Fructooligosaccharides and mannose affect Clostridium difficile adhesion and biofilm formation in a concentration-dependent manner.

The aim of this study was to investigate the effects that prebiotic and candidates for prebiotics on Clostridium difficile strains to adhere to various human epithelial cell lines and to compare the adhesive properties of specific C. difficile strains. We also sought to examine the effect of different concentrations of fructooligosaccharides and mannose on the formation of biofilms by C. difficile strains. The influence of cellobiose, fructooligosaccharides, inulin, mannose, and raffinose on the adherence properties of various C. difficile strains, including motile 630, non-motile M120, and 10 clinical motile ribotype 027 strains, to non-mucous secreting HT-29, mucous secreting HT-29 MXT, and CCD 841 CoN cells lines. The most effective prebiotics were used in biofilm formation assays. We demonstrated that all C. difficile strains adhered to all cell lines. However, the C. difficile M120 non-motile strain was statistically more likely to adhere to all three cell lines (CFU median, 40) compared to the motile strains (CFU median, 3; p < 0.001). Furthermore, among the carbohydrates examined, only fructooligosaccharides and mannose were found to significantly decrease adhesion (p < 0.001) of C. difficile strains. Alternatively, using a biofilm assay, we observed, via confocal laser scanning microscopy, that sub-inhibitory concentrations (1%) of fructooligosaccharides and mannose functioned to increase biofilm formation by C. difficile. We demonstrated that specific prebiotics and candidate prebiotics exhibit varying anti-adhesive properties towards C. difficile in vitro and that treatment with sub-inhibitory concentrations of prebiotics can cause an increase in biofilm formation by C. difficile.

Enterotoxigenic Clostridium perfringens infection and pediatric patients with inflammatory bowel disease.

BACKGROUND AND AIMS: Clostridium difficile is the major cause of antibiotic-associated diarrhea and is the most well known bacterial pathogen associated with inflammatory bowel disease (IBD). Enterotoxigenic Clostridium perfringens has also been detected in up to 15% of antibiotic-associated diarrhea cases, and it has not been found in healthy people. The aim of this study was to investigate the prevalence of C. perfringens infection in pediatric patients with IBD. METHODS: This was a prospective, controlled study evaluating pediatric IBD patients in the Department of Pediatric Gastroenterology and Nutrition in Warsaw, Poland. All of the patients were diagnosed according to the Porto criteria. There were two control groups: (1) non-IBD patients that were suspected for bacterial diarrhea and (2) healthy children. Stool samples were collected on the day of admission. C. perfringens infection diagnosis was based on a positive stool enzyme immunoassay (C. perfringens enterotoxin test kit TechLab). RESULTS: 91 fecal specimens from patients with IBD were collected. The average patient age was 11.7 years in IBD group, 7.4 years in non-IBD patients with diarrhea, and 7.4 years in healthy children. The prevalence of C. perfringens infection was 9% (8/91; CI 95% 4.6-16.4). There were more Crohn's patients (6/8) in the C. perfringens positive group. There was no C. perfringens infection in the two control groups. CONCLUSION: Our pilot data add evidence to the hypothesis that Clostridia other than C. difficile may play a significant role in the clinical course of IBD. However, further studies are needed to confirm this.

Three-step diagnostic algorithm in diagnosing patients suspected of Clostridium difficile-associated diarrhea.

UNLABELLED: Clostridium difficile is a predominant etiological agent of healthcare-associated infectious diarrhea. Immunoenzymatic tests for detecting toxins A/B from faecal samples are still used in routine diagnosis of Clostridium difficile-associated diseases in a number of healthcare centers in Poland. Recently, however, new diagnostic tests were introduced which allow for detecting toxigenic strains of C. difficile in a more effective and precise manner. It is of importance, especially in the light of hypervirulent strain occurrence. AIM: The aim of the present paper was to evaluate the efficacy of three-step algorithm in the diagnosis of Clostridium difficile-associated diseases (CDAD), considering the occurrence of false negative test results for toxins while using exclusively immunoenzymatic tests. MATERIALS AND METHODS: In the present study, faecal samples collected from patients presenting diarrhea were tested. Immunoenzymatic tests were used for detecting glutamate dehydrogenase (GDH) and toxins A/B. Culture and RT-PCR were also employed. RESULTS: Of 615 study participants, toxigenic strains GDH (+) TOX (+) were identified in 108 patients while for 67 patients, test results remained unspecified GDH (+) TOX (-). Further analysis of unspecified samples revealed 32 patients infected with toxigenic strains, i.e. 22.9% of all positive test results (n=140). CONCLUSION: Three-step diagnostic algorithm is an effective and reliable tool for diagnosing C.difficile- associated diseases.

Prevalence and association of PCR ribotypes of Clostridium difficile isolated from symptomatic patients from Warsaw with macrolide-lincosamide-streptogramin B (MLSB) type resistance.

Isolates (79 in total) of Clostridium difficile obtained over a 2 year period from 785 patients suspected of having C. difficile-associated diarrhoea (CDAD) and being hospitalized in the University Hospital in Warsaw were characterized by toxigenicity profile and PCR ribotyping. Furthermore, their susceptibility to clindamycin and erythromycin was determined. Among the 79 C. difficile isolates, 35 were classified as (A+)B+, 1 as (A+)(B+)CDT+, 36 as (A-)B+ and 7 as (A-)B-. A total of 21 different PCR ribotypes was detected. Two main (A+)B+ strains circulated in our hospital: ribotype 014 and ribotype 046. Unexpectedly, the predominant PCR ribotype was type 017, a known (A-)B+ strain, and this accounted for about 45.5 % of all isolates cultured from patients with CDAD. Isolates belonging to PCR ribotype 017 were found in cases from epidemics of antibiotic-associated diarrhoea in the internal and surgery units. High-level resistance (MIC > or = 256 mg l(-1)) to clindamycin and erythromycin was found in 39 (49 %) of the C. difficile isolates. Interestingly, 34 (94 %) of macrolide-lincosamide-streptogramin B (MLSB) type resistance strains did not produce toxin A, but produced toxin B and were (A-)B+ ribotype 017. Thirty-seven of the high-level resistance strains harboured the erythromycin-resistance methylase gene (ermB). C. difficile isolates (2/29) that had high-level clindamycin and erythromycin resistance, and belonged to PCR ribotype 046, were ermB negative. These investigations revealed that the predominant C. difficile strain isolated from symptomatic patients hospitalized in University Hospital in Warsaw was MLSB-positive clindamycin/erythromycin-resistant PCR ribotype 017.

[Detection of ermB gene responsible for high level resistance to clindamycin (MLS type resistance) among Clostridium difficile strains isolated from antibiotic associated diarrhea (AAD)]. / Wykrywanie genu ermB warunkujacego wysoka opornosc na klindamycyne (opornosc typu MLS) w szczepach Clostridium difficile izolowanych z przypadków biegunki poantybiotykowej (AAD).

In 68 C. difficile strains isolated from feacal samples of patients with antibiotic associated diarrhoea (AAD) investigated presence of ermB gene transferable of high level resistance to clindamycin. The primers set 2980/2981 used for identification of ermB gene amplified a 688 bp segment. We used the Etest to assess all strains for susceptibility to clindamycin. This study demonstrates that 57% of strains isolated from faecal samples of patients with AAD were highly resistant to clindamycin (minimal inhibitory concentration (MIC) of clindamycin, 256 mg/L) and possessed the ermB gene.