The silver lining of antibiotic resistance: Bacterial-mediated reduction of tetracycline plant stress via antibiotrophy.

The reuse of water using effluents containing antibiotics from anthropogenic activities has been mainly linked to the development of antibiotic resistance. However, we report that the development of bacterial tolerance promotes plant growth. In the present study, we aimed to evaluate the efficiency of inoculation of a new antibiotic-degrading bacterium, Erwinia strain S9, in augmenting the tolerance of pea (Pisum sativum L.) plants to tetracycline (TET) (10 and 20 mg/L). Physiological parameters such as tissue elongation and biomass, as well as relative water content, were remarkably lower in plants exposed to TET than in the control. The inhibitory effects of TET were associated with reduced CO2 assimilation, stomatal conductance, transpiration, dark respiration, and light saturation point (LSP). High concentrations of TET-induced oxidative stress are attested by the overproduction of superoxide radicals (O2•-), hydrogen peroxide (H2O2), and hydroxyl radicals (HO•), resulting in increased malondialdehyde content and cell death. The high activity of antioxidant enzymes such as catalase, ascorbate peroxidase, and guaiacol peroxidase validated the proposed mechanism. Under TET stress conditions, supplementation with Erwinia strain S9 was beneficial to pea plants through osmotic adjustment, increased nutrient uptake, gas exchange optimization, and increased antioxidant activities. Its presence not only ensures plant survival and growth during antibiotic stress but also degrades TET via significant antibiotrophy. This strategy is a cost-effective environmental chemical engineering tool that can be used to depollute wastewater or to improve crop resistance in rhizofiltration treatment when treated wastewater is reused for irrigation.

Insights on strain 115 plant growth-promoting bacteria traits and its contribution in lead stress alleviation in pea (Pisum sativum L.) plants.

The present study aims to characterize the plant growth-promoting bacterial traits of Bacillus simplex (strain 115). This bacterium was inoculated in hydroponically conditions to improve pea (Pisum sativum L.) growth submitted to lead (Pb) toxicity. Root nodulation system was developed enough in 23-day-old plants attesting the interaction between the two organisms. In addition to its phosphate solubilization and siderophore production traits that reached 303.8 µg P mL-1 and 49.6 psu respectively, the Bacillus strain 115 exhibited Pb bio-sorption ability. Inoculation of Pb-stressed pea with strain 115 showed roots and shoots biomass recovery (+ 70% and + 61%, respectively). Similarly, water and protein contents were increased in Pb-treated plants after bacterial inoculation. In the presence of strain 115, Pb relative toxicity level decreased (- 39.3% compared to Pb stress only). Moreover, catalase and superoxide dismutase activities were upregulated in Pb-exposed plants (+ 56% and + 51%, respectively). After inoculation with strain 115, catalase and superoxide dismutase activities were restored by - 38% and - 44% respectively. Simultaneously, oxidant stress indicator (H2O2 and 4-hydroxynonenal) and osmo-regulators (proline and glycine-betaine) contents as well as lipoxygenase activity decreased significantly in Pb-treated plants after Bacillus strain's inoculation. Taken together, the results give some evidences for the plant growth-promoting capacity of strain 115 in helping alleviation of Pb stress.

Evidences for antioxidant response and biosorption potential of Bacillus simplex strain 115 against lead.

In this study, we investigated effects of lead on growth response and antioxidant defense protection in a new identified strain isolated from a soil, in the rhizosphere of Sainfoin Hedysarum coronarium L. Different concentrations of lead (0, 0.2, 1.5 and 3 g L-1) added to Bacillus simplex strain 115 cultures surprisingly did not inhibit its growth. However, a resulting oxidative stress as attested by overproduction of H2O2 (+ 6.2 fold) and malondialdehyde (+ 2.3 fold) concomitantly to the enhancement of proteins carbonylation (+ 221%) and lipoxygenase activity (+ 59%) was observed in presence of 3 g L-1 of lead. Intrinsic antioxidant defenses were revealed by the coupled up-regulation of catalase (+ 416%) and superoxide dismutase (+ 4 fold) activities, with a more important Fe-SOD increase in comparison to the other isoforms. Bioaccumulation assays showed both intracellular and extracellular lead accumulation. Biosorption was confirmed as a particularly lead resistance mechanism for Bacillus simplex strain 115 as the metal sequestration in cell wall accounted for 88.5% to 98.5% of the total endogenous metal accumulation. Potentiality of this new isolated microorganism as a biotechnological tool for agricultural soil lead bioremediation was thus proposed.

Molecular analysis of methanogen populations and their interactions within anaerobic sludge digesters.

Knowledge of archaeal population structure, function and interactions is of great interest for a deeper understanding of the anaerobic digestion step in wastewater treatment process, that represents a bottle neck in the optimization of digesters performance. Although culture-independent techniques have enabled the exploration of archaeal population in such systems, their population dynamics and interactions still require further investigation. In the present study, 2646 almost full archaeal 16S rRNA gene sequences retrieved from 22 anaerobic digesters located worldwide were analyzed and classified into 83 Operational Taxonomic Units (OTUs) for Euryarchaeotes and 2 OTUs for Crenarchaeotes. Among the Euryarchaeotes, Methanosarcinales represent the predominant archaeal population (47.5% of total sequences), followed by the ARC I (WSA2) lineage (25.3%), Methanomicrobiales (19.9%) and Methanobacteriales (1.9%). Theses lineages are predominant in nine, five, two and one digesters respectively. However, the remaining 5 digesters show no predominance of any methanogenic group. According to the predominance of theses lineages, 5 digester profiles were distinguished. This study revealed a clear interaction between the 4 methanogenic lineages. A core of 12 OTUs represented by five, four, two and one OTU for Methanosarcinales, Methanomicrobiales, ARC I and Methanobacteriales respectively were quantitatively abundant in at least 50% of the analyzed digesters. 16S rRNA targeted hybridization oligonucleotide probes targeting the predominant OTUs are being developed to follow their population dynamics under various parameters.

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.

Microbial community structure associated with the high loading anaerobic codigestion of olive mill and abattoir wastewaters.

The effect of increasing the organic loading rates (OLRs) on the performance of the anaerobic codigestion of olive mill (OMW) and abattoir wastewaters (AW) was investigated under mesophilic and thermophilic conditions. The structure of the microbial community was also monitored. Increasing OLR to 9g of chemical oxygen demand (COD) L(-1)d(-1) affected significantly the biogas yield and microbial diversity at 35°C. However, at 55°C digester remained stable until OLR of 12g of CODL(-1)d(-1) with higher COD removal (80%) and biogas yield (0.52Lg(-1) COD removed). Significant differences in the bacterial communities were detected between mesophilic and thermophilic conditions. The dominant phyla detected in the digester at both phases were the Firmicutes, Actinobacteria, Bacteroidetes, Synergistetes and Spirochaete. However, Verrucomicrobia, Proteobacteria and the candidate division BRC1 were only detected at thermophilic conditions. The Methanobacteriales and the Thermoplasmales were found as a high predominant archaeal member in the anaerobic sludge.

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.

Members of the uncultured bacterial candidate division WWE1 are implicated in anaerobic digestion of cellulose.

Clones of the WWE1 (Waste Water of Evry 1) candidate division were retrieved during the exploration of the bacterial diversity of an anaerobic mesophilic (35 ± 0.5°C) digester. In order to investigate the metabolic function of WWE1 members, a 16S rRNA gene -based stable isotope probing (SIP) method was used. Eighty-seven percent of 16S r rRNA gene sequences affiliated to WWE1 candidate division were retrieved in a clone library obtained after polymerase chain reaction (PCR) amplification of enriched DNA fraction from anaerobic municipal solid waste samples incubated with (13) C-cellulose, at the end of the incubation (day 63) using a Pla46F-1390R primer pair. The design of a specific WWE1 probe associated with the fluorescence in situ hybridization (FISH) technique corroborated the abundant representation of WWE1 members in our (13) C-cellulose incubations. Secondary ion mass spectrometry-in situ hybridization (SIMSISH) using an iodine-labeled oligonucleotide probe combined with high-resolution nanometer-scale SIMS (NanoSIMS) observation confirmed the isotopic enrichment of members of WWE1 candidate division. The (13) C apparent isotopic composition of hybridized WWE1 cells reached the value of about 40% early during the cellulose degradation process, suggesting that these bacteria play a role either in an extracellular cellulose hydrolysis process and/or in the uptake fermentation products.

Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate.

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.

Molecular analyses of the microbial community composition of an anoxic basin of a municipal wastewater treatment plant reveal a novel lineage of proteobacteria.

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anoxic activated sludge from a municipal wastewater treatment plant. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by polymerase chain reaction using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 132 and 249 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was done using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota (93.8% of the operational taxonomic units [OTUs]) and Crenarchaeota (6.2% of the OTUs). Within the bacterial library, 84.8% of the OTUs represent novel putative phylotypes never described before and affiliated with ten divisions. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 60.4% of the OTUs, followed by Bacteroidetes (22.1%) and gram-positives (6.1%). Interestingly, we detected a novel Proteobacteria monophyletic group distinct from the five known subclasses, which we named New Lineage of Proteobacteria (NLP) lineage, and it is composed of eight clones representing 4.6% of the Proteobacteria. A new 16S rRNA-targeted hybridization probe was designed and fluorescent in situ hybridization analyses shows representatives of NLP as cocci-shaped microorganisms. The Chloroflexi, Acidobacterium, and Nitrospira phyla and TM7 candidate division are each represented by ≤3% of clone sequences. A comprehensive set of eight 16S and 23S rRNA-targeted oligonucleotide probes was used to quantify these major groups by dot blot hybridization within 12 samples. The Proteobacteria accounted for 82.5 ± 4.9%, representing the most abundant phyla. The Bacteroidetes and Planctomycetales groups accounted for 4.9 ± 1.3% and 4 ± 1.7%, respectively. Firmicutes and Actinobacteria together accounted for only 1.9 ± 0.5%. The set of probes covers 93.4 ± 14% of the total bacterial population rRNA within the anoxic basin.

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.

"Candidatus Cloacamonas acidaminovorans": genome sequence reconstruction provides a first glimpse of a new bacterial division.

Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as "Candidatus Cloacamonas acidaminovorans." "Candidatus Cloacamonas acidaminovorans" is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.

Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester.

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by <5% of the total OTUs. A comprehensive set of 15 16S and 23S rRNA-targeted oligonucleotide hybridization probes was used to quantify these major groups by dot blot hybridization within 12 digester samples. In contrast to the clone library, Firmicutes and Actinobacteria together accounted for 21.8 +/- 14.9% representing the most abundant phyla. They were surprisingly followed by the Chloroflexi representing 20.2 +/- 4.6% of the total 16S rRNA. The Proteobacteria and the Bacteroidetes group accounted for 14.4 +/- 4.9% and 14.5 +/- 4.3%, respectively, WWE1, a novel lineage, accounted for 11.9 +/- 3.1% while Planctomycetes and Synergistes represented <2% each. Using the novel set of probes we extended the coverage of bacterial populations from 52% to 85.3% of the total rRNA within the digester samples.

Novel major bacterial candidate division within a municipal anaerobic sludge digester.

In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.

Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant.

We examined anoxic and aerobic basins and an anaerobic digestor of a municipal wastewater treatment plant for the presence of novel planctomycete-like diversity. Three 16S rRNA gene libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for Planctomyces: Phylogenetic analysis of 234 16S rRNA gene sequences defined 110 operational taxonomic units. The majority of these sequences clustered with the four known genera, Pirellula (32%), Planctomyces (18.4%), Gemmata (3.8%), and Isosphaera (0.4%). More interestingly, 42.3% of the sequences appeared to define two distantly separated monophyletic groups. The first group, represented by 35.5% of the sequences, was related to the Planctomyces group and branched as a monophyletic cluster. It exhibited between 11.9 and 20.3% 16S rRNA gene sequence dissimilarity in comparisons with cultivated planctomycetes. The second group, represented by 6.8% of the sequences, was deeply rooted within the Planctomycetales tree. It was distantly related to the anammox sequences (level of dissimilarity, 20.3 to 24.4%) and was a monophyletic cluster. The retrieved sequences extended the intralineage phylogenetic depth of the Plantomycetales from 23 to 30.6%. The lineages described here may have a broad diversity of undiscovered biochemical and metabolic novelty. We developed a new 16S rRNA-targeted oligonucleotide probe and localized members of one of the phylogenetic groups using the fluorescent in situ hybridization technique. Our results indicate that activated sludge contains very diverse representatives of this group, which grow under aerobic and anoxic conditions and even under anaerobic conditions. The majority of species in this group remain poorly characterized.