- Volume 54, Issue 2, 2005
Volume 54, Issue 2, 2005
- Editorial
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- Clostridium Difficile
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Alternative treatments for Clostridium difficile disease: what really works?
More LessVancomycin and metronidazole have been used for treating Clostridium difficile-associated disease (CDAD) for the past 25 years, but approximately 20 % of patients develop recurrent disease. The increasing incidence of nosocomial outbreaks, cases of recurrent CDAD and other complications (toxic megacolon, ileus, sepsis) has fuelled the search for different types of treatments. As the understanding of the pathogenesis of this disease has matured, newer treatment strategies that take advantage of these mechanisms have been developed. This review will describe such treatments and examine the evidence for each strategy.
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Revised nomenclature of Clostridium difficile toxins and associated genes
Several different nomenclatures have been applied to the Clostridium difficile toxins and their associated genes. This paper summarizes the new nomenclature that has been agreed to by the research groups currently active in the field. The revised nomenclature includes C. difficile toxins and other related large clostridial toxins produced by Clostridium sordellii and Clostridium novyi, and corresponding toxin genes, as well as toxin production types of C. difficile strains.
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Quorum sensing in Clostridium difficile: analysis of a luxS-type signalling system
More LessThe increasing incidence of Clostridium difficile-associated disease, and the problems associated with its control, highlight the need for additional countermeasures. The attenuation of virulence through the blockade of bacterial cell-to-cell communication (quorum sensing) is one potential therapeutic target. Preliminary studies have shown that C. difficile produces at least one potential signalling molecule. Through the molecule's ability to induce bioluminescence in a Vibrio harveyi luxS reporter strain, it has been shown to correspond to autoinducer 2 (AI-2). In keeping with this observation, a homologue of luxS has been identified in the genome of C. difficile. Adjacent to luxS Cd a potential transcriptional regulator and sensor kinase, rolA and rolB, have been located. RT-PCR has been used to confirm the genetic organization of the luxS Cd locus. While AI-2 production has not been blocked so far using antisense technology, AI-2 levels could be modulated by controlling expression of the putative transcriptional regulator rolA. RolA, therefore, acts as a negative regulator of AI-2 production. Finally, it has been shown that the exogenous addition of AI-2 or 4-hydroxy-5-methyl-3(2H) furanone has no discernible effect on the production of toxins by C. difficile.
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Effect of phage infection on toxin production by Clostridium difficile
More LessInfection with Clostridium difficile and subsequent production of toxins A and B may result in C. difficile-associated diarrhoea and pseudomembranous colitis in hospital patients. The effect of four temperate phages, obtained by induction of clinical C. difficile isolates, on toxin production by C. difficile was determined. None of these phages converted a lysogenized non-toxigenic C. difficile strain to toxin production. One of the accessory toxin genes, tcdE, was detected in three phages, ϕC2, ϕC6 and ϕC8; however, the non-repeating regions of tcdA and tcdB encoding the enzymic domains were not carried on phage DNA. Phage infection of toxigenic strains increased toxin B production in four of six lysogens, although the level of tcdB transcription as determined by real-time RT-PCR was not significantly altered. However, levels of toxin A transcription in two lysogens were significantly altered without any corresponding differences in toxin A production.
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Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Δerm) and demonstration that the conjugative transposon Tn916ΔE enters the genome of this strain at multiple sites
More LessErythromycin resistance in Clostridium difficile strain 630 is conferred by a genetic element termed Tn5398 which contains two erm(B) genes: erm1(B) and erm2(B). An erythromycin-sensitive derivative of strain 630 (designated 630Δerm) was generated by spontaneous mutation after continuous subculture for 30 days. This strain had lost the erm2(B) gene from within Tn5398 but retained erm1(B). However, the strain could revert to erythromycin resistance at a frequency of 2.79 × 10−8, although it still contained the deletion of erm2(B). The availability of C. difficile 630Δerm allowed the behaviour of Tn916ΔE to be investigated in this strain. This element entered the genome at multiple sites indicating that it could be useful as an insertional mutagen.
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Detection of binary-toxin genes (cdtA and cdtB) among Clostridium difficile strains isolated from patients with C. difficile-associated diarrhoea (CDAD) in Poland
Clostridium difficile A+B+ and A−B+ strains isolated from stool samples of patients with C. difficile-associated diarrhoea (CDAD) were selected from the University Hospital Warsaw collection. The binary-toxin genes cdtA and cdtB were detected by PCR in five of the 41 A+B+ strains tested, but in none of the 17 A−B+ strains tested, giving 8.6 % prevalence (5/58) of binary-toxin-positive strains. All of the strains that were positive for binary-toxin genes were grouped into toxinotype IV, suggesting that in this institution toxinotype IV might dominate among the population of C. difficile with binary-toxin genes.
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Prevalence and characteristics of bacteria and host factors in an outbreak situation of antibiotic-associated diarrhoea
Antibiotic-associated diarrhoea (AAD) represents a clinical entity leading to prolonged hospital stays and diagnostic and therapeutic procedures, and results in additional costs. The aim of the present study was to assess the prevalence and characteristics of different bacteria in stools of patients with AAD. The reliability of diagnostic procedures under routine conditions was evaluated. Host factors were also analysed. From June 2002 to April 2003 89 cases of diarrhoea were reported at a hospital unit for internal medicine. Clostridium difficile and Clostridium perfringens toxin enzyme-immunoassays (EIAs), and culture for C. difficile, C. perfringens and Staphylococcus aureus were performed on stool samples from all patients. Toxin production was determined in isolated S. aureus strains. In vitro susceptibility of S. aureus for oxacillin and of C. difficile for vancomycin, metronidazole, linezolid, fusidic acid and tetracycline was tested. Host factors, such as age, comorbidities, antibiotic exposure and contact with other patients, were evaluated. Twenty-six stools were positive for C. difficile toxins by an EIA technique, while C. difficile was cultured from 39. C. difficile was isolated from 21 stools that were EIA negative. Additionally, from 28 stools S. aureus and/or C. perfringens could be isolated. Nine samples contained only S. aureus and/or C. perfringens. Thirty-one stools were negative in all tests. All C. difficile isolates were susceptible to vancomycin and metronidazole. Age >60 years, and diseases of the vascular system, the heart, the kidneys and the lungs were identified as risk factors for acquiring C. difficile in this setting (P values < 0.05). Stool culture for C. difficile was shown to be more sensitive than toxin EIA in this study. Risk factors for the acquisition of C. difficile in outbreak situations seem to differ from risk factors in the normal hospital setting. The role of toxin-producing S. aureus in cases of AAD needs further investigation.
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An improved protocol for pulsed-field gel electrophoresis typing of Clostridium difficile
More LessPulsed-field gel electrophoresis (PFGE) is the ‘gold standard’ technique for bacterial typing and has proved to be discriminatory and reproducible for typing Clostridium difficile. Nevertheless, a high proportion of strains are non-typable by this technique due to the degradation of the DNA during the process. The introduction of several modifications in the PFGE standard procedure increased typability from 40 % (90 isolates) to 100 % (220 isolates) while maintaining the high degree of discrimination and reproducibility of the technique.
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Toxigenic status of Clostridium difficile in a large Spanish teaching hospital
More LessThe aim of this study was to evaluate the toxigenic status of circulating strains of Clostridium difficile in a large teaching hospital. Overall 220 isolates were studied of which 199 (90.5 %) produced both large clostridial toxins detected by conventional methods. Ten more strains (4.5 %) had toxin A and B genes detectable by PCR. Eleven (5.0 %) variant strains (A−B+) were detected among the isolates studied and 10 strains (4.5 %) had the binary toxin genes (cdtA and cdtB).
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PCR ribotyping of Clostridium difficile isolates originating from human and animal sources
More LessMolecular typing of Clostridium difficile isolates from animals and humans may be useful for evaluation of the possibility for interspecies transmission. The objective of this study was to evaluate C. difficile isolates from domestic animals and humans using PCR ribotyping. Isolates were also tested using PCR for the presence of genes encoding toxins A and B. One hundred and thirty-three isolates of C. difficile from dogs (n = 92), horses (n = 21) and humans (n = 20), plus one each from a cat and a calf, were evaluated. Overall, 23 ribotypes were identified. Of these, nine were identified from dogs, 12 from horses, seven from humans and one each from the cat and calf. In dogs, humans and horses, one or two different ribotypes predominated. Overall, 25 % of isolates from humans were indistinguishable from isolates from one or more animal species. Genes encoding C. difficile toxins A and B were detected in all human, equine and bovine isolates, and in 69 % of canine isolates. While different ribotypes appear to predominate in different mammalian species, several indistinguishable strains may be found in multiple species. This suggests that there is a potential for interspecies transmission of C. difficile and epidemiological studies are warranted.
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Typing by sequencing the slpA gene of Clostridium difficile strains causing multiple outbreaks in Japan
More LessPrevious reports have documented that a surface layer protein (SlpA) varies among Clostridium difficile isolates. The typing system by sequencing the variable region of the slpA gene was applied to typing C. difficile strains belonging to one PCR ribotype, type smz, which has been identified as frequently causing outbreaks in Japan. The PCR ribotype smz strains recovered from patients at different hospitals in Japan were examined. Among 10 type smz strains tested, three subtypes, smz-1, -2 and -3, were identified that differed from each other by one nucleotide. slpA sequence typing was also applied to direct typing on DNA extracted from stool specimens. Of 22 stool specimens examined, 17 were PCR positive for slpA; eight were typed as slpA sequence type smz-1 and nine as type smz-2. C. difficile was cultured from 12 of these 17 stool specimens, and the sequence results of the recovered isolates were compared with those from the DNA extracted from the stool specimens. In all 12 of these stool specimens, the sequence results of DNA from recovered C. difficile isolates completely agreed with those of DNA extracted directly from stool specimens. The remaining five stool specimens were culture-negative for C. difficile. Sequence typing has the advantage of enabling easy comparison of typing results among multiple laboratories via the Internet without exchanging reference strains as is required in typing systems which depend on banding-pattern analyses. slpA sequence typing appears to be a reproducible and reliable typing system for C. difficile as well as being useful for the typing of C. difficile when stool specimens contain only small numbers of C. difficile or are inappropriate for culturing.
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Coexistence of multiple PCR-ribotype strains of Clostridium difficile in faecal samples limits epidemiological studies
Clostridium difficile is an important cause of antibiotic-associated diarrhoea. The simultaneous presence of different strains in individual faecal samples has not yet been established, but is important for epidemiological studies. Recurrences of Clostridium difficile-associated diarrhoea (CDAD) are observed in 15–20 % of patients and have been reported as relapses or reinfections with a new strain. In a period of 1 year, 28 faecal samples from 23 patients with a first episode of CDAD were collected at the Leiden University Medical Centre. In addition, 52 faecal samples from 23 patients, from three different hospitals, with one (n = 19), two (n = 2) or three (n = 2) recurrences were studied. PCR-ribotyping was applied as the standard typing method for the isolates. The toxinogenic and clindamycin-resistance profiles of the isolates was determined by PCR. Of 23 patients with a first episode of CDAD, two (8.7 %) harboured two different types, with no differences in toxinogenicity or clindamycin resistance, within one faecal sample. One of these 23 patients showed two types in three faecal samples from the same episode. Of the 23 patients with recurrences, six (26 %) showed a different strain type isolated in a recurrent episode. The number of cases of multiple C. difficile strains in faecal samples from patients with a first episode of CDAD did not differ significantly from the number of different strains present in recurrent episodes (chi-square test, P ⩽ 0.2). This observation limits the application of typing methods for studying the epidemiology of CDAD.
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Clinical features of Clostridium difficile-associated diarrhoea due to binary toxin (actin-specific ADP-ribosyltransferase)-producing strains
Toxins A and B are known to be the primary virulence factors of Clostridium difficile. Other potential virulence factors have been identified such as binary toxin (actin-specific ADP-ribosyltransferase toxin, or CDT). A retrospective case–control study was performed in order to identify clinical features and risk factors of C. difficile-associated diarrhoea due to binary toxin-producing strains. Each case (a patient with diarrhoea due to binary toxin-producing strain) was compared with two controls (patients with diarrhoea due to a C. difficile strain that did not produce binary toxin) matched for ward and date of hospitalization. cdtA and cdtB genes were screened by PCR. Production of CDT was studied by Western blotting using an antiserum against Ia and Ib from the Clostridium perfringens iota toxin, and the activity of the binary toxin was assessed using an ADP-ribosyltransferase assay. Twenty-six cases (14 males and 12 females) were identified in 1999 and 2000. Cases and controls did not differ significantly for sex, age, previous administration of antibiotics or frequency of endoscopic examination. Diarrhoea was community-acquired more often in cases than in controls (65.4 vs 35.7 %, P = 0.017) and more often represented the cause of hospitalization (61.5 vs 26.2 %, P = 0.003). Moreover, diarrhoea in cases was more frequently associated with abdominal pain (63.6 vs 39.4 %, P = 0.07) and with liquid stools (76.9 vs 59.5 %, P = 0.14) than in controls. These results suggest that there could be a correlation between the production of binary toxin and the severity of diarrhoea.
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Laboratory diagnosis of Clostridium difficile-associated diarrhoea: a plea for culture
More LessA routine protocol for diagnosing Clostridium difficile-associated diarrhoea (CDAD) based on both faecal-cytotoxin detection and toxigenic culture was adopted by the microbiology laboratory of the St Luc-UCL University Hospital in Brussels in 1997. A toxigenic culture is a faecal culture followed, in the case of positivity, by a direct immunoassay on colonies to detect toxin A production. The results obtained over the past 7 years in the hospital are reviewed here. A total of 10 552 diarrhoeal stools from 7042 patients were analysed, of which 9494 were negative for all tests. A total of 1058 samples (10 %) from 794 patients were culture-positive, of which 460 (4.4 %) were positive for a faecal cytotoxin. The remaining 598 cultures were tested for toxin A on colonies; 355 of them were positive, which is 3.4 % of the total, and the remaining 243 (2.3 %) were negative. The positivity of the faecal-cytotoxin assay was statistically linked to the number of colonies observed on the culture plate. In conclusion, over a 7 year period, toxigenic culture allowed the diagnosis of 355 cases of CDAD that would have been missed by a protocol using a faecal-cytotoxin assay alone. In terms of both patient care, prevention of environmental contamination and prevention of risk of a hospital outbreak, it is proposed that these results justify the recommendation to perform both faecal-toxin assay and culture in routine medical practice.
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Immunological properties of surface proteins of Clostridium difficile
Sera from patients with Clostridium difficile-associated disease (CDAD) and sera from a control group were analysed by an ELISA to detect antibodies directed against four surface proteins and toxins A and B of C. difficile. The surface proteins were the flagellar cap protein FliD, the flagellin FliC, the adhesin Cwp66 divided into two domains, Cwp66-Nterminal and Cwp66-Cterminal, and the fibronectin-binding protein Fbp68. For each antigen, antibody levels in the CDAD patient group and in the control group were compared. In the CDAD patient group, the mean of the antibody levels decreased from Cwp66-Cterminal to Fbp68, FliD, toxins B and A, Cwp66-Nterminal and finally FliC, suggesting different immunogenic properties among these adhesins. For Cwp66-Nterminal, FliC, FliD and Fbp68, the antibody level observed in the control group was higher than in the CDAD group with a statistically significant difference whereas the antibody level for toxins A and B was not statistically different. In conclusion, this study suggests that during the clinical course of disease, C. difficile adhesins are able to induce an immune response which could play a role in the defence mechanism of the host.
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Bovine antibody-enriched whey to aid in the prevention of a relapse of Clostridium difficile-associated diarrhoea: preclinical and preliminary clinical data
In a pilot study, the feasibility of immune whey protein concentrate (40 %; immune WPC-40) to aid the prevention of relapse of Clostridium difficile diarrhoea was evaluated. Immune WPC-40 was made from milk after immunization of Holstein–Frisian cows with C. difficile-inactivated toxins and killed whole-cell C. difficile. Immune WPC-40 contained a high concentration of specific sIgA antibodies, and was effective in neutralizing the cytotoxic effect of C. difficile toxins in cell assays in vitro. Immune WPC-40 conferred protection from otherwise lethal C. difficile-associated caecitis in hamsters. To obtain preliminary data in humans, 16 patients (10 male; median 57 years) with toxin- and culture-confirmed C. difficile diarrhoea were enrolled in an uncontrolled cohort study. Nine had a history of relapsing C. difficile diarrhoea. After completion of standard antibiotic treatment, the patients received immune WPC-40 TID for 2 weeks; it was well tolerated and no treatment-related adverse effects were observed. In all but one case, C. difficile toxins had disappeared from the faeces upon completion of treatment. During a follow-up period of median 333 days (range 35 days to 1 year), none of the patients had suffered another episode of C. difficile diarrhoea. These preliminary data suggest that immune whey protein concentrate-40 may be of help in the prevention of relapse of C. difficile diarrhoea.
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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