Biology subjects

Chapagain, C.

Publications and source records attributed to Chapagain, C..

3 recordsLinked to original sources

bioRxiv2025

Optimizing Sample Preparation for Direct Nanopore Sequencing to Enable Rapid Pathogen and Antimicrobial Resistance Profiling in Bovine Mastitis

Long-read metagenomic sequencing allows for the rapid, culture-independent, and accurate identification of causative pathogens and antimicrobial resistance (AMR) profiles, supporting precise antibiotic use and reducing the spread of resistance. However, its application to mastitis milk is challenging due to the complex milk matrix, low bacterial count, and high somatic cell content. This study primarily aimed to further optimize our previously developed direct sequencing protocol for milk samples from mastitis cases. Additional optimizations included combining centrifugation, gradient centrifugation, and fat fraction treatment with Tween 20 and citric acid. Subsequently, four DNA extraction kits (Blood and Tissue, Molysis Complete5, HostZero, and SPINeasy Host depletion) were evaluated for their ability to remove host DNA and enrich bacterial DNA for long-read sequencing with Oxford Nanopore technologies. qPCR was used to quantify bacterial and bovine DNA, allowing comparison of host depletion efficiency among the kits. Our results show that simple centrifugation effectively concentrates bacterial cells, removing the need for chemical treatments. The HostZero kit consistently produced higher DNA yields, better DNA integrity, and more effective host DNA depletion. Using nanopore sequencing, both Gram-positive and Gram-negative mastitis pathogens, along with their AMR genes, were successfully detected. Overall, this study underscores the importance of an effective DNA extraction method for the direct sequencing of mastitis milk samples. Additionally, our findings support the potential of direct metagenomic sequencing as a rapid, culture-free approach for identifying mastitis pathogens and their resistance profiles.

microbiology↗

bioRxiv2025

Development and optimization of the host DNA depletion in blood cultures using a saponin and SAN nucleases-based method

Bloodstream infections and sepsis are major health issues causing millions of deaths annually. Early and accurate diagnosis of sepsis is crucial for reducing mortality and combating antimicrobial resistance. However, current diagnostic methods are slow and time-consuming. Nanopore sequencing has the potential to serve as a rapid diagnostic method for sepsis. However, extracting bacterial DNA from blood samples is challenging due to the high content of host DNA. This study aimed to develop and optimize a method using saponin and SAN nucleases for effective host DNA depletion in blood cultures. Different concentrations of saponin, HL-SAN, and M-SAN nucleases, combined with various salt conditions (NaCl and MgCl2), were tested for their ability to deplete host DNA in blood cultures spiked with E. coli and S. aureus. The impact of different bead beating durations on DNA fragment sizes was also examined. The efficiency of host DNA depletion and bacterial DNA extraction was assessed using quantitative PCR (qPCR) and nanopore sequencing. Results indicated that 4% of saponin effectively lysed host cells, releasing DNA while preserving bacterial cells. The host DNA is then efficiently depleted using 250 units (10 L) of HL-SAN and M-SAN nucleases. Although SAN in combination with higher salt concentrations (2.5 M NaCl and 50 mM MgCl2) showed somewhat better host DNA removal, the difference was not statistically significant. Reducing bead beating time to 6 minutes improved the recovery of longer DNA fragments compared to 10 minutes. These results demonstrate an effective method for host DNA depletion using saponin and SAN nucleases, which is compatible with downstream processes, such as nanopore sequencing and qPCR.

microbiology↗

bioRxiv2024

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

PurposeUrinary Tract Infection (UTI) is a prevalent global health concern accounting for 1-3% of primary healthcare visits. The current methods for UTI diagnosis have a high turnaround time of 3-5 days for pathogen identification and susceptibility testing. This work is a proof-of-concept study aimed at determining the detection limit by establishing a culture and amplification-free DNA extraction methodology from spiked urine samples followed by real-time Nanopore sequencing and data analysis. MethodsThis study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Pathogens were spiked at two clinically significant concentrations of 103 and 105 CFU/ml. Three commercial DNA extraction kits were investigated based on the quantity of isolated DNA, average processing time, elution volume and the average cost incurred per extraction. The outperforming kit was used for direct DNA extraction and subsequent sequencing on MinION and Flongle flowcells. ResultsThe Blood and Tissue kit outperformed the other kits. All pathogens were identified at a concentration of 105 CFU/ml within ten minutes, and the corresponding AMR genes were detected within three hours of the sequencing start. The overall turnaround time including the DNA extraction and sequencing steps was five hours. Moreover, we also demonstrate that the identification of some pathogens and antibiotic-resistance genes was possible at a spike concentration of 103 CFU/mL. ConclusionThis study shows great promise toward reducing the time required for making an informed antibiotic administration from approximately 48 hours to five hours thereby reducing the number of empirical doses and saving lives.

microbiology↗