Antimicrobial resistance is a growing global concern for animals and humans. During the past decade, few studies have investigated the MICs of M. synoviae isolates in vitro. At present, the commonly used clinical antimicrobial medicines for the treatment of mycoplasma disease are macrolides (e.g., TY and TIL), pleuromutilins (e.g., valnemulin and TIF), tetracyclines (e.g., DO and OT), fluoroquinolones (e.g., ENR), as well as LS. In this study, we investigated the antimicrobial susceptibility of 32 M. synoviae strains isolated from China from 2016 to 2019.

Our results showed that the MIC values of M. synoviae isolates were generally low for LS, pleuromutilin, and macrolides. Similar results were observed in a recent study in Asia, except for TIL [21]. When spectinomycin was applied in combination with lincomycin, it improved the efficacy of two antimicrobials against most M. synoviae [22]. In this study, 31/32 isolates were sensitive to low concentrations of LS (MICs 0.063 ~ 1 μg/mL). A previous study investigated the antibiotic susceptibility of 41 M. synoviae strains originating from Central and Eastern Europe between 2002 and 2016, including Hungary, Austria, the Czech Republic, Slovenia, Ukraine, Russia, and Serbia. Overall, similar low MIC values (0.25 ~ 2 μg/mL) were detected for LS [13]. The macrolides showed good activity against M. synoviae strains worldwide, but higher MIC values (> 2 μg/mL) were also identified in Europe [13, 23, 24]. In the current study, all isolates were sensitive to low concentrations of TY and TIL with MICs of only 2/32 and 3/32 isolates = 1 μg/mL. In contrast, TIL MICs clearly showed a time-dependent gradual transition to high concentrations in 154 M. synoviae isolates from Italy collected from 2012 to 2017. Seven M. synoviae isolates showed an MIC > 32 μg/mL for TIL between 2013 and 2016 [25]. High MIC values were also detected in another study, which showed 25/87 M. synoviae strains with high MIC values (> 8 μg/ml for TIL and/or > 1 μg/ml for TY and/or > 0.5 μg/ml for tylvalosin) from 18 different countries from 1982 to 2019 [26]. As mentioned above, increased TIL MICs (≥64 μg/ml MIC90 values) were also detected in M. synoviae isolates collected from China, India, Indonesia, Malaysia, the Philippines, the Republic of Korea, and Thailand [21]. Our results confirmed the high efficiency of TY and TIL against M. synoviae in China. Previous research has shown that pleuromutilins display high efficacy against avian mycoplasmas [27]. To date, the MIC values for TIF in the Europe mentioned above are relatively low (0.004 ~ 2.5 μg/mL). All M. synoviae isolates remained sensitive to TIF with MICs ranging from 0.12 to 2.5 μg/mL in South Africa between 2003 and 2015 [16]. The MIC values of VA were ≤ 0.039 μg/mL in Central and Eastern Europe [26]. The M. synoviae isolates examined in this study showed high susceptibility to VA (MIC < 0.016 ~ 0.031 μg/mL) and TIF (MIC < 0.016 ~ 0.063 μg/mL). Especially, 31 M. synoviae isolates had MIC values equal or lower than the lowest concentration of VA (0.016 μg/mL). Therefore, pleuromutilins are supposed to be preferable in the treatment of M. synoviae infection.

For tetracyclines, 3/32 of M. synoviae isolates showed intermediate MIC values for DO, and only one strain showed an intermediate MIC value for OT. The MIC50 and MIC90 values for DO were same as OT, being 2 and 4 μg/mL respectively. The finding of tetracycline resistance was not unexpected because of the long-term widespread use of tetracyclines in feed in China. Aureomycin, DO and OT are the most widely used antimicrobials. It can not only prevent bacterial infection but also improve the growth performance of animals. Our results indicated that long-term use of tetracycline antimicrobials can reduce the sensitivity of M. synoviae. Interestingly, even though the M. synoviae isolates did not have high MIC values of tetracyclines, our results do not align with previous studies in Europe, which showed that the MIC values of OT were higher than those of DO. For example, MIC values of OT and DO to M. synoviae were 0.031 ~ 32 μg/mL and 0.062 ~ 2 μg/mL, respectively, from six European countries from 2014 to 2016 [24]. In another study, 84 M. synoviae field strains were collected from 18 different countries from 2010 to 2019, and the majority of strains were from Hungary, Italy, the Netherlands, Israel, and Spain. The MIC values of OT and DO were ≤ 0.25 ~ 8 μg/mL and ≤ 0.039 ~ 1.25 μg/mL, respectively [26]. The difference in MIC value may be due to the geographic area, density of poultry flocks, and different quantitative uses among countries. Since 2020, all forms of growth-promoting antimicrobials, except for traditional Chinese medicines have been forbidden to be used as feed additives in China. With the increase in the number of laws and regulations concerning the use of antimicrobials, we speculate that the resistance of M. synoviae to tetracyclines may be decreased in the future.

Previous studies showed that resistance to ENR increased rapidly [17, 28, 29]. In this study, high MIC values (4 ~ 32 μg/mL) for ENR were present in all M. synoviae isolates. The MIC50 and MIC90 values of ENR were the highest, with 12 isolates showing MIC values of 16 μg/mL, 3 isolates showing MIC values of 32 μg/mL. In Israel and Europe, decreased susceptibility to ENR was detected in 59% of M. synoviae field strains, with MICs ranging from 1 to > 16 μg/mL [17]. There is no standardized method for MIC testing in animal mycoplasma, but genetic mutations can determine the presence of antimicrobial resistance. To further investigate the mechanism of ENR resistance, gyrA, gyrB, parC, and parE genes in the QRDRs of 32 MS isolates were sequenced and analyzed. Topoisomerase IV (parC) is considered to be the primary target of ENR in M. synoviae, based on decreased susceptibility after experimental infection in vivo [18]. Amino acid positions 85–89 (80–84 according to Escherichia coli strain K-12 substrain MG1655) of parC were identified as hot spot regions that seem to have a principal role [26]. Amino acid substitutions at positions 80 and 84 of parC are known as important spots for ENR resistance in many bacteria, including mycoplasmas and may be alone or together with a mutation of gyrA [30,31,32,33]. In addition, amino acid substitutions at positions 79 and 81 of parC were also identified in mycoplasma [17, 34]. In the current study, comparison of the parC QRDR identified a mutation at nucleotide position 254 (C254T) resulting in a Thr 85 Ile amino acid change in all M. synoviae isolates and ATCC 25204, which was similar to a previous study [26]. In the gyrB gene, a SNP mutation has been found at position 1250 (G1250A) and resulted in a Ser 417 Asn amino acid change in 11/32 M. synoviae isolates with high MIC values to ENR, which has also been reported [17].

Comparison of the gyrA QRDR found the presence of different amino acid substitutions at positions 686, 804 and 814. Mutations at Glu 804 Gly and Thr 686 Ala were identified in all M. synoviae isolates and ATCC 25204. To our knowledge, these nonsynonymous mutations have not been reported in previous research. Our results revealed that there is a correlation between MIC values and amino acid mutations at positions 804 and 686 of the gyrA QRDR. Indeed, M. synoviae isolates containing these two amino acid substitutions had MICs ranging from 4 to 32 μg/mL. These two amino acid mutations may together affect M. synoviae with decreased susceptibility to ENR. More strains with a broader spectrum of MICs should be identified to prove this conclusion, and the relevance of the mutations that occurred in gyrA QRDR should be further investigated. The mutation in the QRDR of parE at positions 197 (Pro to Ser) in 27/32 M. synoviae isolates has also not been reported. Amino acid substitution at position 420 of parE corresponds to residue 426 Asp of gyrB in E. coli, which is a multiple possible marker for quinolone resistance in many bacteria [35, 36]. The role of position 197 of parE in quinolone resistance needs to be further established.

In conclusion, 32 M. synoviae isolates had low MIC values for the combination of lincomycin and spectinomycin, pleuromutilin and macrolides. However, 3/32 and 1/32 M. synoviae isolates showed intermediate MIC values for DO and OT. High MIC values for ENR were detected in all isolates, with MICs ranging from 4 to 32 μg/mL. Furthermore, mutations at Glu 804 Gly and Thr 686 Ala of gyrA QRDR were identified in all M. synoviae isolates and ATCC 25204. The mutation in the QRDR of the parE gene resulted in amino acid changes at positions 197 (Pro to Ser) in 27/32 M. synoviae isolates. These nonsynonymous mutations were first identified to be related to ENR resistance.

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