Micol Gennari

Presence of T3SS2 and other virulence-related genes in tdh-negative Vibrio parahaemolyticus environmental strains isolated from marine samples in the area of the Venetian Lagoon, Italy

Vibrio parahaemolyticus-mediated disease has traditionally been associated with two virulence factors, thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH), which are present in most clinical isolates. Recently, it has been suggested that other virulence-related factors, such as some type III secretion system (T3SS) proteins, urease and DNA-methyltransferase, among others, might also play a role in disease caused by this bacterial species and have been shown to be carried by clinical, but not by environmental strains. Screening for a number of virulence and virulence-related genes in a collection of V. parahaemolyticus strains isolated from the Italian Adriatic coast indicates that in addition to the trh-positive strains isolated (6%), a significant percentage (18%) of these strains contain one or more genes with a possible role in pathogenicity. Specifically, some of the V. parahaemolyticus strains described in this study are the first environmental strains ever detected carrying T3SS2 genes. Data obtained by reverse transcription-PCR on environmental strain RNA indicate that at least some of these genes are functional. On the basis of the results obtained, it is suggested that such strains might constitute an environmental reservoir of genes possibly contributing to V. parahaemolyticus pathogenicity and to the spread, in the marine environment, of virulence-related genes usually found in clinical strains.

Altered intestinal function precedes the appearance of bacterial DNA in serum and ascites in patients with cirrhosis: a pilot study.

Objectives Bacterial translocation seems to precede the occurrence of overt bacterial infection in patients with cirrhosis. The presence of bacterial DNA in blood and ascites correlates with bacterial translocation and is frequent in patients with advanced cirrhosis without overt infection. Our aim was to search for bacterial DNA in patients with cirrhosis both with and without ascites, and to study its correlation with abnormal intestinal motility or permeability and the presence of bacterial overgrowth. Methods Blood and ascites samples were obtained on day 1, and blood samples were taken twice a day for the following 3 days. Bacterial DNA was assayed by polymerase chain reaction using universal primers for rRNA 16 s. Oro-caecal transit time and bacterial overgrowth were assessed with Lactulose H2 breath testing. Intestinal permeability was assessed by determining urinary lactulose and mannitol excretion with high performance liquid chromatography. Results We studied seven patients (six were male, age range was 42–78 years). Aetiology was alcohol in four, HCV in two, HBV in one; ascites was present in four and Child−Pugh grade was A in four and B in three. All patients had increased intestinal permeability, six had decreased transit time and one had bacterial overgrowth. In only one patient (with ascites), polymerase chain reaction was positive for bacterial DNA both in ascites and serum for all 4 days on which samples were taken. Conclusion Increased intestinal permeability and abnormal motility were frequent without evidence of bacterial translocation in cirrhosis even without ascites. They are likely to be facilitators for bacterial translocation and thus precede it.

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.

Integrated Evaluation of Environmental Parameters Influencing Vibrio Occurrence in the Coastal Northern Adriatic Sea (Italy) Facing the Venetian Lagoon

In the marine environment, the persistence and abundance of Vibrio are related to a number of environmental parameters. The influence of the different environmental variables in determining the Vibrio occurrence could be different in the specific geographic areas around the world. Moreover, oceanographic parameters are generally interdependent and should not be considered separately when their influence on bacterial presence and concentration is tested. In this study, an integrated approach was used to identify key parameters determining the abundance of Vibrio spp in marine samples from the Venetian Lagoon in Italy, which is an important area for fish farming and tourism. Multivariate techniques have been adopted to analyze the dataset: using PCA, it was shown that a relatively high proportion of the total variance in this area was mainly due to two independent variables, namely salinity and temperature. Using cluster analysis, it was possible to categorize different groups with homogeneous features as regards space (“stations”) and time (“seasons”) distribution, as well as to quantify the values of environmental variables and the Vibrio abundances in each category. Furthermore, integrating key environmental factors and bacterial concentration values, it was possible to identify levels of salinity and sea surface temperature which were optimal for Vibrio concentration in water, plankton, and sediment samples. The identification of key environmental variables conditioning Vibrio occurrence should facilitate ocean monitoring, making it possible to predict unexpected variations in marine microflora which determine possible public health risks in coastal areas.

Serological and molecular characterization of Vibrio parahaemolyticus marine strains carrying pandemic genetic markers

In 2005, pandemic Vibrio parahaemolyticus was reported to have been introduced in Europe: O3:K6 strains were isolated from clinical cases in France and Spain, and were found to be associated with consumption of contaminated seafood. On the contrary, pandemic strains were not isolated from seafood or from the environment itself. Analysis of two V. parahaemolyticus strains isolated in May 2007 from Northern Italy seawater and plankton samples revealed the presence of the virulence gene tdh and the pandemic-specific markers orf8 and toxRS/new sequence (group-specific PCR). The two strains showed serotypes not included in the ‘pandemic group’, but their molecular typing proved that they represent a single clone showing a genetic profile very similar to that of pandemic O3:K6 reference isolates. Moreover, the two marine strains carried three virulence-related genes associated with clinical strains and, to date, hardly ever or never detected in environmental strains. The presence, in strains isolated from the marine environment, of genetic pandemic markers and virulence genes normally associated with clinical isolates proves that marine strains might constitute a public health concern.

The use of multiple typing methods allows a more accurate molecular characterization of Vibrio parahaemolyticus strains isolated from the Italian Adriatic Sea.

Vibrio parahaemolyticus is a natural inhabitant of marine environments and constitutes part of the autochthonous microbial communities, but is also associated with human gastroenteritis, wound infections and septicemia. Recently, a number of clinical cases of infection due to ingestion of seafood contaminated with V. parahaemolyticus and potentially pandemic marine strains isolated from water and plankton have been reported in Europe. To identify the source of virulent strains and to analyze the possible persistence, in time and space, of particular clones, the molecular typing of Vibrio strains is of high epidemiological interest. In this study, we applied pulsed-field gel electrophoresis and two PCR-based techniques (enterobacterial repetitive intergenic consensus- and repetitive extragenic palindromic-PCR) to establish the DNA fingerprints for the analysis of genetic variability among the environmental V. parahaemolyticus strains isolated in the area of the Venetian Lagoon. A temporal distribution of the environmental strains in the studied geographical area and, in some cases, a strong association between a certain genetic profile and a specific source have been evidenced. A number of genetic clusters/clones seem to persist over time, reappearing in the marine environment for subsequent months and also at a 1-year gap. The use of multiple typing methods allowed a more accurate characterization of the environmental strain genetic profiles and the identification of clones hardly revealed through common techniques.