Use of Purified Clostridium difficile Spores To Facilitate Evaluation of Health Care Disinfection Regimens

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 10⁶ pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is a major cause of health care-acquired infections and antibiotic-associated diarrhea (2). In recent years, several genetic variants of C. difficile have emerged as important health care pathogens (6). Perhaps most notable is the “hypervirulent” variant, commonly referred to as PCR ribotype 027/restriction endonuclease analysis (REA) group BI, that produces elevated levels of toxins TcdA and TcdB (17, 19). Other virulent ribotypes that display extensive heterogeneity among their toxin protein sequences (26) and gene activities (8) have emerged. Using whole-genome sequencing, we demonstrated that there are broad genetic differences between the entire genomes of several common variants, including ribotype/REA group variants 012/R, 017/CF, and 027/BI used in this study (12, 27, 31). In contrast, phylogeographic analysis of 027/BI isolates from Europe and the United States demonstrates that this clade is extremely clonal and implies recent transcontinental spread of hypervirulent C. difficile (12).C. difficile is distinct from many other health care pathogens because it produces highly infectious spores that are shed into the environment (25, 28). C. difficile spores can resist disinfection regimens that normally inactivate other health care pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, therefore challenging current infection control measures (2). A multifaceted approach is normally used to control C. difficile in health care facilities (32). Interventions include antimicrobial stewardship, increased clinical awareness, patient isolation (11), and enhanced environmental disinfection regimens based on hydrogen peroxide (H₂O₂) vapor (HPV) (4). While attempts to break the spore-mediated infection cycle and interrupt these efficient routes of transmission are important for infection control measures, there is little quantitative evidence indicating which interventions are most effective (7). Here we describe the exploitation of pure C. difficile spores (16) and a murine transmission model (15) in simple, practical methods to quantitatively monitor the impact of health care disinfection regimens on C. difficile viability. These methods can be used to optimize disinfection regimens targeted at C. difficile.     

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community-wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single-locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.     

Effect of carnitine on greening barley leaves

Carnitine increases chlorophyll production in greening barley leaves. [Methyl-¹⁴C]carnitine fed to greening leaves was not utilized as a carbon sou     

Angiogenesis and the role of the endothelial nicotinic acetylcholine receptor

An endothelial nicotinic acetycholine receptor (nAChR) mediates endothelial proliferation, survival, migration and tube formation in vitro, and angiogenesis in vivo. Exogenous nicotine stimulates this angiogenic pathway. This action of nicotine may contribute to tumor angiogenesis and tumor growth; atherosclerotic plaque neovascularization and progression; and other tobacco-related diseases. The endothelial nAChR mediates an angiogenic pathway that is interdependent with growth factor mediated pathways, as shown by pharmacological and molecular studies. The characterization of this new angiogenic pathway may provide a new therapeutic avenue for disorders of insufficient or pathological angiogenesis.     

Cyclic AMP signaling contributes to neural plasticity and hyperexcitability in AH sensory neurons following intestinal Trichinella spiralis-induced inflammation

Trichinella spiralis infection causes hyperexcitability in enteric after-hyperpolarising (AH) sensory neurons that is mimicked by neural, immune or inflammatory mediators known to stimulate adenylyl cyclase (AC)/cyclic 3',5'-adenosine monophosphate (cAMP) signaling. The hypothesis was tested that ongoing modulation and sustained amplification in the AC/cAMP/phosphorylated cAMP related element binding protrein (pCREB) signaling pathway contributes to hyperexcitability and neuronal plasticity in gut sensory neurons after nematode infection. Electrophysiological, immunological, molecular biological or immunochemical studies were done in T. spiralis-infected guinea-pigs (8000 larvae or saline) after acute-inflammation (7 days) or 35 days p.i., after intestinal clearance. Acute-inflammation caused AH-cell hyperexcitability and elevated mucosal and neural tissue levels of myeloperoxidase, mast cell tryptase, prostaglandin E2, leukotrine B4, lipid peroxidation, nitric oxide and gelatinase; lower level inflammation persisted 35 days p.i. Acute exposure to blockers of AC, histamine, cyclooxygenase or leukotriene pathways suppressed AH-cell hyperexcitability in a reversible manner. Basal cAMP responses or those evoked by forskolin (FSK), Ro-20-1724, histamine or substance P in isolated myenteric ganglia were augmented after T. spiralis infection; up-regulation also occurred in AC expression and AC-immunoreactivity in calbindin (AH) neurons. The cAMP-dependent slow excitatory synaptic transmission-like responses to histamine (mast cell mediator) or substance P (neurotransmitter) acting via G-protein coupled receptors (GPCR) in AH neurons were augmented by up to 2.5-fold after T. spiralis infection. FSK, histamine, substance P or T. spiralis acute infection caused a 5- to 30-fold increase in cAMP-dependent nuclear CREB phosphorylation in isolated ganglia or calbindin (AH) neurons. AC and CREB phosphorylation remained elevated 35 days p.i.. Ongoing immune activation, AC up-regulation, enhanced phosphodiesterase IV activity and facilitation of the GPCR-AC/cAMP/pCREB signaling pathway contributes to T. spiralis-induced neuronal plasticity and AH-cell hyperexcitability. This may be relevant in gut nematode infections and inflammatory bowel diseases, and is a potential therapeutic target.     

Inspiratory resistance delays the reporting of symptoms with central hypovolemia: association with cerebral blood flow

We tested the hypothesis that breathing through an inspiratory threshold device (ITD) during progressive central hypovolemia would protect cerebral perfusion and attenuate the reporting of presyncopal symptoms. Eight human subjects were exposed to lower-body negative pressure (LBNP) until the presence of symptoms while breathing through either an active ITD (-7 cmH(2)O impedance) or a sham ITD (0 cmH(2)O). Cerebral blood flow velocity (CBFV) was measured continuously via transcranial Doppler and analyzed in both time and frequency domains. Subjects were asked to report any subjective presyncopal symptoms (e.g., dizziness, nausea) at the conclusion of each LBNP exposure. Symptoms were coincident with physiological evidence of cardiovascular collapse (e.g., hypotension, bradycardia). Breathing on the active ITD increased LBNP tolerance time (mean +/- SE) from 2,014 +/- 106 s to 2,259 +/- 138 s (P = 0.006). We compared CBFV responses at the time of symptoms during the sham ITD trial with those at the same absolute time during the active ITD trial (when there were no symptoms). While there was no difference in mean CBFV at these time points (sham, 44 +/- 4 cm/s vs. active, 47 +/- 4; P = 0.587), total oscillations (sum of high- and low-frequency spectral power) of CBFV were higher (P = 0.004) with the active ITD (45.6 +/- 10.2 cm/s(2)) than the sham ITD (22.1 +/- 5.4 cm/s(2)). We conclude that greater oscillations around the same absolute level of mean CBFV are induced by inspiratory resistance and may contribute to the delay in symptoms and cardiovascular collapse that accompany progressive central hypovolemia.     

A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts

Chloroplast genomes in land plants harbor approximately 20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.     

A comparison of the gene expression profiles of CRL-1807 colonocytes exposed to endogenous AAPH-generated peroxides and exogenous peroxides from heated oil

Oxidation of PUFAs in the diet has the potential to be genotoxic and hence carcinogenic. Such carcinogenic processes originate within stem cells of the colon. These cells appear to be predisposed to the carcinogenic process. In colon cells (CRL-1807) exposed to chemical reactions simulating exogenous and endogenous peroxidation reactions, we have observed that undifferentiated cells could mount an effective recombinational repair/TCR response to an endogenous peroxidative DNA damage insult, but not to an external exogenous peroxidative insult as one would encounter from a dietary source. This may suggest that defects in such specific DNA repair may play a role in tumour development in undifferentiated colonocytes exposed to a diet-derived lipid peroxides.