Antibiofilm Activity of Aloe barbadensis Miller Extract against

Staphylococcus aureus

Kanesti Ismirajna

and Irena Agustiningtyas

Medical Study Program, Faculty of Medicine, Universitas Islam Indonesia, Yogyakarta, Indonesia

Department of Microbiology, Faculty of Medicine, Universitas Islam Indonesia, Yogyakarta, Indonesia

Keywords: Biofilm, Staphylococcus aureus, Aloe barbadensis, microtiter plate, antibiofilm activity.

Abstract: Background: Aloe barbadensis Miller is a type of plant that can potentially inhibit the growth of bacteria

causing infections. Improper use of antibiotics can result in the occurrence of bacterial resistance caused by

the formation of biofilms on a living creature. The goal of this study was to investigate the antibiofilm activity

of Aloe barbadensis Miller against the biofilm produced by Staphylococcus aureus. This study is classified as

an experimental in vitro with the post-test only control group design. The Aloe barbadensis Miller rind

macerated with methanol solvent. The extract was divided into concentrations of 20%, 10%, 5%, 2.5%, 1.25%

and 0.63%, tested against biofilm formation by Staphylococcus aureus ATCC 25923. The antibiofilm activity

was tested by microtiter plate biofilm assay method with 3 replications on a 96 well round-bottom microplate.

Then observed by calculating the value of optical density using a microplate reader (λ = 620 nm). Extract

concentrations 20%, 10%, 5%, 2.5%, 1.25% and 0.63% showed a minimum percentage of inhibition of

biofilm concentration were -30.3%; 10.9%; 10.5%; 45.9%; 19.3% and 16.3% respectively. Statistical analysis

using One-Way ANOVA (p = 0.008) followed by post-hoc LSD which showed a concentration of 2.5% had

no significant difference (p > 0.05) with positive control using ciprofloxacin. Although the methanol extract

of Aloe barbadensis Miller rind showed inhibitory activity against S. aureus biofilms was equivalent to

positive control but had a Minimum Biofilm Inhibition Concentration (MBIC50) percentage <50%.

1 INTRODUCTION

Staphylococcus aureus is one of the most common

causes of bacterial infection in humans worldwide

(Singh et al., 2017; Chen et al., 2014; Tong, et al.,

2015). S. aureus can produce an extracellular polymer

matrix consisting of polysaccharides, air, nucleic

acids, proteins, and extracellular DNA (Archer et al.,

2011). This matrix ensures the survival of the biofilm

colony and protects it against the phagocytic activity

of macrophages, the host immune system,

temperature, and pH fluctuations (Leseigneur et al.,

2020). Antibiotic therapy, in general, will only kill

planktonic bacterial cells, while bacteria that are

tightly arranged in biofilm will remain alive so that

they can cause chronic infections that resistant to

antibiotics (Tong et al., 2015; Zaman et al., 2017).

The Indonesian Ministry of Health (2015) states that

from the 2013 WHO data, the death rate due to

https://orcid.org/0000- 0003-2009-1029

https://orcid.org/0000-0003-4870-9484

resistance is around 700 thousand people per year

(Kemenkes, 2015). It's estimated, by 2050 the death

rate could increase to 10 million per year. The

existence of biofilms has the opportunity to develop

drugs by utilizing the same bioactive plants (Abraham

et al.,2012). Also, Indonesia is mega biodiversity

which is rich in medicinal plants that have the

potential to be developed (Kusmana et al., 2015).

Aloe vera (Aloe barbadensis Miller), is currently

processed as food, drink, and medicine. However,

only part of the gel is used, while the skin of the

leaves becomes waste (Aryanti et a., 2013). The

active ingredients that have been identified in the

extract of the bark of the leaves of Aloe barbadensis

Miller include saponins, sterols, acemannan, and

anthraquinones which are toxic to bacterial cells

(Marimuthu et al., 2012; Benzidia et al., 2018;

Devaraj et al., 2011). Through this research,

hopefully that the incidence of S. aureus resistance

Ismirajna, K. and Agustiningtyas, I.

Antibioﬁlm Activity of Aloe barbadensis Miller Extract Against Staphylococcus aureus.

DOI: 10.5220/0010488200840090

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 84-90

ISBN: 978-989-758-499-2

can be overcome with antibiofilm as an alternative

therapy that can reduce the prevalence of infection.

The purpose of this study was to determine the

antibiofilm activity of the methanol extract of the

Aloe barbadensis Miller leaf bark against the biofilm

of Staphylococcus aureus ATCC 25923..

2 MATERIALS AND METHODS

2.1 Research Design

This type of research is experimental in vitro study

with a post-test only control group design. The effect

of Aloe barbadensis Miller in inhibiting biofilm of S.

aureus ATCC 25923 using the Microtiter Plate

Biofilm Assay method(14). S. aureus ATCC 25923

was bred from the Yogyakarta Health Center.

2.2 Time and Place of Research

The process of determining aloe vera plants is carried

out at the Plant Systematics Laboratory of the Faculty

of Biology, Gadjah Mada University. The plant

extraction process is carried out at the Integrated

Laboratory of the Faculty of Mathematics and Natural

Sciences, Islamic University of Indonesia, in the

division of Pharmaceutical Biology. Testing the

potential of Aloe barbadensis Miller leaf bark extract

against Staphylococcus aureus ATCC 25923 biofilm

was conducted at the Microbiology Laboratory of the

Faculty of Medicine, Islamic University of Indonesia.

The research was conducted in June-July 2020.

2.3 Research Tools and Materials

The tools used in this study include a round-bottom

microplate, microplate reader, analytical scale,

refrigerator, oven, autoclave, incubator, Laminar Air

Flow (LAF), bunsen, loop wire, beaker glass,

Erlenmeyer, petri dish, test tube, test tube rack,

cotton, micropipette, micro tip, dropper pipette,

mortar, pestle, vial, microscope, Staphylococcus

aureus ATCC 25923 bacteria, sterile distilled water,

physiological NaCl, Gram stain, McFarland solution,

nutrient agar (NA) media, nutrient broth (NB) media,

catalase, and coagulase reagents.

2.4 Time and Place of Research

The process of determining aloe vera plants is carried

out at the Plant Systematics Laboratory of the Faculty

of Biology, Gadjah Mada University. The plant

extraction process is carried out at the Integrated

Laboratory of the Faculty of Mathematics and Natural

Sciences, Islamic University of Indonesia, in the

division of Pharmaceutical Biology. Testing the

potential of Aloe barbadensis Miller leaf bark extract

against Staphylococcus aureus ATCC 25923 biofilm

was conducted at the Microbiology Laboratory of the

Faculty of Medicine, Islamic University of Indonesia.

The research was conducted in June-July 2020.

2.5 Preparation of Aloe Barbadensis

Miller Leaf Bark Extract and

Secondary Metabolites Test.

Aloe vera leaves are washed with tap water and then

peeled to separate the gel and the skin of the leaves.

The extraction process starts from chopping the

sample, drying the sample in a dry cabinet,

pollinating the alar miller, macerated dry Simplicia

with methanol solvent, filtering, evaporation, and

packaging then testing phytochemicals.

2.6 Purification and Characterization

of Test Bacteria

Purification and characterization of tested bacteria by

inoculating bacteria on NA media with streak plate

techniques, gram staining, catalase test, and catalase

test on glass objects (Kaiser et al., 2016).

2.7 Determination of the Minimum

Inhibitory Level (MIL) and

Minimum Killing Level (MKL).

The dilution series of extract concentrationstested

were 20%, 10%, 5%, 2.5%, 1.25% and 0.63%

compared to positive control (Ciprofloxacin 0.00125

mg/ml), negative control (bacterial suspension). and

media control which was carried out 3 times

replication (Bazargani et al., 2017)

2.8 The Biofilm Formation Test of

Staphylococcus aureus ATCC

25923

Two bacteria were used in the biofilm formation test,

S. aureus ATCC 25923 and S. epidermidis non-

biofilm as a negative control, tested 3 times. The

microplate was washed 3 times with sterile phosphate

buffer saline (PBS) (Teanpasian et al., 2016). Then

stained with 0.1% crystal violet and then rinsed 3-4

times with sterile distilled water (Lestari et al., 2017).

Then add 275 µL of 33% acetic acid to each well to

dissolve the crystal violet and incubate for 10-15

Antibioﬁlm Activity of Aloe barbadensis Miller Extract Against Staphylococcus aureus

minutes. Then OD (Optical Density) was measured

with a microplate reader with a wavelength of 620

nm. OD cut off is calculated by the formula:

OD cut off = O average negative control +

(3x standard deviation OD negative control)

(Singh et al., 2017)

2.9 The Antibiofilm Performance Test

Antibiofilm testing was done using the microdilution

method with a series of dilutions using a 96 well

round-bottom microplate. The group used was the

same as the test group in the antibacterial test but

incubated for 2 days. Washing was done using the

same as in the biofilm formation test. Then entered

into the formula MBIC50 [Inhibitory% = {((OD-

negative control test group) / (OD negative control)}

x 100}] (Singh et al., 2017).

2.10 Data Analysis Method

The data obtained were analyzed using SPSS. The

inhibition of S. aureus ATCC 25923 biofilm

formation was obtained from the Optical Density

(OD) value measured using a microplate reader. The

data were analyzed using Shapiro Wilk and Levene's

test to determine the distribution and homogeneity of

the data. Data that is normally distributed (p> 0.05) is

followed by the One-Way ANOVA test with testing

criteria based on probability, namely H0 is accepted

if p value> 0.05 and H0 is rejected if p-value <0.05.

Then the post hoc test of Least Significant Difference

(LSD) was carried out to determine the MBIC50

(Minimum Biofilm Inhibitory Concentration 50%).

2.11 Research Ethics

This research received permission from the Ethics

Committee for Medical and Health Research, Faculty

of Medicine, Islamic University of Indonesia with

number 4 / Ka.Kom.Et / 70 / KE / XII / 2019.

3 RESULTS

Based on the results of plant determination, it was

proven that the plant used in this study was Aloe vera

(L.) Burm.f. with the synonym Aloe barbadensis

Miller. Then from 900 grams of fresh material,

6.1223 grams of extract were obtained. The extract

was tested for secondary metabolites and it was found

that it contains active compounds of flavonoids,

alkaloids, saponins, and polyphenols. The results of

purification and characterization of S. aureus ATCC

25923 bacteria on nutrient media to obtain the results

of bacterial colonies that grow yellowish white. Gram

staining obtained the test bacteria showing a purple

colour, cocci-shaped, single or clustered irregularly

like grapes and without spores Figure 1. The catalase

and coagulase

test for bacteria each showed positive results

indicated by the presence of gas bubbles (O2) or foam

and the formation of clots / deposits Figure 2(19)(20).

Figure 1. Purification and characterization of S. aureus bacteria on NA media (left) and microscopic appearance at 100x

magnification (right).

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

Figure 2. Positive catalase test results, there are air bubbles (left); The coagulase test results are positive, there is clamping

(right).

The results of the antibacterial test on NA media

with Petri dish 1 to 4 were clear and no visible

colonies were growing. Meanwhile, in Petri dish 5

and 6, the growth of S. aureus was shown Figure 3.

The results of the S. aureus biofilm formation test

were formed purple biofilm rings on the good walls.

The absorbance / OD values of S. aureus and S.

epidermidis non-biofilm formation tests were

obtained in Table 1 and the antibiofilm test in Table

The absorbance value was analyzed using

statistical tests. The results of the distribution

normality test using the Shapiro-Wilk test, obtained a

p value> 0.05 in each test solution group, meaning

that the data were normally distributed. Levene's test

obtained a p-value <0.05 in each test solution group,

meaning that the data did not have the same variance.

Obtained normal data distribution and variance which

are not the same. One-Way ANOVA test obtained a

p-value of 0.008 which indicates that there is a

significant difference in the test solution group. In the

post-hoc LSD test (Table 3) it was found that some

groups showed significant differences with p-value

<0.05 (significant result) at a concentration of 20%

with a concentration of 2.5%, a concentration of 20%

with 1.25%, a concentration of 20 % with the positive

control, 20% concentration with media control, 10%

concentration with media control, 5% concentration

with media control, 1.25% concentration with media

control, 0.63% concentration with media control,

negative control with the positive control, control

media with a concentration of 5% and control media

with the negative control.

Figure 3. Antibacterial test result in A) microplate and B) nutrient agar.

Information

1. 20% Concentrate

2. 10% Concentrate

3. 5% Concentrate

4. 2,5% Concentrate

5. 1,25% Concentrate

6. 0,625% Concentrate

7. Positive Control (Ciprofloxacin)

8. Negative Control (bacterial suspension

9. Media Control

Antibioﬁlm Activity of Aloe barbadensis Miller Extract Against Staphylococcus aureus

Table 1. Biofilm Inhibition Optical Density Staphylococcus aureus ATCC 25923

Treatment

Absorbance Average %

Inhibition

lication 1 Re

lication 2 Re

lication 3

Extract 20% 0.5495 0.6840 1.6010 0.9448 -30.35

Extract 10% 0.6053 0.5537 0.7781 0.6457 10.92

Extract 5% 0.7637 0.6089 0.5734 0.6487 10.51

Extract 2.5% 0.4006 0.3621 0.4122 0.3916 45.97

Extract 1.25% 0.4969 0.5847 0.6720 0.5845 19.36

Extract 0.625% 0.5231 0.7644 0.5318 0.6064 16.33

Positive Control 0.2443 0.2894 0.3663 0.30 58.61

Negative Control 0.6326 0.8336 0.7083 0.7248

Media Control 0.1214 0.1721 0.1502 0.1479

Table 2.Post-hoc analysis result LSD type

E20% E10% E5% E2.5% E1.25% E0.625% KP KN KM

E20% - 0.097 0.100 0.005 0.049 0.063 0.001 0.214 0.000

E10% 0.097 - 0.986 0.154 0.724 0.821 0.058 0.648 0.009

E5% 0.100 0.198 - 0.149 0.712 0.807 0.056 0.661 0.009

E2.5% 0.005 0.154 0.149 - 0.273 0.224 0.598 0.067 0.170

E1.25% 0.049 0.724 0.712 0.273 - 0.899 0.113 0.422 0.020

E0.625% 0.063 0.821 0.807 0.224 0.899 - 0.089 0.497 0.015

KP 0.001 0.058 0.056 0.598 0.113 0.089 - 0.023 0.385

KN 0.214 0.648 0.661 0.067 0.422 0.497 0.023 - 0.003

KM 0.000 0.009 0.009 0.170 0.020 0.015 0.385 0.003 -

Information: E = extract consentration; KP = Positive Control; KN = Negative Control; KM = Media Control.

4 DISCUSSION

Table 1 shows the ODcut value calculated from the

negative control (S. epidermidis non-biofilm) of

0.1341. The average S. aureus biofilm with a value of

0.3991 is a value that is between 2xODcut and

4xODcut. So it can be concluded that the S. aureus

bacteria used in this study were moderate biofilm-

former (Singh et al., 2020).

Based on the microplate reader result in Table 2.

Higher OD score indicates the increasing survived

biofilm microorganism amount (Locke et al., 2012).

From the percentage inhibition formula, it was found

that the higher the absorbance value / OD of the test

solution, the lower the percentage value of inhibition

(inversely proportional). The absorbance/OD value of

Aloe vera leaf extract which was the best in inhibiting

the growth of S. aureus biofilm was produced at a

concentration of 2.5%, namely 0.3918 and had an

inhibition percentage of 45.9% (Teanpaisan et al.,

2017). When compared with a positive control

containing Ciprofloxacin and bacterial suspension, it

had an absorbance / OD value of 0.3000 and an

inhibition percentage of 58.6%. This shows that the

percentage of inhibition in the positive control is

higher than the series test solution for the dilution of

the extract concentration. The percentage value of

inhibition by the series dilution test solution with the

concentration of 2.5% extract did not reach the

MBIC50 requirement, called the minimum inhibitory

concentration of biofilm of 50% (Pirbalouti et al,

2010; Pratiwi et al., 2015).

The results of statistical analysis, the post hoc

LSD test showed that the extract concentration of

2.5% had the best value based on the percentage of

MBIC50 compared to the concentration of other

extracts. The extract concentration of 2.5% had a

significance value of 0.113 against the positive

control group, the antibiotic Ciprofloxacin. This

indicates a meaningless relationship. Thus, the extract

concentration of 2.5% had the same ability as the

positive control in inhibiting S. aureus biofilm. So, it

can be concluded that although the methanol extract

of aloe vera leaf bark (Aloe barbadensis Miller) has

statistically shown inhibitory activity against S.

aureus biofilms is equivalent to that of positive

controls, it has a Minimum Biofilm Inhibition

Concentration (MBIC50) percentage <50%.

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

Several factors can cause the methanol extract of

the aloe leaf bark (Aloe barbadensis Miller) to have

MBIC50 <50%. First, the methanol solvent was used

in the extraction of the test plants. The methanol has

a polar group that is stronger than its nonpolar group,

this can be seen from the chemical structure of

methanol which contains a hydroxyl group (polar)

and a carbon group (nonpolar). The methanol can

extract a greater amount of phytochemical

compounds so that it can extract more bioactive

components that have higher polarity properties

(Seidel et al., 2012).

According to Seidel (2012), the high polarity

index in methanol solvents can extract secondary

metabolites that have polar properties such as

flavonoids glycosides, tannins, and some alkaloids.

This solvent is also effective for extracting phenolic

compounds with low molecular weight and moderate

polarity levels (Lin et al., 2009) flavonoid aglycones

(Dehkarghanian et al., 2010) anthocyanins,

terpenoids, saponins, flavones, and polyphenol

compounds. Non-polar solvents such as n-hexane

which has a zero polarity index are effective in

dissolving lipophilic compounds, such as alcanas,

waxes, colour pigments, sterols, some terpenoids, and

alkaloids (Romadanu et al., 2014).

Lawrence et al, shows that the ethanol extract of

Aloe vera gel has a wider diameter of inhibition zone

against S. aureus bacteria than the methanol extract

and acetone extract of Aloe vera gel. It is suspected

that the methanol extract of Aloe barbadensis Miller

leaf bark used in this study has not been able to

dissolve other secondary compounds/metabolites that

are lipophilic (Lawrance et al., 2009).

Also, considering the effect produced by the

methanol extract of the Aloe barbadensis Miller is

still the result of the combined work of various

secondary compounds/metabolites that can affect the

mechanism of action of one compound with another.

So for further development, it is necessary to isolate

pure compounds for antibiofilm activity tests.

5 CONCLUSION

The methanol extract of aloe vera leaf bark (Aloe

barbadensis Miller) has an inhibitory activity against

S. aureus biofilms equivalent to positive controls but

has a Minimum Biofilm Inhibition Concentration

(MBIC50) <50%.

ACKNOWLEDGMENTS

Thank you to the author, to the dean of the UII

Medical Faculty who has permitted to research at the

Microbiology Laboratory.

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(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )