Harmonization of antimicrobial susceptibility testing by broth microdilution for Rhodococcus equi of animal origin

Riesenberg, Anne; Feßler, Andrea T.; Frömke, Cornelia; Kadlec, Kristina; Klarmann, Dieter; Kreienbrock, Lothar; Werckenthin, Christiane Susanne; Schwarz, Štefan

doi:10.1093/jac/dkt134

Cited by 15 publications

(3 citation statements)

References 5 publications

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“…Antimicrobial susceptibility testing. Inocula were prepared from overnight cultures in MH broth by the direct colony suspension method according to the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) (36). The MIC of erythromycin was determined using Etest strips (bioMérieux, Durham, NC) according to the manufacturer's recommendations at concentrations between 0.016 and 256 mg/liter.…”

Section: Methodsmentioning

confidence: 99%

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi

Willingham-Lane

Berghaus

et al. 2019

Appl Environ Microbiol

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Rhodococcus equi is a leading cause of severe pneumonia in foals. Standard treatment is dual antimicrobial therapy with a macrolide and rifampin, but the emergence of macrolide- and rifampin-resistant R. equi isolates is an increasing problem. The objective of this study was to determine the effect of macrolide and/or rifampin resistance on fitness of R. equi. Three unique isogenic sets were created, each consisting of four R. equi strains, as follows: a susceptible parent isolate, strains resistant to macrolides or rifampin, and a dual macrolide- and rifampin-resistant strain. Each isogenic set’s bacterial growth curve was generated in enriched medium, minimal medium (MM), and minimal medium without iron (MM-I). Bacterial survival in soil was analyzed over 12 months at −20°C, 4°C, 25°C, and 37°C, and the ability of these strains to retain antimicrobial resistance during sequential subculturing was determined. Insertion of the mobile element conferring macrolide resistance had minimal effect on in vitro growth. However, two of three rpoB mutations conferring rifampin resistance resulted in a decreased growth rate in MM. In soil, macrolide- or rifampin-resistant R. equi strains exhibited limited growth compared to that of the susceptible R. equi isolate at all temperatures except −20°C. During subculturing, macrolide resistance was lost over time, and two of three rpoB mutations reverted to the wild-type form. The growth of rifampin-resistant R. equi colonies is delayed under nutrient restriction. In soil, possession of rifampin or macrolide resistance results in decreased fitness. Both macrolide and rifampin resistance can be lost after repeated subculturing. IMPORTANCE This work advances our understanding of the opportunistic environmental pathogen Rhodococcus equi, a disease agent affecting horses and immunocompromised people. R. equi is one of the most common causes of severe pneumonia in young horses. For decades, the standard treatment for R. equi pneumonia in horses has been dual antimicrobial therapy with a macrolide and rifampin; effective alternatives to this combination are lacking. The World Health Organization classifies these antimicrobial agents as critically important for human medicine. Widespread macrolide and rifampin resistance in R. equi isolates is a major emerging problem for the horse-breeding industry and might also adversely impact human health if resistant strains infect people or transfer resistance mechanisms to other pathogens. This study details the impact of antimicrobial resistance on R. equi fitness, a vital step for understanding the ecology and epidemiology of resistant R. equi isolates, and will support development of novel strategies to combat antimicrobial resistance.

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Section: Methodsmentioning

confidence: 99%

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi

Willingham-Lane

Berghaus

et al. 2019

Appl Environ Microbiol

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“…There are multiple possible source for mistakes with the broth macrodilution method, and difficulties in reading broth dilution assays at 24 h for some isolates of R. equi have been reported previously (25). A recent publication demonstrated that addition of 2% (vol/vol) lysed horse blood to the cation-adjusted Mueller-Hinton broth facilitates the discrimination between growth and no growth of R. equi (26). Lysed horse blood was not used in the present study because this information was not available at the time this study was conducted.…”

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confidence: 99%

Comparison of Etest, Disk Diffusion, and Broth Macrodilution for In Vitro Susceptibility Testing of Rhodococcus equi

et al. 2015

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“…Positive and negative control wells were tested on each plate then incubated at 37 °C. The standard reading was located at 24 h 17 . The assay was validated if the visible growth in positive controls was retained acceptable.…”

Section: Methodsmentioning

confidence: 99%

In vitro performances of novel co-spray-dried azithromycin/rifampicin microparticles for Rhodococcus equi disease treatment

Rampacci

Marenzoni

Chiaradia

et al. 2018

Sci Rep

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This work was aimed at providing clues on the in vitro performances of novel azithromycin/rifampicin combinations, in the form of co-spray-dried microparticles (AZM/RIF MP), against Rhodococcus equi, an animal and emerging human pathogen found responsible for worrying zoonosis. Various AZM/RIF combinations were spray-dried and characterized for their morphology and size. Susceptibility studies included determination of MIC, MBC, Fractional Inhibitory/Bactericidal Concentration Indexes and intracellular activity in R. equi-infected THP-1 cells. Cytotoxicity was tested on BEAS-2B cells through MTT assay and combination index assessment for drug interaction. Spray-dried MP were collapsed and 3–10 times smaller than commercial powders. Drug combinations showed an enhancement of in vitro antibacterial activity with a remarkable synergistic bactericidal effect. Azithromycin MP and AZM/RIF MP 2:1 led to a CFU reduction of >90% up to 4 days after treatment at all tested concentrations (p = 0.001) but AZM/RIF MP 2:1 were at least four-fold more potent than AZM MP alone. IC50 values of >100 mg/L supported low cytotoxicity of drug combinations and the combination index suggested an antagonistic toxic effect. Co-spray-drying enhanced powder dispersibility and solubility, which may improve bioavailability as well as provide administration alternatives. The novel AZM/RIF MP combinations could result a valid platform to develop new treatment strategies against R. equi infections in animals and humans.

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Harmonization of antimicrobial susceptibility testing by broth microdilution for Rhodococcus equi of animal origin

Cited by 15 publications

References 5 publications

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi

Comparison of Etest, Disk Diffusion, and Broth Macrodilution for In Vitro Susceptibility Testing of Rhodococcus equi

In vitro performances of novel co-spray-dried azithromycin/rifampicin microparticles for Rhodococcus equi disease treatment

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