Isoeugenol as a potential antifungal molecule against azole resistant

environmental isolates of Aspergillus fumigatus and their biofilm

Lovely Gupta*, Pooja Sen and Pooja Vijayaraghavan

Lab-120, Antimycotic and Drug Susceptibility Laboratory, J3 block, Amity Institute of

Biotechnology, Amity University, Uttar Pradesh, NOIDA-201301

Email: lgupta@amity.edu; vrpooja@amity.edu

Introduction

Methodology and Results

Conclusion

1. Clinical and Laboratory Standards Institute (CLSI). Reference method

for broth dilution testing of filamentous fungi, Approved standard- 2nd

Ed. CLSI document M38-A2, Wayne, Pennsylvannia, USA, 2008.

2. Ferreira SB, Dantas TB, Silva DF, de Melo TR, Lima EO. In vitro

antifungal effect of isoeugenol against Penicillium citrinum strains.

MOL2NET, 2017; 3: 1-7.

3. Nett JE and Andes D. Fungal Biofilms: In vivo models for discovery of

anti-biofilm drugs. Microbiol. Spectr., 2015; 3(3): E30.

4. Boominathan M and Ramamurthy V. Antimicrobial activity of

Heliotropium indicum and Coldenia procumbens. J. Ecobiol., 2009;

24(1): 11-15.

Acknowledgement

1. The present work was funded by Department of Science

and Technology (DST) under DST-SERB project (Grant

order- EMR/2016/005752) to PV.

2. We would like to thank Amity University, NOIDA for the

facilities and infrastructure provided to perform experiments

and;

3. 9th Advances against Aspergillosis and Mucormycosis 2020

organizing members for awarding partial scholarship to attend

and present our research work.

Figure 1: Biofilm eradication percentage after treating the pre-formed

biofilm with isoeugenol at various concentrations (range 39 – 2000 µg/ml).

The effective concentration range from 312-625 µg/ml against azole

resistant strains (p >0.05).

4. In-silico ADME/Tox study of phenolic compound

Isoeugenol:

Phenolic compound isoeugenol has properties to

become a drug, its drug likeliness score is -0.76.

Isoeugenol showed drug-likeliness properties with no

side effects on cardiovascular, lungs, liver,

gastrointestinal systems. Similar results were reported

by National Toxicology Program Report. It has been

approved for food use by the Food and Drug

Administration when used in the minimum quantity

required to produce its intended effect.

References

The antifungal activity of isoeugenol against azole resistant strains of A. fumigatus was observed to be more effective and non-

toxic as compared to azole drugs and thus, can be explored further. The use of plant phenolic compound alone or in combination

with traditional drugs represents an important alternative to conventional therapy. Further, expression of genes involved in the

ECM formation will be studied. However, the results of present in-vitro drug testing study need to be mutually substantiated with

in-vivo therapeutic aspects.

At a global level, most of the fungal infections are being caused by Aspergillus fumigatus in millions of cases of chronic pulmonary aspergillosis and allergic bronchopulmonary

aspergillosis. In the present scenario, acquired azole resistance in A. fumigatus is an emerging problem that compromises the clinical efficacy of azole antifungals. There are several

mutations reported in the cyp51A gene of A. fumigatus that affects the activity of azole drugs. A. fumigatus strain also forms hydrophobic biofilm comprising of numerous intertwined

hyphae covered with extracellular matrix (ECM) (Nett and Andes, 2015). It forms a protective shield against antifungals, thereby reducing their efficacy drastically. The treatment of

biofilm related Aspergillus infection is also a serious clinical challenge, emphasising the need for new therapeutic agents (Boominathan and Ramamurthy, 2009). There are various

naturally occurring phenolic compounds identified as antimicrobial agents. Essential oil isoeugenol is one of the naturally occurring phenolic constituent of clove oil, nutmeg, basil

(Ferreira et al., 2017). The objective of the present study is to elucidate the antifungal efficacy of isoeugenol against azole resistant environmental isolates of A. fumigatus and their

biofilm.

1. Isolation of 11 azole resistant A. fumigatus strains from 80 soil samples

across North India and further identified by 18S ITS region sequencing

via Sanger’s method.

2. Minimum inhibitory concentrations (MIC100) of isoeugenol against azole

resistant A. fumigatus with reference to ATCC strain of A. fumigatus was

calculated using micro-broth dilution method of CLSI.

Table 1: MIC100 of isoeugenol and azole drugs against

azole resistant strains of A. fumigatus

Strains Isoeugenol (µg/ml)

Itraconazole (µg/ml)

Ketoconazole (µg/ml)

MIC 100

OF10 625 < 2 >5

B15 625 >2 >5

RT28 625 < 2 >5

B08 312 >2 >5

Y29 625 < 2 >5

B19 625 >2 >5

RB01 625 >2 >5

B03 625 < 2 >5

OF26 625 < 2 >5

R01 625 >2 >5

R03 625 >2 >5

ATCC 46645 312 0.195 0.32

Wild type ATCC 46645 biofilm

(Untreated)

20 µm

ECM with

intermingled

hyphae

Wild type ATCC 46645 biofilm

(Isoeugenol treated MBEC80)

No ECM

20 µm

Azole resistant Y29 strain biofilm

(Isoeugenol treated MBEC80)

20 µm

No ECM

20 µm

ECM with

intermingled

hyphae

Azole resistant Y29 strain

biofilm (Untreated)

3. Experimental design of the efficacy assessment of phenolic compound on pre-formed biofilm of azole resistant

A. fumigatus strains

Aspergillus

fumigatus strainsBiofilm of azole resistant strains of

A. fumigatus was grown upto 11th

column at 37 ºC for 48 hrs on RPMI

1640 medium with l-glutamine and

without bicarbonate (pH

7.0) followed by 24 hrs isoeugenol

treatment with reference ATCC

46645 strain.

MBEC80 was calculated by MTT

assay at 570nm

Analysis of the biofilm morphology was

conducted under scanning electron microscope

(SEM).

Azo

le

resistan

t

strain

AT

CC

strain

S. No. Properties Criterion in-silico

analysis of

Isoeugenol

1. Molecular weight < 500 164.08

2. Hydrogen donor < 5.0 1

3. Hydrogen acceptor < 5.0 2

4.Log P

(lipophilicity)< 5 2.64

5. Log S (Solubility) - -2.47

6. Oral bioavailability - >70%

7. Drug likeness score - -0.76

Lipinski Violations 0

Table 2: In silico prediction of druglikeness via ACD

labs, freeware

Figure 2: Biofilm morphology treated and

untreated samples under scanning electron

microscope.

Chemical structure

of isoeugenol