I. Sanders

Whole genome sequencing reveals potential spread of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011

Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.

Comparison of real-time PCR techniques to cytotoxigenic culture methods for diagnosing Clostridium difficile infection.

ABSTRACT In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027.

Epidemiology of Clostridium difficile infections in a tertiary-care hospital in Korea.

To survey healthcare-associated Clostridium difficile infection (HA-CDI) in a 900-bed tertiary-care hospital, we prospectively investigated the epidemiology of CDI and distribution of PCR-ribotypes. From February 2009 through January 2010, all patients with HA-CDI were enrolled. Epidemiological information and prescription records for antibiotics were collected. The C. difficile isolates were characterized using reference strains and were tested for antibiotic susceptibility. During the survey, incidence of HA-CDI was 71.6 per 100 000 patient-days. In total, 140 C. difficile isolates were obtained from 166 patients with HA-CDI. The PCR-ribotyping yielded 38 distinct ribotypes. The three most frequently found ribotypes made up 56.4% of all isolates; they comprised 37 isolates (26.4%) of PCR-ribotype 018, 22 (15.7%) of toxin A-negative PCR-ribotype 017, and 20 (14.3%) of PCR-ribotype 001. Clostridium difficile PCR-ribotype 018 was present in all departments throughout the hospital during the 11 months, whereas ribotype 017 and ribotype 001 appeared mostly in the pulmonary department. Hypervirulent C. difficile PCR-ribotype 027 was detected in 1 month on two wards. The incidence of CDI in each department showed a seven-fold difference, which correlated significantly with the amount of prescribed clindamycin (R = 0.783, p 0.013) or moxifloxacin (R = 0.733, p 0.025) in the departments. The rates of resistance of the three commonest ribotypes to clindamycin and moxifloxacin were significantly higher than those of other strains (92.1% versus 38.2% and 89.5% versus 27.3%, respectively). CDI is an important nosocomially acquired infection and this study emphasizes the importance of implementing country-wide surveillance to detect and control CDI in Korea.

Antimicrobial activity of LFF571 and three treatment agents against Clostridium difficile isolates collected for a pan-European survey in 2008: clinical and therapeutic implications

OBJECTIVES In November 2008, a study was performed with support from the European Centre for Disease Prevention and Control (ECDC) to obtain an overview of Clostridium difficile infections (CDIs) in European hospitals. A collection of 398 C. difficile isolates obtained from this hospital-based survey was utilized to identify antimicrobial susceptibility patterns of common C. difficile PCR ribotypes across Europe. METHODS The MICs of three approved therapeutic agents (vancomycin, metronidazole and fidaxomicin) and LFF571 (a novel semi-synthetic thiopeptide antibiotic) were determined by the agar dilution method. RESULTS MICs of fidaxomicin and LFF571 were in general 2-4-fold lower than those of vancomycin and metronidazole. Isolates belonging to clade 2, including the hypervirulent ribotype 027, had one-dilution higher MIC50 and MIC90 values for fidaxomicin and metronidazole, whereas similar MIC values were observed for vancomycin and LFF571. Isolates belonging to C. difficile PCR ribotype 001 were more susceptible to fidaxomicin than other frequently found PCR ribotypes 014/020 and 078. Six isolates from three different countries had a metronidazole MIC of 2 mg/L. Four of the six isolates were characterized as PCR ribotype 001. CONCLUSIONS There was no evidence of in vitro resistance of C. difficile to any of the four agents tested. However, the results suggest type-specific differences in susceptibility for the treatment agents we investigated. Continuous surveillance of C. difficile isolates in Europe is needed to determine the possible clinical implications of ribotype-specific changes in susceptibility to therapeutic agents.

Detection of Clostridium difficile in feces of asymptomatic patients admitted to the hospital

ABSTRACT Recent evidence shows that patients asymptomatically colonized with Clostridium difficile may contribute to the transmission of C. difficile in health care facilities. Additionally, these patients may have a higher risk of developing C. difficile infection. The aim of this study was to compare a commercially available PCR directed to both toxin A and B (artusC. difficile QS-RGQ kit CE; Qiagen), an enzyme-linked fluorescent assay to glutamate dehydrogenase (GDH ELFA) (Vidas, bioMérieux), and an in-house-developed PCR to tcdB, with (toxigenic) culture of C. difficile as the gold standard to detect asymptomatic colonization. Test performances were evaluated in a collection of 765 stool samples obtained from asymptomatic patients at admission to the hospital. The C. difficile prevalence in this collection was 5.1%, and 3.1% contained toxigenic C. difficile. Compared to C. difficile culture, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the C. difficile GDH ELFA were 87.2%, 91.2%, 34.7%, and 99.3%, respectively. Compared with results of toxigenic culture, the sensitivity, specificity, PPV, and NPV of the commercially available PCR and the in-house PCR were 95.8%, 93.4%, 31.9%, 99.9%, and 87.5%, 98.8%, 70%, and 99.6%, respectively. We conclude that in a low-prevalence setting of asymptomatically colonized patients, both GDH ELFA and a nucleic acid amplification test can be applied as a first screening test, as they both display a high NPV. However, the low PPV of the tests hinders the use of these assays as stand-alone tests.

Toxigenic Clostridium difficile PCR ribotypes in edible marine bivalve molluscs in Italy

Even though food of animal sources and different foodstuffs are well known to be potentially carrier of Clostridium difficile, few data are available on the occurrence of C. difficile in seafood. This work investigated the occurrence of C. difficile in edible bivalve molluscs in southern Italy. Out of the 925 investigated samples, 3.9% contained C. difficile. Eighteen strains harboured both genes for toxins A and B whereas 1 only had toxin B gene. Binary toxin genes were found in 22.2% of the isolates. The most frequently ribotypes found were 078/126 (22.2%), 010 (19.4%), and 001 (8.3%). All isolates were susceptible to metronidazole, vancomycin, fidaxomicin, and to the new semisynthetic thiopeptide antibiotic LFF571, whereas 19.4% of them were resistant to moxifloxacin, 30.5% to clindamycin, 38.8% to erythromycin, and 100% to ciprofloxacin. This study points out that edible molluscs could be a potential source of toxigenic C. difficile ribotypes and a potential risk for human health.

High occurrence of various Clostridium difficile PCR ribotypes in pigs arriving at the slaughterhouse

Background: Clostridium difficile is recognized as an important cause of nosocomial diarrhoea in humans, especially in association with the administration of antibiotics. Furthermore, C. difficile can not only cause neonatal enteritis in pigs but can also be found in pigs without any clinical disease symptoms. Clostridium difficile had been found on pork samples destined for human consumption. However, little is known about the risk of food-borne transmission. Objective: To elaborate the risk of food-borne transmission of C. difficile via pigs. Animals and methods: The occurrence of C. difficile was assessed in pigs arriving at a slaughterhouse in the Netherlands. Rectal faecal samples from 50 pigs originating from 10 different farms were taken just after the pigs were stunned and bled. These samples were examined using a real-time PCR (BD GeneOhm™ Cdiff Assay) combined with culturing following enrichment. Results: Using real-time PCR, none of the faecal samples were found positive for C. difficile while after culturing following enrichment, 14 out of 50 samples (28%) contained C. difficile. The positive samples were derived from nine different farms and encompassed seven different PCR ribotypes (015 predominant). All isolated C. difficile strains were positive for the toxin A and B genes. Conclusion: These results indicate that C. difficile can be found in faecal samples obtained from pigs after they were stunned and bled in a slaughterhouse. Clinical importance: The potential risk of these findings on food-borne transmission via pigs and associated impact on human health cannot be excluded and needs further study.

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

ABSTRACT 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.

Ribotype 078 Clostridium difficile infection incidence in Dutch hospitals is not associated with provincial pig farming: Results from a national sentinel surveillance, 2009-2015.

Background It has been suggested that the high incidence of ribotype 078 Clostridium difficile infections (CDI) in the Netherlands is related to pig farming. Methods We used data of hospitalised CDI patients (>2yrs of age) diagnosed between May 2009 and May 2015 in 26 hospitals participating in a national sentinel surveillance. We compared clinical and geographical characteristics of 078 CDI to other CDI. We investigated the association between 078 CDI incidence and four indicators of pig farming (piglet, pig, piglet farm and pig farm density) by mixed-effects Poisson regression. We used a space-time permutation model to search for community-onset 078 CDI clusters (using SaTScan). Results A total of 4,691 CDI were identified. Ribotype 078 was isolated in 493 of 3,756 patients (13.1%) including a typing result. These patients had slightly higher community-onset disease and a 35% increase of 30-day mortality compared to non-078 CDI patients. The pooled overall and 078 incidence rates were 2.82 (95% CI, 2.42–3.29) and 0.26 (95% CI, 0.21–0.31) CDI per 10,000 patients-days respectively. Hospital 078 CDI incidence was not associated with provincial pig (IRR, 0.98; 95% CI, 0.89–1.08), piglet (IRR, 0.95; 95% CI, 0.75–1.19), pig farm (IRR, 1.08; 95% CI, 0.84–1.39), or piglet farm density (IRR, 1.00; 95% CI, 0.56–1.79). No clusters of community-onset ribotype 078 CDI were found. Conclusions Our results do not indicate that the ribotype 078 CDI incidence in hospitals is related to pig (farm) or piglet (farm) density. However, transmission beyond provincial borders or in non-hospitalised patients cannot be excluded.

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of Clostridium difficile isolates from three Czech hospitals in Eastern Bohemia in 2011–2012

In 2011–2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.