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.

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.

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.

Toxic phospholipases D of Corynebacterium pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticum: cloning and sequence homology.

The genes encoding toxic phospholipases D (PLD) from Corynebacterium pseudotuberculosis (Cp)biovar equi and C. ulcerans (Cu) have been cloned and sequenced. The deduced proteins are 307 amino acids (aa) in length and include a putative signal sequences of 26-aa. A molecular mass of 31.2 and 31.0 kDa and pI values of 8.84 and 6.73 are predicted for the secreted (mature) proteins from Cp and Cu, respectively. Comparison of the deduced primary structure of the two proteins to those of the PLD produced by Cp biovar ovis and Arcanobacterium haemolyticum (Ah) revealed that the four enzymes share 64-97% identity. The aa sequences of this group of proteins were unique when compared to the sequences of other phospholipases in GenBank and were found to share only small regions of homology with other proteins, including two conserved domains of glyceraldehyde-3-phosphate dehydrogenase (G3PD). The similarity of PLD from Cp biovar equi, Cu and Ah to the PLD of Cp biovar ovis suggests that these enzymes may act as virulence determinants.

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.

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.

Corynebacterium pseudotuberculosis: in vitro susceptibility to 39 antimicrobial agents.

The minimal inhibitory concentrations of 39 antimicrobial agents for 54 isolates of Corynebacterium pseudotuberculosis in vitro have been determined. The most active agents were penicillins, macrolides, tetracyclines, cephalosporins, lincomycin, chloramphenicol, and rifampicin. Most isolates were resistant to aminoglycosides, nitrofurans, polymyxins, nalidixic acid, and cycloheximide.

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.

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.

Characterization of detergent-soluble proteins of Corynebacterium pseudotuberculosis.

Whole cells and culture supernatant of isolates of Corynebacterium pseudotuberculosis were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting). SDS-PAGE analysis of detergent-solubilized whole cells revealed more than 20 bands in silver-stained gels. However, the SDS-soluble proteins that are present in all the isolates of the bacterium can be separated into four groups, as follows, (i) high molecular mass proteins that are greater than 119 kDa, (ii) a set of three proteins with molecular mass of 84, 64 and 58 kDa, (iii) a doublet consisting of proteins of molecular mass 33 to 30 kDa, and (iv) low molecular mass proteins of 25 to 20 kDa. SDS-PAGE analysis of ammonium sulphate concentrated culture supernatant demonstrated more than seven bands in silver-stained gels ranging in molecular mass from 64-14 kDa. Sera from goats with C. pseudotuberculosis-induced disease were used to probe immunoblots of electrophoresed SDS-soluble proteins. Ten or more SDS-soluble proteins from whole cells, ranging in molecular mass from 119-20 kDa were recognized by antibodies in sera of naturally infected goats. These sera also recognized up to five molecules ranging from 64-30 kDa, on immunoblots of ammonium sulfate concentrated culture supernatant.

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.

Effect of Corynebacterium pseudotuberculosis phospholipase D on viability and chemotactic responses of ovine neutrophils.

Corynebacterium pseudotuberculosis phospholipase D (PLD) significantly affected viability of ovine neutrophils after 24 hours' exposure. This effect was more marked in cells that ingested PLD emulsified in oil. Treatment of neutrophils with PLD significantly (P < 0.05) reduced the ability of these cells to migrate toward activated sheep serum. The PLD was not chemotactic, but it activated normal sheep serum, producing factors that were chemotactic for neutrophils.

Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens.

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay to detect the genes for the major toxins of Clostridium perfringens (cpa [alpha toxin], cpb [beta toxin], etx [epsilon toxin], iA [iota toxin], and cpe [enterotoxin]). SAMPLE POPULATION: Cultures of C perfringens obtained from collections and diagnosticians throughout North America. PROCEDURE: PCR primers were derived from published sequences of the genes for the major toxins (the "typing" toxins and enterotoxin). The concentration of each primer was titrated in a PCR assay to allow concurrent amplification of multiple target sequences, and other parameters of the assay were optimized (including concentrations of other reagents and times and temperatures for denaturation of template, annealing of primers, and primer extension). Specificity of the assay was measured by comparing genotype with phenotype (where it was known). RESULTS: The genotype, determined by multiplex PCR assay, agreed with phenotype in 99% (86/87) of strains where phenotype had been determined. Applied to 361 isolates from domestic animals and human beings, 95% (n = 344) were type A, and 12.8% (n = 44) of these contained cpe. The remaining 5% (n = 17) of the isolates were type B (n = 1), type C (n = 11), type D (n = 2), or type E (n = 4). CONCLUSION AND CLINICAL RELEVANCE: Previous studies have documented usefulness of PCR in genotyping C perfringens. The multiplex assay is as effective, but simpler, and may be a useful alternative to standard in vivo typing methods. Results of genotyping of field isolates suggested the need for further epidemiologic study of clostridial enteritis, particularly as this pertains to predominant etiologic toxin types, and documented the presence of the reportedly rare genotypes B and E.

Transmission of swine dysentery by carrier pigs.

Swine dysentery (SD) was transmitted to healthy pigs by contact with experimentally-induced carrier pigs. Carrier pigs were produced by exposure of specific pathogen-free (SPF) swine to swine acutely affected with SD. When carrier pigs became acutely affected with SD, they were allowed to recover naturally or were treated with dimetridazole or ronidazole. Recovery was based on disappearance of clinical signs of SD. At a given time after recovery, normal SPF swine were housed with the carriers in a disinfected isolation unit to determine the ability of carriers to transmit SD. In each of 3 experiments, carriers that had recovered and remained asymptomatic for 11 to 25 days transmitted SD to contacts in 14 to 51 days. In 2 experiments, pathogenic Treponema hyodysenteriae was isolated from carriers 12 days before clinical SD was observed in contacts. Carriers which had recovered and remained asymptomatic for 70 and 90 days transmitted SD to contacts in 1 of 3 experiments. Treponema hyodysenteriae was isolated only from contacts with clinical signs of SD. In 4 experiments, carriers that had been treated with nitroimidazole compounds and subsequently recovered for 19 to 44 days failed to transmit SD. Culture of fecal samples on trypticase soy agar with 5% bovine blood and 400 microgram of spectinomycin/ml was helpful in predicting the carrier state, but phase contact contact microscopy of wet fecal smears was not.

Experimentally induced pneumonic pasteurellosis: dose-response relationships and protection against natural reinfection in calves.

Calves were inoculated intranasally with 2 X 10(6.2) tissue culture infective doses of infectious bovine rhinotracheitis virus, followed in 7 days by intratracheal inoculations with 1 of 4 challenge doses of pathogenic Pasteurella haemolytica. Severity and duration of the ensuing clinical signs of respiratory tract disease were correlated with the challenge dose of bacteria. Calves given 1 X 10(6) colony-forming units (CFU) of bacteria did not develop reliable clinical evidence of disease, whereas those given 1 X 10(8) CFU or 1 X 10(10) CFU of bacteria developed clinical signs of pneumonic pasteurellosis within 12 to 24 hours of bacterial challenge. Severity of clinical signs was equal at the 10(8) and 10(10) doses of bacteria, but duration of clinical signs was greater in calves given the 10(10) dose. Calves given 1 X 10(12) CFU of bacteria developed relatively severe respiratory tract disease in excess of what was necessary for positive clinical detection. Positive correlations were found between the bacterial challenge dose and the height and duration of increased rectal temperature, amount and duration of increases in ocular and nasal discharges, and the subjective evaluation of depressed attitude and appetite. Correlations were not found between challenge dose and respiratory rate or character, or between challenge dose and complete blood cell count. Convalescent calves were resistant to naturally occurring pneumonic pasteurellosis, which caused severe disease in nontreated calves. Adverse effects of P haemolytica were not observed after the first 4 to 15 days after bacterial administration; however, the bacteria were isolated from nasal secretions of convalescent calves 89 to 116 days after bacterial inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of Corynebacterium pseudotuberculosis by electroporation.

Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistance plasmid from C diphtheriae. Corynebacterium pseudotuberculosis cultivated in brain-heart infusion broth was washed 3 times with water, and resuspended to a final concentration of about 5 x 10(13) colony-forming units/ml. An electroporator constructed in our laboratory incorporated an electrode with 0.8-mm interelectrode gap, using disposable spectrophotometer cuvettes as containers for electroporation. The pNG2 was prepared in Escherichia coli and 4 to 16 micrograms of pNG2 DNA was mixed with 400-microliters amounts of cell suspension in prechilled cuvettes. After incubation on ice for 5 to 10 minutes, the mixture was electroporated at field strengths of up to 18 kV/cm, mixed with 1.5 ml of brain-heart infusion broth, and incubated at 37 C for 2 hours with agitation. Aliquots were then plated on brain-heart infusion blood agar with 15 micrograms of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of approximately 4 x 10(4) transformants/micrograms of pNG2 DNA. Most total transformants and most transformants per microgram of pNG2 were generated at a field strength of 18 kV/cm. When the concentration of pNG2 DNA was varied, the average total number of transformants increased through a concentration of 30 micrograms/ml, but the efficiency of transformation was highest at the lowest DNA concentration. Transformants contained unmodified pNG2.

Characterization of lectin-binding lymphocytes in goats with caseous lymphadenitis.

Numbers of lymphoid cells bearing markers for peanut agglutinin, wheat germ agglutinin, and anti-immunoglobulin G in healthy goats and in goats with caseous lymphadenitis were determined by use of immunofluorescent techniques. Results indicated that, when compared with healthy goats, diseased goats had normal numbers of anti-immunoglobulin G-positive cells (B cells), had significantly reduced numbers of peanut agglutinin-positive cells (T cells), and had fewer cells with receptor sites for Lens culinaris agglutinin and concanavalin A. Compared with healthy goats, numbers of peanut agglutinin-positive cells in goats with caseous lymphadenitis indicated that the goats may have had a compromised cell-mediated immunity.