Kin discrimination modifies strain distribution, spatial segregation and incorporation of extracellular matrix polysaccharide mutants of Bacillus subtilis strains into mixed floating biofilms

Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivate Bacillus subtilis strains as mixtures of isogenic, kin and nonkin strain combinations in the biofilm-promoting medium (MSgg) in static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtili

Feasibility of droplet digital PCR analysis of plasma cell-free DNA from kidney transplant patients

Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals. The assays are capable of accurate quantification of down to 0.1% minor allele content when analyzing 165 ng of human DNA. We found no significant differences in the yield of extracted cfDNA using the three different commercial kits tested. More cfDNA was extracted from the plasma of KTx patients than from healthy volunteers, especially early after transplantation. The median level of donor-derived minor alleles in KTx samples was 0.35%. We found that ddPCR using the evaluated assays within specific range is suitable for analysis of KTx patientsʼ plasma but recommend prior genotyping of donor DNA and performing reliable preamplification of cfDNA

Metagenomic characterization of parental and production CHO cell lines for detection of adventitious viruses

Viral contamination is a major concern for biological products. Therefore, virus testing of raw materials and cells is essential for the safety of the final product. We used high-throughput sequencing to detect viral-like sequences in selected CHO cell lines. Our aim was to test various approaches of sample preparation, to establish a pipeline for metagenomic analysis and to characterize standard viral metagenome of production and parental CHO cell lines. The comparison of the metagenomics composition of the differently prepared samples showed that among four tested approaches sequencing of ribosomal RNA depleted total RNA is the most promising approach. The metagenomics investigation of one production and three parental CHO cell lines of diverse origin did not indicate the presence of adventitious viral agents in the investigated samples. The study revealed an expected background of virus-like nucleic acids in the samples, which originate from remains of expression vectors, endogenized viral elements and residuals of bacteriophages