A. Pianetti

Occurrence and expression of virulence-related properties of Vibrio species isolated from widely consumed seafood products

In this study, widely consumed fresh seafood products were examined for the presence of Vibrio spp. Thirteen percent of the samples examined were found to be contaminated with halophilic vibrios belonging to the species V. alginolyticus (81.48%), V. parahaemolyticus (14.8%) and V. cholerae non 0:1 (3.7%). A greater isolation frequency (18.9%) was found for mussels. Significant adhesiveness and strong cytotoxicity factors were revealed in a significant number of the Vibrio spp. isolated. These results confirm that the presence of Vibrio spp. in seafood products is common, and suggest that routine examination of such products for these pathogenic agents would be advisable.

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 cuttlefish, 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.

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste‐water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae, 46 (22%) as Aer. sobria, 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii. Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP‐types) based on their biochemical phenotypes. Several different PhP‐types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP‐types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico‐chemical conditions. The production of toxins capable of causing cytoskeletal‐dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: A comparative study

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.

Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

ABSTRACT The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.

Faecal contamination indicators, salmonella, vibrio and aeromonas in water used for the irrigation of agricultural products

The faecal contamination indicators (total coliforms, faecal coliforms, Escherichia coli, enterococci) and the genera Salmonella, Vibrio, Aeromonas were investigated in water samples used for irrigation. During 4 months, 52 samples were taken. The methods used were: multiple tube fermentation method for faecal contamination indicators and membrane filtration techniques for salmonella, aeromonas and vibrio. Two samples were positive for Salmonella spp., fourteen for Aeromonas spp. and no samples for Vibrio spp. No correlation was found between aeromonas and the indicators of faecal contamination. Regarding Aeromonas spp., 21.6% of the strains were adhesive and 12.6% cytotoxic: this confirms the possible role of aeromonas in human pathologies. These results are important to determine the quality of irrigation water in relation to human health. In fact, the spray or sprinkler irrigation produces bioaerosol, which can contaminate the crops that are likely to be eaten uncooked. In addition, the flood or furrow irrigation represents a risk to field workers.

Microbial characteristics of marine sediments in bathing area along Pesaro-Gabicce coast (Italy): a preliminary study

Aims:  This paper evaluates the presence of human pathogen micro‐organisms in marine sediments in a coastal area suitable for bathing. In addition, the grain size analysis was performed in order to correlate the characteristics of the sediments and the microbial content.

Presence of enteroviruses and reoviruses in the waters of the Italian coast of the Adriatic Sea

EEC directive 76/160 requires member states to apply microbiological and chemico-physical standards for the quality of recreational waters. In observation of this regulation, in the present study 144 samples of seawater were taken over a 12-month period and tested to determine viral contamination. The samples were collected from the coastal waters of the Italian town of Pesaro, which is located on the Adriatic Sea. Using cell culture techniques, 32.6% of the seawater samples were found to be contaminated with enteroviruses. Isolation of these viruses was most frequent in the summer months. Thus, our results indicate the need to increase the frequency of monitoring of these waters and to eliminate the sources of contamination.

Determination of viability of Aeromonas hydrophila in increasing concentrations of sodium chloride at different temperatures by flow cytometry and plate count technique

Aeromonads in waters and foods can represent a risk to human health. Factors such as sodium chloride concentration and temperature can affect growth and viability of several food and water-borne pathogens. The behaviour of an Aeromonas hydrophila strain in the presence of 1.7%, 3.4% and 6% NaCl concentrations at 24 degrees C and 4 degrees C was studied over a 188 day period. Viability and membrane potential were assessed by flow cytometry; growth was evaluated by plate count technique. Flow cytometry evidenced that A. hydrophila retained viability over the period although varying according to temperature and salt concentrations. Colony Forming Units were generally lower in number than viable cells especially in the presence of 6% NaCl, indicating the occurrence of stressed cells which maintain metabolic activity yet are not able to grow on agar plates. In conclusion, A. hydrophila showed a long-term halotolerance even at elevated (6%) NaCl concentrations and a lesser sensitivity to salt at low temperature; therefore, low temperature and salt, which are two important factors limiting bacterial growth, do not assure safety in the case of high initial contamination. Finally, cytometry appears a valid tool for the rapid detection of the viability of pathogenic bacteria in food and environmental matrices to control and prevent health risks.

Presence of several pathogenic bacteria in the Metauro and Foglia Rivers (Pesaro-Urbino, Italy)

Abstract The presence of bacteria of the genera Salmonella, Campylobacter , Aeromonas and Yersinia in the Metauro and Foglia Rivers and the possible relationships with faecal indicators were studied. Monthly sampling was carried out and a total of 168 samples was obtained. Thirty-eight (22.6%) samples were positive for Salmonella spp., 52 (30.9%) for Campylobacter spp., 39 (23.2%) for Aeromonas spp. and 8 (4.7%) for Yersinia spp. No correlation was found between the microorganisms of the different bacterial genera and the indicators of faecal contamination.