Investigating streptococcal biodiversity in sepsis using next-generation sequencing

Sepsis is one of the leading causes for fatalities in the intensive care unit, and also one of the biggest health problems worldwide. It is a disease caused primarily by bacterial infections but can also be caused by viral or fungal infections. Since it is such a big health problem being associated with increased risk of sepsis, coupled with longer stays in the intensive care unit, the need for fast diagnosis and treatment is very important. Currently, culture is the leading diagnostic method for identification of bacteria, although other methods are currently being tested to improve identification time and decrease cost and workload. Next generation sequencing (NGS) has the capacity to output several million reads in a single experiment, making it very fast and relatively cheap compared to other older sequencing methods such as Sanger sequencing. The ability to analyze genes and even whole genomes, opens the possibilities to identify factors such as bacterial species, virulence genes and antibiotic resistance genes. The aim of this study was to find any possible correlations between 16 species of streptococci and clinical data in patients with suspected sepsis. Initial species identification was performed using MALDI-TOF before the samples were sequenced using NGS. Sequence files were then quality controlled and trimmed before being assembled. Following assembly, coverage was controlled for all assembled genomes before the downstream analysis started. Different tools such as 16S RNA species identification, multi locus sequence typing and antibiotic resistance finder were used, among other tools. The results were extremely mixed, with the overall quality of the data being of good quality, but the assembly and downstream analysis being worse. The most consistent species was S. pyogenes. No correlation between sepsis patients and relevant clinical data was found. The mixed quality of results from assembly and downstream analysis were most likely contributed to difficulties in culturing and sequencing of the streptococci. Finding ways to circumvent these problems would most likely aid in general sequencing of streptococcal species, and hopefully in clinical applications as well