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      Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

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          Abstract

          To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.

          Author Summary

          Staphylococcus aureus is a major cause of human disease, made even more notable due to the spread of antibiotic resistance. We used a combination of animal models to study the spread of bacteria between organs during an infection and the resulting effect of antibiotic intervention. We found that S. aureus infection is highly clonal, following a “bottleneck” in which very few bacterial cells found each abscess. Despite previous in vitro research, the effect of antibiotics on S. aureus infection was poorly understood. We utilized our systemic infection models to study intervention with sub-curative antibiotic doses, such as one might encounter upon failing to complete an antibiotic course. We have shown that such doses are able to support the preferential expansion of antibiotic-resistant organisms during a mixed infection. This selection is due to the clonal pattern of infection, occurring despite a lack of effect on growth rate or on the spontaneous generation of resistance. Furthermore, it is generic to multiple pathogen species, including Pseudomonas aeruginosa, and antibiotic classes, such as with methicillin-resistant S. aureus (MRSA) in the presence of oxacillin. Given the current debate in the field, our results have important implications for the design of properly-controlled treatment regimes.

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          Most cited references54

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          A common mechanism of cellular death induced by bactericidal antibiotics.

          Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.
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            The biological cost of antibiotic resistance.

            The frequency and rates of ascent and dissemination of antibiotic resistance in bacterial populations are anticipated to be directly related to the volume of antibiotic use and inversely related to the cost that resistance imposes on the fitness of bacteria. The data available from recent laboratory studies suggest that most, but not all, resistance-determining mutations and accessory elements engender some fitness cost, but those costs are likely to be ameliorated by subsequent evolution.
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              Cell death from antibiotics without the involvement of reactive oxygen species.

              Recent observations have suggested that classic antibiotics kill bacteria by stimulating the formation of reactive oxygen species (ROS). If true, this notion might guide new strategies to improve antibiotic efficacy. In this study, the model was directly tested. Contrary to the hypothesis, antibiotic treatment did not accelerate the formation of hydrogen peroxide in Escherichia coli and did not elevate intracellular free iron, an essential reactant for the production of lethal damage. Lethality persisted in the absence of oxygen, and DNA repair mutants were not hypersensitive, undermining the idea that toxicity arose from oxidative DNA lesions. We conclude that these antibiotic exposures did not produce ROS and that lethality more likely resulted from the direct inhibition of cell-wall assembly, protein synthesis, and DNA replication.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                February 2014
                27 February 2014
                : 10
                : 2
                : e1003959
                Affiliations
                [1 ]Krebs Institute, University of Sheffield, Western Bank, Sheffield, United Kingdom
                [2 ]Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom
                [3 ]MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Western Bank, Sheffield, United Kingdom
                [4 ]Department of Infection and Immunity, University of Sheffield, Western Bank, Sheffield, United Kingdom
                [5 ]Biosciences, University of Exeter, Cornwall Campus, Penryn, United Kingdom
                University of Tubingen, Germany
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: GM TKP AW SAR SJF. Performed the experiments: GM TKP AW NLW. Analyzed the data: GM MB SJF. Wrote the paper: GM SAR SJF.

                Article
                PPATHOGENS-D-13-02845
                10.1371/journal.ppat.1003959
                3937288
                24586163
                fc5076ff-0733-41de-ba0c-13d8d30b3b9f
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 3 November 2013
                : 14 January 2014
                Page count
                Pages: 11
                Funding
                This work was funded by a Wellcome Trust Project Grant (Reference Number WT089981MA), an EU project: Predicting Antibiotic Resistance (PAR, Reference Number 241476) and from the European Community's Seventh Framework Programme [FP7-PEOPLE-2011-ITN] under grant agreement no. PITN-GA-2011-289209 for the Marie-Curie Initial Training Network FishForPharma. SAR is supported by an MRC Senior Clinical Fellowship (Reference Number: G0701932). Aquarium staff were supported by MRC Centre grant G0700091. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Bacterial pathogens
                Gram positive
                Gram negative
                Staphylococci
                Immunity
                Innate immunity
                Host-pathogen interaction
                Microbial pathogens
                Medicine
                Infectious diseases
                Bacterial diseases
                Pseudomonas infections
                Staphylococcal infection

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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