The Role of Transport Channels in the Mechanism of Action of a Novel Antimicrobial Formulation

Globally, antimicrobial resistance (AMR) is emerging as an increasing health threat without available effective antimicrobials. This creates a critical need for novel and effective antimicrobials with new mechanisms of action to circumvent AMR. Enterococcus faecium is one of the main causes of nosocomial infections exhibiting resistance to almost all available antibiotics. A potential formulation to combat resistance in both vancomycin-sensitive and vancomycin-resistant E. faecium (VSE, VRE), is the formula of 1.98 mM carvacrol and 4.20 mM cuminaldehyde with 0.031 mg/l vancomycin. The mechanism of action of this formulation is yet to be determined. Transcriptomic analysis showed that many genes were differentially regulated when exposed to the formulation; including genes related to transportation in the bacterial cell. Five of these genes were chosen for further investigations; bcr, ecfA1, ecsA-1, yloB and nhaC_2. The current study aims to investigate the involvement of transport channels in the antimicrobial mechanism of action of a novel antimicrobial formulation. The involvement of channels similar to TRPV1 mammalian channels in the mechanism of action of the formulation was investigated by conducting growth assays and viable counts with all treatment conditions (carvacrol, cuminaldehyde, carvacrol and cuminaldehyde together, vancomycin and the formulation) and with either 1.6 mM capsaicin or 30 nM AMG517. The effects of the formulation/components on the expression levels of bcr, ecfA1, ecsA-1, yloB and nhaC_2 were established by conducting qPCR under the effect of all treatment conditions and in the presence of either 1 mM calcium or 0.1 mM EDTA over the time points 10, 30, 60, 120 or 360 minutes. The development of strains of E. faecium containing gene knockout of either bcr or yloB were attempted. The data obtained showed that AMG517 did not reverse the action of either capsaicin, carvacrol or cuminaldehyde on VSE elucidating that the channels investigated in this study are not similar to TRPV1 mammalian channels. Of the five genes analysed by qPCR, the main changes happened at 10 and 30 minutes with significant alterations in the expression level of yloB by -5.67 and 3.68-fold, and bcr by -13.5 and 2.16-fold, respectively, and in the expression levels of ecfA1 by 2.85-fold at 120 minutes, ecsA-1 by 25.87-fold at 360 minutes, and nhaC_2 by 4.7-fold at 120 minutes. Ca2+ has induced a significant increase in the expression of yloB by 11.18-fold with capsaicin at 10 minutes; and by -50, -4.4, 6.6 and 3.08-fold with the formulation at 10, 30, 60 and 120 minutes, respectively, all in comparison to those in the absence of Ca2+. The addition of EDTA to the formulation induced non-significant downregulation by 1.06-fold and significant downregulation by 10-fold of bcr at later time points of 120 and 360 minutes, respectively, in comparison to being upregulated by 15.03 and 15.71-fold when with the formulation alone at these similar time points. Construction of plasmids containing ΔyloB and Δbcr was successfully established in Escherichia coli TG1 (repA+); however, isolation of chloramphenicol/ampicillin-resistant ΔyloB and Δbcr VRE strains was not successful following electroporation of pGhost9-cat/bla constructs. Overall, the findings of the current study suggest the involvement of transportation channels in the mechanism of action of the formulation against E. faecium VRE; however, it is yet to be established if the novel antimicrobial formulation will be effective in combating VRE in both hospital and community environments.