Yajarayma J. Tang

Apparent Outbreaks of Clostridium Difficile-Associated Diarrhea in Horses in a Veterinary Medical Teaching Hospital

Intestinal colonization with toxigenic strains of Clostridium difficile was documented in 9 of 10 horses with acute onset diarrhea in a veterinary medical teaching hospital, whereas a similar isolate was detected in only 1 of 23 other horses without diarrhea in the hospital. One horse with diarrhea was infected simultaneously with both C. difficile and Salmonella krefeld. Clostridium difficile was detected by fecal culture on selective medium, confirmed with a latex particle agglutination test, and identified as toxigenic by polymerase chain reaction amplification of toxin A and toxin B gene sequences. Using an arbitrarily-primed polymerase chain reaction, 6 distinct C. difficile isolates were detected in the feces of the 9 affected horses at the time of the outbreak of diarrhea.

Resistance to moxifloxacin in toxigenic Clostridium difficile isolates is associated with mutations in gyrA.

ABSTRACT Clostridium difficile is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea. Fluoroquinolones such as ciprofloxacin are associated with lower risks of C. difficile-associated diarrhea. In this study, we have analyzed 72C. difficile isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses. They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN). Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF. The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (gyrA), the production of toxins A and B, and the epidemiological relationship of these isolates. These factors were also investigated using PCR-based methods. All strains tested were susceptible to MEO and VAN. Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC ≥ 16 μg/ml). Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the gyrAprotein. Resistant strains selected in vitro did not contain mutations at that position. These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in thegyrA gene.

Fecal shedding of Clostridium difficile in dogs: a period prevalence survey in a veterinary medical teaching hospital

The goal of this study was to determine the fecal prevalence of Clostridium difficile in dogs who were patients at a veterinary medical teaching hospital. Stool specimens collected from 152 dogs (in- and outpatients) were analyzed for the presence of C. difficile. An additional 42 stool specimens were collected and examined from dogs recently housed at local animal shelters. Following culture on selective medium, C. difficile was identified by a latex agglutination test, and the presence of the toxin A and B genes was determined individually by polymerase chain reaction. Clostridium difficile was isolated from the feces of 28 of the veterinary hospital patients (18.4%); isolates from 14 of these patients (50.0%) were toxigenic. Diarrhea was a clinical finding in 5 (35.7%) of the dogs carrying toxigenic isolates of C. difficile, whereas diarrhea was noted in only 2 of 14 dogs (14.3%) shedding nontoxigenic isolates. Three of 14 dogs (2 1.4%) shedding toxigenic isolates of C. difficile were receiving antibiotics at the time of stool collection, whereas 5 of 14 dogs (37.5%) shedding non-toxigenic strains of C. difficile were receiving antibiotics. The carriage rate of C. difficile was significantly higher for animals categorized as inpatients of the veterinary hospital. The carriage rate also provided evidence for an increased risk for fecal shedding with increasing age. Clostridium difficile was not isolated from any of the 42 dogs recently housed at local animal shelters. This study confirms the presence of toxigenic C. difficile in dogs at a veterinary teaching hospital. Additional studies will be required to determine whether prior antibiotic treatment increases the frequency of C. difficile fecal shedding from dogs and whether colonized dogs are a risk for transmission of the organism to susceptible human populations.

Isolation of a Toxin B-Deficient Mutant Strain of Clostridium difficile in a Case of Recurrent C. difficile-Associated Diarrhea

Clostridium difficile-associated diarrhea (CDAD) recurs in approximately 15%-20% of patients after discontinuation of metronidazole or vancomycin therapy. Most recurrences are believed to be endogenous relapses due to the persistence of spores. However, there is evidence that reinfection with a different strain is a cause of recurrence. We report the case of a patient with a history of multiple episodes of C. difficile colitis. The patient, a 56-year-old female, has had 5 years of repeated recurrences, each shortly after discontinuing vancomycin therapy. During the course of these episodes, three isolates were cultured from her stools at different times. These isolates were analyzed for the presence of toxin A and B gene sequences and genotyped by means of arbitrarily primed polymerase chain reaction (AP-PCR). The original two isolates contained the toxin A and B genes, as determined by PCR, and were of the same AP-PCR type. During her last relapse, a C. difficile strain lacking at least a portion of the toxin B gene was isolated. AP-PCR analysis of this isolate showed a different DNA banding pattern from that of the previous isolates. A vancomycin susceptibility assay revealed a slight decrease in vancomycin activity as compared with that against the prior isolate. This case demonstrates two unique features: (1) recurrent infections can be due to reinfections and (2) toxin B mutants can possibly cause CDAD. This study also raises concerns about long-term vancomycin use and the development of resistance of C. difficile to vancomycin.

Molecular typing methods for the epidemiological identification of Clostridium difficile strains

Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhea (CDAD), the most common cause of nosocomial diarrhea. Cross-infection between patients and transmission through the environment and medical personnel are important factors in the acquisition of CDAD. In order to understand differences in epidemiology and pathogenesis, a number of typing schemes have been developed. We will review the typing methods used to study the epidemiology of C. difficile infections and how they have evolved from a phenotypic identification to state of the art molecular methods, detecting genetic polymorphisms among strains. These molecular methods include PCR-based methods (arbitrarily primed-PCR [AP-PCR] and PCR ribotyping), restriction endonuclease analysis (REA) and pulse field gel electrophoresis (PFGE). The application, usefulness and feasibility of these methods are compared and discussed. Finally, the role of genomics as a tool to investigate CDAD is introduced.

Persistence of an Endemic (Toxigenic) Isolate of Clostridium difficile in the Environment of a General Medicine Ward

The epidemiology of Clostridium difficile-associated diarrhea (CDAD) in an endemic setting was investigated by use of DNA typing methods to determine the strain identity of C. difficile isolates. Two predominant toxigenic clones were found in the environment and accounted for 29.8% (type 1) and 15.5% (type 2) of CDAD cases, respectively. In endemic settings, the environment and cross-transmission may play a role in acquisition of CDAD.

Genotyping of Bacteroides fragilis isolates from stool specimens by arbitrarily-primed-PCR

In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a distinct and unique DNA banding pattern indicating a high degree of genotypic variability. Eleven types were identified among the foal isolates. Type 20, a nonenterotoxigenic type, was present in 30% of the foals. No correlation was found between the human and horse isolates. No clear relationship between a disease state (diarrhea or IBD) and specific types was observed. AP-PCR will be useful as a rapid method to determine genetic relatedness and in future epidemiologic studies of diarrheal diseases due to B. fragilis.

Isolation of Clostridium innocuum from cases of recurrent diarrhea in patients with prior Clostridium difficile associated diarrhea

Clostridium innocuum isolates resistant to vancomycin (MIC values of 16-24 microg/mL) were isolated from three patients with recurrent Clostridium difficile -associated diarrhea (CDAD). We discuss the clinical significance and problems associated with the identification and differentiation of these two clostridial species, which may result in misdiagnosis of patients.

Genotyping of Clostridium difficile isolates from a hospital in Warsaw: A preliminary study

Abstract Background: Toxigenic Clostridium difficile is the etiologic agent of C. difficile -associated diarrhea. It is believed that patient-to-patient transmission and environmental contamination are risk factors for the spread of this disease in hospitals. The development of precise typing schemes has provided some understanding of the epidemiology of this nosocomial infection. Objective: The purpose of this study was to identify strain types of C. difficile present in the environment of a maternity ward and surgical wards of a hospital in Warsaw, and to correlate these types with those isolated from neonates and surgical patients. Methods: Arbitrarily primed polymerase chain reaction (APPCR), with the arbitrary primer PG-05, was used to genotype 14 isolates of C. difficile obtained from different sources in a hospital in Warsaw. These included clinical isolates from neonates and surgical patients, as well as environmental isolates from the maternity and surgical wards. Results: A predominant toxigenic isolate was found in the maternity environment, and this strain type was also isolated from the stools of two of five newborns. Unique and different DNA banding patterns were observed in the remaining three isolates from neonates. In addition, isolates from an infant with diarrhea also showed a unique AP-PCR type. The isolates from three surgical patients analyzed had the same AP-PCR profile. This type was also found in the surgery ward environment. Conclusion: These data support environmental contamination as an important factor in the etiology of C. difficile -associated diarrhea in this hospital.

Polymerase chain reaction for identification and typing of Clostridium difficile isolated from Chinese patients

Abstract Objective: Toxigenic Clostridium difficile , the etiologic agent of C. difficile -associated diarrhea (CDAD), is the most common cause of hospital-acquired diarrhea in many developed countries. In spite of the endemic nature of diarrhea in China, few reports of C. difficile infection in this country have been documented in the literature. Methods: In this study, a polymerase chain reaction (PCR) assay was used to determine the presence of toxigenic C. difficile in the stools of 19 patients who developed diarrhea after antibiotic use in a hospital in China. Clostridium difficile was also cultured from the stool of these patients. After DNA extraction, PCR was performed with primers targeting C. difficile toxin A and toxin B gene sequences. Results: Toxin sequences were detected in 5 of 19 (26%) specimens analyzed. To determine strain identity, these isolates were genotyped using arbitrarily primed PCR (AP-PCR). Three APPCR profiles were identified among these isolates. Three of these isolates had the same DNA banding pattern, and they were isolated from patients who were at the same institution. Conclusions: These preliminary results indicate that C. difficile may be involved in some diarrhea cases in China. This study may aid in the understanding of the nature of endemic diarrhea in this country.