Influence of selected Lactobacillus sp. on Clostridium difficile strains with different toxigenicity profile

This study was performed for determination of antagonistic activity of Lactobacillus spp. (L. plantarum 2017405, L. rhamnosus GG, L. acidophilus DSM 21007 and L. fernmentumn 353) on Clostridiunl difficile strains belonging to different toxigenicity profiles. Forty strains C. difficile isolated from patients suffering from antibiotic associated diarrhea (AAD) were used. Among C. difficile strains 13 produced toxin A and B (A+B+CDT-), 14 produced only toxin B (AB'CDT), 9 produced toxins A and B and possessing of binary toxin genes (A+B+CDT-) and 4 were non-toxigenic (A-B-CDT-). We did not observe relationship between degree of antagonistic activity Lactobacillus spp. and profile of toxigenicity of C. difficile strains.     

Clostridium difficile strains not producing Toxin A,but producing Toxin B [toxA(-)tox B(+)]--etiologic agent of antibiotic associated diarrhoea (AAD) in Poland

Previously, toxin A-negative/toxin B-positive Clostridium difficile strains were not thought to be associated with clinically significant diseases. In our study among 159 tested C. difficile strains isolated from feacal samples from 413 patients with antibiotic associated diarrhoea (AAD) 17 strains (11%) were negative in the "Culturette Brand Toxin" CD (Becton-Dickinson) for detection toxin A and positive in the TOX A/B test, designed for detection of both toxins. The conserved regions of both toxin genes were detectable in all of isolates studied by the PCR. Nine of these C. difficile strains had a deletion in the A gene and remaining 8 strains, revealed an amplicon with the expected size of approximately 2500 bp. In this paper we described the first time the toxin A-negative/toxin B-positive C. difficile strains with deletion in toxin A gene, isolated from the faecal samples of patient with AAD in Poland.     

Comparison of AP-PCR methods,ribotyping (PCR-ribotyping) and pulsed-field electrophoresis (PFGE) for strains of Clostridium difficile producing toxin B and not producing toxin A

In this study were used AP-PCR, PCR-ribotyping and pulsed-field elecrophoresis (PFGE) for comparative study of toxin A-negative/toxin B-posi-tive Clostridium difficile strains with deletion in toxin A gen. We investigated nine unrelated clinical strains, isolated from different units and different time from patients suffering to antibiotic associated diarrhea (AAD). We found that toxin A-negative/toxin B-positive C. difficile strains isolated in Poland belonging to a single genotype A, are being similar to the Japanese strains.     

Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.     

Profile of toxigenicity of Clostridium difficile strains isolated from paediatric patients with clinical diagnosis of antibiotic associated diarrhea (AAD)

This study was performed to determine profile of toxigenicity of 18 Clostridium difficile strains isolated from paeditric patients suffering from antibiotic associated diarrhea (AAD). Toxigenicity of C. difficile strains was tested for detection toxin A and toxin B by phenotypic methods and for detection of the tcdA and tcdB genes using of PCR. Changes in the repeating regions of the tcdA genes were detected with the NK9/NKV011 primer pairs. For detection of binary toxin (CDT) cdtA and cdtB genes, cdtApos/cdtArev i cdtBpos/cdtBrev two pair primers in PCR was used. Among C. difficile strains was detected three profiles of toxigenicity: C. difficile strains possesing of tcdA and tcdB genes but not possesing cdtA and cdtB genes of binary toxin (A+B+CDT-), strains possesing tcdA and tcdB and cdtA and cdtB genes (A+B+CDT+), strains with deletion of toxin A gene (A-B+CDT-). This is the first report on the occurence of binary positive C. difficile strains isolated from paediatric patients.     

Efficacy of Clostridium butyricum preparation concomitantly with Helicobacter pylori eradication therapy in relation to changes in the intestinal microbiota

Antibiotic associated diarrhea due to human intestinal microbiota abnormalities is a side effect of H. pylori eradication therapy. We examined intestinal microbiota changes during H. pylori eradication therapy and the preventive effect of CBM588 as a probiotic agent. Nineteen patients with gastro-duodenal ulcer were randomly divided into three groups: group A (without probiotics), group B (with regular doses of CBM588) and group C (with double doses of CBM588). The incidence of diarrhea and soft stools during H. pylori eradication therapy was 43% in group A and 14% in group B, while none of the patients in group C reported diarrhea or soft stools. Both bacterial counts and detection rates of bifidobacteria and/or obligate anaerobe were decreased by eradication therapy. However, bacterial counts of obligate anaerobes in group C were significantly higher than in group A ( P  < 0.05). Additionally, during eradication therapy C. difficile toxin A was detected in both group A and group B but not in group C.
In conclusion, these results indicate that H. pylori eradication therapy induces antibiotic associated diarrhea due to abnormalities in intestinal microbiota and/or C. difficile . However, these side effects might be prevented by probiotics.     

Clostridium difficile-associated typhlitis in specific pathogen free guineapigs in the absence of antimicrobial treatment

Clostridium difficile (toxin) associated typhlitis was diagnosed in untreated barrier-maintained specific pathogen free guineapigs. It resembled the pathological lesions of antibiotic induced enterocolitis. The possible role of limited colonization resistance to C. difficile provided by mouse enteric microflora in the pathogenesis of the disease is discussed.     

Clostridium difficile-associated diarrhea from a general hospital in Argentina

Clostridium difficile is responsible for 15-25% of all cases of antibiotic associated diarrhea. The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics. Although the clinical and financial impact of nosocomial C. difficile infection is believed to be significant, only limited information is available on the importance of C. difficile as a cause of diarrhea in Argentina. The aim of the study was to evaluate the impact and diagnosis methods of CDAD from symptomatic patients in a general hospital from Argentina. Consecutive diarrheal stool samples from symptomatic patients from a General Hospital in Argentina were screened for toxigenic C. difficile between April 2000 and April 2001. Toxins were detected in stools by the Premier Cytoclone A+B EIA. Each specimen was examined for toxigenic C. difficile strains by culture. From 104 specimens, 40 (38.5%) [32 of 87 patients (36.8%)] were positive and 64 (61.5%) [55 of 87 patients (63.2%)] were negative by stool toxin assay and/or toxigenic culture. In 11 of 40 positives samples C. difficile toxins were detected only by toxigenic culture. Five (15.6%) patients presented with symptomatic recurrences. Toxin-negative strains were not isolated. This data indicates that the high prevalence of toxigenic strains of C. difficile is of concern in routine diagnostic testing for C. difficile toxins in our study population. Detection of toxins in stools by EIA, coupled with testing strains for toxigenicity only in those cases in which direct toxin assay produces negative results, may be a satisfactory strategy. CDAD is an emerging nosocomial problem in our hospital. It will be necessary to evaluate the epidemiology and measures to control nosocomial spread.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

悉生小鼠体内大肠杆菌和青春型双歧杆菌对艰难梭菌的拮抗作用

本文应用悉生小鼠做模型,研究了大肠杆菌(E.coli)和青春型双歧杆菌(Bifidobacterium adolescentis)对艰难梭菌(Clostridium diffi-cile)的拮抗作用。E.coli和B.adolescentis预先接种无菌SSB小鼠,再用C.difficile攻击。结果表明,E.coli和E.coli B.adolescentis对小鼠均有保护作用,保护平分别为87.5%(7/8)和100%(8/8)。B.adolescentis定值后数量达10~(10.28)CFU/g,且对E.coli数量和小鼠本身无影响。E.coli和B.adolescentis联合比E.coli单独抑制C.difficile在肠道中繁殖的作用更强(0.02>P>0.01),但对其毒素产生和粘附力的作用无明显差异。C.difficile攻击后的1～14天,小鼠粪便中C.difficile菌数在10~4至10~8CFU/g内变化,细胞毒素为10~3CFU/g,A毒素滴度为10~2/g,B.adolescentis也一度下降10~2CFU/g。接种C.difficile后,小鼠虽无明显的腹泻症状,但组织学仍可观察到肠粘膜有充血和分泌增加等轻度损害。扫描电镜和普通光镜均发现E.coli单独或与B.adolescentis共同吸附在肠粘膜微绒毛表面,未见有C.difficile吸附。     

Prevention of Clostridium difficile-induced experimental pseudomembranous colitis by Saccharomyces boulardii: a scanning electron microscopic and microbiological study

The ability of Saccharomyces boulardii to protect mice against intestinal pathology caused by toxinogenic Clostridium difficile was studied. Different regions of the intestine of experimental mice were prepared for observation by scanning electron microscopy or homogenized for C. difficile enumeration and quantification of toxin A by enzyme immunoassay and toxin B by cytotoxicity. The test group was treated for 6 d with an S. boulardii suspension in drinking water and challenged with C. difficule on day 4. The three control groups were: axenic mice, mice treated with only S. boulardii and mice only challenged with C. difficile. The results showed that: (i) 70% of the mice infected by C. difficile survived when treated with S. boulardii; (ii) the C. difficile-induced lesions on the small and large intestinal mucosa were absent or markedly less severe in S. boulardii-treated mice; and (iii) there was no decrease in the number of C. difficile but rather a reduction in the amount of toxins A and B in S. boulardii-treated mice.     

Molecular analysis of the digestive microbiota in a gnotobiotic mouse model during antibiotic treatment: Influence of Saccharomyces boulardii

The probiotic Saccharomyces boulardii is a non-pathogenic yeast that has been proven efficient in the prevention of antimicrobial-associated diarrhea and of Clostridium difficile associated colitis. We evaluated the influence of the administration of S. boulardii on the composition of the fecal microbiota in a human microbiota-associated mouse model. This evaluation was run before, during and after a 7-day oral treatment with amoxicillin clavulanic acid. Predominant groups of bacteria were quantified with fluorescence in situ hybridization combined with flow cytometry using group-specific 16S rRNA targeted oligonucleotide probes designed for the Eubacteria, Bacteroides-Porphyromonas-Prevotella, Clostridium coccoides-Eubacterium rectale, Faecalibacterium prausnitzii, Clostridium histolyticum, Lactobacillus-Enterococcus and Enterobacteriaceae groups and Bifidobacterium species. S. boulardii did not quantitatively alter the total anaerobic microbiota nor the dominant bacterial groups. During the antibiotic treatment in the two groups of mice receiving the yeast or not, the level of Enterobacteriaceae and Bacteroides groups increased when the C. coccoides-E. rectale group decreased dramatically. After the antibiotic treatment was discontinued, the return to the initial level was reached more rapidly in the S. boulardii-treated mice than in the control mice (p<0.05) for the C. coccoides-E. rectale and Bacteroides-Porphyromonas-Prevotella groups. This quicker recovery of normal intestinal microbiota equilibrium after antibiotic therapy could be a mechanism for S. boulardii preventive effect on antibiotic-associated diarrhea in humans.     

兰州市婴幼儿艰难梭菌带菌状况

在兰州市随机采集的２１６名健康婴幼儿粪标本经分离并对其培养特性、菌落菌体形态特征、生化反应特性以及毒素原性等进行一系列检查,检出了３７人的粪便含有艰难梭菌（１０４─１０８／ｇ）,检出总阳性率为１７．１％,新生儿、婴儿及幼儿各年龄组的检出阳性率分别为１３．５％（１９／４１）、３３．３％（１３／３９）、１３．９％（５／３６）。３７株分离菌中毒素原性阳性者仅有８株（２１．６％）,均分布于婴儿与幼儿两组。新生儿１４１人中２７人的粪标本为胎粪,只有１人（３．７％）含艰难梭菌（２×１０６／ｇ）,但不产毒素。所有婴幼儿中有２２人因各种原因曾用过抗菌药物,但仅有２人的粪便含艰难梭菌,而且均为非产毒株,表明艰难梭菌检出率与药物服用之间似无显著的相关性。     

Toxin binding of tolevamer, a polyanionic drug that protects against antibiotic-associated diarrhea

Tolevamer, (GT160-246), is a sodium salt of styrene sulfonate polymer that is under development for the treatment of diarrhea caused by infection with Clostridium difficile. Pulsed ultrafiltration binding experiments in phosphate buffer containing 0.15 M Na(+) provide per polymer chain dissociation constants of 133 nM and 8.7 microM for the binding of tolevamer to C. difficile toxins A and B, respectively. At 0.05 M Na(+), the binding of toxin A to tolevamer is irreversible, whereas the dissociation constant to toxin B under these conditions is 120 nM. Binding constants obtained from fluorescence polarization data for toxin A binding to tolevamer at 0.15 M Na(+) agree substantially with those obtained by pulsed ultrafiltration. The binding activity of tolevamer reported here correlates well with previously reported results for the inhibition of the biological activity of C. difficile toxins A and B. From the fluorescence polarization data, it is estimated that one toxin A molecule interacts with between 600 to 1000 monomer units on tolevamer at 0.15 M Na(+). Thus, the data suggest a very large interaction surface between polymer and toxin A.     

Antibiotics and Clostridium difficile

Clostridium difficile is now established as a major nosocomial pathogen. C. difficile infection is seen almost exclusively as a complication of antibiotic therapy, and is particularly associated with clindamycin and third-generation cephalosporins. Depletion of the indigenous gut microflora by antibiotic therapy has long been established as a major factor in the disease. However, the direct influence of antimicrobials upon virulence mechanisms such as toxin production and adhesion in the bowel, and the exact mechanisms by which the organism causes disease remain to be elucidated.     

The anti-sigma factor TcdC modulates hypervirulence in an epidemic BI/NAP1/027 clinical isolate of Clostridium difficile

Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.     

Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.     

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).     

Clostridium difficile toxin and faecal lactoferrin assays in adult patients

Clostridium difficile is the primary aetiological agent of antibiotic-associated diarrhoea. The faecal lactoferrin (FL) assay is a simple in vitro test which is highly sensitive to the presence of a marker of polymorphonuclear cells. We evaluated the use of the FL assay in conjunction with the C. difficile toxin assay in faecal samples obtained from 231 adult patients. The relationship between C. difficile toxin and FL in both negative and positive status was highly significant statistically (P < 0.001). Therefore, the FL assay performed simultaneously with the C. difficile toxin assay can help rule out asymptomatic carriage of C. difficile.     

Transient fecal shedding and limited animal-to-animal transmission of Clostridium difficile by naturally infected finishing feedlot cattle

To longitudinally assess fecal shedding and animal-to-animal transmission of Clostridium difficile among finishing feedlot cattle as a risk for beef carcass contamination, we tested 186 ± 12 steers (mean ± standard deviation; 1,369 samples) in an experimental feedlot facility during the finishing period and at harvest. Clostridium difficile was isolated from 12.9% of steers on arrival (24/186; 0 to 33% among five suppliers). Shedding decreased to undetectable levels a week later (0%; P < 0.001), and remained low (< 3.6%) until immediately prior to shipment for harvest (1.2%). Antimicrobial use did not increase fecal shedding, despite treatment of 53% of animals for signs of respiratory disease. Animals shedding C. difficile on arrival, however, had 4.6 times higher odds of receiving antimicrobials for respiratory signs than nonshedders (95% confidence interval for the odds ratio, 1.4 to 14.8; P = 0.01). Neither the toxin genes nor toxin A or B was detected in most (39/42) isolates based on two complementary multiplex PCRs and enzyme-linked immunosorbent assay testing, respectively. Two linezolid- and clindamycin-resistant PCR ribotype 078 (tcdA+/tcdB+/cdtB+/39-bp-type deletion in tcdC) isolates were identified from two steers (at arrival and week 20), but these ribotypes did not become endemic. The other toxigenic isolate (tcdA+/tcdB+/cdtB+/classic tcdC; PCR ribotype 078-like) was identified in the cecum of one steer at harvest. Spatio-temporal analysis indicated transient shedding with no evidence of animal-to-animal transmission. The association between C. difficile shedding upon arrival and the subsequent need for antimicrobials for respiratory disease might indicate common predisposing factors. The isolation of toxigenic C. difficile from bovine intestines at harvest highlights the potential for food contamination in meat processing plants.