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.     

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.     

An outbreak of Clostridium difficile infections due to new PCR ribotype 826: epidemiologic and microbiologic analyses

ObjectivesTo investigate an unusual outbreak of five patients with a total of eight episodes of a Clostridium difficile infection on a gastrointestinal surgical ward of a Dutch tertiary-care, university-affiliated hospital.MethodsClinical case investigations and laboratory analyses were performed. Laboratory analyses included PCR ribotyping, multiple-locus variable-number tandem repeat analysis typing, toxin typing, antimicrobial susceptibility testing and whole genome sequencing.ResultsThe outbreak was associated with recurrent and severe disease in two of five patients. All episodes were due to a unique ribotype that was not recognized in the collection of an international network of reference laboratories and was assigned PCR ribotype 826. PCR ribotype 826 is a toxin A–, toxin B– and binary toxin–positive ribotype which according to molecular typing belongs to clade 5 and resembles the so-called hypervirulent ribotype 078. The presence of a clonal outbreak was confirmed by whole genome sequencing, yet the source of this newly identified ribotype remained unclear.ConclusionsThis newly identified C. difficile PCR ribotype 826 is part of clade 5 and might also have increased virulence. The recognition of this outbreak highlights the need for ongoing C. difficile infection surveillance to monitor new circulating ribotypes with assumed increased virulence.     

Impact of ribotype 027 on Clostridium difficile infection in a geriatric department

The purposes of this study were to describe the epidemiology (2001–2009) of Clostridium difficile infections (CDI) in a geriatric department and to compare the clinical data of patients infected with a 027 or non-027 strain. We retrospectively identified all geriatric patients with CDI and analysed the clinical and microbiological data of 133 patients for whom a ribotype was available between March 2003 and December 2009. The incidence of CDI in our geriatric department increased from 0.2 per 100 admissions in 2001 to 8.1 in 2004 and decreased to 1.3 in 2008 before a new rise to 2.1 in 2009. The percentage of ribotype 027 decreased from 2007 but it remained the most prevalent ribotype during the years 2007–2009, with a greater dispersion of ribotypes. The mean age of the patients was 84 years and the median Charlson index was 6.0. Previous use of fluoroquinolones was a significant risk factor for developing a CDI with an 027 strain (p?=?0.001). Cure was significantly lower in the 027 group (p?=?0.003). The total attributable mortality was 24.1 %. A multiparametric model showed that attributable mortality was influenced by the ribotype 027 (p?=?0.037), the severity of clinical symptoms (p?=?0.001) and the type of treatment (p?=?0.002). Oral vancomycin had a protective effect against mortality. Attention should be paid to elderly patients developing a CDI, especially after the administration of fluoroquinolones. Oral vancomycin could be recommended as the first-line agent not only to protect against recurrence or severe CDI, but to diminish the attributable mortality risk.     

The emergence of Clostridium difficile PCR ribotype 078 in piglets in the Czech Republic clusters with Clostridium difficile PCR ribotype 078 isolates from Germany,Japan and Taiwan

Clostridium difficile is a major nosocomial pathogen in humans with an increasing incidence in the community. The “one-health” approach of research is needed to investigate possible reservoirs of C. difficile and route of its transmission. The objective of this study is to investigate the occurrence of C. difficile in pigs in the Czech Republic with characterisation of the isolates to determine their genetic relatedness to C. difficile isolates from European and Asian pigs. A total of 198 pig faeces samples from 23 farms were investigated and of those 57 samples (55 piglets, 2 sows) from 11 farms were confirmed as C. difficile positive. The majority of C. difficile isolates belonged to the sequence type 11 and clade 5. The predominant ribotypes were 078 (n = 23), 078-variant (n = 5), 033 (n = 10) followed by RTs 150 (n = 7), 011 (n = 5), 045 (n = 4), 126, 014, 002 (n = 1, each). All isolates were susceptible to metronidazole, vancomycin and tetracycline. Isolates of RTs 150 and 078-variant were moxifloxacin resistant (MIC≥32 mg/L) and carried the amino acid substitution Thr82Ile in the GyrA. A multi-locus variable number tandem-repeats analysis (MLVA) revealed a clonal relatedness of isolates within individual farms and in C. difficile RT078 isolates between two Czech farms. Czech C. difficile RT078 isolates clustered with German C. difficile RT078 isolates and Czech C. difficile 078-variant isolates clustered with C. difficile RT078 isolates from Japan and Taiwan. This study found an emergence of C. difficile RT078 in Czech piglets that was related genetically to C. difficile RT078 isolates from Germany, Japan and Taiwan.     

The Clostridium difficile PCR ribotype 027 lineage: a pathogen on the move

Clostridium difficile is a Gram-positive, spore-forming, human and animal pathogen that is the major cause of antibiotic-associated diarrhoea worldwide. The past decade has seen the rapid emergence of the hypervirulent PCR ribotype (RT) 027 complex, which has been associated with increases in the incidence and severity of disease and mortality. In this review, we describe the potential virulence factors that have been reported in strains from the RT 027 complex. We review the emergence, population structure, dissemination and evolution of this lineage.     

An enhanced DNA fingerprinting service to investigate potential Clostridium difficile infection case clusters sharing the same PCR ribotype

Of 53 potential Clostridium difficile infection (CDI) case clusters/outbreaks, affecting 2 to 41 patients in 27 institutions, 19% comprised unrelated isolates and 34% had highly related and distinct isolates as shown by multilocus variable-number tandem-repeat analysis, despite sharing a common ribotype. These findings emphasize the value of enhanced fingerprinting to confirm or refute suspected CDI case clusters.     

Successful combat of an outbreak due to Clostridium difficile PCR ribotype 027 and recognition of specific risk factors

In the period April–September 2005, an outbreak of Clostridium difficile infection (CDI) due to PCR ribotype  027 occurred among 50 patients in a 341-bed community hospital in Harderwijk, The Netherlands. A retrospective case–control study was performed to identify risk factors specific for CDI, using a group of patients with CDI ( n  = 45), a group of randomly selected control patients without diarrhoea ( n  = 90), and a group of patients with non-infectious diarrhoea ( n  = 109). Risk factors for CDI and for non-CDI diarrhoea were identified using multiple logistic regression analysis. Independent risk factors for CDI were: age above 65 years (OR 2.6; 95% CI  1.0–5.7), duration of hospitalization (OR 1.04 per additional day; 95% CI  1.0–1.1), and antibiotic use (OR 12.5; 95% CI  3.2–48.1). Of the antibiotics used, cephalosporins and fluoroquinolones were identified as the major risk factors for development of CDI. The risk of developing CDI was particularly high in people receiving a combination of a cephalosporin and a fluoroquinolone (OR 57.5; 95% CI  6.8–483.6). The main factors affecting the risk of non-CDI diarrhoea were proton-pump inhibitors, immunosuppressive drugs, underlying digestive system disease, previous surgery, and gastric tube feeding. The outbreak ended only after implementation of restricted use of cephalosporins and a complete ban on fluoroquinolones, in addition to general hygienic measures, cohorting of patients in a separate ward, education of staff, and intensified environmental cleaning. The results of this study support the importance of appropriate antimicrobial stewardship in the control of hospital outbreaks with C. difficile PCR ribotype  027.     

Clostridium difficile infection in children hospitalized due to diarrhea

The frequency of Clostridium difficile infection (CDI)-related hospitalizations is increasing. The aim of this study was to determine the extent of CDI among children hospitalized with diarrhea, risk factors or predictors for severe CDI, the prevalence of NAP1, and to compare the course of CDI depending on bacteria toxicity profile. A retrospective analysis of case records of 64 children (age range 3 months–16 years, median age 2.12 years) with CDI as defined by diarrheal disease and positive polymerase chain reaction (PCR) test (Xpert C. difficile) was conducted. Modified national adult guidelines were used to assess the severity of CDI. CDIs represented 2.7 % of patients with diarrhea (13.5 cases per 1,000 admissions). Thirty-three CDIs (52 %) were community-associated. Antibacterial use preceded CDI in 61 patients (95 %). Seventeen cases (27 %) were binary toxin-positive (CDT+), 13 of which were NAP1 (20.5 %). Over 75 % of CDIs with NAP1 was hospital-acquired, and more often proceeded with generalized infection (p?<?0.05). Risk factors for severe CDI (34 %) included NAP1 [odds ratio (OR), 4.85; 95 % confidence interval (Cl), 1.23, 21.86) and co-morbidities (OR, 4.25; 95 % Cl, 1.34, 14.38). Diarrhea ≥10 stools daily was associated with severe CDI (p?=?0.01). Recurrence occurred in three patients (4.5 %). There was no mortality. C. difficile is an important factor of antibiotic-associated diarrhea in children. Co-morbidities and NAP1 predispose to severe CDI.     

Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany

Clostridium (Clostridioides) difficile is the main cause of nosocomial diarrhoea. Ribotype 018 (RT018) has been recognized as the predominant strain responsible for C. difficile infection (CDI) in Italy, whereas in most other European countries only sporadic RT018 cases occur.Between August and October 2015, a suspected C. difficile outbreak at two associated hospitals in Southern Germany was investigated by comprehensive molecular typing. Surprisingly, RT018 was detected in 9/82 CDI patients, which has never been described before in a German outbreak. Phenotypic analysis revealed fluoroquinolone and macrolide resistance. Genetic subtyping using multiple-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS) was performed and outbreak isolates were directly compared to sporadic German RT018 isolates and to epidemic ones from Milan, Northern Italy. Molecular typing confirmed a hospital outbreak with closely related RT018 isolates. Both, MLVA and WGS revealed high similarity of outbreak strains with epidemic isolates from Italy, but low similarity to other German isolates. Comparison between both typing strategies showed that ribotyping in combination with MLVA was appropriate to identify related isolates and clonal complexes, whereas WGS provided a better discrimination with more detailed information about the phylogenetic relationship of isolates. This is the first hospital outbreak in Germany presumably caused by cross-national transmission of an Italian epidemic RT018 strain.     

Molecular analysis of Clostridium difficile PCR ribotype 027 isolates from Eastern and Western Canada

The prevalence and characteristics of PCR ribotype 027 strains of Clostridium difficile have come into question following recent outbreaks in Eastern Canada and elsewhere. In order to determine the distribution of this strain in other regions in Canada, we screened a bank of 1,419 isolates recovered from three different Canadian health regions between 2000 and 2004. Among isolates from a Montreal area hospital, PCR ribotype 027 strains represented 115/153 strains (75.2%) from 2003 to 2004, but ribotype 027 strains were absent in 2000 and 2001. In Calgary, by contrast, ribotype 027 rates have remained relatively stable over 4 years of surveillance, representing 51/685 (7.4%) hospital isolates and 62/373 (16.6%) strains from the community (P < 0.001). PCR ribotype 027 accounted for 8/135 (5.9%) hospital isolates in the Fraser Health Region in 2004. repetitive extragenic palindromic PCR was used to subtype a random selection of 027 isolates from each region. All 10 of the isolates from Quebec were of a single subtype, which was also dominant among isolates from Alberta (8/10 isolates) and British Columbia (6/8 isolates). Comparative sequencing of the tcdC repressor gene confirmed the documented 18-bp deletion and identified a second, single-base-pair deletion at position 117. Both deletions were conserved across all three provinces and were identified in a United Kingdom reference strain. The presence of a frameshift in the early portion of the tcdC gene implies serious functional disruption and may contribute to the hypervirulence of the 027 phenotype. PCR ribotype 027 strains appear to be widely distributed, to predate the Montreal outbreak, and to have measurable community presence in Western Canada.     

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 isolates with increased sporulation: emergence of PCR ribotype 002 in Hong Kong

We identified a predominant clone of Clostridium difficile PCR ribotype 002, which was associated with an increased sporulation frequency. In 2009, 3,528 stool samples from 2,440 patients were tested for toxigenic C. difficile in a healthcare region in Hong Kong. A total of 345 toxigenic strains from 307 (13.3%) patients were found. Ribotype 002 was the predominant ribotype, which constituted 35 samples from 29 (9.4%) patients. The mean sporulation frequency of ribotype 002 was 20.2%, which was significantly higher than that of the 56 randomly selected ribotypes other than 002 as concurrent controls (3.7%, p?<?0.001). Patients carrying toxigenic ribotype 002 were more frequently admitted from an elderly home (p?=?0.01) and received more ??-lactam antibiotics in the preceding 3 months compared with the controls (p?=?0.04) . The identification of toxigenic ribotype 002 in 2009 was temporally related to a significant increase in both the incidence of toxigenic C. difficile from 0.53 to 0.95 per 1,000 admissions (p?<?0.001) and the rate of positive detection from 4.17% to 6.28% (p?<?0.001) between period 1 (2004?C2008) and period 2 (2009). This finding should alert both the physician and the infection control team to the establishment of and possible outbreaks by ribotype 002 in our hospitals, as in the case of ribotype 027.     

Clostridium difficile Isolates Resistant to Fluoroquinolones in Italy: Emergence of PCR Ribotype 018

Recent evidence strongly suggests an association between the use of fluoroquinolones and Clostridium difficile infection (CDI). Resistance to fluoroquinolones has been described not only in the hypervirulent strain 027, but also in other important PCR ribotypes circulating in hospital settings. In a European prospective study conducted in 2005, strains resistant to moxifloxacin represented 37.5% of C. difficile clinical isolates. In this study, we investigated a sample of 147 toxigenic C. difficile isolates, collected in Italy from 1985 to 2008, for the presence of mutations in gyr genes that conferred resistance to fluoroquinolones based on a LightCycler assay. Results were confirmed by the determination of MICs for moxifloxacin. Strains resistant to moxifloxacin were also investigated for resistance to three other fluoroquinolones and for a possible association between fluoroquinolone and macrolide-lincosamide-streptogramin B resistance. C. difficile isolates were typed by PCR ribotyping. In total, 50 clinical isolates showed substitutions in gyr genes and were resistant to fluoroquinolones. Ninety-six percent of the C. difficile resistant isolates showed the substitution Thr82-to-Ile in GyrA, as already observed in the majority of resistant strains worldwide. A significant increase of resistance (P < 0.001) was observed in the period 2002 to 2008 (56% resistant) compared to the period 1985 to 2001 (10% resistant). Coresistance with erythromycin and/or clindamycin was found in 96% (48/50) of the isolates analyzed and, interestingly, 84% of resistant strains were erm(B) negative. The majority of the fluoroquinolone-resistant isolates belonged to PCR ribotype 126 or 018. PCR ribotype 126 was the most frequently found from 2002 to 2005, whereas PCR ribotype 018 was predominant in 2007 and 2008 and still represents the majority of strains typed in our laboratory. Overall, the results demonstrate an increasing number of C. difficile strains resistant to fluoroquinolones in Italy and changes in the prevalence and type of C. difficile isolates resistant to fluoroquinolones circulating over time.Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacillus that may cause a spectrum of diseases, ranging from uncomplicated mild diarrhea to pseudomembranous colitis, collectively known as C. difficile infections (CDIs) (3, 39). The risk of CDI appears to be greater with certain antimicrobial agents and increases if strains are resistant to administered antimicrobials (30).Recently, the new hypervirulent C. difficile strain, typed as PCR ribotype 027, toxinotype III, pulsed-field gel electrophoresis pattern NAP1, has been associated with more severe and fatal cases in the United States, Canada, Japan, and Europe (4, 16, 18, 20, 21, 22). Strain 027 isolates are characterized by a hyperproduction of toxins A and B, production of binary toxin, and resistance to erythromycin and fluoroquinolones (12, 18, 34). Resistance to these antibiotics characterizes not only strain 027 but the majority of C. difficile strains circulating in hospital settings and responsible for disease (2, 34).The use of macrolide-lincosamide-streptogramin B (MLSB) antibiotics has long been known to be one of the major risk factors for CDI (15, 25). With C. difficile, resistance to these antibiotics, in particular to erythromycin and clindamycin, is due to an erm(B) gene carried by Tn5398, a mobile element that shows heterogeneous genetic organization. Nevertheless, an increased number of C. difficile erm(B)-negative isolates resistant to MLSB has been described, including strains characterized as PCR ribotype 027 (1, 19).Historically, fluoroquinolones were considered a low risk in C. difficile diseases, but recent evidence strongly suggests an association between their use and CDI (6, 24, 26). Resistance to fluoroquinolones has been described not only in the epidemic strain 027 but also in other important PCR ribotypes, and it is increasing, since recent data have shown that resistant strains represent 37.5% of C. difficile clinical isolates in Europe (2). Two main mechanisms of fluoroquinolone resistance have been described: alterations in the targets of antibiotics, DNA gyrase and topoisomerase IV, and decreased accumulation inside bacteria (13, 29). In C. difficile, as in many other bacterial species, resistance is determined by amino acid substitutions in the quinolone resistance-determining region (QRDR) of the target enzymes (27). Since this bacterium does not have genes for topoisomerase IV, these alterations are located in the QRDR of either GyrA or GyrB, the DNA gyrase subunits (11, 34). Recent studies indicated that the replacement of Thr82 with Ile in GyrA characterized 93% of European toxigenic C. difficile isolates resistant to fluoroquinolones, including the hypervirulent epidemic clone 027/NAP1/III (12, 34). The remaining 7% showed a substitution in position 426 (Asp to Asn or Val) of GyrB (34).Few data on fluoroquinolone resistance of C. difficile clinical isolates are available for Italy (34) and, for this reason, we analyzed a convenient sample of toxigenic isolates, collected by the Istituto Superiore di Sanità (ISS) from 1985 to 2008, for substitutions in GyrA and GyrB and for their MIC values to moxifloxacin. All C. difficile resistant strains were also tested for their resistance to ciprofloxacin, gatifloxacin, and levofloxacin and investigated for a possible association between fluoroquinolone and MLSB resistance. Typing of resistant isolates was carried out using the already-described PCR ribotyping method (5).