In vitro antimicrobial inhibition of Mycoplasma bovis isolates submitted to the Pennsylvania Animal Diagnostic Laboratory using flow cytometry and a broth microdilution method

Abstract

Mycoplasma bovis, a pathogenic mollicute first reported in 1961, is associated with pneumonia, mastitis, conjunctivitis, otitis, and arthritis in cattle. 8 Reports from Europe suggest that M. bovis is found in 13-50% of animals with disease. 13 It is unclear what percentage of disease in the United States may be attributed to M. bovis since large-scale epidemiological studies have not been performed. Poor efficacy of M. bovis vaccines produces few clinical options. Mycoplasma bovis infections are either treated with antimicrobials, which frequently do not clear the infection, or infected animals are culled. There is concern over the increasing resistance to frequently administered antimicrobials, including spectinomycin, tetracycline, and tilmicosin.14 The unique characteristics of M. bovis, such as the lack of a cell wall, cause concern regarding treatment options due to the ineffectiveness of cell wall-targeting antimicrobial therapies. Clinical Laboratory Standards Institute (CLSI)-approved and standardized levels of minimum inhibitory concentrations (MIC) for antimicrobials have not been established regarding Mycoplasma species.9,11,12 Multiple methods have been employed in order to determine MICs in many species of Mycoplasma. These methods have included broth dilutions, solid agar testing, E-strip testing, and most recently, flow cytometry. 2,6,9,10,15,16,18 The MIC values have been determined for Mycoplasma agalactiae, Mycoplasma hyopneumoniae, Mycoplasma putrefaciens, Mycoplasma capricolum, and Mycoplasma mycoides using a selection of antimicrobials with flow cytometry for one isolate of each. 1-3The objectives of the current study were to employ a microdilution method to determine the MIC of 7 antimicrobials on M. bovis isolates (n = 192) submitted to the Pennsylvania Animal Diagnostic Laboratory (PA-ADL; University Park, (n = 192) were tested for antimicrobial susceptibility to enrofloxacin, erythromycin, florfenicol, spectinomycin, ceftiofur, tetracycline, and oxytetracycline using a broth microdilution method. The most effective antimicrobials against M. bovis determined by using the broth microdilution method were florfenicol, enrofloxacin, and tetracycline with minimum inhibitory concentration (MIC) ranges of 2-32 µg/ml, 0.1-3.2 µg/ml, and 0.05 to >12.8 µg/ml, respectively. Spectinomycin, oxytetracycline, and tetracycline showed a wide-ranging level of efficacy in isolate inhibition with broth microdilution with MIC ranges of 4 to >256 µg/ml, 0.05 to >12.8 µg/ml, and 0.05 to >12.8 µg/ml, respectively. A significant difference in the susceptibility levels between quarter milk and lung isolates was found for spectinomycin. When MIC values of a subset of the M. bovis isolates (n = 12) were tested using a flow cytometric technique, the MIC ranges of enrofloxacin, spectinomycin, ceftiofur, erythromycin, tetracycline, oxytetracycline, and florfenicol ranges were 0.1-0.4 µg/ml, 4 to >256 µg/ml, >125 µg/ ml, >3.2 µg/ml, <0.025 to >6.4 µg/ml, 0.8 to >12.8 µg/ml, and <2-4 µg/ml, respectively. Flow cytometr...

Treatment of naturally occurring bovine respiratory disease in juvenile calves with a single administration of a florfenicol plus flunixin meglumine formulation

“…Therefore, MIC determined for M bovis should be interpreted with caution. However, a recent study demonstrated florfenicol is one of the most effective antimicrobials against M bovis strains isolated in the USA (Soehnlen and others 2011) and resistance to florfenicol appears uncommon (Maunsell and others 2011, Vet Path programme, MSD Animal Health, data on file). Overall, these data allow the conclusion that no significant development of resistance in the target pathogens has been observed despite the widespread use of florfenicol in domestic animals for more than 15 years.…”

“…The microbiological examination of the samples included culture, identification and florfenicol minimum inhibitory concentration (MIC) determination. All microbiological procedures were performed in accordance with Clinical Laboratory Standards Institute (CLSI) recommendations with the exception of those used for M bovis and any other Mycoplasma species for which the broth microdilution method described by Soehnlen and collaborators was performed (Rosenbusch and others 2005, Soehnlen and others 2011). Blood samples were serologically analysed for paired titres of infectious bovine rhinotracheitis (IBR) virus, bovine respiratory syncytial virus (BRSV), bovine parainfluenza three virus (PI3), bovine ­adenovirus and bovine viral diarrhoea virus (BVD) to obtain background information on concomitant viral infections.…”

Quantitative Assessment of Mycoplasma Hemadsorption Activity by Flow Cytometry

“…Despite this, there is an increasing number of works showing that FC is an accurate method to detect and quantify mycoplasma cells in broth medium [23], [24] or in natural samples [25]. FC has been used also to quantify the effect of several antibacterial agents [26], [27] and to detect hemotropic mycoplasmas on red blood cells (RBCs) samples [28]. Here we show that FC could be used to measure rapidly and accurately the HA activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve RBCs and mycoplasma cells by their different size and fluorescence.…”

16S rRNA Gene Mutations Associated with Decreased Susceptibility to Tetracycline in Mycoplasma bovis

“…of veterinary importance (reviewed in reference 8), including M. bovis (9)(10)(11)(12)(13)(14)(15), the genetic background for decreased susceptibility has not been elucidated in either field isolates or mutants selected in vitro. M. bovis contains one or two rRNA operons (rrn).…”