Toxigenic Clostridium difficile PCR ribotypes from wastewater treatment plants in southern Switzerland.

The occurrence of Clostridium difficile in nine wastewater treatment plants in the Ticino Canton (southern Switzerland) was investigated. The samples were collected from raw sewage influents and from treated effluents. Forty-seven out of 55 characterized C. difficile strains belonged to 13 different reference PCR ribotypes (009, 010, 014, 015, 039, 052, 053, 066, 070, 078, 101, 106, and 117), whereas 8 strains did not match any of those available in our libraries. The most frequently isolated ribotype (40%) was 078, isolated from six wastewater treatment plants, whereas ribotype 066, a toxigenic emerging ribotype isolated from patients admitted to hospitals in Europe and Switzerland, was isolated from the outgoing effluent of one plant. The majority of the isolates (85%) were toxigenic. Forty-nine percent of them produced toxin A, toxin B, and the binary toxin (toxigenic profile A(+) B(+) CDT(+)), whereas 51% showed the profile A(+) B(+) CDT(-). Interestingly, eight ribotypes (010, 014, 015, 039, 066, 078, 101, and 106) were among the riboprofiles isolated from symptomatic patients admitted to the hospitals of the Ticino Canton in 2010. Despite the limitation of sampling, this study highlights that toxigenic ribotypes of C. difficile involved in human infections may occur in both incoming and outgoing biological wastewater treatment plants. Such a finding raises concern about the possible contamination of water bodies that receive wastewater treatment plant effluents and about the safe reuse of treated wastewater.

Low occurrence of Clostridium difficile in retail ground meat in Sweden.

This pilot study was conducted to evaluate the occurrence of Clostridium difficile in samples of ground meat in Sweden. From April to September 2008, 82 meat samples were collected from randomly selected retail shops in Uppsala County (central Sweden). C. difficile was isolated from 2 (2.4%; both ground beef) of the 82 meat samples. No C. difficile was detected in pork, hamburger, sheep, poultry, or other type of meat samples. The two C. difficile isolates produced both toxin A and toxin B. These findings indicate that C. difficile might be present in ground meat samples in Sweden. However, further studies are necessary to confirm these preliminary data and to elucidate the public health significance of meat contamination by C. difficile in Sweden.

Antibiotic susceptibility profile of Aeromonas spp. isolates from food in Abu Dhabi, United Arab Emirates.

A total of 57 Aeromonas isolates from food samples such as fresh and frozen chicken, game birds, pasteurized milk, baby food, bakery products, fruit and vegetables, fish, and water from Abu Dahbi, UAE were investigated for antibiotic susceptibility profile. Most strains were resistant to penicillins (ticarcillin, mezlocillin, oxacillin, piperacillin), sulfamethoxazole, trimethoprim and macrolides (erythromycin, vancomycin, clindamycin) but sensitive to tetracycline, chloramphenicol, nitrofurantoin, aminoglycosides (amikacin, gentamicin, tobramycin), cephalosporins (cefuroxime, ceftrioxone, cefazolin, cephalexin, cephalothin, cefoxitin, cefotaxime), quinolone (ciprofloxacin), colistin sulphate and SXT (trimethoprim-sulfamethoxazole). On the other hand, many antibiotics showed excellent inhibitory activity (>75% strains were sensitive to them) against all the strains tested. These include cefuroxime, ceftrioxone, ciprofloxacin, colistin, amikacin, gentamicin, tetracycline, chloramphenicol, nitrofurantoin, cefotaxime and tobramycin. In conclusion, the results show a detailed pattern of sensitivity of the various Aeromonas spp. isolates to a variety of antibiotics and provide useful information in the context of selective isolation and phenotypic identification of the aeromonads from food.

Toxin production by and adhesive properties of Clostridium difficile isolated from humans and horses with antibiotic-associated diarrhea.

Clostridium difficile is a common nosocomial pathogen in humans and animals that causes diarrhea and colitis following antibiotic therapy. Isolates of C. difficile obtained from faecal material from 20 human patients and 6 equine subjects with antibiotic-associated diarrhea were investigated regarding production of toxins A and B, their capacity to adhere to the human intestinal Caco-2 cell line and equine intestinal cells, and for the presence of fimbriae. The results showed that most (17/20) of the human clinical isolates produced both toxins A and B. One of the human isolates proved toxin A-negative/toxin B-positive. All (6/6) horse isolates were positive for both toxins A and B. Both the human and horse isolates possessed the capacity to adhere, to varying degree, to human and equine intestinal cells. It appeared that human isolates produced greater amounts of toxin B, and that there was a host-species dependency on ability to attach to intestinal epithelial cells. No fimbriae were found in any of the investigated isolates.

Occurrence of enterotoxic Staphylococcus aureus in raw milk from yaks and cattle in Mongolia.

Staphylococcal food poisoning is considered one of the leading foodborne illnesses in humans worldwide and is associated with contaminated foods of animal origin, such as milk and dairy products. In this study, we investigated the occurrence of staphylococci and the enterotoxigenic properties of Staphylococcus aureus isolated from raw milk from yaks (Bos mutus) and cattle in Mongolia. Staphylococci were isolated from 72 (74%) of the 97 raw milk samples. Of the samples containing staphylococci, 69% (50 of 72) were from yaks and 30.5% (22 of 72) were from cattle. S. aureus was detected in 10% of yak (7 of 72) and 21% of cattle (15 of 72) milk samples. Staphylococcal enterotoxin C was detected in 23% (5 of 22) of the S. aureus strains investigated, based on the reverse passive latex agglutination technique. Three of the five enterotoxigenic strains were from yaks and two were from cattle. None of the S. aureus strains tested produced staphylococcal enterotoxins A, B, or D. To our knowledge, this is the first report of the occurrence of staphylococci and enterotoxigenic S. aureus in milk from yaks and cattle in Mongolia.

Putative virulence factors of the Aeromonas spp. isolated from food and environment in Abu Dhabi, United Arab Emirates.

Thirty randomly selected Aeromonas isolates from food and the environment in Abu Dhabi, United Arab Emirates, were characterized for putative virulence determinants, such as production of cytotoxin, cytotonic toxin, and hemolysin and their capacity to adhere to and invade Henle 407 cells in vitro. Seventy percent of the tested isolates were cytotoxin producers, and 80% were hemolytic. Cytotoxin was produced by 6 of 7 A. hydrophila strains, 6 of 13 A. caviae strains, and 6 of 7 A. veronii bv. sobria strains, mostly from food sources. A. schubertii, A. jandaei, and A. trota also produced both cytotoxin and hemolysin. All of the 30 isolates tested adhered to Henle 407 cells, but none were able to invade the cells, as determined with the in vitro assay. However, no significant correlation of the presence of these putative virulence factors was found among these aeromonad food isolates.

Serotypes and anti-microbial susceptibility of Plesiomonas shigelloides isolates from humans, animals and aquatic environments in different countries.

A total of 73 strains of Plesiomonas shigelloides isolated from humans (24 strains) animals (21 strains) and aquatic environment (28 strains) were determined for their O:H serotype and susceptibility to 18 anti-microbial substances and to the vibriostatic agent O/129. Of all strains, 86.3% were typeable by the O and 94.5% by the H anti-sera used. The serotype distribution was heterogeneous within a country and between the countries. Of the 57 different serotypes identified, O11:H2 (2 strains), O22:H3 (4 strains), O35:HH11 (2 strains), O52:H3 (2 strains) and O90:H6 (2 strains) were found among isolates from humans and animals (mainly in cats) in Finland and Cuba, and O23:H1a1b (3 strains) among isolates from environmental sources in Slovak Republic and Italy. Most (93-100%) of all strains were susceptible to all anti-microbials tested but resistant (92-96%) to the broad-spectrum penicillins (ampicillin, mezlocillin). No correlation between anti-microbial resistance patterns and serotypes was found.

Clostridium difficile in faeces from healthy dogs and dogs with diarrhea.

BACKGROUND: This study was conducted to evaluate the faecal occurrence and characterization of Clostridium difficile in clinically healthy dogs (N = 50) and in dogs with diarrhea (N = 20) in the Stockholm-Uppsala region of Sweden. FINDINGS: Clostridium difficile was isolated from 2/50 healthy dogs and from 2/20 diarrheic dogs. Isolates from healthy dogs were negative for toxin A and B and for the tcdA and tcdB genes. Both isolates from diarrheic dogs were positive for toxin B and for the tcdA and tcdB genes. The C. difficile isolates from healthy dogs had PCR ribotype 009 (SE-type 6) and 010 (SE-type 3) whereas both isolates from dogs with diarrhoea had the toxigenic ribotype 014 (SE-type 21). One of the isolates from healthy dogs was initially resistant to metronidazole. CONCLUSIONS: This study revealed presence of toxigenic C. difficile in faecal samples of diarrheic dogs and low number of non- toxigenic isolates in healthy dogs from Uppsala-Stockholm region in Sweden. However, more comprehensive studies are warranted to investigate the role of C. difficile in gastrointestinal disease in dogs.

Isolation and characterization of Clostridium difficile 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.

Molecular evidence of Plesiomonas shigelloides as a possible zoonotic agent.

The most frequently used method for establishing epidemiological relationships between Plesiomonas shigelloides strains is O:H serotyping. However, a number of strains are not serotypeable and isolates from diverse sources can display the same serovar. Moreover, since the zoonotic nature of Plesiomonas has been suggested and this hypothesis is based on the identical serovars found in animals and humans, we intend to use four DNA-based techniques: random amplified polymorphic DNA-PCR, enterobacterial repetitive intergenic consensus-PCR, repetitive extragenic palindromic-PCR, and pulsed field gel electrophoresis in order to screen 24 strains belonging to nine O:H serovars isolated from humans, animals, and the environment. In general, P. shigelloides showed a high genetic heterogeneity. Three pairs of strains, each containing a human and an animal isolate, displayed similar genotypes. This is the first report that provides molecular evidence that P. shigelloides may be zoonotic.

Recombining population structure of Plesiomonas shigelloides (Enterobacteriaceae) revealed by multilocus sequence typing.

Plesiomonas shigelloides is an emerging pathogen that is widespread in the aquatic environment and is responsible for intestinal diseases and extraintestinal infections in humans and other animals. Virtually nothing is known about its genetic diversity, population structure, and evolution, which severely limits epidemiological control. We addressed these questions by developing a multilocus sequence typing (MLST) system based on five genes (fusA, leuS, pyrG, recG, and rpoB) and analyzing 77 epidemiologically unrelated strains from several countries and several ecological sources. The phylogenetic position of P. shigelloides within family Enterobacteriaceae was precisely defined by phylogenetic analysis of the same gene portions in other family members. Within P. shigelloides, high levels of nucleotide diversity (average percentage of nucleotide differences between strains, 1.49%) and genotypic diversity (64 distinct sequence types; Simpson's index, 99.7%) were found, with no salient internal phylogenetic structure. We estimated that homologous recombination in housekeeping genes affects P. shigelloides alleles and nucleotides 7 and 77 times more frequently than mutation, respectively. These ratios are similar to those observed in the naturally transformable species Streptococcus pneumoniae with a high rate of recombination. In contrast, recombination within Salmonella enterica, Escherichia coli, and Yersinia enterocolitica was much less frequent. P. shigelloides thus stands out among members of the Enterobacteriaceae. Its high rate of recombination results in a lack of association between genomic background and O and H antigenic factors, as observed for the 51 serotypes found in our sample. Given its robustness and discriminatory power, we recommend MLST as a reference method for population biology studies and epidemiological tracking of P. shigelloides strains.