Inaccuracies of the ISO 11731 Method for Environmental Validation of Legionella in Building Water Systems: Opportunities to Improve Sensitivity and Detect Viable but Non-Culturable Legionella.

Current environmental diagnostics for the detection of Legionella fail to detect viable but non-culturable Legionella, have sensitivity limitations and are time-consuming (10-14 days to results). The objective of this study was to compare Legionella detection results between the standard ISO 11731 and an innovative Legionella detection method that utilizes a hybrid methodology of traditional microbiology and molecular detection. In this study, four hundred and seventy-six (476) potable building water samples were analyzed with ISO 11731 and the novel method in parallel. Of the 476 total samples that were tested, a discrepancy of 21% was observed when comparing the ISO 11731 method to the novel method. Separating the samples based on hazard control methods yielded a 15.4% discrepancy for chlorinated systems (n = 284) and a 29% discrepancy for monochloraminated systems (n = 192). The data presented here conclusively show inaccuracies in environmental validation for building water systems based on results returned by the standard ISO 11731 method. This is especially evident in systems primarily disinfected with monochloramines. Overall, these data highlight the need for new and innovative methods to overcome the inaccuracies of the traditional ISO 11731 spread plates to prevent disease and injury caused by Legionella.

Validation of the MYChrOme™ Culture Plate for Detection and Differentiation of Rapid-Growing Nontuberculous Mycobacteria in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 062101.

BACKGROUND: The MYChrOme™ Culture Plate is a chromogenic media for the detection and differentiation of rapid-growing nontuberculous mycobacteria (NTM) in water, aided by MYCOn™ decontamination to reduce background microbiota. OBJECTIVE: Evaluate the MYChrOme Culture Plates for the detection of rapid-growing NTM in potable and non-potable water as part of the AOAC Performance Tested Method(s)SM program. METHODS: Inclusivity and exclusivity of MYChrOme were evaluated with 50 target and 30 non-target organisms. Method robustness and lot stability of MYChrOme were analyzed. The candidate method was compared to a modified US Food and Drug Administration (FDA) Method: U.S. FDA-Isolation and Identification of Nontuberculous Mycobacteria in Tattoo Inks using an equivalency test. The matrix study consisted of artificially contaminated potable water and naturally contaminated non-potable water. Independent laboratory testing was conducted to verify method performance in non-potable water. RESULTS: The inclusivity of MYChrOme was 94% within one week, and 98% within two weeks. The exclusivity was 96% for untreated samples and 100% for treated samples. The candidate method remained statistically equivalent for robustness and a three-month shelf-life was confirmed. For both matrixes, the candidate and reference methods were not equivalent, with more colonies enumerated on the candidate method except for one contamination level of the potable matrix. CONCLUSION: The MYChrOme culture method can successfully detect and differentiate rapid-growing NTM in the matrixes tested, with sensitivity equivalent or higher than the reference method. HIGHLIGHTS: The MYChrOme culture plate offers differentiation of rapid-growing NTM colonies, improved detection in non-potable samples with MYCOn decontamination, and results within 7 days.

RNA-Seq used to identify ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production.

The pinyon ips beetle, Ips confusus (LeConte) is a highly destructive pest in pine forests in western North America. When colonizing a new host tree, I. confusus beetles coordinate a mass attack to overcome the tree's defenses using aggregation pheromones. Ips confusus, as with other Ips spp. beetles, biosynthesize ipsdienol and ipsenol in a specific enantiomeric blend and ratio as aggregation pheromones. While several of the initial steps in the pheromone biosynthetic pathway have been well defined, the final steps were unknown. We used comparative RNA-Seq analysis between fed and unfed male I. confusus midgut tissue to identify candidate genes involved in pheromone biosynthesis. The 12,995 potentially unique transcripts showed a clear separation based on feeding state. Differential expression analysis identified gene groups that were tightly connected. This analysis identified all known pheromone biosynthetic genes and suggested a novel monoterpene double bond reductase, ipsdienone reductase (IDONER), with pheromone biosynthetic gene expression patterns. IDONER cDNA was cloned, expressed, and functionally characterized. The coding DNA sequence has an ORF of 1101 nt with a predicted translation product of 336 amino acids. The enzyme has a molecular weight of 36.7 kDa with conserved motifs of the medium chain dehydrogenases/reductase (MDR) superfamily in the leukotriene B4 dehydrogenases/reductases (LTB4R) family. Tagged recombinant protein was expressed and purified. Enzyme assays and GC/MS analysis showed IDONER catalyzed the reduction of ipsdienone to form ipsenone. This study shows that IDONER is a monoterpene double bond reductase involved in I. confusus pheromone biosynthesis.

Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment.

The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires' disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires' disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10-14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at >1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples.

Identification of multiple 5-HT4 partial agonist clinical candidates for the treatment of Alzheimer's disease.

The cognitive impairments observed in Alzheimer's disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT(4)) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT(4) agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure-response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT(4) partial agonist candidates 2d and 3 were chosen for clinical development.

The 5-hydroxytryptamine4 receptor agonists prucalopride and PRX-03140 increase acetylcholine and histamine levels in the rat prefrontal cortex and the power of stimulated hippocampal θ oscillations.

5-Hydroxytryptamine (5-HT)(4) receptor agonists reportedly stimulate brain acetylcholine (ACh) release, a property that might provide a new pharmacological approach for treating cognitive deficits associated with Alzheimer's disease. The purpose of this study was to compare the binding affinities, functional activities, and effects on neuropharmacological responses associated with cognition of two highly selective 5-HT(4) receptor agonists, prucalopride and 6,7-dihydro-4-hydroxy-7-isopropyl-6-oxo-N-[3-(piperidin-1-yl)propyl]thieno[2,3-b]pyridine-5-carboxamide (PRX-03140). In vitro, prucalopride and PRX-03140 bound to native rat brain 5-HT(4) receptors with K(i) values of 30 nM and 110 nM, respectively, and increased cAMP production in human embryonic kidney-293 cells expressing recombinant rat 5-HT(4) receptors. In vivo receptor occupancy studies established that prucalopride and PRX-03140 were able to penetrate the brain and bound to 5-HT(4) receptors in rat brain, achieving 50% receptor occupancy at free brain exposures of 330 nM and 130 nM, respectively. Rat microdialysis studies revealed that prucalopride maximally increased ACh and histamine levels in the prefrontal cortex at 5 and 10 mg/kg, whereas PRX-03140 significantly increased cortical histamine levels at 50 mg/kg, failing to affect ACh release at doses lower than 150 mg/kg. In combination studies, donepezil-induced increases in cortical ACh levels were potentiated by prucalopride and PRX-03140. Electrophysiological studies in rats demonstrated that both compounds increased the power of brainstem-stimulated hippocampal θ oscillations at 5.6 mg/kg. These findings show for the first time that the 5-HT(4) receptor agonists prucalopride and PRX-03140 can increase cortical ACh and histamine levels, augment donepezil-induced ACh increases, and increase stimulated-hippocampal θ power, all neuropharmacological parameters consistent with potential positive effects on cognitive processes.

Expression of rat I-TAC/CXCL11/SCYA11 during central nervous system inflammation: comparison with other CXCR3 ligands.

The chemokines are a large gene superfamily with critical roles in development and immunity. The chemokine receptor CXCR3 appears to play a major role in the trafficking of activated Th1 lymphocytes. There are at least three major ligands for CXCR3: mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, and of these three ligands, CXCL11 is the least well-characterized. In this study, we have cloned a rat ortholog of CXCL11, evaluated its function, and examined its expression in the Th-1-mediated disease, experimental autoimmune encephalomyelitis (EAE) in the rat. Based on its predicted primary amino-acid sequence, rat I-TAC/CXCL11 was synthesized and shown to induce chemotaxis of activated rat T lymphocytes in vitro and the in vivo migration of T lymphocytes when injected into the skin. I-TAC/CXCL11 expression, as determined by RT-PCR, increased in lymph node and spinal cord tissue collected from rats in which EAE had been actively induced, and in spinal cord tissue from rats in which EAE had been passively induced. The kinetics of expression were similar to that of CXCR3 and IP-10/CXCL10, although expression of both CXCR3 and IP-10/CXCL10 was more intense than that of I-TAC/CXCL11 and increased more rapidly in both lymph nodes and the spinal cord. Only minor levels of expression of the related chemokine mig/CXCL9 were observed. Immunohistochemistry revealed that the major cellular source of I-TAC/CXCL11 in the central nervous system (CNS) during EAE is likely to be the astrocyte. Together, these data indicate that I-TAC/CXCL11 is expressed in the CNS during the clinical phase of EAE. However, the observation that I-TAC/CXCL11 is expressed after receptor expression is detected suggests that it is not essential for the initial migration of CXCR3-bearing cells into the CNS.

Engineering autoactivating forms of matrix metalloproteinase-9 and expression of the active enzyme in cultured cells and transgenic mouse brain.

Matrix metalloproteinases (MMPs) are hypothesized to play an important role in the pathogenesis of several central nervous system disorders. Increased levels of expression of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) have been observed in Alzheimer's disease, stroke, multiple sclerosis, and amyotrophic lateral sclerosis. This suggests an aberrant regulation of MMPs that could lead to inappropriate expression of MMP activity. To allow us to evaluate the effect of increased levels of active MMP-9 in the central nervous system, mutant forms of the enzyme were designed to autocatalytically remove the pro domain, yielding active enzyme. This was accomplished by modifying residues in the cysteine switch autoinhibitor region of the propeptide. Stable cell lines and transgenic mice that express G100L and D103N autoactive forms of human MMP-9 were developed to study the role of dysregulation of MMP-9 in disease.