Antibiotic resistance is a global health emergency, with resistance detected to all antibiotics currently in clinical use and only a few novel drugs in the pipeline. Understanding the molecular mechanisms that bacteria use to resist the action of antimicrobials is critical to recognize global patterns of resistance and to improve the use of current drugs, as well as for the design of new drugs less susceptible to resistance development and novel strategies to combat resistance. In this Review, we explore recent advances in understanding how resistance genes contribute to the biology of the host, new structural details of relevant molecular events underpinning resistance, the identification of new resistance gene families and the interactions between different resistance mechanisms. Finally, we discuss how we can use this information to develop the next generation of antimicrobial therapies.

Rresistance-nodulation-division (RND) efflux pumps in Gram-negative bacteria remove multiple, structurally distinct classes of antimicrobials from inside bacterial cells therefore directly contributing to multidrug resistance. There is also emerging evidence that many other mechanisms of antibiotic resistance rely on the intrinsic resistance conferred by RND efflux. In addition to their role in antibiotic resistance, new information has become available about the natural role of RND pumps including their established role in virulence of many Gram-negative organisms. This review also discusses the recent advances in understanding the regulation and structure of RND efflux pumps.

Introduction. We are becoming increasingly reliant on the effectiveness of biocides to combat the spread of Gram-negative multi-drug-resistant (MDR) pathogens, including Klebsiella pneumoniae . It has been shown that chlorhexidine exposure can lead to mutations in the efflux pump repressor regulators SmvR and RamR, but the contribution of each individual efflux pump to biocide tolerance is unknown. Hypothesis. Multiple efflux pumps, including SmvA and AcrAB-TolC, are involved in increased tolerance to biocides. However, strains with upregulated AcrAB-TolC caused by biocide exposure are more problematic due to their increased MDR phenotype. Aim. To investigate the role of AcrAB-TolC in the tolerance to several biocides, including chlorhexidine, and the potential threat of cross-resistance to antibiotics through increased expression of this efflux pump. Methodology. Antimicrobial susceptibility testing was performed on K. pneumoniae isolates with ramR mutations selected for after exposure to chlorhexidine, as well as transposon mutants in components and regulators of AcrAB-TolC. RTPCR was used to detect the expression levels of this pump after biocide exposure. Strains from the globally important ST258 clade were compared for genetic differences in acrAB-TolC and its regulators and for phenotypic differences in antimicrobial susceptibility. Results. Cross-resistance to antimicrobials was observed following mutations in ramR. Exposure to chlorhexidine led to increased expression of acrA and its activator ramA, and transposon mutants in AcrAB-TolC have increased susceptibility to several biocides, including chlorhexidine. Variations in ramR within the ST258 clade led to an increase in tolerance to certain biocides, although this was strain dependent. One strain, MKP103, that had increased levels of biocide tolerance showed a unique mutation in ramR that was reflected in enhanced expression of acrA and ramA. MKP103 transposon variants were able to further enhance their tolerance to specific biocides with mutations affecting SmvA. Conclusions. Biocide tolerance in K. pneumoniae is dependent upon several components, with increased efflux through AcrAB-TolC being an important one.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter , such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter . AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter , treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is extensively drug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes 6 RND efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, however the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-three species making up the Acinetobacter genus were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually orthologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the Acinetobacter genus. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH were only found in a subset of Acinetobacter, that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.