Plasmid Dynamics in KPC-Positive Klebsiella pneumoniae during Long-Term Patient Colonization.

UNLABELLED: Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists' actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms. IMPORTANCE: In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care.

Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods.

Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of adapter sequences to both the 5' and 3' ends of small RNAs. Studies have shown that adapter ligation introduces a significant but widely unappreciated bias in the results of high-throughput small RNA sequencing. We show that due to this bias the two widely used Illumina library preparation protocols produce strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that are >5-fold differentially detected and some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. In an attempt to mitigate this bias, the new Bioo Scientific NEXTflex V2 protocol utilizes a pool of adapters with random nucleotides at the ligation boundary. We show that this protocol is able to detect robustly several miRNAs that evade capture by the Illumina-based methods. While these analyses do not indicate a definitive gold standard for small RNA library preparation, the results of the NEXTflex protocol do correlate best with RT-qPCR. As increasingly more laboratories seek to study small RNAs, researchers should be aware of the extent to which the results may differ with different protocols, and should make an informed decision about the protocol that best fits their study.