Only viable is reliable? – microbial cell-free DNA (mcfDNA) represents a precise marker for pathogen determination

A current dogma in diagnosis of clinical infections states that only the identification of viable species from patient specimens delivers reliable information on relevant pathogens in critically ill patients. Therefore, microbiological approaches including blood culture still represent the gold standard for pathogen diagnosis . Although established and further Improved for decades, microbiological approaches still lack sufficient sensitivity and specificity. Blood cultures from septic patients remain negative in up to 70-90% of cases, for example, leaving the treating physician with unspecific empiric therapy of undefined pathogens.

To evaluate the eligibility of microbial cell-free DNA for a more reliable determination of relevant pathogens we characterized dynamics of mcfDNA in blood by next-generation sequencing (NGS) and congruence of mcfDNA pathogen identification with classical blood culture results in a mouse model for abdominal sepsis (cecal ligation and puncture-/CLP-model). Accordingly, we isolated mcfDNA from CLP-mice at different time points following CLP surgery (between 6 h and up to 72 h) and analyzed for pathogenic species by NGS and a bioinformatics workflow for quantitative and relevance assessment. In this context, we established protocols that are compatible with minimal amounts of input material facilitating time series analyses in individual mice. Species identified by NGS revealed a high concordance to species identified from classical microbiological cultivation, but also revealed additional species, due to higher sensitivity of mcfDNA analyses. Furthermore, we found a close resemblance of identified species from CLP mice, including E. coli and Enterococci, with those from a human patient cohort comprising 50 patients suffering from mainly abdominal sepsis, indicating the aptitude of the CLP model for studying human sepsis. Dynamics of mcfDNA concentrations also accurately reflect disease progression thus qualifiying this class of circulating DNA as a precise and sensitive marker for diagnosing the status and causative organism of infection in critically ill patients.