Using genomics to improve Bacillus anthracis diagnostics and outbreak investigations

Sammanfattning: The bacterium Bacillus anthracis causes the disease anthrax, primarily in herbivores but many mammals are susceptible to the disease. Its infective form is as a dormant spore that can lie in the soil for decades. Thus, in its cycle of infection, it spends most of the time in an inactive state and replication-induced DNA-mutations are therefore kept at a minimum. Partly due to these long periods of inactivity, all B. anthracis isolates found in the world are genetically very similar. This makes strain characterization difficult and requires high-resolution technologies. Bacillus anthracis also has similar DNA-content as other Bacillus spp. and therefore diagnostic cross-reactions are not uncommon. Anthrax incidence has steadily declined in the world during the last century but there are still endemic areas. In 2008 and in 2011 Sweden suffered two large and costly outbreaks, most likely caused by the disturbance of old anthrax epizootic graves from the 1940s and 1950s. Several studies emanated from these outbreaks including how the bacteria in cows treated with penicillin developed penicillin resistance. Next-generation sequencing (NGS) has revolutionized the way DNA is sequenced and the whole genome (i.e., all the DNA) of a bacterium can now be sequenced in only a few days. In this doctoral thesis, NGS and genomics were used to improve our capability to deal with anthrax outbreaks. Genomic and genetic studies were applied to identify the non-anthrax Bacillus spp. available that were most closely related to B. anthracis. How well the strains could mimic B. anthracis in a model system for B. anthracis spores was evaluated and the best model strains found have since been used in exercises and as controls in real samples. For genome comparisons, a software was created that can compare the genetic content of several hundreds of bacterial genomes. This ensures a rapid characterization of an outbreak pathogen’s genome. The software was also used to in silico-compare all published anthrax PCR-assays to determine which assays that had the highest specificity. This workflow ensures that our molecular diagnostics are as specific for B. anthracis as possible. By using isolates from an anthrax outbreak, the mechanisms of beta-lactam resistance in B. anthracis were studied and chromosomal mutations in a negative sigma factor were found to be the cause. The genomic divergence of a strain during an outbreak was also studied to gain knowledge of the strengths and limitations of using NGS for epidemiological investigations. In summary, this thesis describes different genomic approaches that have improved diagnostic methods, explained ambiguous antimicrobial resistance findings and enhanced the resolution of genomic epidemiological investigation to ensure a robust handling of future anthrax outbreaks.

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