In Vitro Study of Reduction of Oral Enterococcus faecalis Biofilm on

Application of Combination of Chrysomya megacephala Maggot

Extract and Sodium Hypochlorite

Rizka Hidayati

1 a

, Ari Asnani

, Muhamad Salman Fareza

and Dwi Utami Anjarwati

Magister of Biomedicine, Faculty of Medicine, Universitas Jenderal Soedirman, Purwokerto,Indonesia

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Jenderal

Soedirman, Purwokerto, Indonesia

Department of Biochemistry, Department of Pharmacy, Faculty of Health Sciences, Universitas

Jenderal Soedirman, Purwokerto, Indonesia

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

Keywords: Antibiofilm, Root Canal Irrigant, Blowfly Larvae, Endodontic Treatment

Abstract: The tooth's infected root canal relates to the bacteria invasion, such as Enterococcus faecalis. The eradication

of the bacteria using single root canal irrigants becomes difficult because of the formed biofilm. We aimed to

investigate the effectiveness of a combination of C. megacephala maggot extract with the common irrigant,

sodium hypochlorite 3%, on the biofilm reduction of E. faecalis. The C. megacephala maggot extract was

tested at concentrations 25%, 50%, and 100%, and the combination of each extract concentration with sodium

hypochlorite in a volume ratio of 1:1; 1:2, 1:3, 2:1, and 3:1. All treatments were performed three times of

replication with incubation time for 1 hour and 3 hours. Antibiofilm effect was measured with crystal violet

staining and the optical density reading. Data was analyzed with the Statistical Package for the Social

Sciences Statistic Version 22. The least biofilm formation was observed in combination of maggot extract

25% with sodium hypochlorite (2:1) for 1 hour incubation (p=0,05) and combination of maggot extract 25%

(1:1) for 3 hours incubation (p=0,000). This combination effectively inhibits the biofilm of E. faecalis. This

study identified the protease enzymes in C. megacephala maggot extract and investigated C. megacephala

maggot extract's antibiofilm effect combine with the other root canal irrigant.

1 INTRODUCTION

One of the bacteria found in the root canal of the

infected tooth is Enterococcus faecalis. These

bacteria are persistent and can form biofilms.

Therefore they are difficult to be removed. The

bacteria in the root canal treatment are eradicated by

applying root canal irrigant. The agent commonly

used as root canal irrigant is sodium hypochlorite

(NaOCl), with 1,25-5% (Mulyawati, 2011). NaOCl

effectively removes planktonic bacteria, but it is less

effective in reducing biofilms produced by bacteria

(Dunavant et al., 2006). NaOCl is usually combined

with other irrigation solution such as chlorhexidine

https://orcid.org/0000-0001-5351-752X

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

https://orcid.org/0000-0003-2414-1249

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

(CHX) to increase the antibacterial and antibiofilm

effects.

Several researchers have also carried out the

combination of root canal irrigant with natural agents.

Geethapriya et al. (2016) combined chitosan with

EDTA against E. faecalis biofilms with a ratio of 1: 1

in their study, which showed effective results in

inhibiting E. faecalis biofilm formation (Geethapriya

et al., 2016). Other studies combined chitosan with

chlorhexidine (CHX), whose results showed the same

effect with 5% NaOCl in inhibiting E. faecalis biofilm

(Jaiswal et al., 2017). Other studies regarding the

combination of NaOCl with natural agents have never

been done.

Hidayati, R., Asnani, A., Fareza, M. and Anjarwati, D.

In Vitro Study of Reduction of Oral Enterococcus faecalis Bioﬁlm on Application of Combination of Chrysomya megacephala Maggot Extract and Sodium Hypochlorite.

DOI: 10.5220/0010488400980103

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 98-103

ISBN: 978-989-758-499-2

Maggot extract is material from natural sources

that have not been studied as root canal irrigant. Some

maggots, such as maggot Lucilia sericata contain

proteolytic enzyme components that can degrade

bacterial biofilms' extracellular matrix (Chen et al.,

2012). Chymotrypsin in L. sericata secretion can

affect bacterial biofilms' adhesion (Harris et al.,

2013). Other types of maggots, such as Chloroprocta

sp. contain protease enzymes that can reduce the

extracellular biofilm matrix in Staphylococcus

epidermidis (Anjarwati et al., 2017). Another maggot

extract, such as Chrysomya megacephala maggot

extract, has an excretory and secretory. This product

contains serine (trypsin and chymotrypsin), an

antibacterial effect on Escherichia coli,

Staphylococcus aureus, and Bacillus subtilis (El-

Ebiarie et al., 2012; Mohamed 2015a, 2015b).

In this study, the maggot was taken from the

maggot Chrysomya megacephala, a local maggot that

is quite common in Indonesia (Putri, 2018).

Combination of maggot extract C. megacephala with

3% NaOCl as root canal irrigant is expected to

increase the reduction of E. faecalis biofilm.

2 METHODS

2.1 Material

This experimental laboratory study conducted the E.

faecalis biofilm test. The materials used were C.

megacephala maggot extract with a concentration of

25%, 50%, 100% and a combination of each

concentration of maggot extract with 3% NaOCl with

a volume ratio of 1: 1, 1: 2, 1: 3, 2: 1 and 3: 1, 3%

NaOCl as a positive control, tryptic soy broth (TSB)

and sterile phosphate-buffered saline (PBS) as

negative controls. Each treatment was replicated three

times. Biofilm reduction was measured by added 1%

crystal violet with optical density measured at a

wavelength of 595 nm (OD595) using a microtiter

plate reader at the Cancer and Stem Cell Research

Center Laboratory, Muhammadiyah University,

Purwokerto.

2.2 Bacterial Strains

The sample used in this study is that the colony is E.

faecalis ATCC 29212, a moderate biofilm-producing

bacteria. The colony has been isolated and cultured

with Mueller Hinton Agar (MHA) media in the

Microbiology Laboratory of the Faculty of Medicine,

Universitas Jenderal Soedirman, Purwokerto. The

powder of MHA with 12 grams was solved with 240

mL of aquadest and poured into the culture tray (20

mL/tray). The media was sterilized in an autoclave for

15 minutes (2 atm, 121oC), then put in an incubator

for 24 hours to check if there is contamination or not.

The E. faecalis were cultured in sterile media with an

anaerobic environment. The bacteria's growth colony

was taken and diluted with NaCl 0,9% until the

concentration equal to 106 CFU/mL (CFU: Colony

Forming Unit) or 0,5 Mc Farland Standard (Howarto

et al., 2015).

2.3 Rearing Maggot and Collecting

Maggot Extract

The process of collecting C. megacephala maggot

extract was carried out at the Faculty of Medicine,

Universitas Jenderal Soedirman. The rearing of the

maggot C. megacephala was done by installing traps

of flies that have been given raw fish (fish waste).

After one day, the fly eggs that have been collected in

fish waste were transferred to fresh raw fish and left

to grow into a maggot. After growing into a maggot,

the maggot at the end of the second instar and the

beginning of the third instar was collected in a vessel

and washed using ethanol and distilled water three

times (Arora et al., 2010).

Figure 1: Soaking C. megacephala maggot in Phosphate

Buffered Saline (PBS).

Every 1 gram of maggot was immersed in 1 ml of

sterile PBS solution for 1 hour at room temperature in

a dark room (Figure 1). Continued Soaking was for

24 hours (moved to room light). Then maggot in PBS

was incubated at 37

C for 48 hours. Then the maggot

and liquid were separated, then the liquid was

centrifuged at 25

C, with 10,000 rpm for 15 minutes.

In Vitro Study of Reduction of Oral Enterococcus faecalis Bioﬁlm on Application of Combination of Chrysomya megacephala Maggot

Extract and Sodium Hypochlorite

The supernatant obtained from the centrifuge process

was collected and sterilized with a 0.2 µm membrane

filter. Maggot extract obtained from this process was

stored at -20

C (Honda et al., 2011).

2.4 Biofilm Reduction Measurement by

Administering Chrysomya

megacephala Maggot Extract and

Its Combination with Sodium

Hypochlorite

The biofilm of E. faecalis (ATCC 29212) was

measured using 96-well microtiter plates. The

bacteria were transferred from Mueller Hinton Agar

(MHA) media into Tryptic Soy Broth (TSB) and

incubated for 24 hours under anaerobic conditions,

which remained at 37 oC. The culture was diluted

with 1: 100 on the medium. Then 20 µl of bacterial

culture was inoculated with 200 µl TSB in each well,

dispensed into 96-well microtiter plates with a flat

bottom. After anaerobic incubation at 37 °C for 24

hours, the planktonic bacteria from each well were

disposed of carefully by using a micropipette slowly

until the bottom of the well looked clear. Each well

was washed with 300 µl phosphate-buffered saline

(PBS) 2 times slowly.

As many as 100 μL of maggot extract filled into

each well of the 96-well microtiter plate was filled

with at different concentrations (25%, 50%, 100%)

and a combination of maggot extract and 3% NaOCl

with a ratio of 1: 1, 1: 2, 2: 1, 1: 3, and 3: 1. The 96-

well microtiter plate that has been inserted maggot

extract and a combination of maggot extract and

NaOCl are then incubated for 1 hour and 3 hours.

After incubation, the biofilm is examined by giving a

dye of 200 µL of 1% crystal violet solution in water

for 30 minutes and washed with distilled water. Wells

are reversed and dried on paper towels and dry air.

Then, 200 µL of 5% acid isopropanol was added to

each well to remove biofilm colour. The optical

density was measured at 595 nm (OD595) using a

microtiter plate reader (Pierce et al., 2010).

Table 1: The result of optical density reading in 1 hour and 3 hours incubation.

No. Treatment

Mean of absorbance

1 hour 3 hours

1. TSB with E. faecalis (control of bacteria) 0,851 3,064

2. C. megacephala maggot extract 25% 0,253 2,966

3. C. megacephala maggot extract 50% 0,319 2,902

4. C. megacephala maggot extract 100% 0,424 2,927

5. Combination of C. megacephala maggot extract 25%, and NaOCl 3% (1:1) 0,214

0,176

6. Combination of C. megacephala maggot extract 50%, and NaOCl 3% (1:1) 0,756 2,809

7. Combination of C. megacephala maggot extract 100%, and NaOCl 3% (1:1) 0,619 2,915

8. Combination of C. megacephala maggot extract 25%, and NaOCl 3% (1:2) 0,268 0,294

9. Combination of C. megacephala maggot extract 50%, and NaOCl 3% (1:2) 0,501 0,211

10. Combination of C. megacephala maggot extract 100%, and NaOCl 3% (1:2) 0,190 2,998

11. Combination of C. megacephala maggot extract 25%, and NaOCl 3% (1:3) 0,209 0,212

12. Combination of C. megacephala maggot extract 50%, and NaOCl 3% (1:3) 0,232 0,216

13. Combination of C. megacephala maggot extract 100%, and NaOCl 3% (1:3) 0,176 2,830

14. Combination of C. megacephala maggot extract 25%, and NaOCl 3% (2:1)

0,155 0,294

15. Combination of C. megacephala maggot extract 50%, and NaOCl 3% (2:1) 0,498 2,925

16. Combination of C. megacephala maggot extract 100%, and NaOCl 3% (2:1) 0,664 2,273

17. Combination of C. megacephala maggot extract 25%, and NaOCl 3% (3:1) 0,343 2,918

18. Combination of C. megacephala maggot extract 50%, and NaOCl 3% (3:1) 0,273 2,962

19. Combination of C. megacephala maggot extract 100%, and NaOCl 3% (3:1) 1,395 2,920

20. PBS with E. faecalis 0,317 2,982

21. NaOCl 3% with E. faecalis 0,157 0,194

Table 2: The calculating of the value of MBRC

and MBRC

in biofilm test with 1 hour and 3 hours incubation.

Time of

incubation

OD of

Bacterial

control

OD of

Blank

MBRC

(OD of bacterial control –OD of

blank) x 50%

MBRC

(OD of bacterial control –OD of

blank) x 20%

1 hour 0,851 0,097 0,394 0,151

3 hours 3,064 0,109 1,477 0,591

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 )

100

2.5 Statistical Analysis

The effects of various maggot extract concentrations

and their combinations with sodium hypochlorite in

reducing biofilms were analyzed using one-way

ANOVA and Post hoc LSD (Least Significant

Difference) tests by using The Statistical Package for

the Social Sciences Statistic Version 22.

3 RESULTS

From all treatment groups with 1-hour incubation, the

most extensive treatment group in reducing E.

faecalis biofilm was a combination of 25% maggot

extract and sodium hypochlorite with a ratio of 2: 1

(reducing 81.75% of biofilm production produced

from the bacterial control, p < 0,05) ). The

combination of maggot extract (25%) and sodium

hypochlorite (2:1) reduced biofilm better than 3%

sodium hypochlorite only. The Post hoc LSD test

showed that the combination of 25% maggot extract

and sodium hypochlorite with ratio 2:1 does not have

a different effect than 3% sodium hypochlorite in

reducing the biofilm of E. faecalis (p>0,05).

In 3 hours of incubation, the most extensive

treatment group in reducing E. faecalis biofilm was a

combination of 25% maggot extract and sodium

hypochlorite with a ratio of 1: 1 (reducing 94.25% of

biofilm production produced from bacterial control, p

<0.001) (Table 1). The 25% maggot extract and

sodium hypochlorite application (ratio 1:1) showed

more effect than 3% sodium hypochlorite only. The

Post hoc LSD test also showed that the combination

of 25% maggot extract and sodium hypochlorite with

ratio 1:1 does not have a different effect than 3%

sodium hypochlorite in reducing the biofilm of E.

faecalis (p>0,05).

In 1 hour incubation, the value of MBRC50 in

this research is 0,394 and MBRC80 is 0,151 (Table

2). In 1 hour incubation, MBRC50 could be found in

maggot extract 25%, 50%, combination of maggot

extract 25% (1:1), 25% (1:2), 100% (1:2), 25% (1:3),

50% (1:3), 25% (2:1), 25% (3:1), and50% (3:1).

Meanwhile, the value of MBRC80 could not be found

in all the treatment in 1 hour incubation (Figure 1). In

3 hours incubation, the value of MBRC50 is 1,477

and the value of MBRC80 is 0,591 (Table 2). In 3

hours incubation, MBRC50 and MBRC80 could be

found in the same treatment which are the

combination of maggot extract 25% (1:1), 25% (1:2),

50% (1:2), 25% (1:3), 50% (1:3), and 25% (2:1)

(Figure 2).

Figure 2 showed that in 1-hour incubation,

MBRC50 could be reached of maggot extract

application (concentration 25%, 50%, the

Figure 2. The effect of Chrysomya megacephala maggot extract on biofilm reduction of Enterococcus faecalis ATCC

29212 for 1-hour incubation (a) and 3 hours incubation (b)

In Vitro Study of Reduction of Oral Enterococcus faecalis Bioﬁlm on Application of Combination of Chrysomya megacephala Maggot

Extract and Sodium Hypochlorite

101

combination of 25% maggot extract (1:1), 25%

maggot extract (1:2), 100% maggot extract (1:2),

25% maggot extract (1:3), 50% maggot extract (1:3),

25% maggot extract (2:1), 25% maggot extract (3:1),

and 50% maggot extract (3:1)). Meanwhile,

MBRC80 could not be found in 1-hour of incubation.

4 DISCUSSION

Compared to the results of biofilm reduction at 1-hour

incubation (maggot extract 25% (2:1)), there was a

decrease in the concentration of maggot extract

needed to reduce biofilms for a longer time (3 hours),

namely with extract concentration of 25% (1:1). Due

to a decrease in maggot extract protease activity, the

possibility is as time increases, therefore at 3 hours

incubation. The possibility of more significant

antibiofilm activity is due to the combination of 3%

sodium hypochlorite and maggot extract (Łaba et al.,

2010).

Compared with the effect of giving 3% sodium

hypochlorite, the combination of 25% (1:1) maggot

extract (1 hour) and a combination of 25% (2: 1) (3

hours) maggot extract had no different effects on

Enterococcus faecalis biofilm reduction (p>0.05).

This result shows that an evaluation in the research

process is needed to increase the antibiofilm effect to

be more maximal. In PBS solvent with E. faecalis,

compared with a combination of 25% 2: 1 maggot

extract (1-hour incubation) and 25% (1:1) (3 hours

incubation) combination of maggot extract, there

were no significant differences in antibiofilm effects.

This result showed that the presence of PBS as a

solvent in making maggot extract is not a factor that

influences the reduction of bacterial biofilms.

However, the antibiofilm effect produced comes from

the extract of the maggot.

This study also showed that the C. megacephala

maggot extract's antibiofilm effect was more effective

at 3 hours incubation (p <0.001). In other studies,

incubation time also significantly affected the

reduction of extracellular biofilm matrix after maggot

extract. This is thought to be due to proteases

requiring time to break down proteins into dissolved

proteins in exopolysaccharide (EPS) on bacterial

walls. Protease is one type of enzyme contained in

maggot extract. The effect of enzyme damage is

directly proportional to the length of time the

interaction of environmental exposure to the enzyme.

The longer the exposure to the environment will

damage the enzyme's structure, so that a decrease in

enzyme activity Łaba et al., 2010)

5 CONCLUSIONS

C. megacephala maggot extract and its combination

with sodium hypochlorite affect the reduction of

biofilms produced by E. faecalis. Further research is

needed to develop C. megacephala maggot extract,

such as identifying other protease enzymes in C.

megacephala maggot extract to reduce other bacterial

biofilms and determine the antibacterial and

antibiofilm effects of C. megacephala maggot extract

with a combination of other root canal irrigation

materials.

ACKNOWLEDGEMENTS

This paper can be arranged by support from many

people. Therefore, we are grateful to Dr. Warsinah,

M.Si., Apt, Dr. Oedjijono, M.Sc, and Dr. dr. Lantip

Rujito, M.Si. Med for the advice to make this paper

better.

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In Vitro Study of Reduction of Oral Enterococcus faecalis Bioﬁlm on Application of Combination of Chrysomya megacephala Maggot

Extract and Sodium Hypochlorite

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