Revista de Ciencias Tecnológicas (RECIT). Volumen 3 (1): 10-22.

Revista de Ciencias Tecnológicas (RECIT). Universidad Autónoma de Baja California ISSN 2594-1925

Volumen 1 (2): 69-71 Octubre-Diciembre 2018 https://doi.org/10.37636/recit.v126971

ISSN: 2594-1925

Effect on antimicrobial activity of antifungal

agents-silver nanoparticles combined treatments

Efecto sobre la actividad antimicrobiana de los agentes antimicóticos:

tratamientos combinados con nanopartículas de plata

Pérez Arriola Yarithza

, Vázquez Muñoz Roberto

2,3

, Bogdanchikova Nina

, Huerta Saquero

Alejandro

Universidad Autónoma de Baja California (UABC); Carretera Tijuana-Ensenada 3917, Playitas, 22860. Ensenada, Baja

California, México

Centro de Nanociencias y Nanotecnología (CNyN); Universidad Nacional Autónoma de México,

Apartado Postal 14, CP 22800; Ensenada, Baja California, México

Centro de Investigación Científica y de Educación Superior de Ensenada, Carretera Tijuana-Ensenada

3918; CP 22860, Ensenada, Baja California, México

Autor de correspondencia: Alejandro Huerta Saquero, Centro de Nanociencias y Nanotecnología (CNyN); Universidad

Nacional Autónoma de México, Apartado Postal 14, CP 22800; Ensenada, Baja California, México. E-mail:

saquero@cnyn.unam.mx. ORCID: 0000-0002-0156-6773

Recibido: 30 de Junio del 2017 Aceptado: 12 de Diciembre del 2017 Publicado: 01 de Enero del 2018

Abstract. - Infectious diseases are a global public health problem; they are among the leading causes

of death worldwide and consume a significant amount of resources. Fungi are the most common

pathogens in humans and animals. Fungal infections incidence has increased more than 200%.

Currently, antifungal agents are used for combating fungal infections, but there are many problems

associated with their use, such as the emergence of resistant organisms, as well as the complexity

related to the development of new antibiotics. In that sense, silver nanoparticles have proven to be an

alternative solution to fight fungal infections due their capacity to inhibit fungal growth. In this work,

we studied the antimicrobial activity of silver nanoparticles (AgNPs), antifungal agents (Amphotericin

B and Fluconazole) and combinations of both, AgNPs-antifungals over the pathogenic dimorphic

yeast Candida albicans. The antimicrobial tests were performed according to the CLSI ́s M27-A3

protocols. Combined AgNPs-antifungal treatments showed a different effect on antimicrobial activity.

We found a synergistic effect of specific combined treatment, whereas antagonistic effect was observed

with other combination.

Keywords: Nanoantibiotics; Silver Nanoparticles; Infectious Diseases; Synergy; Candida albicans.

Resumen. - En este trabajo se plantea la importancia de la estrategia en la organización como factor

detonante para los procesos de innovación y competitividad empresarial. ¿Cuáles son las estrategias

de las empresas para lograr innovación organizacional? “La pregunta fundamental en el campo de

la dirección estratégica es cómo las firmas consiguen y sostienen la ventaja competitiva”. La

investigación se orienta hacia factores de innovación organización como son la productividad y la

flexibilidad laboral, explorando en las organizaciones la relación entre la capacidad tecnológica y

los sistemas de información como variables de la productividad. Así mismo se propone un modelo

matemático para estimar la productividad en base a las variables de capacidad tecnológica y los

sistemas de información.

Palabras clave: Nanoantibióticos; Nanopartículas de plata; Enfermedades infecciosas; Sinergia; Candida albicans.

Revista de Ciencias Tecnológicas (RECIT). Volumen 1 (2): 69-71

ISSN: 2594-1925

1. Introducción

Infectious diseases (ID) –one of the global first causes of death-

are among the most relevant health problems worldwide,

leading to social and economic issues. Also, incidence and

number of ID increase every year[1]. Recently, fungal

infections have increased their morbidity and mortality in

immunocompromised patients, requiring intensive treatment

and broad spectrum antibiotics.

Candida sp., is the most common opportunistic pathogenic

fungi in humans, causing a mortality rate of up to 40 %.

Currently, infection treatments are based on polyenes (i.e.

amphotericin B), triazoles (i.e. fluconazole) and echinocandins

(i.e. caspofungin). Nevertheless, antibiotics face several

problems, such as the evolution of resistant organisms and the

difficulty regarding the development of new antimicrobials.

Silver nanoparticles (AgNPs) exhibit antifungal properties

against both mycelial fungi and yeasts [2]. In that sense,

combined AgNPs-antibiotic treatments could be useful to

eliminate multiresistant microbes.

Understanding the mechanisms of AgNPs antifungal properties

and synergy with antibiotics may lead to improving current

treatments. In this work, we studied the effect on the

antimicrobial activity of AgNPs –when combined with

antifungal agents-, against Candida albicans.

2. Methodology

2.1. Silver nanoparticles (AgNPs)

Argovit AgNPs, functionalized with polyvinylpyrrolidone,

were supplied by Investigation and Production Center Vector-

Vita, Novosibirsk, Russia. Silver nitrate (Sigma-Aldrich®),

was used as a reference solution. Treatments were diluted in

culture medium, on a concentration range of 0.01-1 μg*ml

-1

2.2. Antifungal agents

Commercial antifungal agents were used: Amphotericin B

(ApB) and Fluconazole (Flu), from Sigma-Aldrich®. For

antimicrobial tests, antifungals were diluted in sterile culture

medium, in a concentration range of 0.1-20 μg*ml

-1

Antifungals were selected according their mode of action.

2.3. Fungal strains and culture conditions

The pathogenic, dimorphic yeast Candida albicans strain

(ATCC SC5614) was obtained from the Centro de

Investigación Científica y de Educación Superior de Ensenada

(CICESE). Candida was cultured in RPMI 1640 media for 48

h at 37 ° C, at 180 rpm (standard conditions).

2.4. Determination of Inhibitory Concentrations of

AgNPs and antimicrobials in C. albicans

The antimicrobial activity of all treatments –AgNPs, AgNO

and antifungals- was performed according to the Clinical and

Laboratory Standards Institute (CLSI) M27-A03 protocol,

with some modifications [3]. UV-Vis spectrophotometry was

used to determine partial inhibitory concentrations.

2.5. Effect of AgNPs-antifungal combined treatments

on Candida´s growth

Antimicrobial activity of AgNPs-Antifungal agents

combined treatments was evaluated. C. albicans was

exposed to sub-lethal concentrations of AgNPs, antifungal

agents, and the combination of these. Then, cultures were

incubated into multiwell plates at standard conditions.

Microbial growth was evaluated by UV-Vis

spectrophotometry at λ=530 nm, in a Multiskan Go (Thermo

Scientific®) spectrophotometer.

2.6. Effect of combined AgNPs-antifungal treatments

on the dimorphic transition of C. albicans

C. albicans was exposed to sub-lethal concentrations of

AgNPs, antifungals, and the combination of these. After

cultures incubation under standard conditions, the effects on

dimorphic transition was evaluated by bright-field optical

microscopy.

3. Results and Discussion

The minimal inhibitory concentration (MIC) values,

calculated for μg of metallic silver (active component), are

shown in Table 1. We found that AgNPs MIC is like those of

ionic silver and ten times lower than antifungal tested. The

AgNPs MIC values reported for C. albicans –at similar

culture conditions-, range from 10

-1

-10

μg*ml

-1

[4][5].

Table 1. Minimal Inhibitory Concentrations

Treatments

MIC (μg*ml

-1

)

AgNPs

AgNO

0.5

Flu

ApB

Some reports have suggested that AgNPs improve the

antifungal activity of antibiotics, but no reliable evidence has

been provided. We found that AgNPs-antibiotics combined

treatments activity was different for each antibiotic. For

AgNPs/Flu, we observed an antagonistic effect, while for

AgNPs/ApB, the effect was synergistic (figure 1). It is

important to note that mechanisms of action of these

antifungals are different; while Fluconazole’s target is

Revista de Ciencias Tecnológicas (RECIT). Volumen 1 (2): 69-71

ISSN: 2594-1925

intracellular, Amphotericin B´s target is extracellular. We

assume that changes in antimicrobial potency –in combined

treatments- are due to the mechanism of action of the antifungal

agents influenced by a chemical interaction between them and

AgNPs. Research in this field is being performed by our group.

Figure 1. Antimicrobial activity of combined treatments. A synergistic effect

in the AgNPs+ApB combined treatment was found, meanwhile AgNPs+Flu

combined treatment showed an antagonistic effect.

We observed that none treatment influenced the dimorphic

capacity of C. albicans. The cells treated with both AgNPs and

antifungals –combined and not-combined- kept their ability to

shift into hyphae or pseudohyphae (figure 2). Although it was

reported that AgNPs prevent the dimorphic transition [5]. Our

findings show that C. albicans keeps its ability to change its

form, even in the presence of AgNPs. This is important to

evaluate, because the hyphal phase is its pathogenic stage.

Figure 2. The effect on the dimorphic capacity of C. albicans, treated with sub-

lethal concentration of the antimicrobial agents. A) Control; B) Cells with

AgNPs; C) Cells with ApB; D) Cells with ApB-AgNPs; E) Cells with Flu; and

F) Cells with Flu-AgNPs

4. Conclusions

AgNPs alter the antimicrobial capacity of antifungals.

Synergistic effect was found in the combined treatment of

AgNPs and Amphotericin B, while it was antagonist in AgNPs-

Fluconazole. On the other hand, AgNPs, antifungals, neither

their combination alter the dimorphic capacity of C. albicans

under standard culture conditions.

Acknowledgments

The authors thank Dr. Katrin Quester for her technical

assistance. We also thank PAPIIT project IN204815,

CONACYT project N279889 and to the Red Internacional de

Bionanotecnología for financial.

Referencias

[1] R. Vazquez-Muñoz and A. Huerta-Saquero, "Nanomateriales con

actividad microbicida: una alternativa al uso de antibióticos," Mundo

Nano. Rev. …, vol. 7, no. 13, pp. 37-47, 2014.

https://doi.org/10.22201/ceiich.24485691e.2014.13.48707

[2] O. Brandt, M. Mildner, A. E. Egger, M. Groessl, U. Rix, M. Posch,

B. K. Keppler, C. Strupp, B. Mueller, and G. Stingl, "Nanoscalic silver

possesses broad-spectrum antimicrobial activities and exhibits fewer

toxicological side effects than silver sulfadiazine," Nanomedicine

Nanotechnology, Biol. Med., vol. 8, no. 4, pp. 478-488, 2012.

https://doi.org/10.1016/j.nano.2011.07.005

[3] R. John H. and G. Mahmoud A., M27-A3: Reference Method for

Broth Dilution AST of Yeasts, 3rd ed. AstraZeneca; Case Western

Reserve University, 2008.

https://www.medicine.wisc.edu/sites/default/files/MIC.pdf

[4] C. Longhi, J. P. Santos, A. T. Morey, P. D. Marcato, N. Duran, P.

Pinge-Filho, G. Nakazato, S. F. Yamada-Ogatta, and L. M. Yamauchi,

"Combination of fluconazole with silver nanoparticles produced by

Fusarium oxysporum improves antifungal effect against planktonic

cells and biofilm of drug-resistant Candida albicans," Med. Mycol.,

vol. 54, no. 4, pp. 428-432, 2016.

https://doi.org/10.1093/mmy/myv036

[5] K. Keuk-Jun, S. Sung, S. Moon, J. Choi, J. G. Kim, and D. G. Lee,

"Antifungal Effect of Silver Nanoparticles on Dermatophytes," vol.

18, pp. 1482-1484, 2008.

https://europepmc.org/article/med/18756112

Este texto está protegido por una licencia Creative Commons 4.0

Usted es libre para Compartir —copiar y redistribuir el material en cualquier medio o formato — y Adaptar el documento —remezclar, transformar y crear a partir del material—

para cualquier propósito, incluso para fines comerciales, siempre que cumpla la condición de:

Atribución: Usted debe dar crédito a la obra original de manera adecuada, proporcionar un enlace a la licencia, e indicar si se han realizado cambios. Puede hacerlo en cualquier

forma razonable, pero no de forma tal que sugiera que tiene el apoyo del licenciante o lo recibe por el uso que hace de la obra.

Resumen de licencia - Texto completo de la licencia