We report the results of an international Clostridium difficile typing study to cross reference strain designations for seven typing methodologies and facilitate inter-laboratory communication. Four genotypic and three phenotypic methods were used to type 100 isolates and compare the results to 39 PCR ribotypes identified among the collection.

ABSTRACT PCR ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR ribotyping.

The prevalence of Clostridium difficile infection (CDI) in pediatric patients with inflammatory bowel disease (IBD) is still not sufficiently recognized. We assessed the prevalence of CDI and recurrences in outpatients with IBD. In addition, the influence of IBD therapy on CDI and antimicrobial susceptibility of the potentially causative C. difficile strains was assessed. This was a prospective, single-center, observational study. All specimens were obtained between January 2005 and January 2007 from the IBD outpatient service and screened for C. difficile and its toxins. C. difficile isolates were genotyped by PCR ribotyping. Diagnosis of Crohn’s disease (CD) and ulcerative colitis (UC) was based on Porto criteria. Severity of disease was assessed using the Hyams scale (for Crohn’s disease) and the Truelove–Witts scale (for ulcerative colitis). One hundred and forty-three fecal samples from 58 pediatric IBD patients (21 with Crohn’s disease and 37 with ulcerative colitis) were screened. The risk of C. difficile infection was 60% and was independent of disease type (CD or UC) (χ2 = 2.5821, df = 3, p = 0.4606). About 17% of pediatric IBD patients experienced a recurrence of CDI. All C. difficile strains were susceptible to metronidazole, vancomycin and rifampin. A high prevalence of C. difficile infection and recurrences in pediatric outpatients with IBD was observed, independent of disease type. There was no significant correlation between C. difficile infection and IBD therapy. PCR ribotyping revealed C. difficile re-infection and relapses during episodes of IBD in pediatric outpatients.

Isolates from patients with Clostridium difficile infection (CDI) usually produce both toxin A (TcdA) and toxin B (TcdB), but an increasing number of reports from Europe and Asia mention infections with TcdA-negative, TcdB-positive (A-/B+) strains, usually characterized as PCR ribotype 017 (type 017). Incidence rates of CDI per 10 000 admissions in a 200-bed Argentinean general hospital were 37, 84, 67, 43, 48 and 42 for the years 2000 to 2005, respectively. The annual percentages of type 017 CDI were 7.7%, 64.6%, 91.4%, 92.0%, 75.0% and 86.4%, respectively. Comparison of 112 017-CDI patients with 41 non-017-CDI patients revealed that 017-CDI patients were more often male (68.8% vs. 46.3%; odds ratio 2.55, 95% confidence interval 1.23-5.50). All type 017 strains tested belonged to toxinotype VIII and had a 1.8-kb deletion in tcdA. In addition, 90% of tested type 017 isolates had high-level resistance to clindamycin and erythromycin, determined by the presence of the ermB gene. Multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to 56 Argentinean isolates and 15 isolates from seven other countries. Country-specific clonal complexes were found in each country. Among 56 Argentinean isolates, four clonal complexes were recognized, accounting for 61% of all isolates. These clonal complexes did not show correlation over time, but seemed to be restricted to specific wards, mainly internal medicine and pulmonology wards. A total of 56% of recurrent infections were caused by a different isolate, despite identification of an identical PCR-ribotype. We conclude that C. difficile type 017 gradually replaced other circulating PCR ribotypes and that MLVA provides detailed insight into nosocomial spread.

A recent Supplement to Clinical Microbiology and Infection entitled 'Infection control measures to limit the spread of C. difficile' pointed out that the incidence of C. difficile-associated diarrhoea (CDAD) has been increasing worldwide, and stressed the importance of research in the fields of epidemiology and infection control [1]. Since 2003, one of the main causes of the increasing prevalence of CDAD has been claimed to be the emergence of PCR ribotype 027/NAP1, which has caused epidemics in North America, the UK, the Netherlands, Belgium and France. The presence of PCR ribotype 027 in Austria, Japan, Ireland, Germany and Switzerland has also been reported recently [2,3]. The majority of publications have emphasized that the presence of this strain is usually associated with more severe symptoms and signs than those associated with the other more common toxin-positive strains [4,5]. Whereas PCR ribotype 027 was present in the population earlier, the majority of the historic strains were fluoroquinolone sensitive [6]. The overuse of antibiotics such as fluoroquinolones may lead to the selection and emergence of resistant strains, and may contribute to the spread of PCR ribotype 027, which is usually resistant to erythromycin. Here, the Eastern European spread of C. difficile PCR ribotype 027 is reported.

Clostridium difficile is a common cause of nosocomial diarrhea. its role in community-acquired diarrhea is also becoming an important public health concern. Hardly any studies have correlated strain ribotypes, toxinotypes and multidrug resistant (MDR) profiles. To investigate these characteristics, 65 C. difficile isolates obtained from stool samples of patients whose cultures were negative on admission but became positive after 48 h of admission to the ICUs of our hospitals were studied to determine the prevalent ribotypes, toxinotypes and their relationship with the MDR profiles using ELISA/cytotoxicity assays, PCR and Etest methods. The toxin-producing strains were toxinotyped by the PCR-RFLP technique. Of the 65 isolates, 42 (64.6%) were toxigenic (T). The isolates were of diverse ribotypes but types 097, 078, 056 and 039 (NT) were predominant. thirty (71.4%) of 42 T and 13 (56.5%) of 23 NT strains were multiresistant to 3 or more antibiotics. Only 3 toxinotypes (0, "V-like" and XII) were encountered. Of the 42 t strains, 30 (71.4%) were of toxinotype 0, and 12 belonged to variant toxinotypes: 4 (9.4%) to toxinotype XII and 8 (19%) to "V-like" toxinotype in which amplified B1 PCR fragments was amplified as expected for toxinotype V but the A3 PCR fragment could not be amplified. The 43 mDR strains were assigned to 3 arbitrary resistance groups; groups 1, 11 and III. the most prevalent isolates (37; 86.1%) were in group II. Of the predominant T ribotypes (097, 078 and 056), c. 62% clustered in group II. Although the number of strains toxinotyped was small, ribotyping and toxinotyping correlated well with the published literature, except for 078 with a novel "V-like" toxinotype. Antibiogram was not as clear-cut.

A surveillance study designed to provide a representative sample of the strains of Clostridium difficile causing infections in hospitals in England was in operation from April 2007 to the end of March 2008. Six hundred and seventy-seven isolates were obtained from 186 hospitals in the nine geographical regions of England as recognised by the Health Protection Agency's Regional Microbiology Network. Typing studies revealed that PCR ribotype 027 is now the most common strain isolated from symptomatic patients, accounting for over 41.3% of isolates in English hospitals. Type 106 was the second most common strain (20.2%) and Type 001, which was once the most common strain associated with hospital outbreaks, has now been reduced to only 7.8% of the total. A mixture of 44 other PCR ribotypes accounted for the remaining 28.9% of isolates. This represents a changing distribution of strains when compared to a previous study performed two years earlier which showed roughly equal proportions of types 106, 001 and 027. Antimicrobial susceptibility testing by the E test method revealed significantly lower susceptibility to metronidazole in the more common strains when compared to the less common ribotypes, although none were classified as clinically resistant. Similarly, no resistance to vancomycin was detected. However, common PCR ribotypes were more resistant to moxifloxacin and erythromycin than the less common strains, which may indicate a selective advantage for resistance to these agents, and combined resistance to these two agents was a good indicator of a common ribotype.

E J Kuijper (ejkuijper@gmail.com)1, F Barbut2, J S Brazier3, N Kleinkauf4, T Eckmanns4, M L Lambert5, D Drudy6, F Fitzpatrick7, C Wiuff8, D J Brown9, J E Coia9, H Pituch10, P Reichert11, J Even11, J Mossong11, A F Widmer12, K E Olsen13, F Allerberger14, D W Notermans15, M Delmée16, B Coignard17, M Wilcox18, B Patel19, R Frei20, E Nagy21, E Bouza22, M Marin10, T Åkerlund23, A Virolainen-Julkunen24, O Lyytikäinen24, S Kotila24, A Ingebretsen25, B Smyth26, P Rooney27, I R Poxton28, D. L. Monnet29 1. National Reference Laboratory for Clostridium difficile. Leiden University Medical Center, Leiden, The Netherlands 2. National Reference Center for Clostridium difficile, Saint-Antoine Hospital, Paris, France 3. Anaerobe Reference Laboratory, National Public Health Service for Wales Cardiff, University Hospital of Wales, Cardiff, United Kingdom 4. Department for Infectious Disease Epidemiology, Robert Koch Institute, Berlin, Germany 5. Epidemiology Unit, Scientific Institute of Public Health, Brussels, Belgium 6. Centre for Food Safety, Food Science and Veterinary Medicine, University College Dublin, Dublin, Ireland 7. Health Protection Surveillance Centre and Beaumont Hospital, Dublin, Ireland 8. Health Protection Scotland, Section for Healthcare Associated Infection and Infection Control, Glasgow, United Kingdom 9. Scottish Clostridium difficile Reference Service, Stobhill Hospital, Glasgow, United Kingdom 10. Department of Medical Microbiology, Medical University of Warsaw, Warsaw, Poland 11. Division of Microbiology, National Public Health Laboratory, Luxembourg 12. Division of Infectious diseases and Hospital Epidemiology, University Hospital, Basel, Switzerland 13. National Reference Laboratory for Enteropathogens, Statens Serum Institut, Copenhagen, Denmark 14. Österreichische Agentur für Gesundheit und Ernährungssicherheit (Austrian Agency for Health and Food Safety; AGES), Vienna, Austria 15. Centrum Infectieziektebestrijding (Centre for Infectious Disease Control; CIb), Rijksinstituut voor Volksgezondheid en Milieu (National Institute for Public Health and the Environment; RIVM), Bilthoven, The Netherlands 16. Microbiology Department, Saint-Luc University Hospital, Brussels, Belgium 17. Departement of Infectious Diseases, Institut de Veille Sanitaire (National Public Health Institute; InVS), Saint-Maurice, France 18. Clostridium difficile Ribotyping Network for England (CDRNE), Health Protection Agency, Reference Leeds General Infirmary, Leeds, United Kingdom 19. 0 Health Protection Agency, London, United Kingdom 20. Microbiology Laboratory, University Hospital, Basel, Switzerland 21. Department of Clinical Microbiology, Faculty of Medicine, University of Szeged, Szeged, Hungary 22. Department of Medical Microbiology, University General Hospital Gregorio Maranon, Madrid, Spain 23. Smittskyddsinstitutet (Swedish Institute for Infectious Disease Control; SMI), Solna, Sweden 24. Kansanterveyslaitos (National Public Health Institute; KTL), Helsinki, Finland 25. Department of Infection Prevention, Rikshospitalet, Oslo, Norway 26. Health Protection Agency, Communicable Disease Surveillance Centre (Northern Ireland), Belfast, United Kingdom 27. Microbiology Laboratory Belfast City Hospital, Belfast, United Kingdom 28. Medical Microbiology, Centre for Infectious Diseases, University of Edinburgh College of Medicine and Veterinary Medicine, Edinburgh, United Kingdom 29. European Centre for Disease Prevention and Control, Stockholm, Sweden

Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of microorganisms frequently isolated from local and systemic infections. In this study, the antimicrobial susceptibilities of clinical strains isolated in 10 European countries were investigated. After identification of 299 GPAC to species level, the minimum inhibitory concentrations of penicillin, imipenem, clindamycin, metronidazole, vancomycin and linezolid were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute. The majority of isolates were identified as Finegoldia magna and Parvimonas micra (formerly Peptostreptococcus micros), isolated from skin and soft tissue infections. All isolates were susceptible to imipenem, metronidazole, vancomycin and linezolid. Twenty-one isolates (7%) were resistant to penicillin (n=13) and/or to clindamycin (n=12). Four isolates were resistant to both agents. The majority of resistant isolates were identified as F. magna and originated from blood, abscesses and soft tissue infections.

OBJECTIVES:Antimicrobial treatment for Clostridium difficile infection (CDI) has typically been metronidazole, although reports have questioned the efficacy of this option. We screened recently isolated C. difficile (2005-06) for susceptibility to metronidazole and compared results for historic isolates (1995-2001).METHODS:C. difficile ribotypes 001 (n = 86), 106 (n = 81) and 027 (n = 48) and isolates from the 10 other most prevalent ribotypes in Leeds (n = 57) were screened using spiral gradient endpoint analysis (SGE). C. difficile with metronidazole SGE MICs > or = 6 mg/L were analysed further by agar incorporation and Etest. Multiple-locus variable-number tandem-repeat analysis (MLVA) typing was performed for 28 C. difficile isolates.RESULTS:No reduced metronidazole susceptibility was observed in C. difficile ribotypes 106 and 027 (geometric mean SGE MICs 1.11 and 0.90 mg/L, respectively). In contrast, 21 (24.4%) C. difficile ribotype 001 demonstrated reduced susceptibility to metronidazole (geometric mean SGE MICs 3.51 mg/L, P < 0.001). Variations in susceptibility were observed relating to the method and media, but increased metronidazole MICs were confirmed by an agar incorporation method. Geometric mean agar incorporation MICs for historic C. difficile ribotype 001 (n = 72) were 1.03 (range 0.25-2) mg/L compared with 5.94 (4-8) mg/L (P < 0.001) for recent isolates displaying reduced metronidazole susceptibility. MLVA typing revealed two clonal complexes of C. difficile with reduced susceptibility to metronidazole.CONCLUSIONS:We have demonstrated the emergence of reduced susceptibility to metronidazole in 24.4% of the recent C. difficile ribotype 001 isolates from our institution. Our observations could have implications in the clinical setting due to the poor penetration of metronidazole into the colon.

According to the UK media and popular press, Clostridium difficile is now a fully fledged member of that notorious but ill-defined group of microorganisms portrayed to the general public as superbugs. Following the trail blazed by methicillin-resistant Staphylococcus aureus (MRSA), C. difficile has made the transition from being an obscure anaerobic bacterium, mainly of interest to specialist anaerobic microbiologists, to that of an infamous superbug responsible for outbreaks of hospital-acquired infection that commonly result in serious disease and death. This review tracks the rise in scientific knowledge and public awareness of this organism.

Outbreaks of Clostridium difficile infections (CDI) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America and Europe. This emerging strain is referred to as PCR ribotype 027 (Type 027). Since 2005, individual countries have developed surveillance studies about the spread of type 027.C. difficile Type 027 has been reported in 16 European countries. It has been responsible for outbreaks in Belgium, Germany, Finland, France, Ireland, Luxembourg, The Netherlands, Switzerland and the United Kingdom (England, Wales, Northern Ireland and Scotland). It has also been detected in Austria, Denmark, Sweden, Norway, Hungary, Poland and Spain. Three countries experienced imported patients with CDI due to Type 027 who acquired the infection abroad.The antimicrobial resistance pattern is changing, and outbreaks due to clindamycin-resistant ermB positive Type 027 strains have occurred in three European countries. Ongoing epidemiological surveillance of cases of CDI, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of new, highly virulent clones.

In recent years, Clostridium difficile infection (CDI) has emerged as an increasing problem, both in in- and outpatients. In a rural region of southern Germany, the annual number of C. difficile toxin (Tcd)-positive patients has increased from 95 to 796 in the period from 2000 to 2007. Simultaneously, the proportion of positive tests among all Tcd examinations has risen from 7.0% to 12.8%, indicating that the higher number of affected patients was not solely due to an increase in the number of assays. Elevated numbers of CDI have recently been associated with outbreaks of the ribotype 027 strain, particularly in North America. This strain has also been isolated in Europe, including in Germany. Ribotyping and PCR testing for binary toxin genes of C. difficile strains isolated from in- and outpatients demonstrate a predominance (59%) of C. difficile ribotype 001, which exhibits antibiotic resistance to erythromycin, ciprofloxacin, and moxifloxacin, but lacks binary toxin genes. In summary, in our region of Germany, the number of patients affected by CDI has increased, probably due to spread of C. difficile ribotype 001.

Honey is used as a therapy to aid wound healing. Previous data indicate that honey can stimulate cytokine production from human monocytes. The present study further examines this phenomenon in manuka honey. As inflammatory cytokine production in innate immune cells is classically mediated by pattern recognition receptors in response to microorganisms, bacterial contamination of honey and the effect of blocking TLR2 and -4 on stimulatory activity were assessed. No vegetative bacteria were isolated from honey; however, bacterial spores were cultured from one-third of samples, and low levels of LPS were detected. Blocking TLR4 but not TLR2 inhibited honey-stimulated cytokine production significantly. Cytokine production did not correlate with LPS levels in honey and was not inhibited by polymyxin B. Further, the activity was reduced significantly following heat treatment, indicating that component(s) other than LPS are responsible for the stimulatory activity of manuka honey. To identify the component responsible for inducing cytokine production, honey was separated by molecular weight using microcon centrifugal filtration and fractions assessed for stimulatory activity. The active fraction was analyzed by MALDI-TOF mass spectroscopy, which demonstrated the presence of a number of components of varying molecular weights. Additional fractionation using miniaturized, reverse-phase solid-phase extraction resulted in the isolation of a 5.8-kDa component, which stimulated production of TNF-alpha via TLR4. These findings reveal mechanisms and components involved in honey stimulation of cytokine induction and could potentially lead to the development of novel therapeutics to improve wound healing for patients with acute and chronic wounds.

This study investigated the mechanisms of multidrug resistance (MDR) in an isolate of Bacteroides fragilis (WI1) from a patient with anaerobic sepsis. The MDR of WI1 affected susceptibility to beta-lactams, clindamycin, fluoroquinolones, metronidazole and tetracycline. In addition to its 5.31-Mb chromosome, WI1 possessed two low-copy-number plasmids, pHagl (5.6 kb) and pHag2 (9.9 kb), that were absent from B. fragilis NCTC 9343. Restriction digestion with EcoRV, HindIII and SstI, combined with DNA sequencing, revealed that pHAG2 contained a tet(Q) gene at base position 3689 that resided on the conjugative transposon CTn341. Genes cfiA (encoding a metallo-beta-lactamase) and erm(F) (encoding a macrolide-lincosamide-streptogramin B resistance determinant) were also found in WI1, but were absent from B. fragilis NCTC 9343. Nitrocefin hydrolysis revealed that WI1 had high beta-lactamase activity. Sequencing of the gyrA quinolone resistance-determining region revealed a mutation causing a Ser82 -> Phe substitution, and comparative quantitative real-time RT-PCR revealed that the cfiA, erm(F) and tet(Q) genes were all expressed in WI1. In addition, the resistance-nodulation-division efflux pump genes bmeB9 and bmeB15 were significantly over-expressed (12.30 +/- 0.42-fold and 3541.1 +/- 95.4-fold, respectively), and the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone and reserpine significantly increased the susceptibility of the isolate to several unrelated antibiotics (p < 0.005). These data suggested that WI1 was highly multidrug-resistant because of the additive effects of chromosome- and plasmid-encoded resistance determinants.

Background:Clostridium difficile is the major cause of antibiotic associated diarrhea (CDAD) and pseudomembranous colitis. Incidence and severity of C. difficile disease have increased in Canada and USA. The main strain responsible is PCR ribotype 027. Surveillance Centre's in the UK identified an increased incidence of CDAD in 2003, predominantly ribotype 027. The 027 strain has now spread to other European countries. Surveillance and control of C. difficile is now a high priority.

Recent outbreaks of Clostridium difficile-associated diarrhoea (CDAD) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America, Japan and Europe. Definitions have been proposed by the European Centre of Disease Prevention and Control (ECDC) to identify severe cases of CDAD and to differentiate community-acquired cases from nosocomial CDAD (http://www.ecdc.europa.eu/documents/pdf/Cl_dif_v2.pdf). CDAD is mainly known as a healthcare-associated disease, but it is also increasingly recognised as a community-associated disease. The emerging strain is referred to as North American pulsed-field type 1 (NAP1) and PCR ribotype 027. Since 2005, individual countries have developed surveillance studies to monitor the spread of this strain. C. difficile type 027 has caused outbreaks in England and Wales, Ireland, the Netherlands, Belgium, Luxembourg, and France, and has also been detected in Austria, Scotland, Switzerland, Poland and Denmark. Preliminary data indicated that type 027 was already present in historical isolates collected in Sweden between 1997 and 2001.

An outbreak of Clostridium difficile infection in Stoke Mandeville hospital in south-east England [1] in 2004/2005 was primarily due to a new and possibly more virulent strain known in the United Kingdom (UK) as PCR ribotype 027. Coinciding with this outbreak, a surveillance programme of C. difficile isolates from symptomatic patients in England with additional results of outbreak investigation requests to the Anaerobe Reference Laboratory (ARL) in Cardiff has established the true extent of its spread throughout British hospitals.