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.

The topical treatment of old world cutaneous leishmaniasis with gentian violet along with cryotherapy: a pilot single-blind randomized controlled clinical trial

Abstract INTRODUCTION: The promising non-clinical antileishmanial effects of gentian violet (GV) encouraged us to evaluate the additive effect of GV on cryotherapy. METHODS: For 8 weeks, 59/161 cutaneous leishmaniasis patients/lesions underwent cryotherapy alone (group 1) or cryotherapy accompanied by 1% GV application (group 2). The primary endpoint was clinical response. RESULTS: Ultimately, 54.7% and 45.3% of the significantly cured lesions belonged to groups 1 and 2, respectively, which was not statistically significant. The clinical response was significantly different between the two groups at the end of the fourth week. CONCLUSIONS: Although the clinical response of the two groups was significantly different at the end of the fourth week, application of GV did not increase the efficacy of cryotherapy.

Comparison of primary human gingival fibroblasts from an older and a young donor on the evaluation of cytotoxicity of denture adhesives

Abstract Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. Objective To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). Material and Methods Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. Results While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. Conclusions All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.

Effect of γ-lactones and γ-lactams compounds on Streptococcus mutans biofilms

Abstract Considering oral diseases, antibiofilm compounds can decrease the accumulation of pathogenic species such as Streptococcus mutans at micro-areas of teeth, dental restorations or implant-supported prostheses. Objective To assess the effect of thirteen different novel lactam-based compounds on the inhibition of S. mutans biofilm formation. Material and methods We synthesized compounds based on γ-lactones analogues from rubrolides by a mucochloric acid process and converted them into their corresponding γ-hydroxy-γ-lactams by a reaction with isobutylamine and propylamine. Compounds concentrations ranging from 0.17 up to 87.5 μg mL-1 were tested against S. mutans. We diluted the exponential cultures in TSB and incubated them (37°C) in the presence of different γ-lactones or γ-lactams dilutions. Afterwards, we measured the planktonic growth by optical density at 630 nm and therefore assessed the biofilm density by the crystal violet staining method. Results Twelve compounds were active against biofilm formation, showing no effect on bacterial viability. Only one compound was inactive against both planktonic and biofilm growth. The highest biofilm inhibition (inhibition rate above 60%) was obtained for two compounds while three other compounds revealed an inhibition rate above 40%. Conclusions Twelve of the thirteen compounds revealed effective inhibition of S. mutans biofilm formation, with eight of them showing a specific antibiofilm effect.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

Influência de diferentes densidades de energia do laser de baixa intensidade em fibroblastos derivados da polpa de dentes decíduos / Influence of low-level laser with different energy densities on pulp fibroblasts from human primary teeth

O objetivo deste trabalho foi comparar os efeitos de diferentes densidades de energia e irradiâncias do Laser de Baixa Intensidade (LBI), variando em função do tempo de irradiação e potência, na viabilidade e proliferação de fibroblastos derivados da polpa de dentes decíduos humanos (HPF). HPF foram cultivados em DMEM e usados entre a 4ª e 8ª passagem. Os grupos foram divididos de acordo com diferentes densidades de energia, variando: Tempo de irradiação - Grupo I Ia (1,2 J/cm2 - 5 mW - 10 s), Ib (2,5 J/cm2 - 5 mW - 20 s), Ic (3,7 J/cm2 - 5 mW - 30 s), Id (5,0 J/cm2 - 5 mW - 40 s), e Ie (6,2 J/cm2 - 5 mW - 50 s); ou potência - Grupo II IIa (1,2 J/cm2 - 5 mW - 10 s), IIb (2,5 J/cm2 - 10 mW - 10 s), IIc (3,7 J/cm2 - 15 mW - 10 s), IId (5,0 J/cm2 - 20 mW - 10 s), e IIe (6,2 J/cm2 - 25 mW - 10 s). Células não irradiadas - cultivadas em condições nutricionais regulares - 10% Soro Fetal Bovino (SFB) (If e IIf) e células não irradiadas - cultivadas em déficit nutricional - 1% SFB (Ig e IIg), foram consideradas controles positivos e negativos, respectivamente. A viabilidade e proliferação celular foram avaliadas, repesctivamente, pelas técnicas MTT e Cristal violeta (CV), nos períodos de 24, 48 e 72 horas após a irradiação. Os dados obtidos foram submetidos à análise estatística por ANOVA 2 critérios, seguido pelo teste de Tukey (P<0,05). No ensaio MTT, os controles negativos, Ig e IIg, apresentaram significativamente menor viabilidade em relação aos correspondentes grupos experimentais: IIa e IIb, 24 horas após a irradiação; Ia, Ib, Ie, If e IIf no período de 48 horas; e Ib-If, assim como, IIa-IIf após 72 horas. Nos diferentes períodos de avaliação do ensaio CV, todos os grupos, exceto Ie, IIe e If, exibiram significativamente maior proliferação em comparação aos respectivos controles negativos. Dentro de um mesmo grupo nos diferentes períodos, os grupos If e IIe apresentaram menor viabilidade durante o período de 24 horas em comparação ao período de 72 horas pelo ensaio MTT. Na avaliação intragrupos, o ensaio CV revelou menor proliferação no período de 24 horas em comparação aos períodos de 48 e 72 horas, independente do grupo avaliado. Os diferentes protocolos de irradiação, grupos I e II, não apresentaram diferença estatisticamente significativa na viabilidade e proliferação celular entre densidades de energia iguais com irradiâncias diferentes durante os períodos avaliados. De acordo com os resultados obtidos, as diferentes densidades de energia e irradiâncias propostas não prejudicaram a viabilidade e proliferação de fibroblastos pulpares de dentes decíduos humanos. A variação do protocolo de irradiação LBI, em função do tempo ou da potência, não interferiram nas respostas celulares após a aplicação da mesma densidade de energia com irradiâncias diferentes.(AU)

The aim of this study was to compare the effects of Low-level laser (LLL) with different energy densities and irradiances, varying according to the irradiation time and power, on cell viability and proliferation of pulp fibloblasts from human primary teeth (HPF). HPF were culture in DMEM and used between 4th and 8th passages. Groups were divided according to different energy densities, varying: Time of irradiation Ia (1.2 J/cm2 - 5 mW - 10 s), Ib (2.5 J/cm2 - 5 mW - 20 s), Ic (3.7 J/cm2 - 5 mW - 30 s), Id (5.0 J/cm2 - 5 mW - 40 s), and Ie (6.2 J/cm2 - 5 mW - 50 s); or output power - Grupo II IIa (1.2 J/cm2 - 5 mW - 10 s), IIb (2.5 J/cm2 - 10 mW - 10 s), IIc (3.7 J/cm2 - 15 mW - 10 s), IId (5.0 J/cm2 - 20 mW - 10 s), e IIe (6.2 J/cm2 - 25 mW - 10 s). Non-irradiated cells - grown in regular nutritional conditions - 10% Fetal Bovine Serum (FSB) (If and IIf) and non-irradiated cells - grown in nutritional deficit - 1% FBS (Ig and IIg) were considered positive and negative controls, respectively. Cell viability and proliferation were respectively assessed through MTT and Crystal violet (CV) assays at 24, 48 and 72h after irradiation. Data were submitted to statistical analysis by ANOVA 2 criteria, followed by Tukey test (P<0.05). In the MTT assay, the negative controls, Ig and IIg, showed significantly lower viability in relation to the corresponding groups: IIa and IIb 24 hours after irradiation; Ia, Ib, Ie, If and IIf at 48 hours period; and Ib-If, as IIa-IIf, after 72 hours. At different periods of evaluation of CV assay, all groups, except Ie, IIe and If, exhibited significantly higher proliferation compared to the respective negative controls. Within the same group at different periods, groups If and IIe showed lower viability during 24 hours compared to 72 hours period by MTT assay. In the intragroup evaluation, CV assay revealed lower proliferation at 24 hours compared to 48 and 72 hours periods, regardless of the evaluated group. Different irradiation protocols, groups I and II, showed no statistically significant differences on cell viability and proliferation among equals energy densities with different irradiances at the evaluated periods. According to these findings, different LLL energy densities and irradiances proposed did not impair viability and proliferation of pulp fibloblasts from human primary teeth. The variation of the LLL irradiation protocol, by the time or power, did not interfere in cellular responses after the application of the same energy density with different irradiances.(AU)

The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization RESULTS: We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain CONCLUSIONS: MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

Enfermedad de Chagas: Contribuciones del Centro de Investigaciones Toxicológicas / Chagas disease: The contribution of the Center for Toxicological Research

La quimioterapia de la enfermedad de Chagas cuenta en la actualidad con el empleo de dos fármacos solamente: Nifurtimox y Benznidazol. Nifurtimox es un nitrofurano y Benznidazol es un compuesto nitroimidazólico. El uso de estas drogas para tratar la fase aguda de la enfermedad se acepta ampliamente. Sin embargo, su utilización en el tratamiento de la fase crónica no está exenta de cuestionamientos serios. Los efectos colaterales de ambas son un inconveniente mayor en su uso, y frecuentemente fuerza a los médicos a detener el tratamiento. Los estudios de toxicidad experimentales con Nifurtimox evidenciaron neurotoxicidad, daño testicular, toxicidad ovárica y efectos deletéreos en corazón, tejido mamario, adrenales, colon y esófago. Para el Benznidazol, se observaron efectos deletéreos en adrenales, colon y esófago. También inhibe el metabolismo de varios xenobióticos transformados por el sistema del citocromo P450 y sus metabolitos reaccionan con los componentes fetales in vivo. Ambas drogas exhibieron efectos mutagénicos significativos y se demostró en algunos estudios que eran carcinogénicas o tumorigénicas. Los efectos tóxicos de ambos fármacos dependen de la reducción enzimática de su grupo nitro. En este trabajo se resume la actividad de este laboratorio en el esfuerzo por comprender los mecanismos de la acción tóxica de estos fármacos...

Proteus mirabilis biofilm - Qualitative and quantitative colorimetric methods-based evaluation

Proteus mirabilis strains ability to form biofilm is a current topic of a number of research worldwide. In this study the biofilm formation of P. mirabilis strains derived from urine of the catheterized and non-catheterized patients has been investigated. A total number of 39 P. mirabilis strains isolated from the urine samples of the patients of dr Antoni Jurasz University Hospital No. 1 in Bydgoszcz clinics between 2011 and 2012 was used. Biofilm formation was evaluated using two independent quantitative and qualitative methods with TTC (2,3,5-triphenyl-tetrazolium chloride) and CV (crystal violet) application. The obtained results confirmed biofilm formation by all the examined strains, except quantitative method with TTC, in which 7.7% of the strains did not have this ability. It was shown that P. mirabilis rods have the ability to form biofilm on the surfaces of both biomaterials applied, polystyrene and polyvinyl chloride (Nelaton catheters). The differences in ability to form biofilm observed between P. mirabilis strains derived from the urine of the catheterized and non-catheterized patients were not statistically significant.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

Genotoxicidad en leucocitos por la quimioprofilaxis de sangre con Violeta de Genciana y su prevención con antioxidantes / Genotoxicity in leukocytes by blood chemoprophylaxis with Gentian Violet and its prevention with antioxidants / Genotoxicidade em leucócitos pela quimioprofilaxia de sangue com Violeta Genciana e sua prevenção com antioxidantes

El Violeta de Genciana (GV) se usa como aditivo en la sangre para eliminar el Trypanosoma cruzi en la quimioprofilaxis de la infección por enfermedad de Chagas vía transfusión sanguínea, cuando no es posible un control previo de laboratorio o bajo situaciones de emergencia. En estos estudios se encontraron efectos genotóxicos del GV con el ensayo Cometa, cuando se lo agregó a la sangre bajo las condiciones empleadas para esterilizarla para transfusión. El efecto genotóxico fue aún más intenso si la sangre se mantenía con GV por 48 horas. Los resultados obtenidos con el ensayo Cometa sugieren la formación de bases hidroxiladas de ADN como resultado de un ataque de especies reactivas de oxígeno y apoyan la genotoxicidad del GV y su potencial carcinogénico ya informado previamente. Los efectos genotóxicos observados en el ensayo Cometa fueron parcialmente prevenidos por administración de antioxidantes que ya tienen uso clínico seguro, como á-tocoferol, ácido lipoico o N-acetilcisteína. El ácido lipoico fue capaz también de reaccionar in vitro con GV. Los resultados sugieren un uso potencial de estos antioxidantes para prevenir los efectos secundarios no deseados del GV para el individuo receptor de la sangre.

Gentian violet (GV) is being used as blood additive to eliminate Trypanosoma cruzi in the chemoprophylaxis of Chagas disease infection via blood transfusion when prior laboratory control is not possible or under emergency circumstances in endemic areas. In these studies genotoxic effects of GV were found employing the Comet assay when GV was added to rat blood under the cisconditions employed to sterilize it for transfusion. The genotoxic effect was even more intense if blood was kept with GV for 48 hours. The positive results obtained in the Comet assay suggest the formation of DNA hydroxylated bases as result of a reactive oxygen species (ROS) attack and further confirm GV genotoxicity and its potential carcinogenic effects previously reported. Genotoxicity effects observed in the Comet assay were partially but significantly prevented by prior administration of antioxidants having safe clinical use such as á-tocopherol; lipoic acid or N-acetylcysteine. Lipoic acid was also able to chemically react in vitro with GV (eg. the one remaining in the transfusion mixture after it had enough time to eliminate the parasite from blood). Results would suggest the potential use of these antioxidants to prevent unwanted side effects of GV for the blood recipient.

O Violeta de Genciana (GV) é utilizado como aditivo no sangue para remover o Trypanosoma cruzi da sangue em quimioprofilaxia da infecção por doença de Chagas através de transfusão de sangue, quando não é possível controle prévio de laboratório ou em situações de emergência. Nestes estudos encontraram-se efeitos genotóxicos do GV utilizando o ensaio Cometa, quando o GV foi adicionado ao sangue sob as condições utilizadas para a esterilização para transfusão. O efeito genotóxico foi ainda mais intenso se o sangue era mantido durante 48 horas com GV. Os resultados obtidos com o ensaio Cometa sugerem a formação de bases de DNA hidroxiladas, como resultado de um ataque de espécies reativas de oxigênio e apoiam a genotoxicidade do GV e seu potencial carcinogênico já informado anteriormente. Efeitos genotóxicos observados no ensaio do Cometa foram parcialmente prevenidos por administração de antioxidantes que já têm uso clínico seguro, como á-tocoferol, o ácido lipoico ou N-acetilcisteína. O ácido lipoico também foi capaz de reagir in vitro com GV. Os resultados sugerem um uso potencial destes antioxidantes para prevenir os efeitos colaterais não desejados de GV para o indivíduo receptor do sangue.

Induced maturation of hepatic progenitor cells in vitro

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

Antibiofilm and Antibacterial Activities of Farnesol and Xylitol as Potential Endodontic Irrigants

This study investigated the antibiofilm and antibacterial effects of farnesol and xylitol in a series of experiments in order to evaluate their potential use as root canal irrigants. The following substances were tested: 0.2% farnesol; 5% and 20% xylitol; 0.2% farnesol plus 20% xylitol; and saline (control). For comparison with an established endodontic irrigant, 2.5% NaOCl was included in each test. Three experiments were conducted: the crystal violet assay, to evaluate the effects on the biofilm biomass; the dentin disinfection test, to evaluate the effects on bacterial viability in biofilms; and the root canal disinfection test, to simulate the use in the root canal environment. Farnesol was the most effective substance in reducing the biofilm biomass, followed by 20% xylitol. All substances affected bacterial viability in biofilms; farnesol showed the best results followed by the farnesol/xylitol combination. Irrigation with all substances significantly reduced the bacterial load (p<0.001), but only the farnesol/xylitol combination was significantly more effective than saline (p=0.02). NaOCl was more effective than any other substance tested in the three experiments (p<0.001). The findings demonstrated that farnesol affected both the biofilm biomass and the viability of cells in the biofilm, while 20% xylitol affected only the biofilm biomass. Although not more effective than NaOCl, the combination of these two antibiofilm substances has potential to be used in endodontics in certain situations.

Este estudo investigou os efeitos antibiofilme e antibacteriano de farnesol e xilitol em uma série de experimentos para avaliar seu uso potencial como irrigante de canais radiculares. As seguintes substâncias foram testadas: farnesol a 0,2%; xilitol a 5% e 20%; farnesol a 0,2% combinado com xilitol a 20%; e solução salina (controle). NaOCl foi testado para comparação. Três experimentos foram conduzidos: o teste do cristal violeta para avaliar os efeitos sobre a biomassa de biofilme, o teste da desinfecção de fragmentos de dentina para avaliar os efeitos na viabilidade bacteriana nos biofilmes e o teste da desinfecção de canal radicular para simular o uso no ambiente do canal radicular. Farnesol foi o mais eficaz, seguido por xylitol a 20%. Todas as substâncias afetaram a viabilidade bacteriana nos biofilmes; farnesol mostrou os melhores resultados, seguido pela combinação farnesol/xilitol. A irrigação com todas as substâncias reduziu significativamente a carga bacteriana (p<0,001), mas somente a combinação farnesol/xilitol foi significativamente mais eficaz que a solução salina (p=0,02). NaOCl foi mais eficaz que qualquer outra substância testada nos três experimentos (p<0,001). Os achados demonstraram que farnesol afetou tanto a biomassa de biofilme quanto a viabilidade das células no biofilme, enquanto que xilitol a 20% afetou a biomassa de biofilme. Embora não mais eficazes que NaOCl, combinações dessas duas substâncias antibiofilmes têm o potencial de ser usadas na Endodontia, em determinadas situações.

Biofilm biomass disruption by natural substances with potential for endodontic use

This study evaluated the in vitro effects of four natural substances on the biomass of bacterial biofilms to assess their potential use as root canal irrigants. The following substances and their combinations were tested: 0.2% farnesol; 5% xylitol; 20% xylitol; 0.2% farnesol and 5% xylitol; 0.2% farnesol, 5% xylitol, and 0.1% lactoferrin; 5% xylitol and 0.1% lactoferrin; and 20 mM salicylic acid. The crystal violet assay was used to evaluate the effects of these substances on the biomass of biofilms formed by Enterococcus faecalis and Staphylococcus epidermidis. All substances except for 20 mM salicylic acid and 20% xylitol reduced biofilm mass when compared to controls. The combination of farnesol and xylitol was the most effective agent against E. faecalis ATCC 29212 (p < 0.05). Farnesol combined with xylitol and lactoferrin was the most effective against biofilms of the endodontic strain of E. faecalis MB35 (p < 0.05). Similarly, combinations involving farnesol, xylitol, and lactoferrin reduced the biomass of S. epidermidis biofilms. In general, farnesol, xylitol, and lactoferrin or farnesol and xylitol reduced biofilm biomass most effectively. Therefore, it was concluded that combinations of antibiofilm substances have potential use in endodontic treatment to combat biofilms.

Estudo bacteriológico do lavado broncoalveolar na gestão de antibióticos na pneumonia associada à ventilação mecânica de pacientes em unidades de terapia intensiva cirúrgica / Bacteriological study of bronchoalveolar lavage in the antibiotic management of suspected ventilator-associated pneumonia of patients in surgical intensive care units

OBJETIVO: determinar a correlação da coloração de Gram com o resultado final das culturas de LBA em pacientes cirúrgicos sob ventilação mecânica com PAV clínica. MÉTODOS: Estudo retrospectivo de 252 amostras de lavado broncoalveolar em pacientes com clínica de pneumonia associada à ventilação mecânica com trauma ou cuidados de pós-operatório. As amostras de coloração de Gram foram classificadas como cocos Gram-positivos e bacilos Gram-negativos, todos os outros resultados foram excluídos. Culturas de lavado broncoalveolar foram comparadas aos resultados da coloração de Gram. RESULTADOS: A correlação entre a coloração de Gram e a cultura do lavado broncoalveolar apresentou índice kappa de 0,27. A sensibilidade da coloração de Gram foi 53,9% e a especificidade de 80,6%. Considerando a identificação de cocos Gram-positivos comparada com os outros resultados (negativos e bacilos Gram-negativos), o valor preditivo negativo foi 94,8%. Na avaliação de bacilos Gram-negativos comparada com os outros resultados (negativos e cocos Gram-positivos), a sensibilidade foi 27,1% e a especificidade foi 95,4%. CONCLUSÃO: O valor preditivo negativo para cocos Gram-positivos parece ser aceitável, mas a sensibilidade da coloração de Gram na etiologia de pneumonia associada à ventilação mecânica não permite prever qual é o micro-organismo antes da cultura.

OBJECTIVE: To determine the correlation of Gram staining with the culture of bronchoalveolar lavage in patients with clinical features of ventilator-associated pneumonia from two adult trauma and surgical intensive care units. METHODS: We conducted a retrospective study of 252 samples of bronchoalveolar lavage from patients with clinical ventilator-associated pneumonia in trauma or surgical postoperative care. Gram staining samples were classified as Gram-positive cocci and Gram-negative bacilli, all other results being excluded. Cultures from bronchoalveolar lavage were compared with Gram staining results. RESULTS: The correlation between Gram staining and culture from the bronchoalveolar lavage showed a kappa index of 0.27. The sensitivity of Gram staining was 53.9% and the specificity, 80.6%. Considering the identification of Gram-positive cocci against other results (negative and Gram-negative bacilli), the negative predictive value was 94.8%. The evaluation of Gram-negative bacilli against other results (negative and Gram-positive cocci) rendered a sensitivity of 27.1% and a specificity of 95.4%. CONCLUSION: It appears that the negative predictive value for Gram-positive cocci is acceptable, but the sensitivity of Gram staining in the etiology of ventilator-associated pneumonia was not able to identify the microorganism before culture.

Análisis de la monitorización de cinco indicadores de calidad del hemocultivo en un hospital universitario en Chile 2009-2011 / Quality indicators for blood culture: three years of monitoring at a university hospital in Chile

Blood culture is considered the "gold standard" for the diagnosis of bacteremia, critical condition with high morbidity and mortality. Because of its importance, it is estimated that the blood culture is a critical test that requires close monitoring on the quality with which the process is performed. The objective of this work is to show the results of the monitoring carried out during the past three years, of 5 quality indicators of blood cultures in the laboratory of the Hospital Clínico de la Pontificia Universidad Católica de Chile, considering pre-analytical, analytical and post-analytical aspects. In the 3 years monitored the mean contamination was 0,7%, 46% of adult bottles had adequate volume, match between Gram stain with final identification was 99.4%, 100% of correct participations were achieved in surveys of external quality control and Gram staining notification before 1 hour was 88.7%. With regard to proposed aims, in 2011 the laboratory complies with all, except the percentage of bottles with appropriate volume of blood inoculated. This indicator is very low and should be corrected as soon as possible since it is known that it is an important condition for optimum performance of blood cultures.

El hemocultivo es el "estándar de oro" para el diagnóstico de bacteriemia, condición grave de alta morbi-mortalidad. Por esto, se estima que el hemocultivo es un examen crítico, que requiere monitorización de su calidad. El objetivo de este trabajo es mostrar los resultados de la monitorización de 3 años de 5 indicadores de calidad del hemocultivo implementados en nuestro hospital. El porcentaje promedio de contaminación de las botellas para hemocultivo fue 0,7%, el porcentaje de botellas de adultos con volumen adecuado fue 46%, la concordancia de la tinción de Gram con la identificación final correspondió a 99,4%, el 100% de las participaciones en encuestas de control de calidad externo fueron correctas y 88,7% de los avisos de valores de alerta de la tinción de Gram fueron realizados antes de 1 hora. En el año 2011 se cumplió con las metas propuestas por el laboratorio para todos los indicadores, excepto con el porcentaje de botellas con volumen apropiado de sangre. Este último, se encuentra muy por debajo de la meta y debe ser mejorado a la brevedad, ya que el volumen de sangre cultivada es el factor más importante para obtener un rendimiento óptimo del hemocultivo.

Espiroquetosis intestinal humana: serie clínica y revisión de la literatura / Human intestinal spirochetosis: clinical series and literature review

Introduction: Human intestinal spirochetosis (HIE) is defined as colonization by spirochetes of the large intestine. Is associated with chronic diarrhea. The incidence and prevalence ranges from 0.4% to 12%. Objective: To determine the prevalence of HIE in the Salvador's Hospital, between 2003 and 2008 in patients with a history of chronic diarrhea and without abnormalities in colonoscopy, in 2 separate groups: patients with and without a history of HIV infection. Material and Methods: Retrospective morphology evaluation of the large bowel endoscopic biopsies to the selected groups. Results: We reviewed 115 biopsies, 98 were from HIV-negative and 17 HIV from positive patients. Two cases of intestinal spirochetosis were detected, both HIV negative, with a prevalence of 1.7%. Comment: The prevalence of HIE is similar to that reported in Western countries. Population studies are needed to determine the real epidemiological impact in our environment.

Introducción: La espiroquetosis intestinal humana (EIH) se define como la colonización del intestino grueso por espiroquetas. Se asocia a diarrea crónica. Su incidencia y prevalencia van desde 0,4 a 12% Objetivo: Determinar la prevalencia de EIH en el Hospital Del Salvador, de Santiago, Chile, entre los años 2003 y 2008, en pacientes con antecedentes clínicos de diarrea crónica y colonoscopia sin hallazgos patológicos, separados en dos grupos: pacientes con y sin antecedentes de infección por VIH. Material y Método: Evaluación morfológica retrospectiva de las biopsias endoscópicas de intestino grueso de los grupos seleccionados. Resultados: Se revisaron 115 biopsias, 98 correspondieron a pacientes sin infección por VIH y 17 a pacientes seropositivos para VIH. Se detectaron dos casos de espiroquetosis intestinal, ambos en pacientes sin infección por VIH, con una prevalencia de 1,7 %. Comentario: La prevalencia de EIH es similar a la publicada en países occidentales. Se requieren estudios poblacionales para determinar el real impacto epidemiológico en nuestro medio.

Formação in vitro de bioflme por Pseudomonas aeruginosa e Staphylococcus aureus na superfície de canetas odontológicas de alta rotação / In vitro biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus on the surface of high-speed dental handpieces

Em consultórios odontológicos, são utilizadas canetas de alta rotação que funcionam conectadas a circuitos de água. Estudos já demonstraram contaminação microbiana em amostras de água coletada de tubulações desses circuitos. Durante sua utilização, essas canetas entram em contato com a microbiota oral, o que pode favorecer a formação de biofilme em sua superfície e influir na qualidade e na segurança dos procedimentos. O objetivo deste trabalho foi avaliar a formação in vitro de biofilme na superfície de canetas odontológicas utilizando-se Pseudomonas aeruginosa e Staphylococcus aureus. Os experimentos foram executados em fragmentos de alumínio provenientesdo corte de canetas odontológicas e a formação do biofilme foi verificada pela contagem de bactérias viáveis (CBV)e por microscopia eletrônica de varredura (MEV). O número de células aderidas atingiu 9 × 106 ufc.cm–2 paraPseudomonas aeruginosa e 6 × 108 ufc.cm–2 para Staphylococcus aureus. A MEV mostrou a adesão de Pseudomonasaeruginosa e Staphylococcus aureus aos fragmentos e a presença de matriz polimérica a partir do sexto dia deincubação. Também foi testada a produção de biofilme por essas bactérias na superfície de placas de poliestireno, pelo método do Cristal Violeta. Ambos os micro-organismos exibiram valores de absorbância superiores ao ponto de corte estabelecido, indicando resultados positivos. Foi demonstrada, ainda, a capacidade de ambas as bactériasproduzirem cápsula, utilizando-se o método do Ágar Vermelho Congo. Nas condições testadas, os experimentosrealizados neste trabalho mostraram a formação in vitro de biofilme na superfície de material proveniente decanetas odontológicas, um evento importante considerando-se que sua presença representa um potencial risco para o estabelecimento de contaminação cruzada.

High-speed handpieces connected to running water circuits are used in dental offices. Studies have shown microbial contamination in water samples collected from the tubing of these circuits. Handpieces come in contact with oral microorganisms during use, which can promote the formation of biofilm on the handpiece and affect procedural quality and safety. The aim of this study was to evaluate in vitro biofilm formation on the surface of high-speed dental handpieces using Pseudomonas aeruginosa and Staphylococcus aureus. The assays were performed on aluminum fragments cut from dental handpieces and biofilm formation assessed by viable bacteria counting (VBC) and scanning electron microscopy (SEM). The number of adhered cells was 9 × 10(6)ufc.cm(-2) for Pseudomonas aeruginosa and 6 × 10(8)ufc.cm(-2) for Staphylococcus aureus. SEM showed the adherence of Pseudomonas aeruginosa and Staphylococcus aureus to handpiece fragments and the presence of a polymer matrix after six days of incubation. We also tested biofilm production by these bacteria on the surface of polystyrene plates using the Crystal Violet method. Both microorganisms displayed absorbance values above the established cut-off point, indicating positive results. The ability of these bacteria for capsule (slime) production was shown using the Congo Red Agar method. Within the limits of these experiments, this study demonstrated in vitro biofilm formation on the surface of material from dental handpieces, indicating a potential cross contamination risk.

Da violeta de genciana à sorologia: o controle da transmissão da doença de chagas nos bancos de sangue / Gentian violet to serology: the control of transmission of Chagas disease in blood banks

Revisões históricas aos avanços científicos para o controle da doença, o Simpósio Internacional Comemorativo do Centenário da Descoberta da Doença de Chagas (1909-2009).