Clostridium difficile presence in Spanish and Belgian hospitals.

Clostridium difficile is recognised worldwide as the main cause of infectious bacterial antibiotic-associated diarrhoea in hospitals and other healthcare settings. The aim of this study was to first survey C. difficile prevalence during the summer of 2014 at the Central University Hospital of Asturias (Spain). By typing the isolates obtained, it was then possible to compare the ribotype distribution at the Spanish hospital with results from the St Luc University Hospital in Belgium over the same period. The prevalence of positive cases reported in Spain and Belgium was 12.3% and 9.3% respectively. The main PCR-ribotypes previously described in Europe were found in both hospitals, including 078, 014, 012, 020 and 002. In the Spanish hospital, most of the C. difficile-positive samples were referred from oncology, acute care and general medicine services. In the Belgian hospital the majority of positive samples were referred from the paediatric service. However, a high percentage of isolates from this service were non-toxigenic. This study finds that the presence and detection of C. difficile in paediatric and oncology services requires further investigation.

Investigation of Clostridium difficile interspecies relatedness using multilocus sequence typing, multilocus variable-number tandem-repeat analysis and antimicrobial susceptibility testing.

Multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA) and antimicrobial susceptibility were performed on 37 animal and human C. difficile isolates belonging to 15 different PCR-ribotypes in order to investigate the relatedness of human and animal isolates and to identify possible transmission routes. MLVA identified a total of 21 different types while MLST only distinguished 12 types. Identical C. difficile strains were detected in the same animal species for PCR-ribotypes 014, 078, UCL 16U and UCL 36, irrespective of their origin or the isolation date. Non clonal strains were found among different hosts; however, a high genetic association between pig and cattle isolates belonging to PCR-ribotype 078 was revealed. MLVA also showed genetic differences that clearly distinguished human from animal strains. For a given PCR-ribotype, human and animal strains presented a similar susceptibility to the antimicrobials tested. All strains were susceptible to vancomycin, metronidazole, chloramphenicol and rifampicin, while PCR-ribotypes 078, UCL 5a, UCL 36 and UCL 103 were associated with erythromycin resistance. The data suggest a wide dissemination of clones at hospitals and breeding-farms or a contamination at the slaughterhouse, but less probability of interspecies transmission. However, further highly discriminatory genotyping methods are necessary to elucidate interspecies and zoonotic transmission of C. difficile.

Clostridium difficile from food and surface samples in a Belgian nursing home: an unlikely source of contamination.

This study investigates the contamination of foods and surfaces with Clostridium difficile in a single nursing home. C. difficile PCR-ribotype 078 was found in one food sample and in none of the tested surfaces. These results indicate that food and surfaces are an unlikely source of C. difficile infection in this setting.

Clostridium difficile infection in elderly nursing home residents.

Age-related changes in intestinal flora and host defences, the receipt of antibiotic treatment, and the presence of underlying diseases are some of the most common risk factors associated with Clostridium difficile infection. Therefore, retirement care facilities for elderly people have been pinpointed as frequent sources of contamination. There is only limited data regarding the presence and epidemiology of C. difficile in nursing homes, and this gap in the current literature emphasises the need to gain a better understanding of the situation in order to prevent the emergence of new outbreaks among this population group.

Multilocus sequence typing analysis and antibiotic resistance of Clostridium difficile strains isolated from retail meat and humans in Belgium.

Clostridium difficile has been isolated from food animals and meat, specially ground pork and ground beef. The recovered isolates were closely related to C. difficile human strains, indicating that animals and food are possible transmission routes of human C. difficile infection. The main objective of this study was to characterize C. difficile isolates from retail meat and to compare with human isolates recovered from hospital patients in Belgium. Raw meat (beef and pork) was obtained from the retail trade. C. difficile was recovered from 2.3% of the beef samples and from 4.7% of the pork samples. A total of 4 different PCR-ribotypes were identified with a large percentage of types 078 and 014. Resistance to moxifloxacin and erythromycin was detected. The multi-locus sequence typing (MLST) analysis showed that meat and human isolates cluster in the same lineage. This study reveals the presence of toxigenic C. difficile in retail meat in Belgium with predominance PCR-ribotypes 078 and 014, which are among the four most prevalent ribotypes of C. difficile isolated from humans in Europe.

Carriage and acquisition rates of Clostridium difficile in hospitalized horses, including molecular characterization, multilocus sequence typing and antimicrobial susceptibility of bacterial isolates.

Clostridium difficile has been identified as a significant agent of diarrhoea and enterocolitis in both foals and adult horses. Hospitalization, antibiotic therapy or changes in diet may contribute to the development of C. difficile infection. Horses admitted to a care unit are therefore at greater risk of being colonized. The aim of this study was to investigate the carriage of C. difficile in hospitalized horses and the possible influence of some risk factors in colonization. During a seven-month period, faecal samples and data relating the clinical history of horses admitted to a veterinary teaching hospital were collected. C. difficile isolates were characterized through toxin profiles, cytotoxicity activity, PCR-ribotyping, antimicrobial resistance and multilocus sequence typing (MLST). Ten isolates were obtained with a total of seven different PCR-ribotypes, including PCR-ribotype 014. Five of them were identified as toxinogenic. A high resistance to gentamicin, clindamycin and ceftiofur was found. MLST revealed four different sequencing types (ST), which included ST11, ST26, ST2 and ST15, and phylogenetic analysis showed that most of the isolates clustered in the same lineage. Clinical history suggests that horses frequently harbour toxigenic and non-toxigenic C. difficile and that in most cases they are colonized regardless of the reason for hospitalization; the development of diarrhoea is more unusual.

Presence of Clostridium difficile in pigs and cattle intestinal contents and carcass contamination at the slaughterhouse in Belgium.

The objective of this study was to evaluate the presence of Clostridium difficile in intestinal and carcass samples collected from pigs and cattle at a single slaughterhouse. C. difficile was isolated in 1% and 9.9% of the pig and cattle intestinal contents and in 7.9% and 7% of cattle and pig carcass samples respectively. A total of 19 different PCR-ribotypes were identified, among them types 078 and 014. Seven of 19 ribotypes correlated with the PCR-ribotypes involved in human C. difficile infections in Belgium. This study confirms that animals are carriers of C. difficile at slaughter and ribotypes are identical than those in humans, and that carcass contamination occurs inside the slaughterhouse.

Clostridium difficile in young farm animals and slaughter animals in Belgium.

Faecal carriage of Clostridium difficile in healthy animals has been reported recently, especially in piglets and calves. However there is limited data about carriage in animals just prior to slaughter in Europe. The main objective of this study was to determine the presence of C. difficile in pigs and cattle at the slaughterhouse. C. difficile was isolated in 6.9% of the cattle at the slaughterhouse. None of the pig slaughter samples were positive for C. difficile after an enrichment time of 72 h. For complementary data, a short study was conducted in piglets and calves at farms. C. difficile was more prevalent in piglets (78.3%) than in calves (22.2%) on the farms. Regarding the piglet samples, 27.8% of the positive samples were detected without enrichment of stools. The PCR ribotype 078 was predominant in farm animals. Samples isolated from slaughter cattle presented the widest range in PCR-ribotype variety, and the most prevalent PCR ribotype was 118a UCL. The results of this study confirm that C. difficile is present in slaughter animals in Belgium with a large percentage of toxigenic strains also commonly found in humans.

Prevalence and genetic heterogeneity of Borrelia burgdorferi sensu lato in Ixodes ticks in Belgium.

Borrelia burgdorferi sensu lato is a genetically diverse group of spirochetes that includes the agent of Lyme borreliosis in which genospecies tend to be associated with specific clinical features. The aim of the study was to determine the prevalence and genetic diversity of Borrelia burgdorferi sensu lato in 524 ticks collected in woods of a western province of Belgium. Presence of spirochetes in ticks was determined by phase contrast microscopy. The mean infection rate of ticks was 12.0%. Variability was observed in the prevalence of infection among the five sites examined, ranging from 2.8 to 21.6%. Identification to genospecies was determined by PCR and sequencing. The most common genomospecies were Borrelia afzelii (55%) and Borrelia garinii (21%). For the first time in Belgium, we detected Borrelia valaisiana and Borrelia spielmanii, representing 14% and 2%, respectively. Borrelia burgdorferisensu stricto counted only for 2%. Co-infections were present in 8% of ticks. We emphasize the need for clinical studies to assess the prevalence of specific genospecies-related clinical manifestations of Lyme borreliosis in Belgium.

Septicemia due to Solobacterium moorei in a patient with multiple myeloma.

We report a case of bacteremia caused by Solobacterium moorei, an anaerobic, non-sporulated Gram-positive bacillus in a patient with a multiple myeloma. The source of infection was presumably related to multiple dento-alveolar abscesses. This is the first recovery of S. moorei from blood cultures.

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.

Pasteurella multocida in peritoneal dialysis: a rare cause of peritonitis associated with exposure to domestic cats.

Pasteurella multocida is a rare cause of peritonitis in peritoneal dialysis patients with only 10 cases reported in the literature so far. All cases were observed in patients with close contact with cats, usually with a direct puncture of the dialysis tubing. We report a case of Pasteurella multocida peritonitis in a patient maintained under continuous cycling peritoneal dialysis (CCPD), who had frequent and close contact with cats. Patients should be made aware of this potential complication and advised to keep domestic animals away from the location of their peritoneal exchanges.

Molecular characterization of fliD gene encoding flagellar cap and its expression among Clostridium difficile isolates from different serogroups.

The fliD gene encoding the flagellar cap protein (FliD) of Clostridium difficile was studied in 46 isolates belonging to serogroups A, B, C, D, F, G, H, I, K, X, and S3, including 30 flagellated strains and 16 nonflagellated strains. In all but three isolates, amplification by PCR and reverse transcription-PCR demonstrated that the fliD gene is present and transcribed in both flagellated and nonflagellated strains. PCR-restriction fragment length polymorphism (RFLP) analysis of amplified fliD gene products revealed interstrain homogeneity, with one of two major patterns (a and b) found in all but one of the strains, which had pattern c. A polyclonal monospecific antiserum raised to the recombinant FliD protein reacted in immunoblots with crude flagellar preparations from 28 of 30 flagellated strains but did not recognize FliD from nonflagellated strains. The fliD genes from five strains representative of the three different RFLP groups were sequenced, and sequencing revealed 100% identity between the strains with the same pattern and 88% identity among strains with different patterns. Our results show that even though FliD is a structure exposed to the outer environment, the flagellar cap protein is very well conserved, and this high degree of conservation suggests that it has a very specific function in attachment to cell or mucus receptors.

Peritonitis due to Brevibacterium otitidis in a patient undergoing continuous ambulatory peritoneal dialysis.

Brevibacterium otitidis is a coryneform rod and, as far as is known, is isolated only from infected ears. We report the first known case of peritonitis caused by B. otitidis in a patient undergoing continuous ambulatory peritoneal dialysis.

Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different serogroups.

Phenotypic and genotypic diversity of the flagellin gene (fliC) of Clostridium difficile was studied in 47 isolates from various origins belonging to the serogroups A, B, C, D, F, G, H, I, K, X, and S3. Electron microscopy revealed 17 nonflagellated strains and 30 flagellated strains. PCR and reverse transcription-PCR demonstrated that the flagellin gene was present in all strains and that the fliC gene was expressed in both flagellated and nonflagellated strains. Southern blotting showed the presence of only one copy of the gene and three different hybridization patterns. DNA sequence analysis of fliC from the strains belonging to serogroups C, D, and X, representative of each profile, disclosed great variability in the central domain, whereas the N- and C-terminal domains were conserved. The variability of the flagellin gene fliC was further studied in the isolates by PCR-restriction fragment length polymorphism (RFLP) analysis. Nine different RFLP groups were identified (I to IX), among which three (I, VII, and VIII) corresponded to numerous serogroups whereas the six others (II, III, IV, V, VI, and IX) belonged to a single serogroup. Flagellin gene RFLP analysis could constitute an additional typing method employable in conjunction with other typing methods currently available.

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.

Antimicrobial susceptibilities and serogroups of clinical strains of Clostridium difficile isolated in France in 1991 and 1997.

Glycopeptides (vancomycin and teicoplanin) and metronidazole are the drugs of choice for the treatment of Clostridium difficile infections, but trends in susceptibility patterns have not been assessed in the past few years. The objective was to study the MICs of glycopeptides and metronidazole for unrelated C. difficile strains isolated in 1991 (n = 100) and in 1997 (n = 98) by the agar macrodilution, the E-test, and the disk diffusion methods. Strain susceptibilities to erythromycin, clindamycin, tetracycline, rifampin, and chloramphenicol were also determined by the ATB ANA gallery (bioMérieux, La Balme-les-Grottes, France). The MICs at which 50% of isolates are inhibited (MIC(50)s) and MIC(90)s of glycopeptides and metronidazole remained stable between 1991 and 1997. All the strains were inhibited by concentrations that did not exceed 2 microgram/ml for vancomycin and 1 microg/ml for teicoplanin. Comparison of MICs determined by the agar dilution method recommended by the National Committee for Clinical Laboratory Standards and the E test showed correlations (+/-2 dilutions) of 86. 6, 95.9, and 99% for metronidazole, vancomycin, and teicoplanin, respectively. The E test always underestimated the MICs. Strains with decreased susceptibility to metronidazole (MICs, >/=8 microgram/ml) were isolated from six patients (n = 4 in 1991 and n = 2 in 1997). These strains were also detected by the disk diffusion method (zone inhibition diameter, /=1 microgram/ml), clindamycin (MICs, >/=2 microgram/ml), tetracycline (MICs, >/=8 microgram/ml), rifampin (MICs, >/=4 microgram/ml), and chloramphenicol (MICs, >/=16 microgram/ml) was observed in 64.2, 80.3, 23.7, 22.7, and 14.6% of strains, respectively. Strains isolated in 1997 were more susceptible than those isolated in 1991, and this trend was correlated to a major change in serogroup distribution. Periodic studies are needed in order to detect changes in serogroups and the emergence of strains with decreased susceptibility to therapeutic drugs.

A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates.

Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10,463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortened tcdA genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability in C. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.

Clostridium difficile-associated diarrhea in HIV-infected patients: epidemiology and risk factors.

A retrospective analysis of all the cases of Clostridium difficile-associated diarrhea (CDAD) in hospitalized patients infected with HIV was performed over a 52-month period to assess the incidence, epidemiology, and risk factors of CDAD. A case of CDAD was defined as a patient with diarrhea and a positive stool cytotoxin B assay. Sixty-seven cases of CDAD were recorded in HIV-infected patients between January 1991 and April 1995. The annual incidence of CDAD ranged from 1.7 to 6.4 per 100 HIV-infected patients discharged from hospital. The 67 CDAD cases included 48 (72%) first episodes and 19 (28%) relapses. Serogroup C accounted for 69% of strains from initial episodes of CDAD. To identify risk factors for CDAD, 34 HIV-infected patients with a first episode were compared with 66 HIV-infected controls matched for the length of hospital stay. Three independent factors remained significantly associated with CDAD among HIV-infected patients: CD4+ cell counts <50/mm3 (OR = 5.2; 95% CI = 1.4-19.3; p = 0.01), clindamycin use (OR = 5.0; 95% CI = 1.3-18.3; p = 0.02) and penicillin use (OR = 4.6; 95% CI = 1.1-18.8; p = 0.03). C. difficile is a common enteric pathogen responsible for nosocomial diarrhea in HIV-infected patients. Clinicians should keep this pathogen in mind when searching for the cause of diarrhea in these patients, especially those who are severely immunocompromised or have received clindamycin or penicillin.