In Vitro Study of Antibacterial Activity of Snake Fruit Extract against Extended Spectrum Beta Lactamase (ESBL) Escherichia coli

Snake fruit is a tropical fruit that is currently in great interest by the public. The This fruit is known to have high antioxidants so it can prevent an increase in cholesterol and other plasma lipids. Moreover, it can also prevent diarrhea. This study investigated the effective which one of the effective doses (25%, 50%, and 100%) from snake fruit extract against the ESBL-Escherichia coli (E. coli). This study was an experimental study with a post-test-only control group design in Microbiology Laboratory, Universitas Prima Indonesia. To investigate the antibacterial activity of snake fruit extract, this study was used disc diffusion methods which used five different groups including positive control (meropenem), negative control (DMSO), 100%, 50%, and 25% of snake fruit extract. The antibacterial assay showed that it required 100% snake fruit extract to inhibit the growth of ESBL-E. coli with average inhibition zone diameter of 7.33 ± 0.58 mm. The lower concentration extract did not show any antibacterial activity like the negative control group. The positive control group showed the most potent antibacterial activity with an average inhibition zone diameter of 36.33 ± 0.58 mm. Thus, it can be concluded that the snake fruit has antibacterial activity


INTRODUCTION
Humans have various activities, lifestyles, diets, and direct contact with the environment. Hence, humans are prone to infect by various bacteria. One of these infections, diarrhea, was caused by bacteria that is also the most common disease in some developing countries, one of these, Indonesia. Moreover, diarrhea has become a global infectious health problem that causes relatively high mortality and morbidity in the short term (WL Ragil & PS Dyah, 2017).
Diarrhea is a disease characterized by more than three defecations in a day occupied by loose stool consistency with or without mucous or blood. Various factors like poor environmental sanitation, inadequate water supplies, poverty, and lower levels of education may affect the morbidity and mortality of diarrhea in some developing countries (Rahman et al., 2016).
Data from the local (Padang City) Health Institute in 2014 showed that diarrhea was the most common infectious disease in Padang, accounting for 80,272 cases in Padan Timur District. Moreover, this disease was more common among men (39,975 people) than women (40.927 people). It may be due to poor water quality contaminated by various bacteria, Escherichia coli (Zikra et al., 2018). This disease was found at a high rate not only in Padang but also in Medan. Badan Pusat Statistik (BPS) in North Sumatera (2017) reported that diarrhea cases were 10,225 cases. On the other hand, Tarigan et al. (2020) reported that around 26,025 people from 2,229,408 in Medan suffered from diarrhea diseases (Tarigan et al., 2020).
Escherichia coli (E. coli) is a coliform group and a member of the Enterobacteriaceae family. This bacterium was a normal intestinal resident from Homoiterm, including humans; thus, it can become an indicator for faecal contamination as water pollution. When this bacterium is found in a certain concentration, it may cause diarrhoea, and in severe cases, it may cause death. Due to this reason, it can be used to evaluate the quality and safety of the water (Cho et al., 2018). Recently, some strains of E. coli can secrete Extended-Spectrum Beta-Lactamase (ESBL), and it was identified as a major multidrug-resistant bacteria in hospital and community settings worldwide. On the other hand, this ESBL-E. coli expand high medical costs and minimize antibiotic choices (Mahmud et al., 2020).
Many pharmacologic modalities have been developed to treat diarrhoea. However, these modalities were insufficient to increase the quality outcome because the chemical-based drug has some side effects and becomes nephrotoxic. So, it is recommended to use herbs for diarrhoea management. Since the ancient period, the herb as traditional medicine has been used in Indonesia. It was due to affordability, cheapness, and efficiency. Moreover, traditional medicine also has safer and fewer side effects than chemical-based drugs (Alviana, 2019).
Indonesia, a tropical region, has an advantage richead by various natural tropical herbs used as traditional medicine. For a long time, Indonesian people have been used snake fruit to treat diarrhoea. However, these people have not yet known the active compound responsible for treating diarrhoea. Therefore, some studies have been performed to explore the antimicrobial activity from the snake fruit extract against the ESBL-E. coli, which caused diarrhoea.
Hence, this study was aimed to look for the antimicrobial activity from various concentrations of snake fruit extract against ESBL-E. coli, which cause diarrhoea by the measurement diameter of inhibition zone as killed ESBL-E. coli zone.

Study Design
This study was an experimental study with post-test only group control design in Microbiology Laboratory, Universitas Prima Indonesia at September 2021-November 2021. All study procedure has been approved by the Health Research Ethics Committee from Universitas Prima Indonesia.

Snake fruit extract process
At the beginning, a kilogram of snake fruit was peeled and cut into small pieces and weighed as much as 500-600 grams. This snake fruit could not be dried because it was wet and easily oxidized. Hence, this snake fruit was put into oneliter methanol as the solvent in a glass jar for one week, and it was stirred regularly every 8 hours by flipping the jar three to five times. After that, it was filtered by a paper filter and evaporated by hotplate and water bath to form a concentrated form of snake fruit extract.

Characterization of ESBL-E. coli
Characterization of ESBL-E. coli was performed by some methods including gram staining, Inoculation on EMBA media, and biochemical test.

Gram staining
Gram staining was performed to evaluate the morphology of ESBL-E. coli bacteria. At the initial, the objectglass was clean with alcohol. After that, the clean object glass was dropped with distilled water and the ESBL-E. coli was suspended into the distilled water. Then, it was fixed into a thin layer by heating the object-glass over the Bunsen burner. Fixed ESBL-E. coli was drop by gram staining reagent included crystal violet as primer stain (30 seconds), Lugol solution as mordant (1 minute), 93% ethyl alcohol as decolorizing agent (15-25 seconds), and safranin as a counter stain (45 seconds). Between these drops, the object-glass was rinsed with distilled water. After that, it was observed under a microscope with 100x magnification, using immerse oil (Pelt et al., 2016).

Inoculation on EMBA media
Fifteen milliliters of EMBA (Eosin Methylene Blue Agar) was poured into a petri dish. An inoculum of ESBL-E. Coli was streaked into the surface of EMBA, and it was incubated for 24-48 hours at 37ºC. The metallic green colour indicated positive test results. (Sari et al., 2019) Biochemical assay The biochemical assay was performed to identify the biochemical reaction from the metabolism process of ESBL-E. coli as family of Enterobacteriaceae. This assay included  (Sari et al., 2019). g. Motility Test: An inoculum from ESBL-E. coli colony in the slant agar was inoculated into the middle part of SIM media in a tube reaction. After that, it was incubated for 24 hours at 37oC. When the media became fog-like turbidity, it indicated the bacteria movement (Pelt et al., 2016).

Serial dilution of extract
The amount of 5 grams of a concentrated form of snake fruit extract was dissolved into 5 ml DMSO by volumetric flask, and it was labeled as 100% snake fruit extract. After that, 2.5 ml of 100% snake fruit extract was pipetted by microliter-pippet to dissolve into 5 ml DMSO by volumetric, and it was labeled as 50% snake fruit extract. Finally, 2.5 ml of 50% snake fruit extract was dissolved in the same way to form a 25% snake fruit extract.

Bacteria Suspension
An inoculum of ESBL-E. coli was suspended into 3-5 ml of normal saline solution in the sterile test tube at LAFC. After that, it was homogenized and incubated at room temperature for five minutes (show a specific turbidity colour) and compared the turbidity of the suspension to 0.5 McFarland standard (total of colon number 1.5 x 10 8 CFU/mL).

Antibacterial Assay
ESBL-E. coli in bacterial suspension was obtained by sterile cotton swab to be spread into the surface of MHA media. After that, each petri dish was divided into four quadrants, and a paper disc was placed in each quadrant that represented a different group of treatment. Each group of treatments was repeated three times. These paper discs have been soaked into 100%, 50%, and 35% snake fruit extract. Meanwhile, the positive and negative control was used Meropenem and DMSO, respectively. Moreover, these Petri dishes were incubated for 24 hours; then, the form inhibition zones were measured by caliper in millimeter. Furthermore, ESBL-E. coli also underwent antibiotic resistance assay against some antibiotics including amikacin, amoxicillin, ampicillin, ampxicillin+clavulanic acid, Ceftriaxone, Cefotaxime, Cefepime, Chloramphenicol, Gentamycin, Levofloxacin, Meropenem, Piperacillin-Tazobactam, and Tetracycline by disc diffusion methods as described above. The formed inhibition zone in antibiotic resistance assay was measured and interpreted as resistance or sensitive based on the CLSI (Clinical and Laboratory Standards Institute) protocol.

Characteristic of ESBL-E. coli
At the beginning, the used ESBL-E. coli from slant agar was identified by some methods included gram staining, EMBA media culture, and biochemical test.

Gram staining
An inoculum of ESBL-E. Coli was stained by gram staining to ensure the ESBL-E. coli morphology under the light microscope. The microscopic view of these colony was shown in Figure 1. According to this gram stanning, this ESBL-E. coli colony was a gram-negative bacterium. When this colony was stained by the primer stain (crystal violet), this primer stain was decolorized by the ethyl alcohol because this colony does not have any peptidoglycan layer which keep the primer stain inside. Moreover, this colony was stain by counter-stain (safranin) that has red color and it caused the colony look red under the microscope (Pelt et al., 2016).

Bacterial Inoculation on EMBA Media
An inoculum of ESBL-E. coli was culture into EMBA Media by streaking it at the surface of EMBA media. The growth colony in the EMBA media was shown by Figure 2. EMBA media color becomes metallic green color after ESBL-E. coli was cultured that indicated lactose fermenter bacteria. EMBA media was used to differentiate Enterobacteriaceae, especially E. coli. EMBA media was selective and differential media, which was used to differentiate lactose fermenters from non-fermenter, while the E. coli was lactose fermenter bacteria. The color change of EMBA media was used to differentiate the fermentation activity from the colony. When the EMBA media become metallic green color, it indicates the colony is lactose fermenter bacteria. EMBA media contains some compounds included contains lactose, sucrose, peptone, eosin Y, and methylene blue; methylene blue inhibits the growth of gram-positive bacteria (Harijani et al., 2013;Juwita et al., 2014).

Biochemical Tests
ESBL-E. coli colony used in this study also underwent some biochemical tests, including IMViC test (Indole, Methyl Red, Voges Proskauer, Simmon Citrate, and Motility), Urease test, and TSIA test. This test observed color-changed media due to the reaction between the bacteria and some substrate in the media. The biochemical tests result was showed by the Figure 3.  Based on Table 1, Indole, TSIA, and motility test indicated this colony has E. coli characteristics. Although other biochemical tests did not show any E. coli characteristics, other tests included gram staining and EMBA media culture indicated the E. coli characteristics in this colony.
The indole test aimed to identify a bacteria's ability to degrade a tryptophan to indole . Fig 3 showed that the colony formed a red color due to the reaction between the Kovac reagent against the formed indole.
MR-VP (Methyl red-Voges-Proskauer) test aimed to identify a bacteria's ability to ferment glucose. MR-VP media contents peptone, glucose, and buffer phosphate. These tests qualitatively determined the degree of acidity in the media.
Fig 3 showed that both MR and VP tests indicated the negative results, which means that this colony could not produce acid properly from glucose fermentation reaction and was unable to react with alpha-naphthol and potassium hydroxide (KOH) (Hemraj et al., 2013).
Simmon Citrate Test used Simmon Citrate media to evaluate the ability of bacteria to convert nitrate to nitrite. Fig 3 showed that this colony did not have any ability to convert nitrate to nitrite because there was no color change in the Simmon citrate media (green) (Sari et al., 2019).
The urease test was aimed to evaluate the bacteria's ability to secrete urease enzyme. When the bacteria in this media had urease enzyme, this bacteria would break the carbon and nitrogen bond in the urea to form ammonia that changed the acid condition to alkaline condition in the urease broth media and colored the media into pink color. Fig 3 above did not show any color changes in urease broth media and it indicates that the colony did not have any urease enzymes (Wahyuni et al., 2018).
The TSIA test was done by using TSIA (Triple Sugar Iron Agar) media in determining carbohydrate fermentation and producing H2S gas. The TSIA test can distinguish bacteria based on their ability to ferment lactose, glucose, and sucrose as well as the ability of the bacteria to produce H2S gas. (Amelia et al., 2016) TSIA test used TSIA media to identify carbohydrate fermentation activity and formation of H2S gas. Fig 3 above showed that the colony formed a yellow color in both the slant and butt of the test tube, which means that the colony could ferment lactose, glucose and sucrose in TSIA media; it was labeled acid/ acid. However, this colony did not form any precipitations, which means that this colony did not form any hydrogen sulfide gas. Moreover, this colony showed the formation of oxygen in the TSIA media. This oxygen formed a gap between the TSIA agar and the butt tube.
The motility test is the most common method to differentiate each type of bacteria from the Enterobacteriaceae family. This test used semi-solid media or SIM (Sulfur, Indole, and Motility) media to identify the bacteria movement. Fig 3 showed that the media become fog-like turbidity along the puncture area of the inoculum loop, which indicated the movement of the bacteria from the inoculum colony. This turbidity was caused by reducing concentration in media from 1.5% to 0.4% when the bacteria was moving to keep its shape (Leboffe & Pierce, 2011

Antimicrobial Assay
Finally, some concentrations of snake fruit extract underwent an antimicrobil activity assay against ESBL-E. coli that has been characterized before, The result of antimicrobial assay was described in Table 3. Table 3 Antimicrobial Assay on Snake fruit Extract against ESBL-E. coli

Groups
Diameter of Inhibition Zone (mm), Mean ± SD 100 % snake fruit extract 7.33 ± 0.58 mm 50 % snake fruit extract 6.00 ± 0.00 mm 25 % snake fruit extract 6.00 ± 0.00 mm Positive control 36.33 ± 0.58 mm Negative control 6.00 ± 0.00 mm Based on Table 3, only the highest concentration of snake fruit extract showed antibacterial activity against the ESBL-E. coli. This antibacterial activity was shown by the average inhibition zone, which was 7.3 ± 0.58 mm. Meanwhile, the lower concentration snake fruit extract did not show any antibacterial activity because of an absence of formed inhibition zone like the negative control group, the average diameter inhibition zone of 6.00 mm was the diameter of paper disc without inhibition zone. However, the antibacterial activity of meropenem as a positive group still had the most potent antibacterial than other groups with an average inhibition zone of 36.3 ± 0.58 mm. Furthermore, the average diameter inhibition zone from 100% snake fruit extract indicated the moderate antibacterial activity against the ESBL-E. coli.

CONCLUSIONS
It can be concluded that the highest concentration of snake fruit extract (100%) had a moderate antibacterial activity for ESBL-E. coli. However, the inhibition zone formed in the control positive (Meropenem) was wider (36.33 ± 0.58 mm) than the highest concentration of snake fruit extract (7.33 ± 0.58 mm).