Macro and micro diversity of Clostridium difficile isolates from diverse sources and geographical locations

Clostridium difficile has emerged rapidly as the leading cause of antibiotic-associated diarrheal disease, with the temporal and geographical appearance of dominant PCR ribotypes such as 017, 027 and 078. Despite this continued threat, we have a poor understanding of how or why particular variants emerge and the sources of strains that dominate different human populations. We have undertaken a breadth genotyping study using multilocus sequence typing (MLST) analysis of 385 C. difficile strains from diverse sources by host (human, animal and food), geographical locations (North America, Europe and Australia) and PCR ribotypes. Results identified 18 novel sequence types (STs) and 3 new allele sequences and confirmed the presence of five distinct clonal lineages generally associated with outbreaks of C. difficile infection in humans. Strains of animal and food origin were found of both ST-1 and ST-11 that are frequently associated with human disease. An in depth MLST analysis of the evolutionary distant ST-11/PCR ribotype 078 clonal lineage revealed that ST-11 can be found in alternative but closely related PCR ribotypes and PCR ribotype 078 alleles contain mutations generating novel STs. PCR ribotype 027 and 017 lineages may consist of two divergent subclades. Furthermore evidence of microdiversity was present within the heterogeneous clade 1. This study helps to define the evolutionary origin of dominant C. difficile lineages and demonstrates that C. difficile is continuing to evolve in concert with human activity.     

Sphingomyelinase D,a novel probe for cellular sphingomyelin: effects on cholesterol homeostasis in human skin fibroblasts

Sphingomyelin (SM) and free cholesterol (FC) are concentrated in the plasma membranes of eukaryotes; however, the physiological significance of their association is unclear. A common tool for studying the role of membrane SM is digestion with bacterial sphingomyelinase (SMase) C, which hydrolyzes SM to ceramide. However, it is not known whether the observed effects of SMase C treatment are due to the loss of SM per se or to the signaling effects of ceramide. In this study, we tested SMase D from Corynebacterium pseudotuberculosis, which hydrolyzes SM to ceramide phosphate, as an alternative probe. This enzyme specifically hydrolyzed SM in fibroblasts without causing accumulation of ceramide. Treatment of fibroblasts with SMase D stimulated translocation of PM FC to intracellular sites by <20% of the rate observed after SMase C digestion. The cells regenerated SM nearly completely within 5 h after SMase C treatment. However, even after 20 h, no regeneration occurred following SMase D digestion. These findings suggest that the translocation of PM FC caused by SMase C digestion is due to the cellular effects of ceramide rather than the loss of SM. Since ceramide phosphate does not appear to have such effects, we suggest that SMase D is a useful probe of membrane SM.     

The adaptation of enzymes to temperature: catalytic characterization of glucosephosphate isomerase homologues isolated from Mytilus edulis and Isognomon alatus, bivalve molluscs inhabiting different thermal environments

Homologues of glucosephosphate isomerase (GPI, EC 5.3.1.9) were purified to homogeneity and kinetically characterized from Mytilus edulis and Isognomon alatus, two bivalve molluscs experiencing contrasting thermal environments. The enzyme isolated from I. alatus functions at warmer temperatures (25-35 C) than GPI from M. edulis, a species that inhabits colder marine littoral habitats (5-20 C). The former exhibits apparent first-order (with respect to substrate) catalytic rate constants (Vmax/KM) in vitro that become progressively greater than the mussel enzyme as the assay temperature is raised. Apparent zero-order catalytic rate constants (Vmax) are relatively less differentiated. Catalytic efficiency, defined as the rate at which a catalytic event occurs in either reaction direction for reference standard states (substrate concentrations), is greater for the enzyme from the tropical species (I. alatus) at all realistic combinations of temperature and substrate concentration except for the lowest temperatures and highest substrate concentrations, where the GPI from the boreal/temperate M. edulis is more efficient. This pattern of catalytic divergence appears to be due primarily to differentiation in Vmax/KM. These results and other published data are reviewed and shown to be inconsistent with claims that adaptation of enzymes to higher cell temperatures requires a loss in catalytic efficiency.      

Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Testing Air-Filtering Systems: I. Procedure for Testing High-Efficiency Air Filters on Exhaust Systems

A procedure was developed for evaluating high-efficiency filters mounted in exhaust ducts at the National Animal Disease Laboratory. An aerosol of the test organism, Escherichia coli B T₃ bacteriophage, was generated in a chamber attached to a ceiling exhaust register in concentrations of at least 1000 viable organisms per ft³ of air. Samples were collected from both the pre- and postfilter areas, and the number of organisms per ft³ of air was determined. The efficiency of the filter was calculated from these figures. A total of 269 high-efficiency filters were tested. Of these, 249 had efficiencies of 98% or greater. The remaining 20, with efficiencies of less than 98%, were repaired and retested. No filter was accepted with an efficiency of less than 98%.     

The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family.

Arcanobacterium (Actinomyces) pyogenes, an animal pathogen, produces a hemolytic exotoxin, pyolysin (PLO). The gene encoding PLO was cloned, and sequence analysis revealed an open reading frame of 1,605 bp encoding a protein of 57.9 kDa. PLO has 30 to 40% identity with the thiol-activated cytolysins (TACYs) of a number of gram-positive bacteria. The activity of PLO was found to be very similar to those of other TACYs, except that it was not thiol activated. The highly conserved TACY undecapeptide is divergent in PLO; in particular, the cysteine residue required for thiol activation has been replaced with alanine. However, mutagenesis of the alanine residue to cysteine did not confer thiol activation on PLO, suggesting a conformational difference in the undecapeptide region of this toxin. Specific antibodies against purified, recombinant PLO completely neutralized the hemolytic activity of A. pyogenes, suggesting that this organism produces a single hemolysin. Furthermore, these antibodies could passively protect mice against lethal challenge with A. pyogenes, suggesting that like other TACYs PLO is an important virulence factor in the pathogenesis of this organism.     

Modeling population viability of captive elephants in Myanmar (Burma): implications for wild populations

Captive Asian elephants Elephas maximus , used as work animals, constitute up to 22–30% of remaining Asian elephants. Myanmar has the largest captive population worldwide (∼6000), maintained at this level for over a century. We used published demographic data to assess the viability of this captive population. We tested how this population can be self-sustained, how many elephants must be supplemented from the wild to maintain it, and what consequences live capture may have for Myanmar's wild population. Our results demonstrate that the current captive population is not self-sustaining because mortality is too high and birth rates are too low. Our models also suggest ∼100 elephants year⁻¹ have been captured in the wild to supplement the captive population. Such supplementation cannot be supported by a wild population of fewer than 4000 elephants. Given the most recent expert estimate of ∼2000 wild elephants remaining in Myanmar, a harvest of 100 elephants year⁻¹ could result in extinction of the wild population in 31 years. Continued live capture threatens the survival of wild and captive populations and must stop. In addition, captive breeding should be increased. These measures are essential to slow the decline and extinction of all of Myanmar's elephants.