Mecanismos de expressão de resistência aos antibióticos e saúde pública / Mechanisms of expression of resistance to antibiotics and public health / Mecanismos de expresión de la resistencia a los antibióticos y salud pública

Com o avanço da medicina e o aumento do uso de antimicrobianos, a resistência microbiana vem se tornando um problema sério na saúde pública. Para que uma bactéria se torne resistente, são necessários vários fatores, entre eles, o uso indiscriminado e prolongado de antimicrobianos e as resistências intrínsecas e adquiridas. Nesse contexto, o objetivo do trabalho foi explorar os mecanismos de ação dos antimicrobianos, de resistência e a sua importância na saúde pública. Foram utilizadas para a presente pesquisa, as bases de dados Pubmed, Google acadêmico e Scielo. Segundo a Organização Mundial da Saúde define-se resistência ao antibiótico quando o mesmo não produz mais efeito. A inserção cada vez mais frequente de antimicrobianos favorece a resistência, onde provocam uma pressão seletiva sobre os microrganismos, tornando-os resistentes a diversas drogas. O uso indiscriminado de antimicrobianos é o principal fator de resistência microbiana, assim como o uso de antimicrobianos sem exame de cultura e teste de sensibilidade. Neste sentido, conclui-se que é de suma importância a atualização de protocolos que contenham os mecanismos de resistência bacteriana a fim de minimizar o uso indiscriminado de antimicrobianos, assim como capacitar os profissionais da saúde para este problema na saúde pública.

With the advance of medicine and the increase in the use of antimicrobials, microbial resistance has become a serious problem in public health. For a bacterium to become resistant, several factors are necessary, among them, the indiscriminate and prolonged use of antimicrobials and the intrinsic and acquired resistance. In this context, the objective of the work was to explore the mechanisms of action of antimicrobials, resistance and their importance in public health. Pubmed, Google academic and Scielo databases were used for this research. According to the World Health Organization, resistance to antibiotics is defined when it no longer has an effect. The increasingly frequent insertion of antimicrobials favors resistance, where they put selective pressure on microorganisms, making them resistant to various drugs. The indiscriminate use of antimicrobials is the main factor of microbial resistance, as well as the use of antimicrobials without culture examination and sensitivity test. In this sense, it is concluded that it is extremely important to update protocols that contain the mechanisms of bacterial resistance in order to minimize the indiscriminate use of antimicrobials, as well as to train health professionals for this problem in public health.

Con los avances de la medicina y el mayor uso de antimicrobianos, la resistencia microbiana se ha convertido en un grave problema de salud pública. Para que una bacteria se vuelva resistente son necesarios varios factores, entre ellos, el uso indiscriminado y prolongado de antimicrobianos y la resistencia intrínseca y adquirida. En este contexto, el objetivo de este trabajo fue explorar los mecanismos de acción de los antimicrobianos, la resistencia y su importancia en la salud pública. Para esta investigación se utilizaron las bases de datos Pubmed, Google Scholar y Scielo. Según la Organización Mundial de la Salud, la resistencia a un antibiótico se define cuando deja de producir efecto. El uso cada vez más frecuente de antimicrobianos favorece la resistencia, ya que provocan una presión selectiva sobre los microorganismos, haciéndolos resistentes a varios fármacos. El uso indiscriminado de antimicrobianos es el principal factor de resistencia microbiana, así como el uso de antimicrobianos sin pruebas de cultivo y sensibilidad. En este sentido, se concluye que es de suma importancia actualizar los protocolos que contienen los mecanismos de resistencia bacteriana para minimizar el uso indiscriminado de antimicrobianos, así como capacitar a los profesionales de la salud para este problema en la salud pública.

Susceptibility to Vancomycin of Biofilm Producing Staphylococci Isolated from Tertiary Care Hospital of Nepal / Susceptibilidad a la vancomicina de estafilococos productores de biopelículas aislados del hospital de atención terciaria de Nepal

Background: Methicillin resistance and biofilm-producing Staphylococci are emerging as multidrug-resistant strains narrowing the efficacy of antimicrobial therapy. Although vancomycin is used as the drug of choice to treat such isolates, different studies worldwide have documented the emergence of strains that are intermediately susceptible or resistant to this antibiotic. Objective: The study aimed to determine the minimum inhibitory concentration of vancomycin to methicillin-resistant and biofilm-producing staphylococci isolated from different clinical specimens. Methods: 375 staphylococci isolated from different clinical specimens over one year were included in the study. Biofilm formation was determined by the Tissue culture plate method (TCP), and ica genes were identified by Polymerase Chain Reaction (PCR). Antibiotic susceptibility and methicillin resistance were done following Clinical and Laboratory Standards Institute (CLSI) guidelines. The minimum inhibitory concentration (MIC) of vancomycin in all isolates was determined by the agar dilution method. Results:Among 375 Staphylococci studied, 43% and 57% represented S. aureus and Coagulase-Negative Staphylococci (CNS), respectively. The rate of Methicillin-Resistant S. aureus (MRSA) and Methicillin-Resistant Coagulase Negative Staphylococci (MRCNS) were 81.4% and 66.8% respectively and determined by the disc diffusion method. The most potential antibiotics were tetracycline and chloramphenicol showing sensitivity to more than 90% isolates. The Minimum Inhibitory Concentration (MIC) value of oxacillin for staphylococci ranged from 0.125-32 µg/ml. Oxacillin agar diffusion method showed 51.6% and 79.9% isolates as MRSA and MRCNS, respectively, revealing a very high percentage of S. aureus and CNS isolates as methicillin-resistant. All isolates had susceptible vancomycin MICs that ranged from 0.125-2 µg/ml. Two S. aureus isolated from Central Venous Catheter (CVC) and catheter specimens were detected with intermediate susceptibility to vancomycin. Similarly, three CNS isolated from blood, CVC, and wound/pus (w/p) were intermediately susceptible to vancomycin. Strong biofilm formation was observed in 22.1% of clinical isolates, and the ica gene was detected among 22.9% of isolates. Only one S. aureus detected as a biofilm producer by the TCP method was found to have intermediate susceptibility to vancomycin. Conclusions: The increment in vancomycin MIC among methicillin-resistant and biofilm-producing staphylococci is alarming. Strict control measures to prevent methicillin-resistant isolates spread and routine surveillance for vancomycin-resistant isolates must be incorporated in hospitals to prevent antimicrobial treatment failure

Antecedentes: Los estafilococos resistentes a la meticilina y productores de biopelículas están surgiendo como cepas multirresistentes que reducen la eficacia del tratamiento antimicrobiano. Aunque la vancomicina se utiliza como fármaco de elección para tratar dichos aislados, diferentes estudios realizados en todo el mundo han documentado la aparición de cepas intermedias susceptibles o resistentes a este antibiótico. Objetivo: El estudio tenía como objetivo determinar la concentración mínima inhibitoria de la vancomicina para los estafilococos resistentes a la meticilina y productores de biofilm aislados de diferentes muestras clínicas. Métodos: Se incluyeron en el estudio 375 estafilococos aislados de diferentes muestras clínicas durante un año. La formación de biopelículas se determinó mediante el método de la placa de cultivo de tejidos (TCP), y los genes ica se identificaron mediante la reacción en cadena de la polimerasa (PCR). La susceptibilidad a los antibióticos y la resistencia a la meticilina se realizaron siguiendo las directrices del Clinical and Laboratory Standards Institute (CLSI). La concentración inhibitoria mínima (MIC) de vancomicina en todos los aislados se determinó por el método de dilución en agar. Resultados:Entre los 375 estafilococos estudiados, el 43% y el 57% representaban S. aureus y estafilococos coagulasa-negativos (ECN), respectivamente. La tasa de S. aureus resistente a la meticilina (SARM) y de estafilococos coagulasa negativos resistentes a la meticilina (ECNM) fue del 81,4% y el 66,8%, respectivamente, y se determinó por el método de difusión de discos. Los antibióticos más potenciales fueron la tetraciclina y el cloranfenicol, que mostraron una sensibilidad superior al 90% de los aislados. El valor de la concentración inhibitoria mínima (CIM) de la oxacilina para los estafilococos osciló entre 0,125-32 µg/ml. El método de difusión en agar de la oxacilina mostró que el 51,6% y el 79,9% de los aislados eran SARM y MRCNS, respectivamente, lo que revela que un porcentaje muy elevado de los aislados de S. aureus y CNS son resistentes a la meticilina. Todos los aislados tenían MIC de vancomicina susceptibles que oscilaban entre 0,125-2 µg/ml. Se detectaron dos S. aureus aislados de muestras de catéteres venosos centrales (CVC) y catéteres con una susceptibilidad intermedia a la vancomicina. Del mismo modo, tres S. aureus aislados de sangre, CVC y herida/pus (w/p) fueron intermedianamente susceptibles a la vancomicina. Se observó una fuerte formación de biopelículas en el 22,1% de los aislados clínicos, y se detectó el gen ica en el 22,9% de los aislados. Sólo un S. aureus detectado como productor de biopelículas por el método TCP resultó tener una susceptibilidad intermedia a la vancomicina. Conclusiones: El incremento de la MIC de vancomicina entre los estafilococos resistentes a la meticilina y productores de biofilm es alarmante. Para evitar el fracaso del tratamiento antimicrobiano, deben incorporarse en los hospitales medidas de control estrictas para prevenir la propagación de los aislados resistentes a la meticilina y una vigilancia rutinaria de los aislados resistentes a la vancomicina

Antibacterial and anti-biofilm potential of Plectranthus amboinicus (Lour) Spreng essential oil and Carvacrol against Staphylococcus aureus and Escherichia coli / Potencial antibacteriano e anti-biofilme do óleo essencial de Plectranthus amboinicus (Lour) Spreng e carvacrol frente a Staphylococcus aureus e Escherichia coli

Introduction: Staphylococcus aureus and Escherichia coli are pathogens that cause chronic infections due to antibiotic resistance mechanisms and their ability to adhere to surfaces and to form biofilms. The search for new agents from natural resources to counter microbial biofilms is an urgent priority in healthcare. Objetive: chemical composition, antibacterial and anti-biofilm activity of Plectranthus amboinicus essential oil (PAEO) and carvacrol were investigated against E. coli and S. aureus. Methodology: PAEO was chemically analyzed using gas chromatography coupled to a mass spectrometer. Antimicrobial activity was assessed by the disc diffusion method and broth microdilution method to determine minimum inhibitory concentrations (MICs). Antibiofilm activity was investigated using 96-well plates with a crystal violet assay. Results: carvacrol (85.25%) was the major component of PAEO. The disc diffusion test confirmed the ability of PAEO and carvacrol in inhibiting bacteria in their planktonic form. The MICs of PAEO against S. aureus and E. coli were 0.31 and 1.25%, respectively, with bactericidal effect. Carvacrol demonstrated a significant antibacterial property (MIC = 0.31%), exhibiting bacteriostatic effects against S. aureus and bactericidal effects against E. coli. Carvacrol considerably inhibited E. coli biofilm formations (58.9%). Moreover, carvacrol inactivated the mature biofilms. Conclusion: the data obtained are promising, and facilitates the development of new therapeutic alternatives. These results indicate the potential of carvacrol in treating diseases caused by E. coli and S. aureus

Introdução: Escherichia coli e Staphylococcus aureus são patógenos que podem causar infecções persistentes devido aos mecanismos de resistência e à sua capacidade de aderir e formar biofilme. A busca por novos agentes a partir de recursos naturais para combater o biofilme é uma prioridade na área da saúde. Objetivo: a composição química, a atividade antibacteriana e anti-biofilme do óleo essencial de Plectranthus amboinicus (PAEO) e do carvacrol foram investigadas frente a S. aureus e E. coli. Metodologia: a análise química do PAEO foi realizada por cromatografia gasosa acoplada a espectrômetro de massa. A atividade antimicrobiana foi avaliada por difusão em disco e microdiluição em caldo para determinar as concentrações inibitórias mínimas (CIMs). As atividades do anti-biofilme foram investigadas usando placas de 96 poços pelo ensaio de cristal violeta. Resultados: o carvacrol (85,25%) foi o principal componente do PAEO. A capacidade do PAEO e do carvacrol para inibir bactérias planctônica foi confirmada por difusão em disco. A CIM do PAEO para S. aureus e E. coli foi de 0,31 e 1,25%, respectivamente, com efeito bactericida. O carvacrol demonstrou uma propriedade antibacteriana significativa (CIM=0,31%), exibindo efeito bacteriostático frente a S. aureus e efeito bactericida para E. coli. O carvacrol inibiu significativamente a formação de biofilme de E. coli (58,9%) e promoveu a desestabilização do biofilme maduro. Conclusão: os dados obtidos são promissores, possibilitando o desenvolvimento de novas alternativas terapêuticas e revelam a potencialidade do carvacrol como fonte para o tratamento de doenças causadas por E. coli e S. aureus.

Antibacterial potential of essential oils against planktonic and sessile cells of Escherichia coli isolated from diarrhea cases in swine / Potencial antibacteriano de aceites esenciales contra células de Escherichia coli planctónicas y sésiles aisladas de diarrea porcina

Escherichia coli is a pathogen associated with infections in piglets in the post-weaning phase, its pathogenicity is related to the animal's susceptibility to bacterial enterotoxins. The objective of the present study was to determine the EOs activity against E. colistrain, in the form planktonic and sessile. Although the Disc-Diffusion tests to determine the Minimum Inhibitory Concentration, do not fully corroborate with the other analyzes of this study, it was noticed bacteria inhibition. The EOs were prepared at 0.4%, 0.8% and 1.0% for tests. The tested EOs were effective against E. coliplanktonic cells (p<0.05). As for the sessile cells, the most significant result was inhibition and 100% sessile cells at the concentration of 1.0% of Cymbopogon citratusEO. Although there was resistance in some treatments, the tested EOs demonstrated inhibition capacity, constituting promising alternatives for the control of E. coli, especially of planktonic cells.

Escherichia coli es un patógeno asociado con infecciones en lechones en la fase posterior al destete, su patogenicidad está relacionada con la susceptibilidad del animal a las enterotoxinas bacterianas. El objetivo del presente estudio fue determinar la actividad de contra E. coli, en la forma planctónico y sésil. Aunque las pruebas de difusión de disco para determinar la concentración inhibitoria mínima, no corroboran completamente con los otros análisis de este estudio, se observó inhibición de la bacteria. Las soluciones basadas en AE se prepararon al 0.4%, 0.8% y 1.0% para pruebas. Los AEs probados fueron efectivos contra las células planctónicas (p<0.05). En cuanto a las células sésiles, el resultado más significativo fue la inhibición y el 100% de las células sésiles a la concentración de 1,0% de Cymbopogon citratus. Aunque hubo resistencia en algunos tratamientos, los AEs probados demostraron capacidad de inhibición, constituyendo alternativas prometedoras para el control de E. coli, especialmente de células planctónicas.

Avaliação in vitro da formação de biofilme por cepas de Candida spp. provenientes de indivíduos diabéticos e não diabéticos em ambiente sem e com suplementação de glicose / In vitro evaluation of biofilm formation by Candida spp. strains from diabetic and non-diabetic individuals in environment without and with glucose supplementation

É notório o papel do diabetes mellitus como fator de risco para o desenvolvimento de doenças bucais, como a candidíase bucal, cárie e a periodontite. Assim, este estudo tem como objetivo avaliar a formação de biofilme por cepas de Candida spp. provenientes de diabéticos e não diabéticos em ambiente sem e com suplementação de glicose. Trata-se de um estudo experimental laboratorial in vitro, em três etapas. Etapas um e dois, de obtenção e identificação de 48 cepas de Candida spp., sendo que 32 de C. albicans e 16 de C. glabrata, com auxílio da técnica de PCR. Ainda, a etapa três, de processamento microbiológico, com a avaliação da capacidade de formação de biofilme por três ensaios distintos: I) determinação do número de unidades formadoras de colônia (UFC/mL); II) matéria seca dos biofilmes; III) taxa de crescimento de biofilme em fundo de placa de poliestireno. Inicialmente, objetivando simular as características observadas in vivo, o fundo das placas de cultivo recebeu 400 µL de saliva humana para formação da película adquirida. Decorrida a incubação a 37 °C por 24 h, a saliva foi descartada e cada poço de cultura recebeu suspensão padronizada das leveduras (106 UFC/mL) em Saubouraud Dextrose Broth sem suplementação e com suplementação de glicose a 2 e 10 mg/mL, e as placas foram incubadas a 37 °C por 48 h. Para avaliação do número de UFC/mL, o biofilme aderido foi coletado, diluído seriamente e cultivado em placas de Petri com Sabouraud Dextrose Agar. Após incubação os resultados foram expressos em log UFC/mL. Para a avaliação da matéria seca, a solução remanescente foi liofilizada e mensurada em balança de precisão. A taxa de crescimento de biofilme foi avaliada por microscopia Operetta CLS High Content e o FilmTracer(TM) LIVE/DEAD Biofilm Viability kit, conforme o protocolo do fabricante. Posteriormente, 10 imagens por poço foram obtidas e digitalizadas com ampliação de 40 ×. A área recoberta por biofilme (µm2) das imagens foi avaliada com auxílio do software Harmony High Content Imaging. Os dados apresentaram distribuição não normal, e a comparação entre as cepas de diabéticos e não diabéticos foi realizada pelo teste U Mann-Whitney. O teste de Kruskal-Wallis one way foi utilizado para verificar diferenças entre as condições de suplementação de glicose. O nível de significância estatística adotado foi de α = 5%. Os valores de UFC/mL mostraram um maior crescimento das cepas de C. albicans dos pacientes diabéticos em relação aos não diabéticos nas três suplementações (p < 0,001). Por outro lado, acerca da matéria seca em 10 mg/mL e da taxa de crescimento de biofilme sem suplementação de glicose e a 2 mg/mL, os resultados indicaram uma formação de biofilme maior para cepas de C. albicans dos não diabéticos (p < 0,001). Em conclusão, cepas de C. albicans e C. glabrata provenientes de diabéticos e não diabéticos em ambiente sem e com suplementação de glicose apresentaram resultados distintos quanto à formação de biofilme, por diferentes técnicas

The role of diabetes mellitus is notorious as a risk factor for development of oral diseases, such as oral candidiasis, dental caries, and periodontitis. Thus, this study aimed to evaluate biofilm formation by Candida spp. strains from diabetic and non-diabetic individuals in environment without and with glucose supplementation. This is an in vitro experimental laboratory study, in three stages. Stages one and two of obtainment and identification of 48 Candida spp. strains, with 32 of C. albicans and 16 of C. glabrata, with the help of PCR technique. Also, stage three, of microbiological processing, with evaluation of biofilm formation capacity by three different assays: I) determination of the number of colony forming units (CFU/mL); II) biofilm dry matter; III) biofilm growth rate on the bottom of polystyrene plates. Initially, aiming to simulate the characteristics observed in vivo, the bottom of the cultivation plates received 400 µL of human saliva for formation of acquired pellicle. After the incubation at 37 °C for 24 h, the saliva was discarded and each culture well received standardized suspension of yeast (106 CFU/mL) in Saubouraud Dextrose Broth without supplementation and with glucose supplementation at 2 and 10 mg/mL, and the plates were incubated at 37 °C for 48 h. To assess the number of CFU/mL, the adhered biofilm was collected, seriously diluted, and cultivated in Petri dishes with Sabouraud Dextrose Agar. After the incubation, the results were expressed in log CFU/mL. To assess the dry matter, the remaining solution was lyophilized and measured on a precision scale. The biofilm growth rate was evaluated by Operetta CLS High Content microscopy and FilmTracer(TM) LIVE/DEAD Biofilm Viability kit, according to manufacturer's protocol. Later, 10 images per well were obtained and digitalized with 40 × magnification. The area covered by biofilm (µm2) of the images was assessed with the help of Harmony High Content Imaging software. Data showed non-normal distribution, and the comparison among the diabetic and non-diabetic strains was performed by Mann-Whitney U test. Kruskal-Wallis one-way test was used to verify differences between conditions of glucose supplementation. The level of statistical significance adopted was α = 5%. The values of CFU/mL showed greater growth of the diabetic patient's strains in relation to the non-diabetic ones (p < 0.001). On the other hand, regarding dry matter at 10 mg/mL and the growth rate of biofilm without glucose supplementation and at 2 mg/mL, the results indicated a higher biofilm formation for strains of C. albicans from non-diabetic individuals (p <0.001). In conclusion, C. albicans and C. glabrata strains from diabetic and non-diabetic individuals in environment without and with glucose supplementation showed different results concerning the biofilm formation, using different techniques

Influence of EDTA and its Association with Benzalkonium Chloride on Enterococcus faecalis Adhesion to Dentin / Influencia del EDTA y su Asociación con Cloruro de Benzalconio en la Adhesión de Enterococcus faecalis a la Dentina

ABSTRACT: The aim of this in vitro study was to investigate the influence of ethylenediaminetetraacetic acid (EDTA) associated with the benzalkonium chloride (BAK) on the adhesion and formation of Enterococcus faecalis biofilms attached to coated dentin. Discs standard bovine dentin blocks were treated with the coating materials evaluated: Saline solution (control), 17 % EDTA, 17 % EDTA associated with 1 % BAK for 5 minutes and subsequently washed with saline solution. Afterwards, biofilms of E. faecalis (ATCC 29212) were grown on the surface of coated dentin blocks for time intervals of 1 hour and 7 days (n = 20) and were subsequently washed with phosphate-buffered saline (PBS). Bacterial viability and total biovolume were analyzed by confocal laser scanning microscopy (CLSM) using the Live/Dead technique. Nonparametric Kruskal-Wallis followed by Dunn tests were used to determine statistical differences (a = 5 %). The 17 % EDTA + 1 % BAK group showed significantly lower biovolume and bacterial viability values at the end of 1 hour (p < 0.05). After 7 days of contamination, the 17 % EDTA and 17 % EDTA + 1 % BAK groups showed similar results that differed statistically from those of the control group (p < 0.05). The saline solution group showed higher values. The use of BAK associated with EDTA on dentin blocks surfaces before exposure to contamination was able to interfere in the adhesion of E. faecalis to dentin. Also, dentin treatment by BAK associated with a chelating agent influences the secondary biofilm formation, which could have important effects on the long-term success of root canal treatment.

RESUMEN: El objetivo del estudio consistió en investigar in vitro, la influencia del ácido etilendiamino-tetraacético (EDTA) con cloruro de benzalconio (BAK) en la adhesión y formación de biopelículas de Enterococcus faecalis a la dentina. Discos de dentina bovina fueron tratadas con solución salina (control), 17 % de EDTA, 17% de EDTA asociado con 1 % de BAK durante 5 minutos y lavadas con solución salina. Las biopelículas de E. faecalis (ATCC 29212) se cultivaron sobre los discos de dentina durante intervalos de tiempo de 1 hora y 7 días (n = 20), lavados con solución salina tamponada con fosfato (PBS). La viabilidad bacteriana y el biovolumen total se analizaron mediante microscopía de barrido por láser (CLSM) utilizando la técnica Live / Dead. Se realizó prueba no paramétrica de Kruskal-Wallis, seguida por Dunn con una diferencia estadística (a = 5 %). El grupo de 17 % EDTA + 1 % BAK mostró valores significativamente menores de biovolumen y viabilidad bacteriana al final de 1 hora (p < 0,05). Después de 7 días de contaminación, los grupos de 17 % EDTA y 17 % EDTA + 1 % BAK mostraron resultados similares que diferían estadísticamente del grupo control (p < 0,05). La solución salina mostró valores más altos. La asociación de BAK con EDTA antes de la contaminación interfirió en la adhesión de E. faecalis. Además, el tratamiento de la dentina por BAK asociado con EDTA influye en la formación de biopelículas secundarias, lo que podría tener efectos importantes sobre el éxito a largo plazo del tratamiento del conducto radicular.

Ação inibitória de dentifrícios sobre Streptococcus mutans e Candida albicans: estudo in vitro / Inhibitory effect of dentifrices on Streptococcus mutans and Candida albicans: in vitro study

Introdução: este estudo avaliou a ação antimicrobiana de sete dentifrícios nacionais com ação antiplaca ou antimicrobiana, conforme os fabricantes, sobre os patógenos orais Streptococcus mutans e Candida albicans. Métodos: a avaliação dos dentifrícios foi realizada pelo teste de difusão em ágar. Placas de Petri com meios de cultura ágar cérebro coração e ágar Sabourad foram semeadas com suspensão microbiana padronizada de S. mutans e C. albicans. Poços foram confeccionados no ágar semeado e preenchidos com as soluções dos dentifrícios diluídas em 1:1, 1:2, 1:4 e 1:8, além dos controles positivo (solução de clorexidina 2%) e negativo (dentifrício sem princípios ativos). Após incubação por 48h/37°C, os halos de inibição do crescimento microbiano foram medidos com paquímetro e régua milimetrada, e os resultados finais dos halos obtidos em milímetros. Resultados e conclusão: verificou-se que todos os dentifrícios, na diluição de 1:1, foram efetivos sobre C. albicans e S. mutans, exceto o composto de bicarbonato de sódio e extratos vegetais, o qual não inibiu S. mutans. Na diluição de 1:2, somente o dentifrício contendo triclosan inibiu C. albicans, enquanto todos mostraram algum potencial de inibição ao S. mutans. Nas demais diluições, não foram observados halos de inibição de ambos os microrganismos. A C. albicans demonstrou ser mais resistente à ação inibitória dos antimicrobianos dos dentifrícios testados neste estudo. S. mutans demonstrou ser mais sensível à ação dos dentifrícios, no entanto, a diluição durante a escovação dentária reduz a ação inibitória esperada, indicando a importância da remoção mecânica do biofilme.(AU)

Introduction: this study analyzed the antimicrobial activity in seven national dentifrices with antiplaque or antimicrobial substances in their composition, according the manufacturer, on the oral pathogens Streptococcus mutans and Candida albicans. Methods: the dentifrices antimicrobial effect evaluation was carried out by agar diffusion test. S. mutans and C. albicans. Padronized microbial suspension was cultured on Petri dishes containing Brain Hearth Agar and Sabourad Agar, respectively. Wells were prepared in the cultured agar and filled with 1:1, 1:2, 1:4 and 1:8 dilutions of dentifrice solutions, and besides positive (2% chlorhexidine solutions) and negative controls (dentifrices without active ingredients). After 48h/37°C incubation, the microbial growing inhibition zones were measured with a paquimeter and a millimeter rule, being the final zone results obtained in millimeters. Results and conclusion: on 1:1 dilution, all dentifrices were effective on C. albicans and S. mutans, except one with vegetal extract and sodium bicarbonate composition, which did not inhibit S. mutans. In 1:2 dilution, only tricolsan-containing dentifrice inhibited C. albicans, while all dentifrices presented some inhibition potential on S. mutans. There were no inhibition zones for both microorganims in other dilutions. We concluded that C. albicans showed to be more resistant to antimicrobial agents tested in the dentifrices, in this study. S. mutans showed to be more sensitive to tested dentifrices, however, the product dilution during toothbrushing reduces the expected inhibitory action, strengthening the importance of the mechanical removal of dental biofilm.(AU)

Evaluation of anti-biofouling potential of Viburnum opulus extracts / Evaluación del potencial anti bioincrustante de extractos de Viburnum opulus

In this study the in vitro investigation of the inhibitory effect of ethanol extract of Viburnum opulus L. bark sample on Streptococcus mutans planctonic cells and biofilm has been intended. A Scanning electron microscopy analysis has been performed in order to investigate the inhibitory effect of the extract on Streptococcus mutans biofilms. Furthermore, the Exopolysaccharide and dextran production of this bacteria have been identified in the presence of the extract. It has been found out that the bark extract with the concentration of 2,5 mg/mL is able to inhibit more than 50% of the cells in the different times development phases. According to this, the exopolymeric matrix on the biofilm surface disperses and the Exopolysaccharide and dextran production get lowered in the presence of bark extract compared to the control group. It is considered that this extract can be used as an alternative approach for the new chemotherapeutic strategies against tooth decay.

En este estudio se investigó el efecto inhibitorio in vitro del extracto de etanólico de una muestra de corteza de Viburnum opulus L. en biopelículas de células planctónicas de Streptococcus mutans. Se realizó un análisis de microscopía electrónica de barrido para investigar el efecto inhibitorio del extracto sobre las biopelículas de Streptococcus mutans. Además, se identificó la producción de exopolisacárido y dextrano de esta bacteria en presencia del extracto. Se descubrió que el extracto de corteza con una concentración de 2,5 mg/ml inhibió más del 50% de las células en las diferentes fases de desarrollo. Consecuentemente, la matriz exopolimérica en la superficie de la biopelícula se dispersa y la producción de exopolisacárido y dextrano se reduce en presencia de extracto de corteza en comparación con el grupo de control. Se sugiere que este extracto puede ser usado como un enfoque alternativo para las nuevas estrategias quimioterapéuticas contra la carie dental.

Efeito do extrato da própolis e do digluconato de clorexidina sobre a formação de biofilme por Candida albicans em resina acrílica / Effect of propolis extract and chlorhexidine digluconate on the biofilm formation by Candida albicans in acrylic resin

Objetivo: avaliar in vitro a ação antimicrobiana do extrato da própolis (EP) e do digluconato de clorexidina (DCHX) na formação de biofilme por Candida albicans em resina acrílica termopolimerizada. Métodos: o efeito do EP e DCHX em biofilmes de C. albicans foi avaliado pela quantificação de Unidades Formadoras de Colônias (UFCs), pela quantificação da biomassa por cristal violeta e de polissacarídeos por safranina. Para tanto, C. albicans foram pré-aderidas em corpos de prova e somente em microplacas de poliestireno, posteriormente foi realizado o tratamento com diferentes concentrações de EP (221 µg/mL e 443 µg/mL) e DCHX (0,25% e 0,5%). Resultados: foi demonstrada uma redução significativa na formação de biofilme por C. albicans com ambas as substâncias testadas e em todas as concentrações. Conclusão: de acordo com os resultados, observou-se que ambos os tratamentos foram eficazes na redução do biofilme e que o EP, por ser um produto natural, de baixo custo e sem afeitos colaterais, representa uma alternativa inovadora para o tratamento da candidose oral em usuários de próteses removíveis.(AU)

Objective: to evaluate in vitro the antimicrobial action of propolis extract (EP) and chlorhexidine digluconate (DCHX) in the biofilm formation by Candida albicans in thermopolymerized acrylic resin. Methods: the effect of EP and DCHX on C. albicans biofilms were evaluated through the quantification of Colony Forming Units (CFU's), the quantification of biomass by violet crystal and polysaccharides by safranin. For this purpose, C. albicans were pre-adhered to specimens and only on polystyrene microplates and subsequently the treatment was performed with different concentrations of EP (221 µg/mL and 443 µg/mL) and DCHX (0.25% and 0,5%). Results: a significant reduction in the biofilm formation by C. albicans was demonstrated, with both substances tested and in all concentrations. Conclusion: according to the results, it was observed that both treatments were effective in reducing biofilm and EP, being a natural product, low cost and without side effects, represents an innovative alternative for the treatment of oral candidiasis in users removable dentures.(AU)

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans

Abstract Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Quorum Sensing Interfering Strategies and Their Implications in the Management of Biofilm-Associated Bacterial Infections

Abstract The bacterial species employ various types of molecular communication systems recognized as quorum sensing for the synchronization of differential gene expression to regulate virulence traits and biofilm formation. A variety of quorum sensing inhibitors; molecules that interfere with quorum sensing among bacteria have been examined which can block the action of autoinducers. Moreover, the studies have scrutinized various enzymes for their quorum quenching activity resulting in the degradation of signaling molecules or blocking of gene expression. So far, the studies have found that these approaches are not only capable to reduce the pathogenicity and biofilm formation but also resulted in increased bacterial susceptibility to antibiotics and bacteriophages. The effectiveness of these strategies has been validated in different animal models and it seems that these practices will be transformed in near future to develop the medical devices including catheters, implants, and dressings for the prevention of bacterial infections. Although many of these approaches are still in the research stage, the increasing library of quorum quenching molecules and enzymes will open innovative perspectives for the development of antibacterial approaches which will extend the therapeutic arsenal against the pathogenic bacterial species.

Effect of a calcium hydroxide-based intracanal medicament containing N-2-methyl pyrrolidone as a vehicle against Enterococcus faecalis biofilm

Abstract This study investigated the effect of a calcium hydroxide (CH) paste (CleaniCal®) containing N-2-methyl pyrrolidone (NMP) as a vehicle on Enterococcus faecalis (E. faecalis) biofilms compared with other products containing saline (Calasept Plus™) or propylene glycol (PG) (Calcipex II®). Methodology Standardized bovine root canal specimens were used. The antibacterial effects were measured by colony-forming unit counting. The thickness of bacterial microcolonies and exopolysaccharides was assessed using confocal laser scanning microscopy. Morphological features of the biofilms were observed using field-emission scanning electron microscopy (FE-SEM). Bovine tooth blocks covered with nail polish were immersed into the vehicles and dispelling was observed. The data were analyzed using one-way analysis of variance and Tukey tests (p<0.05). Results CleaniCal® showed the highest antibacterial activity, followed by Calcipex II® (p<0.05). Moreover, NMP showed a higher antibacterial effect compared with PG (p<0.05). The thickness of bacteria and EPS in the CleaniCal® group was significantly lower than that of other materials tested (p<0.05). FE-SEM images showed the specimens treated with Calasept Plus™ were covered with biofilms, whereas the specimens treated with other medicaments were not. Notably, the specimen treated with CleaniCal® was cleaner than the one treated with Calcipex II®. Furthermore, the nail polish on the bovine tooth block immersed in NMP was completely dispelled. Conclusions CleaniCal® performed better than Calasept Plus™ and Calcipex II® in the removal efficacy of E. faecalis biofilms. The results suggest the effect might be due to the potent dissolving effect of NMP on organic substances.

Efectos del medio de cultivo y de la metodología aplicada sobre la formación de biopelículas de 2 cepas de Escherichia coli diarreagénicas / Effects of the culture medium and the methodology applied on the biofilm formation of 2 diarrheagenic Escherichia coli strains

La capacidad de formar biopelículas de los microorganismos patógenos en gran variedad de ambientes, superficies y condiciones trae consigo un importante riesgo, tanto para la industria alimentaria como para la salud pública. Este trabajo tuvo como objetivo evaluar y comparar los efectos de la metodología empleada y de los medios de cultivo utilizados, sobre la capacidad de una cepa de Escherichia coli verotoxigénica no O157 y una enteropatogénica de formar biopelículas sobre una superficie de poliestireno. Se ensayaron 2 variantes metodológicas en cultivo estático y se utilizaron medios de cultivo con diferente composición. Los resultados mostraron que ambas cepas formaron una mayor cantidad de biopelícula en cultivo en LB suplementado con glucosa, con recambio del medio a las 24 h y la cuantificación de la biopelícula realizada a las 48 h de incubación. Dichas condiciones podrían ser utilizadas en futuros estudios sobre formación de biopelícula.

The ability to form biofilms of pathogenic microorganisms in a wide variety of environments, surfaces and conditions constitute an important risk, both for the food industry and for public health. The aim of this work was to evaluate and to compare the effects of the methodology applied and the culture medium used on the ability of a non-O157 verotoxigenic Escherichia coli strain and an enteropathogenic strain to form biofilm on polystyrene surface. Two methodological variants were tested in static culture and culture mediums with different composition were used. The results showed that both strains were able to form a greater biofilm under culture in LB supplemented with glucose, with medium replacement at 24 h and the quantification of the biofilm carried out at 48 h of incubation. These conditions could be used in future studies on biofilm formation.

Effect of thymol and environmental factors on growth and biofilm formation by Listeria monocytogenes / Efecto del timol y factores ambientales sobre el crecimiento y formación de biopelícula por Listeria monocytogenes

Thymol (2-isopropyl-5-methylphenol) is an aromatic monoterpene found in essential oils extracted from plants belonging to the Lamiaceae family, such as Thymus, Ocimum, Origanum, Satureja, Thymbra and Monarda genera. Growth and biofilm formation by Listeria monocytogenes CLIP 74902 were evaluate using three carbon sources in the presence of thymol. Specific growth rate (h-1) values at 37o with glucose, trehalose and cellobiose with the addition of thymol (µg/mL) 0 (control) and 750, were respectively: 0.22, 0.07; 0.14, 0.04; 0.11, 0.04. Lag periods obtained under the same conditions were (h): 8.19, 13.2; 22.5, 27.5; 23.1, 28.1. A marked antibiofilm activity was observed against the exposure with 750 µg/mL of thymol, showing a high percentage of inhibition: glucose (99 %), trehalose (97 %) and cellobiose (98%), compared to the control. The results suggest that thymol could be used to inhibit the growth and production of biofilms by L. monocytogenes in the food industry.

Timol (2-isopropil-5-metilfenol) es un monoterpeno aromático presente en los aceites esenciales extraídos de plantas pertenecientes a la familia Lamiaceae, como los géneros Thymus, Ocimum, Origanum, Satureja, Thymbra y Monarda. El crecimiento y formación de biopelícula por Listeria monocytogenes CLIP 74902 fueron evaluados utilizando tres fuentes de carbono en presencia de timol. La velocidad específica de crecimiento (h-1) a 37o con glucosa, trehalosa y celobiosa con la adición de timol (µg/mL) 0 (control) y 750, fueron respectivamente: 0.22, 0.07; 0.14, 0.04, 0.11, 0,04. Los períodos lag obtenidos en las mismas condiciones fueron (h): 8.19, 13.2; 22.5, 27.5; 23.1, 28.1. Una marcada actividad antibiofilm fue obtenida con 750 µg/mL de timol, mostrando un alto porcentaje de inhibición con glucosa (99%), trehalosa (97%) y celobiosa (98%), respecto al control. Los resultados sugieren que timol podría ser usado para inhibir el crecimiento y producción de biopelículas por L. monocytogenes en la industria alimentaria.

Efeito da adição de óleo de licuri (Syagrus coronata) e Tween 80 em filme de amido de araruta (Maranta arundinacea L. ) / Effect of the addition of licuri oil (Syagrus coronata) and Tween 80 in a film of araruta starch (Maranta arundinacea L. )

O objetivo de trabalho foi avaliar o efeito da adição de óleo de licuri nas propriedades do filme a base de amido de araruta (bioplástico). Foram elaborados dois filmes, um conteúdo óleo de licuri e outro sem o óleo na formulação. Os filmes foram caraterizados através de analises de espessura, solubilidade, atividade de água, permeabilidade a vapor de água, ensaios de tração e analise de cor. Os resultados apontaram que o óleo de licuri reduziu a resistência á tração, o modulo de Young, a atividade de água, a solubilidade e a luminosidade dos filmes de amido de araruta. Além disso, a adição do óleo levou à um aumento da espessura e permeabilidade ao vapor de água. A adição de óleo de licuri influenciou de forma favorável algumas propriedades do filme a base de amido de araruta.

Ação de diferentes sanitizantes em biofilmes de Salmonella Minnesota / Action of different sanitizers in biofilmes of Salmonella Minnesota

Objetivou-se avaliar a eficiência do contato por 15 minutos do hipoclorito de sódio 1%, e ácido peracético 0,8% na inibição de biofilmes formad os por três cepas distintas de Salmonella Minnesota em aço, poliuretano e polipropileno e determinar a recuperação das células remanescentes. Qualitativamente houve influência na classificação dos biofilmes de acordo com a superfície, que foram diferentes dependendo do tipo de cepa e de material, porém a quantidade de células sésseis permanece constante, pois independente da cepa e da superfície, os biofilmes mantiveram uma contagem de 5,18 Log UFC, indicando que as diferenças referem-se à matriz polimérica. O uso do hipoclorito foi capaz de destruir as células, que não foram recuperadas após reincubação. Já o ácido peracético não promoveu redução significativa. O uso do hipoclorito de sódio 1% é eficiente na sua remoção.

Ação do extrato do resíduo de Caryocar brasiliense frente a formação de biofilme dos principais microrganismos veiculados por produtos de origem animal / Action of the Caryocar brasiliense waste extract against the biofilm formation of the main microorganisms carried by products of animal origin

Objetivou-se com o presente estudo avaliar a inibição da formação de biofilme pelo extrato do resíduo do pequi frente a microrganismos patogênicos e deteriorantes de alimentos. A casca do pequi foi obtida de frutas coletadas na cidade de Montes Claros/MG, foi seca em estufa de ventilação forçada, moída e peneirada em 250 Mesh. Seu extrato foi obtido em micro-ondas analítico. Foram utilizadas cinco cepas padrão e cinco isolados de produtos de origem animal, que foram reativadas e confirmado a sua pureza em ágar específico. Para avaliar a formação do biofilme foram utilizadas microplacas esterilizadas, sendo adicionados 90µL de BHI com 1% de glicose, 10µL da suspensão de cada bactéria e o extrato em diferentes concentrações. Concluiu-se que o extrato da casca do pequi inibe a formação de biofilme por bactérias patogênicas e deteriorantes.

Pseudomonas aeruginosa: patogenicidad y resistencia antimicrobiana en la infección urinaria / Pseudomonas aeruginosa: pathogenicity and antimicrobial resistance in urinary tract infection

Resumen Dentro de las infecciones nosocomiales más frecuentes asociadas a bacterias multi-resistentes y de peor pronóstico, se encuentran las producidas por Pseudomonas aeruginosa. Esta bacteria posee una alta capacidad de adaptación a condiciones adversas como por ejemplo el pH y la osmolaridad de la orina. Pseudomonas aeruginosa es uno de los principales patógenos implicados en infecciones nosocomiales y de pacientes inmunosuprimidos. Esta bacteria se considera un agente infeccioso oportunista que posee diversos mecanismos de patogenicidad, así como de resistencia a antimicrobianos, lo que contribuye a la dificultad en el tratamiento de estas infecciones. En la presente revisión bibliográfica se analizan la taxonomía, los mecanismos de patogenicidad y genes de resistencia de P. aeruginosa. Así también, se abordan los factores microambientales de la infección urinaria producida por esta bacteria, haciendo un acercamiento al entendimiento de las bases fisiopatológicas de esta infección.

Among the most frequent nosocomial infections associated with polyresistant bacteria and with a worse prognosis, are those produced by Pseudomonas aeruginosa. This bacterium has a high capacity to adapt to adverse conditions such as pH and osmolarity of urine. Pseudomonas aeruginosa is one of the main pathogens involved in nosocomial infections and immunosuppressed patients. This bacterium is considered an opportunistic infectious agent that has diverse mechanisms of pathogenicity, as well as resistance to antimicrobials, which contributes to the difficulty in the treatment of these infections. In the present bibliographic review, the taxonomy, pathogenicity mechanisms and resistance genes of P. aeruginosa are analyzed. Likewise, the micro-environmental factors of the urinary infection produced by this bacterium are approached, making an approach to the understanding of the pathophysiological bases of this infection.

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on biofilm formation and virulence factors of Escherichia coli

ABSTRACT Objective: To evaluate the influence of sub-minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) on biofilm formation and virulence factors of Escherichia coli clinical isolates. Methods: Sub-MICs of CIP were determined using growth curve experiments. The biofilm-forming capacity of E. coli clinical isolates and E. coli ATCC 25922 treated or untreated with sub-MICs of CIP was assessed using a crystal violet staining assay. The biofilm structure of E. coli isolate was assessed with scanning electron microscopy (SEM). The expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were measured using quantification RT-PCR (qRT-PCR) in E. coli isolates and E. coli ATCC 25922. Results: Based on our results, the sub-MICs of CIP were 1/4 MICs. Sub-MICs of CIP significantly inhibited biofilm formation of E. coli clinical isolates and E. coli ATCC 25922 (p < 0.01). SEM analyses indicated that the biofilm structure of the E. coli changed significantly after treatment with sub-MICs of CIP. Expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were also suppressed. Conclusions: The results revealed that treatment with sub-MICs of CIP for 24 h inhibited biofilm formation and reduced the expression of virulence genes and biofilm formation genes in E. coli.