Occurrence of Clostridium difficile in fecal samples of HIV-infected children in Poland

The prevalence of Clostridium difficile and its toxins (A and B) in HIV-positive children in Poland was investigated in a group of 18 children, aged 6 months to 8 1/2 years. Stool samples were tested using an antigen detection method for toxin A/B, cytotoxicity-neutralization and culture. In 3 cases (17%) C. difficile toxins were detected in both stool samples and strains recovered from culture. The three strains isolated were shown by PCR methods to contain toxins A and B genes. All children had been treated previously with antimicrobial and antiviral agents. All three C. difficile-positive children had mild diarrhea that resolved without specific therapy. Further studies involving a large number of children and molecular analyses of isolated C. difficile strains are necessary to determine the frequency and rate of carriage of C. difficile strains among HIV-positive children in Poland.     

Detection of the ADP-ribosyltransferase toxin gene (cdtA) and its activity in Clostridium difficile isolates from Equidae

Clostridium difficile is an antibiotic-associated emerging pathogen of humans and animals. Thus far three toxins of C. difficile have been described: an enterotoxin (ToxA), a cytotoxin (ToxB) and an ADP-ribosyltransferase (CDT). In the present work we describe the first isolation of CDT producing C. difficile from Equidae with gastro-intestinal disease. Out of 17 C. difficile strains isolated from Equidae, 11 were positive for the genes tcdA and tcdB encoding ToxA and ToxB. In addition four of these 11 isolates were positive for the cdtA gene encoding the catalytic subunit of the ADP-ribosyltransferase CDT. Interestingly none of the isolates derived from canines (41 isolates) and felines (4 isolates) harboured the cdtA gene. In C. difficile field isolates which contained the cdtA gene, ADP-ribosyltransferase activity could also be detected in culture supernatants indicating expression and secretion of CDT. All strains were associated with intestinal disorders, but no association was found for the occurrence of toxins with a specific clinical diagnosis.     

Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator

Clostridium difficile binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, here designated cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR(+) and cdtR-negative strains of C. difficile, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.     

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.     

Toxins A and B of Clostridium difficile

Abstract: The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.     

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.     

Cultivation of fungi from fecal specimens in cases of antibiotic associated diarrhea (AAD)

The aim of performed examinations was to isolate, identify and determine a drug susceptibility of fungi cultured from faecal specimens submitted for detection of Clostridium difficile in cases of antibiotic-associated diarrhoea (AAD). One hundred samples of diarrhoeic faeces were examined using routine bacteriological methods (isolation and identification of C. difficile), serological test (detection of C. difficile toxins A/B) and mycological methods (isolation, identification and drug susceptibility testing of fungi). Out of twenty seven specimens of diarrhoeic faeces fungal strains were isolated, in 20 samples C. difficile strain and/or C. difficile toxins A/B were detected, in 23 specimens fungal strains, C. difficile strains and/or toxins A/B of this species were present. The most active in vitro agent against cultured fungal strains was nystatin. In conclusion it can be stated, that fungal strains are responsible for some cases of antibiotic-associated diarrhoea. So, mycological diagnostics of faecal samples from patients with diarrhoea after antibiotic therapy is necessary. Cases of diarrhoea with mixed bacterial and fungal aetiology (C. difficile + yeast-like fungus) were observed.     

Clostridium difficile toxin expression is inhibited by the novel regulator TcdC

Clostridium difficile, an emerging nosocomial pathogen of increasing clinical significance, produces two large protein toxins that are responsible for the cellular damage associated with the disease. The precise mechanisms by which toxin synthesis is regulated in response to environmental change have yet to be discovered. The toxin genes (tcdA and tcdB) are located in a pathogenicity locus (PaLoc), along with tcdR and tcdC. TcdR is an alternative RNA polymerase sigma factor that directly activates toxin gene expression, while the inverse relationship between expression of tcdR, tcdA and tcdB genes on the one hand and tcdC on the other has led to the suggestion that TcdC somehow interferes with toxin gene expression. This idea is further supported by the finding that many recent C. difficile epidemic strains in which toxin production is increased carry a common tcdC deletion mutation. In this report we demonstrate that TcdC negatively regulates toxin synthesis both in vivo and in vitro. TcdC destabilizes the TcdR-containing holoenzyme before open complex formation, apparently by interaction with TcdR or TcdR-containing RNA polymerase holoenzyme or both. In addition, we show that the hypertoxigenicity phenotype of C. difficile epidemic strains is not due to their common 18 bp in-frame deletion in tcdC.     

Evaluation of different methods for detection of Clostridium difficile toxins in Poland

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.     

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.     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

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.     

Structure and mode of action of clostridial glucosylating toxins: the ABCD model

Toxins A and B, which are the major virulence factors of antibiotic-associated diarrhea and pseudomembranous colitis caused by Clostridium difficile, are the prototypes of the family of clostridial glucosylating toxins. The toxins inactivate Rho and Ras proteins by glucosylation. Recent findings on the autocatalytic processing of the toxins and analysis of the crystal structures of their domains have made a revision of the current model of their actions on the eukaryotic target cells necessary.     

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.     

Differentiation of Clostridium difficile toxin from Clostridium botulinum toxin by the mouse lethality test

The mouse lethality test is the most sensitive method for confirming the diagnosis of infant botulism. Both Clostridium difficile and Clostridium botulinum produce heat-labile toxins which are lethal for mice and can be found in the feces of infants. These two toxins can be distinguished from one another in this assay when both are present in the same fecal specimen because they appear to be immunologically distinct toxins.     

Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins

Rho proteins, which are involved in recepto-mediated regulation of the actin cytoskeleton, are substrates for ADP-ribosylation by Clostridium botulinum C3 toxins. Recently, it was shown that Rho and other members of the Rho subfamily of low-molecular-mass GTP-binding proteins are glucosylated by C. difficile toxins A and B. Glucosylation occurs at threonine-37, which is a crucial amino acid residue for the regulatory functions of the small GTP-binding proteins. These toxins should prove useful as tools for studying the functions of Rho proteins.     

Structural characterization of the cell wall binding domains of Clostridium difficile toxins A and B; evidence that Ca2+ plays a role in toxin A cell surface association

Clostridium difficile (C.difficile) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca2+-dependent fashion. Millimolar Ca2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca2+ in the sensitization of toxin A/cell-surface interactions.     

Morphological and genetic diversity of temperate phages in Clostridium difficile

Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages phiC2, phiC5, and phiCD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.