Effect of N-acetyl cysteine, rifampicin, and ozone on biofilm formation in pan-resistant Klebsiella pneumoniae: an experimental study

ABSTRACT BACKGROUND: To the best of our knowledge, this is the first study to evaluate the effectiveness of specific concentrations of antibiofilm agents, such as N-acetyl cysteine (NAC), rifampicin, and ozone, for the treatment of pan-resistant Klebsiella pneumoniae (PRKp). OBJECTIVES: We evaluated the effectiveness of antibiofilm agents, such as NAC, rifampicin, and ozone, on biofilm formation in PRKp at 2, 6, 24, and 72 h. DESIGN AND SETTING: This single-center experimental study was conducted on June 15, 2017, and July 15, 2018, at Istanbul Faculty of Medicine, Istanbul University, Turkey. METHODS: Biofilm formation and the efficacy of these agents on the biofilm layer were demonstrated using colony counting and laser-screened confocal microscopy. RESULTS: NAC at a final concentration of 2 μg/mL was administered to bacteria that formed biofilms (24 h), and no significant decrease was detected in the bacterial counts of all isolates (all P > 0.05). Rifampicin with a final concentration of 0.1 μg/mL was administered to bacteria that formed biofilm (24 h), and no significant decrease was detected in bacterial count (all P > 0.05). Notably, ozonated water of even 4.78 mg/L concentration for 72 h decreased the bacterial count by ≥ 2 log10. CONCLUSION: Different approaches are needed for treating PRKp isolates. We demonstrate that PRKp isolates can be successfully treated with higher concentrations of ozone.

Antiadhesion and antibiofilm potential of Fagonia indica from Cholistan desert against clinical multidrug resistant bacteria / Potencial de antiadesão e antibiofilme de Fagonia indica do deserto do Cholistão contra bactérias multirresistentes clínicas

High resistance to antimicrobials is associated with biofilm formation responsible for infectious microbes to withstand severe conditions. Therefore, new alternatives are necessary as biofilm inhibitors to control infections. In this study, the antimicrobial and antibiofilm activities of Fagonia indica extracts were evaluated against MDR clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica has antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against multidrug resistant (MDR) clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica had antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against MDR isolates. The maximum inhibitory effects of Fagonia indica chloroform extract on biofilm formation was observed on Staphylococcus aureus (71.84%) followed by Klebsiella pneumoniae (70.83%) after 48 hrs showing that inhibition is also time dependent. Our results about bacterial cell protein leakage indicated that MDR isolates treated with chloroform extract of Fagonia indica showed maximum protein leakage of K. pneumoniae (59.14 µg mL-1) followed by S. aureus (56.7 µg mL-1). Cell attachment assays indicated that chloroform extract resulted in a 43.5-53.5% inhibition of cell adherence to a polystyrene surface. Our results revealed that extracts of Fagonia indica significantly inhibited biofilm formation among MDR clinical isolates, therefore, could be applied as antimicrobial agents and cost effective biofilm inhibitor against these MDR isolates.

A alta resistência aos antimicrobianos está associada à formação de biofilme responsável por micróbios infecciosos para suportar condições severas. Portanto, novas alternativas são necessárias como inibidores de biofilme para controlar infecções. Neste estudo, as atividades antimicrobiana e antibiofilme dos extratos de Fagonia indica foram avaliadas contra isolados clínicos MDR. O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica tem efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e valor de concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados clínicos multirresistentes (MDR). O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica teve efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados MDR. Os efeitos inibitórios máximos do extrato de clorofórmio Fagonia indica na formação de biofilme foi observada em Staphylococcus aureus (71,84%), seguido por Klebsiella pneumoniae (70,83%) após 48 horas, mostrando que a inibição também é dependente do tempo. Nossos resultados sobre extravasamento de proteínas de células bacterianas indicaram que isolados MDR tratados com extrato clorofórmico de Fagonia indica apresentaram vazamento máximo de proteínas de K. pneumoniae (59,14 µg mL-1), seguido por S. aureus (56,7 µg mL-1). Ensaios de fixação de células indicaram que o extrato de clorofórmio resultou em uma inibição de 43,5-53,5% da aderência das células a uma superfície de poliestireno. Nossos resultados revelaram que extratos de Fagonia indica inibiram significativamente a formação de biofilme entre isolados clínicos MDR, portanto, poderiam ser aplicados como agentes antimicrobianos e inibidores de biofilme de baixo custo contra esses isolados MDR.

Antiadhesion and antibiofilm potential of Fagonia indica from Cholistan desert against clinical multidrug resistant bacteria / Potencial de antiadesão e antibiofilme de Fagonia indica do deserto do Cholistão contra bactérias multirresistentes clínicas

High resistance to antimicrobials is associated with biofilm formation responsible for infectious microbes to withstand severe conditions. Therefore, new alternatives are necessary as biofilm inhibitors to control infections. In this study, the antimicrobial and antibiofilm activities of Fagonia indica extracts were evaluated against MDR clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica has antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-¹ against multidrug resistant (MDR) clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica had antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-¹ and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-¹ against MDR isolates. The maximum inhibitory effects of Fagonia indica chloroform extract on biofilm formation was observed on Staphylococcus aureus (71.84%) followed by Klebsiella pneumoniae (70.83%) after 48 hrs showing that inhibition is also time dependent. Our results about bacterial cell protein leakage indicated that MDR isolates treated with chloroform extract of Fagonia indica showed maximum protein leakage of K. pneumoniae (59.14 µg mL-¹) followed by S. aureus (56.7 µg mL-1). Cell attachment assays indicated that chloroform extract resulted in a 43.5-53.5% inhibition of cell adherence to a polystyrene surface. Our results revealed that extracts of Fagonia indica significantly inhibited biofilm formation among MDR clinical isolates, therefore, could be applied as antimicrobial agents and cost effective biofilm inhibitor against these MDR isolates.

A alta resistência aos antimicrobianos está associada à formação de biofilme responsável por micróbios infecciosos para suportar condições severas. Portanto, novas alternativas são necessárias como inibidores de biofilme para controlar infecções. Neste estudo, as atividades antimicrobiana e antibiofilme dos extratos de Fagonia indica foram avaliadas contra isolados clínicos MDR. O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica tem efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-¹, e valor de concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados clínicos multirresistentes (MDR). O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica teve efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-¹, e concentração bactericida mínima (MBC) de 500-3000 µg mL-¹ contra isolados MDR. Os efeitos inibitórios máximos do extrato de clorofórmio Fagonia indica na formação de biofilme foi observada em Staphylococcus aureus (71,84%), seguido por Klebsiella pneumoniae (70,83%) após 48 horas, mostrando que a inibição também é dependente do tempo. Nossos resultados sobre extravasamento de proteínas de células bacterianas indicaram que isolados MDR tratados com extrato clorofórmico de Fagonia indica apresentaram vazamento máximo de proteínas de K. pneumoniae (59,14 µg mL-¹), seguido por S. aureus(56,7 µg mL-¹). Ensaios de fixação de células indicaram que o extrato de clorofórmio resultou em uma inibição de 43,5-53,5% da aderência das células a uma superfície de poliestireno. Nossos resultados revelaram que extratos de Fagonia indica inibiram [...].

Antiadhesion and antibiofilm potential of Fagonia indica from Cholistan desert against clinical multidrug resistant bacteria

Abstract High resistance to antimicrobials is associated with biofilm formation responsible for infectious microbes to withstand severe conditions. Therefore, new alternatives are necessary as biofilm inhibitors to control infections. In this study, the antimicrobial and antibiofilm activities of Fagonia indica extracts were evaluated against MDR clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica has antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against multidrug resistant (MDR) clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica had antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against MDR isolates. The maximum inhibitory effects of Fagonia indica chloroform extract on biofilm formation was observed on Staphylococcus aureus (71.84%) followed by Klebsiella pneumoniae (70.83%) after 48 hrs showing that inhibition is also time dependent. Our results about bacterial cell protein leakage indicated that MDR isolates treated with chloroform extract of Fagonia indica showed maximum protein leakage of K. pneumoniae (59.14 µg mL-1) followed by S. aureus (56.7 µg mL-1). Cell attachment assays indicated that chloroform extract resulted in a 43.5-53.5% inhibition of cell adherence to a polystyrene surface. Our results revealed that extracts of Fagonia indica significantly inhibited biofilm formation among MDR clinical isolates, therefore, could be applied as antimicrobial agents and cost effective biofilm inhibitor against these MDR isolates.

Resumo A alta resistência aos antimicrobianos está associada à formação de biofilme responsável por micróbios infecciosos para suportar condições severas. Portanto, novas alternativas são necessárias como inibidores de biofilme para controlar infecções. Neste estudo, as atividades antimicrobiana e antibiofilme dos extratos de Fagonia indica foram avaliadas contra isolados clínicos MDR. O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica tem efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e valor de concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados clínicos multirresistentes (MDR). O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica teve efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados MDR. Os efeitos inibitórios máximos do extrato de clorofórmio Fagonia indica na formação de biofilme foi observada em Staphylococcus aureus (71,84%), seguido por Klebsiella pneumoniae (70,83%) após 48 horas, mostrando que a inibição também é dependente do tempo. Nossos resultados sobre extravasamento de proteínas de células bacterianas indicaram que isolados MDR tratados com extrato clorofórmico de Fagonia indica apresentaram vazamento máximo de proteínas de K. pneumoniae (59,14 µg mL-1), seguido por S. aureus (56,7 µg mL-1). Ensaios de fixação de células indicaram que o extrato de clorofórmio resultou em uma inibição de 43,5-53,5% da aderência das células a uma superfície de poliestireno. Nossos resultados revelaram que extratos de Fagonia indica inibiram significativamente a formação de biofilme entre isolados clínicos MDR, portanto, poderiam ser aplicados como agentes antimicrobianos e inibidores de biofilme de baixo custo contra esses isolados MDR.

Suppression of Xanthomonas oryzae pv. oryzae biofilm formation by Acacia mangium methanol leaf extract / Supressão de Xanthomonas oryzae pv. oryzae: formação do biofilme oryzae pelo extrato de folhas de metanol de Acacia mangium

Abstract Xanthomonas oryzae pv. oryzae (Xoo), a pathogen responsible for rice bacterial leaf blight, produces biofilm to protect viable Xoo cells from antimicrobial agents. A study was conducted to determine the potency of Acacia mangium methanol (AMMH) leaf extract as a Xoo biofilm inhibitor. Four concentrations (3.13, 6.25, 9.38, and 12.5 mg/mL) of AMMH leaf extract were tested for their ability to inhibit Xoo biofilm formation on a 96-well microtiter plate. The results showed that the negative controls had the highest O.D. values from other treatments, indicating the intense formation of biofilm. This was followed by the positive control (Streptomycin sulfate, 0.2 mg/mL) and AMMH leaf extract at concentration 3.13 mg/mL, which showed no significant differences in their O.D. values (1.96 and 1.57, respectively). All other treatments at concentrations of 6.25, 9.38, and 12.5 mg/mL showed no significant differences in their O.D. values (0.91, 0.79, and 0.53, respectively). For inhibition percentages, treatment with concentration 12.5 mg/mL gave the highest result (81.25%) followed by treatment at concentrations 6.25 and 9.38 mg/mL that showed no significant differences in their inhibition percentage (67.75% and 72.23%, respectively). Concentration 3.13 mg/mL resulted in 44.49% of biofilm inhibition and the positive control resulted in 30.75% of biofilm inhibition. Confocal laser scanning microscopy (CLSM) analysis of Xoo biofilm inhibition and breakdown showed the presence of non-viable Xoo cells and changes in aggregation size due to increase in AMMH leaf extract concentration. Control slides showed the absence of Xoo dead cells.

Resumo Xanthomonas oryzae pv. oryzae (Xoo), um patogênico responsável pela influência bacteriana na folha do arroz, produz biofilme para proteger células Xoo viáveis de agentes antimicrobianos. Foi conduzido um estudo para determinar a potência do extrato de folha de Acacia mangium methanol (AMMH) como um inibidor de biofilme Xoo. Quatro concentrações (3,13, 6,25, 9,38 e 12,5 mg/mL) de extrato de folha de AMMH foram testadas quanto à sua capacidade de inibir a formação de biofilme Xoo em uma placa de microtitulação de 96 poços. Os resultados mostraram que os controles negativos tiveram o maior valor de OD do que os outros tratamentos, indicando a intensa formação de biofilme. Isso foi seguido do controle positivo (sulfato de estreptomicina, com concentração de 0,2 mg/mL, e extrato de folha de AMMH, com concentração de 3,13 mg/mL), que não apresentou diferenças significativas nos seus valores OD (1,96 e 1,57, respectivamente). Todos os outros tratamentos com concentrações de 6,25, 9,38, e 12,5 mg/mL não tiveram diferenças significativas nos seus valores OD (0,91, 0,79, e 0,53, respectivamente). Para percentagens de inibição, o tratamento com concentração 12,5 mg/mL apresentou o maior resultado (81,25%), seguido do tratamento em concentrações de 6,25 e 9,38 mg/mL, que não mostraram diferenças significativas na sua percentagem de inibição (67,75 e 72,23%, respectivamente). Concentração 3,13 mg/mL resultou em 44,49% de inibição do biofilme, e o controle positivo resultou em 30,75% de inibição do biofilme. Análise por microscopia confocal de leitura a laser de inibição e separação de biofilme Xoo revelou a presença de células Xoo não viáveis e alterações no tamanho da agregação por causa do aumento na concentração de extrato de folha de AMMH. Slides de controle mostraram a ausência de células Xoo mortas.

Inhibitory effect of baicalin on Streptococcus mutans UA159 in vitro / 口腔疾病防治

Objective @# To investigate the inhibitory effect of baicalin on Streptococcus mutans UA159 in vitro.@*Methods @#The minimum inhibitory concentration (MIC) of baicalin on Streptococcus mutans UA159 was determined by the liquid multiple dilution method combined with the OD600 value measured by microplate. The OD600 value of Streptococcus mutans UA159 in different concentrations of baicalin was measured by an enzyme mapping instrument. A growth curve was drawn, and the adhesion rate and adhesion inhibition rate were calculated. The effect of baicalin on the formation of Streptococcus mutans UA159 biofilms was observed by the crystal violet quantitative method and scanning electron microscopy. The effect of baicalin on the total number of Streptococcus mutans UA159 bacteria was observed by scanning electron microscopy.@*Results@#The MIC of baicalin on Streptococcus mutans UA159 was 12 mg/mL. With increasing baicalin concentration, the growth rate of Streptococcus mutans UA159 was slowed, the adhesion rate of Streptococcus mutans UA159 decreased and the adhesion inhibition rate increased（P < 0.05）. The results of crystal violet quantitative method showed that compared with the bacterial control group, the biofilm formation of Streptococcus mutans UA159 was significantly reduced after adding baicalin at 0 h, 6 h and 12 h (P < 0.001). Under a scanning electron microscope, the total number of bacteria decreased significantly after adding baicalin at 0 h, 6 h and 12 h.@*Conclusion@# baicalin ; natural medicine ; Streptococcus mutans UA159 ; caries ; minimum inhibitory concentration ; growth curve ; adhesion rate ; adhesion inhibition rate ; biofilm formation ; in vitro study

Inhibitory effects of epigallocatechin-3-gallate on the pathogenic properties of P. gingivalis in vitro / 口腔疾病防治

Objective @#To explore the antibacterial activity of epigallocatechin-3-gallate (EGCG) on P. gingivalis and the inhibitory effects on matrix metalloproteinases (MMPs) production induced by P. gingivalis.@*Methods@# The antimicrobial effect of EGCG against planktonic cultures and biofilms of P. gingivalis was evaluated using microplate dilution assays. The microstructural changes in biofilms were studied using scanning electron microscopy (SEM). The inhibitory effect of EGCG on arginine gingipain (Rgp) and lysine gingipain (Kgp) activity of P. gingivalis was evaluated using synthetic chromogenic peptides and fluorogenic substrates. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR analysis were used to assess MMP-1 and MMP-2 mRNA expression and secretion by human gingival fibroblasts (HGFs) stimulated with P. gingivalis in the presence or absence of EGCG, respectively. @*Results @# The MIC and MBC of EGCG against P. gingivalis were 62.5 μg/mL and 500 μg/mL, respectively. EGCG can not only inhibit the biofilm formation of P. gingivalis but also has a scavenging effect on mature biofilms and can affect their viability. Additionally, 10 μg/mL and 50 μg/mL of EGCG inhibited the proteinase activities of Rgp and Kgp, respectively (P < 0.05). Finally, the mRNA expression and secretion of MMP-1 and MMP-2 by HGFs stimulated by P. gingivalis were significantly inhibited by 50 μg/mL of EGCG (P < 0.05). @*Conclusion@#EGCG exhibits antimicrobial effects against P. gingivalis and reduces the expression of MMPs by HGFs.

Research Advance on the Factors Influencing / 中国医学科学院学报

The biofilm formation is a dynamic process of bacterial growth,and the extracellular components can encase these microorganisms,making them more resistant to antibiotics and host immune attack.The formation of antibiotic-resistant bacterial biofilms will be a major challenge for the treatment and control of clinical infections.

Advances on the mechanisms regulating the formation of the biofilm of Listeria monocytogenes / 生物工程学报

Listeria monocytogenes is an important food-borne pathogen. The distribution and survival of L. monocytogenes are related to its ability to form biofilms. Biofilms are resistant to adverse environments, and bacteria separated from the biofilms may lead to persistent food contaminations. The formation, maturation and structure of biofilms depend on a variety of external and internal factors, among which a variety of regulatory mechanisms play important roles. This review summarizes the regulatory mechanisms (including intracellular, intercellular and interspecific interactions) involved in the biofilm formation of L. monocytogenes in order to control the biofilm formation in food processing environments, thus providing new intervention strategy for food safety.

Comparative analysis of biofilm formation on materials used for the fabrication of implant-supported prostheses

Abstract The purpose of this study was to compare biofilm formation on materials used for the fabrication of implant-supported dental prostheses. Twenty discs (D=15 mm, H=3 mm) were fabricated from one of the following restorative materials: yttria tetragonal zirconia polycrystal (Y-TZP); commercially pure titanium (CP-Ti); or heat-cured polymethyl methacrylate (PMMA). Specimens were polished following standard protocols. A non-contact profilometer (NPFLEX, Bruker, UK) was used to assess the surface roughness of each disk; results were reported as Ra (µm). Five strains of Gram-negative bacteria frequently associated with peri-implantitis, Aggregatibacter actinomycetemcomitans, Candida. albicans, Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia, were cultured on hand-polished discs fabricated from heat-cured PMMA, Y-TZP, or CP-Ti to compare biofilm formation on each type of material. The results were reported as colony-forming units per milliliter (CFU/mL). One-way ANOVA and post hoc tests were used to compare surface roughness and bacterial colonization on the respective materials. Statistical significance was set at a = 0.05. Discs fabricated from Y-TZP had a significantly higher Ra value (350 ± 30 µm) than either PMMA, or CP-Ti discs. Discs fabricated from either Y-TZP and CP-Ti may exhibit less colonization by bacteria associated with peri-mucositis and peri-implantitis. Y-TZP and CP-Ti are suggested materials for fabrication of implant-supported prostheses, considering biofilm formation.

Resumo O objetivo deste estudo foi comparar a formação de biofilme em materiais utilizados na confecção de próteses dentárias implantossuportadas. Vinte discos (D = 15 mm, H = 3 mm) foram confeccionados com um dos seguintes materiais restauradores: zircônia tetragonal policristalina estabilizada por ítrio (Y-TZP); titânio comercialmente puro (CP-Ti); ou polimetilmetacrilato (PMMA). As amostras foram polidas seguindo protocolos padrão. Um perfilômetro sem contato (NPFLEX, Bruker, UK) foi usado para avaliar a rugosidade da superfície de cada disco; os resultados foram relatados como Ra (µm). Cinco cepas de bactérias Gram-negativas freqüentemente associadas a peri-implantite, Aggregatibacter actinomycetemcomitans, Candida. albicans, Porphyromonas gingivalis, Prevotella intermedia e Tannerella forsythia, foram cultivadas em discos polidos à mão feitos de PMMA, Y-TZP ou CP-Ti para comparar a formação de biofilme em cada tipo de material. Os resultados foram relatados como unidades formadoras de colônias por mililitro (UFC/mL). Análise de variância a um fator e testes post hoc foram usados ​​para comparar a rugosidade da superfície e a colonização bacteriana nos respectivos materiais. A significância estatística foi estabelecida em a=0,05. Os discos feitos de Y-TZP tiveram um valor Ra significativamente mais alto (350 ± 30 µm) do que os discos de PMMA ou CP-Ti. Os discos fabricados com Y-TZP e CP-Ti podem apresentar menor colonização por bactérias associadas à perimucosite e peri-implantite. Considerando O Y-TZP e CP-Ti são materiais indicados para a confecção de próteses implantossuportadas, considerando a formação de biofilme

Orostachys japonicus ethyl acetate fraction suppresses MRSA biofilm formation

To investigate the effect of Orostachys (O.) japonicus, a perennial herbaceous plant of the Family Crassulaceae, on biofilm formed by methicillin-resistant Staphylococcus aureus (MRSA). Methods: Powdered O. japonicus was extracted by 95% methanol, concentrated, and then, systematically fractionated with n-heane, dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol, and H2O according to polarity. Among them, the flavonoid-rich EtOAc fraction demonstrated the highest antibacterial activity and was used in this study. Using the biofilm inhibition assay, cell-surface attachment assay, confocal laser scanning microscopy, latex agglutination assay, and real time qRT-PCR, we examined whether the EtOAc fraction inhibited the formation of MRSA biofilm. Results: The EtOAc fraction exhibited distinct activity against biofilm formation and cell-surface attachment of MRSA up to 1 mg/mL through down-regulating the expression of mecA gene and the production and agglutination of penicillin-binding protein 2a as solidly observed in biofilm inhibition assay, cell-suface attachment assay, confocal laser scanning microscopy, latex agglutination assay, and real time qRT-PCR analysis. Conclusions: These results suggest that O. japonicus could be utilized as a potential resource for the development of new antibiofilm formation of MRSA and antibacterial agents in the future.

Orostachys japonicus ethyl acetate fraction suppresses MRSA biofilm formation

To investigate the effect of Orostachys (O.) japonicus, a perennial herbaceous plant of the Family Crassulaceae, on biofilm formed by methicillin-resistant Staphylococcus aureus (MRSA). Methods: Powdered O. japonicus was extracted by 95% methanol, concentrated, and then, systematically fractionated with n-heane, dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol, and H2O according to polarity. Among them, the flavonoid-rich EtOAc fraction demonstrated the highest antibacterial activity and was used in this study. Using the biofilm inhibition assay, cell-surface attachment assay, confocal laser scanning microscopy, latex agglutination assay, and real time qRT-PCR, we examined whether the EtOAc fraction inhibited the formation of MRSA biofilm. Results: The EtOAc fraction exhibited distinct activity against biofilm formation and cell-surface attachment of MRSA up to 1 mg/mL through down-regulating the expression of mecA gene and the production and agglutination of penicillin-binding protein 2a as solidly observed in biofilm inhibition assay, cell-suface attachment assay, confocal laser scanning microscopy, latex agglutination assay, and real time qRT-PCR analysis. Conclusions: These results suggest that O. japonicus could be utilized as a potential resource for the development of new antibiofilm formation of MRSA and antibacterial agents in the future.

Beneficial effects of ethyl-cyanoacrylate coating against candida albicans biofilm formation

Abstract The aim of this study was to verify whether modifications made in a hard chairside reline resin by an ethyl-cyanoacrylate adhesive, ECA (Super Bonder®, Loctite, Itapevi, SP, Brazil) would be able to inhibit or reduce Candida albicans biofilm formation on its surface, comparing to a commercial surface sealant (BisCover®, Bisco, Schaumburg, USA). Reline resin specimens were fabricated and randomly divided into 6 groups (n=8): CG (control group), no surface treatment; ECA1, ECA coating on the surface before sterilization; ECA2, ECA coating after sterilization; ECA3, ECA incorporated in the resin bulk; DPE1, BisCover® coating before sterilization; DPE2, BisCover® coating after sterilization. Specimens were inoculated with C. albicans SC5314 (1x107 cells/mL) and incubated for 24 h. Then, the biofilm were stained with LIVE/DEAD® BaclightTM L7007 Kit and analyzed by Confocal Laser Scanning Microscopy. The images were evaluated by bioImageL® v.2.0 software and total biovolume (µm3), viable cells (%), and covered area (%) were calculated. Data were statistically analyzed by Kruskal-Wallis and Dunn tests (p<0.05). Results showed that ECA-coated groups presented better results, reducing C. albicans biofilm formation. Acquired images revealed that these groups (ECA1 and ECA2) presented a reduced number of cells, mostly in yeast form (less pathogenic), while the other groups presented higher number of cells, mostly in hyphae form (more pathogenic). Based on these findings, a beneficial effect of Super Bonder® coating reline resins surface could be demonstrated, suggesting a promising way to prevent fungal biofilm formation on dentures.

Resumo O objetivo deste estudo foi verificar se as modificações feitas com o adesivo etil cianoacrilato, ECA (Super Bonder ®, Loctite, Itapevi, SP, Brasil) sobre as resinas acrílicas para reembasamento, poderiam inibir ou reduzir a formação de biofilmes de C.albicans sobre sua superfície quando comparado com um selante de superfície comercial (BisCover®, Bisco, Schaumburg, EUA). Amostras de resina acrílica para reembasamento foram fabricadas e divididas aleatoriamente em 6 grupos (n=8): CG (grupo controle), sem tratamento superficial; ECA1, revestimento de ECA na superfície antes da esterilização; ECA2, revestimento de ECA após esterilização; ECA3, ECA incorporado no volume da resina; DPE1, revestimento de BisCover® antes da esterilização; DPE2, revestimento de BisCover® após esterilização. Os espécimes foram inoculados com C. albicans SC5314 (1x107 células/mL) e incubados durante 24 h. Seguidamente, o biofilme foi corado com LIVE/DEAD® BaclightTM L7007 Kit e analisado no microscópio confocal de varredura a laser. As imagens foram avaliadas pelo software bioImageL® v.2.0, no qual foram calculados o biovolume total (μm3), as células viáveis (%) e a área coberta (%). Os dados foram analisados estatisticamente pelos testes de Kruskal-Wallis e Dunn (p<0,05). Os resultados mostraram que os grupos revestidos com ECA apresentaram os melhores resultados, reduzindo a formação do biofilme de C. albicans. As imagens adquiridas revelaram que esses grupos (ECA1 e ECA2) apresentaram um número reduzido de células, principalmente na forma de levedura (menos patogênico), enquanto os outros grupos apresentaram um maior número de células, principalmente na forma de hifas (mais patogênicas). Com base nessas descobertas, encontra-se um efeito benéfico na aplicação do adesivo ECA sobre as superfícies das resinas acrílicas para reembasamento, sugerindo assim uma nova alternativa de prevenir a formação de biofilme fúngico em próteses dentárias.

Medically Important Coagulase Negative Staphylococci

In recent years, coagulase negative Staphylococci(CONS) are increasingly recognized as one of the major causes of health care associated infections (HCAI). CONS infections are one of the major causes of morbidity and mortality in hospitalized patients. As CONS colonize various parts of the skin and mucous membranes, most of infections are en-dogenous in origin. However, they are also transmitted exogenously via various medical/ surgical procedures. The aim of present review article is to update and summarize the information available on medically important CONS with special reference to epidemiology, laboratory diagnosis and antibiotic resistance.

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.

Distribution of clinical isolates of Candida spp. and antifungal susceptibility of high biofilm-forming Candida isolates

Abstract INTRODUCTION: The increase in the incidence of fungal infections, especially those caused by Candida albicans and other Candida species, necessitates the understanding and treatment of Candida-associated infections. In this study, we aimed to investigate the identification, distribution, and biofilm formation ability of different clinical Candida isolates and evaluate the distribution and antifungal susceptibilities of high biofilm-forming (HBF) Candida isolates. METHODS: For identification, carbohydrate fermentation, carbohydrate assimilation, and ChromAgar tests were used. Biofilm formation was assessed using crystal violet binding assay, while the susceptibility to antifungal agents was determined using ATBTM Fungus 3 test kits. RESULTS: The majority of Candida species were C. parapsilosis (31.3%; 31/99) and C. tropicalis (30.3%; 30/99). C. tropicalis was found to be the most frequently isolated species among all HBF Candida species. HBF Candida isolates were more frequently isolated from vaginal swab (35.7%; 10/28), tracheal aspirate (17.9%; 5/28), and urine (17.9%; 5/28). The majority of tested isolates were resistant to itraconazole and voriconazole, whereas no isolate was deemed resistant to 5-flucytosine. CONCLUSIONS: C. tropicalis displays the highest biofilm formation ability among all the Candida species evaluated, and HBF Candida isolates were more frequently seen in vaginal swab, tracheal aspirate, and urine samples. Our findings revealed that 5-flucytosine is the most efficient antifungal agent against HBF Candida isolates.

Association of furanone C-30 with biofilm formation & antibiotic resistance in Pseudomonas aeruginosa

Background & objectives: Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial bloodstream infections in humans. This study was aimed to explore the association of furanone C-30 with biofilm formation, quorum sensing (QS) system and antibiotic resistance in P. aeruginosa. Methods: An in vitro model of P. aeruginosa bacterial biofilm was established using the standard P. aeruginosa strain (PAO-1). After treatment with 2.5 and 5 ?g/ml of furanone C-30, the change of biofilm morphology of PAO-1 was observed, and the expression levels of QS-regulated virulence genes (lasB, rhlA and phzA2), QS receptor genes (lasR, rhlR and pqsR) as well as QS signal molecule synthase genes (lasI, rhlI, pqsE and pqsH) were determined. Besides, the AmpC expression was quantified in planktonic and mature biofilm induced by antibiotics. Results: Furanone C-30 treatment significantly inhibited biofilm formation in a dose-dependent manner. With the increase of furanone C-30 concentration, the expression levels of lasB, rhlA, phzA2, pqsR, lasI, rhlI pqsE and pqsH significantly decreased in mature biofilm bacteria while the expression levels of lasR and rhlR markedly increased. The AmpC expression was significantly decreased in both planktonic and biofilm bacteria induced by imipenem and ceftazidime. Interpretation & conclusions: Furanone C-30 may inhibit biofilm formation and antibiotic resistance in P. aeruginosa through regulating QS genes. The inhibitory effect of furanone C-30 on las system appeared to be stronger than that on rhl system. Further studies need to be done with different strains of P. aeruginosa to confirm our findings.

Biofilm-forming capacity of blood-borne Candida albicans strains and effects of antifungal agents / Capacidad de formación de biopelículas de las cepas de Candida albicans de transmisión sanguínea y efectos de los agentes antifúngicos

Infections related to Candida albicans biofilms and subsequent antifungal resistance have become more common with the increased use of indwelling medical devices. Regimens for preventing fungal biofilm formation are needed, particularly in high-risk patients. In this study, we investigated the biofilm formation rate of multiple strains of Candida albicans (n = 162 clinical isolates), their antifungal susceptibility patterns, and the efficacy of certain antifungals for preventing biofilm formation. Biofilm formation was graded using a modified Christensen's 96-well plate method. We further analyzed 30 randomly chosen intense biofilm-forming iso lates using the XTT method. Minimum biofilm inhibition concentrations (MBIC) of caspofungin, micafungin, anidulafungin, fluconazole, voriconazole, posaconazole, itraconazole, and amphotericin B were determined using the modified Calgary biofilm method. In addition, the inhibitory effects of antifungal agents on biofilm formation were investigated. Our study showed weak, moderate, and extensive biofilm formation in 29% (n = 47), 38% (n = 61), and 23% (n = 37) of the isolates, respectively. We found that echinocandins had the lowest MBIC values and that itraconazole inhibited biofilm formation in more isolates (26/32; 81.3%) than other tested agents. In conclusion, echinocandins were most effective against formed biofilms, while itraconazole was most effective for preventing biofilm formation. Standardized methods are needed for biofilm antifungal sensitivity tests when determining the treatment and prophylaxis of C. albicans infections.

Las infecciones relacionadas con las biopelículas de Candida albicans y la consiguiente resistencia antifúngica se han vuelto fenómenos habituales con el uso creciente de dispositivos médicos permanentes. Son necesarios regímenes para prevenir la formación de biopelículas fúngicas, en especial en los pacientes de alto riesgo. En este estudio se investigó la tasa de formación de biopelículas de numerosas cepas de Candida albicans (162 aislados clínicos), sus patrones de sensibilidad a los antifúngicos y la eficacia de algunos de estos agentes para prevenir la formación de biopelículas. La formación de biopelículas se clasificó utilizando el método de Christensen modificado de 96 pocillos. Posteriormente se analizaron 30 aislados de formación intensa de biopelículas elegidos al azar, utilizando el método XTT. Se calcularon las concentraciones mínimas de inhibición de biopelículas (minimum biofilm inhibition concentrations, MBIC) de la caspofungina, la micafungina, la anidulafungina, el fluconazol, el voriconazol, el posaconazol, el itraconazol y la anfotericina B, utilizando el método modificado de biopelículas de Calgary. Además, se investigaron los efectos inhibitorios de los agentes antifúngicos sobre la formación de biopelículas. Nuestro estudio encontró una formación débil, moderada e intensa de biopelículas en el 29% (n = 47), 38% (n = 61) y 23% (n = 37) de los aislados, respectivamente. Encontramos que las equinocandinas mostraron los menores valores MBIC, y que el itraconazol inhibió la formación de biopelículas en más aislados (26/32; 81,3%) que otros agentes ensayados. En conclusión, las equinocandinas resultaron más eficaces frente a las biopelículas formadas, mientras que el itraconazol resultó más eficaz para prevenir la formación de biopelículas. Se necesita contar con métodos estandarizados para efectuar las pruebas de sensibilidad a los antifúngicos en términos de formación de biopelículas a la hora de determinar el tratamiento y la profilaxis de las infecciones por C. albicans.

Anti-quorum sensing and anti-biofilm formation activities of plant extracts from South Korea

Objective: To investigate anti-quorum sensing (anti-QS) and anti-biofilm formation (anti- BF) activities of the ethanol extracts of 388 plants. Methods: The anti-QS activity of the plant extracts was evaluated by disc-diffusion assays using the bio-reporter strain, Chromobacterium violaceum CV017. Pseudomonas aeruginosa PAO1, Yersinia enterocolitica ATCC 9610, and Agrobacterium tumefaciens C58, which possess QS systems, were used to evaluate the anti- BF activity of the plant extracts. Results: Among 388 plant extracts, the Cornus controversa (C. controversa) and Cynanchum wilfordii extracts exhibited the strongest anti-QS activity. The C. controversa extract exhibited anti-BF activity against Pseudomonas aeruginosa, Yersinia enterocolitica and Agrobacterium tumefaciens, whereas the Cynanchum wilfordii extract exhibited no anti-BF activity against Pseudomonas aeruginosa. In addition, the C. controversa extract suppressed soft rot of cabbage. Conclusions: The C. controversa extract inhibits bacterial QS and BF, and is capable of controlling soft rot. Therefore, this extract has potential for the prevention and treatment of bacterial infections and for the development of alternatives to antibiotics.

Inibição do sistema quorum sensing AI-1 por Capsicum frutescens e Capsicum annuum em bactérias Gram-negativas / Inhibition of AI-1 quorum-sensing system by Capsicum frutescens and Capsicum annuum in Gram-negative bacteria

A inibição do quorum sensing (QS) altera a comunicação bacteriana, reduzindo a expressão de fatores de virulência e a formação de biofilmes, o que pode conferir menor pressão seletiva em comparação aos antibióticos tradicionais. As frutas e hortaliças constituem uma fonte rica em compostos com propriedades potenciais de inibição do QS. Entretanto, há pouca referência sobre o potencial de pimentas do gênero Capsicum e de seus compostos isolados como inibidores do QS. Esse trabalho teve como objetivo avaliar o efeito de extratos orgânicos obtidos das variedades de pimenta-malagueta e pimentão vermelho sobre o sistema QS dependente do sinalizador AI-1 (acil homoserina lactona - AHL) em bactérias Gram-negativas. Os extratos foram obtidos por extração em fase sólida e separados em uma fração metanólica e outra amônica; sendo os compostos característicos identificados e quantificados por cromatografia líquida de alta eficiência (CLAE). A atividade antimicrobiana dos extratos foi avaliada pela determinação da concentração inibitória mínima (MIC) e pela curva de crescimento de Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 e Pseudomonas aeruginosa PAO1. O efeito anti-QS dos extratos foi avaliado pelos testes de difusão em ágar e quantificação da produção de violaceína em meio líquido por C. violaceum e sobre a formação de biofilme, avaliado pelo ensaio de cristal violeta e microscopia confocal, em S. liquefaciens e P. aeruginosa nas temperaturas 30 ºC e 37 ºC. Os resultados obtidos pela CLAE indicaram que o extrato metanólico de pimenta-malagueta (EMPM) continha capsaicinoides como a capsaicina e dihidrocapsaicina, luteolina e outros compostos não identificados; já o extrato amônico desta não continha os compostos capsaicinoides. Ambos os extratos de pimentão vermelho continham luteolina e compostos não identificados, mas não apresentaram capsaicinoides. Como o EMPM era representativo dos demais extratos, por conter tanto capsaicinóides quanto luteolina, o foco deste trabalho foi avaliar os efeitos do EMPM sobre fenótipos microbianos nas concentrações 5; 2,5; 1,25 e 0,625 mg/ml, além de utilizar a capsaicina como controle comparativo em concentrações equivalentes às do extrato (25, 50 e 100 µg/ml). Os resultados da atividade antimicrobiana mostraram inibição parcial do crescimento das bactérias nas concentrações sub-MIC (MIC >5 mg/ml) de 5 e 2,5 mg/ml de EMPM. A capsaicina também inibiu parcialmente o crescimento das bactérias a 100 µg/ml, com exceção de S. liquefaciens a 37 ºC, cujo crescimento foi induzido em 50 e 25 µg/ml. A produção de violaceína foi reduzida pelo EMPM a 1,25 e 0,625 mg/ml, sem afetar o crescimento de C. violaceum. Ensaios com C. violaceum CV026, estirpe biosensora capaz de produzir o pigmento na presença de AI-1 exógeno, sugerem que o possível mecanismo de atuação do extrato sobre o sistema QS em C. violaceum 12472 é sobre a síntese do sinalizador, já que não foi observada inibição da produção de violaceína em CV026 pelo extrato. Contrariamente, a capsaicina incrementou a produção do pigmento na estirpe 12472, mas ensaios com a estirpe CV026 indicaram que a capsaicina não atua como sinalizador do QS, uma vez que esta não induziu a produção de violaceína nesta estirpe. Já a formação de biofilme foi incrementada na presença do EMPM, sendo consideravelmente maior em P. aeruginosa a 30 ºC. Igualmente, observou-se indução da formação de biofilme por capsaicina em S. liquefaciens (37 ºC) e P. aeruginosa (30 ºC). Porém, a capsaicina não teve efeito sobre a formação de biofilme de S. liquefaciens quando cultivada a 30 ºC, nem P. aeruginosa a 37 ºC. Os resultados revelam que a produção de violaceína em C. violaceum ATCC 12472 é inibida pelo EMPM, mas não pela capsaicina. Já, o EMPM e a capsaicina, de forma geral, não inibem a formação de biofilme de S. liquefaciens MG1 nem P. aeruginosa PAO1. Outros estudos são necessários para elucidar os mecanismos pelos quais o EMPM e a capsaicina agem sobre os fenótipos avaliados neste trabalho

Quorum sensing inhibition alters bacterial communication by reducing virulence factors expression and biofilm formation, exerting less selective pressure compared to antibiotics. Fruits and vegetables are rich sources of compounds with potential QS-inhibition properties. However, there are few references about the potential of peppers belonging to the genus Capsicum and its isolated compounds as QS inhibitors. This study aimed to assess the effect of organic extracts obtained from Capsicum varieties, pimenta-malagueta (red chili) and pimentão vermelho (red bell pepper), on the AI-1 dependent QS system. The extracts were obtained by solid phase extraction and split into a methanolic and an ammonic fraction. Characteristic compounds were identified and quantified by high performance liquid chromatography (HPLC). The antimicrobial activity of the extracts was assessed by determining the minimal inhibitory concentration (MIC) and the growth curve of Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 and Pseudomonas aeruginosa PAO1. The anti-QS effect of the extracts was evaluated by the agar diffusion assay and the quantification of violacein production was assessed in liquid medium by C. violaceum, as well as in the biofilm formation test determined by the crystal violet assay and confocal microscopy with S. liquefaciens and P. aeruginosa at 30 ºC and 37 ºC. HPLC results showed that the methanolic extract of pimenta-malagueta (EMPM) contained capsaicinoids such as capsaicin and dihidrocapsaicin, luteolin and other unidentified compounds in lower concentrations; while its ammonic extract did not have capsaicinoids. Both pimentão vermelho extracts contained luteolin and other unidentified compounds in low concentrations, but they did not contain capsaicinoids. As EMPM was representative among the extracts because it contained capsaicinoids and luteolin, the focus of this work was to assess the effect of EMPM over microbial phenotypes at concentrations of 5, 2.5, 1.25 and 0.625 mg/ml, using capsaicin as a comparative control at equivalent concentrations to those in EMPM (25, 50 and 100 µg/ml). Antimicrobial activity assays showed a partial inhibition growth of bacteria at sub-MIC concentrations (MIC >5 mg/ml) of EMPM at 5 and 2.5 mg/ml. Similarly, capsaicin partially inhibited bacterial growth at 100 µg/ml, except for S. liquefaciens at 37 ºC in which growth was induced at 50 and 25 µg/ml. Violacein production was reduced by EMPM at 1,25 and 0,625 mg/ml without affecting C. violaceum growth. Assays with C. violaceum CV026, a biosensor strain that produces violacein in the presence of exogenous AI-1, suggest that EMPM reduced violacein production in C. violaceum 12472 by interfering with the AI-1 synthesis. In contrast, capsaicin incremented violacein synthesis in strain 12472, but experiments with strain CV026 revealed that capsaicin does not function as an analog of AI-1. Biofilm formation was increased in EMPM presence, being remarkably superior in P. aeruginosa cultivated at 30 ºC, as opposed to cultivation at 37 ºC. Similarly, capsaicin induced biofilm formation in S. liquefaciens (37 ºC) and P. aeruginosa (30 ºC). However, capsaicin did not affect biofilm formation on S. liquefaciens cultured at 30 ºC, neither on P. aeruginosa at 37 ºC. These results show that violacein production in C. violaceum ATCC 12472 is inhibited by EMPM, but not by capsaicin. In general, EMPM and capsaicin did not inhibit biofilm formation in S. liquefaciens MG1 neither in P. aeruginosa PAO1. More studies are necessary to elucidate the mechanisms by which EMPM and capsaicin affect the studied phenotypes in this work