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Background: Asymptomatic faecal carriage of Clostridioides difficile has been widely evaluated, but its prevalence across a wide range of clinical departments and related risk factors are not well described. The objectives of the PORTADIFF study were to evaluate the prevalence and identifying risk factors leading to asymptomatic carriage of both toxigenic and non-toxigenic C. difficile., Methods: The PORTADIFF study was a 1-day prevalence study carried out in 10 different French hospitals. Adult patients, who agreed to participate, were included in this study and provided a fresh stool sample. C. difficile strains isolated from carriage were characterized by polymerase chain reaction (PCR) detection of tcdA, tcdB, cdtA and cdtB, and PCR ribotyping., Results: In total, 721 patients were included in this study. The median age was 73 years (range 18-101 years) and the male/female ratio was 1.06. C. difficile (either toxigenic or non-toxigenic strains) was isolated from 79 (11%) patients; 42 (5.8%) strains were toxigenic. The prevalence rates of asymptomatic carriage ranged from 5% on surgical wards to 19% on long-term care wards. The main risk factors associated with asymptomatic carriage were antibiotic treatment within the preceding 3 months (81.8% vs 53.7%; P<0.01), hospitalization within the preceding 2 months (55.8% vs 33%; P<0.01), cumulative duration of hospital stay before study inclusion (mean 50.1 vs 34.5 days; P<0.047), and hospitalization on a ward with high global incidence of C. difficile infection., Conclusion: Eleven percent of hospitalized patients were asymptomatic carriers of toxigenic or non-toxigenic C. difficile, and may constitute a potential reservoir of C. difficile strains., (Copyright © 2022 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Background: Clostridium difficile has long been considered to be a nosocomial pathogen but has emerged in the community in recent years. During infancy, asymptomatic C. difficile colonization is common. However, knowledge of colonization determinants and strain characteristics is limited. We studied the dynamics of C. difficile colonization in healthy infants from the community. Determinants of colonization and strain genotypes were also determined in a cohort of infants attending day nurseries., Methods: A 1-year follow-up study involving 10 healthy infants was performed to determine the incidence and kinetics of intestinal C. difficile colonization. In addition, a 1-point study involving 85 healthy infants (age, 0-3 years) from 2 day nurseries was performed. C. difficile isolates were typed by polymerase chain reaction-ribotyping and analyzed for the presence of toxin genes., Results: During the follow-up study, all infants acquired C. difficile and were colonized for several months. An early (neonatal) and a late (4-6 months of age) acquisition period were identified. In day nurseries, 38 infants (45%) carried C. difficile, with 11 (13%) carrying a toxigenic isolate. Age and several environmental factors were associated with the C. difficile carrier state. Strains causing disease in adults were identified in infants. Interestingly, no infant carried the common epidemic 027 or 078 strains., Conclusions: This study provides information on the dynamics of colonization in infants in the community and on the genotype of involved strains. C. difficile colonization appears mainly as an age-dependent process. Pathogenic strains circulate in asymptomatic infants from the community, who represent a potential reservoir of pathogenic strains.

Three commercial molecular assays were evaluated for toxigenic Clostridium difficile detection in stools. As compared to toxigenic culture, BD GeneOhm Cdiff (BD Diagnostics), XPert C. difficile (Cepheid) and illumigene C. difficile (Meridian Bioscience) demonstrated respectively a sensitivity of 95.5%, 97.8% and 86.7% and a specificity of 97.9%, 97.9% and 100%., (Copyright © 2012 Elsevier B.V. All rights reserved.)

During early infancy asymptomatic intestinal colonization by Clostridium difficile is frequent. To update information on infant colonization prevalence and to characterize infant strains, in terms of their virulence factors and their phylogenetic diversity, a prospective screening of C. difficile in the stools of infants 0 to 2 years old was conducted at Jean Verdier Hospital (Hôpital Jean Verdier) over an 18 month period. C. difficile was screened by toxigenic culture, and molecular characterization was performed by PCR-ribotyping and multilocus sequence typing (MLST). The overall C. difficile colonization prevalence was 33.7 % (99/294). The colonization rate by a toxigenic strain was 7.1 % (21/294). Community-acquired C. difficile accounted for 66.7 % (66/99) of cases. Molecular typing was performed on 90 isolates from Jean Verdier Hospital and 8 additional isolates from another hospital in Versailles (Centre Hospitalier de Versailles). Among these isolates, 23 were toxigenic (21 tcdA(+)/tcdB(+) and 2 tcdA(-)/tcdB(+)). All the isolates were negative for the binary toxin genes. Seventeen PCR ribotypes (PRs) were identified, with five PRs accounting for 82.7 % (81/98) of the isolates. MLST generated 15 different sequence types (STs). The predominant genotype, PRJV11-ST38 (33.7 %), included only non-toxigenic strains. Toxigenic strains were distributed in eight genotypes. Neither PR027-ST3, nor PR078/126-ST49 were identified but some PRs/STs corresponded to well-known adult infectious strains. These results indicate that infants are widely colonized by non-toxigenic strains. However, toxigenic adult infectious strains circulate in asymptomatic infants even in the community; thus, infants may be a reservoir for adult infectious strains.

Aim: Clostridium difficile is the most common agent of postantibiotic and nosocomial bacterial diarrhoea. Since the emergence of the highly virulent and epidemic strain NAP1/027 in Europe, it appears necessary to isolate C. difficile strains to realize an epidemiologic follow-up by molecular typing. The aim of this work was to compare three selective culture conditions for the isolation of C. difficile., Methods: One hundred and thirty stools collected from patients hospitalized at Jean Verdier were swabbed on the commercial medium CLO (BioMérieux) and on a medium prepared at the laboratory (CCTa: Columbia, cefoxitine 8 mg/l, cycloserine 250 mg/l, horse blood 5 %, sodium taurocholate 0.1 %) with and without preliminary alcoholic shock (EtOH). C. difficile was isolated from 38 stools and colonies were counted on each medium., Results: The fluorescence intensity of C. difficile colonies is comparable on CLO and CCTa-EtOH media, however their aspect is more characteristic on CLO. This medium appears very selective contrary to the CCTa medium on which an associated flora obstructs the fluorescence reading and requires a new isolation of the suspect strains. On average 30 times more colonies of C. difficile are counted on CCTa+/-EtOH than on CLO, suggesting the presence of great proportions of spores in the stools., Conclusions: The medium CLO is successful for the isolation of C. difficile despite of its selectivity. Nevertheless, it appears interesting to associate a medium enhancing spore germination as the CCTa medium inoculated after alcoholic shock to increase the sensitivity of detection while being freed from conservation and transport conditions., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

The disease spectrum caused by Clostridium difficile infection ranges from antibiotic-associated diarrhoea to life-threatening clinical manifestations such as pseudomembranous colitis. C. difficile infection is precipitated by antimicrobial therapy that causes a disruption of the normal colonic microbiota, predisposing to C. difficile intestinal colonisation. The pathogenicity of C. difficile is mediated by two exotoxins, TcdA and TcdB, both of which damage the human colonic mucosa and are potent cytotoxic enzymes. C. difficile must first be implanted in the gut and attach to epithelial cells, which are protected by a layer of dense mucus. Confirmed and putative accessory virulence factors that could play a role in adherence and intestinal colonisation have been identified and include proteolytic enzymes and adhesins. Recently, the epidemiology of C. difficile infection has radically changed and an increased incidence is associated with outbreaks in North America and Europe. Several reports suggest that disease severity is increasing to include sepsis syndrome and toxin megacolon. Elderly, debilitated patients in hospitals and nursing homes are particularly vulnerable. A hypervirulent, epidemic strain has been associated with the changing epidemiology and severity of disease. Here, we review the characteristics of the epidemic NAP1, PCR ribotype 027 C. difficile strain that could explain its hypervirulence and epidemic spread.

Clostridium difficile is the most common agent of nosocomial bacterial diarrhoea in adults. In 2006, C. difficile outbreaks were described in France with the highly virulent strain PCR-ribotype 027, which is also resistant to moxifloxacin and erythromycin. The aim of this study is to perform a phenotypic and molecular characterization of C. difficile strains isolated in Jean-Verdier-René-Muret hospitals. Thirty three C. difficile toxigenic strains isolated in symptomatic patients from 2001 to 2007 were studied. Toxins A and B detection was performed with an immunoenzymatic method (ICTAB, Meridian). The agar diffusion method was performed for determination of antibiotic susceptibility for metronidazole, vancomycin, erythromycin and moxifloxacin. The E-test was performed for determination of metronidazole, vancomycin and tigecycline MIC. Binary toxin genes cdtA and cdtB were detected by PCR. PCR-ribotyping was performed according to Bidet et al. From 2001 to 2007, all the isolates studied were susceptible to metronidazole, vancomycin and tigecyclin. We observed a significant decrease of susceptibility to moxifloxacin (100% in 2001 versus 28.5% in 2007) and to erythromycin (60% in 2001 versus 14% in 2007). Toxins A/B were detected in all the isolates. Fifteen per cent of the isolates studied produced the binary toxin not correlated with a specific PCR-ribotype. Ribotype 18 was the most prevalent PCR-ribotype detected since 2006. The isolates displaying this PCR-ribotype were resistant to erythromycin and moxifloxacin and were principally isolated in the same ward, suggesting cross infection. This study showed that: (1) over a six-year period, the susceptibility to metronidazole and vancomycin remained stable; (2) different clones of C. difficile circulated during these six years. Recently an epidemic strain resistant to erythromycin and moxifloxacin of ribotype 18 has emerged in the gastroenterology unit where fluoroquinolones are frequently used demonstrating the role of antibiotic selection pressure. The emergence of these isolates could explain the significant decrease of susceptibility to moxifloxacin and erythromycin observed in 2007. However, today, no isolate with a PCR-ribotype 027 was detected.

Aim: In vitro determination of Clostridium difficile susceptibility to antibiotics is not routinely performed. The aim of this study was to evaluate the performance of antibiotic susceptibility determination with the disk diffusion method for screening C. difficile isolates with decreased susceptibility to antibiotics., Methods: Thirty-six C. difficile isolates (toxigenic or not) isolated in 2005 and 2006 from three hospitals Assistance publique-Hôpitaux de Paris (Jean-Verdier, René-Muret, Beaujon) were studied by disk diffusion method with 14 antibiotics. Mueller-Hinton agar supplemented with sheep blood (Bio-Rad*) were swabbed with a C. difficile suspension at 1 McFarland. To check the results obtained with the disk diffusion method, Minimal Inhibitory Concentration (MIC) were performed respectively with E-test for glycopeptides and metronidazole and with the agar dilution reference method and E-test for new molecules with a potential activity against anaerobes: imipenem, ertapenem, linezolid and moxifloxacin., Results: The decreased susceptibility (resistant and intermediate) observed was 40% for amoxicillin-clavulanate, 60% for piperacillin-tazobactam, 100% for ceftriaxone, 81% for imipenem, 61% for ertapenem, 2% for chloramphenicol, 34% for erythromycin, 90% for lincomycin, 2% for linezolid, 98% for levofloxacin, 17% for moxifloxacin and 0% for vancomycin, teicoplanin and metronidazole. The results obtained with the disk diffusion method were compared to MICs obtained with E-test and reference method., Conclusion: The disk diffusion method seems to be a good method to detect isolates suspected to have a decreased susceptibility and consequently to reduce MIC determinations.

The discriminatory potential of a combination of various typing methods was evaluated on a set of 21 Clostridium difficile isolates obtained from symptomatic patients hospitalized in a geriatric unit and 7 non-toxigenic isolates from the same hospital. Isolates were firstly serotyped and toxinotyped. Of the 28 isolates, 19 belonged to serogroup A. PCR-ribotyping and PCR-RFLP on the fliC and slpA genes were then applied to these 19 isolates. The results suggest that the combination of PCR-ribotyping with PCR-RFLP analysis of slpA could be more discriminatory and suitable for studying C. difficile epidemiology.

The treatment of intestinal Clostridium difficile infections rests on administration of either a glycopeptide or metronidazole. Given the current shifts in resistance patterns of anaerobes to antimicrobials, a study of the susceptibility of C. difficile to metronidazole was timely. The objective of this study was to evaluate the influence of the culture medium on the Minimal Inhibitory Concentration (MIC) of metronidazole as determined using the E-test. Thirty-one strains were grown on three different media supplemented with 5% horse blood, namely Columbia agar, Wilkens Chalgren agar, and Brucella agar. Results were compared to those obtained using the reference agar dilution method (ADM). As recommended by the French Society for Microbiology, susceptibility was defined as an MIC < or = 4 mg/L. When used on strains susceptible by the ADM, the E-test yielded lower values than the ADM with all three media. Furthermore, findings suggest that E-test results obtained with strains whose MIC is in the 4 to 8 mg/L range by the ADM should be interpreted with caution and, in some cases, tested using the ADM.

The production of proteolytic enzymes by 10 Clostridium difficile isolates of varying toxigenicity and clinical origin was studied to determine if all isolates secreted proteases. Different protease substrates were studied: gelatin, collagen, phenylazobenzyloxycarbonyl-leucyl-glycyl-L-prolyl-D-arginine (Pz-peptide), casein, azocasein, and azocoll. All isolates degraded gelatin, collagen, and azocoll. The supernatants of all isolates contained an enzyme capable of attacking gelatin incorporated in a polyacrylamide gel (zymograms) and forming two closely spaced lytic bands with an estimated molecular mass of 35-40 kDa. Polyclonal antibodies, produced against the C. difficile gelatinase, revealed in Western blots a 35-kDa protein in the culture supernatants of all C. difficile isolates. In the same manner, Clostridium perfringens collagenase polyclonal antibodies detected a 120-kDa protein in the culture supernatants of all isolates; this suggests that at least two proteases may exist in C. difficile. The protease activities of the 10 strains examined did not seem strikingly different quantitatively but were in general weak and their role in pathogenicity is suspect.