Evaluation of antibacterial activity from different Aloe vera (L.) Burm. F. extracts against Staphylococcus aureus strains

This study aimed to evaluate the susceptibility of Staphylococcus aureus strains isolated from the milk of cows with subclinical mastitis to Aloe vera in natura sap, ethanolic extract and in natura gel. The substances were tested using agar well diffusion and agar disk-diffusion methods. There was no inhibition of bacterial growth in the agar disk-diffusion method. On the other hand, the agar well diffusion method displayed in vitro growth inhibition of S. aureus in ethanolic extract and in natura sap of 91.66% (22/24) and 35.29% (6/17), respectively. Although the gel showed no effect against the strains tested in both methods, the agar well diffusion method was the most efficient to evaluate the antimicrobial activity of Aloe vera extracts.


Introduction
Aloe Vera (L.) Burm.f. belongs to the Asphodelaceae family, genus Aloe and has a viscous gel extracted through its mucilage (Castro & Ramos, 2002;The Linnean Society of London, 2016). This plant originated from the Mediterranean, Canary Island and Madeira Island (Lorenzi & Souza, 2008), and due to its adaptability, today this plant is found worldwide, including many Brazilian regions.
A. vera can be found in households as an ornamental plant, due to the fact that it can be easily cultivated, and is also used for therapeutic and cosmetic purposes (Sartório, 2000;Lorenzi & Souza, 2008;The Linnean Society of London, 2016). This species has a succulent leaf, concave on the top side, grey-green color, grows up to 30-60 centimeters and contains spikes on the leaves' edges (Grandi, 2014;The Linnean Society of London, 2016).
Transverse section of the leaves allows observation of two layers: the external (chlorenchyma), which is dark green, and the internal (parenchyma), which is translucid and mucilaginous from where the aloe gel, also known as mucilage, is obtained. In the zone where parenchyma and chlorenchyma connect, vascular bundles are located. The vascular bundles contain the aloetic cells or aloetic tissue, that produce a yellow viscous latex (sap) known as aloetic liquid or aloetic juice (Brasil, 2010).
Aloe gel is composed of 99,5% water as well as small amounts of hydro-and liposoluble vitamins, minerals, enzymes, polysaccharides, phenolic components, and organics acids. The therapeutic components attributed to aloe gel and its derivatives are related to the diversity of its components (Hamman, 2008). A. vera contains barbalodine, aloin, alloylodine, aloethine, aloeferon, picric acid, resins, mucilage, vitamins B and C as active components (Sartório, 2000). The plant has been used for medicinal purposes due to its bactericidal action, healing properties and antiinflammatory action (Habeeb et al., 2007;Semenoff Segundo et al., 2007;Semenoff et al., 2008;Ramos & Pimentel, 2011;Stanley et al., 2014). The antibacterial activity of this plant has been attributed to anthraquinones, which are natural components found in the A. vera, and present in the sap extracted from its leaves (Hamman, 2008;Sociedade Brasileira de Farmacognosia, 2015;The International Aloe Science Council, 2018).
In veterinary medicine, the use of A. vera is not well described as it is primarily a human medicine. Studies performed showed that aqueous extract could be used as post-milking teat dipping in goats as an alternative to glycerin iodine solution. This has been shown to keep the somatic cell count in the reference value and did not affect the milk composition. Furthermore, A. vera contains antimicrobial proprieties, which can be valuable to control causative agents of mastitis such as S. aureus, Streptococcus agalactiae, and E. coli (Amaro et al., 2011;Chikwanda et al., 2013).
Following the rise of antimicrobial resistance, there is a need to identify substances with antimicrobial properties (Silveira et al., 2009;Maddison et al., 2010). This study aims to compare the inhibitory potential from three A. vera extracts against Staphylococcus aureus strains isolated from milk of cows with subclinical mastitis.

Methods
For this study twenty-four (24) Staphylococcus aureus samples were previously isolated from the milk of cows with subclinical mastitis and stored in the Laboratory of Veterinary Microbiology Federal University of Mato Grosso (UFMT), Sinop extension (Koneman, 2008).
The A. vera extract was prepared in the Laboratory of Veterinary Microbiology, Federal University of Mato Grosso (UFMT), Sinop extension. From the A. vera leaves, three extracts were obtained: in natura gel, in natura sap and ethanolic extract from dehydrated leaf lamina adapting the methodology described by Bussmann et al., 2010. In order to obtain in natura sap, A. vera leaves were washed with distilled water and neutral detergent and then disinfected with 70% alcohol. After drying, the leaves were sectioned transversely and the sap was collected and then stored in a sterile plastic tube.
Three leaves of A. vera were collected and sent to the laboratory to obtain ethanolic extract. They were hygenized with neutral detergent and running taper water and then rinsed with distilled water. The chlorenchyma and parenchyma were separated using a sterile scalpel and spoon, as well as a plastic cutting board disinfected with 70% alcohol. Subsequently, the chlorenchyma was kept for 15 minutes in a solution containing 15 mL of 2.5% sodium hypochlorite and 1000 mL of distilled water. Then, the leaves were thoroughly rinsed with distilled water to remove the sanitizing solution. The chlorenchyma was deposited on a paper towel to dry. Afterward, the leaves were placed in a plastic tray and then kept in a drying oven at 45 ° C for 72 hours to dry and concentrate the active compounds.
During the drying period, the drying oven door was opened once a day for five minutes for spontaneous air exchange. After 72 hours, the material was stored in a sterile glass beaker, wrapped in aluminum foil and kept at room temperature. The dehydrated leaf lamina was manually fragmented and then macerated in a porcelain mortar. Five grams of dehydrated leaf lamina were weighed in a petri dish and placed in an Erlenmeyer flask, and 50 mL of 70% alcohol was added thereafter. This mixture was homogenized and the container sealed with aluminum Kraft paper. It was then wrapped in aluminum foil and kept at room temperature for ten days. After this period, the mixture was filtered in a paper filter n° 1 (Melitta®). For the evaporation of the remaining alcohol, the extract was placed in a water bath at 45 ° C for four days and then stored in plastic microtubes type Eppendorf® and then refrigerated.
The mucilage was extracted from the leaves according to Agarry et al. 2005, with the following adaptations: they were triturated with the aid of a sterile stainless-steel spoon and then sieved to promote the release of the gel which was stored in a glass beaker until use.
For the agar disk-diffusion method, the sap and gel were extracted 24 hours prior the experiment. In this case, microdisks of six millimeters in diameter were made using filter paper Nalgon® (reference 3550) and Melitta®, which were stored in plastic microtubes and sterilized before being immersed in the extracts (in natura sap, ethanolic extract, and in natura gel). With the aid of pre-flamed forceps, the disks were inserted into the microtubes containing the respective extracts and kept submerged for 24 hours. Petri dishes containing S. aureus were prepared to perform susceptibility test using A. vera extracts, in accordance to "Clinical and Laboratory Standards Institute" (CLSI, 2012). The observation of the growth inhibition zone was done following the CLSI recommendation (CLSI, 2012). In this procedure, five disks were used: vancomycin 30 micrograms (μg) as positive control (C+); sterile dry filter paper disk as negative control (C-); disk impregnated with A. vera ethanolic extract (E); disk impregnated with A. vera in natura gel (G), and disk impregnated with A. vera in natura sap (S).
For well diffusion method, the sap and gel extraction procedure of both was performed at the moment of use, in order to have minimal changes in its physicochemical properties. In this method, the approach described by Silveira et al. 2009, with adaptations was followed. Five 8 mm wells were made with a 1000 microliter sterile pipet tips on Mueller Hinton Agar plates, which were previously inoculated with a swab containing S. aureus samples corresponding to 0.5 degrees on the McFarland Nephelometric Scale. In these wells, 50 microliters of the following were added: 2% chlorhexidine soap (Higimax®) as a positive control (C +), sterile distilled water as negative control (C-), A. vera in natura gel (G), A. vera ethanolic extract (E), and A. vera in natura sap (S).
In both methods, the plates were incubated at 37 ° C for 24 hours and then the zone of growth inhibition was measured with a millimeter ruler.

Results and discussion
The susceptibility profile of 24 samples of S. aureus from cows with subclinical mastitis was analyzed through the agar well diffusion method in addition to analysis of nine samples applying the agar disk-diffusion method. For these analyses, two types of filter paper were used for both the methods.
In the agar disk-diffusion method, zone of growth inhibition of S. aureus strains was not observed in both filter papers when using in natura gel, ethanolic extract and in natura sap (Table 1).
Although the agar disk-diffusion method is widely used in laboratories to verify the sensitivity profile of bacteria against antimicrobials, in this study this technique could not demonstrate the real effectiveness from the extracts of A. vera tested. This fact may be related to the lack of solubility of substances in the growth medium, which may have compromised diffusibility of the tested product (Semenoff et al., 2008). In addition, the concentration of antimicrobial active component impregnated in the disk may be less than the minimum inhibitory concentration capable of inhibiting the growth of the tested strains. Thus, information obtained from other studies and the present results emphasize that the agar diskdiffusion method is not suitable to evaluate the inhibition of microbial growth in A. vera substrate.
In the agar well diffusion method, there was growth inhibition of 22 S. aureus strains (91.66%) in ethanolic extract. In the case of in natura gel, no inhibition of bacterial growth was observed. However, in natura sap was effective against 35.29 % (6/17) of the S. aureus strains tested (table 2).  Researchers observed a 4mm zone of growth inhibition in S. aureus colony using 100μl A. vera ethanolic extract in wells of 10 mm (Agarry et al., 2005). In another study, A. vera ethanolic extract was tested in concentrations of 100, 25 and 6.25 mg/mL against S. aureus strains, using 60μl of the extract deposited in 8 mm wells, and zone of growth inhibition of 20 ± 0.3 mm was noted in the extract concentration of 100 mg/mL (Moody et al., 2004). In the present study, 8 mm wells were used and 50μl ethanolic extract was added, with inhibition zone ranging from 11 mm to 25 mm in 22 S. aureus samples. The discrepancy evidenced in this study, when comparing the results obtained by other researchers, is due to the difference in the amount of extracts used or could also be associated to a lower concentration of active components in the plant, since several environmental factors may alter the production of secondary metabolites, altering its therapeutic properties (Globbo-Neto & Lopes, 2007). The results obtained in the present study reinforce the efficacy of the ethanolic extract against S. aureus strains and can be used in vivo to establish alternative therapies in the treatment of bovine mastitis caused by this bacterial species.
In the present investigation, when in natura sap was used through the agar well diffusion method, growth inhibition of 35.29% (6/17) was observed in the tested samples. The absence of efficacy of the in natura sap against other strains, however, can be explained by the fact that not all bacterial strains were tested with the same sap sample. This result occurred due to the necessity of extracting the sap at the moment of its use, with minimum change in the physical-chemical properties. Although the samples came from the same plant, concentrations of antimicrobial substances in the sap may have changed since environmental changes could alter the chemical components. In addition, dilution and lower production of the sap's active components can occur if there are a greater water availability and lack of solar radiation, respectively (Globbo-Neto & Lopes, 2007).
In a research conducted with S. aureus strains, growth inhibitions of 18 mm were observed using 100 μl of A. vera in natura gel in 10 mm wells (Agarry et al., 2005). However, other researchers tested the A. vera in natura gel using agar well diffusion method at concentrations of 100%, 25%, and 6.5% and did not found a zone of growth inhibition in any bacteria tested including S. aureusthe same result was observed in the present study (Moody et al., 2004). The divergence regarding the results obtained in the sensibility test to the A. vera gel can be related to the number of active compounds present in each plant that can be influenced by environmental factors, which alter the chemical composition of the therapeutic components (Reynolds & Dweck, 1999;Globbo-Neto & Lopes, 2007). In addition, when comparing the action of sap and gel in natura A. vera against S. aureus strains, the absence of antibacterial activity can be explained by the low concentration of anthraquinone in the gel (Hamman, 2008).
Although the results obtained in this research did not demonstrate the inhibition of the growth of S. aureus strains, A. vera gel was used in vivo to treat chronic ulcers in legs of humans infected with multiresistant microorganisms (Banu et al., 2012). In the study mentioned above, from the 30 patients in the gel treated group, only two presented positive cultures after 11 days of experiment, demonstrating in vivo antibacterial effect of A. vera. The response observed in the use of gel in vivo is due to immunomodulatory properties present in this substance, in which polysaccharides, mainly acenamamin, activate macrophages and increase the number of lymphocytes, boosting the immune system (Reynolds & Dweck, 1999;Ramos & Pimentel 2011). Furthermore, an experiment with aqueous extract of the A. vera gel had shown an antibacterial action similar to iodine glycerine, postulating that it can be used as post-dipping solution in goats (Amaro et al., 2011). Thus, due to the difference between the in vivo and in vitro actions of the A. vera gel, in vivo studies are required to verify the potential of this substance in the treatment of bovine mastitis caused by S. aureus.
Among the methods used, the agar well diffusion method was the most appropriate to test the antimicrobial properties of A. vera extracts. The results obtained are consistent with the results obtained by other researchers, who showed a greater sensitivity of the agar well diffusion method in comparison to the agar disk-diffusion method when determining the antibacterial activity of natural products from plants, fungi, and lichens (Valgas et al., 2007). The difference in the results obtained among both methods, as such in this study, is correlated to the precipitation of insoluble substances present in the extract on the filter paper used (Whatman® n ° 3) in agar disk-diffusion method , thus did not show diffusion of these substances in the agar as was the case in the diffusion well technique. Besides that, the difference between the results obtained in the techniques used may be related to amount of inhibitory substances in which they were used 50 μl of extract in the agar well diffusion method and approximately 10 μl in the agar disk-diffusion method. Thus, considering in vitro antimicrobial activity of A. vera on S. aureus strains isolated from milk of cows with subclinical mastitis, it is possible that this plant can be used an alternative treatment in this disease to reduce the presence of S. aureus in the dairy herd.

Conclusion
Aloe vera ethanolic extract and the sap presented in vitro antimicrobial activity against S. aureus strains isolated from milk of cows with subclinical mastitis. However, A. vera gel did not show growth inhibition of the strains tested. In addition, the agar well diffusion method was the most adequate to evaluate the antimicrobial potential of A. vera extracts.