Thymol: Inhibitory Activity on Escherichia coli and Staphylococcus aureus Adhesion to Human Vaginal Cells

Journal of Essential Oil Research: JEOR, Jul/Aug 2006 by Sasso, Monica Dal, Culici, Maria, Braga, Pier C, Guffanti, Enrico E, Mucci, Manuela

Abstract

Adhesion is an important starting event in the pathogenesis of bacterial infection because the microorganisms must first adhere to host tissue in order to multiply and create a colony or colonies before specific symptoms allow the disease process to be detected. This is particularly true in the case of female urogenital infections, including urinary tract infections, bacterial vaginosis and vaginitis. Thymol is a component of thyme essential oil, which has been reported to possess interesting antimicrobial effects on various microorganisms; however, its ability to interact with the adhesion of bacteria (an important determinant of bacterial virulence) has not been investigated.

The aim of this study was to assess whether thymol interferes with the adhesion of Escherichia coli and Staphylococcus aureus to human vaginal epithelial cells. The adhesiveness of three strains of E. coli to vaginal cells was significantly reduced at thymol concentrations ranging from 1/2 MIC to 1/32 MIC, and in three strains of S. aureus at concentrations ranging from 1/2 MIC to 1/16 MIC.

Sub-MICs down to 1/8 MIC also significantly reduced the hemagglutination of E. coli, which is correlated with fimbriation and thus provides a clue relating to the interference of thymol, a phenolic structure with an hydroxyl group, on the physico-chemical characteristics of the outer surface of bacteria. This is of interest for the strategy of protecting against vaginitis or vaginosis using drugs other than antibiotics.

Key Word Index

Thymol, bacterial adhesiveness, Escherichia coli, Staphylococcus aureus, human vaginal cells.

Introduction

It has been empirically recognized for centuries that plant essential oils and extracts interfere with microorganisms in various ways (1). Interest in the use of natural substances, particularly essential oils and their typical components, for various applications has increased in recent years. In the pharmacological field (1,2), the antimicrobial properties of essential oils and their constituents against various bacteria and fungi have been assessed and confirmed (3-5).

Bacterial strategies to survive, grow and form colonies are based on physico-chemical interactions between the bacteria and surrounding surfaces. Human urogenital, respiratory and intestinal mucosal surfaces are the most common portals of entry for bacteria. The interactions between bacteria and the mucosal surfaces of the body has received much attention because almost all infections caused by bacteria or fungi require the microorganisms to create a colony or colonies before the clinical signs of the disease can be detected; but to create a colony and thus infection, microorganisms must first adhere to host tissue through specific ligand-receptor interactions.

The thyme oil obtained from plants of the genus Thymus, which belongs to the family of Labiatae or Lamiaceae, has been credited with a long list of pharmacological properties (6,7), including antimicrobial effects as described by several authors (8-12). Thymol is a constituent of thyme oil and has considerable antimicrobial effects on various microorganisms (13-15), but its ability to interact with an important determinant of bacterial virulence such as the bacterial adhesion has not been investigated. The aim of this study was to assess whether thymol interferes with the adhesion of Escherichia coli and Staphylococcus aureus to human vaginal epithelial cells.

Materials and Methods

Patients: The vaginal epithelial cells were obtained from healthy, non-pregnant, regularly menstruating women, aged 25-49 years. A clinical examination and microscopy of a wet smear of vaginal contents revealed no signs of genital infections. The cell specimens were obtained on various days of the menstrual cycle except during menstrual bleeding. None of the women used vaginal douches or tablets, nor did any of them use oral or local chemical contraceptives. They were all sexually active.

Collection of vaginal epithelial cells: The cells were obtained from the mucosal surface of the midvaginal wall by gentle scraping with a sterile spatula that was then immediately twirled in 2 mL of phosphate-buffered saline (PBS) (0.02 M phosphate and 0.15 M NaCl, pH 7.3). In order to dislodge the cells, the suspensions were passed four times through a 150 µm diameter needle to break up epithelial cell clumps. The cell suspensions obtained from four subjects were pooled, washed three times to free them of debris and nonadherent bacteria by means of low-speed centrifugation (260 g, 10 min, 25°C), and frozen in aliquots at -70°C. For the adhesion assays, the cells were thawed quickly and washed again, after which PBS was added to the suspensions in order to reach a concentration of 3x10^sup 5^ cells/mL as determined by direct microscopic counts (interference contrast microscopy) in a Biirker chamber (Passoni, Milan, Italy).

Bacterial and culture condition: The effects of different thymol concentrations were tested on two strains of E. coli isolated from human urinary infections, one E. coli ATCC 25922, two strains of S. aureus isolated from human respiratory infections, and one S. aureus ATCC 25923. Suspensions of each organism were prepared from overnight cultures in tryptic soy broth (Sigma, Milan, Italy) at 37°C under static conditions. The organisms were harvested, washed three times in PBS, and adjusted to 3x10^sup 8^ organisms/mL as determined by direct microscopic counts in a Petroff-Hausser chamber (Thomas Scientific, Swedesboro, NJ, USA).