Identification and Characterisation of Two Functional Antibiotic MATE Efflux pumps in the Halophilic Archaeon Halorubrum saccharovorum CSM52

Abstract

Abstract Multidrug efflux pumps have been found to play a crucial role in drug resistance in bacteria and eukaryotes. In this study, we investigated the presence of functional multidrug and toxic compound extrusion (MATE) efflux pumps, inferred from whole genome sequencing, in the halophilic archaeon Halorubrum saccharovorum CSM52 using Hoechst 33342 dye accumulation and antimicrobial sensitivity tests in the presence and absence of efflux pump inhibitors (EPIs). The whole genome sequence of H. saccharovorum CSM52 contained two putative MATE type efflux pump genes, which may contribute the inherent resistance to conventional antimicrobial agents reported in archaea. Antimicrobial susceptibility of the wild type H. saccharovorum CSM52 testing revealed a lack of sensitivity to a wide range of antimicrobials, including glycopeptides, aminoglycosides, macrolides, fluoroquinolones, tetracycline, and chloramphenicol. However, the presence of EPIs, such as thioridazine, fluoxetine, and chlorpromazine, significantly increased the susceptibility of H. saccharovorum CSM52 to a number of these antimicrobials, indicating the potential involvement of efflux pumps in the observed resistance. A molecular modelling study with EPIs and substrate antimicrobials provided important insights into the molecular interactions with the putative transporter. It suggests that the occupancy of the transporter channel by EPIs has the potential to impact the efflux of antimicrobials. Phylogenetic analysis of the amino acid sequences of both MATE pumps showed low similarity with bacterial representatives, suggesting the presence of novel and distinct MATE efflux pumps in archaea. Our findings provide the first evidence of active antibiotic efflux mechanisms in archaea and their potential roles in antimicrobial resistance, broadening our understanding of mechanisms of archaeal antimicrobial resistance, an overlooked aspect of AMR research. IMPORTANCE Antimicrobial resistance poses a significant threat to public health worldwide. By investigating the presence and functionality of MATE efflux pumps in the halophilic archaeon Halorubrum saccharovorum CSM52, the research contributes to a deeper understanding of the mechanisms underlying drug resistance in archaea and archaea as a potential reservoir of antimicrobial resistance genes (ARGs). This is of high significance with the growing body of reports that suggest the involvement of archaea in some human diseases, such as brain abscess and Parkinson’s disease [1, 2]. This is also important due to the high probability that archaea are involved in horizontal gene transfer with both bacteria and eukaryotes domains, which possibly includes antimicrobial resistance genes. The identification of two distinct MATE efflux pump genes and their functional characterisation and involvement in inherent antimicrobial resistance mechanisms is the first demonstration of functional unmodified-antibiotic efflux mechanisms in the Archaea. Furthermore, this study has significant implications for our understanding of the antibiotic resistome, and the potential for archaea to be important, often overlooked, reservoirs of antibiotic resistance genes.