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.

Recovery from mild Escherichia coli O157:H7 infection in young and aged C57BL/6 mice with intact flora estimated by fecal shedding, locomotor activity and grip strength.

Escherichia coli 0157:H7 is a food-borne pathogen that can cause severe complications in vulnerable populations. Mouse infection models of E. coli 0157:H7 are usually developed under severe animal suffering classification by depleting the normal flora, in which age plays a role. OBJECTIVE: To develop a refined method for longitudinal monitoring of E. coli 0157:H7 in young and old mice with intact flora. METHODS: We applied discriminant analysis and computed composite standardized scores from 19 variables obtained from physiological parameters, analysis of locomotor activity, grip strength measurement and fecal shedding in 16 aged and 16 young C57BL/6 mice after two mild oral challenges of E. coli 0157:H7. The resulting scores were validated in another experiment performed in 24 aged and 24 young mice including a group (8 aged and 8 young mice) treated with oxytetracycline. RESULTS: We show that our scores are significantly affected in the post-infection period and that can be used to measure and compare the recovery time after a treatment. The scores are most sensitive when separately developed in young and aged mice. CONCLUSIONS: We developed a method that minimizes the level of animal suffering and that can be applied in preclinical testing of new therapies.

Molecular characterization and drug susceptibility of non-O1/O139 V. cholerae strains of seafood, environmental and clinical origin, Italy.

Toxigenic and antimicrobial susceptibility patterns and genetic relatedness of 42 non-O1/O139 V. cholerae strains, the majority of them isolated from seafood and marine water of the Adriatic sea, Italy, and 9 clinical strains, two of which with seawater of the Adriatic as the source of infection, were studied. All strains had hlyA El Tor gene but lacked ctxA gene. Four and two isolates, respectively, also had stn/sto and tcpA Class genes. More than 90% of strains showed susceptibility to cefotaxime, ciprofloxacin, cloramphenicol, tetracycline, trimethoprim + sulfamethoxazole and intermediate or full resistance to tetracycline and erythromycin. Six strains of seafood and clinical source were multi-drug resistant. PFGE analysis allowed to type all the strains with 50 banding patterns. Twenty-one strains, 11 and 8 from seafood and seawater, respectively, and 2 of clinical origin, were grouped into 9 different clusters. We report the presence of toxigenic and multidrug resistant non-O1/O139 V. cholerae strains in Adriatic, some of which genetically related, and support that they represent a potential reservoir of toxin and antibiotic resistance genes.

Occurrence of Arcobacter spp. and correlation with the bacterial indicator of faecal contamination Escherichia coli in bivalve molluscs from the Central Adriatic, Italy.

A total of 162 samples of bivalve molluscs (45 mussels and 117 clams) collected between December 2012 and 2014 from harvesting areas of the Central Adriatic were analysed by a culturing method for the presence of Arcobacter spp. Species identification was performed by PCR and sequencing analysis of a fragment of the rpoB gene. Overall, Arcobacter species were detected in 30% of samples, specifically 33% clams and 22% mussels. A. butzleri was the most common species (20% of the samples), followed by A. cryaerophilus (9%) and A. skirrowii (1%). A seasonal association of A. butzleri contamination was detected. A. butzleri was significantly more commonly recovered from samples collected during the winter-spring period (29%) than from those of the summer-autumn (8%). A. cryaerophilus was cultured from 6% to 11% of the samples collected in summer-autumn and winter-spring, respectively, but these differences were not statistically significant. A. skirrowii was recovered from a sample of mussels harvested in May 2014. To identify associations between the occurrence of Arcobacter spp. and E. coli levels, samples were divided into groups generating results with E. coli at >230MPN/100g and E. coli at ≤230MPN/100g, the latter corresponding to EU microbiological criteria allowed for live bivalve molluscs at retail level. A. butzleri was significantly more commonly detected in samples with higher E. coli levels (48%) than in those with lower levels of E. coli (10%), providing evidence for considering E. coli as an index organism for A. butzleri contamination in bivalve molluscs.

Genetic diversity of Arcobacter isolated from bivalves of Adriatic and their interactions with Mytilus galloprovincialis hemocytes.

The human food-borne pathogens Arcobacter butzleri and A. cryaerophilus have been frequently isolated from the intestinal tracts and fecal samples of different farm animals and, after excretion, these microorganisms can contaminate the environment, including the aquatic one. In this regard, A. butzleri and A. cryaerophilus have been detected in seawater and bivalves of coastal areas which are affected by fecal contamination. The capability of bivalve hemocytes to interact with bacteria has been proposed as the main factor inversely conditioning their persistence in the bivalve. In this study, 12 strains of Arcobacter spp. were isolated between January and May 2013 from bivalves of Central Adriatic Sea of Italy in order to examine their genetic diversity as well as in vitro interactions with bivalve components of the immune response, such as hemocytes. Of these, seven isolates were A. butzleri and five A. cryaerophilus, and were genetically different. All strains showed ability to induce spreading and respiratory burst of Mytilus galloprovincialis hemocytes. Overall, our data demonstrate the high genetic diversity of these microorganisms circulating in the marine study area. Moreover, the Arcobacter-bivalve interaction suggests that they do not have a potential to persist in the tissues of M. galloprovincialis.

Bioaccumulation experiments in mussels contaminated with the food-borne pathogen Arcobacter butzleri: preliminary data for risk assessment.

The aim of this study was to evaluate, at a laboratory scale, the ability of this microorganism to grow in seawater and bioaccumulate in mussels (Mytilus galloprovincialis) maintained in constantly aerated tanks, containing twenty litres of artificial seawater. Three concentrations of A. butzleri LMG 10828(T) were tested (about 5 × 106 CFU/mL, 5 × 104 CFU/mL, and 5 × 10² CFU/mL). Following contamination, enumeration of A. butzleri was performed from water and mussels each day, for up to 96 h. Three contamination experiments with artificial seawater in absence of mussels were also performed in the same manner. In the experiments with mussels, A. butzleri declined in water of approximately 1 log every 24 h from the contamination. In artificial seawater without mussels the concentration of A. butzleri remained on the same logarithmic level in the first 48 h and then decreased of about 1 log every 24 hours. In mussels, the concentration was approximately 2 log lower than the exposition level after 24 h from the contamination, and then it decreased exponentially of 1 log every 24 h. Our findings suggest that in the experimental conditions tested A. butzleri is neither able to effectively grow in seawater nor bioaccumulate in mussels, at least in the free and cultivable form.