalmente mais segura.

Comparative metagenome of a stream impacted by the urbanization phenomenon

Abstract Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance) and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.

Longitudinal study on clinical and microbial analysis of periodontal status in pregnancy

Abstract This study was aimed to provide a longitudinal overview of the subgingival bacterial microbiome using fluorescence in situ hybridization (FISH) technique, in women in the second trimester of pregnancy (between 14 and 24 weeks), and 48 h and 8 weeks postpartum. Of 31 women evaluated during pregnancy, 24 returned for the 48-h and 18 for their 8-week exams postpartum. Probing depth (PD), bleeding on probing, clinical attachment level, and presence of calculus were recorded. Subgingival plaque samples were collected, and FISH was used to identify the numbers of eight periodontal pathogens. Friedman test was used to compare differences between follow-up examinations, followed by a multiple comparison test for a post hoc pairwise comparison. Clinically, a significantly greater number of teeth with PD = 4-5 mm were found during pregnancy than on postpartum examinations. Microbial analysis showed a statistically significant decrease in cell count over the study period for Prevotella nigrescens. P. intermedia, Campylobacter rectus, and Porphyromonas gingivalis also decrease, although not significantly, and Aggregatibacter actinomycetemcomitans increased. No significant changes were found for Fusobacterium nucleatum, Treponema denticola, or Tannerella forsythia. Our data demonstrate a change in the subgingival microbiota during pregnancy, at least for P. nigrescens.

Detection and enumeration of periodontopathogenic bacteria in subgingival biofilm of pregnant women

The aim of this study was to use the fluorescence in situ hybridization (FISH) technique to test the hypothesis of qualitative and quantitative differences of 8 periodontopathogens between pregnant and non-pregnant women. This cross-sectional study included 20 pregnant women in their second trimester of pregnancy and 20 non-pregnant women. Probing depth, bleeding on probing, clinical attachment level, and presence of calculus were recorded. Subgingival plaque samples were collected and the FISH technique identified the presence and numbers of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia and Prevotella nigrescens. The Mann-Whitney U-test was applied to compare the data between the two groups. The mean age, ethnicity, marital status, education, and economic level in both groups were similar. The clinical parameters showed no significant differences between pregnant and non-pregnant women. The numbers of subgingival periodontopathogens were not found to be significantly different between groups, despite the higher mean counts of P. intermedia in pregnant women. Colonization patterns of the different bacteria most commonly associated with periodontal disease were not different in the subgingival plaque of pregnant and non-pregnant women.