Executive Summary
A collection of 44 reference, clinical and methicillin-resistant S. aureus isolates was obtained. The collection was tested for susceptibility to tea tree oil, and some preliminary tests were conducted to investigate the production of virulence factors.

Tea tree oil susceptibility data showed that minimum inhibitory concentrations (MICs) ranged from 0.12 – 0.5% (v/v), with 90% of isolates inhibited by 0.5%. Minimum bactericidal concentrations (MBCs) ranged from 0.5 - 2.0% with 90% of isolates killed at or below 2.0%.

In addition to susceptibility testing, some simple agar plate assays were used to characterise (or biotype) the isolates by screening for the production of several enzymes or other characteristics. Of the 44 S. aureus isolates, 34% showed haemolysis on blood agar, 73% showed strong lipase reactions, 16% were positive for lecithinase, and assays investigating pigment production demonstrated that 57% showed yellow pigment, 34% showed cream pigment and 9% showed white.

Before complex assays investigating the production of virulence factors were conducted, experiments to determine how low, or sub-inhibitory concentrations of tea tree oil affected the growth of S.

aureus in vitro were performed. These assays showed that growth in the presence of 0.016 and 0.031% tea tree oil was not markedly different from growth in the absence of tea tree oil, whereas the presence of 0.062% tea tree oil inhibited bacterial growth more substantially. From these experiments it was concluded that concentrations of 0.031% or below would be used for assays investigating the effects of tea tree oil on the production of virulence factors. Additional screening assays were conducted to determine the most appropriate isolates for thorough investigation in each of the virulence factor assays. Those isolates producing the relevant virulence factor at high levels in vitro were selected for further investigation.

The first virulence factor to be investigated in depth was the production of the extracellular enzyme protease. Six isolates were investigated by culturing each organism for 24 h with tea tree oil and removing samples at 4, 8 and 24 h to measure levels of protease in the cell-free supernatant.

Although most isolates showed lower levels of protease when cultured with tea tree oil, reductions were only statistically significant for three of the six isolates, and were not significant at all time points. The presence of tea tree oil did not significantly affect the numbers of viable organisms present at any time point.

The second virulence factor to be investigated was the production of coagulase, another extracellular protein. Coagulase levels for a total of 12 isolates were measured after 16 h incubation in the presence of 0.031% tea tree oil. For four isolates, significantly higher levels of coagulase were produced in the presence of tea tree oil when compared to control cells. For the remaining eight isolates, coagulase levels were either increased (n = 4), decreased (n = 1) or equivalent (n = 3), but these differences were not significant.

The third virulence factor assay investigated the production of extracellular toxins by S. aureus. A total of eight isolates was tested for the production of enterotoxins A, B, C or D and another two isolates were tested for the production of toxic shock syndrome toxin. In general, these experiments did not demonstrate any significant reduction in levels of toxin after growth for 24 h in the presence of 0.016 or 0.031% tea tree oil. As a result, toxin levels after growth in the presence of 0.062% tea tree oil were also investigated. For nine of the 10 isolates toxin levels were reduced when cells were grown with 0.062% tea tree oil, but analysis of viable count data demonstrated a parallel decrease in the numbers of cells present. These decreases in toxin levels may therefore have been a function of reduced cell numbers.

The final characteristic of S. aureus to be investigated was the formation of biofilm on polystyrene surfaces. Six isolates were selected for this assay on the basis of preliminary investigations. The effects of tea tree oil, at concentrations ranging in doubling dilutions from 2 – 0.002% was investigated. Results showed that for most isolates, the presence of 2, 1 and 0.5% tea tree oil completely prevented growth, and very few viable organisms were present at these concentrations.

For two isolates, the formation of biofilm was significantly reduced in the presence of 0.016 – 0.25% tea tree oil. For a third isolate, biofilm formation was significantly inhibited by 0.016, 0.031 and 0.062% tea tree oil, but not by 0.12 and 0.25%. For the fourth isolate, biofilm formation was significantly increased in the presence of 0.062, 0.12 and 0.25% tea tree oil, and for the remaining two isolates no significant changes were seen.

To summarise, these assays produced some interesting and disparate findings. The investigation of extracellular proteases and toxins demonstrated that the production of these particular proteins does not appear to be specifically inhibited by tea tree oil. Instead, reductions were for the most part a function of a decrease in cell density. In contrast, investigation of the protein coagulase indicated that levels were increased in the presence of tea tree oil, although the mechanisms by which this occurred remain unknown. Lastly, for some isolates, the formation of biofilm was significantly reduced in the presence of sub-inhibitory tea tree oil, but for other isolates no such effect was seen. This study has demonstrated that the presence of tea tree oil does affect the production of virulence factors by S.

aureus, but does not necessarily cause a reduction. This work was heavily weighted towards investigating the production of virulence factors that are extracellular proteins. Further studies investigating characteristics that are either distantly or unrelated to the production of extracellular proteins, such as the susceptibility of cells to phagocytosis or changes in adherence characteristics, may reveal more promising results.
 

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