The Activities of Streptomyces W-5A as Antibacterial and

Antibiofilm towards Methicillin-resistant Staphylococcus aureus 2983

Annisa Permata Dinda

, Ari Asnani

and Dwi Utami Anjarwati

Department of Chemistry, Faculty of Mathematics and Sciences, Universitas Jenderal Soedirman, Purwokerto, Indonesia

Department of Microbiology, Faculty of Medicine, Universitas Jenderal Soedirman, Purwokerto, Indonesia

Keywords: MRSA, Streptomyces, antibiofilm, antibacterial

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) causes nosocomial infection worldwide. MRSA can

defend itself by forming a biofilm layer, thereby increasing the virulence factor. WHO categorizes MRSA as

high risk on the Priority Pathogen List for searching for new antibiotics. Recently, we have reported the

potency of Streptomyces W-5A as anti-MRSA based on qualitative screening. Thus, this research aimed to

analyze the potency of Streptomyces W-5A as antibacterial and antibiofilm towards MRSA 2983. Cultivation

of Streptomyces W-5A used Starch Casein Nitrate (SCN) agar, and anti-MRSA extract production used SCN

broth. Samples were taken at incubation times of 0, 3, 6, 9, 12, and 15 days. Each sample was tested for

antibacterial activity with the Kirby-Bauer method, whereas inhibition of biofilm formation and biofilm

degradation with microtiter method. The results showed that the optimum antibacterial activity was achieved

after nine days of incubation with an inhibition zone of 9.589 ± 0.521. The optimum % inhibition of biofilm

formation was 77.806 ± 13.595% after 12 days of incubation. The optimum % degradation of biofilm was

80.465 ± 7.586% after nine days of incubation. These findings suggest that Streptomyces W-5A has the

potency to produce antibacterial and antibiofilm compounds against MRSA 2983.

1 INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA)

is an S. aureus bacterium resistant to methicillin and

several other beta-lactam antibiotics (Boucher &

Corey, 2008). MRSA is an infection-causing

bacterium that can defend itself by forming a

protective layer called a biofilm. S. aureus biofilms

develop rapidly and form colonies on moist and

nutrient-rich surfaces (Tarver, 2009). The ability to

form biofilms is one of S. aureus' virulence factors,

which causes antibiotic resistance to bacteria (Høiby

et al., 2010; Lee et al., 2013).

Infection by microbes is estimated to be 80%

related to the formation of biofilms, which contribute

to the nature of antibiotic resistance (Archer et al.,

2011). Biofilms consist of microbial cells and a

matrix of extracellular polymeric substance (EPS). As

much as 50-90% of the EPS matrix's main ingredients

are organic carbon consisting of polysaccharides,

https://orcid.org/0000-0002-1992-6685

https://orcid.org/0000-0002-8569-2565

https://orcid.org/0000-0001-8394-2543

proteins, nucleic acids, lipids, phospholipids, and

humic substances, and 15% are bacterial cells

(Deshpande & Joshi, 2011). These biofilms cause

antibiotics cannot reach the target, bacterial cells;

hence the bacteria that cause infection cannot be

inhibited or destroyed using antibiotics. Therefore,

exploration of antibiofilm compounds becomes

necessary. The antibiofilm compounds are expected

to inhibit biofilm formation and degrade biofilms

(Konai & Haldar, 2017). The inhibitory activity of

biofilms occurs because these compounds have

antibacterial activity, while biofilms' degradation

activity occurs because they can depolymerize

complex compounds in the biofilm matrix.

Actinobacteria, particularly the genus

Streptomyces, has been reported to produce bioactive

compounds with anti-MRSA activity. Streptomyces

albus, granaticin B from Streptomyces violaceoruber,

and streptorubin B from Streptomyces sp. MC11204

has been reported to inhibit biofilm formation and

Dinda, A., Asnani, A. and Anjarwati, D.

The Activities of Streptomyces W-5A as Antibacterial and Antibioﬁlm towards Methicillin-resistant Staphylococcus aureus 2983.

DOI: 10.5220/0010488601090115

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 109-115

ISBN: 978-989-758-499-2

109

damage the S. aureus biofilm (Oja et al., 2015; Suzuki

et al., 2015). Bhakyashree and Krishnan (2018)

reported that Streptomyces sp. Strain VITBKA3

showed anti-MRSA activity. Balasubramanian et al.

(2017) also reported that ethyl acetate extract from

Streptomyces sp. strain SBT343 could reduce biofilm

formation of several Staphylococcal species,

including MRSA USA300.

Recently, Asnani et al. (2020) have reported the

potency of Streptomyces W-5A as anti-MRSA based

on qualitative screening. Streptomyces W-5A was

isolated from mangrove areas in Segara Anakan

Cilacap, a source of indigenous marine actinobacteria

(Asnani et al., 2016). Following that report, this

research aimed to analyze the potency of W-5A

isolates as antibacterial and antibiofilm towards

MRSA 2983.

2 MATERIALS AND METHODS

The research was conducted from January to

September 2020 in the Biochemistry Laboratory and

Research Laboratory in UNSOED, Purwokerto.

Streptomyces W-5A was isolated from mangrove

sediment in Segara Anakan Cilacap. The MRSA 2983

is known as a bacterium capable of producing biofilm

matrix. It was isolated from a clinical specimen from

the pus of female patient in Prof. Dr Margono

Soekarjo Hospital, Banyumas regency, Indonesia.

2.1 Cultivation of Isolate W-5A

Streptomyces W-5A was cultivated using continuous

streak on Starch Casein Nitrate (SCN) following the

procedure described by Asnani et al. (2016). Starch

Casein Nitrate (SCN) agar [starch, casein, KNO

, MgSO

.7H

O, NaCl, FeSO

.7H

O, agar]

was added 1 µL nystatin for every 10 mL of SCN

medium. The cultures were incubated for seven days

at room temperature.

2.2 Production of Anti-MRSA Extract

Production of anti-MRSA extract used SCN broth

following the procedure described by Asnani et al.

(2020). A total of 10 plugs (6 mm diameter) of

Streptomyces W-5A were inoculated into 100 mL of

SCN broth. The culture was incubated at 90 rpm and

room temperature until it reached the exponential

phase for inoculum. Next, 10% of the inoculum was

inoculated into a new SCN broth to produce anti-

MRSA compounds. The cultures were incubated

using an orbital shaker at a speed of 90 rpm. Samples

were taken at incubation times of 0, 3, 6, 9, 12, and

15 days. Each sample was separated by centrifugation

at 4.000 rpm for 10 minutes at 4

C, then filtered to

obtain the crude extract. Each extract was tested for

antibacterial activity, inhibition of biofilm formation,

and biofilm degradation.

2.3 Antibacterial Test

The extract's antibacterial activity was evaluated

using the disc paper diffusion method on the Mueller

Hinton Agar (MHA) medium following CLSI (2019).

A total of 30 μL of the extract was added to disc paper

(6 mm), then placed on the MHA medium that had

been inoculated by MRSA 2984 using the spread

plate method. The test culture was incubated at 37

for 24 hours. A clear zone around the disc paper

indicated a positive result of antibacterial activity

against MRSA. Hence, the parameter observed was

the diameter of the inhibition zone.

2.4 Biofilm Formation Inhibition Test

The potency of the extract to inhibit biofilm

formation was tested using the microtiter plate

method described by Suzuki et al. (2015) with

modification. The MRSA 2983 was inoculated in

Brain Heart Infusion (BHI) medium with 1% glucose

(BHI-Glu) and incubated at 37

C for 24 hours. Then,

the culture was adjusted for its turbidity level using a

0.5 McFarland standard and diluted in BHI-Glu with

a ratio of 1:100. The inhibition of biofilm formation

was carried out by adding a total of 10 µL of diluted

MRSA and 100 µL of extract to the microplate and

incubated at 37

C for 24 hours. After incubation,

planktonic cells were removed carefully, washed

twice with PBS, stained with 0.1% crystal violet

solution, and incubated for 30 minutes. Then, the

microplate was washed with water to remove excess

crystal violet and dried. HCl in isopropanol (1:20)

was then added, and the optical density (OD) value

was measured using a microplate reader at a

wavelength of 595 nm. The percentage of biofilm

formation inhibition was calculated using the

following formula (Pratiwi et al., 2015).

%Inhibition =













 100% (1)

2.5 Biofilm Degradation Test

The potency of extract to degrade biofilms was tested

using the microtiter plate method described by Suzuki

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 )

110

et al. (2015) with modification. The MRSA 2983 was

prepared as the previous procedure. The biofilm

degradation test was carried out by inoculating

diluted MRSA in each microplate well and incubated

at 37

C 24 hours. After incubation, planktonic MRSA

cells were carefully removed, then the extract was

added into each well. The mixtures were incubated at

C for 24 hours, exposing the extract to biofilm

formation at the bottom of the well. After incubation,

the microplate was treated similarly to the previous

procedure, and the OD value was measured at 595

nm. The percentage of biofilm degradation was be

calculated using the following formula (Pratiwi et al.,

2015).

%Degradation =













 100% (2)

2.6 Data Analysis

The research data were the diameter of the inhibition

zone, %inhibition, and %degradation. All data were

analyzed using one-factor analysis of variance

(ANOVA) at a 95% confidence level. If the ANOVA

analysis results have a significant effect, it continued

with the Duncan test.

3 RESULTS

3.1 Antibacterial Activity

The extract was tested for antibacterial activity using

the paper disc diffusion method. This method is

expected to determine the sensitivity of a microbe to

various antibiotics. The experimental parameters

were the presence or absence of a clear zone formed

around the disc paper, which showed MRSA's growth

inhibition. The research result indicated that the

extract has antibacterial activity (Figure 1). The

antibacterial activity of Streptomyces W-5A's extract

increased from day 0 and reached the highest activity

on day 9th with the diameter of the inhibition zone

was 9.589 ± 0.521 mm. The zone of inhibition

showed potent inhibition of extract against MRSA

298.

The one-way ANOVA test analysis results

showed a significant effect (Sig. <0.05) between the

incubation time and the inhibition zone diameter

(Table 1). Further tests using the Duncan test showed

that the 9-day incubation time had an antibacterial

activity significantly different from other incubation

times. This finding indicated that the optimum

incubation time for producing antibacterial

compounds was nine days.

Figure 1: Antibacterial activity of Streptomyces W-5A

extract against MRSA 3983.

3.2 Inhibition of Biofilm Formation

The inhibition of biofilm formation aimed to

determine the potency of extract to inhibit MRSA

biofilms' formation. Compounds with antibacterial

activity against microorganisms that form biofilms

can be used to inhibit biofilm formation (Memariani

et al., 2019). The tests used a microplate and BHI

medium with glucose. BHI medium is often used to

grow Gram-positive and Gram-negative bacteria,

whereas glucose to the BHI medium aimed to induce

MRSA biofilms formation (You et al., 2014).

Figure 2: The inhibition formation of biofilm MRSA 2983

by Streptomyces W-5A extract.

The research result is presented in Figure 2. The

extract inhibited the formation of biofilm MRSA

2983. The percentage inhibition of biofilm formation

by the extract increased from 0 days until it reached

the optimum incubation time, 12 days, with a per cent

inhibition of 77.806% ± 13.595%.

03691215

Diameter of inhibition zone

(mm)

Incubation (Days)

100

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% Inhibition

Incubation (Days)

The Activities of Streptomyces W-5A as Antibacterial and Antibioﬁlm towards Methicillin-resistant Staphylococcus aureus 2983

111

Table 1: Results of one-way ANOVA for antibacterial and antibiofilm activities

Sum of Squares df Mean Square F

Sig.

(1) Antibacterial Activit

Between Groups 17.209 5 3.442 6.307 0.004

Within Grou

s 6.549 12 0.546

Total 23.758 17

(

)

Inhibition of biofilm formation

Between Groups 3766.922 5 753.384 2.856 0.630

Within Groups 3165.493 12 263.791

Total 6932.415 17

(

)

Biofilm de

radation

Between Grou

s 4655.138 5 931.028 3.084 0.051

Within Groups 3622.848 12 301.904

Total 8277.986 17

Significant value = 0.05

After it reached the optimum incubation time, the rate

of inhibition of biofilm formation decreased.

The analysis results with the one-way ANOVA

test showed no significant effect (Sig. <0.05) between

the incubation time treatment on the percentage of

inhibition of biofilm formation (Table 1). However,

50% inhibition of biofilm formation was achieved

after six days of incubation and reached a maximum

of 12 days of incubation.

3.3 Biofilm Degradation

The research result indicated that the extract could

degrade the biofilm of MRSA 2983 (Figure 3). The

percentage of biofilm degradation increased from day

0 until it reached the optimum incubation time, which

was the 9th day, with the percentage of biofilm

degradation was 80.465% ± 7.586%. After it reached

the optimum incubation time, the rate of biofilm

degradation decreased.

Figure 3: The degradation of biofilm MRSA 2983 by

Streptomyces W-5A extract.

The analysis results using the one-way

ANOVA test showed no significant effect (Sig.

<0.05) between the incubation time treatment on the

percentage of biofilm degradation (Table 1).

However, 50% of biofilm degradation was achieved

after three days incubation and reached a maximum

at nine days incubation.

The tests used the microplate method with BHI-

Glu medium. The clinical MRSA 2983 was added to

a well-filled medium, then incubated for biofilm

formation. After incubation, planktonic cells formed

on the surface, while biofilms formed and stuck

tightly to the well-walls. This adhesion was

accompanied by a build-up of organic materials

covered by an extracellular polymer matrix. This

matrix was a structure of threads crossed with each

other and acted as an adhesive for the biofilm. The

planktonic cells formed were removed by washing,

then the Streptomyces W-5A extract was added to the

well. After incubation, all wells were washed to

remove planktonic bacteria and added crystal violet

to colour the biofilm. Besides using crystal violet,

biofilm quantification can also use resazurin,

safranin, and tryptan blue dyes (Peeters et al., 2008;

Sandasi et al., 2010). In this research, crystal violet

dye was used because it was easy to obtain and

economical in price.

4 DISCUSSION

Actinobacteria have been reported to have

antibacterial activity against MRSA. Bister et al.

(2004) wrote that Abyssomicin C obtained from

actinobacteria has potential as anti-MRSA. León et al.

(2011) reported that the dichloromethane extract from

actinobacteria isolates (I-400A, B1-T61, M10-77)

have high antibacterial activity against MRSA ATCC

43300 and VRE ATCC 51299. Rajan & Kannabiran

(2014) also reported 2,4-dichloro-5-sulfamoyl

benzoic acid (DSBA) extracted from marine

100

03691215

% Degradation

Incubation (Days)

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

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Streptomyces sp. VITBRK2 has anti-MRSA activity.

Furthermore, Bhakyashree & Krishnan (2018) wrote

that Streptomyces sp. VITBKA3 is potential as an

anti-MRSA compound against MRSA strains

ATCC43300 and ATCC700699.

Isolate at a particular incubation time that

produces the highest inhibition zone is considered the

optimum time for making antibacterial compounds.

The duration of the production phase of each microbe

varies depending on genetic factors and

environmental conditions. Susilowati et al. (2007)

reported that actinobacteria produced optimal

antibacterial compounds at the optimum incubation

time of 72 hours (isolate A3.5) and 96 hours (isolate

F6.1) against Enteropathogen bacteria. Dhananjeyan

et al. (2012) used an incubation time of five days to

produce antibacterial compounds from actinobacteria

against Escherichia coli MTCC 50, Pseudomonas

aeruginosa MTCC 424, and Bacillus subtilis MTCC

441. Bhakyashree & Krishnan (2018) used an

incubation time of seven days to produce antibacterial

compounds from actinobacteria against MRSA

strains ATCC 43300 and ATCC 700699.

The clinical isolate MRSA 2983 is known as a

biofilm producer. This research used an initial MRSA

concentration of 1.5 × 108 CFU/mL (0.5 McFarland

standard). According to Skogman et al. (2016),

biofilms can be formed with an initial bacterial

concentration of 106-108 CFU/mL. MRSA was

added to the well containing the medium, and then the

extract was added so that the biofilm growth would

coincide with the presence of extract. If the extract

has inhibitory activity, there will be inhibition of

MRSA biofilm formation.

After incubation, the well was washed to remove

planktonic bacteria, then stained with crystal violet.

Crystal violet can bind to proteins and

polysaccharides in the bacterial extracellular matrix

(Peeters et al., 2008). In this research, if, after staining

with crystal violet, a purple colour was formed, the

amount of binding dye was assumed to be the same

as the number of biofilm matrices in the well

(O'Toole, 2011).

Microbes capable of producing biofilms generally

have the potential for resistance to antibiotics.

Bacteria in the biofilm can withstand antibiotics

because the antibiotics fail to penetrate to destroy the

biofilm. Biofilm degradation test is known as an

approach to dealing with biofilms by adding bioactive

compounds that can trigger biofilm destruction

(Belbase et al., 2017; Bjarnsholt et al., 2013; Schierle

et al., 2009). Thus, the biofilm degradation test aims

to determine the potency of extract to degrade biofilm

MRSA.

Bioactive compounds to inhibit biofilm formation

were highly produced at 12 days of incubation in this

research, whereas bioactive compounds to degrade

biofilm were highly made at nine incubation days.

Indeed, the production of anti-MRSA compounds has

various optimum incubation time. Oja et al. (2015)

reported that Streptomyces violaceoruber (DSM-

40701) produced antibiofilm compounds against S.

aureus with an incubation time of four days. Suzuki

et al. (2015) said that Streptomyces sp. strain

MC11024 made antibiofilm compounds against S.

aureus and MRSA N315 with an incubation time of

three days. Balasubramanian et al. (2017) reported

that Streptomyces sp. SBT343 produces antibiofilm

compounds against Staphylococcal bacteria with an

incubation time of ten days.

5 CONCLUSIONS

Streptomyces W-5A isolated from Segara Anakan

Cilacap indicated the potency as anti-MRSA. To our

knowledge, this is the first report of indigenous

Streptomyces with three major anti-MRSA activities,

including antibacterial, biofilm formation inhibition,

and biofilm degradation. This finding highlights

Segara Anakan Cilacap as an essential source of

indigenous microbe for pharmaceutical purposes.

Further research is necessary, particularly for the

isolation and characterization of bioactive

compounds.

ACKNOWLEDGEMENTS

The authors would like to express profound gratitude

for the fundamental research grant contract No.

T/1641/UN23.18/PT.01.03/2020 from the

Directorate of Research and Community Service,

Ministry of Research and Technology/National

Agency for Research and Innovation of the Republic

of Indonesia.

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