Suitability and setup of next-generation sequencing-based method for taxonomic characterization of aquatic microbial biofilm.

A robust and widely applicable method for sampling of aquatic microbial biofilm and further sample processing is presented. The method is based on next-generation sequencing of V4-V5 variable regions of 16S rRNA gene and further statistical analysis of sequencing data, which could be useful not only to investigate taxonomic composition of biofilm bacterial consortia but also to assess aquatic ecosystem health. Five artificial materials commonly used for biofilm growth (glass, stainless steel, aluminum, polypropylene, polyethylene) were tested to determine the one giving most robust and reproducible results. The effect of used sampler material on total microbial composition was not statistically significant; however, the non-plastic materials (glass, metal) gave more stable outputs without irregularities among sample parallels. The bias of the method is assessed with respect to the employment of a non-quantitative step (PCR amplification) to obtain quantitative results (relative abundance of identified taxa). This aspect is often overlooked in ecological and medical studies. We document that sequencing of a mixture of three merged primary PCR reactions for each sample and further evaluation of median values from three technical replicates for each sample enables to overcome this bias and gives robust and repeatable results well distinguishing among sampling localities and seasons.

Biomarker response, health indicators, and intestinal microbiome composition in wild brown trout (Salmo trutta m. fario L.) exposed to a sewage treatment plant effluent-dominated stream.

Concerns about the effect of sewage treatment plant (STP) effluent on the health of freshwater ecosystems have increased. In this study, a unique approach was designed to show the effect of an STP effluent-dominated stream on native wild brown trout (Salmo trutta L.) exposed under fully natural conditions. Zivny stream is located in South Bohemia, Czech Republic. The downstream site of Zivny stream is an STP-affected site, which receives 25% of its water from Prachatice STP effluent. Upstream, however, is a minimally polluted water site and it is considered to be the control site. Native fish were collected from the upstream site, tagged, and distributed to both upstream and downstream sites. After 30, 90, and 180days, fish were recaptured from both sites to determine whether the downstream site of the Zivny stream is associated with the effects of environmental pollution. Several biomarkers indicating the oxidative stress and antioxidant enzyme activities, cytochrome P450 activity, xenoestrogenic effects, bacterial composition, and lipid composition were investigated. Additionally, polar chemical contaminants (pharmaceuticals and personal care products (PPCPs)) were quantified using polar organic chemical integrative samplers (POCIS). Fifty-three PPCPs were detected in the downstream site; 36 of those were constantly present during the 180-day investigation period. Elevated hepatic 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylase (BFCOD) (after 90days) and blood plasma vitellogenin concentrations in males were detected in fish downstream of the STP effluent during all sampling events. An increase in the fishes' total fat content was also observed, but with low levels of ω-3 fatty acid in muscle tissue. Two bacterial taxa related to activated sludge were found in the intestines of fish from downstream. Our results show that Prachatice STP is a major source of PPCPs in the Zivny stream, which has biological consequences on fish physiology.

The effects of sewage treatment plant effluents on hepatic and intestinal biomarkers in common carp (Cyprinus carpio).

Sewage treatment plants (STPs) are one of the major source of pharmaceuticals and personal care products in the aquatic environment. Generally, the effects of individual chemicals on fish are studied under laboratory conditions, which leads to results that are potentially not realistic regarding the effects of these chemicals under environmental conditions. Therefore, in this study, common carps were held in exposed pond that receive water from STP effluents for 360 days under natural conditions. Elimination of xenobiotics starts in the fish intestine, in which the microbial community strongly influences its function. Moreover, the fish intestine functions as crucial organ for absorbing lipids and fatty acids (FA), with consequent transport to the liver where their metabolism occurs. The liver is the primary organ performing xenobiotic metabolism in fish, and therefore, the presence of pollutants may interact with the metabolism of FA. The catalytic activity of CYP1A and CYP3A-like enzymes, their gene expression, FA composition and intestinal microbiome consortia were measured. The catalytic activity of enzymes and their gene and protein expression, were induced in hepatic and intestinal tissues of fish from the exposed pond. Also, fish from the exposed pond had different compositions of FA than those from the control pond: concentration of 18:1 n-9 and 18:2 n-6 were significantly elevated and the longer chain n-3 FA 20:5 n-3, 22:5 n-3 and 22:6 n-3 were significantly lowered. There were clear differences among microbiome consortia in fish intestines across control and exposed groups. Microbiome taxa measured in exposed fish were also associated with those found in STP activated sludge. This study reveals that treated STP water, which is assumed to be clean, affected measured biomarkers in common carp.

Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes.

In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.