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Applied and Environmental Microbiology, February 2005, p. 797-803, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.797-803.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Antimicrobial Action of Carvacrol at Different Stages of Dual-Species Biofilm Development by Staphylococcus aureus and Salmonella enterica Serovar Typhimurium

J. R. Knowles,^1* S. Roller,^1, D. B. Murray,^1, and A. S. Naidu^1,

South Bank University, London, United Kingdom¹

Received 30 June 2004/ Accepted 1 September 2004

The effects of carvacrol, a natural biocide, on dual-species biofilms formed by Staphylococcus aureus and Salmonella enterica serovar Typhimurium were investigated with a constant-depth film fermentor. Biofilm development reached a quasi-steady state in 12 days at 25°C with S. aureus predominance (99%). Cryosectional analysis detected viable S. aureus and S. enterica serovar Typhimurium at depths of 320 and 180 µm from the film surface, respectively. Carvacrol pulses (1.0 mmol/h) inhibited S. aureus by 2.5 log CFU/biofilm during the early stages of film formation, ultimately causing a significant reduction (P < 0.001) of the staphylococcal population at quasi-steady state. Initial carvacrol pulsing elicited a 3 log CFU/biofilm reduction in viable S. enterica serovar Typhimurium, and additional periodic carvacrol pulses instigated significant inhibition of salmonellae (1 to 2 log CFU/biofilm) during biofilm development. Carvacrol pulsing reduced protein levels fivefold (P < 0.001) during initial biofilm development. Comparative studies with a peroxide-based commercial sanitizer (Spor-Klenz RTU) revealed that this commercial sanitizer was more biocidal than carvacrol during early biofilm development. When the biofilm reached quasi-steady state, however, periodic pulses with 1 mmol of carvacrol per h (P = 0.021) elicited a significantly higher inhibition than Spor-Klenz RTU (P = 0.772). Dual-species microcolonies formed under the influence of continuously fed low carvacrol concentrations (1.0 mmol/h) but failed to develop into a mature quasi-steady-state biofilm and did not reach any stage of film formation in the presence of high concentrations (5.0 mmol/h). These data show that carvacrol is an effective natural intervention to control dual-species biofilm formation.

Present address: Thames Valley University, London, United Kingdom.

Present address: Kitano Symbiotic Systems Biology Project, Tokyo, Japan.

Present address: Center for Antimicrobial Research, California State Polytechnic University, Pomona, CA 91768.

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