Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, a contagious respiratory disease, causing significant economic losses worldwide. Antibiotic treatment is commonly utilised in the pig industry to control M. hyopneumoniae infection. Since the conventional antibiotic susceptibility test is time-consuming, taking up to weeks’ period, antibiotics are usually empirically chosen.

Certain single nucleotide polymorphisms in the parC (C239A/T, G250A) and gyrA (G242C, C247 T, A260 G) genes show correlation with decreased fluoroquinolone susceptibility by the change of the target site. Furthermore, the nucleotide alteration A2059 G in the 23S rRNA sequence correlates with significantly decreased macrolide and lincosamide susceptibility of M. hyopneumoniae. Mismatch amplification mutation assays (MAMA) and high resolution melt (HRM) analysis, capable to detect the mentioned resistance markers, were developed in the present study, in order to provide susceptibility data in a considerably shorter time than the conventional methods. The results of the MAMA and HRM assays were congruent with the results of the conventional antibiotic susceptibility method of the tested M. hyopneumoniae field isolates. The sensitivity of the MAMAs was 10³-10⁴ copy numbers, while that of the HRM assay was 10⁵-10⁶ copy numbers.

To the best of our knowledge this was the first time that MAMA and HRM assays were developed for the rapid detection of decreased fluoroquinolone, macrolide or lincosamide susceptibility in M. hyopneumoniae strains.

猪肺炎支原体是猪传染性肺炎的病原体，是一种传染性呼吸道疾病，在世界范围内造成重大经济损失。养猪业通常采用抗生素治疗来控制猪肺炎支原体感染。由于常规的抗生素敏感性测试很费时，需要长达数周的时间，因此通常根据经验选择抗生素。

parC（C239A / T，G250A）和gyrA（G242C，C247 T，A260 G）基因中的某些单核苷酸多态性与靶位点变化引起的氟喹诺酮敏感性降低相关。此外，23S rRNA序列中的核苷酸改变A2059 G与猪肺炎支原体的大环内酯和林可酰胺敏感性显着降低有关。。本研究开发了能够检测上述抗性标记的错配扩增突变分析（MAMA）和高分辨率熔解（HRM）分析，目的是在比传统方法短得多的时间内提供敏感性数据。MAMA和HRM分析的结果与测试的猪肺炎支原体田间分离株的常规抗生素敏感性方法的结果一致。MAMA的灵敏度为10 ³ -10 ⁴拷贝数，而HRM分析的灵敏度为10 ⁵ -10 ⁶拷贝数。

据我们所知，这是首次开发MAMA和HRM检测方法，用于快速检测猪肺炎支原体菌株中氟喹诺酮，大环内酯或林可酰胺敏感性的下降。