Corrigendum: The Resistome of Farmed Fish Feces Contributes to the Enrichment of Antibiotic Resistance Genes in Sediments below Baltic Sea Fish Farms.

[This corrects the article on p. 2137 in vol. 7, PMID: 28111573.].

Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers.

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.

The Resistome of Farmed Fish Feces Contributes to the Enrichment of Antibiotic Resistance Genes in Sediments below Baltic Sea Fish Farms.

Our previous studies showed that particular antibiotic resistance genes (ARGs) were enriched locally in sediments below fish farms in the Northern Baltic Sea, Finland, even when the selection pressure from antibiotics was negligible. We assumed that a constant influx of farmed fish feces could be the plausible source of the ARGs enriched in the farm sediments. In the present study, we analyzed the composition of the antibiotic resistome from the intestinal contents of 20 fish from the Baltic Sea farms. We used a high-throughput method, WaferGen qPCR array with 364 primer sets to detect and quantify ARGs, mobile genetic elements (MGE), and the 16S rRNA gene. Despite a considerably wide selection of qPCR primer sets, only 28 genes were detected in the intestinal contents. The detected genes were ARGs encoding resistance to sulfonamide (sul1), trimethoprim (dfrA1), tetracycline [tet(32), tetM, tetO, tetW], aminoglycoside (aadA1, aadA2), chloramphenicol (catA1), and efflux-pumps resistance genes (emrB, matA, mefA, msrA). The detected genes also included class 1 integron-associated genes (intI1, qacEΔ1) and transposases (tnpA). Importantly, most of the detected genes were the same genes enriched in the farm sediments. This preliminary study suggests that feces from farmed fish contribute to the ARG enrichment in farm sediments despite the lack of contemporaneous antibiotic treatments at the farms. We observed that the intestinal contents of individual farmed fish had their own resistome compositions. Our result also showed that the total relative abundances of transposases and tet genes were significantly correlated (p = 0.001, R2 = 0.71). In addition, we analyzed the mucosal skin and gill filament resistomes of the farmed fish but only one multidrug-efflux resistance gene (emrB) was detected. To our knowledge, this is the first study reporting the resistome of farmed fish using a culture-independent method. Determining the possible sources of ARGs, especially mobilized ARGs, is essential for controlling the occurrence and spread of ARGs at fish farming facilities and for lowering the risk of ARG spread from the farms to surrounding environments.

Fish farming affects the abundance and diversity of the mercury resistance gene merA in marine sediments.

Microbes in sediments below fish farming cages are exposed to a variety of compounds used in aquaculture, such as antifoulants, antimicrobials and mercury. Here, we assessed the abundance of the mercury resistance gene merA in the sediment of four fish farms and four pristine sites using quantitative PCR, and at three fish farms and two pristine sites by constructing clone libraries and sequencing merA amplicons. The abundance of merA was consistently greater at fish farm sites than at pristine sites, and the merA phylotypes were different between fish farm and pristine sites. Mercury concentrations were too low to cause selection pressure and did not differ significantly among samples. The primers designed specifically for this study were capable of detecting two new potential merA clades.