Antimicrobial Properties of Aloe Vera Gel Extracts against Bacterial Isolates from Wound of Donkey

Safia Arbab1, Rehana Buriro1, Shams Uddin Bhugio1, Atta Hussain Shah2, Jamila Soomro3 Qudratullah kalwar4, Saqib Ali Fazilani1 and Waseem Ali Vistro5* 1Department of Veterinary Pharmacology, Sindh Agriculture University, Tando Jam 2Department of Animal Product Technologies, Sindh Agriculture University, Tando Jam 3Department of Animal Physiology and Biochemistry, Sindh Agriculture University, Tando Jam 4Department of Animal Reproduction, Sindh Agriculture University, Tando Jam 5Department of Anatomy and Histology. Sindh Agriculture University, Tando Jam Article Information Received 24 April 2019 Revised 30 June 2019 Accepted 06 July 2019 Available online 29 May 2020


INTRODUCTION
A loe vera (Aloe barbadensis miller) is a cactus like xerophytes plant and about 360 species of this plant have been identified so far. It has been named due to its therapeutic effectiveness among all tested species. It is cultivated in warm climatic areas of the world. Aloe vera (AV) has been known as "secret plant" because it contains photochemical, vitamins and nutrients (Maenthaisong et al., 2007). This plant has elongated, pointed and fleshy leaves which consists of two parts, an outer skin (green rind or latex) and an inner pulp which is colorless mucilaginous gel (Helal et al., 2003;Rodriguez et al., 2005).
Aloe vera has been used for various therapeutic purposes worldwide including Japan, Greece, and China (Rajasekaran et al., 2004;Parthipan, 2011). It has broad range of therapeutic effect and is used traditionally to treat variety of veterinary and human ailments (Blumenhal et al., 1998).

O n l i n e F i r s t A r t i c l e
healing, discoloration, foul odor and friable granulation tissue at the base of the wound and on disruption of these wounds, there is increased pain (Wells et al., 1988;Devrajani et al., 2000;Bowler et al., 2001). Several studies indicated that the aloe vera has a notable effect on wound healing. To the best of our knowledge, so far very little work has been conducted on Staphylococcus aureus, Escherichia coli, Shigella and Salmonella spp. isolated from donkey wounds. Therefore, considering the importance of the donkey as a domesticated member of the horse family, Equidae, which is mainly used for transportation in developing countries like Pakistan, this study has been designed to check the antibacterial effect of aloe vera against Escherichia coli, Staphylococcus aureus, Shigella and Salmonella spp. with the aim to provide the cheap source of medication.

Collection of donkey wound samples
A total of 50 wound samples were collected from veterinary clinics and nomadic animals in the vicinity of Tando jam and taken to the Department of Veterinary Pharmacology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tando Jam. All samples were collected under aseptic condition with the help of Tran'sweb tube.

Isolation and identification of pathogens
For detection of Staphylococcus aureus wound samples were sub cultured on the nutrient agar. Plates were observed for such as yellow golden colonies further confirmation was done by Gram's staining and biochemical reactions of the isolates.
For the detection of E. coli, wound samples were sub cultured on the MacConkey agar which is typical for E. coli growth. Plates were observed for bacterial growth which included pink colonies on MacConkey's agar and further confirmation was done by Gram's staining and biochemical test of the isolates.
For the detection of Salmonella/ Shigella spp., wound samples were sub cultured on the Salmonella/Shigella agar (SS Agar), which is specific for Salmonella and Shigella spp. Growth plates were observed for typical colonies such as for Shigella, as transparent or translucent colorless colonies. Shigella spp were observed whereas colonies of Salmonella spp may appear with or without black centers and further confirmation was done by Gram's staining and biochemical test of the isolates.

Extraction of gel from aloe vera leaves
The aloe vera plant leaves were collected from the local plant nursery, Hyderabad, washed with distilled water and was subjected to surface sterilization with 70% alcohol. The leaves were incised and gel was separated with the help of sterile knife. Further, the parenchymatous covering of the leaves was blended to make it homogenous and filtered with muslin cloth and sterilized in autoclave for 15 min (Agary et al., 2005). The filtered gel was used as stock solution (100% concentration).

Ethanol extraction method
For ethanol extraction, fresh aloe vera gel was dried in the oven at 80 ºC for 48 h and then powdered. Ten grams of this powder was dissolved in 100 ml of ethanol and left for 24 h. The contents were filtered through Whatman filter paper No.1 and filtrate was evaporated to dryness. This dried extract was further powdered and dissolved in 10 ml of distilled water and sterilized. The resultant solution was kept at 4 ºC, and used as stock solution (Chatterjee et al., 2013).

Disc preparation
The 6 mm (diameter) discs were prepared from Whatman No.1 filter paper. The discs were sterilized by autoclaving at 120 ºC for 15 min. After sterilization, the moisture containing discs were dried in hot air oven at 50 ºC for 1 h (Arunkumarand Muthuselvam, 2009). The prepared discs were kept under aseptic condition for further use.

Crude and ethanol extract of aloe vera gel sensitivity tests
To check the susceptibility of the isolated organisms against crude and ethanol extract of aloe vera at different concentrations i-e 30 µl and 60 µl, aloe vera gel was used to check susceptibility and resistance pattern of above four bacterial isolates.
The surface of Muller-Hinton agar was dried by incubating at 37 ºC for 30 min. The isolated colonies were selected and dispended in barium chloride. The sterile cotton swab was dipped in the bacterial solution and then rolled over the surface of the agar medium and evenly with bacterial suspension and placed in incubator for 30 minutes to get dried. Sterile Whatman NO.1 filter paper discs with absorbed aloe vera extracts, crude as well as ethanol extract at different concentrations i-e 30µl and 60µl, and blank/control (without any aloe vera) discs were placed on Muller Hinton agar plate with the help of sterile forceps and slightly pressed to make it adhere to the surface of the medium. The plates were closed, wrapped in polythene bag, kept inverted (medium up and disc downward) and incubated for 24 h at 37 ºC.

RESULTS AND DISCUSSION
The present study was aimed to compare the antibacterial activity of crude and ethanol extract of aloe vera at different concentrations against some common pathogenic bacterial isolated from donkey wounds.

Prevalence of bacteria isolated from wounds
A total of 50 wound samples were examined and all were recorded positive for different organisms. Out of 50 samples, 45, 35, 33 and 25 were found positive for Staphylococcus aureus, Escherichia coli, Shigella and Salmonella Spp, respectively (Table I). All organisms were identified on their morphological, cultural characteristics and staining reactions. Organisms were further confirmed by their biochemical reactions. The incidence recorded 90%, 70%, 66% and 50%, respectively. A similar type of study was done to isolate the bacterial organisms from wound samples of animals by various workers throughout world. However, the comparison of the present figures can be compared with the results of (Rind and Khan, 2000), who isolated and characterized bacterial species from surgical and non-surgical wounds located on skins and hides of domestic animals. Their results showed, Staphylococcus aureusas most prevalent organisms followed by Escherichia coli and Bacillus spp isolates from buffaloes, cattle, sheep and goat wounds. Another study showed that Staphylococcus are the common pathogen isolates from wounds of dogs (Kelly et al., 1993).
The result of the current study is in accordance with the previously reported studies that showed isolated different species of aerobic and anaerobic bacteria from skin wounds of cattle, sheep, goats and dogs and result indicated that among all organisms Staphylococcus aureus was a predominant species, whereas E. coli was on list after Staphylococcus. These studies showed that S. aureus was prevalent among all other isolates from other animals. (Kelly et al., 1993;Dewani et al., 2000). Different species of bacteria were isolated from cattle, sheep and goat and besides other animals Staphylococcus O n l i n e
The current study observed that S. aureus was the most common organism present in wound, as Staphylococcus is prevalent in the environment and form where it can get access to the wounds which are source of bacterial propagation. Similarly, the same species were observed the most dominant and common species in the wounds of camel (Devrajani et al., 2000).
Out of 50 wound samples taken from donkey wounds, 16 (32%) were contaminated with pure bacterial species, whereas 34 (68%) were found having contamination with mixed bacterial species (Table II). It is clear that mixed infection was common in wound sample of donkey. The data of other study showed that out of 46 different animals wound samples 35 were found to be contaminated with mixed organisms, while in 11 samples had single bacterial species. It is concluded that mixed infections are always common in the wound samples (Kamla et al., 2010). In another study in which the bacterial organisms were isolated from human wounds, polymicrobial infection was common and recorded in 59 (27·1%) of the samples (Bessa et al., 2015). It was reported that in wound samples from sheep and goats, more than 80% were with mixed infections, while 20% samples were contaminated with pure infection. It was clear from the results that mixed infections are common in wound samples of animals (Talan et al., 1989).

Susceptibility of bacteria to crude aloe vera extract
Two different concentrations i-e 30 µl and 60 µl of crude aloe vera gel were used to check the susceptibility of the isolated organisms. The results indicated that 30 µl crude extract of aloe vera did not show any result and no clear sensitivity zone was formed around the disc, whereas 60 µl of crude extract of aloe vera produced a clear zone and organisms showed quite sensitive reaction against crude extract (Table II).

Susceptibility of bacteria to ethanol extract of aloe vera
Using ethanol extracts showed the better results as compared to crude aloe vera at both concentrations i-e 30 and 60 µl. S. aureus was recorded as most susceptible among all isolates and recorded highly sensitive (15 to 22 mm), followed by E. coli (14 to 18 mm), Shigella (13 to 15 mm) and Salmonella (11 to 14 mm), respectively. Ethanol extract showed greater susceptibility against gram positive as well as gram negative isolates at 60 µl (Table II).
Study was conducted to check the antibacterial activity of ethanol and chloroform extract of aloe vera, ethanol extract of A. Vera which exhibited maximum inhibition against S. aureus. S. pneumonia and B. subtilis (JothiKarumari et al., 2014). A similar kind of investigation was carried out in which ethanol extract was used and it showed the greatest effect on S. aureus, Escherichia coli, Klebisella pneumoniae and shigella compared to pure Aloe extract. Methanol extract exhibited maximum antibacterial activity against S. aureus 24 mm, followed by B. cereus (Rudrangshu et al., 2015). The result of the current study are in accordance with the previously reported studies showed greater susceptibility against gram positive as well as gram negative isolates while, significantly least inhibition was with chloroform extract (Cera et al., 1980;Azghani et al., 1995). In another study ethanol extract of aloe vera exhibited greater antimicrobial activity besides methanol, chloroform and Di-chloro Methane extracts (Rudrangshu et al., 2015). The current study also showed agreement with the previous studies (Agarry et al., 2005). The result of current study are showed that ethanol extract showed 100% efficacy and susceptibility against gram positive as well as gram negative isolates same as antibiotics. Ethanol extract showed better result as compare to crude extract because ethanol is more polar than water. Apart from that, a lower antimicrobial action against Gram-negative has been recorded as compared to Gram-positive organism because of the presence of additional lipopolysaccharide layer in the Gram-positive bacteria .

CONCLUSION
It could be concluded from the present study that ethanol extract of aloe vera showed greater efficacy against bacterial isolates as compared to crude aloe vera extract.

Statement of conflict of interest
The authors have declared is no conflict of interests.

O n l i n e F i r s t A r t i c l e
Comparative Study of Antimicrobial Properties of Aloe Vera Gel Extracts