Hypervirulent Ribotype Research Articles

P1-85 Epidemiological risk assessment of C difficile outbreaks leads to rapid implementation of a national laboratory based surveillance system and changes in hospitals' hygienic guidelines

IntroductionIn January 2009 the Capital region of Denmark experienced for the first time an outbreak of hypervirulent Clostridium difficile PCR ribotype 027.MethodsAn epidemiological investigation was launched in a regional hospital...

Emergence of Clostridium difficile Ribotype 027 in Korea

BackgroundClostridium difficile infection (CDI) has markedly risen and is associated with hypervirulent ribotype 027 outbreaks in North America and Europe since 2003. The aims of this study were to determine the prevalence of ribotype 027 among C. difficile isolates in Korea, to characterize the ribotype 027 isolates, and to determine the clinical severity of CDI in patients infected with these isolates.MethodsA total of 1,251 isolates of C. difficile recovered from stool specimens of suspected CDI patients at two tertiary-care hospitals and one commercial laboratory between 2002 and 2009. Genes for toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA and cdtB) were detected by PCR. Mutation in the tcdC gene was detected by sequencing after PCR amplification. For molecular genotyping, we performed PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations of moxifloxacin were determined using Etest strips (AB bioMérieux, Sweden).ResultsWe identified 7 isolates as ribotype 027. These isolates had the same tcdC mutation as the epidemic strain, and 6 of them were resistant to moxifloxacin. The isolates were categorized into 3 different PFGE types and 7 different MLVA types. All the 7 cases had occurred sporadically.ConclusionsC. difficile ribotype 027 is uncommon, but it has emerged in Korea. The spread of this ribotype should be closely monitored in order to avoid an outbreak of CDI in Korea.

A Possible Oligosaccharide-Conjugate Vaccine Candidate for Clostridium difficile Is Antigenic and Immunogenic

Nosocomial infections with the Gram-positive pathogen Clostridium difficile pose a major risk for hospitalized patients and result in significant costs to health care systems. Here, we present the chemical synthesis of a PS-II hapten of a cell wall polysaccharide of hypervirulent ribotype 027 of C. difficile. Mice were immunized with a conjugate consisting of the synthetic hexasaccharide and the diphtheria toxoid variant CRM(197). The immunogenicity of the glycan repeating unit was demonstrated by the presence of specific IgG antibodies in the serum of immunized mice. Murine monoclonal antibodies interact with the synthetic hexasaccharide, as determined by microarray analysis. Finally, we found that specific IgA antibodies in the stool of hospital patients infected with C. difficile recognize the synthetic PS-II hexasaccharide hapten.

Decline in Clostridium difficile-associated disease rates in Singapore public hospitals, 2006 to 2008

BackgroundClostridium difficile is the major cause of pseudomembranous colitis associated with antibiotic use, and the spread of the hypervirulent epidemic ribotype 027/NAP-1 strain across hospitals worldwide has re-focused attention on this nosocomial pathogen. The overall incidence and trend of C. difficile-associated disease (CDAD) in Singapore is unknown, and a surveillance program to determine these via formal laboratory-based reporting was established.FindingsLaboratory and pharmacy data were collated from one tertiary and two secondary hospitals on a quarterly basis between 2006 and 2008. All hospitals tested for C. difficile using Immunocard Toxins A&B (Meridian Bioscience Inc., Cincinnati, OH) during this period. Duplicate positive C. difficile results within a 14-day period were removed. The CDAD results were compared with trends in hospital-based prescription of major classes of antibiotics.Overall CDAD incidence-density decreased from 5.16 (95%CI: 4.73 - 5.62) cases per 10,000 inpatient-days in 2006 to 2.99 (95%CI: 2.67 to 3.33) cases per 10,000 inpatient-days in 2008 (p < 0.001), while overall rates for C. difficile testing increased significantly (p < 0.001) within the same period. These trends were mirrored at the individual hospital level. Evaluation of antibiotic prescription data at all hospitals showed increasing use of carbapenems and fluoroquinolones, while cephalosporin and clindamycin prescription remained stable.ConclusionsOur results demonstrate a real decline of CDAD rates in three large local hospitals. The cause is unclear and is not associated with improved infection control measures or reduction in antibiotic prescription. Lack of C. difficile stool cultures as part of routine testing precluded determination of the decline of a major clone as a potential explanation. For more accurate epidemiological trending of CDAD and early detection of epidemic clones, data collection will have to be expanded and resources set in place for reference laboratory culture and typing.

The LMW surface-layer proteins of Clostridium difficile PCR ribotypes 027 and 001 share common immunogenic properties

The aim of this study was to investigate the S-layer proteins (SLPs) of the hypervirulent Clostridium difficile PCR ribotype 027 and compare them with those of PCR ribotype 001 and other PCR ribotypes involved in C. difficile infection and outbreaks, by molecular analysis and immunological assays. It has been demonstrated previously that PCR ribotype 027 SlpA is conserved in C. difficile strains belonging to this PCR ribotype and that it is a new variant, showing 88 % identity with SlpA of PCR ribotype 001. As the low-molecular-weight (LMW) SLPs of C. difficile are immunodominant antigens, attention was focused on this region of the genome. Sequencing of strains of different PCR ribotypes (001, 012, 014, 017, 027 and 078) showed that SlpA was conserved among strains belonging to the same PCR ribotype. Comparison of the LMW SLP region among these strains identified ten regions with sequence identity between PCR ribotypes 027 and 001, and low conservation with the other PCR ribotypes. In particular, two of these regions corresponded to areas predicted to be surface exposed. Three specific peptides, including those of the two surface-exposed regions, were recognized by human sera against PCR ribotypes 027 and 001 and by a rabbit polyclonal serum against the SLPs of PCR ribotype 027. In contrast, these peptides were not recognized by a polyclonal serum against the SLPs of PCR ribotype 012 used as a control. These results confirm the antigenic role of the LMW SLP and suggest that it may have a role in evasion of the host immune response.

Multilocus variable-number tandem repeat analysis: a helpful tool for subtyping French Clostridium difficile PCR ribotype 027 isolates

The objective of this study was to evaluate the usefulness of multilocus variable-number tandem repeat analysis (MLVA) for typing and subtyping of Clostridium difficile. Sixty-eight strains were studied, including strains from PCR ribotypes 027, 078/126, 014/020/077, 017 and 023. The stability of variable-number tandem repeat (VNTR) loci was tested by comparing the MLVA results of two strains subcultured 11 times. After DNA extraction, seven tandem repeat loci (A6, B7, C6, E7, F3, G8, H9) from published MLVA schemes were amplified by PCR and sequenced. The distance between two strains was determined by calculating the summed tandem repeat difference. Genomic diversity was evaluated by using the minimum spanning tree (Bionumerics 5.1 software program; Applied Maths). Among the 68 C. difficile isolates examined, 65 unique MLVA types were identified, suggesting a high discriminatory power. An overall good agreement was observed between MLVA types and PCR ribotypes. The stability of VNTR loci was good. MLVA could separate isolates of the hypervirulent PCR ribotype 027 clone in several clusters; all 027 strains isolated within a hospital were grouped in a specific cluster or were placed very close to each other. Results of MLVA confirmed that strains from PCR ribotypes 078 and 126 were closely related although some were located in different branches of the tree. Similar results were observed for most strains from PCR ribotypes 014, 020 and 077. This highly discriminatory method is time-consuming and expensive, but is a valuable tool for subtyping of C. difficile, especially of 027 strains.

Comparison of toxin and spore production in clinically relevant strains of Clostridium difficile

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins that it produces (TcdA and TcdB) are responsible for the characteristic pathology of C. difficile infection (CDI), while its spores persist in the environment, causing its widespread transmission. Many different strains of C. difficile exist worldwide and the epidemiology of the strains is ever-changing: in Scotland, PCR ribotype 012 was once prevalent, but currently ribotypes 106, 001 and 027 are endemic. This study aimed to identify the differences among these ribotypes with respect to their growth, and toxin and spore production in vitro. It was observed that the hypervirulent ribotype 027 produces significantly more toxin than the other ribotypes in the exponential and stationary phases of growth. Further, the endemic strains produce significantly more toxins and spores than ribotype 012. Of note was the observation that tcdC expression did not decrease into the stationary phase of growth, implying that it may have a modulatory rather than repressive effect on toxin production. Further, the increased expression of tcdE in ribotype 027 suggests its importance in the release of the toxins. It can thus be concluded that several genotypic and phenotypic traits might synergistically contribute to the hypervirulence of ribotype 027. These observations might suggest a changing trend towards increased virulence in the strains currently responsible for CDI.

Comparison of repetitive extragenic palindromic sequence-based PCR with PCR ribotyping and pulsed-field gel electrophoresis in studying the clonality of Clostridium difficile

Clostridium difficile infection is most often induced by antibiotic treatment. Recently, morbidity and mortality resulting especially from C. difficile PCR ribotype 027 have increased significantly. In addition, more severe disease has been associated with C. difficile PCR ribotype 078 strains. Thus, reliable typing methods for epidemic control are needed. In the present study, we compared an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA, USA) to PCR ribotyping and pulsed-field gel electrophoresis (PFGE) typing using 205 isolates of C. difficile (including 24 previously characterized isolates). Among the 181 clinical isolates, a total of 31 different PCR ribotypes, 38 different PFGE types and subtypes and 28 different rep-PCR types were found. Six major rep-PCR groups (DL1–DL6) harboured 86% of the clinical isolates. All isolates belonging to PCR ribotypes 027 and 001 clustered in their own rep-PCR groups, enabling us to screen out the hypervirulent ribotype 027 strain. Within the PCR ribotype 001, four subgroups were found using rep-PCR. Overall, in 75% (135/181) of the isolates, the classification attributed following rep-PCR and PCR ribotyping was comparable. In conclusion, the automated rep-PCR-based typing method represents an option for first-line molecular typing in local clinical microbiology laboratories. The method was easy to use as well as rapid, requiring less hands-on time than PCR ribotyping or PFGE typing. The conventional PCR ribotyping or PFGE, however, are needed for confirmatory molecular epidemiology. In addition, more epidemiology-oriented studies are needed to examine the discriminatory power of automated rep-PCR with isolates collected from a larger geographical area and during a longer period of time.

Rapid detection of the Clostridium difficile ribotype 027 tcdC gene frame shift mutation at position 117 by real-time PCR and melt curve analysis

The emergence of the hypervirulent strain Clostridium difficile PCR ribotype 027 has increased the necessity for rapid C. difficile typing tests for clinical and epidemiological purposes. We developed a rapid real-time polymerase chain reaction (PCR) test for the detection of C. difficile. As the target, we chose the tcdC gene, which encodes for a negative regulator in toxin production. A deletion at position 117 of the tcdC gene, which is associated with severe tcdC truncation, is well conserved in all PCR ribotype 027 isolates. Probe sequences of the real-time PCR test were designed to result in distinct melt profiles for sequence variations at positions 117 to 120 of the tcdC gene. The tcdC gene deletion at position 117 was easily detected with real-time PCR and melt curve analysis in all C. difficile ribotype 027 isolates. In five non-027 strains and 46 hospitalised patient samples, melt curve analysis detected no deletion. PCR results were confirmed by DNA sequencing. The combination of real-time PCR and melt curve analysis is a rapid and accurate method for the detection of C. difficile DNA and simultaneous screening for the tcdC gene deletion at position 117, which is closely related to the C. difficile PCR ribotype 027 strain.

Typing and Subtyping of Clostridium difficile Isolates by Using Multiple-Locus Variable-Number Tandem-Repeat Analysis

Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other. Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters. Additionally, 29 toxin A-negative, toxin B-positive isolates belonging to PCR ribotype 017 from eight different countries revealed eight country-specific clusters. MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.

Clostridium difficilein Clinical Practice: Increasing Rates, More Virulent Organisms and New Therapies on the Horizon

Clostridium difficile-associated diarrhea has recently come under increasing scrutiny because of outbreaks of disease in eastern Canada in association with a hypervirulent ribotype 027 (NAP1) strain. Hospitals in the United States, the United Kingdom and Europe have been affected as well. Epidemiological studies are underway to determine the presence of the more virulent strain ofC difficilein hospitals across Canada. Because this strain is highly quinolone resistant, it is suspected that overuse of that class of antimicrobial agent may be a selection factor for this strain. AlthoughC difficile-associated diarrhea is usually considered a nosocomial infection, approximately one-quarter of cases occur in the community. Recurrence following successful resolution of diarrhea occurs in one in five patients treated with metronidazole or vancomycin; relapse is more common in patients needing retreatment. New therapies that may have equivalent or higher response rates and lower recurrence rates are being investigated. It is hoped that these new treatments will be a substantial improvement over current therapies. In the interim, prudent antimicrobial use and attention to infection control practices are the main preventive strategies. Prompt testing forC difficiletoxin in patients with diarrhea after antibiotic exposure, discontinuation of unnecessary antibiotic therapy and rapid initiation of treatment should minimize complications. With the appearance of more toxigenic strains ofC difficile, careful monitoring of patients to detect suboptimal clinical response is necessary.