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Valutazione dell'impatto ambientale di antimicrobici utilizzati per i trattamenti di massa negli allevamenti
No full textNegli allevamenti intensivi, i trattamenti di massa implicano la somministrazione di grandi quantità di antimicrobici che possono essere non metabolizzati dall’animale o essere metabolizzati ed escreti come metaboliti attivi o inattivi. Poichè le deiezioni provenienti da tali allevamenti vengono comunemente usate per la fertilizzazione dei terreni agricoli, può derivarne una contaminazione ambientale da farmaci. I residui di principi attivi possono poi essere trasferiti dal suolo alle acque superficiali tramite dilavamento o infiltrazione. Le conseguenze ambientali derivanti dalla concimazione del suolo con letame contaminato da farmaci costituiscono un argomento di grande interesse, poiché la persistenza di antimicrobici nell'ambiente può rappresentare un rischio ecotossicologico per popolazioni di organismi non-target e portare alla comparsa e alla diffusione di resistenza agli antimicrobici (AMR). Per valutare gli effetti in organismi non-target, sono stati selezionati i fluorochinoloni (FQs): due farmaci ad uso veterinario (enrofloxacina e flumechina) ed uno ad uso umano (levofloxacina), utilizzati per test di inibizione dell’allungamento radicale su tre piante terrestri (Lycopersicon esculentum, Lactuca sativa e Daucus carota). Queste dicotiledoni hanno mostrato una limitata sensibilità ai 3 FQs. Per tal motivo, successivi studi sono stati effettuati sull’organismo modello Daphnia magna. Sono stati sviluppati e messi a punto dei test innovativi in grado di evidenziare effetti tossici ritardati in seguito ad esposizione embrionale o neonatale. Si è verificata, inoltre, la modalità di interazione tossicologica dei tre composti in miscele binarie e ternarie. I risultati ottenuti hanno mostrato che i test ufficiali presentano dei limiti, in quanto portano a una sovrastima dell’EC50, e che le miscele di questi composti mostrano un grado di tossicità che si giustifica con interazioni di tipo additivo. Parallelamente, disponendo di nanoparticelle magnetiche ingegnerizzate con flumechina che potrebbero trovare futura applicazione in acquacoltura, è stata saggiata anche la biocompatibilità e l’efficacia terapeutica di questo core-shell nanocarrier, su una coltura di D. magna naturalmente infettatasi con batteri sensibili alla flumechina. Altre attività di ricerca concernenti i FQs hanno rappresentato l’avvio di un progetto di ricerca volto a valutare i possibili effetti epigenetici di flumechina su D. magna. Dai primi risultati sono emersi effetti tossici causati dal farmaco in prima generazione (nanismo armonico, mortalità), fenomeni di letargia e minore responsività alle fonti luminose nelle due generazioni successive non esposte al farmaco. Le analisi biomolecolari, attualmente in corso di svolgimento da parte di altri ricercatori che partecipano al progetto, mirano ad evidenziare eventuali alterazioni del genoma che giustifichino i diversi effetti finora osservati a livello fenotipico.
L’altra parte del progetto di tesi ha investigato il fenomeno della diffusione dell’antimicrobico resistenza conseguente ai trattamenti di massa in ambito zootecnico, prendendo in esame le deiezioni degli animali ed i terreni agricoli prima e dopo la fertilizzazione con letami/liquami. Lo scopo di tale progetto è stato quello di valutare le modificazioni nella composizione delle comunità microbiche nelle diverse tipologie di campione mediante Next Generation Sequencing, nonché la presenza e l’abbondanza di geni di antimicrobico resistenza nei confronti di fluorochinoloni, macrolidi, polimixine e β-lattamici, utilizzando Real-Time PCR con SYBRGreen e le curve di melting. A tal fine sono stati impiegati saggi pubblicati in letteratura, ma è anche stato necessario sviluppare dei nuovi saggi per l’identificazione e la quantificazione di geni di resistenza alla colistina (mcr-4 e mcr-5) ed ottimizzare e validare 5 saggi di qPCR, quali ermB, blaCTXM1-like, blaCMY-2, qnrA e qnrS.In intensive animal farming, mass medication involves the administration of large quantities of antimicrobial drugs that may be unmetabolized by the animal or metabolized and excreted as active or inactive metabolites. Since the manure from these farms is commonly used for the fertilization of agricultural soil, these treatments may lead to an environmental drug contamination. Subsequently, the residues of active ingredients can be transferred from the soil to surface water through seepage and/or runoff. The environmental consequences of soil fertilization with drug-contaminated manure are a topic of great interest. Indeed, the persistence of antimicrobials in the environment can: 1.represent an ecotoxicological risk for non-target organisms and 2.lead to the emergence and spread of bacterial resistance to antimicrobials (AMR).
For the evaluation of the possible effects in non-target organisms, fluoroquinolones (FQs) were selected; two drugs used in veterinary medicine (enrofloxacin and flumequine) and one used in human medicine (levofloxacin) were employed in root-elongation inhibition tests on three terrestrial plants (Lycopersicon esculentum, Lactuca sativa and Daucus carota). All these dicotyledons showed limited sensitivity to the 3 FQs; for this reason, subsequent studies with the same antibiotics were carried out on the model-organism Daphnia magna. Innovative tests were developed and fine-tuned to highlight delayed toxic effects after embryonic or neonatal exposure. The toxicological interactions of the 3 drugs, in binary and ternary mixtures, were also verified. The results showed that the official tests have some limitations, since they lead to an overestimation of the EC50, and that the mixtures of these compounds show a degree of toxicity that can be explained by additive interactions. Another test was a minor contribution to a project of other researchers studying the biocompatibility and therapeutic efficacy of magnetic nanoparticles engineered with flumequine. This nano-immobilized antibiotic represents a promising tool for “green aquaculture”. The core-shell nanocarrier was tested on a D. magna culture naturally infected with bacteria known to be sensitive to the drug: it showed to be effective and relatively non-toxic. Lastly, other research activities concerning FQs represented the launch of a new research project aimed at evaluating the possible epigenetic effects of flumequine (transgenerational toxicity) on D. magna. Preliminary results showed, in addition to the already known toxic effects caused by the drug to the first generation (dwarfism, mortality), phenomena of lethargy and less responsiveness to light sources, occurring in the next two generations non-exposed to the drug. The molecular analysis, currently undertaken by other researchers participating to the project, aims to highlight any alterations of the genome that may justify the various effects observed at the phenotypic level.
Regarding the potential spread of antimicrobial resistance following the use of antimicrobials in the livestock sector, animal manure, and agricultural soil before and after fertilization, were investigated. The aim was to evaluate the changes in the composition of the microbial communities in the aforementioned types of samples using NGS analysis and assess the presence and abundance of antimicrobial resistance genes (ARGs) against four antimicrobials classes (i.e., fluoroquinolones, macrolides, polymyxins and β-lactams) by employing gene specific SYBRGreen Real-Time PCRs paired with melting curves analysis. To this end, published assays were used. When not available, new assays were designed and developed (i.e., assays for the detection and quantification of colistin resistance genes, mcr-4 and mcr-5) or optimized and validated with the SYBRGreen chemistry (i.e., assays for the detection and quantification of ermB, blaCTXM1-like, blaCMY-2, qnrA and qnrS genes)
Missense single nucleotide variants affecting CYP3A catalytic activity are present in Limousine cattle
Get PDFCytochrome P450 (CYP) 3A is one of the most important subfamily of drug metabolising enzymes. Genetic factors such as breed and allelic variants might modify CYP3A expression and enzyme activity and lead to inter- and intra-individual variability in clinical response or toxicity. CattleCYP3Agene cluster has been recently re-sequenced in 300 Piedmontese cattle by deep targeted sequencing. Thirteen missense single nucleotide variants (SNVs) were identified, and for five of them an impact on CYP3A activity was demonstratedin vitro. In the present work, we assessed the genetic frequency of these five SNVs in Limousine, a cattle breed widely raised for meat production in Veneto Region. A total of 215 cows were genotyped using specific melting curve assays. Only two missense SNVs out of five, both located onCYP3A28coding sequence, were detected:rs384467435 andrs454167819. The former mutant allele was present in homozygosis in the 4% of tested cows, while in heterozygosis in the 24% of animals. The second SNV was detected only in heterozygosis in 11 cows out of 215 (i.e. 4.9%). These findings suggest the presence of missense SNVs, proved to halve CYP3A28 catalytic activity, in both Limousine and Piedmontese meat cattle breeds. As a consequence, these SNVs might impact on the kinetics of xenobiotics metabolised by CYP3A, including drugs and natural toxins like ivermectin and aflatoxins, thereby resulting in toxicity or accumulation of harmful residues in foodstuffs. This result paves the way for new considerations on the fate of xenobiotics in cattle farming
Fenbendazole and triclabendazole effects on CYP1A1/1A2 and FMO1/3 mRNAs in cattle liver slices: preliminary results
No full textBackground. Fenbendazole (FBZ) and triclabendazole (TCBZ) are benzimidazole drugs (BZDs) widely used in veterinary practice as anthelmintics. Members of cytochrome P450 (CYP) and flavin monooxygenase (FMO) superfamilies of drug metabolizing enzymes are primarily responsible of their biotransformation1. The xenobiotic-dependent up-regulation of CYPs is well documented, while FMOs are generally considered not inducible2. In the present study, the effect of FBZ and TCBZ (alone or in combination) on CYP1A1/2 and FMO1/3 mRNA levels were measured on precision-cut bovine liver slices (bLS).
Methods. Precision-cut bLS from 6 male cattle were obtained according to Maté et al3. Following the fine-tuning of an absolute quantification protocol for target genes4, bLS were incubated for 0, 6 and 12 h with FBZ and TCBZ (50 μM), alone or in combination. β-naphthoflavone (βNF, 25 μM) was used as positive control to confirm gene induction. Target gene mRNA levels were measured by qPCR. The TATA Box binding protein and ribosomal protein lateral stalk subunit P0/β-actin were used as internal control genes to normalize FBZ/TCBZ and βNF data, respectively.
Results. At T0, CYP1A2 mRNA levels were 25-fold higher than CYP1A1; however, FMO1 and 3 were equally represented. βNF up-regulated CYP1A1 (P<0.05; 4-fold vs control) after 6 h of incubation, while increasing amounts (P<0.05) of CYP1A2, FMO1/3 mRNAs were noticed after 12 h (4-, 2.5- and 3.5-fold vs control, respectively). Concerning BZDs, FBZ increased CYP1A1/2 mRNAs (P<0.05) after 12 h of incubation, whereas TCBZ up-regulated only FMO3 (P<0.05) and after 6 h. No transcriptional effect was ever noticed following bLS exposure to BZDs combination.
Conclusion. Likewise to humans, bLS appear to be a reliable model to study CYP1A induction by NF and other potential aryl hydrocarbon receptor agonists. Meanwhile, for the first time we proved cattle FMO1-3 up-regulation by NF. About BZDs, FBZ and TCBZ were shown to affect CYP1A1/2 and FMO3 gene expression. Confirmatory studies on CYP1A and FMOs catalytic activities are actually underway
Functional impact of cytochrome P450 3A (CYP3A) exonic polymorphisms in cattle
No full textBackground. The human cytochrome P450 3A (hCYP3A) subfamily is the most important subfamily involved in human xenobiotic metabolism, on account of its major role in the metabolism of ~30% of clinically used drugs1. Current increasing evidences show that hCYP3A4 and hCYP3A5 genetic variants significantly contribute to inter-individual variability in drug metabolism2. No information are actually available for bovine CYP3A (bCYP3A), despite the need of improving the safe use of veterinary drugs and, in the meantime, avoid the presence of harmful xenobiotic residues in cattle food products. Within this scenario, the aim of this study was to identify the exonic variants of bCYP3A subfamily members known so far3 in Piedmontese cattle breed, and evaluate their potential functional impact in terms of catalytic activity towards marker substrates.
Methods. bCYP3A28, 3A38 and 3A48 exonic variants were identified, through a targeted Illumina sequencing approach, on 16 DNA pools obtained from a total of 300 Piedmontese beef cattle liver samples. Then, each individual genotype was determined using the melting curve genotyping analysis.
Afterwards, wild type and mutated sequences were cloned and heterologously expressed in V79 cells together with CYP oxido-reductase. Their metabolite profiling, following the incubation with two CYP3A marker substrates, i.e. testosterone (TST) and nifedipine (NIF), was assessed by using mass spectrometry. To appraise the functional impact of aforementioned variants in the Piedmontese cattle population, the individual genotype was finally combined with the corresponding CYP3A-dependent catalytic activity (TST hydroxylation) in liver subcellular fractions4.
Results. Thirteen exonic variants were identified in bCYP3A cluster (3, 3 and 7 for bCYP3A28, 3A38 and 3A48, respectively) and validated using HybProbe probes. Five variants out of 13 showed differences in enzyme activity. In particular, all bCYP3A28 variants significantly reduced (-50%) the production of 6β-OH TST; however, one variant of bCYP3A38 and bCYP3A48 increased ~3-fold 16β-OH TST hydroxylation and NIF oxidation, respectively. In Piedmontese cattle breed population, individuals homozygous for rs384467435 SNP (one of identified bCYP3A28 variants), showed a reduced pattern of TST 6β-hydroxylation in liver subcellular fractions.
Conclusion. Overall, these findings suggest that variation in bCYP3A genotype might have a functional relevance, providing evidence of the possible consequences either on animal (kinetics, clinical efficacy/side effects and toxicity) than on human (presence of harmful residues) health
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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