Pharmaceutical nanotechnology
Studies on the kinetics of killing and the proposed mechanism of action of microemulsions against fungi

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Abstract

Microemulsions are physically stable oil/water clear dispersions, spontaneously formed and thermodynamically stable. They are composed in most cases of water, oil, surfactant and cosurfactant. Microemulsions are stable, self-preserving antimicrobial agents in their own right. The observed levels of antimicrobial activity associated with microemulsions may be due to the direct effect of the microemulsions themselves on the bacterial cytoplasmic membrane. The aim of this work is to study the growth behaviour of different microbes in presence of certain prepared physically stable microemulsion formulae over extended periods of time. An experiment was designed to study the kinetics of killing of a microemulsion preparation (17.3% Tween-80, 8.5% n-pentanol, 5% isopropyl myristate and 69.2% sterile distilled water) against selected test microorganisms (Candida albicans, Aspergillus niger, Schizosaccharomyces pombe and Rhodotorula spp.). Secondly, an experiment was designed to study the effects of the microemulsion preparation on the cytoplasmic membrane structure and function of selected fungal species by observation of 260 nm component leakage. Finally, the effects of the microemulsion on the fungal membrane structure and function using S. pombe were studied using transmission electron microscopy. The results showed that the prepared microemulsions are stable, effective antimicrobial systems with effective killing rates against C. albicans, A. niger, S. pombe and Rhodotorula spp. The results indicate a proposed mechanism of action of significant anti-membrane activity, resulting in the gross disturbance and dysfunction of the cytoplasmic membrane structure which is followed by cell wall modifications, cytoplasmic coagulation, disruption of intracellular metabolism and cell death.

Introduction

It has been suggested that microemulsions are self-preserving antimicrobial agents in their own right (Friberg, 1984). This suggestion is made on the basis that bacteria cannot survive in pure fat or oil and that water is necessary for their growth and reproduction (Al-Adham et al., 2000). It is suggested that the molecular structure of microemulsions is harmful to the bacterial cell and in particular, that they adversely affect the structure and function of the bacterial cytoplasmic membrane (Jones et al., 1997, Brotoleto et al., 1998).

The work of Al-Adham et al. (2000) clearly indicates the antimicrobial activity of microemulsions against bacterial cells (Pseudomonas aeruginosa and Staphylococcus aureus) over short periods (<45 s). Such rapid biocidal activity is indicative of direct attack on the structural integrity of the cell, rather than secondary effect via metabolic inhibition (Gilbert, 1984). Al-Adham et al. (2000) tested this hypothesis by the use of transmission electron microscopy and the electron micrographs of the microemulsion-treated cells of P. aeruginosa, exhibited both extra- and intra-cellular effects. These micrographs indicate that, as expected, microemulsions have significant anti-membrane activity, resulting in gross disturbance and dysfunction of cytoplasmic membrane structure leading to subsequent internal damage of the cell.

Subsequent studies suggest that microemulsions are highly effective anti-biofilm agents against P. aeruginosa, Salmonella spp., Escherichia coli, S. aureus and Listeria monocytogenes (Al-Adham et al., 2003, Teixeira et al., 2007) and that the antimicrobial activity is dependent upon the position of the microemulsion within its zone of stability (Al-Adham et al., 2012).

Fu et al. (2006) showed that the antifungal activities of microemulsions prepared with glycerol monolaurate (GML) as the oil and Tween as surfactant had higher antimicrobial activity than GML alone. Later work by the same group on a monolaurin microemulsion system exhibited antimicrobial activity against Bacillus subtilis, E. coli, Aspergillus niger and Penicillium digitatum (Fu et al., 2009). Food-grade microemulsions have been of increasing interest to researchers and exhibit great potential in industrial applications (Flanagan and Singh, 2006) where glycerol monolaurate (GML) microemulsions show enhanced antimicrobial activities against S. aureus (Zhang et al., 2007). Food-grade dilution-stable microemulsions are effective antibacterial systems against B. subtilis (Zhang et al., 2008a), E. coli and S. aureus (Zhang et al., 2009). The antifungal activity of a food-grade dilution-stable microemulsion indicates that microemulsions effectively inhibit the growth of moulds A. niger and Penicillium italicum (Zhang et al., 2008b, Zhang et al., 2008c) and the yeast Candida albicans and Saccharomyces cerevisiae (Zhang et al., 2010). These studies have led to our greater understanding and interest in the antimicrobial nature of microemulsions.

However, further studies are required on the kinetics of killing and the proposed mechanism of action of microemulsions against fungal cells. The objective of this study was to investigate the kinetics of killing of selected fungal species C. albicans, A. niger, Schizosaccharomyces pombe and Rhodotorula spp. by a selected microemulsion formula and to study the effects of microemulsion exposure on the cytoplasmic membrane structure and function of the tested fungal species by observing of 260 nm component leakage and by transmission electron microscopy. The fungi used in this study were selected for their characteristics as budding yeasts (C. albicans and Rhodotorula spp.), spore-forming fungi (A. niger) and fission yeasts (S. pombe). C. albicans is a widely studied opportunistic pathogen of humans and a standard test microorganism in antimicrobials testing regimes. A. niger is an occasional human pathogen and allergen and is also included in many antimicrobials test panels. S. pombe is amongst the simplest of yeasts, a fission yeast, and its inclusion in the study was due to this group having a significant record with this organism in antimicrobials activity trials. Rhodotorula spp. is a wild yeast and was included out of general interest.

Section snippets

Chemical reagents

Tween-80, isopropyl myristate (IPM), n-pentanol (GC assay, 99%), sodium hydroxide (NaOH), orthophosphoric acid (H3PO4), and Tris/HCl buffer were purchased from Promega, USA, 2.5% glutaraldehyde, 4% paraformaldehyde, PIPES buffer, osmium tetraoxide, uranyl acetate, propylene oxide, Reynold lead citrate and phosphate buffer were purchase from Sigma, Poole, Dorset, UK.

Preparation of the microemulsion

Oil-in-water (O/W) microemulsion systems were prepared by slowly titrating a weighed series of three component mixtures

Kinetics of killing

Changes in the viability of fungal cells cultures (C. albicans, A. niger, S. pombe and Rhodotorula spp.) were observed over a short period of time after exposure to known concentration of the microemulsion (Fig. 1), which gives clear evidence of a true biocidal activity. The viability decreased rapidly until no viable cells were observed at 2 min for S. pombe and 4 min for Rhodotorula spp. A 6 log cycle reduction of C. albicans was obtained in less than 6 min. The LT90% value (the time taken to

Discussion

The results of the kinetics of killing of microemulsions formula clearly indicate that microemulsions are effective antimicrobial agents, with a rapid killing rate against budding yeasts (C. albicans and Rhodotorula spp.), spore-forming fungi (A. niger) and fission yeasts (S. pombe). Such rapid biocidal activity is indicative of a direct attack on the structural integrity of the cell rather than a secondary effect through metabolic inhibition (Gilbert, 1984). The LT90% value of 58 s for S. pombe

Conclusions

It is clear from this study that the antifungal mode of action of microemulsions is largely due to an initial periplasmic leakage, followed by gross membrane dysfunction, internalization of the microemulsion and subsequent coagulation of cytoplasmic components and organelles. Our results also indicate that exposure to microemulsion leads to cell wall and cytoplasmic membrane leakage, which occurs in two distinct, but overlapping phases.

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