Antibiotic-resistant bacteria that are difficult or impossible to treat are becoming increasingly common and are causing a global health crisis. Antibiotic resistance is encoded by several genes, many of which can transfer between bacteria. New resistance mechanisms are constantly being described, and new genes and vectors of transmission are identified on a regular basis. This article reviews recent advances in our understanding of the mechanisms by which bacteria are either intrinsically resistant or acquire resistance to antibiotics, including the prevention of access to drug targets, changes in the structure and protection of antibiotic targets and the direct modification or inactivation of antibiotics.

Molecular mechanisms of antibiotic resistance.

It is well established that multidrug-resistance efflux pumps encoded by bacteria can confer clinically relevant resistance to antibiotics. It is now understood that these efflux pumps also have a physiological role(s). They can confer resistance to natural substances produced by the host, including bile, hormones and host-defence molecules. In addition, some efflux pumps of the resistance nodulation division (RND) family have been shown to have a role in the colonization and the persistence of bacteria in the host. Here, I present the accumulating evidence that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and propose that these pumps therefore have greater clinical relevance than is usually attributed to them.

Multidrug-resistance efflux pumps ? not just for resistance

Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.

Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria

We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, α-, β-, and γ-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of less conserved residues are involved in establishing contacts with the phosphate backbone and target bases in the operator. Information related to the TetR family of regulators has been updated in a database that can be accessed at www.bactregulators.org.

The TetR Family of Transcriptional Repressors

The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

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The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

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Yu Fang Jin

Papers

Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.