Overall Decrease in the Susceptibility of Mycoplasma bovis to Antimicrobials over the Past 30 Years in France

et al. (2014) Overall Decrease in the Susceptibility of Mycoplasma bovis to Antimicrobials over the Past 30 Years in France. PLoS ONE 9(2): e87672. doi:10.1371/journal.pone.0087672 Overall Decrease in the Susceptibility of Mycoplasma bovis to Antimicrobials over the Past 30 Years in France Anne V. Gautier-Bouchardon 0 Se verine Ferre 0 Dominique Le Grand 0 Agne` s Paoli 0 Emilie Gay 0 Fran cois Poumarat 0 Mitchell F. Balish, Miami University, United States of America 0 1 ANSES, Laboratoire de Ploufragan/Plouzane , Unite Mycoplasmologie-Bacte riologie, Ploufragan, France, 2 Universite Europe enne de Bretagne , Rennes, France, 3 ANSES , Laboratoire de Lyon, UMR Mycoplasmoses des Ruminants , Lyon , France , 4 Universite de Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants, Marcy L'Etoile, France , 5 ANSES , Laboratoire de Lyon, Unite Epide miologie , Lyon , France Mycoplasma (M.) bovis is frequently implicated in respiratory diseases of young cattle worldwide. Today, to combat M. bovis in Europe, only antimicrobial therapy is available, but often fails, leading to important economical losses. The antimicrobial susceptibility of M. bovis is not covered by antimicrobial resistance surveillance networks. The objectives of this study were to identify resistances that were acquired over the last 30 years in France and to determine their prevalence within comtemporary strains. The minimum inhibition concentration (MIC) values of 12 antimicrobials, considered active on M. bovis, were compared, using an agar dilution method, between 27 and 46 M. bovis isolates respectively obtained in 19781979 and in 2010-2012 from 73 distinct respiratory disease outbreaks in young cattle all over France. For eight antimicrobials, resistances were proven to be acquired over the period and expressed by all contemporary strains. The increase of the MIC value that inhibited 50% of the isolates (MIC50) was: i) substantial for tylosin, tilmicosin, tulathromycin and spectinomycin, from 2 to .64, 2 to .128, 16 to 128 and 4 to .64 mg/mL, respectively, ii) moderate for enrofloxacin, danofloxacin, marbofloxacin and oxytetracycline, from 0.25 to 0.5, 0.25 to 0.5, 0.5 to 1, 32 to .32 mg/mL, respectively. No differences were observed for gamithromycin, tildipirosin, florfenicol and valnemulin with MIC50 of 128, 128, 8, ,0.03 mg/ mL, respectively. If referring to breakpoint MIC values published for respiratory bovine pathogens, all contemporary isolates would be intermediate in vivo for fluoroquinolones and resistant to macrolides, oxytetracycline, spectinomycin and florfenicol. - Formerly the name mycoplasma has commonly denoted bacteria of the class Mollicutes, nowadays it refers exclusively to members of the genus Mycoplasma. This genus comprises the simplest life forms that can self-replicate and includes major human and animal pathogens that cause diseases whose occurrence has long been underestimated [1]. All Mycoplasmas are cellwall less bacteria and therefore are naturally resistant to all antimicrobial families that block cell wall synthesis (e.g. b-lactams and glycopeptides). In cattle, Mycoplasma (M.) bovis causes respiratory disease, mastitis, arthritis and otitis [2]. It is now known that this mycoplasma species is frequently implicated in cases of bovine respiratory disease (BRD) in calves raised in feedlots worldwide [3]: it has been isolated in 40% of BRD outbreaks in the UK [3]; 25 to 80% in Italy [4,5]; 25 to 54% in Israel [6]; and 25% to 90% in France [7,8]. In these cases of BRD, M. bovis mostly occurs in coinfection with viruses and/or other bacteria but is often the only etiological agent in the chronic forms of BRD, which respond poorly to antimicrobials [2,9,10]. Today, only antimicrobials and sanitary controls are available to combat M. bovis infections. Commercial vaccines are only available in a few countries and their efficacy is subject to debate [1113]. Assessing the susceptibility of mycoplasmas to antimicrobials is difficult. Some characteristics of these organisms, such as their slow growth, small size and complex growth media requirements are incompatible with the standard procedures used to test the susceptibility of classic bacteria to antimicrobials such as the disk diffusion method. The Clinical and Laboratory Standards Institute (CLSI) has only recently established standardised antimicrobial susceptibility tests to determine the minimal inhibitory concentrations (MIC) for human mycoplasma pathogens [14]. However, these procedures cannot be used for all mycoplasmas because nutritional requirements, metabolic capacities and fitness vary among species [14]. For veterinary mycoplasma species, recommendations to control the main sources of experimental bias were proposed in 2000 by the International Research Programme on Comparative Mycoplasmology (IRPCM) [15]. Today there is no veterinary reference strain well characterized for MICs to be shared for quality control purposes, which is a major hurdle to compare results from different studies. Moreover, the absence of established antimicrobial breakpoint concentrations for mycoplasmas makes it difficult to evaluate the likely in vivo therapeutic efficacy from MIC data established in vitro. Several studies on the susceptibility of M. bovis to antimicrobials have been published [6,1625] but recent ones are scarce [23,24] or have not been published so far [Gosney and Ayling, unpublished results; Cai et al., unpublished results]. The experimental procedures used vary considerably: MIC tests were carried out using either the liquid broth microdilution method [16,19,20,22,23,25], the solid agar dilution method [24] or the E testH [6,21]. Measuring mycoplasma growth is difficult in liquid and solid media because broth turbidity is difficult to measure in a standardised way and colony size on agar can be microscopic. In broth, growth is measured indirectly by a color change of a pH indicator with the inclusion of a substrate, typically glucose, arginine or urea, according to the species. Because M. bovis does not use any of these substrates, alternative indirect assay methods have been specifically developed based on either tetrazolium reduction [16]; alamarBlueH, a color redox indicator [22,23]; or phosphatase [6]. Growth has also been directly measured either by observing colonies on agar plates under a stereomicroscope [6,21,24] or by observing pellets after centrifuging the cultures [19]. The reference M. bovis type strain ATCC 25523 has often been used as a control [6,15,16,21,22,24]; the large disparities in observed MIC values, from 5 to 8 two-fold dilutions for some antimicrobials, illustrates the difficulty in comparing studies carried out using different methods. Reports, for most antimicrobials except fluoroquinolones, give MICs that are distributed over a large range of dilutions and suggest that strains greatly vary in their susceptibility, but without any clear separation of sub-populations. Comparative stu (...truncated)