Post on 19-Jul-2020
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