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.     

NO-synthase independent NO generation in mammals

Inorganic nitrate () and nitrite () are part of the nitrogen cycle in nature. To the general public these anions are generally known as undesired residues in the food chain with potentially carcinogenic effects. Among biologists, these inorganic anions have merely been viewed as inert oxidative end products of endogenous nitric oxide (NO) metabolism. However, recent studies surprisingly show that nitrate and nitrite can be metabolized in vivo to form nitric oxide (NO) and other bioactive nitrogen oxides. This represents an important alternative source of NO especially during hypoxia when the oxygen-dependent l-arginine-NO pathway can be altered. A picture is now emerging suggesting important biological functions of the nitrate-nitrite-NO pathway with profound implications in relation to the diet and cardiovascular homeostasis. Moreover, an increasing number of studies suggest a therapeutic potential for nitrate and nitrite in diseases such as myocardial infarction, stroke, hypertension, renal failure and gastric ulcers.     

Rapamycin promotes β-amyloid production via ADAM-10 inhibition

Rapamycin is a well known immunosuppressant drug for rejection prevention in organ transplantation. Numerous clinical trials using rapamycin analogs, involving both children and adults with various disorders are currently ongoing worldwide. Most recently, rapamycin gained much attention for what appears to be life-span extending properties when administered to mice. The risk for Alzheimer disease (AD) is strongly and positively correlated with advancing age and is characterized by deposition of β-amyloid peptides (Aβ) as senile plaques in the brain. We report that rapamycin (2.5 μM), significantly increases Aβ generation in murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP). In concert with these observations, we found rapamycin significantly decreases the neuroprotective amino-terminal APP (amyloid precursor protein) cleavage product, soluble APP-α (sAPP-α) while increasing production of the β-carboxyl-terminal fragment of APP (β-CTF). These cleavage events are associated with decreased activation of a disintegrin and metallopeptidase domain-10 (ADAM-10), an important candidate α-secretase which opposes Aβ generation. To validate these findings in vivo, we intraperitoneal (i.p.) injected Tg2576 Aβ-overproducing transgenic mice with rapamycin (3 mg/kg/day) for 2 weeks. We found increased Aβ levels associated with decreased sAPP-α at an average rapamycin plasma concentration of 169.7 ± 23.5 ng/mL by high performance liquid chromatography (HPLC). These data suggest that although rapamycin may increase the lifespan in some mouse models, it may not decrease the risk for age-associated neurodegenerative disorders such as AD.     

Bayesian Methods for Examining Hardy–Weinberg Equilibrium

Summary .  Testing for Hardy–Weinberg equilibrium is ubiquitous and has traditionally been carried out via frequentist approaches. However, the discreteness of the sample space means that uniformity of  p -values under the null cannot be assumed, with enumeration of all possible counts, conditional on the minor allele count, offering a computationally expensive way of  p -value calibration. In addition, the interpretation of the subsequent  p -values, and choice of significance threshold depends critically on sample size, because equilibrium will always be rejected at conventional levels with large sample sizes. We argue for a Bayesian approach using both Bayes factors, and the examination of posterior distributions. We describe simple conjugate approaches, and methods based on importance sampling Monte Carlo. The former are convenient because they yield closed-form expressions for Bayes factors, which allow their application to a large number of single nucleotide polymorphisms (SNPs), in particular in genome-wide contexts. We also describe straightforward direct sampling methods for examining posterior distributions of parameters of interest. For large numbers of alleles at a locus we resort to Markov chain Monte Carlo. We discuss a number of possibilities for prior specification, and apply the suggested methods to a number of real datasets.     

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Co-occurrence and genotypic distribution of Phytophthora species recovered from watersheds and plant nurseries of eastern Tennessee

In 2008 statewide surveys of symptomatic foliage of nursery plants from Tennessee resulted in isolation of 43 isolates of Phytophthora spp. This sample set includes four described species (P. citrophthora, P. citricola, P. nicotianae, P. syringae), and a provisional species of Phytophthora ('P. hydropathica'). At the same time a stream-baiting survey was initiated to recover Phytophthora from eight watersheds in eastern Tennessee, some of which are near plant nurseries. Baiting was accomplished by submerging healthy Rhododendron leaves approximately 1 wk and isolation onto selective media. Six baiting periods were completed, and in total 98 Phytophthora isolates and 45 isolates of Pythium spp. were recovered. Three described species (P. citrophthora, P. citricola and P. irrigata) and the provisional species 'P. hydropathica' were obtained as well as three undescribed Phytophthora taxa and Pythium litorale. Isolates from both surveys were identified to species with morphology and the internal transcribed spacer (ITS) sequence. Isolates from species co-occurring in streams and nurseries (P. citricola, P. citrophthora and 'P. hydropathica') were characterized further with amplified fragment length polymorphism (AFLP) analyses and mefenoxam tolerance assays. Isolates representing a putative clonal genotype of P. citricola were obtained from both environmental and nursery sample sets.     

Development of a Novel Aluminum Tolerance Phenotyping Platform Used for Comparisons of Cereal Aluminum Tolerance and Investigations into Rice Aluminum Tolerance Mechanisms

The genetic and physiological mechanisms of aluminum (Al) tolerance have been well studied in certain cereal crops, and Al tolerance genes have been identified in sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Rice (Oryza sativa) has been reported to be highly Al tolerant; however, a direct comparison of rice and other cereals has not been reported, and the mechanisms of rice Al tolerance are poorly understood. To facilitate Al tolerance phenotyping in rice, a high-throughput imaging system and root quantification computer program was developed, permitting quantification of the entire root system, rather than just the longest root. Additionally, a novel hydroponic solution was developed and optimized for Al tolerance screening in rice and compared with the Yoshida''s rice solution commonly used for rice Al tolerance studies. To gain a better understanding of Al tolerance in cereals, comparisons of Al tolerance across cereal species were conducted at four Al concentrations using seven to nine genetically diverse genotypes of wheat, maize (Zea mays), sorghum, and rice. Rice was significantly more tolerant than maize, wheat, and sorghum at all Al concentrations, with the mean Al tolerance level for rice found to be 2- to 6-fold greater than that in maize, wheat, and sorghum. Physiological experiments were conducted on a genetically diverse panel of more than 20 rice genotypes spanning the range of rice Al tolerance and compared with two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. These results clearly demonstrate that the extremely high levels of rice Al tolerance are mediated by a novel mechanism, which is independent of root tip Al exclusion.Aluminum (Al) is the most abundant metal in the earth''s crust, constituting approximately 7% of the soil (Wolt, 1994). Al is predominately found as a key component of soil clays; however, under highly acidic soil conditions (pH < 5.0), Al³⁺ is solubilized into the soil solution and is highly phytotoxic. Al³⁺ causes a rapid inhibition of root growth that leads to a reduced and stunted root system, thus having a direct effect on the ability of a plant to acquire both water and nutrients. Approximately 30% of the world''s total land area and over 50% of potentially arable lands are acidic, with the majority (60%) found in the tropics and subtropics (von Uexkull and Mutert, 1995). Thus, acidic soils are a major limitation to crop production, particularly in the developing world.As a whole, cereal crops (Poaceae) provide an excellent model for studying Al tolerance because of their abundant genetic resources, large, active research communities, and importance to agriculture. In addition, work in one cereal species can rapidly translate into impact throughout the family. Previous research has focused on understanding the genetic and physiological mechanisms of Al tolerance in maize (Zea mays), sorghum (Sorghum bicolor), and wheat (Triticum aestivum). The most recognized physiological mechanism conferring Al tolerance in plants involves exclusion of Al from the root tip (Miyasaka et al., 1991; Delhaize and Ryan, 1995; Kochian, 1995; Kochian et al., 2004a, 2004b). The exclusion mechanism is primarily mediated by Al-activated exudation of organic acids such as malate, citrate, or oxalate from the root apex, the site of Al toxicity (Ryan et al., 1993, 2001; Ma et al., 2001). These organic acids chelate Al in the rhizosphere, reducing the concentration and toxicity of Al at the growing root tip (Ma et al., 2001). Phosphate has also been identified as a class of root exudates involved in cation chelation and therefore can be considered a potential exudate involved in Al exclusion from the root tip (Pellet et al., 1996).Al-activated malate and citrate anion efflux transporters have been cloned from wheat (ALMT1; Sasaki et al., 2004) and sorghum (SbMATE; Magalhaes et al., 2007), and root citrate efflux transporters have been implicated in Al tolerance in maize (Piñeros and Kochian, 2001; Zhang et al., 2001). Recently, a maize homolog of sorghum SbMATE was shown to be the root citrate efflux transporter that plays a role in maize Al tolerance (Maron et al., 2010). Although organic acids have been shown to play a major role in Al tolerance in these species, another exclusion mechanism has been identified in an Arabidopsis (Arabidopsis thaliana) mutant, where a root-mediated increase in rhizosphere pH lowers the Al³⁺ activity and thus participates in Al exclusion from the root apex (Degenhardt et al., 1998). Furthermore, there is clear evidence that tolerance in maize cannot be fully explained by organic acid release (Piñeros et al., 2005). These types of findings strongly suggest that multiple Al tolerance mechanisms exist in plants.Rice (Oryza sativa) has been reported to be the most Al-tolerant cereal crop under field conditions, capable of withstanding significantly higher concentrations of Al than other major cereals (Foy, 1988). Despite this fact, very little is known about the physiological mechanisms of Al tolerance in rice. Two independent studies have identified increased Al accumulation in the root apex in susceptible compared with Al-tolerant rice varieties, but no differences were observed in organic acid exudation or rhizosphere pH (Ma et al., 2002; Yang et al., 2008). These studies suggest that rice may contain novel physiological and/or genetic mechanisms that confer significantly higher levels of Al tolerance than those found in other cereals. A more thorough analysis is required to clarify the mechanism of Al tolerance in rice.Cultivated rice is characterized by deep genetic divergence between the two major varietal groups: Indica and Japonica (Dally and Second, 1990; Garris et al., 2005; Hu et al., 2006; Londo et al., 2006). Extensive selection pressure over the last 10,000 years has resulted in the formation of five genetically distinct subpopulations: indica and aus within the Indica varietal group, and temperate japonica, tropical japonica, and aromatic/groupV within the Japonica varietal group (Garris et al., 2005; Caicedo et al., 2007; K. Zhao and S. McCouch, personal communication). (Note: When referring to varietal groups, the first letter will be capitalized, while lowercase letters will be used to refer to the subpopulation groups.) Subpopulation differences in trait performance are often significant, particularly with respect to biotic and abiotic stress (Champoux et al., 1995; Lilley et al., 1996; Garris et al. 2003; Xu et al., 2009). This can lead to confusion because trait or performance differences may be confounded with subpopulation structure, leading to false positives (type 1 error; Devlin and Roeder, 1999; Pritchard and Donnelly, 2001; Yu et al., 2006; Zhao et al., 2007). Therefore, it is important to consider the subpopulation origin of genotypes being compared when studying the genetics and physiology of Al tolerance in rice.Al tolerance screening is typically conducted by comparing root growth of seedlings grown in hydroponic solutions, with and without Al (Piñeros and Kochian, 2001; Magalhaes et al., 2004; Sasaki et al., 2004). Sorghum and maize are often screened for Al tolerance in Magnavaca''s nutrient solution (Piñeros and Kochian, 2001; Magalhaes et al., 2004; Piñeros et al., 2005), while rice seedlings are typically grown in Yoshida''s solution (Yoshida et al., 1976). Furthermore, Al concentrations used to screen for Al tolerance in maize (222 μm), sorghum (148 μm), and wheat (100 μm) are significantly lower than those used for screening Al tolerance in rice (1,112–1,482 μm; Wu et al., 2000; Nguyen et al., 2001, 2002, 2003). These differences in chemical composition of the nutrient solutions make it difficult to directly compare plant response to Al across these cereals. In rice, the high Al concentrations required to observe significant differences in root growth between susceptible and resistant varieties also complicate Al tolerance screening due to the precipitation of Al along with other elements. The result is that control (−Al) and treatment (+Al) solutions may differ with regard to essential mineral nutrients that react with Al, leading to differences in growth not directly attributable to Al. Additionally, because the active form of Al that is toxic to root growth is Al³⁺, any Al that precipitates out of solution has no effect on root growth (Kochian et al., 2004a). In a hydroponic solution, Al may be found in one of four forms: (1) as free Al³⁺, where it actively inhibits root growth; (2) precipitated with other elements and essentially unavailable to inhibit plant growth; (3) different hydroxyl monomers of Al, which are not believed to be toxic to roots (Parker et al., 1988); or (4) complexed with other elements in an equilibrium between its active and inactive states. The degree to which Al inhibits root growth is primarily dependent upon the activity of free Al³⁺ in solution (Kochian et al., 2004a).The objectives of this study were to (1) develop and optimize a suitable nutrient solution and high-throughput Al tolerance screening method for rice; (2) quantify and compare differences in Al tolerance between maize, sorghum, wheat, and rice; and (3) use the developed screening methods to determine if rice utilizes the organic acid-mediated Al exclusion mechanism that is observed in maize, sorghum, and wheat.