Dynamic microRNA gene transcription and processing during T cell development.

By disrupting microRNA (miRNA) biogenesis, we previously showed that this pathway is critical for the differentiation and function of T cells. Although various cloning studies have shown that many miRNAs are expressed during T cell development, and in a dynamic manner, it was unclear how comprehensive these earlier analyses were. We therefore decided to profile miRNA expression by next generation sequencing. Furthermore, we profiled miRNA expression starting from the hematopoietic stem cell. This analysis revealed that miRNA expression during T cell development is extremely dynamic, with 645 miRNAs sequenced, and the expression of some varying by as much as 3 orders of magnitude. Furthermore, changes in precursor processing led to altered mature miRNA sequences. We also analyzed the structures of the primary miRNA transcripts expressed in T cells and found that many were extremely long. The longest was pri-mir-29b-1/29a at ∼168 kb. All the long pri-miRNAs also displayed extensive splicing. Our findings indicate that miRNA expression during T cell development is both a highly dynamic and a highly regulated process.

Detection of Klebsiella pneumoniae human gut carriage: a comparison of culture, qPCR, and whole metagenomic sequencing methods.

Klebsiella pneumoniae is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with K. pneumoniae is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of K. pneumoniae at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting K. pneumoniae in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting K. pneumoniae in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified K. pneumoniae to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman's rho = 0.91). WMS also supported accurate intra-sample K. pneumoniae sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of K. pneumoniae from fecal samples with potential to support infection control and enhance insights in K. pneumoniae gastrointestinal ecology.

What is the context?Klebsiella pneumoniae is a major cause of healthcare-associated infections and a key contributor to the spread of resistance to last-line antimicrobials.Gastrointestinal colonization by K. pneumoniae is a risk factor for developing infection and can facilitate the spread of antimicrobial resistance.Culture-based detection may lack sensitivity and is ill-suited to detecting within-sample K. pneumoniae abundance and diversity.Developing molecular methods to improve K. pneumoniae abundance and strain diversity detection are essential in understanding human gut colonization and ecology.What is new? We compared culture-based detection of K. pneumoniae within human fecal samples to two molecular-based techniques: 1) qPCR, which amplifies K. pneumoniae species complex-specific DNA targets with high sensitivity, and 2) whole metagenomic sequencing (WMS), which sequences the entire DNA content of complex microbial communities.Our findings show:qPCR had the highest sensitivity, detecting K. pneumoniae in all (52/52) culture-positive samples and 11/51 and 23/47 culture-negative samples, using a direct-fecal and culture-sweep method, respectively. qPCR could accurately quantify K. pneumoniae to 16 genome copies/reaction.WMS had lower sensitivity, positive in 37/52 culture-positive samples, and demonstrated false positives arising from closely related bacterial species. Relative abundance estimates could be performed to 0.01%.WMS performed accurate strain-level detection of K. pneumoniae to 0.1% relative abundance and could detect within-sample strain diversity.What is the impact?qPCR and WMS are valid methods for the detection and characterization of colonizing K. pneumoniae with potential to enhance our understanding of the gastrointestinal ecology of this important pathogen.