Lack of association between clinical outcome of Clostridium difficile infections, strain type, and virulence-associated phenotypes

Clostridium difficile strain NAP1/027 (North American pulsed-field gel electrophoresis [PFGE] type 1 and PCR ribotype 027 [R027]) has been associated with recent outbreaks in North America and Europe. It has been associated with more severe disease symptoms, higher mortality rates, and greater risk of relapse. This strain is thought to produce more toxins and sporulate to higher levels. However, recent studies suggest that this may not always be the case. The objective of our study was to assess, in a nonoutbreak situation, whether specific strains, such as NAP1/027, were associated with more severe disease symptoms, higher toxin production, and/or greater sporulation in vitro. We isolated and characterized C. difficile strains from 21 patients with mild to moderate, severe, or complicated symptoms of C. difficile infection (CDI). The isolates were characterized by different molecular typing methods, including PCR ribotyping, tandem repeat sequence typing (TRST), and sequencing of the tcdC gene. Fourteen isolates were of PCR ribotype 027 with deletions in tcdC, but no association with severity or clinical outcome was found. We show by immunodot blot detection of toxins with monoclonal antibodies that all R027 isolates produced more TcdA and TcdB than other strains. On the other hand, they consistently produced fewer spores than non-R027 isolates. Taken together, our data suggest that NAP1/027 isolates are not always associated with more severe disease, even though they may produce larger amounts of toxins. Our study also suggests that current assertions regarding the NAP1/027 may not apply to all isolates and that other factors may come into play.     

Molecular analysis of Clostridium difficile at a university teaching hospital in Japan: a shift in the predominant type over a five-year period

Clostridium difficile isolates recovered from patients admitted to a teaching hospital in Japan over a 5-year period were analyzed. Two molecular typing systems, PCR ribotyping and pulsed-field gel electrophoresis (PFGE) analysis, were used. Twenty-six PCR ribotypes were found among the 148 isolates. The predominant type at our hospital appeared to shift during the study period, from PCR ribotype a in 2000 (15/33, 45%) to PCR ribotype f in 2004 (18/28, 64%). By using PFGE with thiourea added to both the gel and running buffer, all 148 Clostridium difficile isolates were successfully classified into 37 types and 61 subtypes. The PCR ribotype f isolates were further classified into four types and 11 subtypes by PFGE. The PFGE patterns of the 11 subtypes differed from each other by only 1 to 4 bands, suggesting that these differences might reflect genetic changes during patient-to-patient transmission over the 5-year period analyzed, and that PCR ribotype f isolates might be outbreak-related. In addition, the PCR ribotype f was identical to the PCR ribotype designated smz, which is reported to have caused multiple outbreaks in Japan. These results confirmed that PCR ribotype f (type smz) has specific virulence or survival factors that make it more likely to cause nosocomial outbreaks at Japanese hospitals. PCR ribotype 027, which has been reported to have caused recent outbreaks in North America and Europe, was recovered from one patient in this study; however, this strain was community-acquired. Our findings emphasize the importance of monitoring specific strains to control and prevent nosocomial infection.     

Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays

Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.     

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.     

Increased sporulation rate of epidemic Clostridium difficile Type 027/NAP1

Clostridium difficile PCR ribotype 027 comprised 0.2% of a collection of Swedish isolates in 1997-2001 (3 of 1,325 isolates). These isolates had lower moxifloxacin MICs than the epidemic type 027 isolates, but they had the same tcdC sequence and toxin yield. Type 027 produced 3- to 13-fold more toxin than did major Swedish types. One epidemic strain (027/NAP1a) sporulated more than did other type 027 isolates, a feature that should contribute to its survival and spread.     

Molecular typing and long-term comparison of clostridium difficile strains by pulsed-field gel electrophoresis and PCR-ribotyping

Thirty-two related and 68 unrelated isolates of Clostridium difficile, isolated in different Italian hospitals since 1987, were analysed by PFGE and PCR-ribotyping to investigate their genetic relatedness. The isolates were classified into 28 groups by PFGE and 20 ribotypes by PCR-ribotyping. A single clone of C. difficile was recognised as the cause of three geographically and chronologically distant outbreaks. The correlation between PFGE and PCR-ribotyping results was good, with agreement for 77 (84%) of the 92 isolates typed by both methods. However, among sporadic isolates the discriminatory power of PFGE was more evident. Eight isolates that were untypable by PFGE could be analysed by PCR-ribotyping. The dendrograms generated showed that the genetic relatedness of the C. difficile isolates obtained by both techniques was comparable. The majority of the isolates in recent years appeared to be genetically unrelated to isolates from past infections. However, two clonal groups identified in all time periods had a common origin and this seems to indicate that they share some advantageous biological characteristics. The constant monitoring of C. difficile epidemiology will allow acquisition of further important data on this nosocomial pathogen.     

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.     

Analysis of Clostridium difficile isolates from nosocomial outbreaks at three hospitals in diverse areas of Japan

Clostridium difficile isolates recovered from patients with C. difficile-associated diarrhea (CDAD) at three hospitals located in diverse areas of Japan were analyzed by three typing systems, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), and Western immunoblotting. At the three hospitals examined, a single PCR ribotype strain (type smz) was predominant and accounted for 22 (65%) of 34, 18 (64%) of 28, and 11 (44%) of 25 isolates, respectively. All of the 51 isolates that represented PCR ribotype smz were nontypeable by PFGE because of DNA degradation. Since the type smz strain did not react with any of the antisera against 10 different serogroups (A, B, C, D, F, G, H, I, K, and X), we prepared a new antiserum against a type smz isolate. All 51 type smz isolates presented identical banding patterns, reacting with the newly prepared antiserum (designated subserogroup JP-0 of serogroup JP). These results were compared with those of a strain from a hospital outbreak that occurred in New York, which has been identified as type J9 by restriction enzyme analysis and type 01/A by arbitrarily primed PCR but was nontypeable by PFGE because of DNA degradation. This strain was reported to be epidemic at multiple hospitals in the United States. The J9 strain represented a PCR ribotype pattern different from that of a type smz strain and was typed as subserogroup G-1 of serogroup G by immunoblot analysis. A single outbreak type causing nosocomial CDAD in Japan was found to be different from the strain causing multiple outbreaks in the United States, even though the outbreak strains from the two countries were nontypeable by PFGE because of DNA degradation.     

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.     

Molecular epidemiology of endemic Clostridium difficile infection and the significance of subtypes of the United Kingdom epidemic strain (PCR ribotype 1)

We previously identified two subtypes of the epidemic strain Clostridium difficile PCR ribotype 1, one clindamycin-sensitive strain (arbitrarily primed PCR [AP-PCR] type Ia) and a closely related clindamycin-resistant strain (AP-PCR type Ib) in our institution. We have now carried out prospective epidemiological surveillance for 4 years, immediately following the relocation of two acute medicine wards for elderly patients (wards A and B), to determine the clinical epidemiology of subtypes of the epidemic C. difficile PCR ribotype 1 group. To maximize the chance of strain discrimination, we used three DNA fingerprinting methods, AP-PCR, ribospacer PCR (RS-PCR), and pulsed-field gel electrophoresis (PFGE), to analyze C. difficile isolates recovered from symptomatic patients and from repeated environmental samplings. On ward B the incidence of C. difficile infection correlated significantly with the prevalence of environmental C. difficile both in ward areas closely associated with patients and health care personnel (r = 0.53; P < 0.05) and in high-reach sites (r = 0.85; P < 0.05). No such relationships were found on ward A. Seventeen distinct C. difficile genotypes were identified, 17 by AP-PCR, 12 by PFGE, and 11 by RS-PCR, but only 4 of 17 genotypes caused patient infection. Isolates recovered from the hospital ward environment were much more diverse (14 genotypes). AP-PCR type Ia represented >90% of the C. difficile isolates. In addition to this genotype, only two others were isolated from both patient feces and environmental surfaces. AP-PCR type Ib (clindamycin-resistant PCR ribotype 1 clone) was not associated with any cases of C. difficile infection and was isolated from the environment on only two occasions, after having been implicated in a cluster of six C. difficile infections 5 months before this study. The disappearance of this strain implies that differences in virulence and/or selective pressures may exist for this strain and the closely related, widespread C. difficile AP-PCR type Ia strain. Our findings emphasize the need to understand the epidemiology and virulence of clinically significant strains to determine successful control measures for C. difficile infections.     

Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.     

Comparison of Multilocus Sequence Typing and the Xpert C. difficile/Epi Assay for Identification of Clostridium difficile 027/NAP1/BI

Clostridium difficile 027/NAP1/BI is the most common C. difficile strain in the United States. The Xpert C. difficile/Epi assay allows rapid, presumptive identification of C. difficile NAP1. We compared Xpert C. difficile/Epi to multilocus sequence typing for identification of C. difficile NAP1 and found “very good” agreement at 97.9% (κ = 0.86; 95% confidence interval, 0.80 to 0.91).     

First isolation of Clostridium difficile PCR ribotype 027 from a patient with severe persistent diarrhoea in Hungary

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 in Dutch animals: their presence, characteristics and similarities with human isolates

The presence and characteristics of Clostridium difficile were investigated in 839 faecal samples from seven different animal species in the Netherlands. The number of positive samples ranged from 3.4% (cattle) to 25.0% (dogs). Twenty-two different PCR ribotypes were identified. Among 96 isolates, 53% harboured toxin genes. All C. difficile isolates from pigs, cattle and poultry were toxinogenic, whereas the majority of isolates from pet animals consisted of non-toxinogenic PCR ribotypes 010 and 039. Ribotype 012 was most prevalent in cattle and ribotype 078 in pigs. No predominant ribotypes were present in horse and poultry samples. Overall, PCR ribotypes 012, 014 and 078 were the most frequently recovered toxinogenic ribotypes from animal samples. Comparison with human isolates from the Dutch Reference Laboratory for C. difficile at Leiden University Medical Centre (LUMC) showed that these types were also recovered from human hospitalized patients in 2009/2010, encompassing 0.8%, 11.4% and 9.8% of all isolates, respectively. Application of multiple-locus variable-number tandem-repeat analysis indicated a genotypic relation of animal and human ribotype 078 strains, but a clear genotypic distinction for ribotypes 012 and 014. We conclude that toxinogenic C. difficile PCR ribotypes found in animals correspond to PCR ribotypes associated with human disease in hospitalized patients in the Netherlands. Contrary to PCR ribotype 078, significant genetic differences were observed between animal and human PCR ribotype 012 and 014 isolates.     

International typing study of toxin A-negative,toxin B-positive Clostridium difficile variants

Clinically important strains of Clostridium difficile that do not produce toxin A but produce toxin B and are cytotoxic (A(-)/B(+)) have been reported from multiple countries. In order to compare the relatedness of these strains, we typed 23 A(-)/B(+) C. difficile isolates from the United Kingdom (6 isolates), Belgium (11 isolates), and the United States (6 isolates) by three well-described typing methods. Restriction endonuclease analysis (REA), PCR ribotyping, and serogrouping differentiated 11, 4, and 3 different strain types, respectively. Twenty-one of the 23 A(-)/B(+) variants had a 1.8-kb truncation of the toxin A gene characteristic of toxinotype VIII strains; 20 of the 21 toxinotype VIII-like strains were PCR type 17. PCR type 17 isolates could be differentiated into two separate strain groups by serogrouping and by REA. REA further discriminated these isolates into eight subgroups (REA types). PCR type 17-serogroup F-REA group CF isolates were recovered from all three countries, and one specific REA type, CF4, was recovered from patients with C. difficile disease in the United Kingdom and the United States. C. difficile A(-)/B(+) variants of apparent clonal origin are widely distributed in Europe and North America.     

Characterization of Clostridium difficile strains isolated from patients in Ontario, Canada, from 2004 to 2006

Clostridium difficile is the bacterium most commonly surmised to cause antimicrobial- and hospital-associated diarrhea in developed countries worldwide, and such infections are thought to be increasing in frequency and severity. A laboratory-based study was carried out to characterize C. difficile strains isolated from persons in Ontario, Canada, during 2004 to 2006 according to toxin type (enterotoxin A, cytotoxin B, and binary toxin [CDT]), tcdC gene characterization, ribotyping, pulsed-field gel electrophoresis, and toxinotyping. Clostridium difficile was isolated from 1,080/1,152 (94%) samples from 21 diagnostic laboratories. Isolates with toxin profiles A⁺ B⁺ CDT⁻, A⁺ B⁺ CDT⁺, A⁻ B⁺ CDT⁻, and A⁻ B⁺ CDT⁺ accounted for 63%, 34%, 2.4%, and 0.6% of isolates, respectively. Alterations in tcdC were detected in six different ribotypes, including ribotype 027. A total of 39 different ribotypes were identified, with ribotype 027/North American pulsotype 1 (NAP1), an internationally recognized outbreak strain associated with severe disease, being the second most common ribotype (19% of isolates). Transient resistance to metronidazole was identified in 19 (1.8%) isolates. While a large number of ribotypes were found, a few predominated across the province. The high prevalence and wide distribution of ribotype 027/NAP1 are disconcerting in view of the severity of disease associated with it.     

Prevalence of the Clostridium difficile BI/NAP1/027 strain across the United States Veterans Health Administration

ObjectivesThe increased incidence and severity of Clostridium difficile infection (CDI) are thought to result partly from the emergence of the hypervirulent BI/NAP1/027 strain. Limited recent data are available on the prevalence of BI/NAP1/027 in the United States (US). The objective of this study was to assess the recent prevalence of BI/NAP1/027 within the US Veterans Health Administration (VHA).MethodsPatients with CDI at any Veterans Affairs Medical Center found to routinely test for the presence of BI/NAP1/027 during the study period were included between 1 June 2011 and 30 June 2016 in this retrospective, observational, nationwide study.ResultsIn total, 7571 patients had 8224 positive C. difficile tests that had a corresponding BI/NAP1/027 test. Of those, there were 1810 (22.0%) presumptive positive for BI/NAP1/027. The overall prevalence of BI/NAP1/027 decreased from a high of 26.2% in 2013 to 16.9% in 2016. Statistically significant reductions in rates from 2012 to 2016 occurred in seven of nine US Census Bureau regions.ConclusionsThe prevalence of C. difficile with the BI/NAP1/027 strain was 22.0% across the VHA between 2012 and 2016. Further studies are needed to confirm these results and for continued monitoring of the trends in BI/NAP1/027 prevalence.     

Detection of virulence genes of Clostridium difficile by multiplex PCR

Antikainen J, Pasanen T, Mero S, Tarkka E, Kirveskari J, Kotila S, Mentula S, Könönen E, Virolainen‐Julkunen A‐R, Vaara M, Tissari P. Detection of virulence genes of Clostridium difficile by multiplex PCR. APMIS 2009; 117: 607–13. Clostridium difficile strains belonging to the PCR ribotype 027, pulse‐field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027.     

Comparison of resistance against erythromycin and moxifloxacin, presence of binary toxin gene and PCR ribotypes in Clostridium difficile isolates from 1990 and 2008

Worldwide increasing rates of Clostridium difficile infections (CDI) with severe courses and outbreaks have been reported. This change in CDI epidemiology has on one hand been related to the spread of specific PCR ribotypes (e.g. 027) and on the other hand to increased prevalence of resistant C. difficile strains. This single-centre retrospective analysis characterized resistance against erythromycin and moxifloxacin, presence of binary toxin gene and ribotypes in 73 C. difficile isolates from 2008 in comparison with 23 isolates from 1990. In 1990, five different PCR ribotypes including 027 were identified. Resistance against erythromycin was detected in 3 of 23 (13%), while 20 of 23 (87%) from all isolates were susceptible to both erythromycin and moxifloxacin. In contrast, in 2008 a significantly increased prevalence of resistant C. difficile strains was observed, with 40 of 73 (54.8%) isolates being resistant against both antibiotics. Resistant C. difficile strains were mainly assigned to PCR ribotype 001. No isolates belonging to PCR ribotype 027 were identified. Our data provide evidence that the increase of resistant C. difficile strains belonging to PCR ribotype 001 rather than the spread of C. difficile PCR ribotype 027 contribute to the changing epidemiology of CDI.     

Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile

Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C. difficile infections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methods-AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)-to differentiate 99 strains of C. difficile that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile.