Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

Identification of accessory genome regions in poultry Clostridium perfringens isolates carrying the netB plasmid.

Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance.

Clostridium perfringens type C and Clostridium difficile co-infection in foals.

Clostridium perfringens type C is one of the most important agents of enteric disease in newborn foals. Clostridium difficile is now recognized as an important cause of enterocolitis in horses of all ages. While infections by C. perfringens type C or C. difficile are frequently seen, we are not aware of any report describing combined infection by these two microorganisms in foals. We present here five cases of foal enterocolitis associated with C. difficile and C. perfringens type C infection. Five foals between one and seven days of age were submitted for necropsy examination to the California Animal Health and Food Safety Laboratory. The five animals had a clinical history of acute hemorrhagic diarrhea followed by death and none had received antimicrobials or been hospitalized. Postmortem examination revealed hemorrhagic and necrotizing entero-typhlo-colitis. Histologically, the mucosa of the small intestine and colon presented diffuse necrosis and hemorrhage and it was often covered by a pseudomembrane. Thrombosis was observed in submucosal and/or mucosal vessels. Immunohistochemistry of intestinal sections of all foals showed that many large bacilli in the sections were C. perfringens. C. perfringens beta toxin was detected by ELISA in intestinal content of all animals and C. difficile toxin A/B was detected in intestinal content of three animals. C. perfringens (identified as type C by PCR) was isolated from the intestinal content of three foals. C. difficile (typed as A(+)/B(+) by PCR) was isolated from the intestinal content in 3 out of the 5 cases. This report suggests a possible synergism of C. perfringens type C and C. difficile in foal enterocolitis. Because none of the foals had received antibiotic therapy, the predisposing factor, if any, for the C. difficile infection remains undetermined; it is possible that the C. perfringens infection acted as a predisposing factor for C. difficile and/or vice versa. This report also stresses the need to perform a complete diagnostic workup in all cases of foal digestive disease.

TpeL-producing strains of Clostridium perfringens type A are highly virulent for broiler chicks.

Clostridium perfringens type A and type C are causative agents of necrotic enteritis (NE) in poultry. TpeL, a recently-described novel member of the family of large clostridial cytotoxins, was found in C. perfringens type C. Others have since reported TpeL in type A isolates from NE outbreaks, suggesting that it may contribute to the pathogenesis of NE. The virulence of TpeL-positive and -negative C. perfringens strains from cases of NE was examined by challenge of broiler chicks. Gross lesions typical of NE were observed in all challenged birds, and those inoculated with TpeL(pos) strains had higher average macroscopic lesion scores than those inoculated with a TpeL(neg) strain. Infection with TpeL(pos) strains may yield disease with a more rapid course and higher case fatality rate. Thus, TpeL may potentiate the effect of other virulence attributes of NE strains of C. perfringens. However, TpeL(pos) and Tpel(neg) strains compared here were not isogenic, and definitive results await the production and testing of specific TpeL mutants.

The attachment, internalization, and time-dependent, intracellular distribution of Clostridium difficile toxin A in porcine intestinal explants.

Toxin A (TcdA), secreted by toxigenic strains of Clostridium difficile, produces lesions typical of C. difficile-associated disease (CDAD) in susceptible mammal species. Porcine colon explants maintained for 2 hours with TcdA developed severe lesions characterized by cell swelling, swelling of mitochondria and other organelles, distension of cytoplasmic vesicles, expansion of paracellular spaces, apoptosis, and necrosis. Severity of lesions was proportional to the dosage of toxin. No lesions were present in uninoculated control tissues after 2 hours. Receptor-mediated endocytosis is the keystone event in the pathogenesis of the toxin, and susceptibility of a given species is thought to depend on the presence of receptors in intestinal epithelial cells. The fate of TcdA applied to viable colon explants was determined by transmission electron microscopy in an anti-toxin-labeled gold assay. At 5 minutes postinoculation, the presence of TcdA was indicated at the membrane of microvilli or in the cytoplasm of epithelial cells. TcdA was also indirectly observed within endosomes or attached at their margin. A 30-minute inoculation period was associated with many more gold particles labeling structures inside the cell, although some were still attached to microvilli. Within the cell, most TcdA was associated with mitochondria of epithelial cells, but some gold particles decorated the nuclei. Endothelial cells of the lamina propria had evidence of TcdA at both their lumenal and basal aspects, as well as in the cytoplasm and, occasionally, nuclei. Gold particles also labeled the lumen of such vessels as well as leucocytes in blood vessels and the lamina propria.

Clostridium perfringens alpha toxin is produced in the intestines of broiler chicks inoculated with an alpha toxin mutant.

Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo[9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish × Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R = 0.89-0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation.

The distribution and density of Clostridium difficile toxin receptors on the intestinal mucosa of neonatal pigs.

Clostridium difficile is an enteric pathogen affecting a variety of mammals, but it has only recently been diagnosed as a cause of neonatal typhlocolitis in pigs. The most important virulence factors of C. difficile are 2 large exotoxins, toxin A (TcdA) and toxin B (TcdB). TcdA is a potent enterotoxin with effects on host tissues that are dependent upon receptor-mediated endocytosis of the intact toxin. TcdB is an effective cytotoxin, but it apparently does not bind receptors on intact mucosal epithelium. TcdB is much less toxic in vivo unless there is underlying damage to the mucosa, and it is not essential for the virulence of C. difficile. One hypothesis to explain the resistance of most species as neonates (e.g., humans and hamsters) is that they may lack significant numbers of TcdA receptors. The susceptibility of neonatal pigs suggests cells of the gastrointestinal mucosa express sufficient numbers of toxin receptors for lesion development. Immunohistochemical (IHC) assays documented specific binding of TcdA, but not TcdB, to the epithelium of the small and large intestine. The carbohydrate Galalpha1-3beta1-4GlcNAc-R has been described as an important receptor for TcdA. However, IHC indicated a distribution on cell surfaces much different from that of TcdA binding, suggesting a specific interaction of toxin with an alternative receptor.

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.

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.

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.

Enteritis as a cause of mortality in the western bluebird (Sialia mexicana).

Increased mortalities in adult western bluebirds utilizing nestboxes were noted in western Oregon during 1998 and 1999. A necrohemorrhagic enteritis was found in 8 of 10 birds submitted for necropsy. Acanthocephalan parasites were present in four of eight birds with enteritis. Microscopic changes consistent with necrotic or ulcerative enteritis were commonly present. Anaerobic culture of the intestine yielded Clostridium perfringens in three of three birds. Genotype analysis of two of these isolates revealed them to be C. perfringens type A. Bacterial enteritis is believed to be the cause of the increased mortality rate, but further investigation is required to prove a definitive link to a clostridial agent.

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.

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.

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.

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.

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.

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.

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.

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.

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.