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.

A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics

There is a vast literature on the generation and effects of reactive oxygen species in biological systems, both in relation to damage they cause and their involvement in cell regulatory and signaling pathways. The biological chemistry of different oxidants is becoming well understood, but it is often unclear how this translates into cellular mechanisms where redox changes have been demonstrated. This review addresses this gap. It examines how target selectivity and antioxidant effectiveness vary for different oxidants. Kinetic considerations of reactivity are used to assess likely targets in cells and how reactions might be influenced by restricted diffusion and compartmentalization. It also highlights areas where greater understanding is required on the fate of oxidants generated by cellular NADPH oxidases and on the identification of oxidant sensors in cell signaling.

Reconciling the chemistry and biology of reactive oxygen species

Photodynamic therapy (PDT) employs a non-toxic dye, termed a photosensitizer (PS), and low intensity visible light which, in the presence of oxygen, combine to produce cytotoxic species. PDT has the advantage of dual selectivity, in that the PS can be targeted to its destination cell or tissue and, in addition, the illumination can be spatially directed to the lesion. PDT has previously been used to kill pathogenic microorganisms in vitro, but its use to treat infections in animal models or patients has not, as yet, been much developed. It is known that Gram-(−) bacteria are resistant to PDT with many commonly used PS that will readily lead to phototoxicity in Gram-(+) species, and that PS bearing a cationic charge or the use of agents that increase the permeability of the outer membrane will increase the efficacy of killing Gram-(−) organisms. All the available evidence suggests that multi-antibiotic resistant strains are as easily killed by PDT as naive strains, and that bacteria will not readily develop resistance to PDT. Treatment of localized infections with PDT requires selectivity of the PS for microbes over host cells, delivery of the PS into the infected area and the ability to effectively illuminate the lesion. Recently, there have been reports of PDT used to treat infections in selected animal models and some clinical trials: mainly for viral lesions, but also for acne, gastric infection by Helicobacter pylori and brain abcesses. Possible future clinical applications include infections in wounds and burns, rapidly spreading and intractable soft-tissue infections and abscesses, infections in body cavities such as the mouth, ear, nasal sinus, bladder and stomach, and surface infections of the cornea and skin.

Photodynamic therapy: a new antimicrobial approach to infectious disease?

Life evolved in an anaerobic world; therefore, fundamental enzymatic mechanisms and biochemical pathways were refined and integrated into metabolism in the absence of any selective pressure to avoid reactivity with oxygen. After photosystem II appeared, environmental oxygen levels rose very slowly. During this time, microorganisms acquired oxygen tolerance by jettisoning enzymes that use glycyl radicals and exposed low-potential iron-sulfur clusters, which can be directly poisoned by oxygen. They also developed mechanisms to defend themselves against superoxide (O2−) and hydrogen peroxide, partially reduced oxygen species that are generated as inadvertent by-products of aerobic metabolism. Contemporary organisms have inherited both the vulnerabilities and the defenses of these ancestral microbes. Current research seeks to identify these, and bacteria comprise an exceptionally accessible experimental system that has provided many of the answers. This manuscript reviews recent developments and identifies re...

/pdf/cellular-defenses-against-superoxide-and-hydrogen-peroxide-30a6vp7gl9.pdf

Cellular Defenses against Superoxide and Hydrogen Peroxide

Escherichia coli responds to oxidative stress by activating sets of coregulated genes that help the cell to maintain homeostasis. Identified previously by genetic and biochemical approaches, the soxRS system mediates the induction of 18 of these redox-inducible genes (including the soxS gene itself). An overlapping set of genes is activated by an assortment of structurally unrelated molecules with antibiotic activities; many genes in this response are controlled by the marRAB system. The activation of either the soxRS or the marRAB system results in enhanced resistance to both superoxide-generating agents and multiple antibiotics. In order to probe the extent of these regulatory networks, we have measured whole-genome transcriptional profiles of the E. coli response to the superoxide-generating agent paraquat (PQ), an inducer of the soxRS system, and to the weak acid salt sodium salicylate (NaSal), an inducer of the marRA system. A total of 112 genes was modulated in response to PQ, while 134 genes were modulated in response to NaSal. We have also obtained transcriptional profiles of the SoxS and MarA regulons in the absence of global stress, in order to establish the regulatory hierarchies within the global responses. Several previously unrelated genes were shown to be under SoxS or MarA control. The genetic responses to both environmental insults revealed several common themes, including the activation of genes coding for functions that replenish reducing potential; regulate iron transport and storage; and participate in sugar and amino acid transport, detoxification, protein modification, osmotic protection, and peptidoglycan synthesis. A large number of PQ- and NaSal-responsive genes have no known function, suggesting that many adaptive metabolic changes that ensue after stress remain uncharacterized.

Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate.

Redox-operated genetic switches : the SoxR and OxyR transcription factors

Salmonella enterica serovar Typhimurium responds to superoxide-generating agents through soxR-mediated activation of the soxS gene, whose product, SoxS, is necessary for resistance to oxidative stress. The S. enterica serovar Typhimurium soxRS system also mediates redox-inducible resistance to diverse antibiotics, which may be relevant to clinical infections. In order to identify SoxS-regulated genes in S. enterica serovar Typhimurium, a lacI-regulated expression system for the S. enterica serovar Typhimurium soxS gene was developed. This system was used to demonstrate that soxS expression is sufficient for the induction of resistance to the superoxide-generating drug paraquat and for the transcriptional activation of the sodA and micF genes. In addition, a library of random lacZ insertions was generated and screened for clones displaying differential β-galactosidase activity in the presence or absence of SoxS. This selection yielded six independent chromosomal lacZ transcriptional fusions that were activated by either artificial expression of SoxS or exposure of wild-type cells to micromolar concentrations of paraquat. Moreover, disruption of the inducible genes by the insertions rendered S. enterica serovar Typhimurium hypersensitive to millimolar concentrations of paraquat. Nucleotide sequence determination identified the disrupted genes as sodA (Mn-containing superoxide dismutase), fpr (NADPH:ferredoxin oxidoreductase), and ydbK (a putative Fe-S-containing reductase).

Identification of SoxS-Regulated Genes in Salmonella enterica Serovar Typhimurium

Oxidation can damage all biological macromolecules, and the survival of a cell therefore depends on its ability to control the level of oxidants. Microbes possess an astonishing variety of antioxidant defences, ranging from small, oxidant-scavenging molecules to self-regulating, homeostatic gene networks. Most often these antioxidant defences are activated by exposure to specific classes of oxidants. Interestingly, the isolation of pleiotropic mutations that impair or exacerbate the expression of subsets of oxidant-responsive genes led to the identification of global regulators. In a few, well-characterized cases, these regulators can transduce oxidative damage into gene regulation. Recently, the application of genomic tools to study the antioxidant responses of E. coli has both confirmed previous observations and provided evidence for a wealth of putative new anti-oxidant functions. Here, we review the remarkable diversity of antioxidant defence mechanisms, with emphasis on signal transduction by global regulator proteins and the corresponding genetic networks that protect the microbial cell against oxidative stress.

Global adjustment of microbial physiology during free radical stress.

The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.

Pablo J. Pomposiello

Papers

Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate.

Redox-operated genetic switches : the SoxR and OxyR transcription factors

Identification of SoxS-Regulated Genes in Salmonella enterica Serovar Typhimurium

Global adjustment of microbial physiology during free radical stress.

Defining a rob Regulon in Escherichia coli by Using Transposon Mutagenesis