Camilla Filippi dos Santos Alves

Antimicrobial activity of Amazonian oils against Paenibacillus species

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the primary bacterial pathogen of honeybee brood and the causative agent of American foulbrood disease (AFB). One of the feasible alternative treatments being used for their control of this disease is essential oils. In this study in vitro antimicrobial activity of Andiroba and Copaíba essential oils against Paenibacillus species, including P. larvae was evaluated. Minimal inhibitory concentration (MIC) in Mueller-Hinton broth by the microdilution method was assessed. Andiroba registered MIC values of 1.56-25%, while the MICs values obtained for Copaíba oil were of 1.56-12.5%. In order to determine the time-response effect of essential oils on P. larvae, this microorganism was exposed to the oils for up to 48 h. After 24 h treatment with Andiroba oil and after 48 h treatment with Copaíba oil no viable cells of P. larvae ATCC 9545 were observed. The possible toxic effect of essential oils were assessed by the spraying application method of the same concentrations of MICs. Bee mortality was evident only in treatment with Andiroba oil and the Copaíba oil shows no toxic effects after 10 days of observation. Taking together ours results showed for the first time that these oils presented a high activity against Paenibacillus species showing that Copaíba oil may be a candidate for the treatment or prevention of AFB.

Chlorhexidine activity against bacterial biofilms

BACKGROUND A biofilm is a complex microbiological ecosystem deposited on surfaces. Microorganisms in form of biofilms are of particular clinical concern because of the poor response to antimicrobial treatments. This study aimed to determine whether bacterial and fungal biofilms are able to resist the antimicrobial activity of chlorhexidine, a powerful antiseptic widely used in the hospital environment. METHODS Disk diffusion and susceptibility tests were conducted in accordance with Clinical and Laboratory Standards Institute standards for the determination of biofilm inhibitory concentration. Chlorhexidine was tested first at a minimum inhibitory concentration and then at higher concentrations when it was not able to destroy the biofilm. The plates were developed with a solution of 0.1% crystal violet, and readings were made at an optical density of 570 nm. RESULTS Chlorhexidine demonstrated excellent antimicrobial activity for most microorganisms tested in their free form, but was less effective against biofilms of Acinetobacter baumannii, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa. CONCLUSION This study confirms that microorganisms in biofilms have greater resistance to chlorhexidine, likely owing to the mechanisms of resistance conferred to the structure of biofilms.

Antimicrobial activity of Scutia buxifolia against the honeybee pathogen Paenibacillus larvae

The honeybee disease American foulbrood (AFB) is a serious problem since its causative agent (Paenibacillus larvae) has become increasingly resistant to conventional antibiotics. One of the feasible alternative treatments being used for control of this disease are plants extracts. The aim of the present work was to evaluate the effect of crude extract and fractions of Scutia buxifolia against six Paenibacillus species, including P. larvae, and its potential use for the control of AFB. In vitro activity of S. buxifolia samples against Paenibacillus species were evaluated by the disk diffusion and microdilution methods, and the minimal inhibitory concentration (MIC) were also determined. All Paenibacillus species were sensitive to crude extract and fractions of S. buxifolia. The dichloromethane (DC) fraction showed the better MIC (1.56 mg/mL), followed by ethyl acetate (EtAc) (6.25 mg/mL), n-butanol (BuOH) (25 mg/mL) and Crude extract (CE) (50 mg/mL). Toxic effect of S. buxifolia crude extracts and fractions against bees were also evaluated by the spraying application method of the same concentrations of MICs. The samples tested showed no toxic effects for the bees after 15 days of observation. These results are first time described for this species and showed that S. buxifolia presented a important activity against Paenibacillus species and proved to be a natural alternative for the prevention/control of AFB.

Antimicrobial activity of Amazon Astrocaryum aculeatum extracts and its association to oxidative metabolism

Several compounds present in fruits as polyphenols are able to kill or inhibit the growth of microorganisms. These proprieties are relevant mainly in tropical areas, as Amazonian region where infectious are highly prevalent. Therefore, this study investigated the antimicrobial activity of tucumã Amazonian fruit against 37 microorganisms. The potential role of oxidative metabolism imbalance was also studied as causal mechanism of antimicrobial activity. The results showed antibacterial effect of pulp and peel tucumã hydro-alcoholic extracts on three Gram-positive bacteria (Enterococcus faecalis, Bacillus cereus, Listeria monocytogenes) and antifungal effect against Candida albicans. The antimicrobial contribution of main chemical compounds (quercetin, rutin, β-carotene and gallic, caffeic and chlorogenic acids) found in tucumã extracts was also investigated showing an inhibitory effect depending of the organism mainly by quercetin in bacteria and rutin in C. albicans. Analysis of kinetic of DNA releasing in extracellular medium by fluorescence using DNA Pico Green assay(®) and reactive oxygen species production (ROS) showed potential oxidative imbalance contribution on tucumã inhibitory effect. In B. cereus and C. albicans this effect was clear since after 24h the ROS levels were higher when compared to negative control group. In conclusion, tucumã extracts present antimicrobial activity to four microorganisms that have large problems of drug resistance, and the possible mechanism of action of this Amazon fruit is related to REDOX imbalance.

In vitro growth-inhibitory effect of Brazilian plants extracts against Paenibacillus larvae and toxicity in bees

American foulbrood (AFB) is a serious worldwide spreading disease in bees caused by Paenibacillus larvae. Plants extracts are known to decrease or inhibit the growth of these bacteria. The purpose of this study was to evaluate the antimicrobial activity of Calendula. officinalis, Cariniana domestica, and Nasturtium officinale extracts against the P. larvae and to evaluate the toxicity of the extracts in bees. In vitro activity against P. larvae of the extracts was evaluated by micro dilution method and the minimal inhibitory concentrations (MICs) were also determined. The concentrations used in the toxicity test were established based on the MIC values and by the spraying application method. The P. larvae was susceptible to the evaluated crude extract of C. officinalis and N. officinale. To C. domestica, only the ethyl acetate (EtAc) fraction and n-butanol (BuOH) fractions had activity against P. larvae. Toxicity analysis in bees showed no toxicity for N. officinale crude extract and for C. domestica BuOH fraction during 15 days of treatment, however, some deaths of bees occurred during the first three days of treatment with C. officinalis and C. domestica EtAc fraction. The results with these species were firstly described and showed that N. officinale crude extract and C. domestica BuOH fraction both presented not toxic effects in the concentration tested by the spraying application method, and can be a useful alternative for treatment or prevention of AFB.

In vitro antimicrobial and antimycobacterial activity and HPLC–DAD screening of phenolics from Chenopodium ambrosioides L.

The main objective of this study was to demonstrate the antimicrobial potential of the crude extract and fractions of Chenopodium ambrosioides L., popularly known as Santa-Maria herb, against microorganisms of clinical interest by the microdilution technique, and also to show the chromatographic profile of the phenolic compounds in the species. The Phytochemical screening revealed the presence of cardiotonic, anthraquinone, alkaloids, tannins and flavonoids. The analysis by HPLC–DAD revealed the presence of rutin in the crude extract (12.5 ± 0.20 mg/g), ethyl acetate (16.5 ± 0.37 mg/g) and n-butanol (8.85 ± 0.11 mg/g), whereas quercetin and chrysin were quantified in chloroform fraction (1.95 ± 0.04 and 1.04 ± 0.01 mg/g), respectively. The most promising results were obtained with the ethyl acetate fraction, which inhibited a greater number of microorganisms and presented the lowest values of MIC against Staphylococcus aureus and Enterococcus faecalis (MIC = 0.42 mg/mL), Pseudomonas aeruginosa (MIC = 34.37 mg/mL), Paenibacillus apiarus (MIC = 4.29 mg/mL) and Paenibacillus thiaminolyticus (MIC = 4.29 mg/mL). Considering mycobacterial inhibition, the best results were obtained by chloroform fraction against M. tuberculosis, M. smegmatis, and M. avium (MIC ranging from 156.25 to 625 μg/mL). This study proves, in part, that the popular use of C. ambrosioides L. can be an effective and sustainable alternative for the prevention and treatment of diseases caused by various infectious agents.

Potential Use of Buddleja Thyrsoides for the Control and Prevention of American Foulbrood Disease in Honey Bees

Abstract Paenibacillus larvae is the causative agent of American Foulbrood (AFB ), a severe disease that affects the larvae of the honeybees. The use of plant extracts are considered to be an alternative way of controlling the disease. In this study, the in vitro antimicrobial activity of Buddleja thyrsoides Lam. against the Paenibacillus species, including P. larvae, was evaluated. In Mueller-Hinton broth, the minimal inhibitory concentration (MI C) was assessed using the microdilution method. All Paenibacillus species were sensitive to the crude extract and the fractions of B. thyrsoides. The ethyl acetate (EA ) fraction showed a better result with MI C values of 1.68 - 3.36 mg/mL, followed by butanolic (BU) (MI C = 2.18 - 6.54 mg/mL), dichloromethane (DCM) (7.40 - 14.80 mg/mL), and crude extract (CE) (7.51 - 16.90 mg/mL). The toxic effect of the CE and fractions of B. thyrsoides against bees were also evaluated using the spraying application method with the same concentrations of MI Cs. Bee mortality was evident in treatment with DCM fractions only, while CE, EA , and BU extracts showed no toxic effects after 15 days of observation. Furthermore, phenolic acids, tannins, and flavonoids were identified and quantified by highperformance liquid chromatography (HPLC), and may be partially responsible for the antimicrobial properties observed. These results show, for the first time, that B. thyrsoides might be a natural alternative for the prevention/control of AFB .

Oxygen tension during biofilm growth influences the efficacy antimicrobial agents

Objective To compare the antimicrobial efficacy of a 0.12% chlorhexidine (CHX) and herbal green tea (Camellia sinensis) solution on established biofilms formed at different oxygen tensions in an in situ model. Method Twenty-five dental students were eligible for the study. In situ devices with standardized enamel specimens (ES) facing the palatal and buccal sides were inserted in the mouths of volunteers for a 7 day period. No agent was applied during the first four days. From the fifth day onward, both agents were applied to the test ES group and no agent was applied to the control ES group. After 7 days the ES fragments were removed from the devices, sonicated, plated on agar, and incubated for 24 h at 37 °C to determine and quantify the colony forming units (CFUs). Result CHX had significantly higher efficacy compared to green tea on the buccal (1330 vs. 2170 CFU/µL) and palatal (2250 vs. 2520 CFU/µL) ES. In addition, intragroup comparisons showed significantly higher efficacy in buccal ES over palatal ES (1330 vs. 2250 CFU/µL for CHX and 2170 vs, 2520 CFU/µL for CV) for both solutions. Analysis of the ES controls showed significantly higher biofilm formation in palatal ES compared to buccal ES. Conclusion CHX has higher efficacy than green tea on 4-day biofilms. The efficacy of both agents was reduced for biofilms grown in a low oxygen tension environment. Therefore, the oxygen tension environment seems to influence the efficacy of the tested agents.

Antioxidant Activity and Phytochemical Composition of Solanum corymbiflorum Fractions (Leaves and Fruits)

The antioxidant activity and phenolic compounds of the chloroform (CHCl3), ethyl acetate (AcOEt) and n-butanol (n-BuOH) fractions from Solanum corymbiflorum leaves and fruits were evaluated. The AcOEt fraction of the leaves presented the highest content of total polyphenols (114.00 mg GAE/g) and the best antioxidant capacity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test (IC50 = 31.90 µg/mL). For the fruits, the same fraction exhibited the highest content of phenolics (99.77 mg GAE/g) and best results in the DPPH test (IC50 = 141.47 µg/mL). In relation to 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and thiobarbituric acid reactive substances (TBARS) assays, the CHCl3 fraction of leaves and fruits showed better results than the other samples analyzed. Besides of the phenolic compounds, the alkaloids contributed in the activity. Rutin, chlorogenic and caffeic acids quantified by HPLC are some of phenolic compounds responsible by this activity S. corymbiflorum can be a promising source of natural antioxidants. However, more in vivo studies are required to stimulate the consumption and its other potentialities.