To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.
Macrophages are the guardians of the innate immune system, recognizing a broad array of pathogen-associated molecular patterns (PAMPs) to initiate immediate defenses and to recruit the adaptive branch of the immune system. Toll-like receptors (TLRs) detect extracellular microbial products, such as lipopolysaccharide, peptidoglycan, lipotechoic acid, and fl agellin (1), whereas surveillance of the cytosol is the task of nucleotide-binding oligomerization domain (NOD) leucine-rich repeat (LRR) proteins. The best-characterized members of the NOD-LRR family are NOD1 and NOD2, which recognize distinct elements of bacterial cell wall peptidoglycan in the cytosol to mount or modulate a proinfl ammatory immune response or to promote apoptosis (2). In mouse macrophages, the NOD-LRR protein Naip5 (Birc1e) restricts intracellular replication of the opportunistic human pathogen Legionella pneumophila (3-5). Naip5 is comprised of three modules: NH 2-terminal baculoviral inhibitor of apoptosis repeats, a central NOD domain, and COOH-terminal LRRs (2). By analogy to other NOD-LRR proteins, the LRR region is thought to recognize microbial products, triggering oligomerization via the NOD domain and activation of a cellular response that is governed by various NH 2terminal eff ector-binding domains (2). Whereas virtually all mice are resistant to L. pneumophila, the A/J strain encodes a naip5 allele that confers susceptibility to infection (3). Whether the
BackgroundThe Binational Border Infectious Disease Surveillance program began surveillance for severe acute respiratory infections (SARI) on the US–Mexico border in 2009. Here, we describe patients in Southern Arizona.MethodsPatients admitted to five acute care hospitals that met the SARI case definition (temperature ≥37·8°C or reported fever or chills with history of cough, sore throat, or shortness of breath in a hospitalized person) were enrolled. Staff completed a standard form and collected a nasopharyngeal swab which was tested for selected respiratory viruses by reverse transcription polymerase chain reaction.ResultsFrom October 2010–September 2014, we enrolled 332 SARI patients. Fifty‐two percent were male and 48% were white non‐Hispanic. The median age was 63 years (47% ≥65 years and 5·2% <5 years). During hospitalization, 51 of 230 (22%) patients required intubation, 120 of 297 (40%) were admitted to intensive care unit, and 28 of 278 (10%) died. Influenza vaccination was 56%. Of 309 cases tested, 49 (16%) were positive for influenza viruses, 25 (8·1%) for human metapneumovirus, 20 (6·5%) for parainfluenza viruses, 16 (5·2%) for coronavirus, 11 (3·6%) for respiratory syncytial virus, 10 (3·2%) for rhinovirus, 4 (1·3%) for rhinovirus/enterovirus, 3 (1·0%) for enteroviruses, and 3 (1·0%) for adenovirus. Among the 49 influenza‐positive specimens, 76% were influenza A (19 H3N2, 17 H1N1pdm09, and 1 not subtyped), and 24% were influenza B.ConclusionInfluenza viruses were a frequent cause of SARI in hospitalized patients in Southern Arizona. Monitoring respiratory illness in border populations will help better understand the etiologies. Improving influenza vaccination coverage may help prevent some SARI cases.
Rapid identification from positive blood cultures is standard of care (SOC) in many clinical microbiology laboratories. The GenMark Dx ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a multiplex nucleic acid amplification assay based on competitive DNA hybridization and electrochemical detection using eSensor technology. This multicenter study compared the investigational-use-only (IUO) BCID-GP Panel to other methods of identification of 20 Gram-positive bacteria, four antimicrobial resistance genes, and both Pan Candida and Pan Gram-Negative targets that are unique to the BCID-GP Panel. Ten microbiology laboratories throughout the United States collected residual, deidentified positive blood culture samples for analysis. Five laboratories tested both clinical and contrived samples with the BCID-GP Panel. Comparator identification methods included each laboratory’s SOC, which included matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and automated identification systems as well as targeted PCR/analytically validated real-time PCR (qPCR) with bidirectional sequencing. A total of 2,342 evaluable samples (1,777 clinical and 565 contrived) were tested with the BCID-GP Panel. The overall sample accuracy for on-panel organisms was 89% before resolution of discordant results. For pathogenic Gram-positive targets (Bacillus cereus group, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Staphylococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Listeria spp., Listeria monocytogenes, Streptococcus spp., Streptococcus agalactiae, Streptococcus anginosus group, Streptococcus pneumoniae, and Streptococcus pyogenes), positive percent agreement (PPA) and negative percent agreement (NPA) ranged from 93.1% to 100% and 98.8% to 100%, respectively. For contamination rule-out targets (Bacillus subtilis group, Corynebacterium, Cutibacterium acnes, Lactobacillus, and Micrococcus), PPA and NPA ranged from 84.5% to 100% and 99.9% to 100%, respectively. Positive percent agreement and NPA for the Pan Candida and Pan Gram-Negative targets were 92.4% and 95.7% for the former and 99.9% and 99.6% for the latter. The PPAs for resistance markers were as follows: mecA, 97.2%; mecC, 100%; vanA, 96.8%; and vanB, 100%. Negative percent agreement ranged from 96.6% to 100%. In conclusion, the ePlex BCID-GP Panel compares favorably to SOC and targeted molecular methods for the identification of 20 Gram-positive pathogens and four antimicrobial resistance genes in positive blood culture bottles. This panel detects a broad range of pathogens and mixed infections with yeast and Gram-negative organisms from the same positive blood culture bottle.
Objective: Understanding bacterial species at highest risk for harboring blaCTX-M genes is necessary to guide antibiotic treatment. We identified the species-specific prevalence of blaCTX-M genes in clinical isolates from the United States. Methods: 24 microbiology laboratories representing 66 hospitals using the GenMark Dx ePlex® Blood Culture Identification Gram-Negative (BCID-GN) Panel extracted blood culture results from April 2019 to July 2020. The BCID-GN Panel includes 21 Gram-negative targets. Along with identifying blaCTX-M genes, it detects major carbapenemase gene families. Results: 4,209 Gram-negative blood cultures were included. blaCTX-M genes were identified in 462 (11%) specimens. The species-specific prevalence of blaCTX-M genes were as follows: Escherichia coli (16%), Klebsiella pneumoniae (14%), Klebsiella oxytoca (6%), Salmonella spp. (6%), Acinetobacter baumannii (5%), Enterobacter species (3%), Proteus mirabilis (2%), Serratia marcenscens (0.6%), and Pseudomonas aeruginosa (0.5%). blaCTX-M prevalence was 26%, 24%, and 22% among participating hospitals in the District of Columbia, New York, and Florida, respectively. Carbapenemase genes were identified in 61 (2%) organisms with the following distribution: blaKPC (59%), blaVIM (16%), blaOXA (10%), blaNDM (8%), and blaIMP (7%). The species-specific prevalence of carbapenemase genes were as follows: A. baumannii (5%), K. pneumoniae (3%), P. mirabilis (3%), Enterobacter species (3%), Citrobacter spp. (3%), P. aeruginosa (2%), E. coli (<1%), K. oxytoca (<1%), and S. marcescens (<1%). Conclusion: Approximately 11% of Gram-negative organisms in our US cohort contain blaCTX-M genes. blaCTX-M genes remain uncommon in organisms beyond E. coli, K. pneumoniae, and K. oxytoca. Future molecular diagnostic panels would benefit from the inclusion of plasmid-mediated ampC and SHV and TEM ESBL targets.
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