Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820, Merelbeke, Belgium.
PathoSense BV, Lier, Belgium.
Vet Res. 2023 Apr 5;54(1):33. doi: 10.1186/s13567-023-01160-2.
Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolates using phenotypic identification and susceptibility testing on the one hand, and long-read nanopore whole genome sequencing on the other hand. This allowed to address strain divergence down to the whole genome single nucleotide polymorphism (SNP) level. While lowest resolution was observed for 16S rRNA gene classification, a new multi-locus sequence typing (MLST) scheme allowed proper classification up to the species level. Nevertheless, a SNP-level analysis was required to distinguish A. equuli subspecies equuli and haemolyticus. Our data provided first WGS data on Actinobacillus genomospecies 1, Actinobacillus genomospecies 2, and A. arthritidis, which allowed the identification of a new Actinobacillus genomospecies 1 field isolate. Also, in-depth characterization of RTX virulence genes provided information on the distribution, completeness, and potential complementary nature of the RTX gene operons within the Actinobacillus genus. Even though overall low prevalence of acquired resistance was observed, two plasmids were identified conferring resistance to penicillin-ampicillin-amoxicillin and chloramphenicol in one A. equuli strain. In conclusion our data delivered new insights in the use of long-read WGS in high resolution identification, virulence gene typing, and antimicrobial resistance (AMR) of equine Actinobacillus species.
马源杀白细胞素不动杆菌主要与马的疾病相关，是昏睡驹病的主要病原体。虽然现有的表型工具，如生化试验、16S rRNA 基因测序和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS），可用于鉴定不动杆菌属的成员，但这些方法难以区分某些种，也无法进行菌株、毒力和抗菌药物敏感性分型。因此，我们一方面对 24 株马源杀白细胞素不动杆菌进行了表型鉴定和药敏试验，另一方面进行了长读长纳米孔全基因组测序。这使得我们能够在全基因组单核苷酸多态性（SNP）水平上解决菌株分歧问题。虽然 16S rRNA 基因分类的分辨率最低，但新的多位点序列分型（MLST）方案允许在种水平上进行适当的分类。然而，需要进行 SNP 水平分析才能区分杀白细胞素不动杆菌亚种 equuli 和 haemolyticus。我们的数据提供了关于不动杆菌基因组种 1、不动杆菌基因组种 2 和 A. arthritidis 的首个 WGS 数据，这些数据允许鉴定一种新的不动杆菌基因组种 1 野外分离株。此外，对 RTX 毒力基因的深入分析提供了有关 RTX 基因操纵子在不动杆菌属中的分布、完整性和潜在互补性的信息。尽管总体上观察到获得性耐药的低流行率，但在一株杀白细胞素不动杆菌中鉴定出两个质粒，赋予了对青霉素-氨苄西林-阿莫西林和氯霉素的耐药性。总之，我们的数据为长读 WGS 在马源不动杆菌种的高分辨率鉴定、毒力基因分型和抗菌药物耐药性（AMR）中的应用提供了新的见解。
