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Molecular Mechanisms of Antibiotic Resistance - Part I

Alison J. Baylay1, Laura J.V. Piddock1, and Mark A. Webber?2

1 Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK

2 Quadram Institute, Norwich, UK

1.1 Introduction

Since the 1940s, pathogens resistant to antibiotics have emerged and spread around the globe, such that antibacterial resistance is one of the greatest challenges to human health in the twenty-first century. The mechanisms underpinning this evolution of resistance are complex and include the mutations in genes that either encode the targets of antibiotics or factors that control production of proteins that influence bacterial susceptibility to antibiotics, as well as the transfer of genes between strains and species including nonpathogenic bacteria. In this chapter, we give an overview of the molecular mechanisms by which bacteria can survive exposure to some of the most clinically important antibiotics currently available.

To exert its antimicrobial effect, a drug must reach and successfully bind to its target. Bacteria have evolved multiple and different antibiotic resistance mechanisms. These can be broadly grouped into four categories (summarized in Figure 1.1):

• Reducing the concentration of drug able to reach its target, either by preventing its entry or actively removing it from the cell,
• Inactivating or modifying the drug before it reaches the target, either extracellularly or intracellularly,
• Changing the target so that the drug can no longer bind,
• Acquiring an alternative route to carry out the cellular process blocked by the drug.

Figure 1.1 Overview of antibiotic resistance mechanisms. (a) In general, antibiotics function by binding to a cellular target such that an important biochemical process is blocked. (b) Bacteria may resist the action of antibiotic by a variety of mechanisms, which are summarized here. These include reduction of the intracellular concentration of the antibiotic by increased efflux or reduced permeability, inactivation of the antibiotic by hydrolysis or modification, modification of the target to prevent antibiotic binding, and metabolic bypass of the cellular process blocked by the antibiotic.

1.2 Molecular Mechanisms of Resistance

1.2.1 Reducing the Intracellular Concentration of a Drug

1.2.1.1 Increased Efflux

All bacterial genomes encode multiple efflux pumps, which extrude a variety of compounds from the cell. Efflux pumps are ancestrally ancient proteins and their original function is often unknown, but some are known to export naturally occurring molecules that are toxic to the cell [1]. In addition, functional efflux pumps have been shown to be important for other cellular processes, such as virulence and biofilm formation in particular [2-5].

Efflux pumps play a major role in determining the intrinsic level of susceptibility of a bacterial species to a particular drug but can also cause further clinically important antibiotic resistance when they are over-expressed. This can occur via mutations in local or global regulators [6-8], or by acquisition of insertion sequence (IS) elements that act as strong promoters upstream of efflux pump genes [9, 10]. Alternatively, new pump genes can be acquired on mobile genetic elements, for example, the mef and msr genes that encode macrolide transporters in Gram-positive bacteria [11, 12].

While some efflux pumps have a narrow specificity, such as Tet pumps that confer high level resistance to tetracyclines [13], others known as multidrug efflux systems export a wide range of substrates, often including multiple antibiotics [1]. There are five known families of multidrug efflux pump (Figure 1.2):

• Efflux pumps of the resistance-nodulation-division () family are tripartite transporters found in Gram-negative bacteria, which consist of an inner membrane pump, an outer membrane channel and a periplasmic adaptor protein that connects the two channels. Substrate export is powered by the proton motive force. The best studied example is the AcrAB-TolC pump which was initially discovered in Escherichia coli, but close homologs are widely distributed among Gram-negative bacteria [14].
• Major facilitator superfamily () pumps are the largest group of solute transporters and are responsible for most efflux-mediated resistance in Gram-positive bacteria [15, 16], although they are also found in Gram-negative bacteria [17]. They consist of a single polypeptide chain with 12 or 14 membrane spanning domains, with substrate efflux powered by the proton motive force. As an example, several members of this family cause clinically relevant resistance in Staphylococcus aureus. NorA confers resistance to fluoroquinolone antibiotics, QacA exports cationic lipophilic drugs, including biocides such as benzalkonium chloride, and LmrS exports a variety of agents such as lincomycin, linezolid, chloramphenicol and trimethoprim [18-20].
• Small multidrug resistance () transporters are, as the name suggests, small, having 110-120 amino acid proteins with four membrane spanning domains [15]. They form functional transporters by oligomerizing in the membrane, where they transport substrates using the proton motive force [21]. Examples include QacC from S. aureus and EmrA from E. coli, both of which transport toxic organic cations such as methyl viologen [22, 23].
• Multidrug and toxic compound extrusion () efflux pumps are commonly found in Gram-negative bacteria. Unlike RND pumps, they are formed from a single polypeptide chain with 12 membrane spanning domains. They obtain power for efflux using the proton motive force or sodium antiport mechanisms. Examples include VcrM from Vibrio cholerae, MepA from S. aureus and PmpM from Pseudomonas aeruginosa, which transport a variety of substrates including fluoroquinolones and benzalkonium chloride [24-26].
• Some ATP-binding cassette () transporters confer antibiotic resistance, such as PatAB from Streptococcus pneumoniae, which transports fluoroquinolones, and MacAB from E. coli which exports macrolides [27-30]. ABC transporters are a very widespread family of transporters found in all three kingdoms of life. They consist of four subunits: two membrane spanning domains and two ATP binding domains. The family of ABC transporters involved in substrate export are usually formed from homo- or heterodimers of two half-transporters, each consisting of one membrane spanning domain and one nucleotide binding domain [31, 32]. Unlike the other classes of multidrug efflux pumps, the ABC pumps are primary transporters, meaning that transport is coupled to ATP hydrolysis instead of ion transport.

Figure 1.2 Multidrug efflux systems. There are five known classes of multidrug efflux systems, summarized here. RND, resistance-nodulation-division family; MATE, multidrug and toxic compound extrusion family; SMR, small multidrug resistance family; MFS, major facilitator superfamily; ABC, ATP-binding cassette superfamily.

The molecular mechanisms of transport differ between the families of transporters and most are not well understood. In general, MDR efflux systems bind multiple substrates, transduce potential energy to power transport, and traffic the substrates in a unidirectional manner.

1.2.1.2 Reduced Entry (Permeability)

The intracellular concentration of an antibiotic can be reduced by preventing its entry into the cell. This mechanism is particularly relevant in Gram-negative bacteria as the outer membrane forms an efficient permeability barrier. Many antibiotics diffuse across the outer membrane via porin proteins, which form relatively large, non-selective channels allowing solutes to move across the membrane. The major porins in E. coli are OmpC and OmpF. Outer membrane permeability can be reduced, resulting in decreased permeability to antibiotics, by two mechanisms:

• reducing expression of porins or replacing them with other porins that form smaller channels,
• mutation of porin genes in ways which alter the permeability of the porin channel.

Pseudomonas aeruginosa is a good example of the effect of reduction of outer membrane permeability on antibiotic resistance. This bacterium is intrinsically resistant to many agents as it does not express many general diffusion porins, and instead produces smaller, dedicated porins to allow acquisition of nutrients [33, 34]. OprF, a homolog of OmpF, is expressed at high levels but mostly exists in a closed confirmation, while the open conformation is present at low levels [35]. Other changes in outer membrane protein expression have also been observed, for example, reduction in OprD expression causes resistance to the carbapenem imipenem [36]. Additionally, the...