Alexandra Meziti

Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis

Investigation of the initial and spoilage microbial diversity of iced stored sea bream was carried out. Culture dependent methods were used for bacterial enumeration and phenotypic identification of bacterial isolates, while culture independent methods, using bacterial 16S rRNA gene amplification, cloning and sequencing of DNA extracted directly from the flesh were also employed. The culture dependent approach revealed that the initial microbiota was dominated by Acinetobacter, Shewanella, Pseudomonas and Flavobacterium, while at the end of shelf-life determined by sensory analysis (16 days), the predominant microbiota was Pseudomonas and Shewanella. Culture independent approach showed that initially the sea bream flesh was strongly dominated by Acinetobacter, while Pseudomonas, Aeromonas salmonicida and Shewanella were the predominant phylotypes at the end of shelf-life. Initial and spoilage microbiota comprised of phylotypes previously identified by others using traditional or molecular techniques. However, Aeromonas has not been reported as part of the dominant microbiota of sea bream at the time of spoilage. Combination of classical and molecular methodologies better reveals the microbiota during storage by revealing bacteria that escape standard approaches and, thus, provides valuable complementary information regarding microbiological spoilage.

Temporal shifts of the Norway lobster (Nephrops norvegicus) gut bacterial communities.

The aim of this study was to investigate the gut bacterial communities of Nephrops norvegicus individuals, using a suite of molecular tools consisting of automated ribosomal intergenic spacer analysis, 16S rRNA gene-internal transcribed spacer clone libraries and FISH. The animals were collected from Pagasitikos Gulf, Greece, during different months of the year. The diversity of the gut bacterial communities was found to mostly vary with sampling time, which could be related to temporal variations in food supply. The 16S rRNA gene diversity analysis showed dominance of specific phylotypes for each month studied. February, May, July, August and October samples were rich in sequences related to the gammaproteobacterial genera Pseudoalteromonas, Psychrobacter and Photobacterium. September and December samples were dominated by phylotypes affiliated with uncultured representatives of Mollicutes, which are generally associated with the intestinal tracts of various animals. The presence of Gammaproteobacteria and uncultured Mollicutes in August and September samples, respectively, was further confirmed by FISH. None of the morphometric parameters considered was related to the temporal pattern of dominant bacterial communities.

Dietary differences are reflected on the gut prokaryotic community structure of wild and commercially reared sea bream (Sparus aurata).

We compared the gut prokaryotic communities in wild, organically‐, and conventionally reared sea bream (Sparus aurata) individuals. Gut microbial communities were identified using tag pyrosequencing of the 16S rRNA genes. There were distinct prokaryotic communities in the three different fish nutritional treatments, with the bacteria dominating over the Archaea. Most of the Bacteria belonged to the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. The number of bacterial operational taxonomic units (OTUs) was reduced from the wild to the conventionally reared fish, implying a response of the gut microorganisms to the supplied food and possibly alterations in food assimilation. The dominant bacterial OTU in all examined fish was closely related to the genus Diaphorobacter. This is the first time that a member of the β‐Proteobacteria, which dominate in freshwaters, are so important in a marine fish gut. In total the majority of the few Archaea OTUs found, were related to methane metabolism. The inferred physiological roles of the dominant prokaryotes are related to the metabolism of carbohydrates and nitrogenous compounds. This study showed the responsive feature of the sea bream gut prokaryotic communities to their diets and also the differences of the conventional in comparison to the organic and wild sea bream gut microbiota.

Characterization of methanogenic and prokaryotic assemblages based on mcrA and 16S rRNA gene diversity in sediments of the Kazan mud volcano (Mediterranean Sea).

The diversity of the methyl-coenzyme reductase A (mcrA) and 16S rRNA genes was investigated in gas hydrate containing sediment from the Kazan mud volcano, eastern Mediterranean Sea. mcrA was detected only at 15 and 20 cm below seafloor (cmbsf) from a 40-cm long push core, while based on chemical profiles of methane, sulfate, and sulfide, possible anaerobic oxidation of methane (AOM) depth was inferred at 12-15 cmbsf. The phylogenetic relationships of the obtained mcrA, archaeal and bacterial 16S rRNA genes, showed that all the found sequences were found in both depths and at similar relative abundances. mcrA diversity was low. All sequences were related to the Methanosarcinales, with the most dominant (77.2%) sequences falling in group mcrA-e. The 16S rRNA-based archaeal diversity also revealed low diversity and clear dominance (72.8% of all archaeal phylotypes) of the Methanosarcinales and, in particular, ANME-2c. Bacteria showed higher diversity but 83.2% of the retrieved phylotypes from both sediment layers belonged to the delta-Proteobacteria. These phylotypes fell in the SEEP-SRB1 putative AOM group. In addition, the rest of the less abundant phylotypes were related to yet-uncultivated representatives of the Actinobacteria, Spirochaetales, and candidate divisions OP11 and WS3 from gas hydrate-bearing habitats. These phylotype patterns indicate that AOM is occurring in the 15 and 20 cmbsf sediment layers.

Gut bacteria associated with different diets in reared Nephrops norvegicus

The impact of different diets on the gut microbiota of reared Nephrops norvegicus was investigated based on bacterial 16S rRNA gene diversity. Specimens were collected from Pagasitikos Gulf (Greece) and kept in experimental rearing tanks, under in situ conditions, for 6months. Treatments included three diets: frozen natural (mussel) food (M), dry formulated pellet (P) and starvation (S). Gut samples were collected at the initiation of the experiment, and after 3 and 6months. Tank water and diet samples were also analyzed for bacterial 16S rRNA gene diversity. Statistical analysis separated the two groups fed or starved (M and P vs. S samples). Most gut bacteria were not related to the water or diet bacteria, while bacterial diversity was higher in the starvation samples. M and P samples were dominated by Gammaproteobacteria, Epsilonproteobacteria and Tenericutes. Phylotypes clustering in Photobacterium leiognathi, Shewanella sp. and Entomoplasmatales had high frequencies in the M and P samples but low sequence frequencies in S samples. The study showed that feeding resulted in the selection of specific species, which also occurs in the natural population, and might be associated with the animals nutrition.

Anthropogenic effects on bacterial diversity and function along a river-to-estuary gradient in Northwest Greece revealed by metagenomics

Studies assessing the effects of anthropogenic inputs on the taxonomic and functional diversity of bacterioplankton communities in lotic ecosystems are limited. Here, we applied 16S rRNA gene amplicon and whole-genome shotgun sequencing to examine the microbial diversity in samples from the Kalamas River (Northwest Greece), a mid-size river that runs through agricultural and NATURA-protected areas, but also receives urban sewage from a large city through a manmade ditch. Samples from three different locations between the exit of the ditch and the estuary, during three different months showed that temporal differences of taxonomic and functional diversity were more pronounced than spatial ones, and <1% of total taxa were shared among all samples, revealing a highly dynamic ecosystem. Comparisons of gene diversity with other aquatic habitats showed that only the high flow winter samples resembled more to freshwater environments while samples during the decreased water flow months were dominated by sewage inputs and soil-related organisms. Notably, microbial human gut signals were detectable over background freshwater and soil/runoff related signals, even at tens of kilometers downstream the city. These findings revealed the significance of allochthonous inputs on the composition and dynamics of river bacterial communities, and highlighted the potential of metagenomics for source tracking purposes.

Spatially uniform but temporally variable bacterioplankton in a semi-enclosed coastal area.

Studies focusing on the temporal and spatial dynamics of bacterioplankton communities within littoral areas undergoing direct influences from the coast are quite limited. In addition, they are more complicated to resolve compared to communities in the open ocean. In order to elucidate the effects of spatial vs. temporal variability on bacterial communities in a highly land-influenced semi-enclosed gulf, surface bacterioplankton communities from five coastal sites in Igoumenitsa Gulf (Ionian Sea, Greece) were analyzed over a nine-month period using 16S rDNA 454-pyrosequencing. Temporal differences were more pronounced than spatial ones, with lower diversity indices observed during the summer months. During winter and early spring, bacterial communities were dominated by SAR11 representatives, while this pattern changed in May when they were abruptly replaced by members of Flavobacteriales, Pseudomonadales, and Alteromonadales. Additionally, correlation analysis showed high negative correlations between the presence of SAR11 OTUs in relation to temperature and sunlight that might have driven, directly or indirectly, the disappearance of these OTUs in the summer months. The dominance of SAR11 during the winter months further supported the global distribution of the clade, not only in the open-sea, but also in coastal systems. This study revealed that specific bacteria exhibited distinct succession patterns in an anthropogenic-impacted coastal system. The major bacterioplankton component was represented by commonly found marine bacteria exhibiting seasonal dynamics, while freshwater and terrestrial-related phylotypes were absent.

Abdominal setae and midgut bacteria of the mudshrimp Pestarella tyrrhena

We investigated the diversity of the bacterial 16S rRNA genes occurring on the abdominal setal tufts and in the emptied midgut of the marine mudshrimp Pestarella tyrrhena (Decapoda: Thalassinidea). There were no dominant phylotypes on the setal tufts. The majority of the phylotypes belonged to the phylum Bacteroidetes, frequently occurring in the water column. The rest of the phylotypes were related to anoxygenic photosynthetic α-Proteobacteria and to Actinobacteria. This bacterial profile seems more of a marine assemblage rather than a specific one suggesting that no specific microbial process can be inferred on the setal tufts. In the emptied midgut, 64 clones were attributed to 16 unique phylotypes with the majority (40.6%) belonging to the γ-Proteobacteria, specifically to the genus Vibrio, a marine group with known symbionts of decapods. The next most abundant group was the ɛ-Proteobacteria (28.1%), with members as likely symbionts related to the processes involving redox reactions occurring in the midgut. In addition, phylotypes related to the Spirochaetes (10.9%) were also present, with relatives capable of symbiosis conducting a nitrite associated metabolism. Entomoplasmatales, Bacteroidetes and Actinobacteria related phylotypes were also found. These results indicate a specific bacterial community dominated by putative symbiotic Bacteria within the P. tyrrhena’s midgut.

Changes of bacterioplankton apparent species richness in two ornamental fish aquaria

We analysed the 16S rRNA gene diversity within the bacterioplankton community in the water column of the ornamental fish Pterophyllum scalare and Archocentrus nigrofasciatus aquaria during a 60-day growth experiment in order to detect any dominant bacterial species and their possible association with the rearing organisms. The basic physical and chemical parameters remained stable but the bacterial community at 0, 30 and 60 days showed marked differences in bacterial cell abundance and diversity. We found high species richness but no dominant phylotypes were detected. Only few of the phylotypes were found in more than one time point per treatment and always with low relative abundance. The majority of the common phylotypes belonged to the Proteobacteria phylum and were closely related to Acinetobacter junii, Pseudomonas sp., Nevskia ramosa, Vogesella perlucida, Chitinomonas taiwanensis, Acidovorax sp., Pelomonas saccharophila and the rest belonged to the α-Proteobacteria, Bacteroidetes, Actinobacteria, candidate division OP11 and one unaffiliated group. Several of these phylotypes were closely related to known taxa including Sphingopyxis chilensis, Flexibacter aurantiacus subsp. excathedrus and Mycobacterium sp. Despite the high phylogenetic diversity most of the inferred ecophysiological roles of the found phylotypes are related to nitrogen metabolism, a key process for fish aquaria.

Comparison of the Norway lobster (Nephrops norvegicus) gut bacterial communities using 16S rDNA clone libraries and pyrosequencing.

By comparing 16S rDNA cloning and 454 pyrosequencing in the Nephrops norvegicus midgut, several common bacterial OTUs were detected. However, when only one method is to be selected, it needs to be considered whether the revealing of rare OTUs or their accurate phylogenetic relationships is mostly preferred.