Epidemiology of arcobacter-related spp. In shellfish exposed to marine and brackish water with different levels of fecal pollution

Resumo

The high prevalence and diversity of Arcobacter species found in sewage contaminated waters and in shellfish were attributed to the parallel use of a direct culture plating medium and an enrichment step in combination with the incubation of the media under aerobic and microaerobic conditions. Considering such results and that shellfish grow in marine and brackish environments, the use of culture media that better mimics the conditions of these habitats (possessing a higher concentration of salts) could favor still further the isolation of Arcobacter species. This became the first hypothesis of this Ph.D. and therefore, the design and evaluation of media that contained an augmented salt concentration was one of the first objectives. It is well known that bivalves have a filter feeding behavior which make them vehicles of concentration, accumulation, and dissemination of a diverse number of microorganisms. Thus, together with the fact that bivalve mollusks are usually consumed raw or slightly cooked, their consumption poses an extra risk for the safety of the consumers. Spain is the main producer of marine mollusks in Europe and ensures the sanitary quality of marketable shellfish following the European legislation EU 854/2004. This regulation assesses if bivalve mollusks are safe for human consumption and is based on the specific load of the fecal indicator bacteria E. coli that bivalves carry. The E. coli concentration is used to determine if shellfish can be directly consumed or will require a depuration process. However, some reports showed that E. coli is not universally suitable for predicting the presence of some pathogens, like viruses of human origin or marine-borne Vibrio spp. Despite the demonstration that presence of E. coli can predict the presence of Arcobacter in fecally contaminated waters, there is only one study, performed in mussels, which correlated the concentration of E. coli with the presence of A. butzleri. Thus, evaluating the potential correlation between the concentrations of E. coli and Arcobacter spp. in both marine water and shellfish, would be useful to determine to which extent the E.U. legislation can protect the consumers from being exposed to Arcobacter contaminated shellfish. Additionally, new strategies for quantifying Arcobacter in shellfish were also required to enable its comparison with the concentrations of E. coli. Furthermore, the lack of studies of depuration strategies for Arcobacter, make necessary an assessment of the conventional depuration times established for E. coli to confirm if they are enough to remove Arcobacter from shellfish before they are placed in the market. The contamination of food products with Arcobacter, has mainly been associated with cross-contamination with fecally polluted waters. However, recent studies have demonstrated that Arcobacter species are part of the shellfish microbiota, nevertheless, they can be concomitant pathogens of oysters in cases of coinfections caused by Vibrio spp. Therefore, studies to address this aspect are of paramount importance to guide epidemiological and surveillance studies with special focus on the distribution within shellfish tissues. The latter will enable differentiation of those species of Arcobacter found within shellfish that are autochthonous from the allochthonous. Moreover, the relationship between the load of Arcobacter species present in shellfish and the time required to remove, or reduce, this bacteriological load during the depuration process has never been explored and required investigation. One of the main problems in the research of Arcobacter is that their isolation and identification by culture is fastidious and time consuming, requiring on average 4 days. Additionally, the current PCR identification methods are unable to unequivocally characterize all known Arcobacter species. Furthermore, the conventional PCR procedures have the handicap that they cannot differentiate between living and dead cells. Therefore, developing a method able to discriminate viable from no viable cells, like the ones that use propidium monoazide (PMA) in combination with qPCR, would represent a step forward in the recognition of this fastidious bacteria for the agro-alimentary industry. These more specific diagnostic methods can also help to prevent illness and to reduce product recalls avoiding economic losses. Furthermore, it has been recently demonstrated that the diversity of Arcobacter is greater than previously considered. In fact, the genomic study of this genus, revealed that Arcobacter genus uncovered at least seven different genera, for which the names Arcobacter, Aliarcobacter gen. nov., Pseudarcobacter gen. nov., Halarcobacter gen. nov., Malacioarcobacter gen. nov., Poseidoniarcobacter gen. nov., and candidatus Arcomarinus gen. nov., have been proposed. In that work the genomic analysis showed the existence of at least 7 potential new species belonging to the new Arcobacter-related genera. However, a complete polyphasic characterization of these 7 potential new species, was not done and it is required for their accurate description and validation and this is going to be done in the present thesis. The general objective of the present thesis is to provide new insights on the relationship between Arcobacter and shellfish and on whether shellfish could be considered a potential source of human infections by these bacteria. To achieve this, the following specific objectives have been defined: 1. To develop new methods for culturing, detection and quantification of Arcobacter spp. from water and shellfish samples. 2. To evaluate if the fecal indicator E. coli predicts the presence of Arcobacter in water and shellfish samples and to study the diversity of Arcobacter species in shellfish and in their surrounding water. 3. To determine the distribution and prevalence of Arcobacter related spp. in the different compartments and shellfish tissues in order to characterize the dynamics of colonization by these bacteria. 4. To study if the depuration time-periods established by the E.U. shellfish legislation for reducing the load of E. coli, would ensure the parallel reduction of Arcobacter- related spp. in mussels and oysters. 5. To improve available molecular tools for discriminating among viable and non-viable Arcobacter cells in shellfish samples. 6. To assess with a polyphasic taxonomic approach, including genomic information, if strains that seem to correspond to potential new phylogenetic lines represent new species of the recently described new Arcobacter-related genera. All the samples in the present thesis were analysed in parallel, with both a commonly used culture method (enrichment in Arcobacter-CAT broth followed by culture on Blood Agar) and the new one developed in the present thesis, that consisted on Arcobacter-CAT enrichment broth with 2.5% NaCl (w/v) followed by culturing on Marine Agar. In both methods two atmosphere conditions (aerobiosis and microaerophilia) were used for incubation. Identification of the isolates was performed by different molecular approaches: ERIC-PCR for genotyping, multiplex-PCR for confirmation of the genus and RFLP-PCR for level species determination. When incongruent results were obtained, partial sequence of the rpoB gene was analyzed using MEGA 6 software. The quantification of Arcobacter and E. coli was performed by means of the Most Probable Number (MPN) approach. For E.coli, the quantification was performed according to ISO/TS 16649-3:2005, five tubes, in triplicates containing Glutamate broth were inoculated with the dilutions of the homogenates and incubated for 24h at 37°C. Confirmation of E.coli was performed onto Tryptone Bile X-glucuronide Agar medium at 44°C for 24h.Thus, growth of typical greenish-blue color colonies confirmed the presence of E. coli. The derived MPN results was calculated using the CEFAS MPN tables. For Arcobacter a two-steps MPN was performed. Briefly, 5 tubes in triplicates containing Arcobacter-CAT broth were inoculated with the previously made dilutions and were incubated at 30°C for 48 h, under aerobic conditions. Confirmation of the presence of Arcobacter-related spp. consisted on culturing at 30 °C for 48 h under aerobic conditions 100µl of the above enrichment tubes by passive filtration on Blood Agar plates. The MPN final values were obtained using the software MPN Build 23. A specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), was developed for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The optimization of PMA protocol consisted on the evaluation of PMA concentration (20 μM), incubation of the samples (10min in the dark) and photoactivation time (15 minutes). Once the conditions were established, the v-qPCR was assessed using SYBRgreen. The results showed that the new approach, consisting on the supplementation of Arcobacter-CAT broth with 2.5% NaCl and posterior culture in marine agar, improved the recovery of Arcobacter-related genera from marine and brackish environments. The correlation between the levels of Escherichia coli and Arcobacter was also examined in shellfish and their surrounding water, showing positive results. However, when shellfish were harvested from water with a temperature above 26.2ºC, E. coli would fail to predict the presence of these pathogens. The distribution of Arcobacter-related genera within the tissues of mussels and oysters showed that the intervalval liquid was the compartment with higher prevalence of Arcobacter. The analyses of the depuration of E. coli, A. butzleri and M. molluscorum in mussels and oysters under different bacterial loads and in two seasons (summer and winter) showed that the efficacy of the conventional depuration process may not fully eliminate Arcobacter from shellfish and, probably, it is a temperature dependent process. A viable qPCR method for the detection of viable Arcobacter spp. cells in different shellfish matrixes was developed with a satisfactory inhibition of DNA amplification from dead cells in 85% of the Arcobacter species tested. As result of all these experiments, together with the use in parallel of the NaCl.enriched and non-enriched approaches, the isolation and description of seven new species belonging to four new different genera was performed.