Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution

Chlorhexidine (CHX) is a broad-spectrum antiseptic that is used in many topical pharmaceutical formulations. Because there is no official microbiological assay reported in the literature that is used to quantify CHX, this paper reports the development and validation of a simple, sensitive, accurate and reproducible agar diffusion method for the dosage of chlorhexidine digluconate (CHX-D) in an aqueous solution. The assay is based on the inhibitory effect of CHX-D upon the strain of Staphylococcus aureus ATCC 25923, which is used as the test microorganism. The design 3x3 parallel-line model was used. The results were treated statistically by analysis of variance (ANOVA), and they were excellent in terms of linearity (r = 0.9999), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 0.5 to 4.5%. The results obtained were precise, having relative standard deviations (RSD) for intra-day and inter-day precision of 2.03% and 2.94%, respectively. The accuracy was 99.03%. The method proved to be very useful and appropriate for the microbiological dosage of CHX-D in pharmaceutical formulations; it might also be used for routine drug analysis during quality control in pharmaceutical industries.

Clorexidina (CHX) é um antisséptico com amplo espectro de ação utilizada em muitos tipos de preparações farmacêuticas para uso tópico. Uma vez que não há na literatura ensaio microbiológico oficial para quantificar a clorexidina, este trabalho objetivou o desenvolvimento e validação de um ensaio microbiológico simples, sensível, exato e reprodutível, por difusão em ágar, para doseamento de digliconato de clorexidina (CHX-D) em solução aquosa. O ensaio é baseado no efeito da inibição de Staphylococcus aureus ATCC 25923, utilizado como microorganismo teste, pela CHX-D. Utilizou-se o delineamento 3x3. Os resultados foram verificados estatisticamente pela análise de variância (ANOVA) e apresentaram excelente linearidade (r = 0,9999), demonstrando que o método segue o modelo linear com regressão significativa entre o diâmetro da zona de inibição e o lagaritmo da concentração no intervalo de 0,5 a 4,5%. Os resultados obtidos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 2,03% e DPR para precisão inter-dias de 2,94%. A exatidão foi 99,03%. O método provou ser muito útil e apropriado para doseamento microbiológico da CHX-D em formas farmacêuticas e pode ser empregado para análise desta substância no controle de qualidade em indústrias farmacêuticas.

Microbicide activity of clove essential oil (Eugenia caryophyllata)

Clove essential oil, used as an antiseptic in oral infections, inhibits Gram-negative and Gram-positive bacteria as well as yeast. The influence of clove essential oil concentration, temperature and organic matter, in the antimicrobial activity of clove essential oil, was studied in this paper, through the determination of bacterial death kinetics. Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were the microorganisms selected for a biological test. To determine the temperature effect, they were assayed at 21° and 37° C. The concentration coefficient was determined with 0.4%, and 0.2% of essential oil. The influence of the presence of organic matter was determined with 0.4% of essential oil. The results obtained demonstrated that Escherichia coli were more sensitive even though the essential oil exerted a satisfactory action in three cases. In the three microbial species, 0.4% of essential oil at 21º C have reduced the bacterial population in 5 logarithmic orders. Organic matter reduces the antibacterial activity even though the bactericide efficacy was not lost. Clove essential oil can be considered as a potential antimicrobial agent for external use.

Chlorhexidine: beta-cyclodextrin inhibits yeast growth by extraction of ergosterol

Chlorhexidine (Cx) augmented with beta-cyclodextrin (β-cd) inclusion compounds, termed Cx:β-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cx:β-cd complexes, prepared at different molecular ratios, with sterol and yeast membranes. The Minimal Inhibitory Concentration (MIC) against the yeast Candida albicans (C.a.) was determined for each complex; the MICs were found to range from 0.5 to 2 µg/mL. To confirm the MIC data, quantitative analysis of viable cells was performed using trypan blue staining. Mechanistic characterization of the interactions that the Cx:β-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cx:β-cd complexes were performed using Sterol Quantification Method analysis (SQM) and scanning electron microscopy (SEM). SQM revealed that sterol extraction increased with increasing β-cd concentrations (1.71 × 10³; 1.4 × 10³; 3.45 × 10³, and 3.74 × 10³ CFU for 1:1, 1:2, 1:3, and 1:4, respectively), likely as a consequence of membrane ergosterol solubilization. SEM images demonstrated that cell membrane damage is a visible and significant mechanism that contributes to the antimicrobial effects of Cx:β-cd complexes. Cell disorganization increased significantly as the proportion of β-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 1:3 and 1:4 molar ratios of Cx:β-cd were observed to have large aggregates mixed with yeast remains, representing more membrane disruption than that observed in cells treated with Cx alone. In conclusion, nanoaggregates of Cx:β-cd complexes block yeast growth via ergosterol extraction, permeabilizing the membrane by creating cluster-like structures within the cell membrane, possibly due to high amounts of hydrogen bonding.

Microbial contamination of antiseptics and disinfectants.

OBJECTIVE: To study the bacterial contamination of antiseptics and disinfections in-use and the risk factors for contamination. MATERIAL AND METHOD: Bacterial contamination of antiseptics and disinfectants was done by culturing in-use solutions. Eight commonly used solutions were studied: alcohol 70%, chlorhexidine 4%, and 0.5%, povidone iodine 7.5% and 10%, tincture iodine 1-2%, lysol 2% and sodium hypochlorite 0.5%. RESULTS: The following risk factors for contamination were found : preparation by unskilled personnel, improper containers and prolonged use. Contamination with bacteria were found in 1.8% of 16,142 samples tested Highest rate of contamination was found in Lysol 2%. There was no contamination of povidone iodine 10% and tincture iodine 1-2%. Bacterial contamination of antiseptics and disinfectants was highest in provincial hospitals and was not found in university hospitals. The rates of contamination correlated with the duration of use. Most bacteria isolated were those found in the environment. CONCLUSION: The contamination of in-use antiseptics and disinfectants was as high as 1.8%. Risk factors for contamination were improper preparation and prolonged use.

Estudo comparativo in vitro da atividade antibacteriana de produtos fitoterápicos sobre bactérias cariogênicas / Comparative study in vitro of the antibacterial activity from phytotherapeutic products against cariogenic bacterias

O objetivo deste estudo foi avaliar a atividade antibacteriana deprodutos fitoterápicos comerciais frente aos microrganismoscariogênicos: S. mutans (ATCC 2575); S. sobrinus (ATCC27607); S. sanguis (ATCC 10557) e L. casei (ATCC 4646). Osprodutos utilizados foram: Água Rabelo« - (A); Malvatricin« - (B);Mel Rosado« - (C); Apis Flora« - (D); Fitogargarejo« - (E )e Óleode Copahyba« - (F). Empregou-se a técnica de Concentração inibitória mínima (CIM); cada linhagem bacteriana foi reativada em caldo Tryptic Soy Broth, incubada a 370C por 24 horas em microaerofilia e semeadas em meio de cultura Ágar Mueller Hintõn pela técnica de inundação. Os produtos fitoterápicos foram empregados em concentrações de 100 por cento à 0,19 por cento e os halos de inibição mensurados. Verificou-se para a linhagem S.mutans as CIMs de 0,19 por cento; 12,5 por cento; 1,56 por cento; 3,12 por cento e 0,78 por cento para os produtos B, C, D, E e F respectivamente. Para a linhagem S.sanguis as CIMs observadas foram de 0,39 por cento; 25 por cento; 1,56 por cento;6,25 por cento e 0,78 por cento em B, C, D, E e F respectivamente. Sobre o S.sobrinus apenas o produto B mostrou atividade antibacterianana concentração de 0,19 porcento. Em relação ao L. casei os produtos B, D, E e F apresentaram respectivamente CIMs de 0,19 por cento;0,78 por cento; 0,78 por cento e 6,25 por cento. O produto B apresentou os maiores halos frente a todas as linhagens. Concluiu-se que os produtos tiveram desempenhos variados, tendo o Malvatricin« obtido amelhor CIM frente a todas as linhagens, bem como os maiores espectros de inibição e Água Rabelo« não apresentou atividade antibacteriana

Antissépticos e desinfetantes: estudo sobre condiçöes dos recipientes, sua manipulaçäo e o tempo de permanência dos líquidos nos recipientes / Antiseptics and desinfectants: study about recipient conditions, their manipulation and the time of permanence of the liquids

O presente estudo sobre antissépticos e desinfetantes teve como objetivo verificar a contaminaçäo dos mesmos, relacionados às condiçöes dos recipientes, tempo de permanência e a manipulaçäo. Utilizou-se o método experimental, submetendo 362 amostras destes líquidos à cultura microbiológica. A partir da análise e discussäo dos resultados, as autoras destacam alguns procedimentos visando a manutençäo dos antissépticos e desinfetantes livres de contaminaçäo através da utilizaçäo de recipientes adequados, tempo de permanência dos líquidos nos recipientes e a manipulaçäo dos mesmos.