Effect of habituation to tea tree (Melaleuca alternifolia) oil on the subsequent susceptibility of Staphylococcus spp. to antimicrobials, triclosan, tea tree oil, terpinen-4-ol and carvacrol

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Abstract

The aim of this study was to seek additional data on the antimicrobial susceptibility of Staphylococcus spp. after habituation to low levels of the topical antimicrobial agent tea tree (Melaleuca alternifolia) oil. Meticillin-susceptible Staphylococcus aureus (MSSA), meticillin-resistant S. aureus (MRSA) and coagulase-negative staphylococci (CoNS) were habituated to 0.075% tea tree oil for 3 days. Subsequently, the susceptibility of five isolates each of MSSA, MRSA and CoNS to fusidic acid, mupirocin, chloramphenicol, linezolid and vancomycin was determined by Etest, and susceptibility to tea tree oil, terpinen-4-ol, carvacrol and triclosan was determined by agar dilution. Following habituation to 0.075% tea tree oil, antimicrobial MICs differed between control and habituated isolates on 33 occasions (out of a possible 150), with MICs being higher in habituated isolates on 22 occasions. Using clinical breakpoint criteria, one MSSA isolate changed susceptibility category from vancomycin-susceptible (MIC = 2 μg/mL) to intermediate susceptibility (MIC = 3 μg/mL) after habituation in one of two replicates. For the non-antibiotic antimicrobial agents, MICs of habituated and control isolates differed on 12 occasions (out of a possible 120); 10 occasions in MRSA and 2 occasions in MSSA. MICs were higher for habituated isolates on five occasions. However, all the differences were one serial dilution only and were not regarded as significant. Habituation to sublethal concentrations of tea tree oil led to minor changes in MICs of antimicrobial agents, only one of which may have been clinically relevant. There is no evidence to suggest that tea tree oil induces resistance to antimicrobial agents.

Introduction

The need for novel antimicrobial agents is particularly pressing owing to the continued occurrence and spread of resistance to existing agents. Most welcome would be agents from different chemical classes that have diverse mechanisms of action. One possibility is tea tree oil, the essential oil of the Australian native plant Melaleuca alternifolia (Myrtaceae). It has broad-spectrum in vitro activity against Gram-positive and Gram-negative bacteria, including antimicrobial-resistant and multiresistant organisms [1], [2], [3]. Minimum inhibitory concentrations (MICs) are generally in the range 0.12–0.5% and minimum bactericidal concentrations range from 0.12% to 1%. It is used as a topical antimicrobial agent [4] and its use for decolonisation of meticillin-resistant Staphylococcus aureus (MRSA) carriers has attracted particular attention [5], [6], [7].

Ironically, two reports have suggested that exposure to tea tree oil may contribute to the development of antimicrobial resistance in human pathogens [8], [9]. In these reports, bacteria exposed to low, subinhibitory levels of tea tree oil displayed subsequent increases in their MICs to tea tree oil and several antimicrobials. Similar issues have dogged the promotion of products containing triclosan (2,4,4′-trichloro-2′-hydroxydiphenylether) and quaternary ammonium compounds for use in domestic and personal care settings [10], [11]. Like triclosan, tea tree oil is also widely available in a large range of personal care, cosmetic and over-the-counter products. Many tea tree oil products are formulated for non-therapeutic uses and the concentrations of oil in them may not inhibit or kill bacteria. It is this low-level exposure that is alleged to promote resistance to tea tree oil and other antimicrobial agents [8], [9]. Therefore, it is important to clarify whether exposure of bacteria to subinhibitory levels of tea tree oil alters susceptibility to other antimicrobial agents. The aim of this study was to provide additional data on the antimicrobial susceptibility of Staphylococcus spp. habituated to tea tree oil.

Section snippets

Antimicrobial agents

Tea tree oil (batch no. 1216), used throughout the study, was kindly provided by P. Guinane Pty. Ltd. (Kingscliff, NSW, Australia). Concentrations of the components determined by gas chromatographic analysis (and the range specified by the international standard [12], shown in parentheses) were as follows: 2.4% α-pinene (1–6%); 0.3% sabinene (trace to 3.5%); 9.0% α-terpinene (5–13%); 1.1% limonene (0.5–1.5%); 3.1% p-cymene (0.5–8%); 3.7% 1,8-cineole (trace to 15%); 20.1% γ-terpinene (10–28%);

Habituation to tea tree oil

After failure of bacteria to grow in 0.25% tea tree oil using the method of McMahon et al. [9], two variations of the method were used. The heavier inoculum and change of medium (variation 1) did not greatly alter the outcome, with most isolates not surviving the serial subculture process. Representative results are shown in Table 1. On the other occasion (data not shown) only four MSSA isolates, one MRSA and two CoNS grew in the third passage with 0.25% tea tree oil yielding a turbid culture.

Discussion

The inability to serially subculture 30 staphylococci in 0.25% tea tree oil as performed previously [9] was unexpected. In marked contrast to their success at culturing 30 staphylococcal isolates in 0.25% tea tree oil, numerous attempts to perform this habituation procedure failed in our hands, mostly resulting in a lack of viable organisms after one or two passages. Although their method was used by us initially, an obvious difference between the tests was the bacterial strains used, which

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    Present address: bioMérieux, La Balme Microbiology Unit, Global Director Microbiology Research, 3 Route de Port Michaud, 38390 La Balme-les-Grottes, France.

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