Avaliação da microbiota bacteriana conjuntival de cães hígidos atendidos no Hospital Veterinário da Universidade de Marília

A microbiota conjuntival dos cães é formada por uma associação de micro-organismos normalmente não patogênicos, que interagem com o sistema imune do animal e possui função de atuar como barreira natural contra entrada de agentes patogênicos. O presente trabalho foi delineado para investigar os micro-organismos presentes na conjuntiva de cães sadios. Foram utilizados 25 cães sadios da rotina clínico cirúrgica do hospital veterinário da Unimar e após instilar uma gota de colírio anestésico em cada olho colheu-se amostra conjuntival superior e inferior de ambos os olhos com swab etéril, preservando a amostra em meio Stuart e logo após cultivada em meios BHI (Brain Heart Infusion), Ágar sangue de carneiro 6%, Ágar MacConkey e Ágar TSA. Após o crescimento bacteriano foi realizada a identificação dos micro-organismos cultivados por meio de testes de triagem bioquímica como oxidase e catalase, além da análise da morfologia bacteriana em lâmina, padrão e coloração de crescimento em ágar. Houve crescimento bacteriano em amostras colhidas de 20 animais das quais 38% dos isolados foram compatíveis com S. intermedius; 30% de Bacilo sp. e 10% S. aureus. A espécie Staphylococcus sp. é natural de membranas mucosas, não sendo patogênica ao animal. Foi, portanto, constatada a predominância de S. intermedius nas amostras da microbiota do olho dos cães sadios examinados.

Investigation of heteroresistant vancomycin intermediate Staphylococcus aureus among MRSA isolates

Introduction: Heteroresistant vancomycin intermediate Staphylococcus aureus (hVISA) testing is recommended when therapeutic failure is suspected in the clinics. In our research, we aimed to investigate the prevalence of hVISA among methicilline-resistant S. aureus (MRSA) isolates in our university hospital and compared three methods for detection of hVISA. Methodology: One hundred MRSA clinical isolates were collected in our medical microbiology laboratory between 01.04.2018 and 01.10.2019. For screening of hVISA, we used two screening agar plates and used one commercial medium; brain heart infusion agar (BHI) plates containing 4 µg/mL vancomycin and 16 g/Lt casein (BHIA-VC; Satola’s test), BHI agar plates containing 4 µg/mLvancomycin (BHIAV), and commercially obtained vancomycin resistant Enterococci (VRE) agar for detetection of hVISA. Colonies which could grow on plates were counted manually at 24th and 48th hours. Results: Among 100 MRSA isolates, 43 (43%) were found as hVISA using Satola’s test. BHIAV and VRE agar screening test results were found 70% and 4%, respectively. Finally, at the step, MIC values of 20 (47%) hVISA isolates reduced to 2 µg/mL after sub culturing for the gradient test. Conclusions: We found higher rates of hVISA comparing other studies in Turkey. Both VRE agar and BHIAV screening test failed to detect hVISA properly. Meropenem in combination with vancomycin inhibited the growth of 90% hVISA isolates in our study.

Bacterial analysis of biofilms from tooth root surfaces presenting different caries activity

This study evaluated the numbers and determined the proportion of mutans streptococci and Lactobacillus spp., which are possible relevant cariogenic organisms, in biofilms recovered from lesions at root surfaces with active caries lesions (ARC), inactive caries lesions, and sound root surfaces (SRS). Samples were cultured in MSB agar for mutans streptococci counts, Rogosa agar for Lactobacillus spp. counts, and brain-heart infusion agar for total viable anaerobic counts. After incubation, the number of colony-forming units (CFUs) was determined and compared between groups by the Mann-Whitney U test with a significance level set at 95%. The proportion of counts of mutans streptococci and Lactobacillus spp. in the total viable microorganisms was also analyzed by Chi-square test. Ninety samples (30 from each surface) from 37 patients were cultured and analyzed. The CFU was similar between mutans streptococci and Lactobacillus spp. These species were present in at least half of the samples and no difference was found in the frequency of isolation of these species. Only 6 samples showed a proportion of more than 10% of mutans streptococci; 4 of the samples were from ARC. Most (93%) SRS samples did not contain viable Lactobacillus spp. The data indicate the low counts of mutans streptococci and Lactobacillus spp. in root surfaces, regardless of the activity of caries lesions.

Groundnut Shell Infusion Agar as a Culture Medium for Moulds

Aim: To carry out a Comparative mould analysis using groundnut shell infusion agar (GSA) and potato dextrose agar (PDA), as the control. Study Design: Laboratory-experimental design was used in this study. Place and Duration of Study: Soil samples were obtained from three different locations (Garden soil beside Biology Main Laboratory, opposite Faculty of Law and Faculty of Agriculture) in Rivers State University, Port Harcourt Rivers State, Nigeria. The study was carried out for three (3) months at the Microbiology Laboratory, Rivers State University, Port Harcourt. Methodology: Groundnut Shell Infusion Agar (GSA) was prepared by weighing 28 g of blended gari and 15 g of agar powder into 1L of groundnut shells filtrate. Potato dextrose Agar (PDA), a conventional medium was prepared according to the manufacturer’s specifications. GSA was prepared by weighing 28 g of blended gari and 15 g of agar powder into 1L of groundnut shells filtrate. Potato dextrose agar, a conventional medium was prepared according to the Manufacturer’s specifications. Results: The mean mould counts from the different locations ranged from 3.7×107 cfu/ml to 7.8×107 cfu/ml on GSA and 3.7×107 cfu/ml to 1.5×109 cfu/ml on PDA following incubation at room temperature (27°c ± 2) for 3-5 days. The moulds identified were Aspergillus niger, Aspergillus flavus, Trichoderma viride, Rhizopus sp. Mucor sp. Botrytis sp. Helminthosporium caryopsidum, and Penicillum sp. Conclusion: From the results obtained, it showed that GSA could be used successfully for quantitative mould counts and other mycological studies. This would proffer solution to the high cost of conventional media used for moulds as well as agro waste pollution in the environment.

Mutation in MPT64 gene influencing diagnostic accuracy of SD Bioline assay (capilia)

Background Success of India’s TB control program relies on rapid case detection, monitoring, care and treatment of drug resistance. Patients on multidrug resistance (MDR) treatment are monitored by follow up cultures. Discordant results (culture and smear positive while capilia negative) are usually declared negative Mycobacterium tuberculosis complex (MTBC). This study was designed to understand the possible causes of discordant results. Methods The capilia kit was evaluated to test its utility among 4737 follow up MDR patients enrolled during a period of 1 year. A total of 889 were liquid culture positive, 3375 were negative and 473 were contaminated. Of the 889 cultures positive, 829 were found positive by ZN smear, capilia test and MTBDR plus assay. The cultures which gave a positive result on Mycobacterium Growth Indicator Tube 960 (MGIT 960) and ZN smear but were negative on capilia test with no growth on Brain Heart Infusion agar (BHI) were included in this study. The conflicting results of capilia were compared with other molecular techniques; MTBDR plus assay and DNA sequence analysis of MPT64 gene. Results Out of 889 culture positive, 60 (6.7%) were found positive on liquid culture and ZN smear but were negative on capilia. Of these 60 cultures, 10 (16.7%) were found positive by both MTBDR plus assay and PCR. The sequencing analysis revealed that all of the capilia negative isolates had mutations within the MPT64 gene. Conclusion Re-evaluation of culture positive but capilia negative isolates should be done before declaring them as Mycobacterium other than tuberculosis (MOTT) because such cases can act as chronic carriers of TB in the population which can lead to the rise of this lethal disease.

Antibacterial Effect of Peruvian Propolis Collected During Different Seasons on the Growth of Streptococcus Mutans

Introduction: Propolis is a gummy, resinous substance made by bees from the buds and exudates of plants. The antibacterial activity of propolis has been widely studied and is known to vary according to its geographical origin, the type of surrounding flora, the collecting bee species, the mode of its collection and even the season in which it is collected. Unfortunately, these observations have not been corroborated experimentally. Aim: To compare the antibacterial activities of ethanolic extracts of propolis collected in the summer and autumn on the growth of Streptococcus mutans ATCC 25175. Materials and Methods: Propolis samples were collected in the summer and autumn and labeled “A” or “B” by an individual who was not directly involved in the study. Then, 5% ethanolic extracts of propolis were prepared for each sample. S. mutans was plated onto brain heart infusion agar plates into which wells were formed, and the plates were divided into four groups to test the antibacterial effectiveness of both the extracts and the positive (0.12% chlorhexidine digluconate) and negative (96% ethanol) controls. Results: Inhibition halos of 26.4±2.6 and 18.2±1.8 mm were observed for the autumn and summer propolis extracts, respectively, while those of the negative and positive controls were 0 and 13 mm, respectively. These differences were statistically analyzed using Student’s t-test. Conclusion: The significantly higher growth of S. mutans in the extracts made from propolis collected in autumn than that grown on extracts collected in summer indicates that the season in which propolis is collected does indeed influence its antibacterial activity.

ANTIBACTERIAL ACTIVITY OF CELERY LEAVES (APIUM GRAVEOLENS L.) FORMULATED IN TOOTHPASTE AGAINST STREPTOCOCCUS MUTANS

Objective: The objective of this research was to determine the antibacterial activity of the toothpaste from an extract of celery leaves on Streptococcusmutans.Methods: The toothpaste was formulated with various concentrations of celery leaves, F1 with concentration of extract (6.25%), F2 (12.5%), andF3 (25%). Each formula was tested the physical characteristics and antibacterial activity toward S. mutans. The antibacterial activity was determinedby the agar well diffusion method using brain heart infusion agar plates. Furthermore, the antibacterial activities were assessed by the presence orabsence of inhibition zones after the plates were incubated at 37°C for 24 h.Results: The results from this test illustrate that all toothpastes under study at various concentrations of celery leaves extract exhibited antibacterialactivity. Maximum inhibition zone in antibacterial activity test was shown by F2 (12.5%). Therefore, we can use these toothpastes as naturalantibacterial on prevention of dental caries caused S. mutans.Conclusion: The toothpaste from an extract of celery leaves showed significant antibacterial activity against S. mutans.

ANTIBACTERIAL EFFICACY OF NISIN AS AN IRRIGANT AGAINST ENTEROCOCCUS FAECALIS BIOFILM

Objective: This study aimed to compare the antibacterial efficacy of 10% nisin, 2% chlorhexidine (ChX), and 2.5% sodium hypochlorite (NaOCl)against Enterococcus faecalis biofilm in vitro.Methods: Petri dishes containing brain heart infusion agar were seeded with E. faecalis (ATCC 29212) and were incubated overnight at 37°C. Thecellulose nitrate filter membrane was inoculated with E. faecalis for 72 h to grow a biofilm, and we performed the direct contact test between the testsolutions and the biofilm for 10 min. The DNA was quantified using real-time polymerase chain reaction with propidium monoazide additive to countthe living cells.Results: The number of E. faecalis bacteria in the 2% ChX group was the lowest (8.36×103 CFU/mL) while the highest number of bacteria - among theantibacterial substances tested - in the nisin 10% group (5.55×106 CFU/mL).Conclusion: The antibacterial effects against E. faecalis biofilm of 10% nisin were not comparable with those of 2% ChX and 2.5% NaOCl.

Análise da atividade antimicrobiana de probióticos e sua adesividade a bráquetes ortodônticos: estudo in vitro

Resumo Introdução A presença de aparelho ortodôntico fixo dificulta a higienização e potencializa o acúmulo de biofilme bacteriano nas superfícies dentárias. O desenvolvimento de produtos que minimize isso é desejo de pesquisadores em todo o mundo. Objetivo Verificar a ação bacterapêutica de produtos lácteos contendo ou não probióticos sob pool de Streptococcus mutans (SM) (ATCC 25175) e S salivarius (SS) (ATCC 7073), além da adesão desses produtos à superfície de bráquetes ortodônticos. Material e método Pool de cepas ATCC de SM e SS foi formado e plaqueado sobre placa de Petri contendo meio de cultura brain heart infusion ágar (BHI). Após formação do meio, um orifício foi feito no centro da placa seguido do seu preenchimento com 150 µL dos produtos a serem testados, formando os seguintes grupos: GL - Leite bovino; GLP - Leite bovino com probiótico; GLF - Leite fermentado; e GLFP - Leite fermentado com probiótico. Na sequência, as placas foram incubadas por 48h, em estufa a 37ºC. A seguir, foi feita a medição do halo formado entre o produto e o meio com régua milimetrada. Já no disco de membrana, foi formado biofilme com o mesmo pool de cepas, sob discos de membrana. Em seguida, foi feita a diluição seriada contendo o produto de acordo com o grupo: P1 (água); P2 (L); P3 (LP); P4 (LFP), seguida do plaqueamento e a contagem total de micro-organismos. Para a adesividade dos produtos lácteos, bráquetes ortodônticos foram submergidos em cada solução (GL, GLP, GLF e GLFP) e foram incubadas a 37°C/24h. Posteriormente, cada bráquete foi transferido para um ependorf contendo solução salina estéril, que foi submetida a diluições seriadas, posteriormente incubadas a 37°C/48h sob microaerofilia para contagem das UFC/mL. Para análise dos dados, utilizaram-se os testes Levene, Shapiro-Wilk e Kruskal-Wallis. O nível de significância adotado foi de 5% (α = 0,05). Resultado Não houve formação de halo de inibição entre os produtos e o meio de cultura (p<0,05); no disco de membrana, não foram observadas diferenças estatísticas entre os grupos (p=0,679); os grupos tratados com leite bovino com probiótico e leite fermentado com probiótico apresentaram adesividade aos bráquetes ortodônticos (p=0,056). Conclusão Os achados do presente estudo permitem concluir que, em estudos in vitro, não foi possível verificar a bacterioterapia a partir de produtos lácteos contendo ou não probióticos em cepas de SM e SS.

Antagonism of Bacteria from Dog Dental Plaque against Human Cariogenic Bacteria

Dental caries are a process of demineralization and destruction of human teeth. They originate through many factors and are associated with biofilm formation, which consists of bacteria adhered to the teeth that form a structurally and functionally organized mass called dental plaque. Both the presence ofStreptococcus mutansand the frequent consumption of sucrose correlate with a higher prevalence of caries in humans. In dogs, however, the incidence of this disease is low, due to factors such as differences in dental microbiota and/or their low consumption of sucrose. This work evaluated the antagonism of bacteria from dog’s dental plaque againstS. mutans, for the identification of producing strains of biotechnological products for use in preventing caries. This study used 95 bacterial isolates of canine dental plaque from the Veterinary Department at the Federal University of Viçosa, Minas Gerais, Brazil. A spot-on-the-lawn method was performed using Brain Heart Infusion agar with catalase for an initial identification of the antagonistic activity. Additional tests were conducted on the isolates classified as antagonists for confirmation of the activity, using modified Mann-Rogosa-Sharpe medium containing low dextrose concentration. These isolates were incubated at 37°C for 24 hours in anaerobiosis. The peptide nature of inhibition was evaluated using the following proteinases: proteinase K fromTritirachium album, bovine pancreatic trypsin, and type XII-Aα-amylase fromBacillus licheniformis. In the initial identification of those strains exhibiting antimicrobial activity, 14 were classified as antagonists. One of the isolates (Bacillussp.) indicated bacteriocinogenic activity, with a deformed inhibition halo onS. mutansby the addition of trypsin. These results suggest that this bacterial isolate may be applicable to biotechnological use to combat the main etiological agent of caries in humans. Further studies are needed to evaluate the bacteriocinogenic nature of the antimicrobial activities of the other 13 antagonistic bacterial isolates.