Phillip J. Collier

Microemulsions are membrane-active, antimicrobial, self-preserving systems

Microemulsions are physically stable oil/water systems that have potential use as delivery systems for many pharmaceuticals which are normally of limited use due to their hydrophobicity, toxicity or inability to access the site of action. It has been suggested that microemulsions are self‐preserving antimicrobials in their own right, although there is little evidence to support this. In this experiment, microemulsions of various compositions were formulated and tested for their stability and antimicrobial action. The physical stability of the different microemulsions was assessed by centrifugation at 4000 g and by storage in a water bath at 37 °C for one month, during which no phase separation was observed. The antimicrobial activity of the microemulsions was tested using the compendial method, observation of the kinetics of killing, and transmission electron microscopy (TEM) of microemulsion‐exposed cultures of Pseudomonas aeruginosa PA01. These latter experiments on Ps. aeruginosa indicated distinct signs of membrane disruption. The results indicated that the microemulsions are self‐preserved, and that their killing of microbial cultures is very rapid and may be the result of membrane activity.

Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1.

Benzisothiazolone (BIT), N‐methylisothiazolone (MIT) and 5‐chloro‐N‐methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub‐minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol‐interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T‐OMP, in SDS‐PAGE preparations. T‐OMP was observed to disappear from the biocide‐exposed preparations and reappear when the resistance‐induced cultures were passaged in the absence of biocide. This reappearance of T‐OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross‐resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T‐OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon™ form (CMIT and MIT).

Triclosan – an update

The discovery in 1998 that triclosan has a site‐specific action in the bacterial cell as an inhibitor of NADH‐ or NADPH‐dependent enoyl‐acyl carrier protein reductase led to a lively debate in the scientific press. The thesis of this debate was that such a mode of action may allow triclosan to induce resistance and cross‐resistance in bacterial cells. The debate last saw review in 2004, and this paper aims at updating our knowledge in this area, given recent research on the topic.

Microemulsions are highly effective anti-biofilm agents

Aims: The demonstration of the antibiofilm effects of pharmaceutical microemulsions.

The effect of subminimal inhibitory concentrations of antibiotics on virulence factors expressed by Staphylococcus aureus biofilms

Aims:  The effect of subminimal inhibitory concentrations (sub‐MICs) of cefalexin, ciprofloxacin and roxithromycin was investigated on some virulence factors [e.g. coagulase, Toxic Shock Syndrome Toxin 1 (TSST‐1) and biofilm formation] expressed by Staphylococcus aureus biofilms.

Carbon and nitrogen removal in a granular bed baffled reactor

The application of an anaerobic five compartment granular bed baffled reactor (GRABBR) was investigated with brewery wastewater for combined carbon and nitrate removal, with a separate downstream nitrification unit for converting ammonia to nitrate. The GRABBR was operated at an organic loading rate of 3.57 kg chemical oxygen demand (COD) m−3 d−1 and ammoniacal nitrogen (NH4-N) loading rate of 0.13 kg NH4-N m−3 d−1 when nitrified effluent from a downstream nitrification unit was recycled to the feed point of the GRABBR. Carbonaceous matter and nitrate were removed simultaneously in the GRABBR at different recycle to influent ratios (from 1 to 2), with nitrogen oxide (nitrate and nitrite nitrogen, NOx-N) loading rates varying from 0.04 to 0.05 kg NOx-N m−3 d−1. At all recycle to influent ratios, COD removal efficiency of 97% to 98% were observed in the GRABBR, and over 99% by the two-stage treatment configuration (i.e. GRABBR and nitrification unit). All the nitrates added to the GRABBR were denitrified in the first three compartments of the system. For all the recycle to influent ratios studied, almost all ammonia was converted to nitrate nitrogen with only small traces of nitrite nitrogen in the nitrification unit. Methane production was observed throughout the experimental period with its composition varying from 25% to 50%, showing that simultaneous methanogenesis and denitrification occurred. This study shows that a GRABBR could bring about a high degree of carbon and nitrate removal, with simultaneous methanogenesis and denitrification, due to plug flow granular bed multi-stage characteristics of the bioreactor.

Antimicrobial susceptibility changes and T-OMP shifts in pyrithione-passaged planktonic cultures of Pseudomonas aeruginosa PAO1.

Aims: The aim of this study was to determine whether passaging Pseudomonas aeruginosa PAO1 with sub‐MICs of the pyrithione biocides results in both the induction of decreased susceptibility towards these antimicrobials and associated outer membrane profile changes.

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

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.

A new chemically‐defined medium for Bacillus subtilis (168) NCIMB 12900

J. LEITCH AND P.J. COLLIER. 1996. Most chemically‐defined media for the growth of Bacillus subtilis 168 in batch culture are unsuitable for use in nutrient limitation experiments due to either their low cell density yield or their high numbers of added amino‐acids. The authors have developed a medium which gives relatively high cell densities and is highly chemically‐defined, containing only one added amino‐acid. Growth of B. subtilis in this new medium was observed over time using optical density measurements at 470 nm. The growth curve exhibited entry into exponential phase after only 1–2 h and stationary phase after 8 h. Cell density yields and comparison of growth rates with B. subtilis grown in Fang and Demain Medium (1989) suggest that this new medium is highly suitable for the growth of this micro‐organism under both normal and nutrient limited conditions.

The Antimicrobial Activity of Oil-in-Water Microemulsions is predicted by their position within the Microemulsion Stability Zone

Background: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Despite the predominance of two capsular polysaccharides, types 5 and 8, on the surface of clinical isolates, molecular and epidemiological variations have been reported among various geographical areas in addition to various clinical outcomes of S. aureus increasing the challenge for preventive and control measurements against staphylococcal infections and diseases. Materials and Methods: A total of 315 S. aureus isolates of various clinical conditions comprised both hospital and community-acquired infections between 2009 and 2011 in Amman and Gaza were studied. All S. aureus clinical isolates included in the present study have been investigated by PCR method to determine the distribution and diversity of capsule polysaccharide expression. Results: Most of the clinical isolates (90%) expressedeither capsular polysaccharide genotypes CP5 (37%) andCP8 (53%), whereas 10% were nontypeable by PCR. Significant predominance of CP8 genotype was reported in clinical isolates of S. aureus from infections in comparison to those which are commonly colonising various normal body sites. THE INTERNATIONAL ARABIC JOURNAL OF ANTIMICROBIAL AGENTS 2014 Vol. 4 No. 2:4 doi: 10.3823/750 This article is available from: www.iajaa.org 2 Received 18 July 2014; accepted 22 August 2014 Introduction Staphylococcus aureus continues to be a major pathogen causing a wide spectrum of infections with high morbidity and mortality (1). Moreover, increased concern on this pathogen emerged from its resistance against virtually all antimicrobial agents available in hospitals and communities (2). The adaptation of S. aureus to the environment has been marked by the acquisition of methicillin-resistant S. aureus (MRSA) and the emergence of multidrug resistance, making the new strategies to manage staphylococcal infections of major importance in clinical practice.Our understanding of virulence and epidemiological behaviour of this species has been greatly enhanced by sequencing of the genomes of several S. aureus strains. For example, the identification of new non-protein coding RNA genes and the presence of mutations within several metabolic genes have been shown to be involved in phenotypic switching and antibiotic resistance of S. aureus(3,4) . S. aureus is equipped with various virulence factors, including surface-associated adhesins, cytotoxins, superantigens, exoenzymes, and capsularpolysaccharides, which contribute to the pathogenesis of staphylococcal infections (5). The capsular polysaccharide is a cell wall bacterial component that protects bacterium from phagocytosis and enhances microbial virulence (6). Approximately 90% of S. aureus isolates produce one of 11 capsular polysaccharides nevertheless, only two types,type 5 and type 8, are clinically relevant since they are predominant among clinical infection isolates of various geographic origins (7-9). The importance and relevance of these capsule types and their role in septic shock were confirmed by the development of a conjugate vaccine, StaphVAX study (10). In addition, the formation of biofilms in S. aureus is enhanced by augmenting adhesiveness due to both intercellular polysaccharide adhesion and capsular polysaccharides (11). The emergence of resistance to antibiotics through the formation of permeability barrier to antimicrobial agents has been reported in S. aureus and other bacteria(12,13).For example, it has been shown that vancomycin-intermediate resistant S. aureus (VISA) strains produce a distinctly thickened cell wall (14). Few studies were carried out to investigate the potential role of S.aureus polysaccharide capsule as a permeability barrier to several antimicrobial agents(15-16). Generally, although the production of thick capsules in certain serotypes of S.aureus showed a permeability barrier potential to common biocides and affected the susceptibility of S.aureus to some antibiotics, the results are still controversial. Due to the lack of epidemiological and clinical Conclusion: Correlation between capsule genotypes and pathogenic behaviour of S. aureus could be established. Further studies on the molecular levels are required to determine the role and mechanisms of capsular expression in the variability of pathogenic potential of S. aureus.It has been shown previously that thermodynamically stable oil-in-water microemulsions have significant antimicrobial activity against planktonic cells and biofilm cells over short periods of exposure. It was the aim of this study to identify whether the position of the microemulsion within the microemulsion stability zone of the pseudo-ternary phase structure predicts the efficiency of the antimicrobial action of the microemulsion. Microemulsions were formulated at different points within the microemulsion stability zone. Experiments were performed to observe the kinetics of killing of these microemulsions against selected test microorganisms ( Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, Staphylococcus aureus ATCC 6538 and Aspergillus niger ATCC 16404). The results indicated that the antimicrobial activity of the microemulsion is dependant upon its position within the zone of stability and is greater nearer the centre of that zone. The results indicate that significant antimicrobial activity can be observed at all points within the zone of microemulsion stability, but that maximal activity is to be found at the centre of that area.