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We have investigated the changes in the microbial communities on the surface of trout eggs and the skin of adult trout in relation to the presence of Saprolegnia parasitica . This pathogen causes saprolegniosis, a disease responsible for significant losses in salmonid farms and hatcheries. It is known from other disease systems that the host-associated microbiome plays a crucial role in the defence against pathogens, but if the pathogen predominates, this can lead to dysbiosis. However, analyses of the effects of S. parasitica on the diversity, composition, and function of microbial communities on fish skin and eggs are scarce. Thus, we have collected skin swabs from injured and healthy trout ( N = 12), which differed in S. parasitica load, from three different fish farms in Croatia (Kostanjevac, Radovan, and Solin), while trout egg samples ( N = 12) were infected with S. parasitica in the laboratory. Illumina sequencing of the V4 region of the 16S rRNA marker gene showed that infection with S. parasitica reduced the microbial diversity on the surface of the eggs, as evidenced by decreased Pielou's evenness and Shannon's indices. We further determined whether the bacterial genera with a relative abundance of >5.0% in the egg/skin samples were present at significantly different abundances in relation to the presence of S. parasitica . The results have shown that some genera, such as Pseudomonas and Flavobacterium , decreased significantly in the presence of the pathogen on the egg surface. On the other hand, some bacterial taxa, such as Acinetobacter and Janthinobacterium , as well as Aeromonas , were more abundant on the diseased eggs and the injured trout skin, respectively. Finally, beta diversity analyses (weighted UniFrac, unweighted UniFrac, Bray-Curtis) have shown that the sampling location (i.e., fish farm), along with S. parasitica infection status, also has a significant influence on the microbial communities' composition on the trout skin and eggs, demonstrating the strong influence of the environment on the shaping of the host surface microbiome. Overall, we have shown that the presence of S. parasitica was associated with changes in the diversity and structure of the trout skin/egg microbiome. The results obtained could support the development of new strategies for the management of saprolegniosis in aquaculture.

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 ( p NP) 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 p NP 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-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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

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.

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.

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.

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. EC 50 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 . EC 50 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.

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., (© 2020 John Wiley & Sons Ltd.)

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.

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., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The conservation of gray wolf (Canis lupus) and its coexistence with humans presents a challenge and requires continuous monitoring and management efforts. One of the non-invasive methods that produces high-quality wolf monitoring datasets is camera trapping. We present a novel monitoring approach where camera traps are positioned on wildlife crossing structures that channel the animals, thereby increasing trapping success and increasing the cost-efficiency of the method. In this way we have followed abundance trends of five wolf packs whose home ranges are intersected by a motorway which spans throughout the wolf distribution range in Croatia. During the five-year monitoring of six green bridges we have recorded 28 250 camera-events, 132 with wolves. Four viaducts were monitored for two years, recording 4914 camera-events, 185 with wolves. We have detected a negative abundance trend of the monitored Croatian wolf packs since 2011, especially severe in the northern part of the study area. Further, we have pinpointed the legal cull as probable major negative influence on the wolf pack abundance trends (linear regression, r2 > 0.75, P < 0.05). Using the same approach we did not find evidence for a negative impact of wolves on the prey populations, both wild ungulates and livestock. We encourage strict protection of wolf in Croatia until there is more data proving population stability. In conclusion, quantitative methods, such as the one presented here, should be used as much as possible when assessing wolf abundance trends.

Background: The massive accumulation of protein sequences arising from the rapid development of high-throughput sequencing, coupled with automatic annotation, results in high levels of incorrect annotations. In this study, we describe an approach to decrease annotation errors of protein families characterized by low overall sequence similarity. The GDSL lipolytic family comprises proteins with multifunctional properties and high potential for pharmaceutical and industrial applications. The number of proteins assigned to this family has increased rapidly over the last few years. In particular, the natural abundance of GDSL enzymes reported recently in plants indicates that they could be a good source of novel GDSL enzymes. We noticed that a significant proportion of annotated sequences lack specific GDSL motif(s) or catalytic residue(s). Here, we applied motif-based sequence analyses to identify enzymes possessing conserved GDSL motifs in selected proteomes across the plant kingdom., Results: Motif-based HMM scanning (Viterbi decoding-VD and posterior decoding-PD) and the here described PD/VD protocol were successfully applied on 12 selected plant proteomes to identify sequences with GDSL motifs. A significant number of identified GDSL sequences were novel. Moreover, our scanning approach successfully detected protein sequences lacking at least one of the essential motifs (171/820) annotated by Pfam profile search (PfamA) as GDSL. Based on these analyses we provide a curated list of GDSL enzymes from the selected plants. CLANS clustering and phylogenetic analysis helped us to gain a better insight into the evolutionary relationship of all identified GDSL sequences. Three novel GDSL subfamilies as well as unreported variations in GDSL motifs were discovered in this study. In addition, analyses of selected proteomes showed a remarkable expansion of GDSL enzymes in the lycophyte, Selaginella moellendorffii. Finally, we provide a general motif-HMM scanner which is easily accessible through the graphical user interface ( http://compbio.math.hr/ )., Conclusions: Our results show that scanning with a carefully parameterized motif-HMM is an effective approach for annotation of protein families with low sequence similarity and conserved motifs. The results of this study expand current knowledge and provide new insights into the evolution of the large GDSL-lipase family in land plants.

Tolerance towards environmental stress has been frequently considered as one of the key determinants of invasion success. However, empirical evidence supporting the assumption that invasive species can better endure unfavorable conditions compared with native species is limited and has yielded opposing results. In this study, we examined the tolerance to different stress conditions (thermal stress and trace metal zinc pollution stress) in two phylogenetically related and functionally similar freshwater bivalve species, the native Anodonta anatina and the invasive Sinanodonta woodiana. We assessed potential differences in response to stress conditions using several cellular response assays: efficiency of the multixenobiotic resistance mechanism, respiration estimate (INT reduction capacity), and enzymatic biomarkers. Our results demonstrated that the invasive species overall coped much better with unfavorable conditions. The higher tolerance of S. woodiana was evident from (i) significantly decreased Rhodamine B accumulation indicating more efficient multixenobiotic resistance mechanism; (ii) significantly higher INT reduction capacity and (iii) less pronounced alterations in the activity of stress-related enzymes (glutathione-S-transferase, catalase) and of a neurotoxicity biomarker (cholinesterase) in the majority of treatment conditions in both stress trials. Higher tolerance to thermal extremes may provide physiological benefit for further invasion success of S. woodiana in European freshwaters, especially in the context of climate change., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The exploration of marine Actinobacteria has as major challenge to answer basic questions of microbial ecology that, in turn, will provide useful information to exploit Actinobacteria metabolisms in biotechnological processes. The ecological functions performed by Actinobacteria in marine sediments are still unclear and belongs to the most burning basic questions. The comparison of Actinobacteria communities inhabiting marine sediments that are under the influence of different contamination types will provide valuable information in the adaptation capacities of Actinobacteria to colonize specific ecological niche. In the present study, the characterization of different Actinobacteria assemblages according to contamination type revealed the ecological importance of Actinobacteria for maintaining both general biogeochemical functions through a "core" Actinobacteria community and specific roles associated with the presence of contaminants. Indeed, the results allowed to distinguish Actinobacteria genera and species operational taxonomic units (OTUs) able to cope with the presence of either (i) As, (ii) metals Ni, Fe, V, Cr, and Mn, or (iii) polycyclic aromatic hydrocarbons (PAHs) and toxic metals (Hg, Cd, Cu, Pb, and Zn). Such observations highlighted the metabolic capacities of Actinobacteria and their potential that should be taken into consideration and advantage during the implementation of bioremediation processes in marine ecosystems.

Integrons are bacterial genetic elements known to be active vectors of antibiotic resistance among clinical bacteria. They are also found in bacterial communities from natural environments. Although integrons have become especially efficient for bacterial adaptation in the particular context of antibiotic usage, their role in natural environments in other contexts is still unknown. Indeed, most studies have focused on integrons and the spread of antibiotic resistance in freshwater or soil impacted by anthropogenic activities, with only few on marine environments. Notably, integrons show a wider diversity of both gene cassettes and integrase gene in natural environments than in clinical environments, suggesting a general role of integrons in bacterial adaptation. This article reviews the current knowledge on integrons in marine environments. We also present conclusions of our studies on polluted and nonpolluted backgrounds.

Green bridges are used to decrease highly negative impact of roads/highways on wildlife populations and their effectiveness is evaluated by various monitoring methods. Based on the 3-year monitoring of four Croatian green bridges, we compared the effectiveness of three indirect monitoring methods: track-pads, camera traps and active infrared (IR) trail monitoring system. The ability of the methods to detect different species and to give good estimation of number of animal crossings was analyzed. The accuracy of species detection by track-pad method was influenced by granulometric composition of track-pad material, with the best results obtained with higher percentage of silt and clay. We compared the species composition determined by track-pad and camera trap methods and found that monitoring by tracks underestimated the ratio of small canids, while camera traps underestimated the ratio of roe deer. Regarding total number of recorder events, active IR detectors recorded from 11 to 19 times more events then camera traps and app. 80% of them were not caused by animal crossings. Camera trap method underestimated the real number of total events. Therefore, an algorithm for filtration of the IR dataset was developed for approximation of the real number of crossings. Presented results are valuable for future monitoring of wildlife crossings in Croatia and elsewhere, since advantages and disadvantages of used monitoring methods are shown. In conclusion, different methods should be chosen/combined depending on the aims of the particular monitoring study.

Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators (verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthworm's MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity.

Oxcarbazepine (OXC) is generally accepted as a drug without risk of severe drug-induced hepatotoxicity, but according to recently reported pharmacovigilance data this statement has been challenged. However, in the literature there have been no reports of acute OXC-induced hepatotoxicity without systemic manifestations of Drug Rash with Eosinophilia and Systemic Symptoms (DRESS) syndrome. We present a female with seizures one month after delivery who had borderline elevated liver enzymes prior to the initiation of OXC treatment. Two weeks after introducing OXC, highly elevated liver enzymes were found. After discontinuation of OXC the enzymes continued to rise for another week, and afterward gradually decreased. The causal relationship with OXC intake was determined to be highly probable. Two years later, the transitory elevation of liver enzymes was observed during the treatment of acute tonsilopharingitis with amoxicillin + clavulanic acid. The repeated elevation of liver enzymes related to use of different drugs might indicate patients susceptibility for drug induced liver injuries. We suggest that monitoring of liver function tests would be clinically rational for early detection of acute OXC-induced liver hepatotoxicity in the patients with clinical and/or laboratory features which might be interpreted as possible risk factors of the increased susceptibility to drug induced liver injuries.

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, alpha- and beta-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20-80). It showed pronounced activity towards p-nitrophenyl and alpha- and beta-naphthyl esters of C(12)-C(16). Higher activity was observed with alpha-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 degrees C) and thermal stability. CD analysis revealed predominance of alpha-helical structure (54% alpha-helix, 21% beta-sheet) and a T(m) value at 66 degrees C. Systematic bioinformatic analysis of deduced amino acid sequence of S. coelicolor enzyme placed it to the SGNH-hydrolase family. Phylogenetic analysis of the predicted protein homologous to the S. coelicolor SGNH-hydrolase generated three distinct groups consisting of proteins from Actinomycetales, Ascomycota and Nematoda. At present it seems that these enzymes are most conserved among soil inhabiting organisms.