The Potential of Single Garlic Oil in Inhibiting The Growth and Damaging The Membrane of Pseudomonas aeruginosa Bacteria

Nosocomial infection caused by Pseudomonas aeruginosa bacteria is hard to be treated since the infection transmission is fast and it is resistance to the antibiotic. Antibiotic resistance in the bacteria can be treated with various natural ingredients from plants and one of the plants is single garlic. Single garlic contains organosulfur compounds in form of alliin, allicin, and ajoene. This research aimed to analyze the potential of single garlic oil (SGO) in inhibiting the growth and the damage of membrane of P. aeruginosa bacteria in in vitro with disc diffusion method by giving treatments of essential oil extract in concentration of 25 mg/ml, 50 mg/ml, 75 mg/ml and 100 mg/ml, and 1% dimethylsulfoxide (DMSO) as the negative control and Ceftazidime of 30 μg/ ml as the positive control. The damage of the bacterial cell membrane was observed with Scanning Electron Microscopy (SEM) with a magnification of 25000X. The result of the measurement of the zone of inhibition was analyzed using one-way ANOVA. The research results indicate that SGO could inhibit the growth of P. aeruginosa bacteria with strong criteria, which was in a concentration of 100 mg/ml. Article history: Received 26/02/2018 Received in revised form 13/10/2018 Accepted 04/11/2018


Introduction
Infection is a big issue in Indonesia (Priyanto, 2008). One of the causes of infectious diseases is a nosocomial infection (Inweregbu, 2005), which is an infection occurred in the hospital that sourced from health facilities. Gram-negative bacteria causing a nosocomial infection that often found is Pseudomonas aeruginosa (Refdanita, et al., 2004). A research by Khan et al. (2015) using 315 sample of patients in a hospital in China indicates that there were 24.9% of all the patient were experiencing nosocomial infection caused by Pseudomonas aeruginosa.
Nosocomial infection incident in developing countries has a fairly high prevalence including in Indonesia, which is about 6-16%.

Infection caused by Pseudomonas aeruginosa
bacteria is hard to be treated since the infection transmission is fast and it is resistance to the antibiotic. This resistance to an antibiotic is a threat to the treatment of infectious diseases in the world. Therefore, in order to overcome it, the development of a new and efficacious alternative treatment is needed that acts as an antibacterial that derives from natural ingredients of plants.
One of the natural ingredients that can be used as antibacterial is single garlic. Single garlic is garlic consisted of one bulb since it grows in an inappropriate environment (Untari, 2010). Compounds in single garlic are mostly containing sulfur responsible for taste, aroma, and pharmacological properties (Ellmore and Feldberg, 1994).
One of the most important organosulfur compounds in single garlic is essential oil that contains allicin, alliin, and DOI: 10.22146/jtbb.33498 ©2018 JTBB ajoene. Allicin is a sulfur component having antibacterial activities that work through a mechanism of inhibiting the formation of the bacterial cell membrane (Dusica, et al. 2011). Damage in bacterial cell membrane due to the interaction of antibacterial compounds can be observed with a Scanning Electron Microscope (Burt & Reinders, 2003).
There is limited information used single garlic as an antimicrobial.

Bacterial Identification
The identification of bacteria was conducted based on the method of Chaskes et al. (2015). The preparation of sterile water and bacterial colonies were put in object glass and air dried. The preparations were fixed above a Bunsen light and stained with violet crystal and safranin (the modification of Chakes et al., 2015). The positive result of observation with a magnification of 100x for Pseudomonas aeruginosa bacteria was red in color and in a rod shape.

Oxidase Test
Oxidase test was conducted by preparing the oxidase test strip by taking one ose (inoculating loop) of Pseudomonas aeruginosa bacteria and engraving it on the oxidase test strip. A positive result was indicated by oxidase strip that has a color of purple-blue.

Biochemical Test
The biochemical test was conducted through the oxidase test using Microbact System. One colony of bacteria that had been incubated for 24 hours at a temperature of 37 0 C was taken aseptically and put into 5 ml NaCl 0.9% and vortexed so the suspense was homogenous. The bacterial suspense of 100ml was piped and put into plate well, for lysine, ornithine and H 2 S wells, 1-2 drops of mineral oil were added. The pate was incubated at a temperature of 37 0 C for 18-24 hours. The reagent of Nitrate A and B of 2 drops were dropped into well 7; 2 drops of Indol Kovach reagent was dropped into well 8; 2 drops of VP I and VP II reagents were dropped into well 10, and 1 drop of TDA reagent was dropped into well 12. Carbohydrate fermentation test was conducted on plate 12B without any addition of reagents.
The positive result of fermentation was indicated by yellow color and it was negative if there was no change occurred in the color, which was it remained blue

The Preparation of Test Bacterial Culture
The culture of test bacteria was conducted by taking one ose of bacteria from Nutrient Agar (Oxoid) and inoculated in a liquid medium of Nutrient Broth (Oxoid), and vortexed to be homogenous. The culture was incubated at a temperature of 37 °C for 24 hour. The suspense of the test bacteria in a liquid medium of Nutrient Broth that had been incubated was measured using spectrophotometer in the wavelength of 625 nm until Optical Density (OD) of 0.1 equal to 10 8 CFU/ml was known (Murray, et al., 1999).

The Activity Test of Extract of Single Garlic Essential Oil on Pseudomonas aeruginosa
The in vitro test of bacterial inhibition was conducted using disc diffusion method. The dosage of SGO used for testing was 25mg/ml, 50mg/ml, 75mg/ml, and 100mg/ml respectively. The cultivation of test bacterial culture was conducted aseptically using sterile cotton swab containing suspense of Pseudomonas aeruginosa bacteria and scrapped it softly on the surface evenly. Paper discs dropped with each concentration of single garlic essential oil were put on the surface of the agar. Test cups were incubated in a reversed position in an incubator at a temperature of 37 0 C for 1x24 hour. The marking of the inhibition zone diameter was conducted using calipers. The effectiveness of active materials was determined by comparing the inhibition zone diameter to the standard value. The activities were grouped into two categories: strong (10-20 mm), very strong (>20-30 mm) (Greenwood, 1995 (1999) by which bacteria at the age of 1x24 hour and had been given a treatment were centrifuged in velocity of 3500 rpm for 15 minutes. Supernatant formed was disposed and sediment was washed with phosphate buffer. Sediment was added with 2% glutaraldehyde at pH of 7.3 and kept for 1 -2 hours. Next, the sediment was added with tannin acid of 2% and kept for 1-2 hours. Buffer cocodylate was added to the sediment and kept for 20 minutes. 1% osmium tetraoxide was added and kept for 1 hour and then 50% alcohol was added to the sediment and kept for 20 minutes. Respectively, 70%, 80%, and 95% alcohol were added and kept for 10 minutes and then absolute alcohol was added and kept for 20 minutes. Centrifuge was conducted at a velocity of 3500 rpm for 10 minutes. t-butanol was added to the sediment and kept for 20 minutes. The process was conducted two times.
Suspense was made in the butanol as well as thin smear of the suspense on the frozen cover slip. The cover slip was air dried and then sample was read using electron microscope with magnification of 25,000x.

Analysis
Data of antibacterial effect analyze using one way Anova and continued by Post Hoc Test of Gomes Howell. The damage of Pseudomonas aeruginosa bacteria could be observed through the morphological change on cell structure due to the application of single garlic essential oil containing antibacterial compounds. The observation was conducted using SEM with magnification of 25,000x. Figure 1 indicates that gram stain obtained rod-shaped bacterial cells, gram-negative was red in color and biochemical test was conducted using Microbact System Test.

Bacterial Identification and Biochemical Test
Based on the result of the identification test, it can be proved that bacteria used were indeed P. aeruginosa bacteria. aeruginosa with diameter of inhibition zone of 14 mm compare to those 1% DMSO (negative control) that resulted no inhibition zone. The result of inhibition zone in concentration of 25 mg/ml, 50 mg/ml, 75 mg/ml, and 100 mg/ml was, respectively, 7.9mm, 8.4mm, 9.3mm, and 11.1mm. The result of inhibition test of the extract of essential oil on P. aeruginosa can be seen in Figure 2.

Figure 2.
The diffusion method for measuring the inhibition zone of P. areuginosa bacteria. A: Single garlic oil inhibition zone of P. aeruginosa in various concentrations (1 = DMSO as a negative control; 2 = 25mg/ml, 3 = 50mg/ml, 4 = 75mg/ml, 5 = 100mg/ml). B: Positive controls using Ceftazidime 30µg/ml. Table 1 shows that the diameter of inhibition zone in each concentration of the extract of single garlic essential oil of 100 mg/ml, 75 mg/ml, 50 mg/ml, 25 mg/ml, and 0 mg/ml on the growth of P. aeruginosa bacteria had different values; however, the criteria of antibacterial strength in concentration of 100 mg/ml and positive control was the same, which was in the strong category since the diameter was in the range of 10-20 mm (Greenwood, 1995). It indicates that the extract of single garlic essential oil in concentration of 100 mg/ml contained strong antibacterial substances in inhibiting the growth of P. aeruginosa bacteria. The damage in the bacteria due to the application of single garlic essential oil can be observed morphologically through scanning electron microscope (Figure 3). The single garlic oil had an influence on the inhibition of the growth of P. aeruginosa bacteria since it contained more than 100 compounds of secondary metabolites that biologically useful (Jakobsen et al, 2012). The compounds were mostly contained sulfur (Zhang, 1999). There were three organosulfur compounds in single garlic with high concentration, which were alliin (41.1 g/mL), allicin (26.8 g/ mL), and ajoene that divided into E-ajoene (10.1g/mL) and Zajoene (25.1g/mL).
The working mechanism of allicin as an antibacterial is by inhibiting the synthesis of RNA and damaging the wall of bacterial cells (Durairaj, 2010). The inhibition of RNA synthesis was conducted by forming a strong bond in the bacterial enzyme, which was DNA Dependent RNA Polymerase thus it could inhibit the synthesis of bacterial RNA (Jawets et al., 2005). The inhibition of the cell wall was conducted by inhibiting the biosynthesis of peptidoglycan that would give strength and rigidity on the cell wall (Brooks et al, 2013). The compounds in Allium cepa and garlic effective used as antimicrobials in Staphylococcus aureus and Salmonella enteritidis bacteria (Benkeblia, 2004), 30 strains of mycobacteria, consisting of 17 species, were inhibited by various concentrations of garlic (Delaha & Garagusi, 1985). lysed cells are partially visible whilst others are visible in full. Davidson and Branen (1980) stated that antibacterial compounds could react with phospholipid components from gram-negative bacterial cells and it causes lysis on the cell wall. The lysed cell wall could cause the cell wall to be completely or partially removed and it is called as spheroplast.
In the surface of cell membrane of P. aeruginosa bacteria with the application of growing media of Nutrient Broth, the cell surface was intact and flat. It indicates that there was no contact between cell membrane and the antibacterial material thus the surface was visible morphologically.

Conclusions
Single garlic oil (SGO) is potential as an antimicrobial of Pseudomonas aeruginosa bacteria by inhibiting the growth and damaging cell membrane. SGO has the same potential as well as the antibiotic of Ceftazidime.