Extended multilocus variable-number tandem-repeat analysis of Clostridium difficile correlates exactly with ribotyping and enables identification of hospital transmission.

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.

Clostridium difficile infection in Polish pediatric outpatients with inflammatory bowel disease.

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.

Distribution and antimicrobial susceptibility patterns of Clostridium difficile PCR ribotypes in English hospitals, 2007-08.

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.

Increased number of Clostridium difficile infections and prevalence of Clostridium difficile PCR ribotype 001 in southern Germany.

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.

Update of Clostridium difficile infection due to PCR ribotype 027 in Europe, 2008.

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.

Clostridium difficile: from obscurity to superbug.

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.

Genetic analysis of mechanisms of multidrug resistance in a clinical isolate of Bacteroides fragilis.

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.

Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe.

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.

Anaerobic sepsis due to multidrug-resistant Bacteroides fragilis: microbiological cure and clinical response with linezolid therapy.

We describe the first reported case of anaerobic sepsis due to Bacteroides fragilis with simultaneous resistance to metronidazole, beta-lactams, beta lactam/beta-lactamase inhibitors, carbapenems, macrolides, and tetracyclines. Microbiological cure and clinical improvement was achieved with linezolid therapy, an agent that may be useful for the treatment of multidrug-resistant anaerobic infections.

Antimicrobial susceptibility of polymerase chain reaction ribotypes of Clostridium difficile commonly isolated from symptomatic hospital patients in the UK.

Two hundred and seventy-one clinical isolates of Clostridium difficile, including the six most common polymerase chain reaction (PCR) ribotypes isolated from symptomatic patients in UK hospitals, were tested against nine antibiotics (imipenem, erythromycin, levofloxacin, piperacillin/tazobactam, ciprofloxacin, co-amoxiclav, cefotaxime, amoxicillin and clindamycin). All 271 strains were susceptible to co-amoxiclav, piperacillin/tazobactam and amoxicillin, and resistant to cefotaxime and ciprofloxacin. Variable degrees of resistance were found to imipenem, erythromycin, levofloxacin and clindamycin. Significantly greater resistance to erythromycin, levofloxacin and imipenem was found in virtually all members of the two most common PCR ribotypes, 001 and 106. Resistance to these agents may have played a part in their selection as the most common strains of C. difficile found in UK hospitals.

A modified pulsed-field gel electrophoresis (PFGE) protocol for subtyping previously non-PFGE typeable isolates of Clostridium difficile polymerase chain reaction ribotype 001.

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to 50 isolates of the UK epidemic strain of Clostridium difficile, polymerase chain reaction (PCR) ribotype 001, to develop a PFGE-based subtyping scheme. This protocol overcame the inherent DNA degradation problems associated with typing this strain of C. difficile by this method, and whole genomic digestion with SmaI restriction enzyme yielded seven distinct and reproducible PFGE banding patterns. Modified PFGE is an appropriate method for subtyping C. difficile PCR ribotype 001 that could be used to improve epidemiological investigations.

Subtyping of Clostridium difficile polymerase chain reaction (PCR) ribotype 001 by repetitive extragenic palindromic PCR genomic fingerprinting.

Fifty isolates of the most common UK strain of Clostridium difficile [polymerase chain reaction (PCR) ribotype 001] were analysed by three PCR-based typing methods in order to determine genomic diversity within this strain that may form the basis of a subtyping method. The three methods used were repetitive extragenic palindromic elements (REP), conserved repetitive DNA elements (BOX), and enterobacterial repetitive PCR intergenic consensus sequences (ERIC). The performance of each typing method was assessed by comparing powers of discrimination, typeability and reproducibility. All methods had satisfactory levels of typeability and reproducibility as determined by blind-coded repeats, but REP-PCR typing proved to be the most discriminatory method, yielding seven distinct amplicon profiles consisting of up to eight major bands. BOX-PCR generated between two and five major amplicons with four distinct BOX profiles. ERIC-PCR primers, however, could not discriminate between isolates. These results suggest that PCR ribotype 001 is not clonal in nature at present, and that REP-PCR subtyping methods offer promise to further our understanding of the epidemiology of C. difficile PCR ribotype 001 disease in UK hospitals.

Appraisal of Anotox, a new anaerobic atmospheric detoxifying agent for use in anaerobic cabinets.

Using Clostridium sporogenes cultures, inhibitory effects of its gaseous metabolic end products on the activity of palladium Deoxo catalyst and on surface growth of some clinically significant anaerobes were demonstrated. A new product "Anotox" was shown to absorb these metabolites, which enabled uninhibited growth of anaerobes, and prolonged the life of the catalyst.

Evaluation of the Anoxomat: a new technique for anaerobic and microaerophilic clinical bacteriology.

A system of automatic jar evacuation-replacement (Anoxomat) for the culture of anaerobes, capnophiles, and microaerophiles was compared with existing methods of anaerobic cabinets, carbon dioxide incubators, and manual evacuation-replacement. Of the 50 species of anaerobes, 29 strains of capnophiles, and 11 strains of microaerophiles tested, equivalent growth was obtained in all but two instances. The Anoxomat system yielded slightly larger colonies in 26 (52%) of anaerobes tested with superior growth in the anerobic cabinets in three (6%) of cases and equal in both in 21 (42%). Of the microaerophiles and capnophiles tested, there was no significant difference between the Anoxomat and the conventional system. The Anoxomat system seems to be a suitable alternative to anaerobic and carbon dioxide incubators.

Analysis of the porphyrin content of fluorescent pus by absorption spectrophotometry and high performance liquid chromatography.

Extracts of 19 samples of pus which showed red fluorescence with ultraviolet light were screened for the presence of porphyrins by absorption spectrophotometry. All those which showed spectra typical of metal-free porphyrins were analysed by high performance liquid chromatography to identify the porphyrins present. These were predominantly the di-carboxylic porphyrins, deuteroporphyrin and mesoporphyrin, and another which was thought to be pemptoporphyrin. This combination matched those reported previously in normal stools. Protoporphyrin IX was shown not to be the most common fluorescent pigment in pus and was never present alone. However, the di-carboxylic porphyrins may be produced by bacterial metabolism of its labile vinyl side-chains. Black-pigmented bacteroides (the melaninogenicus group of Bacteroides spp. and Porphyromonas spp.) were isolated from 12 (63%) of the 19 pus samples; these may produce protoporphyrin IX by the demetallation of haem.

The distribution of Clostridium difficile in the environment of South Wales.

A large study of the distribution of Clostridium difficile in the environment of the Cardiff area of South Wales was performed with a methodology designed to maximise recovery. A total of 2580 samples was taken, with 184 (7.1%) yielding isolates. The highest yield for C. difficile was obtained from river waters, with 14 (87.5%) of 16 samples from four rivers positive, and from sea water samples with 7 (44%) of 15 positive from six beaches on the Bristol Channel. In addition, 7 (46.7%) of 15 samples of lake water were also positive. Twenty-two (21%) of 104 soil samples, taken from random sites in Cardiff, were positive, as were 20% of environmental samples from four Cardiff hospitals. C. difficile was also isolated from 50% of eight swimming pool waters examined and 1 (5.5%) of 18 of mains tap water. Carriage of C. difficile in 524 faecal samples of assorted farm animals was c. 1%, and was 10% in dogs and 2% in cats. In private residences, the organism was present in 12 (2.2%) of 550 samples. While 2.4% of 300 raw vegetable samples were positive, none of 107 assorted fish gut contents was. These findings indicate that C. difficile may be more widely distributed in the general environment, particularly water, than was previously thought.

Fusobacterium necrophorum infections in England and Wales 1990-2000.

In response to a marked increase in both the number of Fusobacterium necrophorum bacteraemia reports to the PHLS Communicable Disease Surveillance Centre and the number of F. necrophorum isolates referred to the PHLS Anaerobe Reference Unit in 1999, the data from both sources on F. necrophorum infections were reviewed for the decade 1990-2000. There were 208 reports of F. necrophorum bacteraemia (average 19/year; range 14-34/year) with a peak in incidence in the late winter months; 68% were from male patients and the peak age range was 16-23 years. Of 205 referred isolates of F. necrophorum, 122 (59%) were from blood cultures and these represented 58% of the bacteraemia reports; the others were from brain and soft tissue abscesses, pleural and joint fluids, eyes, ears and lymphatic tissue. The average number of referrals was 19/year (range 9-37/year). The peak year for bacteraemia reports (34) and isolate referrals (37) was 1999; this increase was not sustained in 2000. All isolates were susceptible to metronidazole, but 2% were resistant to penicillin and 15% to erythromycin. F. necrophorum continues to be a regular but uncommon cause of bacteraemia and metastatic abscesses following an acute sore throat, especially in young, otherwise healthy adults.

A comparative study of Fusobacterium necrophorum strains from human and animal sources by phenotypic reactions, pyrolysis mass spectrometry and SDS-PAGE.

Fusobacterium necrophorum strains from human infection (21) were compared with strains from animals (17 biotype A, 2 biotype AB, 4 biotype B, 1 biotype unknown), and the type strain NCTC 10575 in conventional tests reaction patterns (CTRPs), SDS-PAGE and pyrolysis mass spectrometry (PMS). Classifications from the three approaches showed one major consensus group comprising all human strains, and another comprising animal biotype A strains. Animal biotype B strains and one animal strain, designated with some doubt to biotype A, were outliers of the consensus 'human strain' group. Again, animal biotype AB strains were outliers of the consensus 'animal biotype A group', as was the type strain, which was clearly atypical in conventional tests and PMS. Colonial and microscopic characters showed good discrimination between the major consensus groups. However, only haemagglutination and the API-ZYM leucine arylamidase of the biochemical tests discriminated well between these groups. The 'animal biotype A group' clearly corresponds to F. necrophorum subsp. necrophorum, but synonymy of F. necrophorum subsp. funduliforme with the group of human strains was less certain. The latter subspecies was described solely on the basis of animal strains, all of biotype B, but each of four animal biotype B strains in this study was an outlier of the 'human strain group' in one or more of the characterisation approaches. Strains of F. necrophorum causing human infection were clearly distinct from the biotype A strains commonly found in animal infection. This has implications for the validity of animal models of human necrobacillosis. In view of these differences, it would be useful to have a validated designation for strains causing human infection. However, it would be premature to assume that the definition of F. necrophorum subsp. funduliforme encompasses the human strains in the absence of confirmatory DNA-homology and 16S rRNA-sequencing studies.

Prevalent PCR ribotypes of clinical and environmental strains of Clostridium difficile isolated from intensive-therapy unit patients in Kuwait.

Ninety-five isolates of Clostridium difficile from symptomatic and asymptomatic patients and 18 from their environment in the intensive-therapy units (ITUs) of four teaching hospitals in Kuwait were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping). A total of 32 different ribotypes was detected among the clinical isolates. The predominant ribotypes from the clinical isolates were types 097 and 078, which accounted for approximately 40 % of all isolates in the ITUs in Kuwait. Ribotypes 097 (toxigenic), 078 (toxigenic) and 039 (non-toxigenic) were three distinct clones that were circulating in all four hospitals. Ribotypes 097, 078 and 076 (i.e. 50 % of isolates from symptomatic patients) were the predominant isolates associated with C. difficile-associated disease (CDAD). The environmental isolates belonged to a diverse range of ribotypes, with no particular types common to all the hospitals. Ribotype 078 was found only in the patient environment in Mubarak hospital, while ribotype 097 was restricted to Amiri hospital. The hospital environment occupied by symptomatic as well as symptom-free patients was contaminated with C. difficile. Eight new strains that did not match any in the PCR ribotype library established at the PHLS Anaerobe Reference Unit, Cardiff, UK, were assigned ribotypes 105, 125, 128, 129, 131, 134, 140 and 141. These findings show that the isolates associated with CDAD in Kuwait are different from those found in the UK and some other European countries.

Real-time PCR investigation into the importance of Fusobacterium necrophorum as a cause of acute pharyngitis in general practice.

Fusobacterium necrophorum is recognized as the cause of a severe life-threatening illness characterized by bacteraemia with metastatic abscesses following an acute sore throat (Lemierre's disease). However, the importance of F. necrophorum as a cause of simple sore throat in the community is unknown. Using quantitative real-time PCR with primers targeting the rpoB gene, 100 routine throat swabs collected from patients presenting to general practitioners with pharyngitis were analysed for the presence of F. necrophorum-specific DNA. The results were compared with those obtained from throat swabs collected from 100 healthy subjects. Ten clinical samples were positive for F. necrophorum DNA, identified as F. necrophorum subspecies funduliforme, using a haemagglutinin-related protein gene-specific PCR assay. All the healthy controls were negative (two-tailed P value = 0.0015; Fisher exact test). These findings suggest that F. necrophorum may play a more important role as a cause of simple sore throat in the community than has been previously appreciated.