Multilocus variable-number tandem-repeat analysis and multilocus sequence typing reveal genetic relationships among Clostridium difficile isolates genotyped by restriction endonuclease analysis.

Numbers of Clostridium difficile infections have increased worldwide in the past decade. While infection with C. difficile remains predominantly a health care-associated infection, there may also be an increased incidence of community-associated infections. C. difficile strains of public health significance continue to emerge, and reliable genotyping methods for epidemiological investigations and global surveillance of C. difficile are required. In this study, multilocus sequence typing (MLST) and multilocus variable-number tandem-repeat analysis (MLVA) were performed on a set of 157 spatially and temporally diverse C. difficile isolates that had been previously genotyped by restriction endonuclease analysis (REA) to determine the concordance among these genotyping methods. In addition, sequence analysis of the tcdC genotype was performed to investigate the association of allelic variants with epidemic C. difficile isolates. Overall, the MLST and MLVA data were concordant with REA genotyping data. MLST was less discriminatory than either MLVA or REA, yet this method established C. difficile genetic lineage. MLVA was highly discriminatory and demonstrated relationships among the MLST genetic lineages and REA genotypes that were previously unrecognized. Several tcdC genotypes were specific to epidemic clones, highlighting the possible importance of toxin misregulation in C. difficile disease pathogenesis. This study demonstrates that a combination of MLST and MLVA may prove useful for the investigation and surveillance of emergent C. difficile clones of global public health concern.

High frequency of rifampin resistance identified in an epidemic Clostridium difficile clone from a large teaching hospital.

BACKGROUND: Rifampin is used as adjunctive therapy for Clostridium difficile-associated disease, and the drug's derivative, rifaximin, has emerged as an attractive antimicrobial for treatment of C. difficile-associated disease. Rifampin resistance in C. difficile strains has been reported to be uncommon. METHODS: We examined the prevalence of rifampin resistance among 470 C. difficile isolates (51.1% during 2001-2002 and 48.9% during 2005) from a large teaching hospital. Rifampin sensitivity was performed using E-test. The epidemic BI/NAP1 C. difficile clone was identified by tcdC genotyping and multilocus variable number of tandem repeats analysis. A 200-base pair fragment of the rpoB gene was sequenced for 102 isolates. Data on rifamycin exposures were obtained for all patients. RESULTS: Rifampin resistance was observed in 173 (36.8%) of 470 recovered isolates and 167 (81.5%) of 205 of epidemic clone isolates (P < .001). Six rpoB genotypes were associated with rifampin resistance. Of 8 patients exposed to rifamycins, 7 had rifampin-resistant C. difficile, compared with 166 of 462 unexposed patients (relative risk, 2.4; 95% confidence interval, 1.8-3.3). CONCLUSIONS: Rifampin resistance is common among epidemic clone C. difficile isolates at our institution. Exposure to rifamycins before the development of C. difficile-associated disease was a risk factor for rifampin-resistant C. difficile infection. The use of rifaximin may be limited for treatment of C. difficile-associated disease at our institution.

Control of an outbreak of infection with the hypervirulent Clostridium difficile BI strain in a university hospital using a comprehensive "bundle" approach.

BACKGROUND: In June 2000, the hospital-acquired Clostridium difficile (CD) infection rate in our hospital (University of Pittsburgh Medical Center-Presbyterian, Pittsburgh, PA) increased to 10.4 infections per 1000 hospital discharges (HDs); the annual rate increased from 2.7 infections per 1000 HDs to 7.2 infections per 1000 HDs and was accompanied by an increase in the frequency of severe outcomes. Forty-seven (51%) of 92 HA CD isolates in 2001 were identified as the "epidemic BI strain." A comprehensive CD infection control "bundle" was implemented to control the outbreak of CD infection. METHODS: The CD infection control bundle consisted of education, increased and early case finding, expanded infection-control measures, development of a CD infection management team, and antimicrobial management. Process measures, antimicrobial usage, and hospital-acquired CD infection rates were analyzed, and CD isolates were typed. RESULTS: The rates of compliance with hand hygiene and isolation were 75% and 68%, respectively. The CD management team evaluated a mean of 31 patients per month (11% were evaluated for moderate or severe disease). Use of antimicrobial therapy associated with increased CD infection risk decreased by 41% during the period 2003-2005 (P<.001). The aggregate rate of CD infection during the period 2001-2006 decreased to 4.8 infections per 1000 HDs (odds ratio, 2.2; 95% confidence interval, 1.4-3.1; P<.001) and by 2006, was 3.0 infections per 1000 HDs, a rate reduction of 71% (odds ratio, 3.5; 95% confidence interval, 2.3-5.4; P<.001). During the period 2000-2001, the proportion of severe CD cases peaked at 9.4% (37 of 393 CD infections were severe); the rate decreased to 3.1% in 2002 and further decreased to 1.0% in 2006--a 78% overall reduction (odds ratio, 20.3; 95% confidence interval, 2.8-148.2; P<.001). In 2005, 13% of CD isolates were type BI (20% were hospital acquired), which represented a significant reduction from 2001 (P<.001). CONCLUSIONS: The outbreak of CD infection with the BI strain in our hospital was controlled after implementing a CD infection control "bundle." Early identification, coupled with appropriate control measures, reduces the rate of CD infection and the frequency of adverse events.

Deletion of fetA gene sequences in serogroup B and C Neisseria meningitidis isolates.

The immunogenic iron-regulated FetA outer membrane protein of Neisseria meningitidis is one of various outer membrane proteins that have been considered potential meningococcal vaccine candidates. In this report, we describe the characterization of three meningococcal isolates that have deleted fetA sequences through genetic recombination at repetitive elements.

tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile.

Severe Clostridium difficile associated disease is associated with outbreaks of the recently described BI/NAP1 epidemic clone. This clone is characterized by an 18-bp deletion in the tcdC gene and increased production of toxins A and B in vitro. TcdC is a putative negative regulator of toxin A&B production. We characterized tcdC genotypes from a collection of C. difficile isolates from a hospital that experienced an outbreak caused by the BI/NAP1 epidemic clone. Sequence analysis of tcdC was performed on DNA samples isolated from 199 toxigenic C. difficile isolates (31% BI/NAP1) from 2001 and 2005. Sequences obtained from 36 (18.6%) isolates predicted wild-type TcdC (232 amino acid residues), whereas 12 (6.1%) isolates had tcdC genotypes with previously described 18- or 39-bp deletions. The remaining isolates comprised 15 unique genotypes. Of these, 5 genotypes contain 18- or 36-bp deletions. Of these five genotypes, one is characterized by a single nucleotide deletion at position 117 resulting in a frameshift that introduces a stop codon at position 196, truncating the predicted TcdC to 65 amino acid residues. All 62 of the isolates in this collection comprising the epidemic clone are characterized by this genotype. This result suggests that severe truncation of TcdC is responsible for the increased toxin production observed in strains belonging to the BI/NAP1 clone and that the 18-bp deletion is probably irrelevant to TcdC function. Further investigations are required to determine the effect of this and other tcdC genotypes on toxin production and clinical disease.

Multilocus variable-number tandem-repeat analysis for investigation of Clostridium difficile transmission in Hospitals.

Clostridium difficile is a major cause of antibiotic-associated gastrointestinal illness. Recently, an increased incidence of hospital-acquired infections with severe outcomes has been reported in North America and Europe. Current molecular-typing methods for detection of outbreaks and nosocomial transmission are labor-intensive, subjective, or insufficiently discriminatory to differentiate between closely related strains. This report describes the development of multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for molecular subtyping of C. difficile. Seven VNTR loci were identified from the C. difficile 630 genome by screening an isolate collection of various restriction endonuclease analysis (REA) types. The stability of the loci for short-term epidemiologic investigations was determined by performing MLVA on consecutive isolates of the same REA type from individual patients collected over as many as 90 days. Validation of MLVA for molecular genotyping was performed by direct comparison with REA results obtained from Hines Veterans Affairs Hospital on a combined collection of 40 C. difficile isolates from two different sources. The ability of MLVA to detect outbreaks was demonstrated on a collection of tertiary-care hospital isolates from a defined C. difficile outbreak in 2001. MLVA successfully clustered C. difficile isolates of the same REA type and discriminated isolates of unique REA type. Thus, MLVA is an objective, portable genotyping method that permits reliable detection of C. difficile outbreaks and can aid epidemiologic investigations of nosocomial transmission.