Background. Studies of Clostridium difficile outbreaks suggested that certain ribotypes (eg, 027 and 078) cause more severe disease than other ribotypes. A growing number of studies challenge the ...validity of this hypothesis. Methods. We conducted a cross-sectional study of C. difficile infection (CDI) to test whether ribotype predicted clinical severity when adjusted for the influence of other predictors. Toxigenic C. difficile isolates were cultured from stool samples, screened for genes encoding virulence factors by polymerase chain reaction (PCR) and ribotyped using high-throughput, fluorescent PCR ribotyping. We collected data for 15 covariates (microbiologie, epidemiologic, and laboratory variables) and determined their individual and cumulative influence on the association between C. difficile ribotype and severe disease. We then validated this influence using an independent data set. Results. A total of 34 severe CDI cases were identified among 310 independent cases of disease (11.0%). Eleven covariates, including C. difficile ribotype, were significant predictors of severe CDI in unadjusted analysis. However, the association between ribotypes 027 and 078 and severe CDI was not significant after adjustment for any of the other covariates. After full adjustment, severe cases were significantly predicted only by patients' white blood cell count and albumin level. This result was supported by analysis of a validation data set containing 433 independent CDI cases (45 severe cases; 10.4%). Conclusions. Ribotype is not a significant predictor of severe CDI when adjusted for the influence of any other variables separately or in combination. White blood cell count and albumin level are the most clinically relevant predictors of severe CDI cases.
The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically ...dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes
Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for C. difficile spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.
A clinically reflective model of the human colon was used to investigate the effects of the broad-spectrum antibiotic omadacycline on the gut microbiome and the subsequent potential to induce ...simulated
infection (CDI). Triple-stage chemostat gut models were inoculated with pooled human fecal slurry from healthy volunteers (age, ≥60 years). Models were challenged twice with 10
CFU
spores (PCR ribotype 027). Omadacycline effects were assessed in a single gut model. Observations were confirmed in a parallel study with omadacycline and moxifloxacin. Antibiotic instillation was performed once daily for 7 days. The models were observed for 3 weeks postantibiotic challenge. Gut microbiota populations and
total viable and spore counts were enumerated daily by culture. Cytotoxin titers and antibiotic concentrations were also measured. Gut microbiota populations were stable before antibiotic challenge. Moxifloxacin instillation caused an ∼4 log
CFU/ml decline in enterococci and
group populations and an ∼3 log
CFU/ml decline in bifidobacteria and lactobacilli, followed by simulated CDI (vegetative cell proliferation and detectable toxin). In both models, omadacycline instillation decreased populations of bifidobacteria (∼8 log
CFU/ml),
group populations (7 to 8 log
CFU/ml), lactobacilli (2 to 6 log
CFU/ml), and enterococci (4 to 6 log
CFU/ml). Despite these microbial shifts, there was no evidence of
bacteria germination or toxin production. In contrast to moxifloxacin, omadacycline exposure did not facilitate simulated CDI, suggesting this antibiotic may have a low propensity to induce CDI in the clinical setting.
Clostridium difficile is an important enteric pathogen of humans and the cause of diarrhea and enteritis in neonatal pigs. Outside Australia, prevalence in piglets can be up to 73%, with a single PCR ...ribotype (RT), 078, predominating. We investigated the prevalence and genotype of C. difficile in Australian pig herds. Rectal swabs (n = 229) were collected from piglets aged <7 days from 21 farms across Australia. Selective culture for C. difficile was performed and isolates characterized by PCR for toxin genes and PCR ribotyping. C. difficile was isolated from 52% of samples by direct culture on chromogenic agar and 67% by enrichment culture (P = 0.001). No association between C. difficile recovery or genotype and diarrheic status of either farm or piglets was found. The majority (87%; 130/154) of isolates were toxigenic. Typing revealed 23 different RTs, several of which are known to cause disease in humans, including RT014, which was isolated most commonly (23%; 36/154). RT078 was not detected. This study shows that colonization of Australian neonatal piglets with C. difficile is widespread in the herds sampled.
Clostridium difficile infection is the leading cause of health care-associated diarrhea, and the bacterium can also be carried asymptomatically. The objective of this study was to identify host and ...bacterial factors associated with health care-associated acquisition of C. difficile infection and colonization.
We conducted a 15-month prospective study in six Canadian hospitals in Quebec and Ontario. Demographic information, known risk factors, potential confounding factors, and weekly stool samples or rectal swabs were collected. Pulsed-field gel electrophoresis (PFGE) was performed on C. difficile isolates to determine the genotype. Levels of serum antibodies against C. difficile toxins A and B were measured.
A total of 4143 patients were included in the study; 117 (2.8%) and 123 (3.0%) had health care-associated C. difficile infection and colonization, respectively. Older age and use of antibiotics and proton-pump inhibitors were significantly associated with health care-associated C. difficile infection. Hospitalization in the previous 2 months; use of chemotherapy, proton-pump inhibitors, and H(2) blockers; and antibodies against toxin B were associated with health care-associated C. difficile colonization. Among patients with health care-associated C. difficile infection and those with colonization, 62.7% and 36.1%, respectively, had the North American PFGE type 1 (NAP1) strain.
In this study, health care-associated C. difficile infection and colonization were differentially associated with defined host and pathogen variables. The NAP1 strain was predominant among patients with C. difficile infection, whereas asymptomatic patients were more likely to be colonized with other strains. (Funded by the Consortium de Recherche sur le Clostridium difficile.).
In 2009 the first European Society of Clinical Microbiology and Infection (ESCMID) treatment guidance document for Clostridium difficile infection (CDI) was published. The guideline has been applied ...widely in clinical practice. In this document an update and review on the comparative effectiveness of the currently available treatment modalities of CDI is given, thereby providing evidence-based recommendations on this issue. A computerized literature search was carried out to investigate randomized and non-randomized trials investigating the effect of an intervention on the clinical outcome of CDI. The Grades of Recommendation Assessment, Development and Evaluation (GRADE) system was used to grade the strength of our recommendations and the quality of the evidence. The ESCMID and an international team of experts from 11 European countries supported the process. To improve clinical guidance in the treatment of CDI, recommendations are specified for various patient groups, e.g. initial non-severe disease, severe CDI, first recurrence or risk for recurrent disease, multiple recurrences and treatment of CDI when oral administration is not possible. Treatment options that are reviewed include: antibiotics, toxin-binding resins and polymers, immunotherapy, probiotics, and faecal or bacterial intestinal transplantation. Except for very mild CDI that is clearly induced by antibiotic usage antibiotic treatment is advised. The main antibiotics that are recommended are metronidazole, vancomycin and fidaxomicin. Faecal transplantation is strongly recommended for multiple recurrent CDI. In case of perforation of the colon and/or systemic inflammation and deteriorating clinical condition despite antibiotic therapy, total abdominal colectomy or diverting loop ileostomy combined with colonic lavage is recommended.
Clostridium difficile is now considered to be one of the most important causes of health care-associated infections. C. difficile infections are also emerging in the community and in animals used for ...food, and are no longer viewed simply as unpleasant complications that follow antibiotic therapy. Since 2001, the prevalence and severity of C. difficile infection has increased significantly, which has led to increased research interest and the discovery of new virulence factors, and has expanded and focused the development of new treatment and prevention regimens. This Review summarizes the recent epidemiological changes in C. difficile infection, our current knowledge of C. difficile virulence factors and the clinical outcomes of C. difficile infection.
A robust high-throughput multilocus sequence typing (MLST) scheme for Clostridium difficile was developed and validated using a diverse collection of 50 reference isolates representing 45 different ...PCR ribotypes and 102 isolates from recent clinical samples. A total of 49 PCR ribotypes were represented overall. All isolates were typed by MLST and yielded 40 sequence types (STs). A web-accessible database was set up (http://pubmlst.org/cdifficile/) to facilitate the dissemination and comparison of C. difficile MLST genotyping data among laboratories. MLST and PCR ribotyping were similar in discriminatory abilities, having indices of discrimination of 0.90 and 0.92, respectively. Some STs corresponded to a single PCR ribotype (32/40), other STs corresponded to multiple PCR ribotypes (8/40), and, conversely, the PCR ribotype was not always predictive of the ST. The total number of variable nucleotide sites in the concatenated MLST sequences was 103/3,501 (2.9%). Concatenated MLST sequences were used to construct a neighbor-joining tree which identified four phylogenetic groups of STs and one outlier (ST-11; PCR ribotype 078). These groups apparently correlate with clades identified previously by comparative genomics. The MLST scheme was sufficiently robust to allow direct genotyping of C. difficile in total stool DNA extracts without isolate culture. The direct (nonculture) MLST approach may prove useful as a rapid genotyping method, potentially benefiting individual patients and informing hospital infection control.
Background. An epidemic strain of Clostridium difficile designated by restriction endonuclease analysis (REA) as group BI has caused multiple outbreaks of severe C. difficile infection (CDI). The ...treatment response of patients infected with this strain is uncertain. Methods. Clostridium difficile isolates were collected from 2 phase 3 clinical trials comparing fidaxomicin to vancomycin and typed using REA. Clinical cure and recurrence outcomes were analyzed by strain type of the infecting organism, BI and non-BI, using both univariate and multivariate analyses. Results. From 999 patients, 719 isolates were available for typing (356 fidaxomicin treated and 363 vancomycin treated). BI was the most common REA group (34% of isolates). Patients infected with BI had lower cure rates (86.6%; 214 of 247) than those infected with non-BI strains (94.3%; 445 of 472) (P < .001). The cure rate difference between the BI and non-BI patients was significant for both vancomycin (P = .02) and fidaxomicin (P = .007). BI patients had a recurrence rate of 27.4% (51 of 186), compared with a recurrence rate of 16.6% (66 of 397) in non-BI patients (P = .002). By multivariate analysis, BI infection was statistically significant as a risk factor for reduced cure (odds ratio OR, 0.48; 95% confidence interval CI, .27—.85; P = .030) and for increased recurrence (OR, 1.57; 95% CI, 1.01—2.45; P = .046). Conclusions. The clinical cure rate of patients infected with the epidemic BI C. difficile strain is lower than the cure rate of those infected with non-BI strains whether treated with fidaxomicin or vancomycin. Similarly, the CDI recurrence rate is increased in patients with the BI strain compared with patients with other C. difficile strains.
The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors ...by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile.
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