Antibiotic susceptibility profiles of Mycoplasma sp. 1220 strains isolated from geese in Hungary

Background Mycoplasma sp. 1220 can induce inflammation primarily in the genital and respiratory tracts of waterfowl, leading to serious economic losses. Adequate housing and appropriate antibiotic treatment are promoted in the control of the disease. The aim of the present study was to determine the in vitro susceptibility to thirteen different antibiotics and an antibiotic combination of thirty-eight M. sp. 1220 strains isolated from geese and a duck in several parts of Hungary, Central Europe between 2011 and 2015. Results High MIC50 values were observed in the cases of tilmicosin (>64 μg/ml), oxytetracycline (64 μg/ml), norfloxacin (>10 μg/ml) and difloxacin (10 μg/ml). The examined strains yielded the same MIC50 values with spectinomycin, tylosin and florfenicol (8 μg/ml), while enrofloxacin (MIC50 5 μg/ml), doxycycline (MIC50 5 μg/ml), lincomycin (MIC50 4 μg/ml) and lincomycin-spectinomycin (1:2) combination (MIC50 4 μg/ml) inhibited the growth of the bacteria with lower concentrations. Tylvalosin (MIC50 0.5 μg/ml) and two pleuromutilins (tiamulin MIC50 0.625 μg/ml; valnemulin MIC50 ≤ 0.039 μg/ml) were found to be the most effective drugs against M. sp. 1220. However, strains with elevated MIC values were detected for all applied antibiotics. Conclusions Valnemulin, tiamulin and tylvalosin were found to be the most effective antibiotics in the study. Increasing resistance was observed in the cases of several antibiotics. The results highlight the importance of testing Mycoplasma species for antibiotic susceptibility before therapy.


Background
Mycoplasma sp. 1220 was first described as a new Mycoplasma species by Stipkovits et al. in 1986 [1]. This Mycoplasma species causes cloaca and phallus inflammation and testicular atrophy in the ganders [1,2]. In the infected geese salpingitis and vaginitis are the main symptoms [1,3]. The egg production activates the pathogen and the flared up mycoplasmosis could induce lethal pathological changes in the embryos [1,4]. Airsacculitis and peritonitis are also common, and general symptoms such as changes in thirst, decreased food consumption, body weight losses, weakness, nasal discharge, impaired breathing, conjunctivitis, diarrhoea and nervous signs were also described in the affected waterfowl flocks [2,[5][6][7][8]. Mycoplasma infection of the birds can aggravate diseases caused by other agents and could lead to serious economic losses [3,6]. Since there is no commercially available vaccine against M. sp. 1220, adequate housing and appropriate antibiotic treatment are promoted in the control of the diseases caused by this agent. Prophylactic medication could prevent economic losses if appropriate antibiotics are administered in the early weeks of life and in expected stress periods. Medication of the layers is essential to reduce the vertical transmission of M. sp. 1220 [2].
Mycoplasmas are resistant to β-lactam antimicrobials because of the lack of cell-wall and the bacteria are also resistant to membrane synthesis inhibitors [2,9]. Antibiotics such as quinolones, tetracyclines, macrolides and pleuromutilins which induce DNA fragmentation or inhibition at the level of protein synthesis are the drugs of choice for the therapy of mycoplasmosis. Among the macrolides, erythromycin showed high effectiveness against Mycoplasma strains which could ferment glucose (e.g. M. sp. 1220), while arginine-hydrolysing strains proved to be less susceptible to this compound [2,10]. Mycoplasma infected waterfowl and poultry flocks are usually treated with macrolides, pleuromutilins or with the combination of lincomycin and spectinomycin [3,[11][12][13][14][15][16][17][18].
The aim of this study was to determine the susceptibility of 38 Hungarian M. sp. 1220 isolates to thirteen antibiotics and a drug combination using the microbroth dilution method.

Methods
A total of 38 M. sp. 1220 strains isolated from geese and a duck originating from different parts of Hungary were tested in the study (Table 1, Fig. 1). The samples were collected during routine diagnostic examinations or necropsies between 2011 and 2015, thus ethical approval was not required for the study. Phallus lymph, cloaca swabs, tracheal swabs, follicules and lung samples were washed in 2 ml of Mycoplasma broth medium (pH 7.8) (ThermoFisher 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 incubated at 37°C in a 5 % CO 2 atmosphere. The cultures were inoculated onto solid Mycoplasma media (Thermo Fisher Scientific Inc./Oxoid Inc./) after colour change of the broth, and were incubated at 37°C and 5 % CO 2 until visible colonies appeared (1-2 days). Purification of mixed cultures was performed by one-time filter cloning, minimizing the in vitro mutations of the isolates. The QIAamp DNA Mini Kit (Qiagen Inc., Hilden, Germany) was used for DNA extraction according to the manufacturers' instructions for Gram-negative bacteria. The purity of the cultures was confirmed by a universal Mycoplasma PCR system targeting the 16S/23S rRNA intergenic spacer region in Mycoplasmatales followed by sequencing on an ABI Prism 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA), sequence analysis and BLAST search [19]. The number of colour changing units (CCU) was calculated by microbroth dilution method, from the lowest dilution showing colour change after one week of incubation [9].
The microbroth dilution examinations on 10 4 -10 5 CCU/ ml of the strains were performed according to Hannan [9]. Mycoplasma broth medium was used in the tests as well, and each 96-well microtiter plates contained growth controls (broth medium without antibiotic), sterility controls (broth medium without antibiotic and Mycoplasma inoculum) and pH controls (broth medium adjusted to pH 6.8).
One clinical isolate (strain MYCAV 65) was selected to be used as quality control of minimal inhibitory concentration (MIC) determination throughout the experiments. The duplicates of three clinical isolates and the duplicate of the selected strain (MYCAV 65) were tested on each 96well microtiter plates.
The MIC values were determined from the lowest concentration of the antibiotics where no pH and colour change of the broth was detected after one week of incubation, meaning that the growth of the bacteria was completely inhibited in the broth. MIC 50 and MIC 90 values were defined as the lowest concentrations that inhibited the growth of 50 % or 90 % of the strains [9].

Results
The quality control strain (MYCAV 65) showed consistent results throughout the study. Strains with elevated MIC values were found in the cases of all tested antibiotics (Tables 1 and 2).
Among the fluoroquinolones, the MIC values of enrofloxacin and difloxacin showed a wide range (1.25 to >10 μg/ml), while all strains had very high MIC values for norfloxacin (≥10 μg/ml) (Fig. 2a, b and c). The MIC 50 was 8 μg/ml for spectinomycin and most of the strains yielded the MIC 50 or higher MIC values (Fig. 2d). The MICs for lincomycin clustered around the MIC 50 value (4 μg/ml) as well, but high MIC values (>64 μg/ml) were yielded in the case of three isolates (Fig. 2e) Fig. 2i and j). While the MIC values for tylosin showed diverse distribution, the strains' susceptibility profiles formed three groups in the case of tilmicosin (≤0.25, 4 and >64 μg/ml) (Fig. 2j). Among the examined three macrolides (tylosin, tilmicosin and tylvalosin), tylvalosin showed the lowest MIC 50 value (0.5 μg/ml) against the strains (Fig. 2k). From the pleuromutilins the MIC values of tiamulin were higher than those of valnemulin, and the latter compound was found to be the most active antibiotic in the examinations ( Fig. 2l and m). In the case of florfenicol, the susceptibility profiles of most strains were similar to each other and showed the MIC 50 and MIC 90 value (8 μg/ml) or its twofold lower dilution (4 μg/ml) with few exceptions (Fig. 2n).   year to year in the cases of certain antibiotics. Higher MIC values were detected in subsequent isolates for lincomycin, lincomycin-spectinomycin combination, tetracyclines (both oxytetracycline and doxycycline), macrolides (tylosin, tilmicosin and tylvalosin), tiamulin and for florfenicol as well.

Discussion
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 [3]. 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 [3]. 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 wellstudied 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 (MIC 50 2 μg/ml and MIC 90 4 μg/ml) and other Mycoplasma species of poultry [3,13,20,21]. In addition, the increasing occurrence of quinoloneresistant M. synoviae and M. gallisepticum field isolates were also observed [13,22]. In the current study, the detected MIC 50 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][21][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 [11]. The lincomycinspectinomycin 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 [12]. In vitro effectiveness of lincomycin at 2 μg/ml MIC 50 values against M. sp. 1220, M. anseris and M. anatis species has been reported [3]. 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 MIC 90 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 MIC 50 values using the same antibiotics [3]. 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][13][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 MIC 90 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][13][14][15]. However, M. gallisepticum could develop resistance rapidly to these compounds, especially to tilmicosin [24]. Earlier, the MIC 50 values in M. sp. 1220, M. anatis, M. anseris and M. cloacale strains were defined to be 2 μg/ml for tylosin [3]. In the current study, the MIC 50 value (8 μg/ ml) of tylosin was higher than the previous observation [3], and the MIC 90 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 [24]. Out of the three macrolides examined in the study, tylvalosin proved to be the most effective agent against M. sp. 1220 strains, showing lower MIC 50 value