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Abstract：
This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended.
Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.
Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.
摘要：
本研究旨在开发一种用于Avibacterium（Av。)副鸡，鸡传染性鼻炎的病原体。为此，共83Av。从15个国家收集了副鸡的分离株和菌株。要为方法验证步骤选择不相关的隔离，用15个Av进行宏观限制分析.paragallinarum.可见Av的增长。在六种肉汤培养基中检查了副鸡，并编制了生长曲线。在兽医Fastidious培养基和阳离子调节的Mueller-Hinton肉汤（CAMHB）+1％鸡血清+0.0025％NADH（CAMHBCSNADH）中，检测到所有分离物的可见生长,并且两种培养基都允许足够的细菌生长。由于Av的可读性更好。副鸡在微量滴定板中的生长，选择CAMHB+CS+NADH用于AST。在35±2°C下孵育48小时后，使用一组24种抗菌剂对5种流行病学无关的分离物进行MIC测试的重复导致了96％-100％的高必需MIC协议。因此，剩下的78Av。使用所提出的方法对副鸡进行了测试并证明了易于阅读的MIC。在来自不同大陆的分离株之间检测到中等收入国家的差异，与来自美国和欧洲的分离株相比，来自非洲的分离株显示出更低的中等收入国家，更常见的是氨基糖苷类药物的MIC升高，喹诺酮类药物,四环素,和/或甲氧苄啶/磺胺甲恶唑。用于方法开发的分离株的PCR分析显示，四环素MIC升高的分离株具有四环素抗性基因tet（B），但未检测到其他测试的抗性基因。因此,来自62个Av的全基因组测序数据。对副鸡进行了分析，发现存在与aph（6）-Id基因具有核苷酸序列同一性的序列，aph(3″)-Ib,blaTEM-1B,catA2，sul2，tet(B)，tet(H),和mcr一样。总的来说,拟议的方法使用CAMHBCSNADH进行敏感性测试，在35±2°C的环境空气中孵育48小时，适用于Av。paragallinarum.由于检测到多种抗性基因,强烈建议开发临床断点。
目的：副鸡副细菌是引起鸡传染性鼻炎的重要兽医学病原。由于抗生素通常用于治疗并且已知病原体的抗性，应在病原体的抗性测试后给予靶向治疗。不幸的是,目前，标准中没有可接受的方法可以对这种挑剔的病原体进行敏感性测试。因此,我们已经制定了一种方法，可以对病原体进行统一的敏感性测试。该方法符合CLSI的要求，可用于诊断实验室。
目的：副鸡副细菌是引起鸡传染性鼻炎的重要兽医学病原。由于抗生素通常用于治疗并且已知病原体的抗性，应在病原体的抗性测试后给予靶向治疗。不幸的是,目前，标准中没有可接受的方法可以对这种挑剔的病原体进行敏感性测试。因此,我们已经制定了一种方法，可以对病原体进行统一的敏感性测试。该方法符合CLSI的要求，可用于诊断实验室。
