Information about the susceptibility of M. sp. 1220 strains to antimicrobials is scarce, as until to date the sole published reference concerning the antibiotic susceptibility profile of this species is a review of Stipkovits and Szathmary . Stipkovits and Szathmary determined the values of enrofloxacin, tylosin, chlortetracycline, oxytetracycline, doxycycline, tiamulin and lincomycin in Mycoplasma species affecting waterfowl (M. anatis, M. cloacale, M. anseris and M. sp. 1220), although detailed data of their method is lacking . Thus we are facing the absence of reports about the antibiotic susceptibility of M. sp. 1220 and also of other Mycoplasma species occurring in waterfowl. Therefore, the results of the current study are also compared to data of antibiotic susceptibility of the well-studied Mycoplasma species of poultry: M. synoviae and M. gallisepticum.
Elevated MIC values were reported previously in the case of the fluoroquinolones, especially of enrofloxacin in M. sp. 1220 (MIC50 2 μg/ml and MIC90 4 μg/ml) and other Mycoplasma species of poultry [3, 13, 20, 21]. In addition, the increasing occurrence of quinolone-resistant M. synoviae and M. gallisepticum field isolates were also observed [13, 22]. In the current study, the detected MIC50 values (5 μg/ml for enrofloxacin, 10 μg/ml for difloxacin and ≥10 μg/ml for norfloxacin) were even higher than the ones reported before [3, 13, 20–22], confirming the observation of increasing quinolone-resistance in Mycoplasma species. In order to save these antibiotics for human disease treatment the directive was to reduce the use of these agents in livestock. Former efforts for the prevention of the appearance of quinolone-resistant species are proved to be unsuccessful considering the observed dramatic elevations in the MIC values of these antibiotics in avian Mycoplasma species [13, 21, 23].
Administration of the combination of lincomycin and spectinomycin could reduce the egg infertility rates and increase the hatching rates and the egg production in M. sp. 1220 infected geese . The lincomycin-spectinomycin therapy was proved to be effective against other Mycoplasma species as well; however, application of spectinomycin in monotherapy is not recommended for its insufficient effectiveness and relatively high MIC values in in vitro experiments . In vitro effectiveness of lincomycin at 2 μg/ml MIC50 values against M. sp. 1220, M. anseris and M. anatis species has been reported . In the present study, all isolates showed elevated MIC values for spectinomycin, lincomycin and lincomycin-spectinomycin combination. The growth of a couple of strains was not inhibited even at the highest concentrations used (64 μg/ml) for spectinomycin and lincomycin individually. The combination of the two antibiotics improved their effectiveness, as lincomycin-spectinomycin combination could inhibit the growth of all examined strains within the concentration range used (0.25 to 64 μg/ml) and lower MIC90 value was observed also.
Previously, tetracyclines (chlortetracycline, doxycycline and oxytetracycline) showed 1–2 μg/ml MIC values against M. sp. 1220 strains. Growth of other Mycoplasma species isolated from waterfowl were inhibited at 2–4 μg/ml MIC50 values using the same antibiotics . Previously Mycoplasma species infecting poultry were observed to be inhibited by elevated MIC values, although with exceptions, as M. synoviae strains showed high susceptibility to doxycycline in the Netherlands [12–14]. In the current study, although the M. sp. 1220 strains showed broad ranges of MIC values for oxytetracycline and doxycycline, more than 50 % of the strains were inhibited by only higher antibiotic concentrations (64 and 5 μg/ml, respectively) and MIC90 values exceeded the concentration ranges used for both compounds. These results show a dramatic increase of the MIC values of tetracyclines against M. sp. 1220 strains and reveals the presence of probably highly resistant strains in Hungary.
Macrolides, especially tylvalosin have good in vitro effectiveness against most Mycoplasma species infecting poultry, showing lower MIC values in previous examinations than quinolones and tetracyclines [3, 12–15]. However, M. gallisepticum could develop resistance rapidly to these compounds, especially to tilmicosin . Earlier, the MIC50 values in M. sp. 1220, M. anatis, M. anseris and M. cloacale strains were defined to be 2 μg/ml for tylosin . In the current study, the MIC50 value (8 μg/ml) of tylosin was higher than the previous observation , and the MIC90 value exceeded the concentration range used in the experiment. However, high variability was observed in the susceptibility of the strains to this compound. Similarly, wide range of the MIC values was detected for tilmicosin, highlighting the necessity of susceptibility testing before antibiotic treatments. As opposed to the diverse susceptibility profiles of the strains for tylosin, the MIC values of tilmicosin were categorized into three separate groups. The observed distribution of the MIC values is likely in association with the capability of Mycoplasma sp. 1220 to develop resistance more rapidly to tilmicosin (i.e. with one or two mutations) than to other macrolides. The same phenomenon was described in other Mycoplasma species as well . Out of the three macrolides examined in the study, tylvalosin proved to be the most effective agent against M. sp. 1220 strains, showing lower MIC50 value (0.5 μg/ml) against the pathogen than the majority of the antibiotics tested.
Pleuromutilins showed good in vitro effectiveness against avian Mycoplasma species in previous studies and low tendency of the development of resistance to these agents has been reported [16–18, 21]. Tiamulin was used successfully for the treatment of mycoplasmosis and its effectiveness was similar to spectinomycin therapy in the treated geese . Stipkovits and Szathmary described low MIC values (MIC50: 0.06 μg/ml, MIC90: 0.25 μg/ml) of tiamulin in the case of M. sp. 1220, and similarly low MIC50 values (0.125–1 μg/ml) were observed against M. anseris, M. anatis and M. cloacale . In the present study, pleuromutilins were found to be the most effective antibiotic agents and the examined compounds, especially valnemulin showed high in vitro effectiveness against all tested isolates of the pathogen. However, it is noteworthy, that strains with elevated MIC values were detected for tiamulin (MIC: 2.5–5 μg/ml) and even for valnemulin (MIC: 0.312 μg/ml). Although the low MIC values of valnemulin against M. sp. 1220 strains in vitro are promising for its clinical use, it should be noted that in a previous study only a single mutation in M. gallisepticum could cause elevation in the MIC values of valnemulin . To date, the use of pleuromutilins in humans is limited, as only one commercially available product is authorized containing this active substance. However, bacterial strains resistant to pleuromutilins have already been described and these strains also show multidrug resistance, which warrants the prudent use of these antibiotic agents .
Phenicols showed good in vitro activity against Mycoplasma species of poultry, but information about their effectiveness in waterfowl is lacking [26, 27]. In the present study, most of the M. sp. 1220 isolates yielded the same MIC values (4 or 8 μg/ml) for florfenicol, and only two isolates (originating from the same region) showed elevated MIC values compared to the MIC50 (8 μg/ml), one of them reaching the highest antibiotic concentration (32 μg/ml) used.
The elevated MIC values of several antibiotics detected in subsequent isolates from the same farms from year to year are likely in association with the inconsistent use of antibiotics, the rapid development of antibiotic resistance and highlight the importance of susceptibility testing before therapy and responsible use of antimicrobial drugs.