Hien T. Trinh

Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of genotype with phenotype

Beta2 toxin, encoded by the cpb2 gene, has been implicated in the pathogenesis of porcine, equine and bovine enteritis by type A Clostridium perfringens. By incorporating primers to cpb2 into a multiplex genotyping PCR, we screened 3270 field isolates of C. perfringens. Of these, 37.2% were PCR positive for the cpb2 gene. The majority of isolates from cases of porcine enteritis were positive for cpb2 (>85%), and this was even more true for C. perfringens isolated from cases of porcine neonatal enteritis (91.8%). In contrast, isolates from normal pigs only contained cpb2 in 11.1% of cases. The correlation between enteritis in other animal species and the presence of cpb2 was not so strong. cpb2 was found in 21.4% of C. perfringens isolates from cattle with enteritis, and in 47.3% of isolates from calves with enteritis or abomastitis. The prevalence of cpb2 varied with genotype, with type A isolates being positive for this gene in 35.1% of cases. Furthermore, enterotoxigenic type D or type E strains almost always carried cpb2. We cloned a 6xHIS-tagged beta2 (HIS-beta2) and used this protein to raise antiserum against beta2. Culture supernatants from 68 cpb2-positive and 13 cpb2-negative strains were tested for the presence of beta2 by Western blotting. In cpb2-positive isolates of porcine origin, beta2 was almost always detected (96.9%). However, in cpb2-positive isolates from other animal species, only 50.0% expressed beta2 protein. The high rate of cpb2-positivity among strains from neonatal pigs with enteritis and the high correlation of genotype with phenotype, supports the contention that beta2 toxin plays a role in the pathogenesis of these infections. However, it may be important to consider the use of an additional method for the detection of beta2 toxin in non-porcine cpb2-positive isolates when making claims about the role of beta2 in enteritis in non-porcine species.

A possible role for Clostridium difficile in the etiology of calf enteritis.

Abstract Clostridium difficile was investigated as a possible cause of enteritis in calves. The organism and its toxins (TcdA and TcdB), respectively, were found in 25.3% and 22.9% of stool samples from diarrheic calves. Culture positive samples were more likely than culture negative samples to be toxin positive. However, toxin positive stools were more common among nondiarrheic calves, but diarrheic calves were nearly twice as likely to be culture positive. Ribotype 078 was dominant among isolates. Salmonella sp. was isolated from both diarrheic and nondiarrheic calves, but large numbers of E. coli were found more commonly in diarrheic calves than in nondiarrheic animals. Prevalence rates for coronavirus and Cryptosporidium sp. were substantially higher in nondiarrheic calves than in diarrheic, but rates of detection of rotavirus and Giardia sp. were more nearly equal between groups. Lesions in naturally infected calves included superficial mucosal erosion with associated fibrinous exudates. Neutrophils and eosinophils infiltrated lamina propria. Large Gram-positive rods morphologically compatible with C. difficile were abundant in the colonic lumen and the organism was isolated by bacteriologic culture. Toxins were found throughout the colon. Purified toxins A and B (individually and conjointly) caused comparable lesions, as well as fluid accumulation, in ligated intestinal loops. Our findings are in substantial agreement with those of others [Rodriguez-Palacios, A., Stampfli, H.R., Duffield, T., Peregrine, A.S., Trotz-Williams, L.A., Arroyo, L.G., Brazier, J.S., Weese, J.S., 2006. Clostridium difficile PCR ribotypes in calves, Canada. Emerg. Infect. Dis. 12, 1730–1736; Porter, M.C., Reggiardo, C., Bueschel, D.M., Keel, M.K., Songer, J.G., 2002. Association of Clostridium difficile with bovine neonatal diarrhea. Proc. 45th Ann. Mtg. Amer. Assoc. Vet. Lab. Diagn., St. Louis, MO, U.S.A.] and add strength to a working hypothesis that C. difficile infection and the accompanying intoxication can manifest as diarrhea in calves. It seems clear that calves serve as multiplying hosts for this organism.

Atypical cpb2 Genes, Encoding Beta2-Toxin in Clostridium perfringens Isolates of Nonporcine Origin

ABSTRACT Beta2-toxin, encoded by cpb2, is implicated in the pathogenesis of Clostridium perfringens enteritis. However, cpb2 genes from nonporcine C. perfringens isolates were not always expressed, at least in vitro. Nucleotide sequencing identified atypical cpb2 genes with 70.2 to 70.7% DNA identity to previously identified (consensus) cpb2. Atypical beta2-toxin displayed 62.3% identity and 80.4% similarity to consensus beta2-toxin. No porcine type C isolates (n = 16) and only 3.3% of porcine type A isolates (n = 60) carried atypical cpb2 genes. However, 88.5% of nonporcine isolates carried atypical cpb2 (n = 78), but beta2-toxin was not expressed. Almost half of the nonporcine consensus cpb2 genes (44.4%) carried a frameshift mutation (n = 9), resulting in an absence of beta2-toxin expression. These findings strengthen the role of beta2-toxin in the pathogenesis of enteritis in neonatal pigs. However, the identification of apparently nonexpressed, atypical cpb2 genes raises the question of whether this protein plays the same role in enteritis in other animal species.

Immunization with recombinant alpha toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens.

Necrotic enteritis (NE) in poultry has re-emerged as a concern for poultry producers, due in part to banning, by many countries, of the use of antimicrobial growth promoters in feeds. This re-emergence has led to a search for alternative methods for control of the disease, particularly vaccination. The objective of this work was to determine if vaccination of broiler chicks with recombinant alpha toxin protected against experimental challenge. Broiler chicks were vaccinated subcutaneously at 5 and 15 days of age, followed 10 days later by challenge with Clostridium perfringens. Birds were challenged twice daily on 4 consecutive days by mixing C. perfringens cultures with feed (three parts culture: four parts feed). Non-vaccinated birds challenged with C. perfringens developed NE at the rate of 87.8%, while only 54.9% of vaccinated birds developed lesions. In addition, non-vaccinated birds had lesion scores averaging 2.37, while average scores in vaccinated birds were 1.35. Vaccination produced an antibody response, with post-vaccination anti-alpha toxin IgG (IgY) titers in vaccinated birds more than 5-fold greater than in non-vaccinated birds. After challenge, vaccinated birds had average IgG (IgY) titers>15-fold higher than those in non-vaccinated birds. These results suggest that alpha toxin may serve as an effective immunogen, and, as such, may play a role in pathogenesis.

Tylosin Resistance in Arcanobacterium pyogenes Is Encoded by an Erm X Determinant

ABSTRACT Arcanobacterium pyogenes, a commensal on the mucous membranes of many economically important animal species, is also a pathogen, causing abscesses of the skin, joints, and visceral organs as well as mastitis and abortion. In food animals, A. pyogenes is exposed to antimicrobial agents used for growth promotion, prophylaxis, and therapy, notably tylosin, a macrolide antibiotic used extensively for the prevention of liver abscessation in feedlot cattle in the United States. Of 48 A. pyogenes isolates, 11 (22.9%) exhibited inducible or constitutive resistance to tylosin (MIC of ≥128 μg/ml). These isolates also exhibited resistance to other macrolide and lincosamide antibiotics, suggesting a macrolide-lincosamide resistance phenotype. Of the 11 resistant isolates, genomic DNA from nine hybridized to an erm(X)-specific probe. Cloning and nucleotide sequencing of the A. pyogenes erm(X) gene indicated that it was >95% similar to erm(X) genes from Corynebacterium and Propionibacterium spp. Eight of the erm(X)-containing A. pyogenes isolates exhibited inducible tylosin resistance, which was consistent with the presence of a putative leader peptide upstream of the erm(X) open reading frame. For at least one A. pyogenes isolate, 98-4277-2, erm(X) was present on a plasmid, pAP2, and was associated with the insertion sequence IS6100. pAP2 also carried genes encoding the repressor-regulated tetracycline efflux system determinant Tet 33. The repA gene from pAP2 was nonfunctional in Escherichia coli and at least one A. pyogenes isolate, suggesting that there may be host-encoded factors required for replication of this plasmid.

Equine Colitis X Associated with Infection by Clostridium Difficile NAP1/027

A 14-year-old Quarter Horse with a 48-hr history of colic was euthanized after failure to respond to treatment. At necropsy, cecal and colonic mucosae were congested throughout, and there was segmental edema and significant thickening of the intestinal wall. Excessive numbers of mononuclear cells were found in mucosal lamina propria. Submucosal hemorrhage was diffuse and extensive, and Clostridium difficile toxins A and B were detected. Large numbers of C. difficile were isolated, and genetic characterization revealed them to be North American pulsed-field gel electrophoresis type 1, polymerase chain reaction ribotype 027, and toxinotype III. Genes for the binary toxin were present, and toxin negative–regulator tcdC contained an 18-bp deletion. This genotype comprises the current human “epidemic strain,” which is associated with human C. difficile–associated disease of greater than historical severity. The diagnosis was peracute typhlocolitis, with lesions and history typical of those attributed to colitis X.

Susceptibility of Arcanobacterium pyogenes from different sources to tetracycline, macrolide and lincosamide antimicrobial agents

Chlortetracycline, oxytetracycline, and the macrolide, tylosin, are extensively used for growth promotion and disease prophylaxis in the cattle and swine industries in the US. Arcanobacterium pyogenes, a common inhabitant of the mucosal surfaces of cattle and swine, is also a pathogen associated with a variety of infections in these animals. A broth microdilution technique was used to determine the antimicrobial susceptibility of 48 A. pyogenes isolates to macrolides, lincosamides and tetracyclines. The MIC50 and MIC90 for chlortetracycline were 0.12 and 8 mg/l, respectively. Similarly, the MIC50 and MIC90 for oxytetracycline were 0.25 and 8 mg/l, while the MIC50 and MIC90 for tetracycline were 0.25 and 16 mg/l, respectively. The MIC50 and the MIC90 were < or = 0.06 and >64 mg/l, respectively, for erythromycin, tylosin and clindamycin. This resistance pattern indicated that some of these A. pyogenes isolates may carry an MLS(B) resistance determinant. A. pyogenes isolates (12.5%) were resistant to erythromycin, and this percentage doubled when MICs were performed following induction with erythromycin. Of the 48 A. pyogenes isolates, 25 and 41.7% were resistant to MLS(B) antimicrobial agents and the tetracycline derivatives, respectively. MLS(B) resistance was present in 22.2 and 35.3% of A. pyogenes isolates of bovine (n=27) or porcine (n=17) origin. In contrast, 70.6% of porcine isolates were resistant to the tetracyclines, compared with 25.9% of bovine isolates. These data suggest that a large proportion of A. pyogenes field isolates may be resistant to these commonly used antimicrobial agents.

Evaluation of VT-1161 for Treatment of Coccidioidomycosis in Murine Infection Models

ABSTRACT Coccidioidomycosis, or valley fever, is a growing health concern endemic to the southwestern United States. Safer, more effective, and more easily administered drugs are needed especially for severe, chronic, or unresponsive infections. The novel fungal CYP51 inhibitor VT-1161 demonstrated in vitro antifungal activity, with MIC50 and MIC90 values of 1 and 2 μg/ml, respectively, against 52 Coccidioides clinical isolates. In the initial animal study, oral doses of 10 and 50 mg/kg VT-1161 significantly reduced fungal burdens and increased survival time in a lethal respiratory model in comparison with treatment with a placebo (P < 0.001). Oral doses of 25 and 50 mg/kg VT-1161 were similarly efficacious in the murine central nervous system (CNS) model compared to placebo treatment (P < 0.001). All comparisons with the positive-control drug, fluconazole at 50 mg/kg per day, demonstrated either statistical equivalence or superiority of VT-1161. VT-1161 treatment also prevented dissemination of infection from the original inoculation site to a greater extent than fluconazole. Many of these in vivo results can be explained by the long half-life of VT-1161 leading to sustained high plasma levels. Thus, the efficacy and pharmacokinetics of VT-1161 are attractive characteristics for long-term treatment of this serious fungal infection.

A Second Tylosin Resistance Determinant, Erm B, in Arcanobacterium pyogenes

ABSTRACT Arcanobacterium pyogenes, a common inhabitant of the mucosal surfaces of livestock, is also a pathogen associated with a variety of infections. In livestock, A. pyogenes is exposed to antimicrobial agents used for prophylaxis and therapy, notably tylosin, a macrolide used extensively for the prevention of liver abscessation in feedlot cattle in the United States. Many, but not all, tylosin-resistant A. pyogenes isolates carry erm(X), suggesting the presence of other determinants of tylosin resistance. Oligonucleotide primers designed for conserved regions of erm(B), erm(C), and erm(T) were used to amplify a 404-bp fragment from a tylosin-resistant A. pyogenes isolate, OX-7. DNA sequencing revealed that the PCR product was 100% identical to erm(B) genes, and the erm(B) gene region was cloned in Escherichia coli. The A. pyogenes Erm B determinant had the most DNA identity with an Erm B determinant carried by the Clostridium perfringens plasmid pIP402. However, the A. pyogenes determinant lacked direct repeat DR1 and contained a deletion in DR2. Flanking the A. pyogenes erm(B) gene were partial and entire genes similar to those found on the Enterococcus faecalis multiresistance plasmid pRE25. This novel architecture suggests that the erm(B) element may have arisen by recombination of two distinct genetic elements. Ten of 32 tylosin-resistant isolates carried erm(B), as determined by DNA hybridization, and all 10 isolates carried a similar element. Insertion of the element was site specific, as PCR and Southern blotting analysis revealed that the erm(B) element was inserted into orfY, a gene of unknown function. However, in three strains, this insertion resulted in a partial duplication of orfY.

Modeling Nikkomycin Z Dosing and Pharmacology in Murine Pulmonary Coccidioidomycosis Preparatory to Phase 2 Clinical Trials

Nikkomycin Z (NikZ) is a chitin synthase inhibitor with activity against Coccidioides species that is being developed as a first-in-class orphan product for treatment of coccidioidomycosis. It has previously been shown to reduce lethal respiratory infections in mice to undetectable levels when treatment is begun 48 hours after infection. The studies described here focus on bracketing NikZ doses for phase 2 and 3 clinical trials, using an established mouse respiratory infection as a model and starting treatment 120 hours after infection. A dose of 80 mg/kg/day, divided into 2 doses, nearly eradicated infection, and larger doses did not improve fungal clearance. Increasing the duration of treatment from 1 week to 3 weeks resulted in a greater percentage of culture-negative mice. Comparative data show that plasma levels of NikZ that nearly eradicate Coccidioides in mice are achievable in patients and provide a plausibly effective dose range for initial phase 2 clinical studies.