Biomedical subjects

J G Songer

Publications and source records attributed to J G Songer.

At least 19 recordsLinked to original sources

PubMed2006

Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains.

Clostridium difficile is the most frequent cause of nosocomial diarrhea worldwide, and recent reports suggested the emergence of a hypervirulent strain in North America and Europe. In this study, we applied comparative phylogenomics (whole-genome comparisons using DNA microarrays combined with Bayesian phylogenies) to model the phylogeny of C. difficile, including 75 diverse isolates comprising hypervirulent, toxin-variable, and animal strains. The analysis identified four distinct statistically supported clusters comprising a hypervirulent clade, a toxin A(-) B(+) clade, and two clades with human and animal isolates. Genetic differences among clades revealed several genetic islands relating to virulence and niche adaptation, including antibiotic resistance, motility, adhesion, and enteric metabolism. Only 19.7% of genes were shared by all strains, confirming that this enteric species readily undergoes genetic exchange. This study has provided insight into the possible origins of C. difficile and its evolution that may have implications in disease control strategies.

Animals↗

PubMed2006

The comparative pathology of Clostridium difficile-associated disease.

Clostridium difficile is a confirmed pathogen in a wide variety of mammals, but the incidence of disease varies greatly in relation to host species, age, environmental density of spores, administration of antibiotics, and possibly, other factors. Lesions vary as well, in severity and distribution within individuals, and in some instances, age groups, of a given species. The cecum and colon are principally affected in most species, but foals and rabbits develop severe jejunal lesions. Explanations for variable susceptibility of species, and age groups within a species, are largely speculative. Differences in colonization rates and toxin-receptor densities have been proposed. Clostridium difficile-associated disease is most commonly diagnosed in Syrian hamsters, horses, and neonatal pigs, but it is reported sporadically in many other species. The essential virulence factors of C. difficile are large exotoxins, toxin A (TcdA) and toxin B (TcdB). Receptor-mediated endocytosis of the toxins is followed by endosomal acidification, a necessary step for conversion of the toxin to its active form in the cytosol. Cell-surface receptors have been characterized for TcdA, but remain to be identified for TcdB. Both TcdA and TcdB disrupt the actin cytoskeleton by disrupting Rho-subtype, intracellular signaling molecules. Disruption of the actin cytoskeleton is catastrophic for cellular function, but inflammation and neurogenic stimuli are also involved in the pathogenesis of the disease.

Animal Diseases↗

PubMed2002

Isolation of Arcanobacterium pyogenes from the porcine gastric mucosa.

Arcanobacterium (Actinomyces) pyogenes is an inhabitant of the mucous membranes of the respiratory and genital tracts of a number of domestic animal species. However, following a precipitating physical or microbial insult, A. pyogenes can become an opportunistic pathogen, associated with suppurative infections. The isolation of A. pyogenes from the bovine ruminal wall indicated that this organism may also inhabit the gastrointestinal tract of, at least, cattle. To determine whether A. pyogenes was also present on the gastric mucosa of a monogastric animal, porcine stomachs were cultured for the presence of this organism. Of 13 stomachs sampled, A. pyogenes was isolated from 5 (39%). The identity of the organism was confirmed by PCR with primers specific to the plo gene, which encodes the A. pyogenes haemolytic exotoxin pyolysin. In addition, an isolate from each positive stomach was subjected to 16S rRNA gene sequencing and the identification as A. pyogenes was confirmed. These data indicate that A. pyogenes may be resident on the gastric mucosa of pigs.

Actinomycetaceae↗

PubMed2001

Molecular characterization of the pore-forming toxin, pyolysin, a major virulence determinant of Arcanobacterium pyogenes.

Arcanobacterium pyogenes is a common inhabitant and opportunistic pathogen of domestic animals. The pathogenesis of this organism in a range of suppurative diseases is not well understood. However, the development of genetic techniques to study this organism has allowed advances in the analysis of A. pyogenes virulence factors. A major step in this analysis was the identification and cloning of the A. pyogenes hemolytic exotoxin, pyolysin (PLO). PLO is the most divergent member of the cholesterol-binding pore-forming family of toxins. PLO is also divergent in a C-terminal undecapeptide motif which is almost invariant among other members of the family. This divergent undecapeptide motif is required for the full cytolytic activity of PLO and is also responsible for its oxygen-resistant nature. Insertional inactivation of the plo gene results in a significant reduction in virulence in an intraperitoneal mouse model of infection. The virulence of the plo mutant can be restored by providing PLO in trans, suggesting that PLO is a major virulence factor in A. pyogenes pathogenesis in mice. Results of previous vaccination trials with crude antigens against A. pyogenes infection in domestic animals and mice have been equivocal at best. However, a recombinant PLO-based subunit vaccine protected mice from experimental A. pyogenes infection, indicating that PLO is also an important host protective antigen. These results provide promise that the dogma that domestic animals are recalcitrant to vaccination against A. pyogenes infection may prove false.

Actinomyces↗

PubMed2001

Cloning, expression, and characterization of a neuraminidase gene from Arcanobacterium pyogenes.

Arcanobacterium pyogenes is an opportunistic pathogen, associated with suppurative infections in domestic animals. In addition to pyolysin, a pore-forming, cholesterol-binding toxin, A. pyogenes expresses a number of putative virulence factors, including several proteases and neuraminidase activity. A 3,009-bp gene, nanH, was cloned and sequenced and conferred neuraminidase activity on an Escherichia coli host strain. The predicted 107-kDa NanH protein displayed similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. Recombinant His-tagged NanH was found to have pH and temperature optima of 5.5 to 6.0 and 55 degrees C, respectively. Insertional deletion of the nanH gene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidase gene in A. pyogenes. NanH was localized to the A. pyogenes cell wall. A. pyogenes adhered to HeLa, CHO, and MDBK cells in a washing-resistant manner. However, the nanH mutant was not defective for adherence to epithelial cells. The role of NanH in host epithelial cell adherence may be masked by the presence of a second neuraminidase in A. pyogenes.

Actinomycetaceae↗

PubMed2000

Enteritis necroticans (pigbel) in a diabetic child.

BACKGROUND AND METHODS: Enteritis necroticans (pigbel), an often fatal illness characterized by hemorrhagic, inflammatory, or ischemic necrosis of the jejunum, occurs in developing countries but is rare in developed countries, where its occurrence is confined to adults with chronic illnesses. The causative organism of enteritis necroticans is Clostridium perfringens type C, an anaerobic gram-positive bacillus. In December 1998, enteritis necroticans developed in a 12-year-old boy with poorly controlled diabetes mellitus after he consumed pig intestines (chitterlings). He presented with hematemesis, abdominal distention, and severe diabetic ketoacidosis with hypotension. At laparotomy, extensive jejunal necrosis required bowel resection, jejunostomy, and ileostomy. Samples were obtained for histopathological examination. Polymerase-chain-reaction (PCR) assay was performed on paraffin-embedded bowel tissue with primers specific for the cpa and cpb genes, which code for the alpha and beta toxins produced by C. perfringens. RESULTS: Histologic examination of resected bowel tissue showed extensive mucosal necrosis, the formation of pseudomembrane, pneumatosis, and areas of epithelial regeneration that alternated with necrotic segments--findings consistent with a diagnosis of enteritis necroticans. Gram's staining showed large gram-positive bacilli whose features were consistent with those of clostridium species. Through PCR amplification, we detected products of the cpa and cpb genes, which indicated the presence of C. perfringens type C. Assay of ileal tissue obtained during surgery to restore the continuity of the patient's bowel was negative for C. perfringens. CONCLUSIONS: The preparation or consumption of chitterlings by diabetic patients and other chronically ill persons can result in potentially life-threatening infectious complications.

Animals↗

PubMed2000

Thiol-activated cytolysins: structure, function and role in pathogenesis.

Members of the thiol-activated family of cytolysins are involved in the mechanism of pathogenesis of a number of Gram-positive species. While they are pore-forming toxins, their major pathogenic effects may be more subtle than simple lysis of host cells, and may include interference with immune cell function and cytokine induction. Crystal structure, electron microscopy, mutagenesis and antibody binding studies have led to the modeling of a novel mechanism of pore formation, encompassing membrane-binding, membrane insertion and oligomerization. Despite their designation as thiol-activated cytolysins, it is now clear that thiol activation is not an important property of this group of toxins.

Amino Acid Sequence↗

PubMed2000

Occurrence of Clostridium perfringens beta2-toxin amongst animals, determined using genotyping and subtyping PCR assays.

Clostridium perfringens isolates are currently classified into one of five biotypes on the basis of the differential production of alpha-, beta-, epsilon- and iota-toxins. Different biotypes are associated with different diseases of man and animals. In this study a multiple PCR assay was developed to detect the genes encoding these toxins. In addition, detection of the genes encoding the C. perfringens enterotoxin and beta2-toxin allowed subtyping of the bacteria. C. perfringens isolates taken from a variety of animals, including foals, piglets or lambs, were genotyped using this assay. Most of the isolates were found to be genotype A and the gene encoding beta2-toxin [corrected] was present in 50% of the isolates genotyped. A significant association between C. perfringens possessing the beta2-toxin gene and diarrhoea in piglets was identified, suggesting that beta2-toxin may play a key role in the pathogenesis of the disease.

Animals↗

PubMed2000

Clostridium perfringens beta-toxin forms potential-dependent, cation-selective channels in lipid bilayers.

Recombinant beta-toxin from Clostridium perfringens type C was found to increase the conductance of bilayer lipid membranes (BLMs) by inducing channel activity. The channels exhibited a distribution of conductances within the range of 10 to 380 pS, with the majority of the channels falling into two categories of conductance at 110 and 60 pS. The radii of beta-toxin pores found for the conductance states of 110 and 60 pS were 12.7 and 11.1 A, respectively. The single channels and the steady-state currents induced by beta-toxin across the BLMs exhibited ideal monovalent cation selectivity. Addition of divalent cations (Zn(2+), Cd(2+), or Mg(2+)) at a concentration of 2 mM increased the rate of beta-toxin insertion into BLMs and the single-channel conductance, while application of 5 mM Zn(2+) to a beta-toxin-induced steady-state current decreased the inward current by approximately 45%. The mutation of arginine 212 of beta-toxin to aspartate, previously shown to increase the 50% lethal dose of beta-toxin for mice nearly 13-fold, significantly reduced the ability of beta-toxin to form channels. These data support the hypothesis that the lethal action of beta-toxin is based on the formation of cation-selective pores in susceptible cells.

Animals↗

PubMed1999

An Arcanobacterium (Actinomyces) pyogenes mutant deficient in production of the pore-forming cytolysin pyolysin has reduced virulence.

Pyolysin (PLO), the hemolytic exotoxin expressed by Arcanobacterium (Actinomyces) pyogenes, is a member of the thiol-activated cytolysin family of bacterial toxins. Insertional inactivation of the plo gene results in loss of expression of PLO with a concomitant loss in hemolytic activity. The plo mutant, PLO-1, has an approximately 1. 8-log10 reduction in the 50% infectious dose compared to that for wild-type A. pyogenes in a mouse intraperitoneal infection model. Studies involving cochallenge of wild-type and PLO-1 bacteria resulted in recovery of similar numbers of both strains, suggesting that PLO production is required for survival in vivo. Recombinant, His-tagged PLO (His-PLO) is cytotoxic for mouse peritoneal macrophages and J774 cells in a dose-dependent manner. Protection against challenge with A. pyogenes could be afforded by vaccination with formalin-inactivated His-PLO, suggesting that PLO is a host-protective antigen, as well as a virulence determinant.

Actinomyces↗

PubMed1998

Enterotoxigenic Clostridium perfringens type A necrotic enteritis in a foal.

A Thoroughbred-Quarter Horse crossbred foal developed hemorrhagic enteritis and died < 48 hours after birth. Gross and histologic findings were suggestive of Clostridium perfringens type C infection, and large numbers of C perfringens were isolated from intestinal contents. However, genotyping of isolates indicated that they were enterotoxigenic C perfringens type A, and isolates were found to produce C perfringens enterotoxin in vitro. This case suggests that enterotoxigenic C perfringens type A may cause enteric disease in horses.

Animals↗

PubMed1998

Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed.

A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes (cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptose-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blood agar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reagents from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electrophoresis in a 2% agarose gel containing ethidium bromide. The average sensitivity of the assay, determined on artificially contaminated feces, was 9.2 x 10(4) colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were negative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eight-tenths % of isolates with the phenotype of C. perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture positive, while 48.3% were PCR positive for cpa. None of these were cpe positive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringens.

Animal Feed↗

PubMed1998

Clostridium perfringens type E animal enteritis isolates with highly conserved, silent enterotoxin gene sequences.

Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express alpha and iota toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the iota toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries iota toxin genes, to several different type A C. perfringens isolates.

Animals↗

PubMed1998

The Arcanobacterium (Actinomyces) pyogenes plasmid pAP1 is a member of the pIJ101/pJV1 family of rolling circle replication plasmids.

The 2.4-kb plasmid pAP1 from Arcanobacterium (Actinomyces) pyogenes had sequence similarity within the putative replication protein and double-stranded origin with the pIJ101/pJV1 family of plasmids. pJGS84, a derivative of pAP1 containing a kanamycin resistance gene, was able to replicate in Escherichia coli and Corynebacterium pseudotuberculosis, as well as in A. pyogenes. Detection of single-stranded DNA intermediates of pJGS84 replication suggested that this plasmid replicates by the rolling circle mechanism.

Actinomyces↗

PubMed1997

Electroporation-mediated transformation of Arcanobacterium (Actinomyces) pyogenes.

Plasmids derived from pNG2 or RSF1010 were introduced into strains of Arcanobacterium (Actinomyces) pyogenes by electroporation. Electroporation conditions were varied systematically to give a maximum electroporation frequency of 3.7 x 10(5) CFU/microgram DNA at 1.5 kV/cm and 246 omega, resulting in a time constant of approximately 10 ms. The A. pyogenes transformants expressed plasmid-encoded resistance to chloramphenicol, erythromycin, kanamycin, and streptomycin. The source of incoming DNA affected the growth rate of transformants, but not the electroporation efficiency. This is the first report of genetic transformation of the veterinary pathogen A. pyogenes.

Actinomyces↗

PubMed1997

Human disease associated with Clostridium perfringens enterotoxin.

Clostridium perfringens continues to be a common cause of food-borne disease. Characteristics of this organism that contribute to its ability to cause food-borne illness include the formation of heat-resistant spores that survive normal cooking/heating temperatures, a rapid growth rate in warm food, and the production of enterotoxin (CPE) in the human gut. Time and temperature abuse associated with food preparation contributes to the majority of outbreaks of C. perfringens food-borne disease. CPE-induced diarrhea has been reported in the absence of a defined food vehicle. These cases have been typically associated with the elderly and following a course of antibiotic therapy. The incidence of CPE-induced diarrhea may be expected to increase with the growing population of immunocompromised (disease-, treatment-, or age-induced) individuals. Clostridium perfringens has been implicated as a possible contributor to the development of SIDS in susceptible individuals. Specifically, it has been hypothesized that CPE acts as a triggering agent, initiating the events associated with the development of SIDS. Continued refinement of both immunoassays and molecular methods for toxin and gene detection, respectively, will facilitate their eventual availability as commercial kits, providing rapid and simplified methods for the detection of C. perfringens isolates that produce or have the capacity to produce CPE as well as other toxins associated with this organism.

Clostridium Infections↗

PubMed1997

Bacterial phospholipases and their role in virulence.

Virulence of many bacterial pathogens is based, at least in part, on the action of phospholipases. The consequences may be immediate and direct, as in the action of Clostridium perfringens alpha toxin on red cells or platelets, or subtle, as with phosphatidylinositol-specific phospholipases of Listeria monocytogenes and other bacteria.

Animals↗

PubMed1997

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.

Actinomyces↗