Comparison of the next-generation Xpert MRSA/SA BC assay and the GeneOhm StaphSR assay to routine culture for identification of Staphylococcus aureus and methicillin-resistant S. aureus in positive-blood-culture broths

A bloodstream infection with Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a serious condition that carries a high mortality rate and is also associated with significant hospital costs. The rapid and accurate identification and differentiation of methicillin-susceptible S. aureus (MSSA) and MRSA directly from positive blood cultures has demonstrated benefits in both patient outcome and cost-of-care metrics. We compare the next-generation Xpert MRSA/SA BC (Xpert) assay to the GeneOhm StaphSR (GeneOhm) assay for the identification and detection of S. aureus and methicillin resistance in prospectively collected blood culture broths containing Gram-positive cocci. All results were compared to routine bacterial culture as the gold standard. Across 8 collection and test sites, the Xpert assay demonstrated a sensitivity of 99.6% (range, 96.4% to 100%) and a specificity of 99.5% (range, 98.0% to 100%) for identifying S. aureus, as well as a sensitivity of 98.1% (range, 87.5% to 100%) and a specificity of 99.6% (range, 98.3% to 100%) for identifying MRSA. In comparison, the GeneOhm assay demonstrated a sensitivity of 99.2% (range, 95.2% to 100%) and a specificity of 96.5% (range, 89.2% to 100%) for identifying S. aureus, as well as a sensitivity of 94.3% (range, 87.5% to 100%) and a specificity of 97.8% (range, 96.1% to 100%) for identifying MRSA. Five of six cultures falsely reported as negative for MRSA by the GeneOhm assay were correctly identified as positive by the Xpert assay, while one culture falsely reported as negative for MRSA by the Xpert assay was correctly reported as positive by the GeneOhm assay

C. elegans Germline-Deficient Mutants Respond to Pathogen Infection Using Shared and Distinct Mechanisms

Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to Gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including Gram positive and Gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16

GATA Transcription Factor Required for Immunity to Bacterial and Fungal Pathogens

In the past decade, Caenorhabditis elegans has been used to dissect several genetic pathways involved in immunity; however, little is known about transcription factors that regulate the expression of immune effectors. C. elegans does not appear to have a functional homolog of the key immune transcription factor NF-κB. Here we show that that the intestinal GATA transcription factor ELT-2 is required for both immunity to Salmonella enterica and expression of a C-type lectin gene, clec-67, which is expressed in the intestinal cells and is a good marker of S. enterica infection. We also found that ELT-2 is required for immunity to Pseudomonas aeruginosa, Enterococcus faecalis, and Cryptococcus neoformans. Lack of immune inhibition by DAF-2, which negatively regulates the FOXO transcription factor DAF-16, rescues the hypersusceptibility to pathogens phenotype of elt-2(RNAi) animals. Our results indicate that ELT-2 is part of a multi-pathogen defense pathway that regulates innate immunity independently of the DAF-2/DAF-16 signaling pathway

Immunity in Caenorhabditis Elegans: a Tale of Two Transcription Factors

<p>Recently, the study of invertebrate innate immunity has garnered considerable attention after the discovery that mammalian homologues of the <italic>Drosophila melanogaster</italic> </p><p>Toll pathway play a role in mammalian innate immunity. One invertebrate model system that has begun to be intensely studied is the nematode <italic>Caenorhabditis elegans</italic>. Immunity in <italic>C. elegans</italic> has been shown to be inducible in that it responds uniquely to different pathogens. These changes in gene expression require transcription factors in order for certain genes to be transcribed. We utilized an RNA interference screen of potential transcription factors to identify the GATA transcription factor ELT-2 as a possible transcription factor involved in immunity. We then demonstrated that ELT-2 was required for resistance to a wide range of pathogens and was responsible for regulating expression of the C-type lectin <italic>clec-67</italic>, a marker of immunity. </p><p>We also studied another transcription factor known to play a role in C. elegans immune function, the FOXO transcription factor DAF-16. We specifically focused in on the role of DAF-16 in germline-deficient mutants, and we demonstrated that such mutants are resistant to many different pathogens. This led to further investigation of the germline-deficient mutant glp-4, which should also show broad range resistance to pathogens but fails to do so. Through whole genome sequencing, we identified mutations that may be responsible for the glp-4 phenotype. We also demonstrated that DAF-16 was active in glp-4 mutants, leading to us proposing a model where glp-4 plays a role in influencing <italic>C. elegans</italic> immunity besides its involvement in germline development.</p>Dissertatio

<i>elt-2</i>(RNAi) animals are hypersusceptible to <i>S. enterica-</i>mediated killing and are colonized by <i>E. coli</i>.

<div><p>(<b>A</b>) Wild-type nematodes grown on <i>E. coli</i> carrying a vector control or on <i>E. coli</i> expressing <i>elt-2</i> double-stranded RNA were exposed to <i>S. enterica</i> SL1344 (P<0.0001).</p> <p>60 nematodes were used for each condition. Results are representative of at least 3 independent experiments.</p> <p>(<b>B</b>) Wild-type nematodes grown on <i>E. coli</i> carrying a vector control or on <i>E. coli</i> expressing <i>elt-2</i> double-stranded RNA were placed on FUdR containing plates with lawns of heat-killed <i>E. coli</i> OP50 (P = 0.0011).</p> <p>20 nematodes were used for each condition. Results are representative of at least 3 independent experiments.</p> <p>(<b>C</b>) and (<b>D</b>) Wild-type nematodes grown on <i>E. coli</i> carrying a vector control (C) or on <i>E. coli</i> expressing <i>elt-2</i> double-stranded RNA (D) were exposed to <i>E. coli</i> expressing DSred for 24 hours, and then visualized using a Leica TCS SL spectral confocal microscope (bar = 50 µm).</p> <p>(<b>E</b>) Wild-type nematodes grown on <i>E. coli</i> carrying a vector control or on <i>E. coli</i> expressing <i>elt-2</i> double-stranded RNA were transferred to a clean LB plate either immediately or after 24 hours exposure to <i>E. coli</i> OP50 (+24 hrs), where they were allowed to defecate for 2 hours.</p> <p>Plates then were placed at 37°C overnight and colonies counted.</p> <p>The combined data from 10 individual animals are shown, and data were normalized to the median colony count for the vector control at each timepoint.</p> <p>Error bars represent SEM.</p> <p>Results are representative of at least 3 independent experiments.</p></div

<i>elt-2(RNAi)</i> animals are more susceptible than wild-type nematodes to a variety of pathogens.

<div><p>(<b>A</b>)Wild-type nematodes grown on <i>E. coli</i> carrying a vector control or on <i>E. coli</i> expressing <i>elt-2</i> double-stranded RNA were exposed to <i>P. aeruginosa</i> PA14 (P = 0.0001).</p> <p>(<b>B</b>) Wild-type nematodes grown on <i>E. coli</i> carrying a vector control or on <i>E. coli</i> expressing <i>elt-2</i> double-stranded RNA were exposed to <i>E. faecalis</i> OG1RF (P<0.0001).</p> <p>(<b>C</b>) Wild-type nematodes grown on <i>E. coli</i> carrying a vector control or on <i>E. coli</i> expressing <i>elt-2</i> double-stranded RNA were exposed to <i>C. neoformans</i> H99 (P<0.0001).</p> <p>60–120 nematodes were used for each condition.</p> <p>Results are representative of at least 3 independent experiments.</p></div