International typing study of Clostridium difficile.

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.

Clostridium difficile infection in Europe: a hospital-based survey.

BACKGROUND: Little is known about the extent of Clostridium difficile infection in Europe. Our aim was to obtain a more complete overview of C difficile infection in Europe and build capacity for diagnosis and surveillance. METHODS: We set up a network of 106 laboratories in 34 European countries. In November, 2008, one to six hospitals per country, relative to population size, tested stool samples of patients with suspected C difficile infection or diarrhoea that developed 3 or more days after hospital admission. A case was defined when, subsequently, toxins were identified in stool samples. Detailed clinical data and stool isolates were collected for the first ten cases per hospital. After 3 months, clinical data were followed up. FINDINGS: The incidence of C difficile infection varied across hospitals (weighted mean 4·1 per 10,000 patient-days per hospital, range 0·0-36·3). Detailed information was obtained for 509 patients. For 389 of these patients, isolates were available for characterisation. 65 different PCR ribotypes were identified, of which 014/020 (61 patients [16%]), 001 (37 [9%]), and 078 (31 [8%]) were the most prevalent. The prevalence of PCR-ribotype 027 was 5%. Most patients had a previously identified risk profile of old age, comorbidity, and recent antibiotic use. At follow up, 101 (22%) of 455 patients had died, and C difficile infection played a part in 40 (40%) of deaths. After adjustment for potential confounders, an age of 65 years or older (adjusted odds ratio 3·26, 95% CI 1·08-9·78; p=0·026), and infection by PCR-ribotypes 018 (6·19, 1·28-29·81; p=0·023) and 056 (13·01; 1·14-148·26; p=0·039) were significantly associated with complicated disease outcome. INTERPRETATION: PCR ribotypes other than 027 are prevalent in European hospitals. The data emphasise the importance of multicountry surveillance to detect and control C difficile infection in Europe. FUNDING: European Centre for Disease Prevention and Control.

Clostridium difficile: the anaerobe that made the grade.

Unlike other anaerobic bacteria of clinical importance, Clostridium difficile has managed to enter into the realm of public awareness. 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 report picks out key moments, particularly in the UK, which tracked the rise in both the public and political awareness of this organism.

Detection of cross-infection associated to a Brazilian PCR-ribotype of Clostridium difficile in a university hospital in Rio de Janeiro, Brazil.

Clostridium difficile is an important nosocomial enteric pathogen and is the etiological agent of pseudomembranous colites. Recently, the rates of C. difficile infection (CDI) have increased worldwide, but in Brazil few data about this situation and the incidence of clonal types of C. difficile exist. This study aimed to isolate and characterize C. difficile strains from samples obtained of a university hospital (HUCFF) in Rio de Janeiro city, Brazil. CDI was identified by ELISA in 27.1% of HUCFF-in-patients enrolled in the study, and the bacterium was recovered from eight of these fecal samples. All strains, except one, presented tcdA and tcdB genes and presented neither the cdtA and cdtB genes nor any significant deletions in the tcdC gene. All strains were sensitive to metronidazole, vancomycin and moxifloxacin, and resistant to clindamycin, ciprofloxacin and levofloxacin. PCR-ribotyping and PFGE revealed four different clonal types among the isolates. The Brazilian PCR-ribotype 133 accounted for 50% of strains isolated, and PCR-ribotype 233 strains were obtained from 25% of the in-patients. The prevalence and resurgence of the Brazilian PCR-ribotype 133 among the hospitalized patients of HUCFF was established, and cross-infection of different patients associated to the same PCR-ribotypes was detected. Our results emphasize the importance of the diagnosis and control of CDI in order to prevent the emergence of specific clones that can lead to C. difficile-associated outbreaks in Brazilian hospitals.

First report of Parabacteroides goldsteinii bacteraemia in a patient with complicated intra-abdominal infection.

Using 16S rRNA sequence analysis, we report the first isolation of Parabacteroides goldsteinii as a monobacterial isolate from blood culture in a patient with abdominal sepsis. P. goldsteinii phenotypically resembles Parabacteroides merdae and Parabacteroides distasonis and may be misidentified by commonly used enzymatic systems, suggesting that it may be more frequently present in clinical specimens than previously appreciated but either misidentified or ignored.

Comparison of molecular typing methods applied to Clostridium difficile.

Since the 1980s the epidemiology of Clostridium difficile infection (CDI) has been investigated by the application of many different typing or fingerprinting methods. To study the epidemiology of CDI, a typing method with a high discriminatory power, typeability, and reproducibility is required. Molecular typing methods are generally regarded as having advantages over phenotypic methods in terms of the stability of genomic markers and providing greater levels of typeability. A growing number of molecular methods have been applied to C. difficile. For the early and rapid detection of outbreak situations, methods such as restriction enzyme analysis, arbitrary primed polymerase chain reaction (PCR), and PCR ribotyping are commonly used. For long-term epidemiology, multilocus sequence typing, multilocus variable number of tandem repeats analysis, and amplified fragment length polymorphism are of interest. Currently, the PCR-ribotyping method and the library of PCR ribotypes in Cardiff are the benchmarks to which most typing studies around the world are compared. Multilocus variable number of tandem repeats analysis is the most discriminative typing method and will contribute significantly to our understanding of the epidemiology of this important nosocomial pathogen.

Equine colitis X associated with infection by Clostridium difficile NAP1/027.

A 14-year-old Quarter Horse with a 48-hr history of colic was euthanized after failure to respond to treatment. At necropsy, cecal and colonic mucosae were congested throughout, and there was segmental edema and significant thickening of the intestinal wall. Excessive numbers of mononuclear cells were found in mucosal lamina propria. Submucosal hemorrhage was diffuse and extensive, and Clostridium difficile toxins A and B were detected. Large numbers of C. difficile were isolated, and genetic characterization revealed them to be North American pulsed-field gel electrophoresis type 1, polymerase chain reaction ribotype 027, and toxinotype III. Genes for the binary toxin were present, and toxin negative-regulator tcdC contained an 18-bp deletion. This genotype comprises the current human "epidemic strain," which is associated with human C. difficile-associated disease of greater than historical severity. The diagnosis was peracute typhlocolitis, with lesions and history typical of those attributed to colitis X.

Distribution of Clostridium difficile strains from a North American, European and Australian trial of treatment for C. difficile infections: 2005-2007.

Clostridium difficile is a widely distributed pathogen with multiple strain types as determined by restriction endonuclease analysis (REA) and by PCR ribotyping, two well-characterized typing systems. In this study, REA typing was performed on 894C. difficile isolates from patients enrolled from 16 countries on three continents in two large, recently conducted clinical treatment trials of C. difficile infection. REA group BI (Ribotype 027) isolates were the most common strains identified and were widely distributed throughout North America, but restricted to three of thirteen countries in Europe. REA group J (Ribotype 001) isolates were the most common strains identified in Europe and non-specific REA groups (historically less frequent) were the most common strains identified in Australia. REA groups BI, J, G and CF correlated with specific PCR ribotypes whereas more than one ribotype was found within REA groups Y, BK, and K. International surveillance of C. difficile strains is important to document the changing epidemiology of this enteric pathogen that continues to cause healthcare facility outbreaks and sporadic infections in other settings.

Characterization of Clostridium difficile strains isolated from immunosuppressed inpatients in a hospital in Rio de Janeiro, Brazil.

The aim of this work was to identify and characterize Clostridium difficile strains from fecal and hospital environmental samples. C. difficile toxins were detected by ELISA in 28.5% of the analyzed samples. Four strains were isolated from immunosuppressed inpatients presenting antibiotic-associated diarrhea. All strains possessed tcdA and tcdB genes and did not present neither the cdtA and cdtB genes nor any significant deletions in the tcdC gene. PFGE and PCR-ribotyping analysis showed that two strains belonged to the same clonal type (ribotype 014) and the other two were grouped into ribotype 106, in spite of presenting a similar, but not identical genetic fingerprint. This report shows that for the first time ribotype 106 was found outside the United Kingdom. All isolates were equally sensitive to metronidazole. The ribotype 014 isolates were highly resistant to clindamycin, while the ribotype 106 isolates were resistant to all fluoroquinolones tested. This work reveals the spread of C. difficile in the hospital unit studied and the presence of three genetically related types, two of them presenting resistance to fluoroquinolones.

Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe.

The European Study Group on Clostridium difficile (ESGCD) conducted a prospective study in 2005 to monitor and characterize C. difficile strains circulating in European hospitals, collecting 411 isolates. Eighty-three of these isolates, showing resistance or intermediate resistance to moxifloxacin (MX), were selected for this study to assess susceptibility to other fluoroquinolones (FQs) and to analyse the gyr genes, encoding the DNA gyrase subunits GyrA and GyrB. Twenty MX-susceptible isolates from the surveillance study were included for comparison. Overall, one amino acid substitution in GyrA (Thr82 to Ile) and four different substitutions in GyrB (Ser416 to Ala, Asp426 to Asn, Asp426 to Val and Arg447 to Lys) were identified. A high level of resistance (MIC >or=32 microg ml(-1)) to MX, ciprofloxacin (CI), gatifloxacin (GA) and levofloxacin (LE) was found in 68 isolates showing the amino acid substitution Thr82 to Ile in GyrA, in eight isolates with the substitutions Thr82 to Ile in GyrA and Ser416 to Ala in GyrB, in two isolates showing the substitution Asp426 to Asn in GyrB and in one isolate with Asp426 to Val in GyrB. The remaining four isolates showed high MICs for CI and LE, but different MIC levels for MX and GA. In particular, intermediate levels of resistance to MX were shown by two isolates, one with the substitution Thr82 to Ile in GyrA, and one showing Asp426 to Asn in GyrB. The substitution Arg447 to Lys in GyrB was found in two strains resistant to MX, CI and LE but susceptible to GA. No substitutions in GyrA were found in the FQ-susceptible strains, whereas two strains showed the amino acid change Ser416 to Ala in GyrB. Thr82 to Ile was the most frequent amino acid change identified in the C. difficile isolates examined. In contrast to previous observations, 10% of the isolates showed this substitution in association with Ser416 to Ala in GyrB. The other amino acid changes found were characteristic of a few strains belonging to certain types and/or countries. Two new substitutions for C. difficile, Ser416 to Ala and Arg447 to Lys, were found in GyrB. Whereas the former does not seem to have a key role in resistance, since it was also detected in susceptible strains, the latter substitution occurred in the same position where other amino acid variations take place in resistant Escherichia coli and other C. difficile strains. A large number of C. difficile isolates now show an alarming pattern of resistance to the majority of FQs currently used in hospitals and outpatient settings, therefore judicious use of these antibiotics and continuous monitoring of in vitro resistance are necessary.

Molecular characterization and antimicrobial susceptibility patterns of Clostridium difficile strains isolated from hospitals in south-east Scotland.

Clostridium difficile isolates (n=149) collected in south-east Scotland between August and October 2005 were typed by four different methods and their susceptibility to seven different antibiotics was determined. The aims were to define the types of strain occurring in this region and to determine whether there were any clonal relationships among them with respect to genotype and antibiotic resistance pattern. Ribotyping revealed that 001 was the most common type (n=113, 75.8 %), followed by ribotype 106 (12 isolates, 8.1 %). The majority of the isolates (96.6 %, n=144) were of toxinotype 0, with two toxinotype V isolates and single isolates of toxinotypes I, IV and XIII. PCR and restriction analysis of the fliC gene from 147 isolates gave two restriction patterns: 145 of pattern VII and two of pattern I. Binary toxin genes were detected in only three isolates: two isolates of ribotype 126, toxinotype V, and one isolate of ribotype 023, toxinotype IV. S-types showed more variation, with 64.5 % (n=40) of the common S-type (4,939) and 21 % (n=13) of S-type 4,741, with six other S-types (one to three isolates each). All ribotype 001 isolates were of the same S-type (4,939), with three isolates of other ribotypes being this S-type. No resistance was found to metronidazole or vancomycin, with resistance to tetracycline only found in 4.3 % of the isolates. A high proportion of isolates were resistant to clindamycin (62.9 %), moxifloxacin, ceftriaxone (both 87.1 %) and erythromycin (94.8 %). Resistance to three antibiotics (erythromycin, clindamycin and ceftriaxone) was seen in 66 isolates, with erythromycin, ceftriaxone and moxifloxacin resistance seen in 96 isolates. Resistance to all four of these antibiotics was found in 62 isolates and resistance to five (the above plus tetracycline) in one isolate: a ribotype 001, toxinotype 0 strain. Whilst ribotype 001 was the most commonly encountered type, there was no evidence of clonal relationships when all other typing and antibiotic resistance patterns were taken into account.

Distribution of Clostridium difficile PCR ribotypes in regions of Hungary.

The objective of this survey was to determine the distribution of Clostridium difficile PCR ribotypes present across three Hungarian geographical regions. A total of 105 isolates of C. difficile from diarrhoeal faeces of both inpatients and outpatients were examined. The toxigenic status of the strains was determined by PCR for the tcdA, tcdB, cdtA and cdtB genes in Szeged (Hungary), while strains were subjected to PCR ribotyping in Cardiff (UK). A total of 31 ribotypes were detected among the 105 C. difficile isolates tested. Five PCR ribotypes were distinct from all previously described types, suggesting that they are new. The most common types in Hungary, during the period examined, were PCR ribotype 014 (24.8 %) and PCR ribotype 002 (13.3 %). The distribution of PCR ribotypes differed in the various Hungarian regions: PCR ribotype 012 was frequent (20.7 %) in South Hungary, whereas this type was rare in the Budapest region and was not common to West Hungary. In West Hungary and the Budapest region, PCR ribotype 014 was most frequent (28.9 and 29 %, respectively).

Prevalence and association of PCR ribotypes of Clostridium difficile isolated from symptomatic patients from Warsaw with macrolide-lincosamide-streptogramin B (MLSB) type resistance.

Isolates (79 in total) of Clostridium difficile obtained over a 2 year period from 785 patients suspected of having C. difficile-associated diarrhoea (CDAD) and being hospitalized in the University Hospital in Warsaw were characterized by toxigenicity profile and PCR ribotyping. Furthermore, their susceptibility to clindamycin and erythromycin was determined. Among the 79 C. difficile isolates, 35 were classified as (A+)B+, 1 as (A+)(B+)CDT+, 36 as (A-)B+ and 7 as (A-)B-. A total of 21 different PCR ribotypes was detected. Two main (A+)B+ strains circulated in our hospital: ribotype 014 and ribotype 046. Unexpectedly, the predominant PCR ribotype was type 017, a known (A-)B+ strain, and this accounted for about 45.5 % of all isolates cultured from patients with CDAD. Isolates belonging to PCR ribotype 017 were found in cases from epidemics of antibiotic-associated diarrhoea in the internal and surgery units. High-level resistance (MIC > or = 256 mg l(-1)) to clindamycin and erythromycin was found in 39 (49 %) of the C. difficile isolates. Interestingly, 34 (94 %) of macrolide-lincosamide-streptogramin B (MLSB) type resistance strains did not produce toxin A, but produced toxin B and were (A-)B+ ribotype 017. Thirty-seven of the high-level resistance strains harboured the erythromycin-resistance methylase gene (ermB). C. difficile isolates (2/29) that had high-level clindamycin and erythromycin resistance, and belonged to PCR ribotype 046, were ermB negative. These investigations revealed that the predominant C. difficile strain isolated from symptomatic patients hospitalized in University Hospital in Warsaw was MLSB-positive clindamycin/erythromycin-resistant PCR ribotype 017.

Subtyping of Clostridium difficile PCR ribotype 001 by REP-PCR and PFGE.

The REP-PCR (repetitive sequence-based PCR using repetitive extragenic palindromic primers) typing method and a modified PFGE method were applied to isolates of Clostridium difficile PCR ribotype 001 with the aim of comparing their performance as methods of subtyping this organism. Of 200 isolates from 60 hospitals tested by REP-PCR, eight subtypes were identified and labelled as REP-PCR subtypes 001-008. The predominant subtype, REP-PCR subtype 003, accounted for 47% of the total. Fifty-two of the 200 isolates were analysed by a modified PFGE method and seven subtypes were identified, labelled as PF-A-PF-G. There was excellent correlation between REP-PCR subtypes and PFGE subtypes with both methods displaying broadly similar discriminatory powers. However, REP-PCR subtyping proved to be a much easier, cheaper and more rapid method suitable for application for routine subtyping of C. difficile ribotype 001. Application of REP-PCR subtyping to UK isolates of C. difficile PCR ribotype 001 from 60 different centres revealed a wide distribution of REP-PCR subtype 003 throughout England and Wales, with a regional clustering of REP-PCR subtype 001 around Northwest England and North Wales. Analysis of isolates from a single hospital over a 4-year period revealed a change in predominant subtype over time.

PCR ribotyping of clinically important Clostridium difficile strains from Hungary.

Isolates of Clostridium difficile from different hospital wards at the University Hospital of Szeged in Hungary were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping). A total of 15 different ribotypes was detected among the 65 isolates tested. The predominant type, PCR ribotype 087, accounted for 39% of all isolates, in contrast with an international typing study where ribotype 001 was the most common. Two non-toxigenic C. difficile strains were found to exhibit the same pattern, which was distinct from those of all the ribotypes described previously, suggesting that this is a new type.

New types of toxin A-negative, toxin B-positive strains among clinical isolates of Clostridium difficile in Australia.

A total of 817 human clinical isolates of Clostridium difficile from all Australian states were screened for A(-)B(+) strains by toxin gene PCR assays. Nine (1.1 %) strains were confirmed to be A(-)B(+) by enzyme immunoassay for toxin production. Of these, six (66.7 %) were binary toxin-positive by PCR. Using PCR ribotyping and toxinotyping, the A(-)B(+) strains could be grouped into seven ribotypes and three toxinotypes. Only one of the ribotypes had been reported previously (017). The prevalence of ribotype 017 was low in this study with only two strains detected. Two new A(-)B(+) toxinotypes were also defined (XXX, XXXI). Toxinotype XXX had a toxin B gene similar to that of toxinotype IV (A(+)B(+)) but with a novel cytopathic region. Toxinotype XXXI was similar to other A(-)B(+) types (X, XVII), but had a larger deletion to the toxin A gene than in either of those types. The types of A(-)B(+) strains identified in this study differed markedly from those described in other regions.

Multihospital outbreak of Clostridium difficile ribotype 027 infection: epidemiology and analysis of control measures.

OBJECTIVE: To report a large outbreak of Clostridium difficile infection (CDI; ribotype 027) between June 2007 and August 2008, describe infection control measures, and evaluate the impact of restricting the use of fluoroquinolones in controlling the outbreak. DESIGN: Outbreak investigation in 3 acute care hospitals of the Northern Health and Social Care Trust in Northern Ireland. INTERVENTIONS: Implementation of a series of CDI control measures that targeted high-risk antibiotic agents (ie, restriction of fluoroquinolones), infection control practices, and environmental hygiene. RESULTS: A total of 318 cases of CDI were identified during the outbreak, which was the result of the interaction between C. difficile ribotype 027 being introduced into the affected hospitals for the first time and other predisposing risk factors (ranging from host factors to suboptimal compliance with antibiotic guidelines and infection control policies). The 30-day all-cause mortality rate was 24.5%; however, CDI was the attributable cause of death for only 2.5% of the infected patients. Time series analysis showed that restricting the use of fluoroquinolones was associated with a significant reduction in the incidence of CDI (coefficient, -0.054; lag time, 4 months; P = .003). CONCLUSION: These findings provide additional evidence to support the value of antimicrobial stewardship as an essential element of multifaceted interventions to control CDI outbreaks. The present CDI outbreak was ended following the implementation of an action plan improving communication, antibiotic stewardship, infection control practices, environmental hygiene, and surveillance.

An outbreak case of Clostridium difficile-associated diarrhea among elderly inpatients of an intensive care unit of a tertiary hospital in Rio de Janeiro, Brazil.

The aim of this study was to investigate Clostridium difficile-associated diarrhea (CDAD) in an intensive care unit (ICU) of a tertiary hospital in Rio de Janeiro, Brazil, and to characterize epidemiologically C. difficile strains obtained from an outbreak of CDAD. Within almost a 4-year surveillance period, CDAD incidence was determined for the first time in Brazil, and a 3-fold increase was observed in the average rate of CDAD, featuring an outbreak. About 80% of the patients were over 65 years. The main antibiotic that could be probably associated to CDAD was piperacillin/tazobactam. Four toxigenic strains were isolated, 3 from stools and 1 from environmental samples. They were all resistant to clindamycin and fluoroquinolones. Fingerprinting analysis revealed their distribution between 2 different polymerase chain reaction ribotypes, with one of them being exclusively found in Brazil. It was possible to detect cross-infection and environmental contamination in the ICU. Our results highlight the importance of a continuous CDAD surveillance in the hospitals, especially when a risk group is exposed.