Virulence-associated factors in Vibrio cholerae non-O1/non-O139 and V. mimicus strains isolated in ornamental fish species.

During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non-O1/non-O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South-East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix-assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization-associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR+ , rtxA+ and hlyAET+ genotype. Interestingly, the V. mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.

Effect of high hydrostatic pressure on murine norovirus in Manila clams.

AIMS: Eating raw or insufficiently cooked bivalve molluscs contaminated with human noroviruses (NVs) can result in acute cases of gastroenteritis in humans. Manila clams (Ruditapes philippinarum) are particularly prone to exposure to NVs due to the brackish environment in which they are farmed which is known to be susceptible to human faecal contamination. High hydrostatic pressure processing (HHP) is a food treatment technique that has been shown to inactivate NV. METHODS AND RESULTS: In this study we investigated the ability of HHP to inactivate murine norovirus (MNV-1), a recognised surrogate for NV, in experimentally contaminated manila clams. Pools of contaminated live clams were subjected to hydrostatic pressure ranging from 300 to 500 MPa for different time intervals of between one and 10 min. The trial was repeated three times, at monthly intervals. CONCLUSIONS: Virus vitality post-treatment was assessed and the data obtained indicates that the use of high hydrostatic pressures of at least 500 MPa for 1 min was effective in inactivating MNV-1. SIGNIFICANCE AND IMPACT OF THE STUDY: HHP results to be an effective technique that could be applied to industrial process to obtain safe Manila clams ready to eat.

Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors.

AIMS: To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors. METHODS AND RESULTS: Fortnightly sampling of shellfish was carried out in two lagoon areas (category B production areas) and one sea area (category A). Environmental parameters in the lagoon and hydrometric level of the tributary river were monitored throughout the sampling period. Samples (n = 120) were analysed for bacterial (E. coli and Salmonella) and viral (HAV and NoV) contamination; samples from category B areas were analysed before and after purification treatment. All the samples were negative for HAV whereas 10 samples (8.3%), all harvested in the lagoon areas, were positive for NoV. Sequencing identified the strains as genotypes II.4 and II.b. None of the samples was found to be contaminated after depuration. CONCLUSIONS: The monitoring showed a low frequency of NoV presence; viral contamination, detected exclusively in shellfish collected from the deltaic area (category B), could be influenced by the flow of the tributary river. The data collected are useful for the design of targeted prevention strategies and for the modulation of control plans after meteorological events.

Direct identification of Photobacterium damselae subspecies piscicida by PCR-RFLP analysis.

Fish pasteurellosis is an infectious disease that affects several teleost species living in temperate marine waters. The pathogen responsible, Photobacterium damselae subspecies piscicida, shows high genetic similarity with P. damselae subsp. damselae, making subspecies discrimination extremely laborious. Here we report for the first time a PCR-RFLP method for the identification of P. damselae subsp. piscicida without prior isolation in pure culture. Genomic sequence information was obtained through cloning and sequencing of RAPD products. Two P. damselae-specific primer pairs were developed and tested on 17 strains of P. damselae subsp. piscicida, 10 strains of P. damselae subsp. damselae, and 6 closely related control species. High sensitivity was achieved in PCR amplification on serially diluted samples (<180 fg of pure bacterial DNA or <10 fg, depending on the amplified fragment). Restriction analysis of PCR products showed a unique digestion profile for all P. damselae subsp. piscicida strains. The same PCR-RFLP method was implemented on total DNA samples extracted from experimentally infected sea bream and sea bass. Positive results were obtained on fish with clear signs of the disease as well as on challenged, but asymptomatic, fish. The method presented here might provide a useful tool for both prevention and rapid diagnosis of fish pasteurellosis.

Detection and characterization of vancomycin-resistant enterococci in farm animals and raw meat products in Italy.

The emergence of vancomycin-resistant enterococci (VRE) in Europe has been ascribed to the long-time use of the glycopeptide antibiotic avoparcin as feed additive in food animals, until its ban in 1997 in EU. The pres- ence of VRE in food of animal origin is believed to represent a potential risk for the consumer. We studied the fecal carriage of VRE in broiler chickens and slaughter pigs in Italy before the avoparcin ban and eval- uated the impact of avoparcin withdrawal on the presence of VRE in raw meat products. Broilers and pigs were both found to be frequently colonized by VRE, as 36% and 24.6% of the flocks or the herds, respec- tively, were positive. Molecular typing of VRE strains by PFGE showed that animals housed in different pens within the same farm were colonized by clonally related strains. After the avoparcin ban, a decrease in the rate of VRE contamination in meat products was observed. Such a decrease was statistically significant in poultry (from 18.8% to 9.6%) but not in pork products (from 9.7% to 6.9%). The majority of VRE from all sources carried the vanA resistance gene and included Enterococcus faecium, E. faecalis, E. hirae, E. durans, and E. gallinarum. None of the strains carried the vanB gene, whereas constitutively resistant vanC-positive strains were frequently found. Our results show that avoparcin withdrawal has been successful in reducing VRE contamination in meat products. However, this measure needs to be complemented by a prudent use of glycopeptide antibiotics in human medicine.

High incidence of antibiotic multi-resistant bacteria in coastal areas dedicated to fish farming.

Marine bacteria exposed to antibiotics in fish farms can acquire antimicrobial resistance by mobile genetic elements and horizontal gene transfer. A total of 872 autochthonous marine bacterial strains was isolated from samples collected from four different fish farms located at northern and southern Italian Adriatic Sea. Resistance to only tetracycline (17%) and to trimethoprim-sulfadiazine (7%) were the most frequent patterns obtained, while flumequine resistance has recorded in only 0.3% of the strains. Comparing strains isolated from coastal areas and fish farms, a significant higher incidence (4% versus 10%) of multi-resistant strains in aquaculture centers was found. Significant differences in antibiotic resistance incidence were also detected among the four fish farms due probably to different approaches in farm management and the more or less frequent use of antibiotics. Antibiotic-resistant and multi-resistant strains isolated constitute an environmental reservoir directly involved in the seafood chain and might represent a public health concern.

Signaling crosstalk between TGFß and Dishevelled/Par1b.

Crosstalk of signaling pathways is critical during metazoan development and adult tissue homeostasis. Even though the transforming growth factor-beta (TGFß) transduction cascade is rather simple, in vivo responsiveness to TGFß ligands is tightly regulated at several steps. As such, TGFß represents a paradigm for how the activity of one signaling system is modulated by others. Here, we report an unsuspected regulatory step involving Dishevelled (Dvl) and Par1b (also known as MARK2). Dvl and Par1b cooperate to enable TGFß/bone morphogenetic protein (BMP) signaling in Xenopus mesoderm development and TGFß responsiveness in mammalian cells. Mechanistically, the assembly of the Par1b/Dvl3/Smad4 complex is fostered by Wnt5a. The association of Smad4 to Dvl/Par1 prevents its inhibitory ubiquitination by ectodermin (also known as transcriptional intermediary factor 1 gamma or tripartite motif protein 33). We propose that this crosstalk is relevant to coordinate TGFß responses with Wnt-noncanonical and polarity pathways.

Sodium serum levels in hypoalbuminemic adults at general medical wards.

Hypoalbuminemia may cause interstitial edema and hemodilution, which we hypothesized may influence serum sodium levels. Our purpose was to compare serum sodium levels of hospitalized adults with or without hypoalbuminemia. All sodium and albumin serum levels of 142 adults hospitalized at general medical wards over a six-month period were searched at a University Hospital mainframe computer. Relevant laboratory data and clinical details were also registered. Hypoalbuminemia was defined by serum albumin concentration < 3.3 g/dl Fisher, Mann-Whitney, and Student's t tests were applied to compare groups with or without hypoalbuminemia. Ninety-nine patients, classified as hypoalbuminemic, had lower blood hemoglobin (10.68 +/- 2.62 vs. 13.54 +/- 2.41), and sodium (135.1 +/- 6.44 vs. 139.9 +/- 4.76 mEq/l) and albumin (2.74 +/- 0.35 vs. 3.58 +/- 0.28 g/dl) serum levels than non-hypoalbuminemic (n = 43). Pearson's coefficient showed a significant direct correlation between albumin and sodium serum levels (r = 0.40) and between serum albumin and blood hemoglobin concentration (r = 0.46). Our results suggest that hypoalbuminemic adults have lower serum sodium levels than those without hypoalbuminemia, a phenomenon that may be at least partially attributed to body water retention associated with acute phase response syndrome.