Biology subjects

Hira, J.

Publications and source records attributed to Hira, J..

3 recordsLinked to original sources

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↗

bioRxiv2025

Low-Cost In-House Re-formulated Brain Heart Infusion Medium for Effective Planktonic Growth and Early Detection of Bloodstream Bacterial Pathogens

Sepsis, a clinically defined life-threatening condition, is a global contributor to high morbidity and mortality rates in humans. It is caused by systemic bloodstream bacterial infections, primarily involving aerobic pathogens such as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. Rapid and accurate identification of these pathogens is a high-demand task, as prolonged diagnosis may increase the mortality rate among sepsis patients. Worldwide, commercial blood culture systems, such as BD BACTEC PLUS Aerobic/F /F culture bottles (used in this study), are routinely used to monitor bloodstream infections. However, due to high costs ($10.00-$15.00/bottle), limited availability of culture media (especially in low- and middle-income countries, and war zones), and a lack of customization for antibiotic susceptibility assay and epidemiology research, there is a need for secondary alternatives to facilitate the growth and identification of bloodborne pathogens. Therefore, we developed a low-cost ($4-$5/bottle) in-house culture medium with a newly improved formulation of Brain Heart Infusion media that enhances bacterial growth from spiked human blood tested on a panel of bacteria (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis). The growth dynamics of these microbes in in-house formulated BHI-Blood+ culture media coincide with those in BACTEC Plus Aerobic/F culture vials, which primarily suggests the compatibility of bloodborne pathogens with this media and can be flagged positive <8h based on cellular growth rate. Additionally, conventional qPCR-based early detection (< 24h) and validation with the Oxford Nanopore MinION NGS platform highlight the value of this in-house culture media as an alternative to commercial culture media in terms of low-cost availability.

microbiology↗

bioRxiv2023

High-throughput single-cell phenotypic profiling and backtracing exposes and predicts clinically relevant subpopulations in isogenic Staphylococcus aureus communities.

Isogenic bacterial cell populations are phenotypically heterogenous and may include subpopulations of antibiotic tolerant or heteroresistant cells. The reversible nature of these phenotypes and lack of biomarkers to differentiate functionally different, but morphologically identical cells is a challenge for research and clinical detection. To overcome this, we present Cellular Phenotypic Profiling and backTracing (CPPT), a flexible fluorescence-activated cell sorting platform, that uses optical probes to visualize and quantify cellular traits and connects the resulting phenotypic profile with a cells experimentally determined fate in single cell-derived growth and antibiotic susceptibility analysis. By applying CPPT on Staphylococcus aureus populations we recorded phenotypic signatures for dormant cells, exposed microanatomy-independent bimodal growth patterns in colony-derived cells, and revealed different culturability of single cells on solid compared to liquid media. We demonstrate that vancomycin-bodipyFL marks cellular subpopulations with increased likelihood to survive antibiotic exposure, showcasing the value of CPPT for discovery of clinically relevant biomarkers.

microbiology↗