Detection of Pathogenic Bacteria From Septic Patients Using 16S Ribosomal RNA Gene–Targeted Metagenomic Sequencing

Abstract

Background Conventional blood cultures were compared to plasma cell-free DNA–based 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR)/next-generation sequencing (NGS) for detection and identification of potential pathogens in patients with sepsis. Methods Plasma was prospectively collected from 60 adult patients with sepsis presenting to the Mayo Clinic (Minnesota) Emergency Department from March through August 2019. Results of routine clinical blood cultures were compared to those of 16S rRNA gene NGS. Results Nineteen (32%) subjects had positive blood cultures, of which 13 yielded gram-negative bacilli, 5 gram-positive cocci, and 1 both gram-negative bacilli and gram-positive cocci. 16S rRNA gene NGS findings were concordant in 11. For the remaining 8, 16S rRNA gene NGS results yielded discordant detections (n = 5) or were negative (n = 3). Interestingly, Clostridium species were additionally detected by 16S rRNA gene NGS in 3 of the 6 subjects with gastrointestinal sources of gram-negative bacteremia and none of the 3 subjects with urinary sources of gram-negative bacteremia. In the 41 remaining subjects, 16S rRNA gene NGS detected at least 1 potentially pathogenic organism in 17. In 15, the detected microorganism clinically correlated with the patient’s syndrome. In 17 subjects with a clinically defined infectious syndrome, neither test was positive; in the remaining 7 subjects, a noninfectious cause of clinical presentation was identified. Conclusions 16S rRNA gene NGS may be useful for detecting bacteria in plasma of septic patients. In some cases of gram-negative sepsis, it may be possible to pinpoint a gastrointestinal or urinary source of sepsis based on the profile of bacteria detected in plasma.

Clinical Application of Metagenomic Next-Generation Sequencing in Patients with Hematologic Malignancies Suffering from Sepsis

“…Our study successfully demonstrated the advantage of mNGS in diagnosing pathogens via serum samples among patients with hematologic malignancies who developed sepsis. Previous studies have shown the superiority of NGS methods targeting the 16S ribosomal RNA gene in the diagnosis of bacterial infection among immunocompromised patients [18][19][20]. Metagenomic NGS further provides effective diagnostic approaches in diagnosing atypical pathogens such as virus, fungi, and mycobacterium [7,14], which are more commonly seen among patients with hematologic malignancies than the general population.…”

Combination of Whole Genome Sequencing and Metagenomics for Microbiological Diagnostics

“…Alternative universal marker genes include the bacterial rpoB or the fungal internal transcribed spacer 1/2 (ITS-1/2) [ 84 ]. 16S sequencing has also been applied for clinical diagnostics, e.g., in abscesses [ 85 ], urinary tract infections [ 85 , 86 ], or sepsis [ 87 ], as well as for environmental studies. Oberaune et al profiled the microbiome of intensive care units (ICUs) and found higher microbial diversity compared to culture-dependent techniques [ 88 ].…”

Molecular Methodologies for Improved Polymicrobial Sepsis Diagnosis

“…Rip-seq (Pathogenomix Inc., Santa Cruz, CA, USA) for example, excludes non-informative or erroneous gene references from databases, resulting in rapid (5 min per analysis) and accurate identification of pathogens, even in polymicrobial samples. A recent study demonstrated that 16S rRNA gene sequencing using Illumina MiSeq coupled with Rip-Seq analysis was able to correctly identify pathogens with 58% concordance with blood culture results, including a polymicrobial case with E. coli and S aureus [97]. The discordant results were most likely due to sample preparation and issues with the assays, such as inhibition, interference from human DNA and the use of cell-free DNA, which has a short half-life [97].…”

“…A recent study demonstrated that 16S rRNA gene sequencing using Illumina MiSeq coupled with Rip-Seq analysis was able to correctly identify pathogens with 58% concordance with blood culture results, including a polymicrobial case with E. coli and S aureus [97]. The discordant results were most likely due to sample preparation and issues with the assays, such as inhibition, interference from human DNA and the use of cell-free DNA, which has a short half-life [97]. A Breakthrough Device Designation has recently been granted by the FDA for the Patho-Seq assay (Pathogenomix Inc., Santa Cruz, CA, USA), which combines sequencing and Rip-Seq technologies for the identification of pathogens from different sample types (e.g., whole blood and CSF) and several clinical conditions, including polymicrobial sepsis.…”