Torbjörn Norén

Increased Sporulation Rate of Epidemic Clostridium difficile Type 027/NAP1

ABSTRACT 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 Epidemiology of Hospital-Associated and Community-Acquired Clostridium difficile Infection in a Swedish County

ABSTRACT All episodes of Clostridium difficile associated diarrhea (CDAD) diagnosed in a defined population of 274,000 including one tertiary and two primary hospitals and their catchment areas were studied during 12 months. The annual CDAD incidence in the county was 97 primary episodes per 100,000, and 78% of all episodes were classified as hospital associated with a mean incidence of 5.3 (range, 1.4 to 6.5) primary episodes per 1,000 admissions. The incidence among hospitalized individuals was 1,300-fold higher than that in the community (33,700 versus 25 primary episodes per 100,000 persons per year), reflecting a 37-fold difference in antibiotic consumption (477 versus 13 defined daily doses [DDD]/1,000 persons/day) and other risk factors. Three tertiary hospital wards with the highest incidence (13 to 36 per 1,000) had CDAD patients of high age (median age of 80 years versus 70 years for other wards, P < 0.001), long hospital stay (up to 25 days versus 4 days), or a high antibiotic consumption rate (up to 2,427 versus 421 DDD/1,000 bed days). PCR ribotyping of C. difficile isolates available from 330 of 372 CDAD episodes indicated nosocomial acquisition of the strain in 17 to 27% of hospital-associated cases, depending on the time interval between index and secondary cases allowed (2 months or up to 12 months), and only 10% of recurrences were due to a new strain of C. difficile (apparent reinfection). In other words, most primary and recurring episodes were apparently caused by the patients endogenous strain rather than by one of hospital origin. Typing also indicated that a majority of C. difficile strains belonged to international serotypes, and the distribution of types was similar within and outside hospitals and in primary and relapsing CDAD. However, type SE17 was an exception, comprising 22% of hospital isolates compared to 6% of community isolates (P = 0.008) and causing many minor clusters and a silent nosocomial outbreak including 36 to 44% of the CDAD episodes in the three high-incidence wards.

Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for Clostridium difficile Detection Challenges Cytotoxin B Cell Test and Culture as Gold Standard

ABSTRACT Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings.

In vitro susceptibility to 17 antimicrobials of clinical Clostridium difficile isolates collected in 1993–2007 in Sweden

This study investigated the MICs of 17 antimicrobials, for 606 toxigenic clinical isolates of Clostridium difficile collected between 1993 and 2007 in Sweden. Low MIC(90) values were found for metronidazole (0.5 mg/L), vancomycin (1.0 mg/L), teicoplanin (0.125 mg/L), fusidic acid (1.0 mg/L), linezolid (2.0 mg/L), daptomycin (2.0 mg/L) and tigecycline (0.064 mg/L). Three isolates (0.5%) had elevated MICs for vancomycin (4-8 mg/L); however, these isolates originated from the same patient, who was receiving long-term intravenous vancomycin treatment. High-level clindamycin resistant isolates (MIC >256 mg/L) peaked in 1997 with 39 of 95 (41%) and out of these, 36% were also highly resistant to erythromycin. beta-Lactams such as penicillin V and piperacillin displayed MIC(90)s of 8 and 32 mg/L, respectively, whereas MICs of cefuroxime were >256 mg/L for all isolates. Universal resistance to ciprofloxacin and levofloxacin was found, and resistance to moxifloxacin increased from 4% of isolates in 2004 to 23% in 2007. Notably, these moxifloxacin-resistant isolates did not belong to the recent epidemic PCR ribotype 027, but to the pre-existing epidemic type 012 (82%), and these isolates accounted for the majority of isolates that were resistant to clindamycin (70%), tetracycline (84%) and rifampicin (92%) as well. This investigation of susceptibility data on clinical C. difficile isolates showed variations of multiresistance to be due to a specific PCR ribotype 012, emphasizing the importance of genotyping when evaluating emerging resistance over time.

Frequent Emergence of Resistance in Clostridium difficile during Treatment of C. difficile-Associated Diarrhea with Fusidic Acid

ABSTRACT Samples from patients with Clostridium difficile-associated diarrhea (CDAD) that were randomized to fusidic acid (n = 59) or metronidazole (n = 55) therapy for 7 days were cultured for Clostridium difficile in feces on days 1, 8 to 13, and 35 to 40. Of the patients who were culture positive only before treatment, 77% (36/47) were permanently cured (no treatment failure and no clinical recurrence), compared to 54% (22/41) of those with persistence of C. difficile at one or both follow-ups (P = 0.03). A similar association between bacterial persistence and a worse outcome of therapy was seen in both treatment groups. Resistance to fusidic acid was found in 1 of 88 pretherapy isolates available, plus in at least 1 subsequent isolate from 55% (11/20) of patients who remained culture-positive after fusidic acid therapy. In 10 of these 11 patients, the resistant follow-up isolate(s) belonged to the same PCR ribotype as the susceptible day 1 isolate, confirming frequent emergence of resistance to fusidic acid during treatment. Despite this, 5 of these 11 patients were permanently cured with fusidic acid, relative to 5 of 9 patients with susceptible C. difficile at follow-up (P = 1.0). None of the 36 PCR ribotypes of C. difficile identified was associated with any particular clinical outcome or emergence of fusidic acid resistance. In conclusion, culture positivity for C. difficile was common after both fusidic acid and metronidazole therapy and was associated with treatment failure or recurrence of CDAD. Development of resistance in C. difficile was frequent in patients given fusidic acid, but it was without apparent negative impact on therapeutic efficacy in the actual CDAD episode.

Clostridium difficile and the Disease It Causes

Clostridium difficile is a spore-forming, toxin-producing, anaerobic bacterium abundant in soils and water. Frequent and early colonization of the human intestinal flora is common and often asymptomatic. Antimicrobials given commonly disrupt the intestinal microflora and through proliferation in colon and production of toxin A and B it precipitates C. difficile infection (CDI). The enterocytic detachment and bowel inflammation provoke C. difficile-associated diarrhoea (CDAD) sometimes developing into severe pseudomembranous colitis (PMC) and paralytic ileus. Infection is acquired from an endogenous source or from spores in the environment, most easily facilitated during hospital stay. In the elderly, comorbidity, hospitalization and antimicrobial treatment present as major risk factors and the slow recolonization of the normal flora likely responsible for single or multiple recurrences of CDI (25-50%) post therapy. The key procedure for diagnosis is toxin detection from stool specimens and sometimes in combination with culture to increase sensitivity. In mild cases stopping the offending antimicrobial will lead to resolution (25%) but standard therapy still consist of either oral metronidazole or vancomycin. Alternative agents are presently being developed and fidaxomicin, as well as nitrothiazolide are promising. Furthermore, host factors like low antitoxin A levels in serum relates to increased risk of recurrence and small numbers of patients have received immunoglobulin with good results. An immunogenic toxoid vaccine has been developed and human colostrum rich in specific secretory Ig A also support the future use of immunotherapy. Today we experience a tenfold increase of CDI incidence in the western world and both epidemics and therapeutic failure of metronidazole is contributing to morbidity and mortality. The current epidemic of the C. difficile strain NAP1/027 emerging in 2002 in Canada and the USA has now spread to most parts of Europe and virulence factors like high toxin production and sporulation challenge the therapeutic situation and cause great concern among infection control workers. Excessive use of modern fluoroquinolones is thought to play an important role in facilitating this epidemic since NAP1/027 was shown to have acquired moxifloxacin resistance compared to historical strains of the same genotype. Both the current epidemic like this and other local outbreaks from resistant or virulent strains warrant culture to be routinely performed enabling susceptibility testing and typing of the pathogen. Genotyping is most commonly done today by pulse-field gel electrophoresis (PFGE) or PCR ribotyping but multilocus variable-number tandem-repeat analysis (MLVA) seems promising. Epidemiological surveillance using all these tools will help us to better understand the global spread of C. difficile.

Novel Molecular Type of Clostridium difficile in Neonatal Pigs, Western Australia

Clostridium difficile causes neonatal enteritis in piglets; strains of PCR ribotype 078 are most commonly identified. We investigated C. difficile prevalence in piglets in Australia and isolated a novel strain with a unique pathogenicity locus. In a mouse infection model, this strain produced more weight loss than did a ribotype 078 strain.

Clostridium difficile PCR ribotype 046 is common among neonatal pigs and humans in Sweden

Clostridium difficile PCR ribotype 046 was found in 67% of neonatal piglets (45/67) sampled from three separate pig-breeding farms in Sweden. Sows from the same farms were tested and 50% were colonized in faeces and 30% were colonized on skin. An environmental source was suggested because identical PCR ribotypes were isolated from faeces as well as externally. Human C. difficile infection outbreaks in southern Sweden by the identical PCR ribotype 046 indicate its zoonotic potential.

IgG Antibody Response to Toxins A and B in Patients with Clostridium difficile Infection.

ABSTRACT IgG antibodies against Clostridium difficile toxins A and B were followed in controls and in patients with an initial C. difficile infection (CDI). Of the 50 CDI patients, 38 were cured and 12 developed recurrence. Compared to controls, patients had significantly lower anti-toxin A and B IgGs at inclusion, but the subsequent levels rose slightly regardless of clinical outcome. The results imply that the general serum reactivity against toxins A and B in the population reduces the risk of CDI, which suggests implications for vaccine strategies.

Mutations in fusA Associated with Posttherapy Fusidic Acid Resistance in Clostridium difficile

ABSTRACT In silico, we identified fusA (2,067 bp) in Clostridium difficile 630. Sequencing of fusA in posttherapy fusidic acid-resistant C. difficile isolates from 12 patients with C. difficile-associated diarrhea (CDAD) identified fusA mutations, one or two nonsynonymous substitutions, or in one case a deletion of one codon associated with resistance. Five of these mutations have previously been described in fusA of fusidic acid-resistant Staphylococcus aureus, but seven were novel fusA mutations. Fusidic acid monotherapy for CDAD seemed to rapidly select conserved resistant mutants.