Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. The rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcomes in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we targeted eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested in an experimental research study on the first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be additionally detected. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time-to-results to 11–13 h and suggesting diagnostic potential in sepsis.

Background Liquid biopsy based on cell-free DNA (cfDNA) is an established approach in clinical diagnostics. In recent years, a fraction of cfDNA comprising short fragments has been discovered, that is enriched at gene promoters and binding sites of DNA-binding proteins. However, the diagnostic potential of such short double-stranded cell-free DNA (footprint DNA) remains to be fully explored. Therefore, we characterized the clinical utility of footprint DNA in septic patients. Methods We enriched for footprint DNA based on size selection and subsequent high-throughput DNA sequencing to receive an unbiased, genome-wide picture of the host response to the infection. Footprint DNA occupancies were analyzed for correlation with clinical metrics including urea, hemoglobin, or alanine aminotransferase (ALT). Additionally, footprint DNA markers were benchmarked by read and receiver operating curve (ROC) analysis against procalcitonin (PCT) as an established marker for infection status as well as against clinical parameters for early death prediction. Findings We found that levels of occupancy of footprint DNA at defined genomic loci semi-quantitatively correlated with physiological markers like ALT or urea from major organ systems including liver or kidney. In a small proof-of-concept cohort, differential signatures of DNA footprints distinguished between patient groups with bacterial and viral infections with an area under the ROC (AUROC) of 1.0, which is considerably better than PCT with an AUROC of 0.75. Likewise, footprint DNA could also predict early death in septic patients with an AUROC of 0.983, compared to the SOFA (Sepsis-related organ failure assessment) score with an AUROC of 0.76. Interpretation Our findings show that footprint DNA delivers quantitative information on physiology at the DNA level, demonstrating its diagnostic and prognostic potential. Identified footprint biomarker regions could be helpful in the clinical assessment of septic patients and other complex diseases outperforming current state-of-the-art clinical diagnostics. Funding This study was financed with internal funds from Fraunhofer society.

Water is essential for human survival and socioeconomic development, yet its overconsumption threatens global food security and ecosystem integrity. This necessitates a 60% increase in food production, further straining water resources. Hydroponic systems represent a promising solution, utilizing up to 90% less water than traditional methods while providing optimal growing conditions for crops. This study was aimed at developing a PCR‐based detection system for main human and plant pathogens in hydroponic systems using treated domestic wastewater. Metagenomic analysis of wastewater samples revealed significant microbial diversity, identifying human pathogens such as Pseudomonas aeruginosa and Yersinia enterocolitica , alongside plant pathogens including Rhodococcus fascians . Specific primer pairs for the most abundant species found in a domestic municipal wastewater sample of target pathogens ( Streptococcus mutans , P. aeruginosa , Acinetobacter baumannii , Y. enterocolitica , Enterococcus faecalis , Pseudomonas viridiflava , R. fascians , Xanthomonas vesicatoria , and Pseudomonas syringae ) were designed and validated, ensuring high specificity and efficiency. Future research should focus on enhancing detection methods and optimizing DNA extraction techniques to improve pathogen quantification and management in hydroponic systems. This approach is crucial for sustainable agricultural practices that minimize water usage while ensuring food safety and environmental health.

Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. Rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcome in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a Suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we target eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested on first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be detected additionally. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time to results to 11 - 13 hours and suggesting diagnostic potential in sepsis.