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One hundred and thirty-eight C. difficile isolates from different sources (66 from the environment, 36 from animals, 9 from food and 27 from humans) were ribotyped by capillary electrophoresis PCR ribotyping (CE-PCR). A multilocus variable tandem repeat analysis (MLVA) was carried out on a sample subset. The most frequently isolated PCR ribotypes were 126 (15.9%), 078 (14.5%), 011/018 (11.6%), 014/020/077 (10.1%), and 010 (2.8%). In particular, strains of PCR ribotype 011/018 were isolated from human, raw milk and environmental samples. The hypervirulent PCR ribotype 027 was isolated from two human samples. The majority of the strains were toxigenic (34.1% showed the toxigenic profile A + B + CDT + and 38.9% the profile A + B + CDT - ). MLVA allowed to identify 4 clonal complexes of genetically related isolates: complex n. 1 grouped together human, environmental and food strains, whereas complex n. 3 included human and environmental isolates. The use of MLVA gave further evidence to the possible role of environment, animals and food as routes of transmission of C. difficile infections to human., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Even though food of animal sources and different foodstuffs are well known to be potentially carrier of Clostridium difficile, few data are available on the occurrence of C. difficile in seafood. This work investigated the occurrence of C. difficile in edible bivalve molluscs in southern Italy. Out of the 925 investigated samples, 3.9% contained C. difficile. Eighteen strains harboured both genes for toxins A and B whereas 1 only had toxin B gene. Binary toxin genes were found in 22.2% of the isolates. The most frequently ribotypes found were 078/126 (22.2%), 010 (19.4%), and 001 (8.3%). All isolates were susceptible to metronidazole, vancomycin, fidaxomicin, and to the new semisynthetic thiopeptide antibiotic LFF571, whereas 19.4% of them were resistant to moxifloxacin, 30.5% to clindamycin, 38.8% to erythromycin, and 100% to ciprofloxacin. This study points out that edible molluscs could be a potential source of toxigenic C. difficile ribotypes and a potential risk for human health., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

The incidence and characterisation of Aeromonas species in human and environmental samples in southern Italy were investigated. The results emphasize that 12.3% of the 210 examined patients carried Aeromonas spp. in their faeces. These results underline the need to include Aeromonas spp. in the list of routinely analysed enteropathogens in all diarrhoeal stool samples, especially in children below 10 years of age, elderly persons and immunocompromised patients.

A survey was undertaken to examine sea water and sediment for the presence of Vibrio and Aeromonas spp. along approximately 900 km of coast in Southern Italy during early and late summer. A quantitative analysis was also done to evaluate the water fecal contamination at the stations examined. The results indicate that all the investigated areas were submitted to a wide spatial fluctuation of fecal contamination and that Vibrio and Aeromonas spp. were present in both high and low fecal-contaminated stations. Sixty two percent of the investigated samples were positive for Aeromonas spp., while 42% of samples were positive for Vibrio spp. It was interesting to note that 38% of the positive stations for both Aeromonas and Vibrio spp. showed a fecal coliform contamination of water at < 10(2) cells 100 ml(-1). Thus, these findings support the hypothesis that the bacterial indicators (such as fecal coliforms) do not always satisfactorily reflect the hygienic quality of water. The presence of Vibrionaceae on copepods was also investigated. Copepods were sampled at a station located inside the harbour of the city of Naples and were found contaminated by V. cholerae non-O1, V. alginolyticus, V. fluvialis and A. caviae. Furthermore, the antibiotic resistance patterns of isolated bacteria showed the presence of a number of resistant strains among the isolates. In order to discriminate the isolates on the basis of their biochemical profiles and/or antibiotic resistance patterns, cluster analysis was carried out which showed that no unique assay could fully discern these isolates. However, the best discrimination resulted from complete pattern profile based on both biochemical profiles and antibiotic resistance patterns.

The colonisation of planktonic copepod integument by bacteria belonging to the family of Vibrionaceae is a well described phenomenon. In this study, besides reporting on the occurrence of Vibrionaceae and other enteropathogens, we further report on the bacterial attachment to the Estuarine copepod Acartia margalefi in a faecal polluted coastal lagoon near Naples, Southern Italy. In addition, we also performed a laboratory experiment to study the ability of 7 bacterial strains (Vibrio cholerae non-Ol, V. mimicus, V. parahaemolyticus, V. alginolyticus, Aeromonas hydrophila, Escherichia coli and Pseudomonas sp.) to colonise the copepod integument. For this laboratory study, 4 different species of copepods, namely Temora stylifera, A. clausi, Centropages typicus and Paracalanus parvus sampled from the Gulf of Naples (Southern Italy) were used. Scanning electron microscope (SEM) studies on the copepods sampled from the lagoon indicated that the bacterial attachment on the integument of Acartia margalefi was mainly on the ventro-lateral body region of the host and in the joints of the thoracic segments, as well as on the swimming and feeding appendages. This infestation, made by rod-like bacteria, was absent in winter but reached peak values of 70% frequency in June. The laboratory studies showed that while V. cholerae non-O1 and A. hydrophila attached on live and dead copepods, respectively, the V. parahaemolyticus, V. alginolyticus, V. mimicus, E. coli and Pseudomonas sp. failed to colonise either live or dead copepods. Thus, this study provides further valuable information about the ecological relationship between different microorganisms (pathogens) and copepods in the coastal marine environment in Southern Italy.

Aeromonas hydrophila strains isolated from the same geographical region (southern Italy) but from different sources (sea sediments and human diarrhea cases) were characterized for the production of potential virulence determinants, such as production of cytotoxins, cytotonic toxins, hemolysin, and dermonecrotic factors and their capacity to adhere to human intestinal 407 cells in vitro. The results showed that isolates from both the sources produced all or some of the virulence factors which may be involved in the pathogenesis of Aeromonas-associated infections. Our study indicates that further epidemiological studies are necessary to elucidate the public health significance of infections caused by Aeromonas spp.