Toxicity evaluation of the ethanolic Jambu bol [Syzygium malaccense (L.) Merr. and Perry] leaves extract and mechanisms underlying its antibacterial action

Aims@#Many plants and their derivatives are widely used in food manufacturing because of their biological activities. They play a significant role as food additives to control microbial growth and the occurrence of oxidation reactions. Syzygium malaccense L. is a well-known plant with biological activities such as antimicrobial and antioxidant activities. Thus, the aims of this study were to evaluate the toxicity of the ethanolic leaves extract of S. malaccense and to study its antibacterial mode of action.@*Methodology and results@#The toxicity assessment of S. malaccense leaves extract was determined using the brine-shrimp larvae model. The action mechanisms against bacterial membrane were determined by studying the intracellular material leakage by means of nucleic acid (DNA and RNA) release, crystal violet dye uptake and cellular protein leakage. The present findings proved the extract's safety as indicated by a high dose of 7.402 mg/mL for lethal concentration (LC50) against brine-shrimp larvae. On the other hand, the ethanolic extract caused a severe membrane permeability towards all the tested bacteria as indicated by the increased intracellular material leakage in a concentration-dependent manner.@*Conclusion, significance and impact of study@#The current study provides valuable information regarding the safety and antibacterial action mechanism of S. malaccense ethanolic leaves extract, thus paving the way for its utilization as a natural preservative in a wide range of food products.

Evaluation antibacterial and antibiofilm activity of the antimicrobial peptide P34 against Staphylococcus aureus and Enterococcus faecalis

ABSTRACT The adhesion ability of bacteria to abiotic surfaces has important implications in food industries, because these organisms can survive for long periods through the biofilm formation. They can be transferred from one place to another in the industry causing contamination of the food processing environment. In this study, the antibacterial and antibiofilm activities of the antimicrobial peptide P34, characterized as a bacteriocin-like substance (BLS P34) were tested against planktonic and sessile cells of Staphylococcus aureus and Enterococcus faecalis isolated from foods. The BLS P34 showed inhibitory effect against all planktonic cells of E. faecalis. The inhibition of biofilm formation and the eradication of pre-formed biofilm were evaluated with the crystal violet assay and with the reduction of 3-bromide [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. The BLS P34 promoted a reduction of percentage of adhered microbial cells on the surface, not being able to perform the complete elimination of biofilm formation. The metabolic activity of S. aureus biofilms decreased considerably between 41-95%. However, E. faecalis cells showed up metabolically stimulated. The BLS P34 has the potential antibiofilm for the species S. aureus. Studies suggest more detailed approaches to a better understanding of the interactions between the antimicrobial and bacterial cells within the biofilm structure.

Effects of aspirin on formation and dispersion of Pseudomonas aeruginosa biofilm / 临床检验杂志

Objective To explore the effects of aspirin on formation and dispersion of Pseudomonas aeruginosa (P.aeruginosa) biofilm.Methods The broth microdilution method was used to detect the minimal inhibitory concentration(MIC) of aspirin for P.aeruginosa.The anti-biofilm effects of aspirin on P.aeruginosa were determined on the microtiter plates combined with crystal violet staining.The serial dilution method for counting colony number on microtiter plate was used to explore the effects of aspirin on initial adherence of P.aeruginosa.Results The MIC values of aspirin against PAO1,PA18,PA53 and PA67 strains of Pseudomonas aeruginosa were 5,2.5,5 and 5 mg/mL respectively.Aspirin significantly inhibited the formation and dispersion of the biofilm of PAO1 and PA18 strains (t =5.65,P ＜ 0.05 and t =5.06,P ＜ 0.05 for inhibition;t =6.45,P ＜ 0.05 and t =6.26,P ＜ 0.05 for dispersion) at the concentration of 2.5 mg/mL.Similar effects were also found in the determination for PA67 and PA53 strains(t =6.45,P ＜0.05 and t =6.26,P ＜ 0.05 for inhibition;t =7.82,P ＜ 0.05;t =9.18,P ＜ 0.05 for dispersion) at aspirin concentration of 1.25 and 0.313 mg/mL respectively.Aspirin inhibited the initial adherence of P.aeruginosa at the concentration of 2.5 mg/mL(P ＜0.05).Conclusion Aspirin could significantly inhibit the initial adherence and biofilm formation of P.aeruginosa and disperse the 24 hour-formed mature bioiflm.

Partial removal and detoxification of Malachite Green and Crystal Violet from laboratory artificially contaminated water by Pleurotus ostreatus / Eliminación parcial y desintoxicación de Verde Malaquita y Cristal Violeta del laboratorio con agua contaminada artificialmente por Pleurotus ostreatus / Extração e desintoxicação de Verde Malaquita e Cristal Violeta de água contaminada artificialmente em laboratório por Pleurotus ostreatus

Abstract The triphenylmethane Malachite Green (MG) and Crystal Violet (CV) dyes are cationic dyes and mix with domestic wastewater when dumped; increasing, among others, the chemical and biological oxygen demand and can cause acute toxicity at different trophic levels. Promoting the removal (decolorization) of MG and CV, and laccase activity (54.8 ± 8.9 and 30.6 ± 2.9 UL-1 respectively) by using P. ostreatus viable biomass needed parameters such as pH (4.5 and 6.0), temperature (25 to 30 °C), stirring speed (120 rpm), percentage of inoculum (2% v/v), and dye concentration (20 and 10 mg L-1). In adsorption studies, it was showed that an acidic pH favors the adsorption of both dyes and the model of pseudo-second order describes best the phenomenon of adsorption. Finally, the germination index (GI), using Lactuca sativa seeds for the initial dyes solutions, was < 50%; demonstrating its high phytotoxic effect. When dye solutions were treated with viable biomass, the GI increased, leaving open the possibility to perform future research to determine if the aqueous solutions, post-treated with P. ostreatus, could be used in treatments that generate less toxic water which could be used in processes that do not require potable water.

Resumen Los colorantes trifenilmetánicos Verde Malaquita (MG) y Crystal Violeta (CV) son catiónicos y al ser vertidos se mezclan con aguas residuales domésticas, incrementando, entre otros, la demanda química y biológica de oxígeno; pudiendo causar toxicidad aguda en diferentes niveles tróficos. En este estudio se encontró que los parámetros pH (4,5 y 6,0), temperatura (25 y 30 °C), velocidad de agitación (120 r.p.m.), porcentaje de inóculo (2 % v/v) y concentración de colorante (20 y 10 mgL-1), presentaron un efecto significativo (p < 0.05) para favorecer la remoción (decoloración) de MG y CV, así como la actividad lacasa (54,76 ± 8,91 y 30,59 ± 2,89 UL-1 respectivamente) al utilizar biomasa viable de P. ostreatus. En los estudios de adsorción se evidenció que pH ácidos favorecen la adsorción de ambos colorantes y que el modelo de Pseudo-segundo orden describe mejor el fenómeno de quimisorción. Finalmente los índices de germinación (IG) empleando semillas de Lactuca sativa, para los colorantes iniciales fueron < 50 %; demostrando su efecto fitotóxico elevado. Cuando las soluciones de colorantes fueron tratadas con biomasa viable, el IG aumentó, dejando abierta la puerta para la realización de investigaciones futuras con la intensión de determinar si las soluciones acuosas, postratadas con P ostreatus, pueden ser utilizadas en tratamientos que generen aguas menos tóxicas y que estas puedan ser empleadas en otros procesos que no requieran agua potable.

Resumen Os corantes de tipo trifenilmetano Verde Malaquita (VM) e Cristal Violeta (CV) são corantes catiônicos e se misturam com águas residuais domésticas quando descartadas; aumentando, entre outros, as demandas químicas e biológicas de oxigênio, podendo causar toxicidade aguda em diferentes níveis tróficos. Promoveu-se a remoção (descoloração) de VM e CV, e atividade da lacase (54.8 ± 8.9 e 30.6 ± 2.9 UL-1 respectivamente) utilizando como parâmetros necessários para a biomassa viável de P. ostreatus como pH (4,5 e 6,0), temperatura (25 a 30 °C), velocidade de agitação (120 RPM), porcentagem de inócuo (2 % v/v), e concentração de corante (20 e 10 mg L-1). Em estudos de absorção, se demonstrou que um pH mais ácido favorece a absorção de ambos corantes e o modelo de pseudo-segunda ordem descreve melhor o fenômeno da absorção. Finalmente, o índice de germinação (IG), utilizando sementes de Lactuca sativa para as soluções iniciais dos corantes, foi < 50 %; demonstrando assim seu alto efeito fitotóxico. Quando as soluções de corante foram tratadas com a biomassa viável, o IG aumentou, deixando em aberto a possibilidade de realizar futuras investigações para determinar se as soluções aquosas, tratadas com P. ostreatus, poderiam ser utilizadas em tratamentos que gerem águas menos tóxicas, que poderia ser utilizada em processos que não requerem água potável.

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.

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosisisolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosisisolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

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.

Biofilm formation of nontypeable Haemophilus influenzae in vitro and morphology of biofilm under scanning electron microscope / 吉林大学学报(医学版)

Objective To investigate the biofilm (BF)formation rule of nontypeable Haemophilus influenzae (NTHi)in vitro, and to observe the internal structure of BF by scanning electron microscope (SEM). Methods NTHi ATCC49247 was investigated in the present study,Pseudomonas aeruginosa (PA)PAO1 was cultured as positive control,at the same time blank control group was set up.The BF of the bacteria were cultured and then collected on day 1,2,3,4,5,6,and 7.The BF formation was detected by crystal violet staining and plate counting and the structure of BF formed by ATCC49247 was observed under SEM on day 3.Results The plate colony counting of biofilm BF by ATCC49247 and PAO1 raised during first 3 d, and then declined to (0.823 6±0.007 5)×107 cfu·mL-1 and (0.942 6±0.019 9)×107cfu·mL-1 respectively on day 7. The differences between two groups were statistically significant on day 3,4,5,and 6 (P<0.05).The differences between different time points in the same bacteria group were statistically significant (P<0.05).The densities of BF formed by ATCC49247 and PAO1 raised during the first 3 d.The absorbances on 570 nm wavelength (A570 )in two groups were 2.717 4±0.017 2 and 2.885 3±0.039 0 ,respectively;and then the A570 values in two groups declined to 0.151 7±0.074 5 and 1.196 9±1.108 5,respectively on day 7;the differences between bacteria groups and blank control were statistically significant (P<0.05 );the differences between two bacteria groups were statistically significant on day 3,4,5,and 6 (P<0.05);the differences between different time points in the same bacteria group were statistically significant (P<0.05).On day 3,the obvious BF formed by ATCC49247 were observed under SEM.Conclusion BF could be formed by NTHi in vitro;crystal violet staining,plate colony counting and SEM could be taken as conventional methods to detect BF.

Crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dysplasia and oral squamous cell carcinoma.

Background: Assessment of mitotic figures (MFs) is routinely practiced as prognostic indicator in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), but identification of MFs poses a problem in terms of staining characteristics. Aim: To evaluate effectiveness of crystal violet stain for staining of MFs and its comparison with hematoxylin and eosin (H and E) stain. Materials and Methods: Study sample includes archival tissues embedded in paraffin blocks diagnosed as OED (n = 30) and OSCC (n = 30). The control group comprised of tissue specimen from oral mucosa of healthy volunteers (n = 30). Two serial sections of each tissue specimen were stained separately with H and E stain and 1% crystal violet stain. The stained sections were observed under microscope for identification and counting of MFs. Data obtained was statistically analyzed by using the Man-Whitney U test. Results: A significant increase in number of MFs was observed in OED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in crystal violet stained tissue sections when compared with H and E stain. Metaphase is the most commonly observed phase of mitosis in crystal violet stain when compared with H and E stain for all three groups. Conclusion: Crystal violet stain can be considered as selective stain for mitotic figures.

Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate

OBJECTIVE: In polymicrobial biofilms bacteria extensively interact with Candida species, but the interaction among the different species of the Candida is yet to be completely evaluated. In the present study, the difference in biofilm formation ability of clinical isolates of four species of Candida in both single-species and multi-species combinations on the surface of dental acrylic resin strips was evaluated. MATERIAL AND METHODS: The species of Candida, isolated from multiple species oral candidiasis of the neutropenic patients, were used for the experiment. Organisms were cultured on Sabouraud dextrose broth with 8 percent glucose (SDB). Biofilm production on the acrylic resins strips was determined by crystal violet assay. Student's t-test and ANOVA were used to compare in vitro biofilm formation for the individual species of Candida and its different multi-species combinations. RESULTS: In the present study, differences between the mean values of the biofilm-forming ability of individual species (C. glabrata>C. krusei>C. tropicalis>C. albicans) and in its multi-species' combinations (the highest for C. albicans with C. glabrata and the lowest for all the four species combination) were reported. CONCLUSIONS: The findings of this study showed that biofilm-forming ability was found greater for non-Candida albicans Candida species (NCAC) than for C. albicans species with intra-species variation. Presence of C. albicans in multi-species biofilms increased, whereas; C. tropicalis decreased the biofilm production with all other NCAC species.

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.

Two Cases of Cutaneous Protothecosis : Unique Histopathological Findings with Crystal Violet Staining and the Therapeutic Effect of Itraconazole

Cutaneous protothecosis sometimes poses diagnostic and therapeutic problems. Isolation of the causative organism may not be successful and spores may be mistaken for other skin diseases unless the characteristic sporangia are detected in tissue sections. Because there are few cases, the optimal therapy is still being debated. On Liebs crystal violet staining we found charateristic purplish dots in Prototheca spores; these correspond to the amyloplasts or dense bodies found under electron microscopy. The isolated organisms were inhibited in vitro by itraconazole, amphotericin B, ketoconazole, and amorolfine and we were able to successfully treat two patients with itraconazole.