Luis G. Arroyo

Clostridium difficile PCR Ribotypes in Calves, Canada

C. difficile, including epidemic PCR ribotypes 017 and 027, were isolated from dairy calves in Canada.

Comparison of the Fecal Microbiota of Healthy Horses and Horses with Colitis by High Throughput Sequencing of the V3-V5 Region of the 16S rRNA Gene

The intestinal tract houses one of the richest and most complex microbial populations on the planet, and plays a critical role in health and a wide range of diseases. Limited studies using new sequencing technologies in horses are available. The objective of this study was to characterize the fecal microbiome of healthy horses and to compare the fecal microbiome of healthy horses to that of horses with undifferentiated colitis. A total of 195,748 sequences obtained from 6 healthy horses and 10 horses affected by undifferentiated colitis were analyzed. Firmicutes predominated (68%) among healthy horses followed by Bacteroidetes (14%) and Proteobacteria (10%). In contrast, Bacteroidetes (40%) was the most abundant phylum among horses with colitis, followed by Firmicutes (30%) and Proteobacteria (18%). Healthy horses had a significantly higher relative abundance of Actinobacteria and Spirochaetes while horses with colitis had significantly more Fusobacteria. Members of the Clostridia class were more abundant in healthy horses. Members of the Lachnospiraceae family were the most frequently shared among healthy individuals. The species richness reported here indicates the complexity of the equine intestinal microbiome. The predominance of Clostridia demonstrates the importance of this group of bacteria in healthy horses. The marked differences in the microbiome between healthy horses and horses with colitis indicate that colitis may be a disease of gut dysbiosis, rather than one that occurs simply through overgrowth of an individual pathogen.

Use of a Selective Enrichment Broth To Recover Clostridium difficile from Stool Swabs Stored under Different Conditions

ABSTRACT The recovery of Clostridium difficile from the stools of patients with C. difficile-associated diarrhea was evaluated by use of an enrichment broth (cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate [TCCFB]) and was compared to that from selective agar (cycloserine-cefoxitin fructose agar [CCFA]) and alcohol shock followed by inoculation onto blood agar (AS-BA). TCCFB was superior to CCFA and AS-BA, and neither the storage time nor the storage temperature affected the recovery rate.

Prevalence and risk factors for Clostridium difficile colonization in dogs and cats hospitalized in an intensive care unit

Clostridium difficile is the most common cause of hospital- and antimicrobial-associated diarrhea in hospitalized humans however the role of C. difficile in diarrhea in dogs has not been defined. A prospective study of C. difficile colonization in dogs and cats was conducted in a veterinary teaching hospital intensive care unit (ICU). Rectal swabs were taken from patients upon admission to the ICU and every third day of hospitalization until discharge or death. C. difficile was isolated from 73/402 (18%) animals; 69% of isolates were toxigenic. Community-associated colonization was identified in 39/366 (11%) of animals that were sampled at the time of admission, while C. difficile was subsequently isolated from 27 of the remaining 327 (8.3%) animals that had a negative admission swab. C. difficile was isolated from seven other dogs during hospitalization, but the origin was unclear because the admission swab was not collected. Administration of antimicrobials prior to admission and administration of immunosuppressive drugs during hospitalization were risk factors for hospital-associated colonization (P=0.006, OR 4.05, 95% CI 1.4-10.8). Acquisition of C. difficile during hospitalization in the ICU was associated with the development of diarrhea (P=0.004). Two ribotypes, one toxigenic and one non-toxigenic, predominated.

Epidemic Clostridium difficile Strain in Hospital Visitation Dog

To the Editor: Rates of illness and death from Clostridium difficile–associated disease (CDAD) and reports of CDAD in persons without traditional risk factors (1) have been increasing. One particular strain of C. difficile has been implicated in outbreaks of CDAD in hospitals in North America and Europe and appears to be spreading internationally at an alarming rate. This strain is classified as ribotype 027, toxinotype III, and possesses genes encoding toxins A, B, and CDT (binary toxin) as well as a deletion in the tcdC gene, which is believed to increase virulence (2). We report this toxin-variant strain of C. difficile in a healthy, 4-year-old toy poodle that visits persons in hospitals and long-term care facilities in Ontario on a weekly basis. C. difficile was isolated from a fecal sample collected in the summer of 2004 as part of a cross-sectional study evaluating pathogen carriage by visitation dogs (3). The isolate was subsequently characterized by ribotyping (4) and by polymerase chain reaction (PCR) detection of genes that encode production of toxins A and B (5). Toxin CDT was confirmed by amplifying the portion of the gene (cdtB) that encodes for the receptor-binding component of the toxin, according to a previously reported protocol (6). As a result, the isolate was classified as ribotype 027, toxinotype III (7), and was found to possess all 3 toxin genes. The tcdC gene deletion was also confirmed with PCR (8). These results indicate that this canine isolate is indistinguishable from the major strain implicated in outbreaks of highly virulent CDAD around the world. According to the infection control practitioner at the hospital the dog visited, CDAD cases were occurring at increased frequency in the facility around the time the dog’s fecal specimen was collected. However, patient diagnosis was made solely through fecal toxin testing, and strains were not characterized. The facility has reported only sporadic cases of CDAD in the past few years. This is the first report of this human, epidemic strain of C. difficile in a dog. Many C. difficile strains isolated from animals, including dogs, are indistinguishable from strains associated with disease in humans (9). To date, no study, including this one, has shown that interspecies transmission occurs; however, that possibility exists, as is becoming apparent with other pathogens, such as methicillin-resistant Staphylococcus aureus. The recurrent exposure of this dog to human healthcare settings suggests that the animal acquired this strain during visits to the hospital or long-term care facility, either from the healthcare environment or contaminated hands of human contacts. We recommend that future studies evaluating the dissemination of this strain and investigations of the movement of C. difficile into the community consider the role of animals.

Changes in the equine fecal microbiota associated with the use of systemic antimicrobial drugs

BackgroundThe intestinal tract is a rich and complex environment and its microbiota has been shown to have an important role in health and disease in the host. Several factors can cause disruption of the normal intestinal microbiota, including antimicrobial therapy, which is an important cause of diarrhea in horses. This study aimed to characterize changes in the fecal bacterial populations of healthy horses associated with the administration of frequently used antimicrobial drugs.ResultsTwenty-four adult mares were assigned to receive procaine penicillin intramuscularly (IM), ceftiofur sodium IM, trimethoprim sulfadiazine (TMS) orally or to a control group. Treatment was given for 5 consecutive days and fecal samples were collected before drug administration (Day 1), at the end of treatment (Days 5), and on Days 14 and 30 of the trial. High throughput sequencing of the V4 region of the 16S rRNA gene was performed using an Illumina MiSeq sequencer. Significant changes of population structure and community membership were observed after the use of all drugs. TMS caused the most marked changes on fecal microbiota even at higher taxonomic levels including a significant decrease of richness and diversity. Those changes were mainly due to a drastic decrease of Verrucomicrobia, specifically the “5 genus incertae sedis”. Changes in structure and membership caused by antimicrobial administration were specific for each drug and may be predictable. Twenty-five days after the end of treatment, bacterial profiles were more similar to pre-treatment patterns indicating a recovery from changes caused by antimicrobial administration, but differences were still evident, especially regarding community membership.ConclusionsThe use of systemic antimicrobials leads to changes in the intestinal microbiota, with different and specific responses to different antimicrobials. All antimicrobials tested here had some impact on the microbiota, but TMS significantly reduced bacterial species richness and diversity and had the greatest apparent impact on population structure, specifically targeting members of the Verrucomicrobia phylum.

Comparison of microbial populations in the small intestine, large intestine and feces of healthy horses using terminal restriction fragment length polymorphism

BackgroundThe composition of the microbiota of the equine intestinal tract is complex. Determining whether the microbial composition of fecal samples is representative of proximal compartments of the digestive tract could greatly simplify future studies. The objectives of this study were to compare the microbial populations of the duodenum, ileum, cecum, colon and rectum (feces) within and between healthy horses, and to determine whether rectal (fecal) samples are representative of proximal segments of the gastrointestinal tract. Intestinal samples were collected from ten euthanized horses. 16S rRNA gene PCR-based TRFLP was used to investigate microbiota richness in various segments of the gastrointestinal tract, and dice similarity indices were calculated to compare the samples.ResultsWithin horses large variations of microbial populations along the gastrointestinal tract were seen. The microbiota in rectal samples was only partially representative of other intestinal compartments. The highest similarity was obtained when feces were compared to the cecum. Large compartmental variations were also seen when microbial populations were compared between six horses with similar dietary and housing management.ConclusionRectal samples were not entirely representative of intestinal compartments in the small or large intestine. This should be taken into account when designing studies using fecal sampling to assess other intestinal compartments. Similarity between horses with similar dietary and husbandry management was also limited, suggesting that parts of the intestinal microbiota were unique to each animal in this study.

Longitudinal study of Clostridium difficile and antimicrobial susceptibility of Escherichia coli in healthy horses in a community setting

Point prevalence studies have reported carriage rates of enteric pathogens in healthy horses, but longitudinal data are lacking. Commensal E. coli is an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data for horses. The objectives of this study were to investigate and molecularly characterize isolates of Clostridium difficile, Clostridium perfringens and Salmonella, collected sequentially over a one year period, and to determine the antibiotic susceptibility profile for E. coli. Fecal samples were collected monthly from 25 adult horses for one year. Selective cultures were performed for all above bacteria. C. difficile isolates were characterized via PCR toxin gene profiling and ribotyping. Broth microdilution was performed to assess antimicrobial susceptibility profiles of E. coli. Toxigenic Clostridium difficile was isolated from 15/275 (5.45%) samples from 10/25 (40%) horses. Four horses were positive at multiple sampling times but different ribotypes were found in three. Ribotypes included 078 (n=6), 001 (n=6) and C (n=3). C. perfringens was not isolated, nor was Salmonella. E. coli was isolated from 232/300 (77%) fecal samples. Resistance to ≥ 1 and ≥ 3 antimicrobials was present in 31/232 (13.4%) and 6/232 (2.6%) respectively. Only two horses shed the same strain of toxigenic C. difficile for more than one month, indicating that shedding is transient. The high number of ribotype 078 is consistent with recent emergence of this strain in the local horse population. The low prevalence of antibiotic resistance in commensal E. coli suggests that healthy horses are not likely a major reservoir of resistance for enteric bacteria.

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

BackgroundClostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status.ResultsToxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, β2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good.ConclusionsClostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.

Effects of unfocused extracorporeal shock wave therapy on healing of wounds of the distal portion of the forelimb in horses

OBJECTIVE To determine effects of extracorporeal shock wave therapy (ESWT) on healing of wounds in the distal portion of the forelimb in horses. ANIMALS 6 horses. PROCEDURES Five 6.25-cm2 superficial wounds were created over both third metacarpi of 6 horses. Forelimbs were randomly assigned to treatment (ESWT and bandage) or control (bandage only) groups. In treated limbs, each wound was treated with 625 shock wave pulses from an unfocused electrohydraulic shock wave generator. In control limbs, each wound received sham treatment. Wound appearance was recorded weekly as inflamed or healthy and scored for the amount of protruding granulation tissue. Standardized digital photographs were used to determine the area of neoepithelialization and absolute wound area. Biopsy was performed on 1 wound on each limb every week for 6 weeks to evaluate epithelialization, fibroplasia, neovascularization, and inflammation. Immunohistochemical staining for A smooth muscle actin was used to label myofibroblasts. RESULTS Control wounds were 1.9 times as likely to appear inflamed, compared with treated wounds. Control wounds had significantly higher scores for exuberant granulation tissue. Treatment did not affect wound size or area of neoepithelialization. No significant difference was found for any of the histologic or immunohistochemical variables between groups. CONCLUSIONS AND CLINICAL RELEVANCE Treatment with ESWT did not accelerate healing of equine distal limb wounds, but treated wounds had less exuberant granulation tissue and appeared healthier than controls. Therefore, ESWT may be useful to prevent exuberant granulation tissue formation and chronic inflammation of such wounds, but further studies are necessary before recommending ESWT for clinical application.