Antibiotic susceptibility profiles of Mycoplasma bovis strains isolated from cattle in Hungary, Central Europe

BMC Veterinary Research Antibiotic susceptibility profiles of Mycoplasma bovis strains isolated from cattle in Hungary, Central Europe Kinga M Sulyok 0 Zsuzsa Kreizinger 0 Lilla Fekete 0 Veronika Hrivnk 0 Tibor Magyar 0 Szilrd Jnosi Nra Schweitzer Ibolya Turcsnyi Lszl Makrai Kroly Erdlyi Mikls Gyuranecz 0 0 Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences , Hungaria korut 21, Budapest 1143 , Hungary Background: Mycoplasma bovis is a worldwide pathogen, causative agent of pneumonia, mastitis, arthritis, and a variety of other symptoms in cattle. The economic losses due to mycoplasma pneumonia could be reduced by antibiotic treatment. The aim of the present study was to determine the in vitro susceptibility of M. bovis strains isolated from cattle in Hungary to eleven antibiotics. Results: Minimal inhibitory concentration (MIC) values of 35 M. bovis strains collected from different parts of Hungary between 2010 and 2013 were determined by the microbroth dilution method. Strains with high MIC values were found in the case of all applied antibiotics. The most effective antibiotics tested in vitro were fluoroquinolones (MIC90 danofloxacin 0.312 g/ml, enrofloxacin 0.312 g/ml, marbofloxacin 0.625 g/ml). Our results confirm the observations of increasing MIC values to antibiotics commonly used in the therapy of mycoplasma infections, primarily to tetracyclines; tetracycline (MIC90 16 g/ml) and oxytetracycline (MIC90 64 g/ml) and macrolides; tylosin (MIC90 128 g/ml) and tilmicosin (MIC90 128 g/ml). The growth of many M. bovis strains was not inhibited by gentamicin (MIC90 8 g/ml), spectinomycin (MIC90 256 g/ml), florfenicol (MIC90 8 g/ml) or lincomycin (MIC90 64 g/ml). Conclusions: Our results emphasize the necessity of periodic testing for antibiotic susceptibility in this geographic region. Based on our in vitro examinations, fluoroquinolones could be the most effective drugs for the therapy of M. bovis infections in Hungary. However, current antimicrobial use policies have to be taken into account to avoid further antibiotic resistance development and to reserve fluoroquinolones for the treatment of severe infections which have responded poorly to other classes of antimicrobials. Antibiotic resistance; MIC; Fluoroquinolones; Microbroth dilution; Mycoplasma bovis - Background Mycoplasma bovis is a widely distributed pathogen, first isolated in the USA in 1961 from a case of severe mastitis in cattle [1]. It is associated with various diseases in cattle including calf pneumonia, mastitis, arthritis, otitis media and genital disorders [2]. M. bovis is considered responsible for a quarter to a third of economic losses in the cattle industry caused by respiratory diseases [3]. In Hungary, the average seropositivity rate of individual animals was found to be 11.3%, in certain herds it even exceeded 50.0%. Tested by enzyme-linked immunosorbent assay the overall rate of seropositive herds was 64.7% [4]. With the exception of seroprevalence on individual level, these values are relatively high in a European context [3,5]. Since no effective vaccine is available against M. bovis, adequate housing and appropriate antibiotic treatment are promoted in the control of the diseases caused by this agent. Antibiotic therapy of mastitis has often failed, but antimicrobial treatment of pneumonia has shown some success and it may help reduce economic losses [3,6]. Mycoplasmas are intrinsically resistant to -lactam antimicrobials and sulphonamides, because they do not possess a cell wall and do not synthesize folic acid. Mycoplasmas are generally susceptible to antibiotics that affect protein (tetracyclines, macrolides, lincosamides, phenicols) or nucleic acid synthesis (fluoroquinolones) [2]. The decreased effectiveness of certain antimicrobial agents (spectinomycin, oxytetracycline and tilmicosin) traditionally used in the therapy of mycoplasma infections was reported in Europe [7]. The aim of this study was to determine the susceptibility of 35 Hungarian M. bovis isolates to eleven antibiotics using the microbroth dilution method. Methods Thirty-five M. bovis strains originating from dairy herds located in different parts of Hungary were tested in this study (Table 1, Figure 1). The samples were collected during routine diagnostic examinations or necropsies between 2010 and 2013. Ethical approval was not required for the study as all samples were collected during routine diagnostic examinations or necropsies. Nasal swabs, lung samples and a single lymph node were homogenized in 2 ml of Mycoplasma broth medium (pH 7.8) (Thermo Fisher Scientific Inc./Oxoid Inc./, Waltham, MA) supplemented with 0.5% (w/v) sodium pyruvate, 0.5% (w/v) glucose and 0.005% (w/v) phenol red and cultured at 37C in a 5% CO2 atmosphere. Following colour change (red to yellow shift) the cultures were inoculated onto solid Mycoplasma media (Thermo Fisher Scientific Inc. /Oxoid Inc./) and were incubated at 37C and 5% CO2 for 3 days, until visible colonies appeared. Mixed cultures were filter cloned only once to exclude contaminant Mycoplasma species and to minimize in vitro mutations of the isolates. DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen Inc., Hilden, Germany) according to the manufacturers instructions for Gram-negative bacteria. All isolates were identified by polymerase chain reaction (PCR) targeting the uvrC gene of M. bovis [8]. The purity of the cultures (e.g. to exclude M. arginini or other Mycoplasma spp. contamination) was confirmed by a universal Mycoplasma PCR system targeting the 16S/23S rRNA intergenic spacer region in Mollicutes [9] followed by sequencing on an ABI Prism 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA), sequence analysis and BLAST search. The same once filter cloned passage of each M. bovis strain was submitted for a 4 gene based multi-locus sequence typing (MLST) and the sequencing data confirmed the purity of the isolates at strain level (i.e. not more than one M. bovis strain in the culture) [10]. Mixed primary cultures which failed to be purified by a single filter cloning were excluded from the study (data not shown). Aliquots of the third passage of purified cultures were stored frozen at 70C until required. The number of colour changing units (CCU) was calculated by microplate dilution method, from the lowest dilution showing colour change after one week of incubation [11,12]. The following antimicrobial agents were examined during the microbroth dilution tests: three fluoroquinolones: danofloxacin (batch SZBA019XV), enrofloxacin (batch SZBA336XV) and marbofloxacin (batch SZBC248XV); two aminoglycosides: gentamicin (batch 051K17475V) and spectinomycin (batch SZBB166XV); two tetracyclines: oxytetracycline (batch SZBC320XV) and tetracycline (batch SZBA140XV); two macrolides: tilmicosin (batch SZBC345XV) and tylosin (batch SZBB160XV); one phenicol: florfenicol (batch (...truncated)