Temporal Resistome and Microbial Community Dynamics in an Intensive Aquaculture Facility with Prophylactic Antimicrobial Treatment.

Excessive use of antimicrobials in aquaculture is concerning, given possible environmental ramifications and the potential contribution to the spread of antimicrobial resistance (AR). In this study, we explored seasonal abundance of antimicrobial resistance genes and bacterial community composition in the water column of an intensive aquaculture pond stocked with Silver Carp (Hypophthalmichthys molitrix) prophylactically treated with sulfamethoprim (25% sulfadiazine; 5% trimethoprim), relative to an adjacent unstocked reservoir. Bacterial community composition was monitored using high-throughput sequencing of 16S rRNA gene amplicons in eight sampling profiles to determine seasonal dynamics, representing principal stages in the fish fattening cycle. In tandem, qPCR was applied to assess relative abundance of selected antimicrobial resistance genes (sul1, sul2, dfrA1, tetA and blaTEM) and class-1 integrons (int1). Concomitantly, resistomes were extrapolated from shotgun metagenomes in representative profiles. Analyses revealed increased relative abundance of sulfonamide and tetracycline resistance genes in fishpond-03, relative to pre-stocking and reservoir levels, whereas no significant differences were observed for genes encoding resistance to antimicrobials that were not used in the fishpond-03. Seasons strongly dictated bacterial community composition, with high abundance of cyanobacteria in summer and increased relative abundance of Flavobacterium in the winter. Our results indicate that prophylactic use of sulfonamides in intensive aquaculture ponds facilitates resistance suggesting that prophylactic use of these antimicrobials in aquaculture should be restricted.

Comparative Metagenomics and Network Analyses Provide Novel Insights Into the Scope and Distribution of ß-Lactamase Homologs in the Environment.

The ß-lactams are the largest group of clinically applied antibiotics, and resistance to these is primarily associated with ß-lactamases. There is increasing understanding that these enzymes are ubiquitous in natural environments and henceforth, elucidating the global diversity, distribution, and mobility of ß-lactamase-encoding genes is crucial for holistically understanding resistance to these antibiotics. In this study, we screened 232 shotgun metagenomes from ten different environments against a custom-designed ß-lactamase database, and subsequently analyzed ß-lactamase homologs with a suite of bioinformatic platforms including cluster and network analyses. Three interrelated ß-lactamase clusters encompassed all of the human and bovine feces metagenomes, while ß-lactamases from soil, freshwater, glacier, marine, and wastewater grouped within a separate "environmental" cluster that displayed high levels of inter-network connectivity. Interestingly, almost no connectivity occurred between the "feces" and "environmental" clusters. We attributed this in part to the divergence in microbial community composition (dominance of Bacteroidetes and Firmicutes vs. Proteobacteria, respectively). The ß-lactamase diversity in the "environmental" cluster was significantly higher than in human and bovine feces microbiomes. Several class A, B, C, and D ß-lactamase homologs (bla CTX-M, bla KPC, bla GES) were ubiquitous in the "environmental" cluster, whereas bovine and human feces metagenomes were dominated by class A (primarily cfxA) ß-lactamases. Collectively, this study highlights the ubiquitous presence and broad diversity of ß-lactamase gene precursors in non-clinical environments. Furthermore, it suggests that horizontal transfer of ß-lactamases to human-associated bacteria may be more plausible from animals than from terrestrial and aquatic microbes, seemingly due to phylogenetic similarities.

High Throughput Analysis of Integron Gene Cassettes in Wastewater Environments.

Integrons are extensively targeted as a proxy for anthropogenic impact in the environment. We developed a novel high-throughput amplicon sequencing pipeline that enables characterization of thousands of integron gene cassette-associated reads, and applied it to acquire a comprehensive overview of gene cassette composition in effluents from wastewater treatment facilities across Europe. Between 38 100 and 172 995 reads per-sample were generated and functionally characterized by screening against nr, SEED, ARDB and ß-lactamase databases. Over 75% of the reads were characterized as hypothetical, but thousands were associated with toxin-antitoxin systems, DNA repair, cell membrane function, detoxification and aminoglycoside and ß-lactam resistance. Among the reads characterized as ß-lactamases, the carbapenemase blaOXA was dominant in most of the effluents, except for Cyprus and Israel where blaGES was also abundant. Quantitative PCR assessment of blaOXA and blaGES genes in the European effluents revealed similar trends to those displayed in the integron amplicon sequencing pipeline described above, corroborating the robustness of this method and suggesting that these integron-associated genes may be excellent targets for source tracking of effluents in downstream environments. Further application of the above analyses revealed several order-of-magnitude reductions in effluent-associated ß-lactamase genes in effluent-saturated soils, suggesting marginal persistence in the soil microbiome.

Phylogenetic diversity of ceftriaxone resistance and the presence of extended-spectrum ß-lactamase genes in the culturable soil resistome.

The aim of this study was to determine the phylogenetic diversity of ceftriaxone resistance and the presence of known extended-spectrum ß-lactamase (ESBL) genes in culturable soil resistomes. Libraries of soil bacterial isolates resistant to ceftriaxone were established from six physicochemically diverse soils collected in Hawaii (USA) and Israel. The phylogenetic affiliation, ceftriaxone and multidrug resistance levels, and presence of known ESBL genes of the isolates were determined. The soil bacterial isolates were phylogenetically grouped with the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Ceftriaxone minimum inhibitory concentrations (MICs) largely followed the phylogeny structure and higher levels of ceftriaxone resistance corresponded to higher multidrug resistance. Three distinct blaTEM variants were detected in soil bacterial isolates belonging to nine different genera. In conclusion, the culturable soil resistomes for ceftriaxone exhibited high phylogenetic diversity and multidrug resistance. blaTEM was the only known ESBL detected in the soil resistomes, and its distribution in different phylogenetic groups suggests its ubiquitous presence and/or possible horizontal gene transfer within the soil microbiomes.

Resistance of Undisturbed Soil Microbiomes to Ceftriaxone Indicates Extended Spectrum ß-Lactamase Activity.

Emergence and spread of antibiotic resistance, and specifically resistance to third generation cephalosporins associated with extended spectrum ß-lactamase (ESBL) activity, is one of the greatest epidemiological challenges of our time. In this study we addressed the impact of the third generation cephalosporin ceftriaxone on microbial activity and bacterial community composition of two physically and chemically distinct undisturbed soils in highly regulated microcosm experiments. Surprisingly, periodical irrigation of the soils with clinical doses of ceftriaxone did not affect their microbial activity; and only moderately impacted the microbial diversity (α and ß) of the two soils. Corresponding slurry experiments demonstrated that the antibiotic capacity of ceftriaxone rapidly diminished in the presence of soil, and ∼70% of this inactivation could be explained by biological activity. The biological nature of ceftriaxone degradation in soil was supported by microcosm experiments that amended model Escherichia coli strains to sterile and non-sterile soils in the presence and absence of ceftriaxone and by the ubiquitous presence of ESBL genes (blaTEM, blaCTX-M, and blaOXA) in soil DNA extracts. Collectively, these results suggest that the resistance of soil microbiomes to ceftriaxone stems from biological activity and even more, from broad-spectrum ß-lactamase activity; raising questions regarding the scope and clinical implications of ESBLs in soil microbiomes.

Spatial and temporal biogeography of soil microbial communities in arid and semiarid regions.

Microbial communities in soils may change in accordance with distance, season, climate, soil texture and other environmental parameters. Microbial diversity patterns have been extensively surveyed in temperate regions, but few such studies attempted to address them with respect to spatial and temporal scales and their correlations to environmental factors, especially in arid ecosystems. In order to fill this gap on a regional scale, the molecular fingerprints and abundance of three taxonomic groups--Bacteria, α-Proteobacteria and Actinobacteria--were sampled from soils 0.5-100 km apart in arid, semi-arid, dry Mediterranean and shoreline Mediterranean regions in Israel. Additionally, on a local scale, the molecular fingerprints of three taxonomic groups--Bacteria, Archaea and Fungi--were sampled from soils 1 cm-500 m apart in the semi-arid region, in both summer and winter. Fingerprints of the Bacteria differentiated between all regions (P<0.02), while those of the α-Proteobacteria differentiated between some of the regions (0.010.05). Locally, fingerprints of archaea and fungi did not display distance-decay relationships (P>0.13), that is, the dissimilarity between communities did not increase with geographic distance. Neither was this phenomenon evident in bacterial samples in summer (P>0.24); in winter, however, differences between bacterial communities significantly increased as the geographic distances between them grew (P<0.01). Microbial community structures, as well as microbial abundance, were both significantly correlated to precipitation and soil characteristics: texture, organic matter and water content (R(2)>0.60, P<0.01). We conclude that on the whole, microbial biogeography in arid and semi-arid soils in Israel is determined more by specific environmental factors than geographic distances and spatial distribution patterns.

Impact of treated wastewater irrigation on antibiotic resistance in the soil microbiome.

The reuse of treated wastewater (TWW) for irrigation is a practical solution for overcoming water scarcity, especially in arid and semiarid regions of the world. However, there are several potential environmental and health-related risks associated with this practice. One such risk stems from the fact that TWW irrigation may increase antibiotic resistance (AR) levels in soil bacteria, potentially contributing to the global propagation of clinical AR. Wastewater treatment plant (WWTP) effluents have been recognized as significant environmental AR reservoirs due to selective pressure generated by antibiotics and other compounds that are frequently detected in effluents. This review summarizes a myriad of recent studies that have assessed the impact of anthropogenic practices on AR in environmental bacterial communities, with specific emphasis on elucidating the potential effects of TWW irrigation on AR in the soil microbiome. Based on the current state of the art, we conclude that contradictory to freshwater environments where WWTP effluent influx tends to expand antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes levels, TWW irrigation does not seem to impact AR levels in the soil microbiome. Although this conclusion is a cause for cautious optimism regarding the future implementation of TWW irrigation, we conclude that further studies aimed at assessing the scope of horizontal gene transfer between effluent-associated ARB and soil bacteria need to be further conducted before ruling out the possible contribution of TWW irrigation to antibiotic-resistant reservoirs in irrigated soils.