Browsing by Author "McKechnie, K."

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    Development and characterization of a long-term murine model of Streptococcus pneumoniae infection of the lower airways
    (American Society of Microbiology, 2014) Haste, L.; Hulland, K.; Bolton, S.; Yesilkaya, H.; McKechnie, K.; Andrew, P. W.
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    Is the antimicrobial mechanism of action of essential oils against bacteria associated with channels similar to TRPV1 channels found in mammalian cells?
    (British Society for Antimicrobial Chemotherapy, 2018-01-18) Alhareth, Z; Owen, L.; Dixon, C. J.; McKechnie, K.; Smith, Laura J.; Laird, Katie
    Objectives: The aim of this investigation was to find out if the antimicrobial mechanism of action of Essential Oils (EOs) against Enterococcus spp. may be associated with channels similar to TRPV1 channels found in mammalian cells. A known antimicrobial combination of EOs compounds (Carvacrol 0.2µl and Cuminaldehyde 25µl) against E. faecium and capsaicin were used to investigate this hypothesis. The bacterial membrane has many porin proteins which act as hydrophilic transmembrane channels and allow small hydrophilic solutes to pass through them1, such channels may be the target for the antimicrobial mechanism of action of EOs in the Gram-positive bacterial cell wall. Methods: Transient receptor potential (TRP) channels, are a group of unique ion channels in mammalian cells, that are effected by a wide spectrum of physical and chemical stimuli2, including high temperature and low pH. Capsaicin the EO compound of chilli peppers is known to be a TRP channel antagonist in mammalian cells2,3 whilst AMG 517 specifically blocks TRP channels, and Ruthenium Red (RR) is a wide spectrum blocker for TRPV1 channels4,5. The Minimum Inhibitory Concentration (MIC) of capsaicin against Vancomycin Sensitive E. faecium (VSE) was defined by agar-broth dilution method, a checkerboard assay was conducted to determine the interactions of AMG517 and RR against E. faecium. A growth curve was also conducted to assess the effect of the EO blend in the presence of AMG517. Results: Capsaicin showed antibacterial activity against E. faecium with an MIC of 1.6mM. The checkerboard data demonstrated that AMG517 has no inhibitory effect on E. faecium and therefore was a good candidate to reverse the effect of EOs blend by potentially blocking channels that were similar to TRPV1. RR was antimicrobial at concentrations above 10µM, however, AMG517 above 60nM reverses the inhibitory effect of RR, demonstrating a competitive effect of AMG517 and RR towards a definite site in the bacterial cell wall. Growth curves showed no reverse effect of the EO blend in the presence of AMG517. Conclusions: This study does not support the existence of a TRPVI homologue in the bacterial cell. Future studies will investigate the genes related to transport channels in E. faecium, that are differentially regulated when exposed to the EO blend, in order to further understand the effect of the EOs on transport channels in bacteria.
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    The mechanism of antibacterial action of Essential Oils against Enterococcus faecium: the role of transport channels
    (2018-11) Alhareth, Z; Owen, L.; Dixon, C. J.; McKechnie, K.; Laird, Katie; Smith, I.
    Aims The overall aim of this study is to establish the mechanisms of action of a novel formulation of plant extracts known to be effective against Vancomycin Resistant E. faecium (VRE) isolates. Channels in the bacterial cell wall were assessed to see if they act in a similar way to mammalian cell Transient Receptor Potential Channels (TRPV1) that are known to be activated by the EOs and capsaicin. Methods and results Growth curves and viable counts were conducted for E. faecium (VSE) in the presence of the TRPV1 channel blocker AMG517, EO blend (carvacrol 0.2µl and cuminaldehyde 25µl) and capsaicin. Microarray data was used to assess which genes associated with ion channels were differentially regulated in the presence of EO blend. AMG517 (10nM to 90nM) did not limit the inhibitory effect of capsaicin (Minimum Inhibitory Concentration:1.6mM) or the EO blend on E. faecium. This suggests that a homologue of TRPV1 does not exist in E. faecium. Microarray data showed that there are genes encoding channels including kup, bcr, gsia_1, mscL, gla_2, ylob that were differentially regulated when exposed to the EO blend. Conclusions The microarray data suggests that cell wall channels are involved in the mechanism of action of EOs; despite the antimicrobial action of capsaicin on E. faecium results are not consistent with the involvement of TRPV1 homologue in bacteria. Significance of study Due to an increase in antibiotic resistance, novel antimicrobial compounds with mechanisms of action that are effective against multi-drug resistant (MDR) microorganisms are of particular interest1,2. Although a homologue of TRPV1 was not identified, microarray data demonstrates that the EO blend is having an effect on genes involved in transport mechanisms. Further investigations are required to ascertain the exact mechanism of action of EOs against bacteria in order for them to be exploited as novel antimicrobials. References 1. Wikaningtyas P, Sukandar EY. The antibacterial activity of selected plants towards resistant bacteria isolated from clinical specimens. Asian Pac J Trop Biomed. 2016;6(1):16-19. doi:10.1016/j.apjtb.2015.08.003 2. Simpkin VL, Renwick MJ, Kelly R, Mossialos E. Incentivising innovation in antibiotic drug discovery and development: Progress, challenges and next steps. J Antibiot (Tokyo). 2017;70(12):1087-1096. doi:10.1038/ja.2017.124