Inhibition and eradication of Staphylococcus aureus biofilms with conventional antibiotic is difficult, and the treatment is further complicated by the rise of antibiotic resistance among staphylococci. Consequently, there is a need for novel antimicrobials that can treat biofilm-related infections and decrease antibiotics burden. Natural compounds such as eugenol with anti-microbial properties are attractive agents that could reduce the use of conventional antibiotics. In this study we evaluated the effect of eugenol on MRSA and MSSA biofilms in vitro and bacterial colonization in vivo. Effect of eugenol on in vitro biofilm and in vivo colonization were studied using microtiter plate assay and otitis media-rat model respectively. The architecture of in vitro biofilms and in vivo colonization of bacteria was viewed with SEM. Real-time RT-PCR was used to study gene expression. Check board method was used to study the synergistic effects of eugenol and carvacrol on established biofilms. Eugenol significantly inhibited biofilms growth of MRSA and MSSA in vitro in a concentration-dependent manner. Eugenol at MIC or 2×MIC effectively eradicated the pre-established biofilms of MRSA and MSSA clinical strains. In vivo, sub-MIC of eugenol significantly decreased 88% S. aureus colonization in rat middle ear. Eugenol was observed to damage the cell-membrane and cause a leakage of the cell contents. At sub-inhibitory concentration, it decreases the expression of biofilm-and enterotoxin-related genes. Eugenol showed a synergistic effect with carvacrol on the eradication of pre-established biofilms. This study demonstrated that eugenol exhibits notable activity against MRSA and MSSA clinical strains biofilms. Eugenol inhibited biofilm formation, disrupted the cell-to-cell connections, detached the existing biofilms, and killed the bacteria in biofilms of both MRSA and MSSA with equal effectiveness. Therefore, eugenol may be used to control or eradicate S. aureus biofilm-related infections.

The alpha 9 and alpha 10 cholinergic nicotinic receptor subunits assemble to form the receptor that mediates efferent inhibition of hair cell function within the auditory sensory organ, a mechanism thought to modulate the dynamic range of hearing. In contrast to all nicotinic receptors, which serve excitatory neurotransmission, the activation of alpha 9 alpha 10 produces hyperpolarization of hair cells. An evolutionary analysis has shown that the alpha 10 subunit exhibits signatures of positive selection only along the mammalian lineage, strongly suggesting the acquisition of a unique function. To establish whether mammalian alpha 9 alpha 10 receptors have acquired distinct functional properties as a consequence of this evolutionary pressure, we compared the properties of rat and chicken recombinant and native alpha 9 alpha 10 receptors. Our main finding in the present work is that, in contrast to the high (pCa(2+)/pMonovalents similar to 10) Ca2+ permeability reported for rat alpha 9 alpha 10 receptors, recombinant and native chicken alpha 9 alpha 10 receptors have a much lower permeability (similar to 2) to this cation, comparable to that of neuronal alpha 4 beta 2 receptors. Moreover, we show that, in contrast to alpha 10, alpha 7 as well as alpha 4 and beta 2 nicotinic subunits are under purifying selection in vertebrates, consistent with the conserved Ca2+ permeability reported across species. These results have important consequences for the activation of signaling cascades that lead to hyperpolarization of hair cells after alpha 9 alpha 10 gating at the cholinergic-hair cell synapse. In addition, they suggest that high Ca2+ permeability of the alpha 9 alpha 10 cholinergic nicotinic receptor might have evolved together with other features that have given the mammalian ear an expanded high-frequency sensitivity.