Microevolution during an Anthrax Outbreak Leading to Clonal Heterogeneity and Penicillin Resistance

Anthrax is a bacterial disease primarily affecting grazing animals but it can also cause severe disease in humans. We have used genomic epidemiology to study microevolution of the bacterium in a confined outbreak in cattle which involved emergence of an antibiotic-resistant phenotype. At the time of death, the animals contained a heterogeneous population of Single Nucleotide Variants (SNVs), some being clonal but most being subclonal. We found that independent isolates from the same carcass had similar levels of SNV differences as isolates from different animals. Furthermore the relative levels of subclonal populations were different in different locations in the same carcass. The heterogeneity appeared to be derived in part from heterogeneity in the infectious dose. The resistance phenotype was linked to clonal mutations in an anti-sigma factor gene and in one case was preceded by an acquisition of a hypermutator phenotype. In another animal, small subclonal populations were observed with counteracting mutations that had turned off the resistance genes. In summary, this study shows the importance of accounting for both acquired and inherited heterogeneity when doing high-resolution infection tracing and when estimating the risks associated with penicillin treatment.

炭疽（Anthrax）是一种主要侵害食草动物的细菌性疾病，亦可引发人类重症感染。本研究采用基因组流行病学（genomic epidemiology）技术，对一次局限性暴发的牛群中炭疽杆菌的微进化（microevolution）过程展开分析，此次暴发伴随抗生素耐药表型（antibiotic-resistant phenotype）的出现。
染疫动物死亡时，其体内存在携带单核苷酸变异（Single Nucleotide Variants, SNVs）的异质性种群：部分为克隆型（clonal）变异，多数则为亚克隆型（subclonal）变异。研究发现，同一病死牛体的独立分离株之间，单核苷酸变异差异水平与不同动物来源的分离株相当。此外，同一病死牛体不同部位的亚克隆种群相对丰度存在差异。这种异质性部分源于感染剂量（infectious dose）本身的异质性。
该耐药表型与抗σ因子基因（anti-sigma factor gene）的克隆突变相关；部分病例在出现耐药表型前，先获得了高突变表型（hypermutator phenotype）。在另一头染疫牛体内，观察到携带拮抗突变的小型亚克隆种群，此类突变可使耐药基因失活。
综上，本研究表明，在开展高分辨率感染溯源（high-resolution infection tracing）以及评估青霉素治疗（penicillin treatment）相关风险时，需同时兼顾获得性与遗传性异质性的影响。