Prevalence and distribution of Clostridium difficile PCR ribotypes in cats and dogs from animal shelters in Thuringia,Germany

Clostridium difficile is an important cause of nosocomial diarrhoea in humans. Pet animals and livestock are discussed as potential natural reservoirs and sources of infection. In this study faecal samples from dogs and cats were collected at 10 animal shelters in Thuringia, Germany. C. difficile was isolated from 9 out of 165 (5.5%) canine and 5 out of 135 (3.7%) feline samples. Five PCR ribotypes (010, 014/020, 039, 045, SLO 066) were identified. PCR ribotypes 010 and 014/020 were detected in more than one shelter and PCR ribotypes 014/020 and 045 were isolated from dogs and cats. MLVA profiles of strains of a PCR ribotype from one shelter were identical or closely related, while strains of the same PCR ribotype from different shelters showed significant differences. This study shows that dogs and cats kept in animal shelters are a reservoir of C. difficile PCR ribotypes which can infect also humans.     

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.     

Macro and micro diversity of Clostridium difficile isolates from diverse sources and geographical locations

Clostridium difficile has emerged rapidly as the leading cause of antibiotic-associated diarrheal disease, with the temporal and geographical appearance of dominant PCR ribotypes such as 017, 027 and 078. Despite this continued threat, we have a poor understanding of how or why particular variants emerge and the sources of strains that dominate different human populations. We have undertaken a breadth genotyping study using multilocus sequence typing (MLST) analysis of 385 C. difficile strains from diverse sources by host (human, animal and food), geographical locations (North America, Europe and Australia) and PCR ribotypes. Results identified 18 novel sequence types (STs) and 3 new allele sequences and confirmed the presence of five distinct clonal lineages generally associated with outbreaks of C. difficile infection in humans. Strains of animal and food origin were found of both ST-1 and ST-11 that are frequently associated with human disease. An in depth MLST analysis of the evolutionary distant ST-11/PCR ribotype 078 clonal lineage revealed that ST-11 can be found in alternative but closely related PCR ribotypes and PCR ribotype 078 alleles contain mutations generating novel STs. PCR ribotype 027 and 017 lineages may consist of two divergent subclades. Furthermore evidence of microdiversity was present within the heterogeneous clade 1. This study helps to define the evolutionary origin of dominant C. difficile lineages and demonstrates that C. difficile is continuing to evolve in concert with human activity.     

Analysis of a Clostridium difficile PCR ribotype 078 100 kilobase island reveals the presence of a novel transposon, Tn6164

ABSTRACT: BACKGROUND: Clostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes, 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases. RESULTS: Analysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn6164), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5'and 3'ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn6164 was not present in this strain. To test the prevalence of Tn6164, a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn6164. Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype. CONCLUSIONS: Tn6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn6164 as a source of antibiotic resistance genes for other gut pathogens.     

Clostridium difficile PCR ribotype 078 toxinotype V found in diarrhoeal pigs identical to isolates from affected humans

In diseased piglets from two Dutch pig-breeding farms with neonatal diarrhoea for more than a year, culture and PCR analyses identified the involved microorganism as Clostridium difficile PCR ribotype 078 harbouring toxin A ( tcdA ) and B ( tcdB ), and binary toxin genes. Isolated strains showed a 39 bp deletion in the tcdC gene and they were ermB gene-negative. A number of 11 porcine and 21 human isolated C. difficile PCR ribotype 078 toxinotype V strains were found genetically related by multiple-locus variable-number tandem-repeat analysis (MLVA). Moreover, a clonal complex was identified, containing both porcine and human isolates. The porcine isolates showed an antimicrobial susceptibility profile overlapping that of isolates from Dutch human patients. On the basis of these pheno- and genotypical analyses results, it was concluded that the strains from affected piglets were indistinguishable from increasingly encountered C. difficile PCR ribotype 078 strains of human C. difficile infections in the Dutch population and that a common origin of animal and humans strains should be considered.     

Clostridium difficile in vegetables, Canada

Background: Clostridium difficile is an important gastrointestinal pathogen of humans and animals. It has been isolated from various foods, including meat and ready‐to‐eat salads, and concern has been expressed regarding food as a possible source of human C. difficile infection (CDI). Aims: We sought to isolate C. difficile from a variety of vegetables obtained from local grocery stores and to characterize these isolates. Materials and Methods: Vegetables were purchased from 11 different grocery stores in Guelph, Ontario, Canada between May and August 2009. Enrichment culture was performed and isolates were characterized by ribotyping, PFGE, toxinotyping and PCR detection of toxin genes. Results: Clostridium difficile was isolated from 4.5% (5/111) of retail vegetables. Two different ribotypes and two different toxinotypes were identified. Three isolates were ribotype 078/NAP 7/toxinotype V, possessing all three toxin genes. The other two isolates shared a ribotype with a toxigenic strain previously found in humans with CDI in this region. Discussion: Contamination of vegetables was found at relatively low levels, however, all isolates were toxigenic and belonging to ribotypes previously associated with CDI. Conclusions: Contamination of vegetables with CDI‐associated isolates can occur and although the implications for food safety practices remain elusive, the presence of toxigenic isolates suggests vegetables could be a source of C. difficile in humans.     

Clostridium difficile ribotypes in humans and animals in Brazil

Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen.     

Laboratory diagnosis of Clostridium difficile associated diarrhoea and molecular characterization of clinical isolates

We evaluated a three-step algorithm for laboratory diagnosis of Clostridium difficile-associated diarrhoea (CDAD). First, stool specimens were screened using an EIA test for glutamate dehydrogenase detection. Screen-positive specimens were tested by a rapid cytotoxintoxin A/B assay and subjected to stool culture. All cultures positive for C. difficile underwent toxigenic culture. The results showed that toxigenic culture allowed us to recover 37/156 (24.4%) stool samples harbouring toxigenic C. difficile that would have been missed by using faecal cytotoxin assay alone. This determined an increase in infection prevalence of 4.2% (from 11.4% to 15.6 %). Furthermore, to characterize the clinical Clostridium difficile isolates and the distribution of PCR ribotypes circulating in the San Carlo Borromeo hospital, molecular typing using semi-automated repetitive-sequence-based PCR (rep- PCR) and PCR ribotyping, and an evaluation of the antibiotic resistance were also performed. Among them, 71 indistinguishable strains were detected by rep-PCR and 83 by PCR-ribotyping revealing C. difficile outbreaks in our hospital. A total of 6 different ribotypes were obtained by PCR ribotyping. The most frequent ribotype was 018 (88.2%) that also showed resistance to moxifloxacin. In one case, uncommon PCR ribotype 186 was also identified.     

Induction of cytokines in a macrophage cell line by proteins of Clostridium difficile

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins produced by C. difficile are responsible for the characteristic pathology observed in C. difficile disease, but several surface-associated proteins of C. difficile are also recognized by the immune system and could modulate the immune response in infection. The aim of this study was to assess the induction of cytokines in a macrophage cell line in response to different antigens prepared from five C. difficile strains: the hypervirulent ribotype 027, ribotypes 001 and 106 and reference strains VPI 10463 and 630 (ribotype 012). PMA-activated THP-1 cells were challenged with surface-layer proteins, flagella, heat-shock proteins induced at 42 and 60 °C and culture supernatants of the five C. difficile strains. The production of the pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and IL-12p70 was observed in response to the surface-associated proteins, and high levels of TNF-α, IL-1β and IL-8 were detected in response to challenge with culture supernatants. The immune response triggered by the surface-associated proteins was independent of the strain from which the antigens were derived, suggesting that these proteins might not be related to the varying virulence of the hypervirulent ribotype 027 or ribotypes 001 and 106. There was no interstrain difference observed in response to the culture supernatants of the tested C. difficile strains, but this was perhaps due to toxicity induced in the macrophages by large amounts of toxin A and toxin B.     

New PCR ribotypes of Clostridium difficile detected in children in Brazil

A total of 35 Brazilian isolates of Clostridium difficile from faecal stools and four isolates from hospital environments were analyzed by PCR ribotyping. A whole cell protein profile (as an alternative for serogrouping), in vitro toxin production and susceptibility to vancomycin, metronidazole and clindamycin were also investigated. All strains were typeable by both phenotypic and genotypic methods, and a total of 13 different PCR ribotypes were identified, of which seven (132, 133, 134, 135, 136, 142 and 143) were considered new types and accounted for 78.5% of all samples evaluated (including hospital environments). A non-toxigenic C. difficile PCR ribotype 133 was detected in all children groups examined (inpatients, outpatients and healthy children), whilst toxigenic PCR ribotypes 015, 131, 134 and 135 were associated mostly with symptomatic children. Serogroups G and D were disseminated both in patients from the community and from the pediatric hospital, with group G prevalent among outpatient children. All strains were susceptible to vancomycin and metronidazole but high levels of resistance to clindamycin were found, especially among serogroups G and D. Co-existence of different ribotypes and serogroups in the same individual was observed. The new seven ribotypes found in this investigation may represent strains characteristic of this region of Brazil.     

Different Antibiotic Resistance and Sporulation Properties within Multiclonal Clostridium difficile PCR Ribotypes 078, 126, and 033 in a Single Calf Farm

Clostridium difficile strains were sampled periodically from 50 animals at a single veal calf farm over a period of 6 months. At arrival, 10% of animals were C. difficile positive, and the peak incidence was determined to occur at the age of 18 days (16%). The prevalence then decreased, and at slaughter, C. difficile could not be isolated. Six different PCR ribotypes were detected, and strains within a single PCR ribotype could be differentiated further by pulsed-field gel electrophoresis (PFGE). The PCR ribotype diversity was high up to the animal age of 18 days, but at later sampling points, PCR ribotype 078 and the highly related PCR ribotype 126 predominated. Resistance to tetracycline, doxycycline, and erythromycin was detected, while all strains were susceptible to amoxicillin and metronidazole. Multiple variations of the resistance gene tet(M) were present at the same sampling point, and these changed over time. We have shown that PCR ribotypes often associated with cattle (ribotypes 078, 126, and 033) were not clonal but differed in PFGE type, sporulation properties, antibiotic sensitivities, and tetracycline resistance determinants, suggesting that multiple strains of the same PCR ribotype infected the calves and that calves were likely to be infected prior to arrival at the farm. Importantly, strains isolated at later time points were more likely to be resistant to tetracycline and erythromycin and showed higher early sporulation efficiencies in vitro, suggesting that these two properties converge to promote the persistence of C. difficile in the environment or in hosts.     

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.     

Secretome analysis of Clostridium difficile strains

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.     

Nationwide Surveillance Study of Clostridium difficile in Australian Neonatal Pigs Shows High Prevalence and Heterogeneity of PCR Ribotypes

Clostridium difficile is an important enteric pathogen of humans and the cause of diarrhea and enteritis in neonatal pigs. Outside Australia, prevalence in piglets can be up to 73%, with a single PCR ribotype (RT), 078, predominating. We investigated the prevalence and genotype of C. difficile in Australian pig herds. Rectal swabs (n = 229) were collected from piglets aged <7 days from 21 farms across Australia. Selective culture for C. difficile was performed and isolates characterized by PCR for toxin genes and PCR ribotyping. C. difficile was isolated from 52% of samples by direct culture on chromogenic agar and 67% by enrichment culture (P = 0.001). No association between C. difficile recovery or genotype and diarrheic status of either farm or piglets was found. The majority (87%; 130/154) of isolates were toxigenic. Typing revealed 23 different RTs, several of which are known to cause disease in humans, including RT014, which was isolated most commonly (23%; 36/154). RT078 was not detected. This study shows that colonization of Australian neonatal piglets with C. difficile is widespread in the herds sampled.     

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.     

Sequence Similarity of Clostridium difficile Strains by Analysis of Conserved Genes and Genome Content Is Reflected by Their Ribotype Affiliation

PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S–23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation.     

Analysis of prevalence, risk factors and molecular epidemiology of Clostridium difficile infection in Kuwait over a 3-year period

We conducted a prospective study to evaluate the prevalence and epidemiology of CDI in Kuwait government hospitals over a 3-year period, January 2003 to December 2005, to determine the ribotypes responsible for CDI and to estimate the prevalence of ribotype 027. We also conducted a case-control study to identify the risk factors in our patient population. A total of 697 stool samples from patients with suspected CDI were obtained and sent to Anaerobe Reference Laboratory, Faculty of Medicine, Kuwait University for Clostridium difficile toxin detection, culture and PCR ribotyping. During the period, 73 (10.5%) out of 697 patients met the case definition of CDI. Of these, 56 (76.7%) were hospital-acquired and 17 (23.3%) were from outpatient clinics. Thus, the prevalence of hospital-acquired CDI amongst patients with diarrhoea was 8% over the study period; the prevalence in 2003, 2004 and 2005 was 9.7%, 7.8% and 7.2%, respectively. Our data showed that 42.9% of the CDI patients were above 60 years, of which >79% were aged 71 years and above. Patients with CDI were more likely than the controls to have been exposed to immunosuppressive drugs and feeding via nasogastric tube. The most common ribotypes isolated during this study were 002, 001, 126 and 140 and they represent 55.1% of all isolates. PCR ribotype 027 was not isolated.     

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.     

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.     

Isolation and characterization of <Emphasis Type="Italic">Clostridium difficile</Emphasis> from shellfish and marine environments

This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater, sediment, and zooplankton were collected at five sampling stations in the Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marine sediment. Majority of the C. difficile isolates were toxin A/B positive. Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.