Recognition of Mycobacterium tuberculosis by the host inflammasome

The NLR gene family mediates host immunity to various acute pathogenic stimuli but its role in chronic infection is not known. This thesis addressed the role of NLRP3 (NALP3), its adaptor protein ASC, and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1[beta] secretion, and this requires the inflammasome components ASC (also PYCARD, TMS1), caspase-1, and NLRP3. However in vivo Mtb aerosol infection of Nlrp3-/-, Casp-1-/- , and WT mice showed no differences in pulmonary IL-1[beta] production, bacterial burden, or long-term survival. In contrast, a significant role was observed for ASC in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of ASC-/- mice. Decreased survival of Mtb-infected ASC-/- animals was associated with defective granuloma formation and reduced CD11c+ CD11bmid/low cells. ASC is known to bind the noninflammasome forming protein Nlrp12in an artificial expression system. Following Mtb infection, Nlrp12-/- mice had similar survival, bacterial burden, and cytokines to the wild-type controls, indicating that Nlrp12 does not protect the host during Mtb infection. These data demonstrate that ASC exerts a novel inflammasome-independent role during chronic Mtb infection

Optimizing identification of clinically relevant gram-positive organisms by use of the bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identification of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical specimens. We evaluated 4 direct-smear methods, including “heavy” (H) and “light” (L) smears, with and without a 1-μl direct formic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or “score.” We found that a heavy smear with a formic acid overlay (H+FA) produced optimal MALDI-TOF MS identification scores and the highest percentage of correctly identified organisms. Using a score of ≥2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of ≥1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identification of Gram-positive bacteria by use of MALDI-TOF MS

Renal abscess caused by a Providencia stuartii isolate biochemically misidentified as Pasteurella

Providencia stuartii is associated with urinary tract infection (UTI) in catheterized patients. Here we report an abscess containing P. stuartii in a patient with a history of UTI, renal stones, and stent placement. This organism was identified by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing following biochemical identification as Pasteurella

Two cases of Kerstersia gyiorum isolated from sites of chronic infection

Kerstersia gyiorum is infrequently associated with human infection. We report the isolation of Kerstersia gyiorum from two patients: the first, a patient with chronic ear infections, and the second, a patient with a chronic leg wound. Both isolates were resistant to ciprofloxacin, which has not been previously reported

De novo meningitis caused by Propionibacterium acnes in a patient with metastatic melanoma

Propionibacterium acnes is a known cause of postneurosurgical meningitis; however, it is rarely implicated in de novo meningitis. Herein we report a case of a 49-year-old male with de novo meningitis caused by P. acnes with metastatic melanoma as the only identified risk factor for his infection

The DNA Sensor AIM2 Maintains Intestinal Homeostasis via Regulation of Epithelial Antimicrobial Host Defense

SummaryMicrobial pattern molecules in the intestine play immunoregulatory roles via diverse pattern recognition receptors. However, the role of the cytosolic DNA sensor AIM2 in the maintenance of intestinal homeostasis is unknown. Here, we show that Aim2−/− mice are highly susceptible to dextran sodium sulfate-induced colitis that is associated with microbial dysbiosis as represented by higher colonic burden of commensal Escherichia coli. Colonization of germ-free mice with Aim2−/− mouse microbiota leads to higher colitis susceptibility. In-depth investigation of AIM2-mediated host defense responses reveals that caspase-1 activation and IL-1β and IL-18 production are compromised in Aim2−/− mouse colons, consistent with defective inflammasome function. Moreover, IL-18 infusion reduces E. coli burden as well as colitis susceptibility in Aim2−/− mice. Altered microbiota in inflammasome-defective mice correlate with reduced expression of several antimicrobial peptides in intestinal epithelial cells. Together, these findings implicate DNA sensing by AIM2 as a regulatory mechanism for maintaining intestinal homeostasis

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

Characterization of NLRP12 during the In Vivo Host Immune Response to Klebsiella pneumoniae and Mycobacterium tuberculosis

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1β production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12−/− mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12−/− mice showed a slight decrease in IL-1β and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12−/− mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12−/− mice. Likewise, Nlrp12−/− mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis

The NLRP3 Inflammasome Mediates In Vivo Innate Immunity to Influenza A Virus through Recognition of Viral RNA

NLR genes mediate host immunity to various pathogenic stimuli. However, in vivo evidence for NLR involvement in viral sensing has not been widely investigated and remains controversial. As an ultimate test of the physiologic role of NLRP3 during RNA viral infection, this work explores the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, ASC, or Caspase-1, but not Nlrc4, exhibit dramatically increased mortality but reduced immune response following influenza virus exposure. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrate that NLRP3-mediated response can be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by influenza virus is dependent upon lysosomal maturation and reactive oxygen species. Inhibition of ROS induction eliminated IL-1β production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA

The Inflammasome Component Nlrp3 Impairs Antitumor Vaccine by Enhancing the Accumulation of Tumor-Associated Myeloid-Derived Suppressor Cells

The inflammasome is a proteolysis complex that generates the active forms of the pro-inflammatory cytokines IL-1β and IL-18. Inflammasome activtation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3−/− mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended upon CD8+ T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3−/− hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSCs). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3−/− MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3−/− mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling