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Marine environment and human health: a whole genomic analysis and evaluation of the pathogenic potential of V.parahaemolyticus strains isolated from Northern Italian seawaters and carrying virulence genes
No full textL’insorgenza di epidemie legate all’ambiente marino sembra esser un fenomeno mondiale in aumento ed un numero crescente di infezioni batteriche umane è stato associato ad aree ricreazionali e legate all’uso commerciale di risorse marine. A causa dell’aumentata dipendenza umana dall’ambiente marino per la pesca, l’acquacoltura, dispositivi di scarico, la potenziale emergenza di patogeni dall’ambiente marino deve esser investigata. Una particolare sfida è costituita dalla potenziale espansione di patogeni marini esistenti nel mare e l’emergenza di nuovi patogeni derivati da ceppi marini autoctoni che hanno acquisito nuovi tratti virulenti via trasferimento genico orizzontale. Alcuni esempi sono l’aumentato consumo di prodotti marini, la comparsa edil diffondersi mondiale di nuovi ceppi di V.parahaemolyticus con potenziale pandemico, e negli ultimi anni alcuni casi di infezioni umane da ceppi di Vibrio species non patogeni.
Lo scopo di questa tesi è stato di comparare ceppi di V.parahaemolyticus ambientali e clinici dal punto di vista genetico e di valutare il potenziale patogeno dei ceppi ambientali.
I ceppi batterici, profondamente studiati nel corso della tesi di Dottorato, sono stati isolati dal Mar Nord Adriatico nel contesto del progetto internazionale “VibrioSea Project”, che aveva come obiettivo quello di sviluppare un sistema di allerta precoce per predire e prevenire le malattie a trasmissione idrica nel Mare Mediterraneo.
La collezione di ceppi ambientali di V.parahaemolyticus è stata caratterizzata tramite una genotipizzazione molecolare per investigare se ceppi isolati nei differenti siti geografici o nei vari periodi dell’anno fossero correlati geneticamente e se fosse possibile identificare cloni persistenti nel tempo o in un sito geografico. Considerando globalmente i dati ottenuti con i 4 metodi di tipizzazione molecolari (ribotyping, PFGE, REP-PCR and ERIC-PCR), e sierotipizzazione, è stato possibile identificare alcuni clusters genetici persistenti in un’area anche per alcuni periodi lunghi. Al contrario non si è potuta stabilire la correlazione tra il tipo di campione o un sito geografico e specifici pattern genici. Il contemporaneo uso di differenti metodi di genotipizzazione si è rivelato essere un approccio efficace permettendo il raggruppamento in ceppi correlati geneticamente e, discriminando, sulla base di piccole differenze, batteri presentanti profili genici molto simili.
Nella seconda parte dello studio è stato valutato il potenziale patogeno dei ceppi ambientali adottando un approccio genomico, sia utilizzando il metodo standard della PCR che un metodo computazionale innovativo, Insignia, che ha permesso il rilevamento di una serie di ceppi ambientali che presentano nel genoma un’siola di patogenicità, includendo diversi geni associati all virulenza. Successivamente l’analisi in vitro della capacità di alcuni ceppi ambientali rappresentativi di aderire e causare danno cellulare a cellule eucarioti ha confermato la patogenicità di ceppi marini.
Sulla base dei dati ottenuti emerge che l’ambiente marino rappresenta un serbatoio di batteri portanti geni di virulenza costituendo così un problema di salute pubblica ed un rischio per la salute umana.Disease outbreaks related to the marine environment appear to be escalating worldwide and growing number of human bacterial infections have been associated with recreational areas and commercial uses of marines resources. Because of the increasing human dependence on marine environments for fisheries, aquaculture, waste disposal and recreation, the potential for pathogen emergence from ocean ecosystems requires investigation.
Particular challenge are the potential expansion of existing-marine indigenous pathogens and the emergence of new pathogens derived from autochthonous marine strains which have acquired new virulence traits via horizontal genetic transfer. The increasing consume of marine food products, the appearance and worldwide spread of new clones of Vibrio parahaemolyticus with pandemic potential and reporting, in very recent years, of some human infection cases caused by non pathogenic vibrios species, are some examples.
The scope of this study has been to compare environmental and clinical Vibrio parahaemolyticus strains from the genetic point of view and to evaluate the pathogenic potential of the environmental strains.
The bacterial strains deeply studied in this doctoral thesis were isolated from the Northern Adriatic Sea in the context of the international VibrioSea Project, aimed to develop a satellite-based early warning system to predict and prevent water-borne diseases in the Mediterranean Sea.
The environmental V. parahaemolyticus strain collection was characterized performing a molecular genotyping to investigate whether the strains isolated in different geographical sites or in different periods of the year were clonally related and if it would be possible to identify clones persisting in time and/or different geographic sites. Considering globally the results obtained with 4 molecular typing methods (ribotyping, PFGE, REP-PCR and ERIC-PCR) and serotyping it has been possible to identify a number of bacterial genetic clusters persisting in the area also for long periods of time. On the contrary, a correlation between the type of sample or a geographic site and specific genetic patterns, has not been established. The contemporary use of different genotyping methods proved to be an effective approach allowing the grouping of genetically related strains also discriminating, on the basis of small differences, bacteria presenting very similar genetic profiles.
In the second part of the study, the pathogenic potential of the environmental strains was evaluated adopting a genomic approach applying standard protocols, such as PCR, but also the innovative computational method Insignia which enabled the detection of a series of environmental strains carrying a pathogenicity island including several virulence-related genes. The subsequent in vitro analysis of the ability of representative environmental strains to adhere to and to cause cell damage in eukaryotic cells confirmed the pathogenic capability of the marine strains.
On the basis of the data obtained it emerges that the marine environment represents a reservoir of bacteria carrying virulence genes thus constituting a public health concern and a risk to human health
Occurrence and molecular characterisation of Vibrio parahaemolyticus in crustaceans commercialised in Venice area, Italy
No full textInfections due to the pathogenic human vibrios, Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus, are mainly associated with consumption of raw or partially cooked bivalve molluscs. At present, little is known about the presence of Vibrio species in crustaceans and the risk of vibriosis associated with the consumption of these products.
The aim of the present study was to evaluate the prevalence and concentration of the main pathogenic Vibrio spp. in samples of crustaceans (n = 143) commonly eaten in Italy, taking into account the effects of different variables such as crustacean species, storage conditions and geographic origin. Subsequently, the potential pathogenicity of V. parahaemolyticus strains isolated from crustaceans (n = 88) was investigated, considering the classic virulence factors (tdh and trh genes) and four genes coding for relevant proteins of the type III secretion systems 2 (T3SS2 alpha and T3SS2 beta).
In this study, the presence of V. cholerae and V. vulnificus was never detected, whereas 40 samples (28%) were positive for V. parahaemolyticus with an overall prevalence of 41% in refrigerated products and 8% in frozen products. The highest prevalence and average contamination levels were detected in Crangon crangon (prevalence 58% and median value 3400 MPN/g) and in products from the northern Adriatic Sea (35%), with the samples from the northern Venetian Lagoon reaching a median value of 1375 MPN/g. While genetic analysis confirmed absence of the tdh gene, three of the isolates contained the trh gene and, simultaneously, the T3SS2 beta genes. Moreover three possibly clonal tdh-negative/trh-negative isolates carried the T3SS2 alpha apparatus.
The detection of both T3SS2 alpha and T3SS2 beta apparatuses in V. parahaemolyticus strains isolated from crustaceans emphasised the importance of considering new genetic markers associated with virulence besides the classical factors. Moreover this study represents the first report dealing with Vibrio spp. in crustaceans in Italy, and it may provide useful information for the development of sanitary surveillance plans to prevent the risk of vibriosis in seafood consumers
Serodiversity and ecological distribution of Vibrio parahaemolyticus in the Venetian Lagoon, Northeast Italy
No full textVibrio parahaemolyticus is a natural inhabitant of estuarine and marine environments constituting part of the autochthonous microflora. This species is associated with human gastroenteritis caused by ingestion of contaminated water and undercooked seafood. During the past several years, the number of V. parahaemolyticus gastroenteritis cases have increased worldwide, causing over half of all food-poisoning outbreaks of bacterial origin. Vibrio populations in water are known to be influenced by environmental factors. Notably, it has been shown that in different parts of the world the distribution of V. parahaemolyticus in the marine environment is related to the water temperature. In this study, we identified environmental determinants affecting distribution of V. parahaemolyticus in the Venetian Lagoon, in the Italian North Adriatic Sea. Data obtained revealed that sea surface temperature constitutes the key factor influencing occurrence of V. parahaemolyticus, but salinity and chlorophyll concentration are also important. Serotyping of a collection of V. parahaemolyticus environmental isolates revealed high serodiversity, with serotypes O3:KUT and O1:KUT, belonging to the 'pandemic group', occurring with higher frequency. From our results, we conclude that there is no correlation between serotype and specific geographic site or season of the year. However, certain serotypes were isolated in the Lagoon during the entire 18 months of the study, strongly suggesting persistence in this environment
Adhesion to medical materials and biofilm formation capability of some species of enterococci in different physiological states.
No full textEnterococci may survive in adverse environments including the human body where bacteriocins, antibiotics, iron-limitation and immune response represent stressing conditions for bacteria that cause division block. In those conditions, bacteria present in the human body would hardly be in an exponentially growing phase but would mostly be in physiological states such as starvation or the viable but nonculturable (VBNC) state. The possibility that the starved and VBNC bacteria can maintain their ability to adhere to living and inanimate substrates is the first mandatory step for them potentially to cause an infection process. In this study it is shown that starved and stationary enterococcal cells are able to form biofilms on plastic material albeit with reduced efficiency as compared to growing cells. Moreover, although VBNC enterococcal forms are not capable of forming biofilms, Enterococcus faecalis and other enterococcal species of medical interest maintain their ability to synthesize the polymeric matrix for a limited period of time under adverse environmental conditions. The data presented, together with those regarding the maintenance of the division recovery potential already proved in nonculturable bacteria, further support the possibility for the VBNC and other nondividing bacterial forms to have a role as infectious agents and to constitute a risk to human health
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
No full textVibrio 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
Isolation of a Vibrio parahaemolyticus pandemic strain from a marine water sample obtained in the northern Adriatic.
No full textEffect on human cells of environmental Vibrio parahaemolyticus strains carrying type III secretion system 2
No full textV. parahaemolyticus is an inhabitant of estuarine and marine environments that causes seafood-borne gastroenteritis worldwide. Recently, a type 3 secretion system (T3SS2) able to secrete and translocate virulence factors into the eukaryotic cell has been identified in a pathogenicity island (VP-PAI) located on the smaller chromosome. These virulence-related genes have previously been detected only in clinical strains. Classical virulence genes for this species (tdh/trh) are rarely detected in environmental strains which are usually considered to lack virulence potential. However, during screening of a collection of environmental V. parahaemolyticus isolates obtained in the North Adriatic Sea in Italy, a number of marine strains carrying virulence-related genes, including genes involved in the T3SS2, were detected. In this study we investigated the pathogenic potential of these marine V. parahaemolyticus strains by studying their adherence ability, their cytotoxicity, their effect on zonula occludin protein1 (ZO-1) of the tight junctions and their effect on transepithelial resistance (TER) in infected Caco-2 cells. By performing a reverse transcription-PCR we also tested the expression of the T3SS2 genes vopT and vopB2 encoding an effector and a translocon protein, respectively. Our results indicate that, similarly to clinical strains, marine V. parahaemolyticus strains carrying vopT, vopB2 and other genes included in the VP-PAI are capable of adhering to human cells and of causing cytoskeletal disruption and loss of membrane integrity in infected cells. On the basis of data here presented, environmental V. parahaemolyticus strains should be included in coastal water surveillance plans as they may represent a risk for human health
Serological and molecular characterization of Vibrio parahaemolyticus marine strains carrying pandemic genetic markers
No full textIn 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.
No full textVibrio 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
Virulence genes and pathogenicity islands in environmental Vibrio strains nonpathogenic to humans
No full textMost Vibrio species in autochthonous marine microbial communities, such as
Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum among others, are considered
nonpathogenic for humans. However, because many bacterial virulence
genes are located in mobile genetic elements, the acquisition of mobile DNA
could mediate the appearance of virulent or more virulent strains even in a
species defined as nonpathogenic. In this study, we screened a collection of
marine nonpathogenic Vibrio strains isolated in the area of the Venetian
Lagoon for the presence of virulence and fitness genes usually present in Vibrio
cholerae and Vibrio parahaemolyticus clinical isolates. More than one-third of
the strains tested positive for the presence of at least one of the potential virulence/
fitness genes with the gene encoding the V. cholerae neuraminidase the
most frequently detected. Moreover, 13 of the environmental strains carried
modified versions of the V. cholerae pathogenicity island VPI-2, and four of
them also contained partial fragments of the V. parahaemolyticus Vp-PAI. The
data obtained support the view of nonpathogenic Vibrio strains as a significant
reservoir of virulence and fitness genes. The emergence of environmental bacteria
with new virulence traits might constitute a direct concern for public health
and a risk for human health
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