ORIGINAL Veterinary Research Forum.

2021; 12 (2) 241 – 246 Veterinary

ARTICLE doi: 10.30466/vrf.2019.103449.2460 Research
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Synergistic effects of dual antimicrobial combinations of synthesized

N-heterocycles or MgO nanoparticles with nisin against the growth of
Aspergillus fumigatus: In vitro study
Hamid Beyzaei1*, Mojtaba Mirzaei2, Narjes Hasan Fakhrabadi3, Behzad Ghasemi4
1 Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran; 2 Young Researchers and Elite Club, Mashhad Branch, Islamic Azad University,

Mashhad, Iran; 3 Department of Pathobiology, Faculty of Veterinary, University of Zabol, Zabol, Iran; 4 Torbat-e Jam Faculty of Medical Sciences, Torbat-e Jam, Iran.

Article Info Abstract

Article history: Introduction of new inhibitory agents such as peptides, heterocyclic derivatives and
nanoparticles (NPs) along with preventive proceedings are effective ways to deal with standard
Received: 06 February 2019 and drug-resistant strains of microorganisms. In this regard, inhibitory activities of some
Accepted: 30 October 2019 recently synthesized 4-thiazolylpyrazoles, imidazolidine- and tetrahydropyrimidine-2-thiones
Available online: 15 June 2021 and magnesium oxide (MgO) NPs alone and in combination with nisin have been assessed
against Aspergillus fumigatus. Antimicrobial susceptibility tests were done via broth
Keywords: microdilution, disk diffusion and streak plate methods according to the modified Clinical and
Laboratory Standards Institute (CLSI) guidelines. Synergistic effects were also determined as
Aspergillus fumigatus fractional inhibitory concentration (FIC) and fractional fungicidal concentration (FFC) values.
MgO nanoparticle Inhibitory potentials of all heterocycles and NPs against A. fumigatus were proved based on
Nisin inhibition zone diameter (IZD) values in the range of 7.72 - 16.85 mm, minimum inhibitory
Synergistic antifungal effect concentration (MIC) values in the range of 64.00 - 512 µg mL-1 and minimum fungicidal
Thiazole concentration (MFC) values in the range of 256 - 2048 µg mL-1. Tetrahydropyrimidine
derivative 3f showed the best inhibitory properties. Inhibitory activity was not significant with
nisin. While antifungal effects of major derivatives were improved by combination with it. The
results indicated that the combined treatment of heterocycles used in the present study with
nisin might be efficient for mold prevention and removal in foodstuffs or other products.
© 2021 Urmia University. All rights reserved.

Introduction rapidly expanding. Researchers recommend identification

and preparation of novel and more efficient antifungal
Aspergillus fumigatus is a saprophytic fungus that agents to treat aspergillosis.3
inhabits in soil and organic residuals. It plays a vital role in Thiazole skeleton is present in many biologically
recycling nitrogen and carbon, and releases many conidia active compounds. This ring exists in vitamin B1, which
in air. Shehu and Bello studied effect of environmental is coenzyme for carboxylase enzyme. Some drugs
factors on the growth of Aspergillus species.1 It was found containing thiazole were applied to treat cancer, high
that the growth of A. fumigatus was increased under blood cholesterol, high blood pressure and AIDS
continuous light, 100% relative humidity and temperature (acquired immune deficiency syndrome).4 Antioxidant,
up to 40.00 ˚C. This pathogenic microorganism is the most anti-inflammatory, antimosquito and antitrypanosomal
important fungal infection risk factor in respiratory properties were observed with thiazole derivatives.5-8
system. Infections can also affect organs such as livers, Antimicrobial potencies of thiazoles were proven
kidneys, eyes, stomach and skin, and increase mortality against bacterial pathogens like Salmonella typhi,
especially in patients with immunodeficiency disorders.2 Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio
Strains of A. fumigatus resistant to traditional antifungal cholerae and Klebsiella pneumoniae and fungi such as
drugs such as itraconazole, isavuconazole, posaconazole, Candida albicans, Cryptococcus neoformans and
voriconazole, isavuconazole and amphotericin B are Aspergillus flavus.9
*Correspondence:
Hamid Beyzaei. PhD
Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran
E-mail: hbeyzaei@uoz.ac.ir
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License which allows users to
read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the
original work is cited properly.
242 H. Beyzaei et al. Veterinary Research Forum. 2021; 12 (2) 241 - 246

There has been a growing interest to synthesize thioamide 4, α-bromocarbonyl compounds 5a-e and
imidazole derivatives due to their inhibitory abilities sodium bicarbonate was stirred in 1 mL N,N-
against tumor cells, Leishmania parasite, Enterococcus dimethylformamide (DMF) at room temperature for 24.00
faecalis, Escherichia coli and S. aureus.10-12 Some - 46.00 hr to afford thiazoles 6a-e .25
imidazolidinyl isoxazole derivatives were prepared, and Synthesis of thiazoles 6a-e:
their fungicidal activities were evaluated on Aspergillus 3-Methyl-4-(4-methylthiazol-2-yl)-1-phenyl-1H-
niger and Rhizopus oryzae.13 pyrazol-5-amine (6a)
Tetrahydropyrimidine derivatives are capable of 1-(2-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-4-
inhibiting growth of Bacillus subtilis, E. coli, Mycobacterium methylthiazol-5-yl)ethan-1-one (6b)
tuberculosis, K. pneumonia and P. aeruginosa.14,15 Several Ethyl 2-(5-amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
derivatives of them were developed as selective muscarinic 4-methylthiazole-5-carboxylate (6c)
agonists for the treatment of Alzheimer's disease.16 In vitro 2-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-
antifungal effects of tetrahydropyrimidine derivatives methylthiazol-4(5H)-one (6d)
were also evaluated on A. niger and C. albicans.17 2-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-
Applications of nanotechnology are expanding in yl)thiazol-4(5H)-one (6e)
various fields of science and extensive amount of Preparation of MgO NPs. Sodium hydroxide (Merck)
researches have been allocated to it.18 The MgO NPs have solution (25.00 mL, 0.008 M) was added dropwise to a
been applied for bone regeneration, pain relieve and the stirred suspension of starch (0.10 g) and magnesium
treatment of cancer and hypertension.19 The MgO NPs are nitrate (12.83 g, 0.10 mol; Merck) in 100 mL distilled
efficient, cost effective and nontoxic antimicrobial agents water. The mixture was left at room temperature for 24 hr
with widespread inhibitory effects on Gram-negative and without stirring. The suspension was centrifuged at 10,000
Gram-positive pathogenic bacteria.20 rpm for 10 min. It was then washed three times using
Nisin is a bacterial peptide with low molecular weight distilled water, then, heated in the furnace at 300 ˚C for 4
of 3510 Dalton. It is used as a food preservative without hr to yield MgO NPs in the range 30.00 - 50.00 nm based
effect on functions of gastrointestinal system and food on the results of X-ray diffraction (XRD) and scanning
flavor. Nisin alone or in combination with other anti- electron microscope (SEM) analysis (Figs. 1 and 2).25
microbial agents can inhibit the growth of microorganisms
like Listeria monocytogenes, S. aureus, Salmonella enterica,
E. coli and Candida lusitaniae.21,22
Biologically importance of N,S-heterocyclic compounds
encouraged us to evaluate inhibitory activities of some
synthesized thiazole, imidazolidine- and tetrahydro-
pyrimidine-2-thione derivatives and MgO NPs alone or in
combination with nisin against A. fumigatus.23

Materials and Methods

General procedure for the synthesis of

imidazolidine- and tetrahydropyrimidine-2-thiones
3a-f. 1.00 mmol of both 1,2- or 1,3-diaminoalkanes 1a-f Fig. 1. XRD spectrum of MgO NPs.
and carbon disulfide (2) and 0.25 mmol of the synthesized
MgO NPs (30 - 50 nm, Zabol, Iran) in 2.00 mL of 96.00%
ethanol (Merck, Darmstadt, Germany) were stirred at
room temperature for 2.50 - 4.00 hr to give imidazolidine-
and tetrahydropyrimidine-2-thiones 3a-f.24
Synthesis of imidazolidine- and tetrahydropyrimidine-
2-thiones 3a-f:
Imidazolidine-2-thione (3a)
4,4-Dimethylimidazolidine-2-thione (3b)
Octahydro-2H-benzo[d]imidazole-2-thione (3c)
Tetrahydropyrimidine-2(1H)-thione (3d)
5,5-Dimethyltetrahydropyrimidine-2(1H)-thione (3e)
4-Ethyltetrahydropyrimidine-2(1H)-thione (3f)
General procedure for the synthesis of thiazoles
6a-e. 1.00 mmol of each compounds including: Fig. 2. SEM image of MgO NPs.
 H. Beyzaei et al. Veterinary Research Forum. 2021; 12 (2) 241 - 246 243

Preparation of initial solutions. Nisin was dissolved Calculation of FIC and FFC values. The synergistic
in sterile 2.00% HCl (Merck) at final concentration 9,011 effect of dual antimicrobial combinations was determined
μg mL-1, incubated in water bath at 80.00 ˚C for 7 min, using the microdilution checkerboard method. Initially,
centrifuged, filtered through a 0.22-μm filter (Millipore, 40.00 μL of SDB was added to all wells of a 64-well plate.
Darmstadt, Germany) and kept at –20.00 ˚C.13 The Then, 25.00 μL of each compound at various concentrations
solutions of all heterocycles were prepared at initial (MIC × 8, MIC × 4, MIC × 2, MIC, MIC / 2, MIC / 4, MIC / 8,
concentration of 9,011 μg mL-1 in 10.00% dimethyl MIC / 16) was added horizontally into all wells of each
sulfoxide (Merck). Ketoconazole (Sigma-Aldrich, Munich, row. Similarly, 25.00 μL of nisin was added vertically into
Germany) as positive control was dissolved in distilled all wells. Finally, 10.00 μL of fungal suspension was added
water at concentration of 17.60 μg mL-1. into them. The plates were incubated under shaking (100
Preparation of the fungal suspension. A. fumigatus rpm) at 37.00 ˚C for 24 hr. The FIC and FFC values were
(PTCC 5009) was prepared from the Persian Type Culture determined and calculated according to MIC and MFC tests
Collection (PTCC), Karaj, Iran. Fungus was cultured on with the following formula:
Sabouraud Dextrose Agar (SDA; HiMedia, Mumbai, India), MIC compound in combination MIC nisin in combination
and incubated for 48 hr at 37.00 ˚C (Fig. 3). Finally, a fungal FIC = +
MIC compound alone MIC nisin alone
suspension with concentration 5.00 × 106 CFU mL-1 in
Sabouraud dextrose broth (SDB; HiMedia) was supplied MFC compound in combination MFC nisin in combination
spectrophotometrically which used as a storage source.25 FFC = +
MFC compound alone MFC nisin alone
Determination of MIC values. 100 μL of SDB was
added into all wells of each row of a 96-well plate. Then, In this experiment, FIC or FFC ≤ 0.50, 0.50 < FIC or
100 μL of initial solutions was added to the first well. After FFC ≤ 0.75, 0.75 < FIC or FFC ≤1.00, 1.00 < FIC or FFC ≤
mixing, serial 2-fold dilutions were continued to the final 4.00 and FIC or FFC ≥ 4.00 indicated synergistic, relative
well of each row. Finally, 10.00 μL of fungal suspension was synergistic, incremental, ineffective and antagonist
added into all wells. As a result, the final concentrations of effects, respectively.27
compounds and ketoconazole were respectively achieved
within range of 4096–32 and 8–0.063 μg mL-1. The plates Results
were incubated under shaking (100 rpm) at 37.00 ˚C for
24 hr. The MICs were detected as the lowest concentration As shown in Table 1, acceptable to good inhibitory
of compounds showing no visible fungal growth.26 effects on A. fumigatus were observed with nisin, MgO NPs
Determination of MFC values. Samples of all invisible and heterocyclic derivatives. The IZD, MIC and MFC values
wells in the MIC test were cultured in SDA and then were found in the range of 7.72 to 16.85 mm, 64.00 to 512
incubated at 37.00 ˚C for another 24 hr. The minimum µg mL-1 and 256 to 2048 µg mL-1, respectively.
fungicidal concentration (MFC) values were determined as Heterocycles 3a-d, 6a, 6c, 6d and MgO NPs displayed
the lowest concentration without colony.26 similar results to block A. fumigatus. The most and the
Measurement of IZD values. 100 μL of fungal least antifungal potentials were belonged to
suspension was spread on SDA. Sterile blank discs were tetrahydropyrimidine 3f and thiazole 6b. Relative
placed on medium. 10.00 μL of initial solutions were poured synergistic effects (FIC and FFC values = 0.75 µg mL-1)
onto disks and the plates were then incubated at 37.00 ˚C were observed by MgO NPs. A variety of interactions was
for 24 hr. Finally, IZDs were measured by caliper.26 observed between heterocycles and nisin according to
their FIC and FFC values. It was determined that antifungal
effects of thiazoles (except 6d) were significantly
improved in combination with nisin.

Discussion

The discovery of new inhibitors against A. fumigatus is

essential. In this study, inhibitory activities of some
synthesized heterocycles and NPs were evaluated against
this pathogen and their interactions were also studied in
combination with nisin.
A variety of interactions was observed between
chemicals and nisin. As expected, nisin alone showed
moderate inhibitory activity against A. fumigatus. Nisin is
often known as an antibacterial agent. It has been found
Fig. 3. Aspergillus fumigatus colonies on SDA plate. that it can block the growth of a variety of Gram-positive
244 H. Beyzaei et al. Veterinary Research Forum. 2021; 12 (2) 241 - 246

Table 1. Antifungal effects of compounds combined with nisin. concentration and pH affect antimicrobial activities of
IZD MIC MFC NPs.35 Inhibitory properties of MgO, CaO and ZnO powders
Compounds FIC FFC
(mm) (μg mL-1) (μg mL-1) have been studied against C. albicans, Saccharomyces
3a 10.22 256 1024 - - cerevisiae, A. niger and Rhizopus stolonifer.36
3b 11.94 256 1024 - - Effective interactions were observed by some thiazole
3c 10.12 256 1024 - - derivatives combined with nisin according to their
3d 11.01 256 1024 - - synergistic effects. These heterocycles as enzyme or
3e 12.10 128 512 - -
protein inhibitors can block the growth of
3f 16.85 64 256 - -
6a 10.35 256 1024 - - microorganisms.37 Substituents such as phenyl, chloro,
6b 7.72 512 2048 - - fluoro, bromo and nitro on thiazole ring improved
6c 10.53 256 1024 - - antimicrobial effects.38 Good to excellent results were
6d 10.61 256 1024 - - reported with thiazoles against A. fumigatus.39,40
6e 14.28 128 512 - - In the present study, synergistic effects on A. fumigatus
MgO NP 10.18 256 1024 - - were recorded with tetrahydropyrimidine derivatives 3d-
Nisin 8.11 512 2048 - - f. They act as channel and surface inhibitors. Some
Ketoconazole 19.68 4.00 8.00 - - synthesized tetrahydropyrimidine derivatives have shown
3a+Nisin - 64.00 256 0.62, R 0.62, R
antifungal effects on A. niger and A. flavus with MICs in the
3b+Nisin - 64.00 512 0.62, R 0.75, R
3c+Nisin - 64.00 512 0.50, S 0.75, R range of 12.50 to 100 µg mL-1.41
3d+Nisin - 64.00 256 0.62, R 0.62, R In our study, imidazolidine derivatives 3a-c also
3e+Nisin - 32.00 256 0.31, S 0.62, R showed synergistic effects on A. fumigatus. It was
3f+Nisin - 16.00 64.00 0.28, S 0.28, S proposed that they could inhibit the synthesis of lipid or
6a+Nisin - 64.00 256 0.37, S 0.37, S dihydrofolate reductase (DHFR) enzyme.42 Antifungal
6b+Nisin - 128 1024 0.50, S 1.00, I effects of some synthetic imidazolidine derivatives on A.
6c+Nisin - 64.00 256 0.37, S 0.37, S fumigatus were increased because of the binding of phenyl
6d+Nisin - 64.00 256 0.62, R 0.62, R groups to their ring.43
6e+Nisin - 32.00 256 0.31, S 0.62, R
To conclude, inhibitory potentials of all tested chemicals
MgO NP+Nisin - 128 512 0.75, R 0.75, R
were proved against standard strains of A. fumigatus;
NP: Nanoparticles, IZD: Inhibition zone diameter, MIC:
Minimum inhibitory concentration, MFC: Minimum fungicidal while their antifungal effects were reinforced in com-
concentration, FIC: Fractional inhibitory concentration, FFC: bination with nisin. These combinations could be used as
Fractional fungicidal concentration, S: Synergistic effect, R: antimicrobial agents to treat fungal infections. In addition,
Relative synergistic effect, I: Incremental effect. nontoxic MgO NPs might be applied as preservatives to
prevent microbial decomposition of food, beverages, bio-
bacteria, however, it is less effective for Gram-negative logical samples, pharmaceutical drugs, cosmetics, paints
bacteria, viruses and fungi due to the presence of outer and wood. The potential of nisin has also been proven to
membrane permeability barrier.28 Antimicrobial effects block one of the most important pathogenic fungi.
of nisin on A. fumigatus have not been studied well so far.
Nisin Z is able to resist oral gingival cells against C. Acknowledgments
albicans.29 Inhibitory properties of nisin and propionic
acid were evaluated on aflatoxin produced by Aspergillus This study was supported by the University of Zabol
parasiticus, Aspergillus ochraceus and Fusarium under Research Project No: IR-UOZ96-10 and Grant No:
moniliforme, and fungistatic activities were improved in a UOZ-GR-9718-9.
special combination of both agents.30 Antifungal effects of
nisin alone and in combination with red ginger essential Conflict of interest
oil (Zingiber officinale var. rubrum) were proved against
A. niger.31 Nisin can reduce or change ATP production The authors declare there are no conflicts of interest.
and the concentration of vital ions through the
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