Delayed toxicity of three fluoroquinolones and their mixtures after neonatal or embryonic exposure, in Daphnia magna

Fluoroquinolones (FQs) are antibacterial drugs, used both in human and veterinary medicine, that are currently considered as emerging micropollutants. This study investigated the delayed toxic effects of enrofloxacin (ENR), flumequine (FLU), levofloxacin (LEV) and their binary mixtures in D. magna. For this purpose, a 10-day follow-up in pure medium was added to the standard D. magna immobilization test. During this follow-up, phenotypic alterations were evidenced, which were related to scarce or zeroed egg production and early mortality. Consequently, the EC50 s recalculated at the end of the follow-up were always remarkably lower than those obtained after the 48 h immobilization test: ENR 3.13 vs. 16.72 mg L 121; FLU 7.18 vs. 25.35 mg L 121; LEV 15.11 vs. > 40 mg L 121. To analyse the possible interactions within the binary mixtures, the method of nonlinear additive isoboles was applied. The three compounds showed invariably to follow the principle of concentration addition. Furthermore, as previous experiments showed toxicity of FLU and ENR after embryonic exposure of D. magna at a concentration of 2 mg L 121, an additional two embryonic tests were conducted with identical design: one with 2 mg L 121 LEV and the other with a ternary mixture containing 0.66 mg L 121 of each of the three FQs. The embryos were exposed for three days in vitro to the drug solutions and were then reconducted to pure medium for 21 days observation. Both the tests ended-up with only non-significant effects on growth and reproduction, confirming the lower toxicity of LEV, when compared to ENR and FLU, and the absence of any evident synergistic interaction among the three FQs. Overall, these studies have shown two relevant features related to the toxicity of the three FQs: (1) they give rise to delayed toxic effects in D. magna that are undetectable by the standard immobilization test; (2) their interaction in mixtures follow the principle of Concentration Addition. Both these indications concern the Environmental Risk Assessment of FQs and may be of interest to regulatory authorities

Detection by real-time PCR of colistin resistance genes (mcr-1 to mcr-3) in animal manure and agricultural soil in Northern Italy

Introduction: The presence of antimicrobials, such as colistin, in animal manure may promote the dissemination of plasmid-mediated colistin resistance (mcr genes) into the environment. Objectives: To assess resistance to colistin mediated by mcr genes in soil fertilized with manure from intensive animal farms. Aim: To provide qualitative data for mcr-1 to mcr-3 genes in manure and soil using a real-time PCR approach. Methods: A total of 84 samples (1 sample of manure/slurry; 1 sample of soil before fertilization, T0; and 1 sample of soil about 30 days after fertilization, T30 per farm) collected from dairy (n = 11), swine (n = 10) and chicken (n = 7) farms were analysed by real-time PCR. Results: Mcr-1, mcr-2 and mcr-3 genes were detected in all three farms and samples. In swine farms, all 3 mcr genes (i.e. 5 slurry, 2 T0 and 3 T30 for mcr-1; 3 slurry for mcr-2; 3 slurry, 2 T0 and 2 T30 for mcr-3) were detected. In chicken and dairy farms, only mcr-1 (i.e. 3 manure in dairy, and 3 T0 and 1 T30 in chicken) and mcr-3 genes (i.e. 2 manure in dairy, and 1 manure and 1 T0 in chicken) were found. Mcr-1 was the most prevalent gene (20%), followed by mcr-3 (13%) and mcr-2 (3.5%). Conclusions: The presence of mcr genes into the environment, following fertilization with animal manure, may be of concern for public health since colistin is considered as last therapeutic option for treating severe human infections

Colistin resistance genes (mcr-1 to mcr-5) in agricultural soil fertilized with animal manure in Northern Italy

Fertilization with animal manure is a common agricultural practice in Italy. Waste products from intensive animal farming, such as cattle, swine and poultry farms, are the most frequently utilized. However, manure may contain residues of antimicrobials used during rearing that may have a negative impact on the environment, including the dissemination of antimicrobial resistance genes [1, 2]. Colistin is an antimicrobial commonly used in animals, and recent studies showed that resistance can be induced by plasmid-mediated mcr genes [3]. The presence of antimicrobials, such as colistin, in animal manure used as fertilizer of agricultural soil may promote the dissemination of mcr genes into the environment. This study aimed at investigating the presence of colistin residues and mcr-1 to mcr-5 genes in agricultural soil before and after fertilization with manure/slurry from intensive animal (i.e. dairy, swine and chicken) farms in Northern Italy. A total of 84 samples (1 sample of manure/slurry per farm; 1 sample of soil before fertilization, T0, per farm; and 1 sample of soil about 30 days after fertilization, T30, per farm) were collected from dairy (n = 11), swine (n = 10) and chicken (n = 7) farms. All samples were analysed by HPLC-MS/MS and by SYBR\uae Green Real-Time PCR for the detection of colistin and mcr-1 to mcr-5 genes, respectively. None of the samples contained colistin residues, whereas all the mcr genes were found. In swine farms, all 5 mcr genes (i.e. 5 slurry, 2 T0 and 3 T30 samples for mcr-1; 3 slurry samples for mcr-2; 3 slurry, 2 T0 and 2 T30 samples for mcr-3; 2 slurry and 1 T30 samples for mcr-4; 1 slurry and 1 T30 samples for mcr-5) were detected. In chicken and dairy farms, mcr-1 (i.e. 3 manure samples in dairy, and 3 T0 and 1 T30 samples in chicken), mcr-3 (i.e. 2 manure in dairy samples, and 1 manure and 1 T0 samples in chicken), mcr-4 (i.e. 1 manure sample in dairy) and mcr-5 (i.e. 1 T0 sample in dairy) genes were found. Overall, mcr-1 was the prevalent gene (20%), followed by mcr-3 (13%), mcr-4 (4.8%), mcr-5 (3.6%), and mcr-2 (3.5%). This study showed that all the mcr genes were present in all types of animal (i.e. dairy, swine and chicken) manure and related fertilized soils regardless of the presence of colistin residues. Furthermore, a higher prevalence and diversity of mcr genes was found in swine manure and related fertilized soils compared to those from dairy and chicken farms. In conclusion, the presence of plasmid-mediated mcr genes both in manure and in agricultural soil is concerning for environmental and public health and further research is needed to understand their ecology into the environment

Protective effects of quercetin towards aflatoxin B1-induced hepatotoxicity in cattle: a whole transcriptomic in vitro study

Introduction: Aflatoxin B1 (AFB1) is a natural feed and food contaminant. In the dairy industry, AFB1 and its derivatives are of concern for the related economic losses and their possible presence in milk and dairy food products. Oxidative stress is one of the prominent toxic effects of AFB1, thus, dietary supplementation with natural antioxidants is a valid strategy to mitigate AFB1 toxicity. In this respect, quercetin (QUE) is a promising bioactive compound. Here we assessed the protective role of QUE in bovine foetal hepatocyte-derived cells (BFH12) exposed to AFB1, by measuring the QUE impact on AFB1-induced cytotoxicity and related whole- transcriptional changes. Methods: to increase cells responsiveness to AFB1, monolayers were pre-treated with 1 nM PCB126 for 24h and then exposed to 3.6 μM AFB1, alone or in combination with sub-cytotoxic concentrations of QUE (10, 20, 30 μM). Cytotoxicity was then assessed. For the whole-transcriptome study, we exposed cells to 30 μM QUE, 3.6 μM AFB1, or their combination. Cells pre-treated with PCB126 and exposed to DMSO were used as control (CTRL). Total RNA was isolated, 12 tagged RNA-seq libraries were prepared and sequenced (50 bp single-end). Reads were quality checked, trimmed and mapped to cow reference genome using validated pipelines. EdgeR and clusterProfiler were used to identify differentially expressed genes (DEGs) and perform the functional enrichment analysis, respectively. Results: 30 μM QUE significantly reduced AFB1-induced cytotoxicity by 13.39%. As to RNA-seq, the differential expression analysis comparing QUE vs CTRL and QUE+AFB1 vs AFB1 identified 1028 and 1890 DEGs, respectively. QUE alone affected the expression of genes related to steroids metabolism, inflammation, immunity, and P450-mediated drug metabolism. When comparing QUE+AFB1 vs AFB1, top upregulated genes possess antiapoptotic and antioxidant activity (e.g. SEMA5A, TF), while top downregulated genes play a role in inflammation and cancer progression (e.g. CXCL5, IL6). If comparing these results with the mRNA changes induced in cells exposed to AFB1 alone, all the top-10 genes upregulated by the cotreatment were inhibited by AFB1; on the opposite, all the top-10 downregulated genes were induced by AFB1 alone. Conclusions: we showed the QUE potential to mitigate AFB1-induced toxicity and the underneath molecular mechanisms. If the beneficial effects will be confirmed in vivo, QUE-supplemented diets might be adopted to improve health status of cattle exposed to AFB1-contaminated feed

Colistin resistance genes (mer-1 to mer-5) in agricultural soil fertilized with animal manure in Northern Italy

Fertilization with animal manure is a common agricultural practice in Italy. Waste products from intensive animal farming, such as cattle, swine and poultry farms, are the most frequently utilized. However, manure may contain residues of antimicrobials used during rearing that may have a negative impact on the environment, including the dissemination of antimicrobial resistance genes [1, 2]. Colistin is an antimicrobial commonly used in animals, and recent studies showed that resistance can be induced by plasmid-mediated mcr genes [3]. The presence of antimicrobials, such as colistin, in animal manure used as fertilizer of agricultural soil may promote the dissemination of mcr genes into the environment. This study aimed at investigating the presence of colistin residues and mer-1 to mcer-5 genes in agricultural soil before and after fertilization with manure/slurry from intensive animal (i.e. dairy, swine and chicken) farms in Northern Italy. A total of 84 samples (1 sample of manure/slurry per farm; 1 sample of soil before fertilization, TO, per farm; and 1 sample of soil about 30 days after fertilization, T30, per farm) were collected from dairy (n = 11), swine (n = 10) and chicken (n = 7) farms. All samples were analysed by HPLC- MS/MS and by SYBR\uae Green Real-Time PCR for the detection of colistin and mer-1 to mer-5 genes, respectively. None of the samples contained colistin residues, whereas all the mcr genes were found. In swine farms, all 5 mer genes (i.e. 5 slurry, 2 TO and 3 T30 samples for mer-1; 3 slurry samples for mer-2; 3 slurry, 2 TO and 2 T30 samples for mer-3; 2 slurry and 1 T30 samples for mcr-4; 1 slurry and 1 T30 samples for mcr-5) were detected. In chicken and dairy farms, mer- 1 (i.e. 3 manure samples in dairy,-and 3 TO and 1 T30 samples in chicken), mcr-3 (i.e. 2 manure in dairy samples, and 1 manure and 1 TO samples in chicken), mcr-4 (i.e. 1 manure sample in dairy) and mcr-S (i.e. 1 TO sample in dairy) genes were found. Overall, mcr-1 was the prevalent gene (20%), followed by mcer-3 (13%), mer-4 (4.8%), mer-5 (3.6%), and mer-2 (3.5%). This study showed that all the mcr genes were present in all types of animal (i.e. dairy, swine and chicken) manure and related fertilized soils regardless of the presence of colistin residues. Furthermore, a higher prevalence and diversity of mer genes was found in swine manure and related fertilized soils compared to those from dairy and chicken farms. In conclusion, the presence of plasmid- mediated mcr genes both in manure and in agricultural soil is concerning for environmental and public health and further research is needed to understand their ecology into the environment

Rapid detection and quantification of plasmid-mediated colistin resistance genes (mcr-1 to mcr-5) by real-time PCR in bacterial and environmental samples

Aim: The aim of the study was to validate a rapid method to detect and quantify colistin resistance genes (mcr-1 to mcr-5) by real-time polymerase chain reaction (RT-PCR) in diverse matrices. Methods and results: The detection limit of two newly designed SYBR Green real-time PCR assays for mcr-4 and mcr-5 and of previously published protocols for mcr-1 to mcr-3 was assessed using serial dilutions of reference strains. The assays could detect all five mcr genes with the lower limit of 102 copy numbers. Escherichia coli isolates (n\ua0=\ua01062) and environmental samples (n\ua0=\ua093) were tested for the presence of mcr genes. The assays enabled the detection of colistin resistance genes both in bacterial isolates and in complex environmental samples. Conclusions: This method represents a set of sensitive, rapid and effective assays for the screening of colistin resistance directly from the environment. Significance and Impact of the Study: Colistin is an antimicrobial commonly used in animals and has recently emerged as a last-resort treatment in humans. Plasmid-mediated mcr genes confer resistance to colistin and represent a major threat for public health since they can be easily disseminated through horizontal gene transfer. The rapid and sensitive detection of mcr genes is of utmost necessity

Insights into Aflatoxin B1 Toxicity in Cattle: An in vitro whole-transcriptomic approach

Aflatoxins, and particularly aflatoxin B1 (AFB1), are toxic mycotoxins to humans and farm animal species, resulting in acute and chronic toxicities. At present, AFB1 is still considered a global concern with negative impacts on health, the economy, and social life. In farm animals, exposure to AFB1-contaminated feed may cause several untoward effects, liver damage being one of the most devastating ones. In the present study, we assessed in vitro the transcriptional changes caused by AFB1 in a bovine fetal hepatocyte-derived cell line (BFH12). To boost the cellular response to AFB1, cells were pre-treated with the co-planar PCB 3,3 ,4,4 ,5-pentachlorobiphenyl (PCB126), a known aryl hydrocarbon receptor agonist. Three experimental groups were considered: cells exposed to the vehicle only, to PCB126, and to PCB126 and AFB1. A total of nine RNA-seq libraries (three replicates/group) were constructed and sequenced. The differential expression analysis showed that PCB126 induced only small transcriptional changes. On the contrary, AFB1 deeply affected the cell transcriptome, the majority of significant genes being associated with cancer, cellular damage and apoptosis, inflammation, bioactivation, and detoxification pathways. Investigating mRNA perturbations induced by AFB1 in cattle BFH12 cells will help us to better understand AFB1 toxicodynamics in this susceptible and economically important food-producing species

Microbial communities in agricultural soil fertilized with animal manure in Northern Italy

Soil is one of the largest reservoir of microbial diversity. Land application of chicken and swine manure is a common agricultural practice worldwide, including Italy. Some studies suggest that livestock manure used as fertilizer in agriculture may cause changes of microbial communities. However, little is known about the impact of manure fertilization on soil microbiomes and potential consequences on the natural environment. This study aimed at investigating the possible influence of fertilization with manure from intensive swine and chicken farms on agricultural soil microbiome. Sixty samples of manure and soil (before manure application and 30 days after) were collected from ten chicken farms and ten swine farms. The microbiome was determined by NGS sequencing employing a multiplexing 16S rRNA gene amplicon sequencing approach based on two-step PCR amplifications with tagged primers. Libraries were pooled and sequenced with Miseq Illumina 2X300 Pair Ends. Raw sequence data were analyzed with QIIME 2, while Silva database and Calypso software were employed for taxonomical and statistical analyses, respectively. Microbial diversity in each sample (\u3b1-diversity) was evaluated: at phylum level, Firmicutes and Bacteroidetes dominated manure microbiome, while Proteobacteria were the most abundant in soil samples. At order level, Clostridiales, Bacteroidales and Bacillales were the most abundant in manure samples, while in soil samples the most abundant order was Chitinophagales. At family level, bacterial families such as Lachnospiraceae, Ruminococcaceae and Rikenellaceae dominated the manure microbiome, whereas Chitinophagaceae were most abundant in soil samples. Manure and soil samples (before and after fertilization) clustered at phylum, order and family levels within two distinct groups. The assessment of the microbial diversity between samples (\u3b2-diversity) confirmed the uniqueness of microbial communities of manure and soil suggesting that manure application may not significantly alter soil microbiome. However, some differences were observed: Enterococcaceae and Lactobacillaceae were more abundant in manure and soil samples after fertilization than in soil samples before manure application. Similarly, Burkholderiaceae, Sphingobacteriaceae, Flavobacteriaceae and in particular Bacillaceae were more abundant in chicken manure and some soil samples after fertilization than before fertilization. This represents one of the first study evaluating the effects of swine and chicken manure fertilization on the agricultural soil microbiome in Italy. Future studies aiming to assess the presence of antimicrobial residues and resistance genes in these samples would be useful to obtain key information regarding the impact of antimicrobial use in intensive animal farming on soil microbial communities

Changes in chicken gut microbiota and antimicrobial resistance genes after amoxicillin and thiamphenicol administration

The aim of this study was to assess the dynamics of microbial communities and antimicrobial resistance genes (ARGs) in the chicken gut following amoxicillin and thiamphenicol treatments and potential co-selection of ARGs. To this purpose, the microbial community composition, using 16S rRNA NGS, and the abundance of ARGs conferring resistance to β-lactams and phenicols, using qPCRs, were determined. Results revealed that the administered antimicrobials did not significantly reduce the gut microbiota diversity, but changed its composition, with taxa (e.g. Gallibacterium and Megamonas) being enriched after treatment and replacing other bacteria (e.g. Streptococcus and Bifidobacterium). Positive correlations were found between ARGs (e.g. cmlA, blaCMY-2, and blaSHV) and the relative abundance of specific taxa (e.g. Lactobacillus and Subdoligranulum). The selective pressure exerted by both amoxicillin and thiamphenicol resulted in an increased abundance of ARGs conferring resistance to β-lactams (e.g. blaTEM-1, blaSHV, and blaCTX-M1-like) and phenicols (e.g. floR and cmlA). These findings, together with the co-occurrence of genes conferring resistance to the two antimicrobial classes (e.g. blaTEM-1 and cmlA), suggest a possible interaction among antimicrobials on resistance emergence, possibly due to the presence of mobile genetic elements (MGEs) carrying multiple resistance determinants