Biology subjects

Stege, P. B.

Publications and source records attributed to Stege, P. B..

3 recordsLinked to original sources

bioRxiv2025

CArP - CApture-based sequencing for Pathogen surveillance in complex matrices

This study highlights the development and application of a novel capture-based long-read sequencing approach, CArP, designed to benefit pathogen surveillance. This combines targeted gene enrichment with the contextual insights of long-read sequencing, providing a highly efficient and flexible approach for pathogen detection. In this initial version, antimicrobial resistance (AMR) genes are utilized as marker genes that improve sequencing sensitivity. This allowed the detection of bacteria in broiler caecal content samples that carried resistance genes for critical antibiotics, including macrolides and quinolones. These specific AMR genes would have remained undetected when traditional metagenomic shotgun sequencing would have been applied, as it failed to detect these genes and therefore putative pathogens. This demonstrates the potential to detect and characterize pathogens more effectively compared to traditional sequencing strategies. The modular design of CArP furthermore demonstrates expansion with additional marker genes, such as viral or fungal markers, that can enable broader pathogen coverage. Taken together, this approach allows to advance pathogen detection and can contribute to global efforts in pandemic preparedness and the subsequent response.

genomics↗

bioRxiv2023

Impact of diet on jejunal microbiota composition during broiler development with special focus on Enterococcus hirae and Enterococcus faecium

Modern broiler breeds allow for high feed efficiency and rapid growth, but come at a cost of increased susceptibility to pathogens and disease. Broiler growth rate, feed efficiency, and health are furthermore affected by the composition of the gut microbiota, which in turn is influenced by diet composition. In this study we therefore assessed how diet composition alters the broiler jejunal gut microbiota. A total of 96 broiler chickens were divided into four diet groups: control, coated butyrate supplementation, medium chain fatty acid supplementation, or a high-fibre low-protein content. Diet groups were sub-divided into age groups (4, 12 and 33 days of age) resulting in groups of 8 broilers per diet per age. The jejunum content jejunum was used for metagenomic shotgun sequencing to determine the microbiota composition on species level. Among all diet groups, a total of 104 differential abundant bacterial species were detected. Most notably were the changes in the jejunal microbiota induced by butyrate supplementation when compared to the control diet, resulting in the reduced relative abundance of mainly Enterococcus faecium and the opportunistic pathogen Enterococcus hirae in broilers 4 days post-hatch. At this early stage of development, the immune system is still immature thereby highlighting the importance to study the relation of diet and the jejunal microbiota. Future studies should furthermore elucidate how diet can be used to promote a beneficial microbiota in the early stages of broiler development.

microbiology↗

bioRxiv2019

CRISPR-Cas9 mediated genome editing in vancomycin resistant Enterococcus faecium

The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. There is thus an urgent need to mechanistically characterize the traits that contribute to the emergence of E. faecium as a multidrug resistant opportunistic pathogen. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants, but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faecium {beta}-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates.

microbiology↗