Eukaryotic molecular diversity at different steps of the wastewater treatment plant process reveals more phylogenetic novel lineages.

Wastewater microbiota represents important actors of organic depollution. Nowadays, some species used as bioindicators of the effluent quality are still identified by microscopy. In the present study, we investigated eukaryotic diversity at the different steps of the treatment process of a wastewater treatment plant (aerobic, anaerobic, clarifier basins and anaerobic digester) using the 18S rRNA gene sequencing approach. Of the 1519 analysed sequences, we identified 160 operational taxonomic units. Interestingly, 56.9% of the phylotypes were assigned to novel phylogenetic molecular species since they show <97% sequence identity with their nearest affiliated representative within public databases. Peritrichia ciliates were the most predominant group, with Epistylis as the most common genus. Although anaerobic, the digester appears to harbor many unclassified phylotypes of protozoa species. Novel lineages such as LKM11 and LKM118 were widely represented in the digester. Diversity values given by Shannon indexes show that the clarifier is the most diversified. This work will help designing molecular tools that are fast, reliable, and reproducible for monitoring wastewater depollution and studying phylogenetic relationships among the wonderful world of protists within this anthropogenic ecosystem.

Co-occurence of Crenarchaeota, Thermoplasmata and methanogens in anaerobic sludge digesters.

16S rRNA Crenarchaeota and Thermoplasmata sequences retrieved from 22 anaerobic digesters were analysed. 4.8 and 0.53 % of archaeal sequences were simultaneously affiliated to these lineages. A core of 2 operational taxonomic units (OTUs) representing 0.6 to -33.6 % of all archaeal sequences were defined for the Crenarchaeotes and identified to already known but not yet cultivable organisms in almost half of the digesters sampled. For the Thermoplasmata, apparently less abundant with 0.7 to -4.7 % of the archaeal sequences, 3 OTUs were identified. We showed here that Crenarchaeotes coexist with methanogens and are particularly abundant when Arch I lineage (also called WSA2 by Hugenholtz) is dominant in digesters. Moreover, Thermoplasmata were detected when Crenarchaeota were present. Interactions between methanogens, Crenarchaeotea and Thermoplamata were thus discussed.

Biological properties of carotenoids extracted from Halobacterium halobium isolated from a Tunisian solar saltern.

BACKGROUND: Bioactive molecules have received increasing attention due to their nutraceutical attributes and anticancer, antioxidant, antiproliferative and apoptosis-inducing properties. This study aimed to investigate the biological properties of carotenoids extracted from Archaea. METHODS: Halophilic Archaea strains were isolated from the brine of a local crystallizer pond (TS7) of a solar saltern at Sfax, Tunisia. The most carotenoid-producing strain (M8) was investigated on heptoma cell line (HepG2), and its viability was assessed by the MTT-test. The cells were incubated with different sub-lethal extract rates, with carotenoid concentrations ranging from 0.2 to 1.5 µM. Antioxidant activity was evaluated through exposing the cells to sub-lethal extract concentrations for 24 hours and then to oxidative stress induced by 60 µM arachidonic acid and 50 µM H2O2. RESULTS: Compared to non-treated cells, bacterial carotenoid extracts inhibited HepG2 cell viability (50%). A time and dose effect was observed, with cell viability undergoing a significant (P < 0.05) decrease with extract concentration. After exposure to oxidative stress, control cells underwent a significant (P < 0.05) decrease in viability as compared to the non-treated cells. CONCLUSIONS: The bacterial extracts under investigation were noted to exhibit the strongest free radical scavenging activity with high carotenoid concentrations. The carotenoid extract also showed significant antiproliferative activity against HepG2 human cancer cell lines.

Virulence and resistance genes profiles and clonal relationships of non-typhoidal food-borne Salmonella strains isolated in Tunisia by whole genome sequencing.

Whole genome sequencing (WGS) has made impressive progress in the field of molecular biology. Its most common application for public health is in the area of surveillance of food-borne diseases. WGS has the potential for providing a large amount of information, such as the identification of the strain type, the characterization of antibiotic resistance and virulence, and phylogeny. In our study, thirty-nine non-typhoidal Salmonella strains were isolated from diverse sources in Tunisia. Non-typhoidal Salmonella are among the most common pathogens contaminating food animals. The presence of virulence and antimicrobial resistance determinants in those strains were investigated using whole genome sequencing (WGS) and appropriate data analysis. The genomes were screened for several Salmonella virulence genes using the Virulence Factor Database VFDB. Twelve different virulence profiles, which correspond to the 12 identified serovars, were recognized. Several antimicrobial resistance genes were also detected: aac (6')-Iaa, sul1, tetA, bla-TEM and qnrS genes. Phylogenetic relationships among the strains were further assessed by a cgMLST analysis. The resulting phylogenetic tree consisted of several clusters consistently with the in silico multilocus sequence typing (MLST) and serotyping. Our findings demonstrated that WGS and subsequent data analysis provided an accurate tool for genetic characterization of bacterial strains compared to usual molecular typing techniques. To the best of our knowledge, this is the first report of an application of WGS for genetic characterization of food-borne Tunisian strains.

Microbial community of salt crystals processed from Mediterranean seawater based on 16S rRNA analysis.

Phylogenetic analysis of 16S rRNA was used to investigate for the first time the structure of the microbial community that inhabits salt crystals retrieved from the bottom of a solar saltern, located in the coastal area of the Mediterranean Sea (Sfax, Tunisia). This community lives in an extremely salty environment of 250-310 g/L total dissolved salt. A total of 78 bacterial 16S rRNA clone sequences making up to 21 operational taxonomic units (OTUs), determined by the DOTUR program to 97% sequence similarity, was analyzed. These OTUs were affiliated to Bacteroidetes (71.4% of OTUs), and gamma-Proteobacteria and alpha-Proteobacteria (equally represented by 14.2% of the OTUs observed). The archaeal community composition appeared more diverse with 68 clones, resulting in 44 OTUs, all affiliated with the Euryarchaeota phylum. Of the bacterial and archaeal clones showing <97% 16S rRNA sequence identity with sequences in public databases, 47.6% and 84.1% respectively were novel clones. Both rarefaction curves and diversity measurements (Simpson, Shannon-Weaver, Chao) showed a more diverse archaeal than bacterial community at the Tunisian solar saltern pond. The analysis of an increasing clone's number may reveal additional local diversity.

Molecular diversity analysis and bacterial population dynamics of an adapted seawater microbiota during the degradation of Tunisian zarzatine oil.

The indigenous microbiota of polluted coastal seawater in Tunisia was enriched by increasing the concentration of zarzatine crude oil. The resulting adapted microbiota was incubated with zarzatine crude oil as the only carbon and energy source. Crude oil biodegradation capacity and bacterial population dynamics of the microbiota were evaluated every week for 28 days (day 7, day 14, day 21, and day 28). Results show that the percentage of petroleum degradation was 23.9, 32.1, 65.3, and 77.8%, respectively. At day 28, non-aromatic and aromatic hydrocarbon degradation rates reached 92.6 and 68.7%, respectively. Bacterial composition of the adapted microflora was analysed by 16S rRNA gene cloning and sequencing, using total genomic DNA extracted from the adapted microflora at days 0, 7, 14, 21, and 28. Five clone libraries were constructed and a total of 430 sequences were generated and grouped into OTUs using the ARB software package. Phylogenetic analysis of the adapted microbiota shows the presence of four phylogenetic groups: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Diversity indices show a clear decrease in bacterial diversity of the adapted microflora according to the incubation time. The Proteobacteria are the most predominant (>80%) at day 7, day 14 and day 21 but not at day 28 for which the microbiota was reduced to only one OTU affiliated with the genus Kocuria of the Actinobacteria. This study shows that the degradation of zarzatine crude oil components depends on the activity of a specialized and dynamic seawater consortium composed of different phylogenetic taxa depending on the substrate complexity.

Occurrence and Phenotypic and Molecular Characterization of Antimicrobial Resistance of Salmonella Isolates from Food in Tunisia.

HIGHLIGHTS: Multidrug-resistant Salmonella isolates have been recovered from food in Tunisia. Salmonella isolates from food are resistant to fluoroquinolones and cephalosporins. Surveillance of the antimicrobial susceptibility of foodborne bacteria is needed in Tunisia.

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach.

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA-DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.

Novel prokaryotic diversity in sediments of Tunisian multipond solar saltern.

The phylogenetic diversity of a sediment microbial community from two ponds having different salinities, 150-200 g/l (M2) and 250-300 g/l (TS38), of an Sfax (Tunisia) solar saltern, was investigated using 16S rRNA clone libraries. The 16S rRNA genes from 135 bacterial clones and 105 archaeal clones were sequenced and phylogenetically analyzed. 32 operational taxonomic units (OTUs) were generated for Bacteria and 64 for Archaea. The bacterial community in M2 sediment was affiliated only with Bacteroidetes, while that in TS38 sediment was dominated by clones affiliated with Bacteroidetes, (gamma, alpha, delta) Proteobacteria and unclassified bacteria; these represented 56.52, 26.08, 4.34, 4.34 and 8.7% of the OTUs, respectively. In the M2 and TS38 sediments, 44.44 and 43.47% of the bacterial OTUs, respectively, were novel. All archaeal sequences fell into the Euryarchaeota phylum. In both sediments, 38.46 and 72.55% of the OTUs had less than 97% 16S rRNA sequence identity, representing novel OTUs. Two sequences, retrieved from TS38 sediment, were found to be affiliated with the candidate division MSBL-1 defining two OTUs. The sediment phylogenetic study revealed the presence of a highly diverse microbial population in highly salty media.

Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge.

The microbial consortium involved in anaerobic digestion has not yet been precisely characterized and this process remains a 'black box' with limited efficiency. In this study, seven anaerobic sludge digesters were selected based on technology, type of sludge, process and water quality. The prokaryotic community of these digesters was examined by constructing and analysing a total of 9890 16S rRNA gene clones. Libraries were constructed using primers specific for the Bacteria and Archaea domains for each digester, respectively. After phylogenetic affiliation, the libraries were compared using statistical tools to determine the similarities or differences among the seven digesters. Results show that the prokaryotic community of an anaerobic digester is composed of phylotypes commonly found in all anaerobic digesters sampled and also of specific phylotypes. The Archaea community is represented by an equilibrium among a restricted number of operational taxonomic units (OTUs). These OTUs are affiliated with Methanosarcinales, Methanomicrobiales and Arc I phylogenetic groups. Statistical analysis revealed that the Bacteria community can be described as a three component model: one-third making up a core group of phylotypes common to most of the digesters, one-third are phylotypes shared among a few digesters and another one-third are specific phylotypes. The core group is composed of only six OTUs affiliated with Chloroflexi, Betaproteobacteria, Bacteroidetes and Synergistetes. Its role in anaerobic degradation appears critical to investigate. This comparison of anaerobic digester populations is a first step towards a future understanding of the relationship among biodiversity, operating conditions and digester efficiency.

Prokaryotic diversity of a Tunisian multipond solar saltern.

16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied.