Analysis of the Effectiveness of Chrysomya sp. Maggot Extract in

Inhibiting the icaA and icaD Genes Regulator Staphylococcus

epidermidis Biofilm

Dylan Tamalsir

, Anton Budhi Darmawan

and Dwi Utami Anjarwati

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

Department of Otorhinolaryngology - Head and Neck Surgery, Faculty of Medicine, Universitas Jenderal Soedirman,

Purwokerto, Indonesia

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

Keywords: Biofilm, Polysaccharide Intercellular Adhesion, icaAD Gene, Maggot, PCR

Abstract: Maggots have proven to be one of the significant ingredients in degrading biofilms by destroying the

polysaccharide intercellular adhesion (PIA) produced by the enzyme encoded by the icaADBC gene. This

experimental study aims to prove the action target of maggot Chrysomya sp. extract on icaAD biofilm

regulatory genes. Biofilm DNA extraction was carried out against bacterial cultures of S. epidermidis ATCC

35984 and S. epidermidis TCC 35983 incubated with Chrysomya sp. maggot extract, using Microtiter plate

(MTP) method. The Chrysomya sp. maggot extract concentration was 0%, 20%, 40%, 60%, 80%, and 100%

with an incubation period of 3 and 24 hours. PCR analyzed gene expression with the primer of icaAD genes.

The qualitative test was carried out by 2% agarose gel electrophoresis. IcaAD genes of both S. epidermidis

strains were detected in all treatments. The icaA band size of ± 980 bp and the icaD gene size of ± 380 bp

can be observed either after the intervention of Chrysomya sp. maggot extract in various concentration (0%,

20%, 40%, 60%, 80%, and 100%) or after the incubation period of 3 and 24 hours. Chrysomya sp. maggot

extract does not affect the icaAD biofilm regulatory genes of Staphylococcus epidermidis.

1 INTRODUCTION

The formation of biofilms is influenced by bacteria

and external factors that produce extracellular

polysaccharide adhesin, called polysaccharide

intercellular adhesin (PIA) or polymeric N-acetyl-

glucosamine (PNAG). PIA is also affected by the ica-

operon regulatory (icaR) enzyme: an operon

containing the icaADBC gene, a known regulator of

biofilm formation in Staphylococcus (O'Gara, 2007).

S. aureus and S. epidermidis contain the

intercellular adhesion operon (ica), which is

responsible for the production of PIA. The icaA and

icaD genes play the most important role among other

ica genes in biofilm formation. The icaA gene

encodes N-acetylglucosaminyltransferase, an enzyme

involved in the synthesis of PIA. Furthermore, the

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icaD gene plays an essential role in N-

acetylglucosaminyltransferase's maximal expression,

leading to complete phenotypic expression of

capsular polysaccharides (Nasr et al., 2012). Several

studies have shown that the formation of biofilms by

Staphylococcus in some invasive medical devices

causing the nosocomial infection is associated with

the presence of both icaA and icaD genes as essential

virulence factors of these bacteria (Arciola et al.,

2001; Ghasemian et al., 2015; Nasr et al., 2012).

Maggots (larvae) of green flies affect biofilms and

their virulence factors (Anjarwati et al., 2017;

Anjarwati and Hapsari, 2014; Bohova et al., 2014;

van der Plas et al., 2007). Maggot extract has different

effectiveness against different bacterial species.

Insects, including flies, can produce antimicrobial

peptides (AMP). AMP has a good effect on Gram-

positive, Gram-negative, and fungal infections. The

Tamalsir, D., Darmawan, A. and Anjarwati, D.

Analysis of the Effectiveness of Chrysomya sp. Maggot Extract in Inhibiting the icaA and icaD Genes Regulator Staphylococcus epidermidis Bioﬁlm.

DOI: 10.5220/0010488801210125

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 121-125

ISBN: 978-989-758-499-2

121

antimicrobial activity by peptides on the defensin

group affects the permeabilization of the target

membrane. This influence is related to inhibition of

RNA, DNA, protein synthesis, and reducing

bacteria's viability. Therefore, the icaA and icaD

genes' expression is expected to be inhibited (Parnés

and Lagan, 2007).

Maggot extract can break down the biofilms of

various bacteria(Cazander et al., 2009). The results

obtained in previous studies concluded that the

maggot extract of Chloroprocta sp. at different

concentrations and incubation times had antibacterial

activity against planktonic bacteria and S. epidermidis

biofilms. The extract can break down the cell

aggregation by destroying the PIA produced by the

enzyme encoded by the icaADBC or Aap genes in the

accumulation phase of biofilm formation (Anjarwati

et al., 2017). This study aims to prove the action target

of maggot Chrysomya sp. extract on icaAD biofilm

regulatory genes.

2 METHODS

This research is a laboratory experiment with a

factorial pattern and a completely randomized design.

The aim was to analyze Chrysomya sp. maggot

extract's ability to inhibit the icaA & icaD, a gene

regulator for the biofilm Staphylococcus epidermidis.

This study's bacterial strains were the strong biofilm

producer S. epidermidis ATCC 35984 and the

intermediate biofilm producer S. epidermidis ATCC

35983.

This study's number of maggots was 1 g/ml PBS,

with one gram of maggots equivalent to ±20 maggots.

In this study, the flies were the green flies Chrysomya

sp. originating from the genus of Chrysomya, family

Challiphoridae, Order Diptera, class Insecta,

Arthropoda division, kingdom Animalia, which is a

type of greenfly that is widely found in the

Purwokerto area(Anjarwati et al., 2017; Hidayati et

al., 2020).

The Chrysomya sp. maggot extract tested was at

concentrations of 0%, 20%, 40%, 60%, 80%, and

100% with an incubation period of 3 hours and 24

hours Microtiter plate (MTP) method. Furthermore,

DNA isolation was first carried out by extracting both

S. epidermidis biofilm by destroying the bacterial cell

walls (Quick DNA Fungal/Bacteria Mini-Prep Kit,

Zymo Research Corp). After obtaining the DNA

isolation sample, Polymerase Chain Reaction (PCR)

optimization was carried out to get optimal PCR

results. The qualitative test was carried out by 2%

agarose gel electrophoresis (Mahardhika et al., 2020).

The primers (Invitrogen Custom DNA Oligos and

design tools) used as follows: Forward icaA primer:

5'CCTAACTAACGAAAGGAG3 ', reverse icaA

primer: 5'AAGATATAGCGATAAGTG3', forward

primer icaD: 5'AAACGTAAGAGGTG3 ', primer

reverse icaD: 5'AGCAATATGATCAAGATAC3',

carried out with a denaturation step for 50 seconds at

94 °C, annealing primer at 49 ° C for both icaA and

icaD genes and a polymerization step at 72 °C during

1 minute. The polymerization was concluded with an

elongation period of 10 minutes at 72 °C. The

amplified gene was then poured into 2% agarose gel

to undergo the electrophoresis process (Mahardhika

et al., 2020).

3 RESULTS

The results of the PCR examination of the icaA and

icaD biofilm regulatory genes of the two

S.epidermidis strains ATCC 35984 and ATCC 35983

have been given Chrysomya sp. maggot extract as the

following figure.

Figure 1: PCR examination results of the icaA gene on the

ATCC35983 S.epidermidis and ATCC35984 S.epidermidis

biofilm samples on maggot extract administration in

different concentrations and incubation times, A.

S.epidermidis ATCC35983, 3 hours; B. S.epidermidis

ATCC35983, 24 hours; C. S.epidermidis ATCC35984, 3

hours; D. S.epidermidis ATCC35984, 24 hours. The icaA

gene appears at 980 bp.

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 )

122

Figure 2: PCR examination results of icaD genes in the

ATCC35983 S.epidermidis and ATCC35984 S.epidermidis

biofilm samples on maggot extract administration in

different concentrations and incubation times, A.

S.epidermidis ATCC35983, 3 hours; B. S.epidermidis

ATCC35983, 24 hours; C. S.epidermidis ATCC35984, 3

hours; D. S.epidermidis ATCC35984, 24 hours. The icaD

gene appears at 380 bp.

4 DISCUSSION

The genetic and molecular basis for S. epidermidis

biofilm formation is quite varied. The broadest theory

in biofilm formation is the involvement of adhesive

polysaccharide capsules (PSA) and adhesin

intracellular polysaccharides (PIA) or polymeric N-

acetyl-glucosamine (PNAG) (Rachmawati et al.,

2020). S. epidermidis contains the intercellular

adhesion operon (ica) responsible for the production

of PIA. This operon contains the icaADBC gene and

the icaR gene that regulates PIA production1. Both

icaA and icaD are prominent supporters of the biofilm

formation mechanism in the S.epidermidis (Zhou et

al., 2013). Figures 1 and 2 described the icaA gene at

980 bp and the icaD gene at 380 bp in the

ATCC35983 S.epidermidis and ATCC35984

S.epidermidis biofilm with/without maggot extract

administration in different concentrations and

incubation times.

Maggot extract has different effectiveness against

different bacterial species. Several studies have

shown that maggot extract can reduce bacterial

biofilms from different bacterial strains through other

mechanisms. Maggot extract is more effective in

preventing biofilm formation and damaging mature

biofilms in E. cloacae bacteria than S. aureus bacteria.

The same study concluded that maggot extracts

significantly affected cell viability in E. cloacae

biofilms while it failed in S. aureus bacteria (Bohova

et al., 2014). Other studies conducted on

Pseudomonas performed that maggots have a low

antimicrobial effect in inhibiting the formation of

pseudomonas biofilms and not inhibiting the growth

of these bacteria (Anders S. Andersen Dorthe

Sandvang et al., 2010; Bexfield et al., 2004). The

Lucilia sericata maggot extract showed no direct

bactericidal or bacteriostatic activity against

planktonic organisms of several different bacteria

types in vitro. However, L.sericata influenced clinical

observations of maggot therapy (Cazander et al.,

2009).

Maggot extract can break down the biofilms of

various bacteria (Cazander et al., 2009). In the present

study, Chrysomya sp. maggot extract does not affect

the icaA & icaD genes regulator biofilm

Staphylococcus epidermidis. The possibility was

maggot extract can only damage the biofilm structure

by damaging PIA in the accumulation process of

biofilms so that it does not directly affect the icaA &

icaD genes in biofilm formation. This result is in line

with the previous research, which concluded that the

extract could break down cell aggregation by

destroying the PIA produced by the enzyme encoded

by the icaADBC or Aap genes in the accumulation

phase of biofilm formation. The underlying

mechanism is maggots' protease activity or

glucosaminidase damaging the polysaccharide

structure (PIA) of the biofilm (Anjarwati et al., 2017).

Downregulation of the icaA gene from the

icaADBC operon can decrease PIA/PNAG

production, leading to a reduction in biofilm

formation. Interestingly, icaA appears to rise during

the Mid-Logarithmic (ML) growth phase but

decreases in the Stationary phase in RT-PCR when

given CCG-2979, a low molecular weight compound

derived from HTS(Ma et al., 2012). For the record,

some genes also play an essential role in the virulence

of Staphylococcus, for example, icaADBC, SigB,

Agr, RNAIII, CodY genes. Changes in the profile of

some genes can cause damage biofilm formation at

different stages and lead to decreased virulence. The

CodY gene is a gene that can suppress the Agr operon

and icaADBC. CodY inhibition can have other effects

on biofilm formation. Activating CodY can increase

biofilm formation in the aureus strain SA564 but

reduces biofilm formation in high biofilm-producing

S30 isolates (Majerczyk et al., 2008; Tu Quoc et al.,

2007), so further research to see the role of maggot

extract on biofilm operon genes, in particular

Analysis of the Effectiveness of Chrysomya sp. Maggot Extract in Inhibiting the icaA and icaD Genes Regulator Staphylococcus

epidermidis Bioﬁlm

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regarding the role of other genes in the biofilm

formation process, needs to be done.

The antimicrobial peptide (AMP), which can be

produced by some insects, including greenfly larvae,

has various mechanisms of action against pathogenic

bacteria, such as permeabilization of cell membranes,

identification of specific protein targets, inhibition of

RNA and DNA. Bacteria can develop resistance to

AMP. Components that can cause bacterial resistance

to AMP include A. Secreted bacterial proteases, for

example, lipopolysaccharide in the outer membrane

of gram-negative bacteria, teichoic wall acid and

lipoteichoic acid in gram-positive bacterial cell walls,

B. Multidrug efflux pumps., And C. Extracellular

biofilm matrix (Bechinger and Gorr, 2017). This

study's weakness is that the extract used is a crude

extract, so further research is needed to see the

content of Chrysomya sp. Maggot extract and its

mechanism of action on genes that play a role in

biofilm formation.

5 CONCLUSION

Chrysomya sp. maggot extract could not inhibit the

icaA & icaD gene regulator of ATCC35983 S.

epidermidis and ATCC35984 S. epidermidis biofilm.

ACKNOWLEDGEMENTS

Many people supported this paper. Therefore, we are

grateful to Ari Asnani, S.Si., M.Sc., Ph.D., Dr. Ir. Sri

Rahayu., M.Si, and Dr. dr. Lantip Rudjito, M.Si. Med

for the advice to make this paper better. The Funding

was from Jenderal Soedirman University and

Directorate General of Research and Development of

the Ministry of Research, Technology, and Higher

Education, Republic of Indonesia.

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