Vibrio parahaemolyticus-specific Halobacteriovorax From Seawater of a Mussel Harvesting Area in the Adriatic Sea: Abundance, Diversity, Efficiency and Relationship With the Prey Natural Level

This research aimed to study the abundance and molecular diversity of Vibrio parahaemolyticus-specific Halobacteriovorax strains isolated from seawater of the Adriatic Sea and the relationship between predator and prey abundances. Moreover, predator efficiency of the Halobacteriovorax isolates toward V. parahaemolyticus and Vibrio cholerae non-O1/O139 strains was tested. V. parahaemolyticus NCTC 10885 was used as primary host for the isolation of Halobacteriovorax from seawater by the plaque assay. Molecular identification was performed by PCR detection of a fragment of the 16S rRNA gene of the Halobacteriovoraceae family members. Moreover, 700 bp PCR products were sequenced and compared between them and to clones described for other sampling sites. Vibrio counts were performed on TCBS agar from 100 ml of filtered water samples and presumptive colonies were confirmed by standard methods. Predatory efficiency of Halobacteriovorax isolates was tested by monitoring abilities of 3-day enrichments to form clear lytic halos on a lawn of Vibrio preys, by the plaque assay. Out of 12 seawater samples monthly collected from June 2017 to May 2018, 10 were positive for V. parahaemolyticus specific Halobacteriovorax with counts ranging from 4 to 1.4 × 103 PFU per 7.5 ml. No significant relationship was found between Halobacteriovorax and Vibrio abundances. The 16SrRNA sequences of our Halobacteriovorax strains, one for each positive sample, were divided into three lineages. Within the lineages, some sequences had 100% similarity. Sequence similarity between lineages was always <94.5% suggesting that they may therefore well belong to three different species. All Halobacteriovorax isolates had the ability to prey all tested Vibrio strains. Additional research is necessary to assess whether stable strains of Halobacteriovorax are present in the Adriatic Sea and to understand the mechanisms by which Halobacteriovorax may modulate the abundance of V. parahaemolyticus and other vibrios in a complex marine ecosystem

Vibrio parahaemolyticus control in mussels by a Halobacteriovorax isolated from the Adriatic sea, Italy

This study evaluated the application of a Halobacteriovorax isolated from water of the Adriatic Sea (Italy) in controlling V. parahaemolyticus in mussels (Mytilus galloprovincialis). Two 72 h laboratory-scale V. parahaemolyticus decontamination experiments of mussels were performed. The test microcosm of experiment 1 was prepared using predator/prey free mussels experimentally contaminated with Halobacteriovorax/V. parahaemolyticus at a ratio of 103 PFU/105 CFU per ml, while that of experiment 2 using mussels naturally harbouring Halobacteriovorax that were experimentally contaminated with 105 CFU per ml of V. parahaemolyticus. For experiment 1, was also tested a control microcosm only contaminated with 105 CFU per ml of V. parahaemolyticus. Double layer agar plating and pour plate techniques were used to enumerate Halobacteriovorax and V. parahaemolyticus, respectively. 16 S rRNA analysis was used to identify Halobacteriovorax. For both experiments in the test microcosm the concentration of prey remained at the same level as that experimentally added, i.e. 5 log for the entire analysis period. In experiment 1, V. parahaemolyticus counts in mussels were significantly lower in the test microcosm than the control with the maximum difference of 2.2 log at 24 h. Results demonstrate that Halobacteriovorax can modulate V. parahaemolyticus level in the mussels. The public impact of V. parahaemolyticus in bivalves is relevant and current decontamination processes are not always effective. Halobacteriovorax is a suitable candidate in the development of a biological approach to the purification of V. parahaemolyticus in mussels

Bdellovibrio bacteriovorus to control Escherichia coli on meat matrices

Bdellovibrio bacteriovorus is a predator micro-organism towards other Gram-negative bacteria. We tested B. bacteriovorus to control Escherichia coli growth on chicken slices and canned beef. Moreover, we analysed B. bacteriovorus's lytic ability on eight toxigenic or multidrug-resistant E. coli strains. In chicken slices, the predator induced the highest prey reduction (4.3 log) respect to control at 6 h. In canned beef, the predator induced the highest prey reduction (2.1 log) respect to control at 6 h. Moreover, B. bacteriovorus showed lytic ability towards all tested E. coli strains. B. bacteriovorus could control E. coli and other pathogenic and spoilage bacteria in those meat-based foods that have a shelf life <10 days. It could integrate modified atmosphere packaging (MAP) to prolong the shelf life and improve the safety of pre-packed fresh meat, meat preparations and meat products. In future applications on meat-based foods, B. bacteriovorus could also minimise the use of additives

CLAMS HARVESTING AREAS IN MARCHE REGION: ANALYSIS OF FECAL CONTAMINATION IN THREE-YEAR PERIOD 2008-2010

n/

PHENOTIPIC AND GENOTIPIC CHARACTERIZATION OF SALMONELLA SPP ISOLATED FROM MOLLUSCAN SHELLFISH IN MARCHE REGION

Salmonella enterica is a major epidemic cause of gastrointestinal infection worldwide. Although the animal host is believed to be the primary habitat of this specie, Salmonella is frequently isolated from water sources and it has been identified in marine environments. In this study the incidence of serotypes of Salmonella in the coastal water of the Italian region of Marche on the Adriatic Sea was evaluated. A total of 3985 samples of molluscan shellfish were analyzed during routine surveillance activity for a period of five years (2002-2007) and 0,95% of the samples were found contaminated with Salmonella. The most prevalent serotypes were Seftenberg (23.5%), Typhimurium (14,7%) and Enteritidis (11.8%) respectively. Pulsed-field electrophoresis and phage typing were used to determine possible genetic relationship (relatedness) between S. Enteritidis strains isolated from bivalve mollusc and those isolated from human cases, animals and foods in Region of Marche. Three isolates from mollusc shellfish, 7 from sporadic human infection and 4 from poultry farms were confirmed as phagetype PT2 and PFGE profile XB0002. These results suggest a molecular fingerprinting relationship among shellfish, human and animal isolates, which could be considered as preliminary evidence of human infections associated with poultry production industry

Selenium response to diverse household cooking methods as applied to 15 species of finfish and shellfish caught in the Mediterranean sea

Total selenium contents were determined in the edible portion of several species of blueback fish (European anchovy, European pilchard, sprat, horse mackerel), whitefish (European hake, common sole, red mullet, flathead grey mullet, tub gurnard), molluscs (cuttlefish, Mediterranean mussel, Manila clam, striped venus clam) and crustaceans (caramote prawn, mantis shrimp). Within each species, specimens were analysed both at the raw state and cooked by the most appropriate technique [selected among the following: oven roasting in a partially covered tin (ORO), pan frying (PAF), cooking in parchment (PAR), cooking in a covered non-stick pan (CNS), pressure cooking (PRE), steam cooking (STM)], to gain knowledge about selenium true retention values. Several considerations prompted and justified this study at the same time: the essentiality of selenium, the medium-to-high level of this element known to be present in raw seafood, the attention recently focused on the influence exerted by processing (cooking in particular) on such issues as selenium bioavailability and speciation, the variety of culinary approaches to finfish and shellfish which have been fine-tuned in centuries in the Mediterranean countries and possibly bears on selenium retention. From October to May, 2-to-5 batches per season were collected for each species, the number of specimens per batch depending on their average size and merceological nature. A species-specific procedure was devised in order to have a representative raw reference for each cooked sample. Within species and batch, both raw and cooked flesh were microwave digested and analysed in duplicate by ICP-MS for total selenium content. True retention values (TRVs) of selenium were determined for each species within its own cooking procedure and cooking yield. Season of catch effect was significant for two-thirds of finfish species and for mantis shrimp only among shellfish, the lower levels being in Autumn. Upon cooking, selenium content increased significantly with the exception of PRE cuttlefish, CNS mussel and CNS Manila clam. The richest sources of selenium were ORO pilchard within blueback fish, ORO tub gurnard and ORO red mullet within whitefish, CNS mussel within molluscs and PAF mantis shrimp within crustaceans. When comparing cooking methods as to the selenium TRVs (%) they generated, significant differences did emerge, the average being 106a, 103a, 100a, 95.1ab, 83.9b, and 46.6c, for ORO, PAF, PAR, STM, CNS, and PRE, respectively

A severe case of Aeromonas veronii biovar sobria travellers’ diarrhoea characterized by Vibrio parahaemolyticus co-isolation

We report a severe case of traveller’s diarrhoea in a patient returning from Ecuador to Italy with the concomitant presence of Aeromonas veronii biovar Sobria and Vibrio parahaemolyticus in their faeces. Based on diagnostic results, epidemiological information and the clinical outcome, we conclude that the real aetiological agent was A. veronii biovar Sobria, while V. parahaemolyticus was only transient in the intestine of the patient

The long noncoding RNA nHOTAIRM1 is necessary for differentiation and activity of iPSC-derived spinal motor neurons

The mammalian nervous system is made up of an extraordinary array of diverse cells that form intricate functional connections. The programs underlying cell lineage specification, identity and function of the neuronal subtypes are managed by regulatory proteins and RNAs, which coordinate the succession of steps in a stereotyped temporal order. In the central nervous system (CNS), motor neurons (MNs) are responsible for controlling essential functions such as movement, breathing, and swallowing by integrating signal transmission from the cortex, brainstem, and spinal cord (SC) towards peripheral muscles. A prime role in guiding the progression of progenitor cells towards the MN fate has been largely attributed to protein factors. More recently, the relevance of a class of regulatory RNAs abundantly expressed in the CNS - the long noncoding RNAs (lncRNAs) - has emerged overwhelmingly. LncRNA-driven gene expression control is key to regulating any step of MN differentiation and function, and its derangement profoundly impacts neuronal pathophysiology. Here, we uncover a novel function for the neuronal isoform of HOTAIRM1 (nHOTAIRM1), a lncRNA specifically expressed in the SC. Using a model system that recapitulates spinal MN (spMN) differentiation, we show that nHOTAIRM1 intervenes in the binary cell fate decision between MNs and interneurons, acting as a pro-MN factor. Furthermore, human iPSC-derived spMNs without nHOTAIRM1 display altered neurite outgrowth, with a significant reduction of both branch and junction numbers. Finally, the expression of genes essential for synaptic connectivity and neurotransmission is also profoundly impaired when nHOTAIRM1 is absent in spMNs. Mechanistically, nHOTAIRM1 establishes both direct and indirect interactions with a number of target genes in the cytoplasm, being a novel post-transcriptional regulator of MN biology. Overall, our results indicate that the lncRNA nHOTAIRM1 is essential for the specification of MN identity and the acquisition of proper morphology and synaptic activity of post-mitotic MNs

Prevalence and virulence properties of non-O1 non-O139 Vibrio cholerae strains from seafood and clinical samples collected in Italy

Seafood and clinical samples collected in Italy during 2006 were analyzed to evaluate prevalence, serological and virulence properties of non-O1 non-O139 Vibrio cholerae (NCV) isolates. Biochemical and serological characterization of the strains was performed by standardized procedures while virulence properties of NCVs were assayed by molecular, in vivo and in vitro toxicological methods. Of the 300 seafood samples examined, including mussel, cod, mackerel, anchovy, clam, prawn and cuttle-fish, 5.6% were positive for NCVs: 4.7% and 8.5% from local and imported seafood, respectively. The prevalence of NCVs was highest in prawn (16.6%) and mussel (7.7%). Of 58 hospitalized patients that presented acute diarrhea, 3.4% eliminated NCVs in stools 24– 48 h after consumption of seafood. All NCVs had ToxR and hlyAET genes but lacked ctxA, zot, and tcpA genes. One isolate from prawn had stn/sto gene. All strains were hemolytic, cytotoxic, and able to induce intestinal and extraintestinal effects on the suckling mouse model. Our results confirm that non-toxigenic NCVs that express the gene encoding El Tor-like hemolysin can be isolated from patients suffering a cholera-like syndrome after consumption of seafood. This evidence along with the virulence and enteropathogenicity of all the ctxA− tcpA− zot- stn/sto− hlyAET+ NCV isolates in the experimental model, suggest that El Tor-like hemolysin may play an important role in human pathogenesis. Moreover, the isolates from seafood showed molecular, biological and enzymatic patterns similar to those isolated from clinical samples, underlining that environmental NCVs are potentially able to induce human infections and confirming the important role of seafood as a vehicle of V. cholerae diseases