Institute for Veterinary Food Science, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.
Clinic for Poultry, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
J Clin Microbiol. 2022 Aug 17;60(8):e0041922. doi: 10.1128/jcm.00419-22. Epub 2022 Jul 19.
() is an opportunistic pathogen in poultry, which, however, has also been associated with human disease. There is currently no approved method for antimicrobial susceptibility testing of this pathogen, so this study aimed at developing a harmonized broth microdilution method for that is suitable for diagnostic laboratories. For this, the CCUG 12391 type strain and 42 field isolates were collected and their species was confirmed by using a species-specific PCR assay and biochemical reactions. To select epidemiologically unrelated isolates, ApaI macrorestriction analysis was performed. Preliminary growth experiments were conducted with six culture media, and based on the results, four media were selected to compile growth curves with four isolates. Independent repetitions of MIC determinations were then performed to evaluate the reproducibility of the values. Cation-adjusted Mueller-Hinton broth (CAMHB) was initially selected as broth medium, but did not show sufficient homogeneity of MICs. Therefore, CAMHB plus 1% chicken serum and 0.0025% NADH was selected and showed a good homogeneity of MICs after 20 h and 24 h of incubation at 35 ± 2°C. This was reflected in essential MIC agreements ranging between 96% and 100%. Testing of a larger collection ( = 43) revealed that easily readable MICs could be obtained for the type strain and all isolates. Some showed elevated MICs of enrofloxacin ( = 35), nalidixic acid ( = 35), penicillin ( = 2), tetracycline ( = 19), and/or trimethoprim-sulfamethoxazole ( = 1). By using PCR analyses, the following antimicrobial resistance genes were detected: , , , (B), (H). The study demonstrated that the proposed medium is suitable for a harmonized broth microdilution susceptibility testing of with a recommended incubation time of 20 to 24 h.
空肠弯曲菌是家禽中的机会致病菌，但也与人类疾病有关。目前，尚无批准的方法可用于该病原体的抗菌药物敏感性测试，因此本研究旨在开发一种适合诊断实验室的空肠弯曲菌协调肉汤微量稀释法。为此，收集了 CCUG 12391 标准株和 42 株临床分离株，并通过种特异性 PCR 检测和生化反应确认其种属。为了选择无关联的分离株，进行了 ApaI 大片段限制分析。用六种培养基进行初步生长实验，根据结果选择四种培养基，并用四种分离株绘制生长曲线。然后进行独立的 MIC 测定重复，以评估值的重现性。最初选择阳离子调整的 Mueller-Hinton 肉汤（CAMHB）作为肉汤培养基，但 MIC 均匀性不理想。因此，选择 CAMHB 加 1%鸡血清和 0.0025%NADH，并在 35±2°C 孵育 20 小时和 24 小时后显示出良好的 MIC 均匀性。这反映在基本 MIC 协议中，范围在 96%至 100%之间。对更大的收集物（n=43）进行测试表明，可获得标准株和所有分离株的可读 MIC。一些分离株对恩诺沙星（n=35）、萘啶酸（n=35）、青霉素（n=2）、四环素（n=19）和/或甲氧苄啶-磺胺甲恶唑（n=1）的 MIC 值升高。通过 PCR 分析，检测到以下抗菌药物耐药基因：,,, (B)，(H)。研究表明，所提出的培养基适合协调肉汤微量稀释法用于空肠弯曲菌的药敏试验，推荐孵育时间为 20 至 24 小时。
