Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment - Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel Congeria jalzici, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches.

Evaluation of the Structure-Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach.

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Host-related traits influence the microbial diversity of the invasive signal crayfish Pacifastacus leniusculus.

The microbiome influences a variety of host-environment interactions, and there is mounting evidence of its significant role in biological invasions. During invasion, shifts in microbial diversity and function can occur due to both changing characteristics of the novel environment and physiological condition of the host. The signal crayfish (Pacifastacus leniusculus) is one of the most successful crayfish invaders in Europe. During range expansion, its populations often exhibit differences in many traits along the invasion range, including sex-composition, size-structure and aggressiveness, but to date it was not studied whether crayfish traits can also drive changes in the host microbiome. Thus, we used 16S rRNA gene amplicon sequencing to examine the effects of host-related traits, namely total length (TL), body condition index (FCF), hepatosomatic index (HSI) and sex on the microbial diversity of the signal crayfish. We examined both external (exoskeletal) and internal (intestinal, hepatopancreatic, hemolymph) microbiomes of 110 signal crayfish individuals from four sites along its invasion range in the Korana River, Croatia. While sex did not exhibit a significant effect on the microbial diversity in any of the examined tissues, exoskeletal, intestinal and hemolymph microbial diversity significantly decreased with increasing crayfish size. Additionally, significant effects of signal crayfish condition (FCF, HSI) on microbial diversity were recorded in the hepatopancreas, a main energy storage organ in crayfish that supports reproduction and growth and also regulates immune response. Our findings provide a baseline for evaluating the contribution of microbiome to an invader's overall health, fitness and subsequent invasion success.

Comparison of exoskeleton microbial communities of co-occurring native and invasive crayfish species.

Host-associated microbial communities are an important determinant of individual fitness and have recently been highlighted as one of the factors influencing the success of invasive species. Invasive hosts introduce their microbes into the new environment, and then both the host and its associated microbes enter into a series of interactions with the native macroscopic and microscopic biota. As these processes are largely unexplored, we aimed to compare the exoskeletal microbial communities of co-occurring and phylogenetically related crayfish: the native narrow-clawed crayfish Pontastacus leptodactylus and the invasive signal crayfish Pacifastacus leniusculus from the recently invaded Korana River, Croatia. The results of high-throughput 16S rRNA sequencing showed that the exoskeletal microbiome of both species is very diverse, significantly influenced by the local environment and dominated by low abundance bacterial families from the phylum Proteobacteria. Furthermore, the exoskeletal microbiomes of the crayfish species differed significantly in the composition and abundance of Amplicon Sequence Variants (ASVs), suggesting that they are to some extent shaped by species-specific intrinsic factors, despite sharing a common habitat. However, over 95% of the bacterial genera associated with the exoskeleton were detected in the exoskeleton samples of both native and invasive crayfish. We paid particular attention to two known crayfish pathogens, Aphanomyces astaci and Saprolegnia parasitica, and find that both species carry low amounts of both pathogens. On the side, we find that a non-standard ddPCR protocol outperforms standard qPCR test for A. astaci under low concentration conditions. Taken together, our results indicate the possibility of bidirectional mixing and homogenisation of exoskeleton microbiome. As such, they can serve as a baseline in future detangling of the processes that act together to shape the microbiomes of co-occuring native and invasive congeners during biological invasions.

Sage, Rosemary, and Bay Laurel Hydrodistillation By-Products as a Source of Bioactive Compounds.

Essential oils from Mediterranean wild plants are widely used, but the hydrodistillation residues produced in parallel with these essential oils are significantly understudied and underexploited. Since there are only fragmentary data in the literature, we have, for the first time, systematically analyzed the chemical composition of the by-products obtained after hydrodistillation of sage, bay laurel, and rosemary leaves, i.e., hydrolates, water residues, and solid residues. The chemical composition of the hydrolates changed compared to their respective essential oils towards the dominance of more hydrophilic, oxygenated compounds, such as camphor in sage, 1,8-cineole in bay laurel, and berbenone in rosemary. However, some compounds, mostly sesquiterpenes, which were present in considerable amounts in essential oils, were absent or only present in very small amounts in the hydrolates. Furthermore, both the water and the solid residues were rich in polyphenols, such as procyanidins in bay laurel and rosmarinic acid in rosemary and sage. In conclusion, we demonstrate the valuable chemical composition of sage, rosemary, and bay laurel hydrodistillation by-products and discuss a wide range of their possible applications.

Impact of nutrients and trace elements on freshwater microbial communities in Croatia: identifying bacterial bioindicator taxa.

Since aquatic microbial communities promptly respond to environmental changes, it is now evident that they can complement traditional taxa such as fish, macroinvertebrates and algae as bioindicators of water quality. The aim of this study was to correlate the physico-chemical parameters of water with the microbial community structure and the occurrence of putative bioindicator taxa. Thirty-five water samples were collected throughout Croatia and their physico-chemical parameters, including the concentration of trace elements using the high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS), and the composition of the microbial communities by high-throughput sequencing of the 16S rRNA marker gene, were analysed in parallel. Partial least squares regression (PLS-R) modelling revealed that a number of microbial taxa were positively correlated with some of the water parameters. For example, some taxa from the phylum Proteobacteria were positively correlated with the ion content of the water (e.g. Erythrobacter, Rhodobacteraceae, Alteromonadaceae), while some Firmicutes taxa, such as the well-known faecal indicators Enterococcus and Clostridium, were correlated with nutrient content (ammonium and total phosphorus). Among the trace elements, uranium was positively correlated with a highest number of microbial taxa. The results obtained will aid in development of protocols for eDNA-based biological assessment of water quality.

The mycobiome of a successful crayfish invader and its changes along the environmental gradient.

BACKGROUND: The microbiome plays an important role in biological invasions, since it affects various interactions between host and environment. However, most studies focus on the bacteriome, insufficiently addressing other components of the microbiome such as the mycobiome. Microbial fungi are among the most damaging pathogens in freshwater crayfish populations, colonizing and infecting both native and invasive crayfish species. Invading crayfish may transmit novel fungal species to native populations, but also, dispersal process and characteristics of the novel environment may affect the invaders' mycobiome composition, directly and indirectly affecting their fitness and invasion success. This study analyzes the mycobiome of a successful invader in Europe, the signal crayfish, using the ITS rRNA amplicon sequencing approach. We explored the mycobiomes of crayfish samples (exoskeletal biofilm, hemolymph, hepatopancreas, intestine), compared them to environmental samples (water, sediment), and examined the differences in fungal diversity and abundance between upstream and downstream segments of the signal crayfish invasion range in the Korana River, Croatia. RESULTS: A low number of ASVs (indicating low abundance and/or diversity of fungal taxa) was obtained in hemolymph and hepatopancreas samples. Thus, only exoskeleton, intestine, sediment and water samples were analyzed further. Significant differences were recorded between their mycobiomes, confirming their uniqueness. Generally, environmental mycobiomes showed higher diversity than crayfish-associated mycobiomes. The intestinal mycobiome showed significantly lower richness compared to other mycobiomes. Significant differences in the diversity of sediment and exoskeletal mycobiomes were recorded between different river segments (but not for water and intestinal mycobiomes). Together with the high observed portion of shared ASVs between sediment and exoskeleton, this indicates that the environment (i.e. sediment mycobiome) at least partly shapes the exoskeletal mycobiome of crayfish. CONCLUSION: This study presents the first data on crayfish-associated fungal communities across different tissues, which is valuable given the lack of studies on the crayfish mycobiome. We demonstrate significant differences in the crayfish exoskeletal mycobiome along the invasion range, suggesting that different local environmental conditions may shape the exoskeletal mycobiome during range expansion, while the mycobiome of the internal organ (intestine) remained more stable. Our results provide a basis for assessing how the mycobiome contributes to the overall health of the signal crayfish and its further invasion success.

Tracing the oomycete pathogen Saprolegnia parasitica in aquaculture and the environment.

Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica.

Variations in the Sporulation Efficiency of Pathogenic Freshwater Oomycetes in Relation to the Physico-Chemical Properties of Natural Waters.

Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation.

Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses.

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader's dispersal.

Microbiome of the Successful Freshwater Invader, the Signal Crayfish, and Its Changes along the Invasion Range.

Increasing evidence denotes the role of the microbiome in biological invasions, since it is known that microbes can affect the fitness of the host. Here, we demonstrate differences in the composition of an invader's microbiome along the invasion range, suggesting that its microbial communities may affect and be affected by range expansion. Using a 16S rRNA gene amplicon sequencing approach, we (i) analyzed the microbiomes of different tissues (exoskeleton, hemolymph, hepatopancreas, and intestine) of a successful freshwater invader, the signal crayfish, (ii) compared them to the surrounding water and sediment, and (iii) explored their changes along the invasion range. Exoskeletal, hepatopancreatic, and intestinal microbiomes varied between invasion core and invasion front populations. This indicates that they may be partly determined by population density, which was higher in the invasion core than in the invasion front. The highly diverse microbiome of exoskeletal biofilm was partly shaped by the environment (due to the similarity with the sediment microbiome) and partly by intrinsic crayfish parameters (due to the high proportion of exoskeleton-unique amplicon sequence variants [ASVs]), including the differences in invasion core and front population structure. Hemolymph had the most distinct microbiome compared to other tissues and differed between upstream (rural) and downstream (urban) river sections, indicating that its microbiome is potentially more driven by the effects of the abiotic environment. Our findings offer an insight into microbiome changes during dispersal of a successful invader and present a baseline for assessment of their contribution to an invader's overall health and its further invasion success. IMPORTANCE Invasive species are among the major drivers of biodiversity loss and impairment of ecosystem services worldwide, but our understanding of their invasion success and dynamics still has many gaps. For instance, although it is known that host-associated microbial communities may significantly affect an individual's health and fitness, the current studies on invasive species are mainly focused on pathogenic microbes, while the effects of the remaining majority of microbial communities on the invasion process are almost completely unexplored. We have analyzed the microbiome of one of the most successful crayfish invaders in Europe, the signal crayfish, and explored its changes along the signal crayfish invasion range in the Korana River, Croatia. Our study sets the perspective for future research required to assess the contribution of these changes to an individual's overall health status and resilience of dispersing populations and their impact on invasion success.

Essential Oils of Sage, Rosemary, and Bay Laurel Inhibit the Life Stages of Oomycete Pathogens Important in Aquaculture.

Saprolegnia parasitica, the causative agent of saprolegniosis in fish, and Aphanomyces astaci, the causative agent of crayfish plague, are oomycete pathogens that cause economic losses in aquaculture. Since toxic chemicals are currently used to control them, we aimed to investigate their inhibition by essential oils of sage, rosemary, and bay laurel as environmentally acceptable alternatives. Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that the essential oils tested were rich in bioactive volatiles, mainly monoterpenes. Mycelium and zoospores of A. astaci were more sensitive compared to those of S. parasitica, where only sage essential oil completely inhibited mycelial growth. EC50 values (i.e., concentrations of samples at which the growth was inhibited by 50%) for mycelial growth determined by the radial growth inhibition assay were 0.031-0.098 µL/mL for A. astaci and 0.040 µL/mL for S. parasitica. EC50 values determined by the zoospore germination inhibition assay were 0.007-0.049 µL/mL for A. astaci and 0.012-0.063 µL/mL for S. parasitica. The observed inhibition, most pronounced for sage essential oil, could be partly due to dominant constituents of the essential oils, such as camphor, but more likely resulted from a synergistic effect of multiple compounds. Our results may serve as a basis for in vivo experiments and the development of environmentally friendly methods to control oomycete pathogens in aquaculture.

Microbial pathogens of freshwater crayfish: A critical review and systematization of the existing data with directions for future research.

Despite important ecological role and growing commercial value of freshwater crayfish, their diseases are underresearched and many studies examining potential crayfish pathogens do not thoroughly address their epizootiology, pathology or biology. This study reviews over 100 publications on potentially pathogenic viruses, bacteria, fungi and fungal-like microorganisms reported in crayfish and systematizes them based on whether pathogenicity has been observed in an analysed species. Conclusions on pathogenicity were based on successful execution of infectivity trials. For 40.6% of examined studies, microbes were successfully systematized, while for more than a half (59.4%) no conclusion on pathogenicity could be made. Fungi and fungal-like microorganisms were the most studied group of microbes with the highest number of analysed hosts, followed by bacteria and viruses. Our analysis demonstrated the need for: (a) inclusion of higher number of potential host species in the case of viruses, (b) research of bacterial effects in tissues other than haemolymph, and (c) more research into potential fungal and fungal-like pathogens other than Aphanomyces astaci. We highlight the encountered methodological challenges and biases and call for a broad but standardized framework for execution of infectivity trials that would enable systematic data acquisition on interactions between microbes and the host.

Effect of Enzymatic, Ultrasound, and Reflux Extraction Pretreatments on the Chemical Composition of Essential Oils.

The effect of different hydrodistillation pretreatments, namely, reflux extraction, reflux extraction with the addition of cell wall-degrading enzymes, and ultrasound, on the yield and chemical composition of essential oils of sage, bay laurel, and rosemary was examined. All pretreatments improved essential oil yield compared to no-pretreatment control (40-64% yield increase), while the oil quality remained mostly unchanged (as shown by statistical analysis of GC-MS results). However, enzyme-assisted reflux extraction pretreatment did not significantly outperform reflux extraction (no-enzyme control), suggesting that the observed yield increase was mostly a consequence of reflux extraction and enzymatic activity had only a minute effect. Thus, we show that ultrasound and reflux extraction pretreatments are beneficial in the production of essential oils of selected Mediterranean plants, but the application of enzymes has to be carefully re-evaluated.

Leitmotif: protein motif scanning 2.0.

MOTIVATION: Motif-HMM (mHMM) scanning has been shown to possess unique advantages over standardly used sequence-profile search methods (e.g. HMMER, PSI-BLAST) since it is particularly well-suited to discriminate proteins with variations inside conserved motifs (e.g. family subtypes) or motifs lacking essential residues (false positives, e.g. pseudoenzymes). RESULTS: In order to make mHMM widely accessible to a broader scientific community, we developed Leitmotif, an mHMM web application with many parametrization options easily accessible through intuitive interface. Substantial improvement of performance (ROC scores) was obtained by using two novel parameters. To the best of our knowledge, Leitmotif is the only available mHMM application. AVAILABILITY AND IMPLEMENTATION: Leitmotif is freely available at https://leitmotif.irb.hr. CONTACT: sinisa@heuristika.hr or ivan.vujaklija@fer.hr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level.

The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a non-destructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.

Corrigendum: Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

[This corrects the article DOI: 10.3389/fmicb.2017.02675.].

Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries.

Effluents from pharmaceutical industries are recognized as significant contributors to aquatic pollution with antibiotics. Although such pollution has been mostly reported in Asia, knowledge on industrial discharges in other regions of the world, including Europe, and on the effects associated with such exposures is still limited. Thus, we performed chemical, microbiological and ecotoxicological analyses of effluents from two Croatian pharmaceutical industries during four seasons. In treated effluents of the company synthesizing macrolide antibiotic azithromycin (AZI), the total concentration of AZI and two macrolide by-products from its synthesis was 1-3 orders of magnitude higher in winter and springtime (up to 10.5 mg/L) than during the other two seasons (up to 638 µg/L). Accordingly, the highest total concentrations (up to 30 µg/L) in the recipient river were measured in winter and spring. Effluents from second company formulating veterinary antibiotics contained fluoroquinolones, trimethoprim, sulfonamides and tetracyclines ranging from low µg/L to approx. 200 µg/L. Low concentrations of these antibiotics, from below the limit of quantification to approx. few µg/L, have also been measured in the recipient stream. High frequency of culturable bacteria resistant to AZI (up to 83%) or sulfamethazine (up to 90%) and oxytetracycline (up to 50%) were also found in studied effluents. Finally, we demonstrated that toxicity to algae and water fleas often exceeded the permitted values. Most highly contaminated effluents induced multiple abnormalities in zebrafish embryos. In conclusion, using a wide array of analyses we have demonstrated that discharges from pharmaceutical industries can pose a significant ecological and public health concern due to their toxicity to aquatic organisms and risks for promoting development and spread of antibiotic resistance.

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs from environmental hotspots such as discharge sites of pharmaceutical effluents.

Multilevel ecotoxicity assessment of environmentally relevant bisphenol A concentrations using the soil invertebrate Eisenia fetida.

Bisphenol A (BPA) presents a serious threat to soil ecosystems, yet its effects on soil-inhabiting organisms are mostly unexplored. Therefore, the impact of environmentally relevant BPA concentrations on a terrestrial model organism, the earthworm Eisenia fetida, was assessed. Animals were cutaneously exposed to 100nM and 10µM BPA up to 10days (10-d). Next, a battery of biomarkers was used for ecotoxicological evaluation on a cellular, tissue and behavioural level. HPLC analysis showed that after a 10-d exposure, BPA accumulation reached a maximum of 2.50µg BPA per g of wet tissue weight. On the cellular level, up to 3-d BPA exposure caused increased lipid oxidation indicating oxidative stress. Histopathological assessment of cell wall and ovaries after 7- and 10-d BPA exposure showed multiple abnormalities, i.e. hyperplasia of epidermis, increased body wall thickness and ovarian atrophy. Detection of these changes was facilitated by a newly proposed semi-quantitative scoring system. Finally, behavioural changes were detected after only 3days of exposure to 100nM BPA. Altogether, the presented multilevel toxicity evaluation indicates high sensitivity of earthworms to low BPA doses.