2010年和2017年匈牙利采集的犬外耳炎相关铜绿假单胞菌的表型敏感性、耐药相关基因组决定因素及可移动遗传元件背景

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中文摘要

背景： 由铜绿假单胞菌引起的犬外耳炎常呈慢性且治疗困难，但整合的历史表型-基因型数据仍然有限。我们对2010年和2017年获得的匈牙利档案菌株收藏进行了表征。
方法： 采用肉汤微量稀释法测定了67株分离株对13种抗菌药物和氯己定的最低抑菌浓度（MIC），其中59株分离株具有匹配的长读长全基因组组装。使用综合抗生素耐药性数据库鉴定耐药相关决定因素，并评估其与可移动遗传元件及质粒样重叠群的预测邻近关系。
结果： 现行犬类临床和实验室标准协会折点将59/67株分离株（88.1%）归类为恩诺沙星耐药，43/67株（64.2%）归类为马波沙星耐药。欧洲抗菌药物敏感性试验委员会流行病学临界值确定了环丙沙星（7/67）、妥布霉素（4/67）、亚胺培南（1/67）和哌拉西林-他唑巴坦（51/67）的非野生型亚群，而所有分离株对头孢他啶、庆大霉素和阿米卡星仍处于可用的野生型分布范围内。经错误发现率校正后，采样年份间无连续MIC分布发生显著变化，尽管马波沙星分类耐药率从2010年的87.5%下降至2017年的42.9%。耐药基因筛查鉴定出64个独特决定因素，包括几乎普遍存在的外排系统组分、PDC和OXA-50样β-内酰胺酶、APH(3')-IIb、arnA和basS。氟喹诺酮相关决定因素crpP存在于37/59个基因组中，并在6株分离株中位于质粒预测重叠群上。经多重检验校正后，无决定因素-MIC关联仍具有显著性。
结论： 保守的耐药基因库与异质性MIC之间的不一致表明，仅凭序列检测不足以进行治疗推断。因此，需要整合表型和基因组监测，以支持犬假单胞菌性耳炎中基于证据的抗菌药物管理。

英文摘要

Background: Canine otitis externa caused by Pseudomonas aeruginosa is frequently chronic and treatment refractory, yet integrated historical phenotype-genotype data remain limited. We characterized an archival Hungarian collection obtained in 2010 and 2017. Methods: Broth microdilution minimum inhibitory concentrations (MICs) were determined for 67 isolates against 13 antimicrobial agents and chlorhexidine, and 59 isolates had matched long-read whole-genome assemblies. Resistance-associated determinants were identified using the Comprehensive Antibiotic Resistance Database, and their predicted proximity to mobile genetic elements and plasmid-like contigs was evaluated. Results: Current canine Clinical and Laboratory Standards Institute breakpoints classified 59/67 isolates (88.1%) as enrofloxacin resistant and 43/67 (64.2%) as marbofloxacin resistant. European Committee on Antimicrobial Susceptibility Testing epidemiological cut-off values identified non-wild-type subsets for ciprofloxacin (7/67), tobramycin (4/67), imipenem (1/67), and piperacillin-tazobactam (51/67), whereas all isolates remained within the available wild-type distributions for ceftazidime, gentamicin, and amikacin. No continuous MIC distribution changed significantly between sampling years after false-discovery-rate correction, although categorical marbofloxacin resistance decreased from 87.5% in 2010 to 42.9% in 2017. Resistance-gene screening identified 64 unique determinants, including near-universal efflux-system components, PDC and OXA-50-like β-lactamases, APH(3')-IIb, arnA, and basS. The fluoroquinolone-associated determinant crpP occurred in 37/59 genomes and was located on plasmid-predicted contigs in six isolates. No determinant-MIC association remained significant after correction for multiple testing. Conclusions: The discordance between conserved resistance-gene repertoires and heterogeneous MICs demonstrates that sequence-based detection alone is insufficient for therapeutic inference. Integrated phenotypic and genomic surveillance is therefore required to support evidence-based antimicrobial stewardship in canine pseudomonal otitis.