Rapid pathogen identification and phenotypic antimicrobial susceptibility directly from urine specimens.

Implementing effective antimicrobial therapy close to the onset of infection lowers morbidity and mortality and attenuates the spread of antimicrobial resistance. Current antimicrobial susceptibility testing (AST) methods, however, require several days to determine optimal therapies. We present technology and an automated platform that identify (ID) Urinary Tract Infection pathogens in 45 min and provide phenotypic AST results in less than 5 h from urine specimens without colony isolation. The ID and AST tests count cells fluorescently labeled with specific rRNA probes using non-magnified digital imaging. The ID test detected five pathogens at ≤ 7,000 CFU/mL and had a linear range of ~ 4 orders of magnitude. For contrived specimens, AST tests gave 93.1% categorical agreement with 1.3% Very Major Errors (VME), 0.3% Major Errors (ME), and 6.3% minor Errors (mE) compared to the broth microdilution (BMD) reference method. For clinical specimens, the ID test had 98.6% agreement and the AST test had 92.3% categorical agreement with 4.2% mE, 3.4% ME and 4.0% VME compared to BMD. Data presented demonstrates that direct-from-specimen AST tests can accurately determine antimicrobial susceptibility/resistance for each pathogen in a specimen containing two pathogens. The method is robust to urine matrix effects and off-target commensal and contaminating bacteria.

Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR.

Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5'→3' nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.

Comparative QTL analysis of early short-time drought tolerance in Polish fodder and malting spring barleys.

KEY MESSAGE: An effective approach for the further evolution of QTL markers, may be to create mapping populations for locally adapted gene pools, and to phenotype the studied trait under local conditions. Mapping populations of Polish fodder and malting spring barleys (Hordeum vulgare L.) were used to analyze traits describing short-time drought response at the seedlings stage. High-throughput genotyping (Diversity Array Technology (DArT) markers) and phenotyping techniques were used. The results showed high genetic diversity of the studied populations which allowed the creation of high-density linkage maps. There was also high diversity in the physiological responses of the barleys. Quantitative trait locus (QTL) analysis revealed 18 QTLs for nine physiological traits on all chromosomes except 1H in malting barley and 15 QTLs for five physiological traits on chromosomes 2H, 4H, 5H and 6H in fodder barley. Chromosomes 4H and 5H contained QTLs which explained most of the observed phenotypic variations in both populations. There was a major QTL for net photosynthetic rate in the malting barley located on chromosome 5H and two major QTLs for overall photochemical performance (PI) located on 5H and 7H. One major QTL related to photochemical quenching of chlorophyll fluorescence was located on chromosome 4H in fodder barley. Three QTL regions were common to both mapping populations but the corresponding regions explained different drought-induced traits. One region was for QTLs related to PSII photosynthetic activity stress index in malting barley, and the corresponding region in fodder barley was related to the water content stress index. These results are in accordance with previous studies which showed that different traits were responsible for drought tolerance variations in fodder and malting barleys.