26Methicillin-resistant Staphylococcus aureus (MRSA) is infrequently reported in mastitis. Yet, 27 as in many other countries, the prevalence of methicillin resistance among S. aureus from 28 mastitis is currently unknown in Belgium. 29To elucidate this, the presence of mecA was investigated in 118 S. aureus strains originating 30 from diagnostic mastitis milk samples from 118 different farms experiencing S. aureus 31 mastitis. MRSA strains were characterized by disk diffusion susceptibility testing, spa-typing, 32MLST and SCCmec-typing. In an additional study, four MRSA-positive farms were selected 33 to assess the in-herd prevalence of MRSA, by sampling all cows in lactation. Isolated MRSA 34 strains were similarly characterized. 35The mecA gene was detected in eleven (9.3%) of the 118 S. aureus isolates, indicating that 36 nearly 10% of the Belgian farms suffering from S. aureus mastitis have an MRSA problem. 37The in-herd prevalence varied between 0% and 7.4%. Characterization of the MRSA strains 38showed that they were all resistant to tetracycline. Additional resistances to macrolides, 39 lincosamides and aminoglycosides were frequently detected. The strains were ST398, spa-40

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The in vitro susceptibilities of 21 Mycoplasma hyopneumoniae field isolates were determined using a broth microdilution technique. One isolate showed acquired resistance to lincomycin, tilmicosin, and tylosin, while five isolates were resistant to flumequine and enrofloxacin. Acquired resistance against these antimicrobials in M. hyopneumoniae field isolates was not reported previously.Mycoplasma hyopneumoniae causes enzootic pneumonia, a chronic respiratory disease in pigs resulting in considerable economic losses. Although appropriate vaccines are available to reduce the consequences of infection, medication with antimicrobials in feed or water is still a common practice. The use of antimicrobials, however, results in the selection of resistant bacteria. The antimicrobial susceptibility of M. hyopneumoniae field isolates has rarely been determined, and limited numbers of strains have been considered in these studies (5,7,8,14,16), mainly due to the fact that M. hyopneumoniae is a fastidious and slowly growing microorganism, making it difficult to obtain large numbers of field strains. In this study, the antimicrobial susceptibility of recently isolated M. hyopneumoniae field strains

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Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is present in almost all swine herds worldwide, but transmission of the pathogen through herds is not yet fully clarified. The aim of this study, performed in 2003, was to investigate and to quantify the transmission of M. hyopneumoniae under experimental conditions by means of an adjusted reproduction ratio (R n ). This R n -value, calculated according to the final size method, expresses the mean number of secondary infections due to one typical infectious piglet during the nursery period. The period lasted from 4 to 10 weeks of age, corresponding with the nursery period used in most European production systems. Additionally, a comparison was made between transmissions of highly virulent and low virulent isolates.Forty-eight weaned piglets, free of M. hyopneumoniae, were housed in six separate pens. During 6 weeks, two animals experimentally infected with M. hyopneumoniae were housed together with six susceptible piglets. At the end of the study, the number of contact-infected animals was determined by the use of nPCR on bronchoalveolar lavage fluid. The R n -values of the highly virulent and the low virulent isolates were estimated to be 1. 47 (0.68-5.38) and 0.85 (0.33-3.39), respectively. No significant difference between the groups was found (P = 0.53). The overall R n was estimated to be 1. 16 (0.94-4.08). Under the present experimental conditions, the transmission of M. hyopneumoniae,

Macrolides and related antibiotics are used to control mycoplasma infections in the pig industry worldwide. Some porcine mycoplasmas, however, survive these treatments by acquiring resistance. The mechanism of acquired resistance to macrolides and lincosamides was studied in more detail for Mycoplasma hyopneumoniae by comparing both the phenotype and genotype of a resistant field isolate to five susceptible isolates. The MICs were significantly higher for the resistant strain for all antibiotics tested. The MICs for the 16-membered macrolide tylosin ranged from 8 to 16 microg for the resistant strain and from 0.03 to 0.125 microg/ml for the five susceptible strains. The MICs for the 15-membered macrolides and lincosamides were higher than 64 microg/ml for the resistant strain while only 0.06 to 0.5 microg/ml for the susceptible strains. Mycoplasma hyopneumoniae strains are intrinsically resistant to the 14-membered macrolides due to a G 2057 A transition (E. coli numbering) in their 23S rDNA. Therefore, high MICs were observed for all strains, although the MICs for the resistant strain were clearly increased. An additional, acquired A 2058 G point mutation was found in the 23S rRNA gene of the resistant strain. No differences linked to resistance were found in the ribosomal proteins L4 and L22. The present study showed that 23S rRNA mutations resulting in resistance to macrolides and lincosamides as described in other Mycoplasma spp. also occur under field conditions in M. hyopneumoniae.

The quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE of ten Mycoplasma hyopneumoniae field isolates that were either sensitive (5) or resistant (5) to the fluoroquinolones flumequine and enrofloxacin were characterized. In all five resistant isolates, one point mutation (C --> A) in parC was found, resulting in an amino acid change from serine to tyrosine at position 80 (Escherichia coli numbering). For four of these isolates, this was the only mutation found. These isolates had a minimum inhibitory concentration (MIC) of enrofloxacin of 0.5 microg/ml, whereas for sensitive isolates the MIC of enrofloxacin was < or =0.06 microg/ml. One resistant isolate (Mh 20) had an extra mutation (C --> T) in gyrA resulting in an amino acid change from alanine to valine at position 83 (E. coli numbering), leading to a further increase in the MIC of enrofloxacin (>1 microg/ml). No mutations resulting in an amino acid change were detected in the QRDR of the gyrB and parE genes of the selected isolates. This is the first description of the mechanism of stepwise resistance against fluoroquinolones in M. hyopneumoniae.