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      Determination of the Mutant Selection Window and Evaluation of the Killing of Mycoplasma gallisepticum by Danofloxacin, Doxycycline, Tilmicosin, Tylvalosin and Valnemulin

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          Abstract

          Mycoplasma gallisepticum is a common etiological cause of a chronic respiratory disease in chickens; its increasing antimicrobial resistance compromises the use of tetracyclines, macrolides and quinolones in the farm environment. Mutant selection window (MSW) determination was used to investigate the propensity for future resistance induction by danofloxacin, doxycycline, tilmicosin, tylvalosin and valnemulin. Killing of M. gallisepticum strain S6 by these antimicrobials was also studied by incubating M. gallisepticum into medium containing the compounds at the minimal concentration that inhibits colony formation by 99% (MIC 99) and the mutant prevention concentration (MPC). Based on the morphology and colony numbers of M. gallisepticum on agar plates, the four kinds of sera in the order of the applicability for culturing M. gallisepticum were swine serum > horse serum > bovine serum > mixed serum. The MPC/MIC 99 values for each agent were as follows: danofloxacin > tilmicosin > tylvalosin > doxycycline > valnemulin. MPC generated more rapid and greater magnitude killing than MIC 99 against M. gallisepticum. Under exposure of 10 5–10 9 CFU/mL at MPC drug levels, valnemulin had the slowest rate of reduction in viable organisms and danofloxacin had the highest rate of reduction.

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          Most cited references35

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          The mutant selection window and antimicrobial resistance.

          The mutant selection window is an antimicrobial concentration range extending from the minimal concentration required to block the growth of wild-type bacteria up to that required to inhibit the growth of the least susceptible, single-step mutant. The upper boundary is also called the mutant prevention concentration (MPC). Placing antimicrobial concentrations inside the window is expected to enrich resistant mutant subpopulations selectively, whereas placing concentrations above the window is expected to restrict selective enrichment. Since window dimensions are characteristic of each pathogen-antimicrobial combination, they can be linked with antimicrobial pharmacokinetics to rank compounds and dosing regimens in terms of their propensity to enrich mutant fractions of bacterial populations. For situations in which antimicrobial concentrations cannot be kept above the window, restricting the enrichment of mutants requires combination therapy.
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            Mutant selection window hypothesis updated.

            The mutant selection window hypothesis postulates that, for each antimicrobial-pathogen combination, an antimicrobial concentration range exists in which selective amplification of single-step, drug-resistant mutants occurs. This hypothesis suggests an antimutant dosing strategy that is keyed to the upper boundary of the selection window: the mutant prevention concentration. Correlations are described between the mutant prevention concentration--a static parameter that is measured with agar plates--and fluctuating drug concentrations that restrict mutant amplification in vitro and in animals. When drug resistance is acquired stepwise, the mutant selection window increases, making the suppression of each successive mutant increasingly more difficult. For agents that kill drug-resistant mutants in a drug concentration-dependent manner, the use of the area under the 24-h time-drug concentration curve value divided by the value of the mutant prevention concentration is suggested as an index for designing antimutant dosing regimens. The need for such regimens is emphasized by a clinical example in which acquisition of drug resistance occurs concurrently with eradication of susceptible bacterial cells. These data support using the mutant selection window to optimize antimicrobial dosing regimens.
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              In vitro cell invasion of Mycoplasma gallisepticum.

              The ability of the widespread avian pathogen Mycoplasma gallisepticum to invade cultured human epithelial cells (HeLa-229) and chicken embryo fibroblasts (CEF) was investigated by using the gentamicin invasion assay and a double immunofluorescence microscopic technique for accurate localization of cell-associated mycoplasmas. The presence of intracellular mycoplasmas in both cell lines was clearly demonstrated, with organisms entering the eukaryotic cells within 20 min. Internalized mycoplasmas have the ability to leave the cell, but also to survive within the intracellular space over a 48-h period. Frequencies of invasion were shown to differ between the two cell lines, but were also considerably dependent on the mycoplasma input population. Of the prototype strain R, a low-passage population in artificial medium, R(low), was capable of active cell invasion, while a high-passage population, R(high), showed adherence to but nearly no uptake into HeLa-229 and CEF. By passaging R(low) and R(high) multiple times through HeLa-229 cells, the invasion frequency was significantly increased. Taken together, these findings demonstrate that M. gallisepticum has the capability of entering nonphagocytic host cells that may provide this pathogen with the opportunity for resisting host defenses and selective antibiotic therapy, establishing chronic infections, and passing through the respiratory mucosal barrier to cause systemic infections.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                4 January 2017
                2017
                : 12
                : 1
                Affiliations
                [1 ]Guangdong Key Laboratory for Veterinary Drug Development and Safety evaluation, South China Agricultural University, Guangzhou, China
                [2 ]Technical Center of Zhuhai Entry-Exit Inspection and Quarantine Bureau, Zhuhai, China
                Miami University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: NZ.

                • Data curation: NZ XS HJ HD.

                • Formal analysis: NZ XS HJ HD.

                • Funding acquisition: NZ.

                • Investigation: NZ XS HJ HD.

                • Methodology: NZ XY YW ZH XG QC.

                • Project administration: NZ.

                • Resources: NZ XS HJ HD.

                • Software: NZ XY YW ZH XG QC.

                • Supervision: NZ.

                • Validation: NZ XY YW ZH XG QC.

                • Visualization: NZ.

                • Writing – original draft: NZ.

                • Writing – review & editing: NZ.

                Article
                PONE-D-16-34938
                10.1371/journal.pone.0169134
                5215565
                28052123
                ca343f12-06b7-4224-8473-3bcb9eff28fd
                © 2017 Zhang et al

                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.

                Page count
                Figures: 4, Tables: 5, Pages: 15
                Product
                Funding
                Funded by: National Key Research and Development Program of China
                Award ID: 2016YFD0501300/2016YFD0501310
                Award Recipient :
                Funded by: 973 program
                Award ID: 2013CB127200/2013CB127203
                Award Recipient :
                This study is supported financially by National Key Research and Development Program of China (2016YFD0501300/2016YFD0501310) and 973 program (2013CB127200/2013CB127203).
                Categories
                Research Article
                Medicine and Health Sciences
                Pharmacology
                Drugs
                Antimicrobials
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobials
                Biology and Life Sciences
                Agriculture
                Livestock
                Swine
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Swine
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Equines
                Horses
                Biology and Life Sciences
                Organisms
                Bacteria
                Mollicutes
                Mycoplasma
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobial Resistance
                Medicine and Health Sciences
                Pharmacology
                Antimicrobial Resistance
                Physical Sciences
                Physics
                States of Matter
                Fluids
                Liquids
                Research and Analysis Methods
                Separation Processes
                Centrifugation
                Medicine and Health Sciences
                Pharmacology
                Drugs
                Antimicrobials
                Antibiotics
                Tetracyclines
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobials
                Antibiotics
                Tetracyclines
                Custom metadata
                All relevant data are within the paper and its Supporting Information file.

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