Elsevier

Microbial Pathogenesis

Volume 93, April 2016, Pages 120-125
Microbial Pathogenesis

Influence of Melaleuca alternifolia oil nanoparticles on aspects of Pseudomonas aeruginosa biofilm

https://doi.org/10.1016/j.micpath.2016.01.019Get rights and content

Highlights

  • Development of nanoparticles with Melaleuca alternifolia oil.

  • Lower minimal inhibitory concentration of nanoparticles against Pseudomonas aeruginosa.

  • Inhibition of adhesion of P. aeruginosa treated with nanoparticles.

  • Decrease of motility of P. aeruginosa exposed to M. alternifolia oil nanoparticles.

  • Antibiofilm activity of nanoparticles against P. aeruginosa.

Abstract

The Pseudomonas aeruginosa is a gram-negative bacillus and frequent cause of infection. This microorganism is resistant intrinsically to various drugs. The P. aeruginosa is associated with the biofilm formation, which causes worsen the prognosis and difficulty the treatment. The influence of Melaleuca alternifolia oil or “tree of tee” oil (TTO) and TTO nanoparticles on adhesion of P. aeruginosa in buccal epithelial cells was investigated. Also was determined the antimicrobial and antibiofilm activity against this microorganism. The TTO nanoparticles were produced by deposition of preformed polymer and the physic-chemical properties of nanoparticles were measured by electrophoresis and dynamic light scattering. The characterization of nanoparticle showed acceptable values for diameter and zeta potential. The evaluation of antimicrobial and antibiofilm activity against P. aeruginosa PAO1 was performed by microdilution indicating the minimal inhibitory concentration, and the potential antibiofilm. It was verified the action on virulence factors such the motility, besides the influence on adhesion in buccal epithelial cells. Both oil and nanoparticles showed a decrease in adhesion of microorganisms to buccal cells, decrease of biofilm and interfering on P. aeruginosa PAO1 motility. The nanostructuration of TTO, shows be a viable alternative against formed biofilm microorganisms.

Introduction

The Pseudomonas aeruginosa is a Gram-negative bacillus no glucose fermenter which have large capacity to adapt and survive in unfavorable environmental conditions, with minimal requirements to survival, became one of the main pathogens associated with nosocomial infections. Even with the progress of antimicrobial therapies, infections by P. aeruginosa still worrying, cause shows a mortality between 18% and 61% of cases [1], [2].

The majorities of microorganisms live in communities known as biofilm, which instead of planktonic cells, the biofilm cells develop in an extracellular matrix that acts like a shelter to the microorganisms, besides protection against the antimicrobials and host defense cells of immunologic system. Are easily developed on damage tissues and abiotic surfaces, such catheter and incubation tubes [3], [4].

The biofilm infections in hospital environment are a serious problem of public health and many methods has been used to try minimize or eliminate them. The great difficult lies in the fact of that many of these methods have important disadvantages, because lead to clinical complications and develop strains multi resistant [5].

The use of medicinal plants such medications have grown in last decades due the increase of resistant microorganisms [6], [7]. The essential oils show a great group of antimicrobial compounds, which are complex mixtures of volatile secondary metabolites. Shows antimicrobial and antifungal properties, used in food industry such preservatives against pathogens of food source. The oil essential components may increase the antimicrobial potential of some drugs, to act for synergy [8].

The Melaleuca alternifolia, also known of “tree of tea” (TTO) was used such anti septic solution during decades, the essential oil contain a diversity of components, being the majority monoterpenes and alcohols. Show a minimum content of 30% of terpinen-4-ol and maximum content of 15% of 1,8-cineol. The terpenen-4-ol shows antimicrobial and anti-inflammatory properties, whereas the 1,8-cineol probably is an undesirable allergen in M. alternifolia products [9]. Studies show that the TTO formulations can be effective on treatment of acne and fungal infections, besides act such antibacterial agent [10]. These antimicrobial activities are attributed by protein denaturation, changing the properties and membrane function of cellular wall, causing the loss of intracellular components, resulting in cellular death [11], [12].

Despite exist a great number of researches to find a solution for the biofilm infections, the pharmaceutical industry is no alternative, because the existing drugs are ineffective on treatment of these infections. However, exist many reports in the literature which show that the nanostructuration technology of some medicinal plants increase the antimicrobial potential of these products [13], [14]. The nanotechnology is considered an important resource in development of new formulations with better capacity of encapsulation, where the drugs act on site of interest and have the capacity to remain for long periods in bloodstream [15]. The nanotechnology has many advantages, especially on development of drug carries and your controlled release. The fact of show a reduced size, the nanoparticles can increase the antimicrobial potent of TTO. Studies performed by Flores et al. [16] found that the TTO have a good nanostructured character and adequate physical-chemical stability. Moreover, the nanoparticles protect the evaporation of oil and increase the stability. In this context, this study aimed to analyze the action of oil and nanoparticles containing TTO (N TTO) in adhesion of P. aeruginosa PA01 on buccal epithelial cells besides evaluate the antibiofilm activity.

Section snippets

TTO and TTO nanoparticles

The TTO was purchased from Delaware (Brazil) and nanoparticles containing TTO were obtained from Inventiva (Porto Alegre, Brazil). Briefly, nanostructured lipid carriers were prepared with 7.5% (w/v) of TTO using a proprietary method from Inventiva, based on high pressure homogenization. Acetyl palmitate was used as solid lipid and polisorbate 80 as surfactant. Total solid content was 18.6% (w/v).

Physical-chemical parameters of TTO nanoparticles

The determination of diameter and polydispersion index of nanoparticles were performed by dynamic

Physicochemical parameters of nanoparticles containing M. alternifolia oil

The formulation was evaluated in relation of physicochemical properties. The measurements showed values of pH, mean diameter, polydispersion index and zeta potential. The values were 6.3 ± 0.3 to pH, mean diameter about 150.2 ± 2 nm, polydispersion index of 0.213 ± 0.017 and zeta potential of −8.69 ± 0.80 mV.

Nanoparticle tracking analysis

The M. alternifolia nanoparticles showed a mean size of 166 ± 29 nm (Fig. 1). This result is in accordance with analysis in Zetasizer (item 3.1.)

Determination of minimal inhibitory concentration (MIC)

After add 2,3,5 tripheniltetrazolium

Discussion

The results of characterization of nanoparticles indicate an adequate homogeneity, because when works with nanometric particles, indifferently of used method for obtainment, all formulations must be monodisperse (PDI < 0.25) and diameter smaller than 300 nm. Moreover, the result of zeta potential is satisfactory, because when the nanostructure show a negative charge, the system stability tend to be superior with a lower probability for particles aggregation [20], [21].

There are advantages of

Conclusion

In conclusion, the nanostructuration of compounds, such M. alternifolia oil, can be a viable alternative against formed biofilm microorganisms. The nanoparticles besides decrease the biofilm production of P. aeruginosa PA01, decreased the bacterial adhesion on epithelial cells. Therefore more studies must be performed to clarify the role of these nanostructures on cell wall and your mechanism of action on specific process during bacterial infection.

Competing interests

The authors declare that they have no competing interests.

Acknowledgment

This work received financial support of PPGPE/Centro Universitário Franciscano-Probic, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and FAPERGS (Fundação de Amparo a Pesquisa do Rio Grande do Sul).

The authors thank NanoBioss Laboratory (SisNANO/MCTI) at University of Campinas for NTA facility.

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