Rabbits as a Reservoir of Multidrug-Resistant Escherichia coli: Clonal Lineages and Public Health Impact.

Escherichia coli, including extended-spectrum ß-lactamases (ESBL)-producing strains, poses a global health threat due to multidrug resistance, compromising food safety and environmental integrity. In industrial settings, rabbits raised for meat have the highest consumption of antimicrobial agents compared to other food-producing animals. The European Union is facing challenges in rabbit farming as rabbit consumption declines and antibiotic-resistant strains of E. coli cause enteric diseases. The aim of this study was to investigate the antibiotic resistance profile, genetic diversity, and biofilm formation in cefotaxime-resistant E. coli strains isolated from twenty rabbit farms in Northern Portugal to address the effect of the pressing issue of antibiotic resistance in the rabbit farming industry. Resistance to critically antibiotics was observed, with high levels of resistance to several categories, such as tetracycline, ampicillin, aztreonam, and streptomycin. However, all isolates were susceptible to cefoxitin and imipenem. Multidrug resistance was common, with strains showing resistance to all antibiotics tested. The blaCTX-M variants (blaCTX-3G and blaCTX-M9), followed by the tetracycline resistance genes, were the most frequent resistance genes found. ST10 clones exhibiting significant resistance to various categories of antibiotics and harboring different resistance genes were detected. ST457 and ST2325 were important sequence types due to their association with ESBL-E. coli isolates and have been widely distributed in a variety of environments and host species. The strains evaluated showed a high capacity for biofilm formation, which varied when they were grouped by the number of classes of antibiotics to which they showed resistance (i.e., seven different classes of antibiotics, six classes of antibiotics, and three/four/five classes of antibiotics). The One Health approach integrates efforts to combat antimicrobial resistance in rabbit farming through interdisciplinary collaboration of human, animal, and environmental health. Our findings are worrisome and raise concerns. The extensive usage of antibiotics in rabbit farming emphasizes the urgent need to establish active surveillance systems.

Unveiling Antibiotic Resistance, Clonal Diversity, and Biofilm Formation in E. coli Isolated from Healthy Swine in Portugal.

Escherichia coli, a commensal microorganism found in the gastrointestinal tract of human and animal hosts, plays a central role in agriculture and public health. Global demand for animal products has promoted increased pig farming, leading to growing concerns about the prevalence of antibiotic-resistant E. coli strains in swine populations. It should be noted that a significant portion of antibiotics deployed in swine management belong to the critically important antibiotics (CIA) class, which should be reserved for human therapeutic applications. This study aimed to characterize the prevalence of antibiotic resistance, genetic diversity, virulence characteristics, and biofilm formation of E. coli strains in healthy pigs from various farms across central Portugal. Our study revealed high levels of antibiotic resistance, with resistance to tetracycline, ampicillin, tobramycin, and trimethoprim-sulfamethoxazole. Multidrug resistance is widespread, with some strains resistant to seven different antibiotics. The ampC gene, responsible for broad-spectrum resistance to cephalosporins and ampicillin, was widespread, as were genes associated with resistance to sulfonamide and beta-lactam antibiotics. The presence of high-risk clones, such as ST10, ST101, and ST48, are a concern due to their increased virulence and multidrug resistance profiles. Regarding biofilm formation, it was observed that biofilm-forming capacity varied significantly across different compartments within pig farming environments. In conclusion, our study highlights the urgent need for surveillance and implementation of antibiotic management measures in the swine sector. These measures are essential to protect public health, ensure animal welfare, and support the swine industry in the face of the growing global demand for animal products.

Antimicrobial Resistance and Clonal Lineages of Escherichia coli from Food-Producing Animals.

Escherichia coli are one of the most important pathogenic bacteria readily found in the livestock and widely studied as an indicator that carries drug-resistant genes between humans, animals, and the environment. The use of antimicrobials in the food chain, particularly in food-producing animals, is recognized as a significant contributor to the development and spread of antimicrobial resistance (AMR) and resistance genes can be transferred from the farm through the food-chain. The objective of this review is to highlight the background of the antimicrobials use in food-producing animals, more specifically, to study clonal lineages and the resistance profiles observed in E. coli, as well as in extended spectrum beta-lactamases (ESBL) producing E. coli, in a set of food-production animals with greater relevance in food consumption, such as pigs, poultry, cattle, fish farming and rabbits. Regarding the prevalence of ESBL-producing E. coli among farm animals, high-to-moderate prevalence was observed, and the highest resistance rates to tetracycline and ampicillin was detected in different farms in all geographic regions. Worldwide pandemic clones and high-risk zoonotic E. coli clones have been identified in most food-producing animals, and some of these clones are already disseminated in different niches, such as the environment and humans. A better understanding of the epidemiology of E. coli and ESBL-producing E. coli in livestock is urgently needed. Animal production is one of the major causes of the antibiotic resistance problem worldwide and a One Health approach is needed.

Grapevine Diversity and Genetic Relationships in Northeast Portugal Old Vineyards.

More than 100 grapevine varieties are registered as suitable for wine production in "Douro" and "Trás-os-Montes" Protected Designations of Origin regions; however, only a few are actually used for winemaking. The identification of varieties cultivated in past times can be an important step to take advantage of all the potential of these regions grape biodiversity. The conservation of the vanishing genetic resources boosts greater product diversification, and it can be considered strategic in the valorisation of these wine regions. Hence, one goal of the present study was to prospect and characterise, through molecular markers, 310 plants of 11 old vineyards that constitute a broad representation of the grape genetic patrimony of "Douro" and "Trás-os-Montes" wine regions; 280 samples, grouped into 52 distinct known varieties, were identified through comparison of their genetic profiles generated via 6 nuclear SSR and 43 informative SNP loci amplification; the remaining 30 samples, accounting for 13 different genotypes, did not match with any profile in the consulted databases and were considered as new genotypes. This study also aimed at evaluating the population structure among the 65 non-redundant genotypes identified, which were grouped into two ancestral genetic groups. The mean probability of identity values of 0.072 and 0.510 (for the 6 SSR and 226 SNP sets, respectively) were determined. Minor differences were observed between frequencies of chlorotypes A and D within the non-redundant genotypes studied. Twenty-seven pedigrees were confirmed and nine new trios were established. Ancestors of eight genotypes remain unknown.

Valorization of Winemaking By-Products as a Novel Source of Antibacterial Properties: New Strategies to Fight Antibiotic Resistance.

The emergence of antibiotic-resistance in bacteria has limited the ability to treat bacterial infections, besides increasing their morbidity and mortality at the global scale. The need for alternative solutions to deal with this problem is urgent and has brought about a renewed interest in natural products as sources of potential antimicrobials. The wine industry is responsible for the production of vast amounts of waste and by-products, with associated environmental problems. These residues are rich in bioactive secondary metabolites, especially phenolic compounds. Some phenolics are bacteriostatic/bactericidal against several pathogenic bacteria and may have a synergistic action towards antibiotics, mitigating or reverting bacterial resistance to these drugs. Complex phenolic mixtures, such as those present in winemaking residues (pomace, skins, stalks, leaves, and especially seeds), are even more effective as antimicrobials and could be used in combined therapy, thereby contributing to management of the antibiotic resistance crisis. This review focuses on the potentialities of winemaking by-products, their extracts, and constituents as chemotherapeutic antibacterial agents.

Silicates of Potassium and Aluminium (Kaolin); Comparative Foliar Mitigation Treatments and Biochemical Insight on Grape Berry Quality in Vitis vinifera L. (cv. Touriga National and Touriga Franca).

Grapevine physiology is influenced by several environmental factors, such as temperature, precipitation, potential evapotranspiration, and sunshine hours. Due to climatic changes, effects in grapevine physiology and consequently on the grape berry composition and quality have been observed. This work aims to make a comparative study of the effect of foliar mitigation treatment with kaolin (5%) and potassium silicates (0.1% and 0.05%) on the grape berry quality; namely on berry weight, pH, probable alcohol, total phenolics, tannins, total anthocyanins, monomeric anthocyanins, calcium, potassium, and magnesium composition from Portuguese grapevines (Vitis vinifera L. cv. Touriga Nacional and Touriga Franca). The results suggested that the phenolic composition and anthocyanin content differs between treatments while other parameters showed distinct behavior among the different applications. Qualitative parameters observed in the present study suggested non-significant changes upon both the applications.

Comparative Insight upon Chitosan Solution and Chitosan Nanoparticles Application on the Phenolic Content, Antioxidant and Antimicrobial Activities of Individual Grape Components of Sousão Variety.

Chitosan, a natural polysaccharide, has been previously proposed as an elicitor in plants to prevent pathogen infections. The present study aimed to analyze the effect of chitosan solution and chitosan nanoparticles treatment applied on the grapevine variety Sousão with respect to the phenolic composition, antioxidant potential and antibacterial activity of its individual grape components. Grapevine plants of selected lines were sprayed with chitosan solution and chitosan nanoparticles, and ethanolic extracts of stems, seeds and skins were prepared from grapevines treated and not treated with chitosan. Total phenolic, anthocyanin and tannin contents were studied, and the identification of the individual phenolic compounds was performed by HPLC-DAD. The antimicrobial susceptibility method was performed using the Kirby-Bauer disc diffusion method against multidrug-resistant bacteria. Overall, there was small increase in the concentration of phenolic compounds, antioxidant and antimicrobial activities in grape components treated with chitosan solution. Seed extracts showed the highest antioxidant and antimicrobial activities. The studied individual components obtained from chitosan-treated grapevines could represent an added value due to the increased antioxidant and antibacterial potentials. The phenolic compounds found in components may be used in food and pharmaceutical industries as natural food preservers and antibiotic adjuvants.

Chitosan Application in Vineyards (Vitis vinifera L. cv. Tinto Cão) Induces Accumulation of Anthocyanins and Other Phenolics in Berries, Mediated by Modifications in the Transcription of Secondary Metabolism Genes.

Despite the numerous beneficial properties and uses of chitosan in agriculture, the molecular mechanisms behind its elicitation potential are still unclear. This study aimed at understanding the effect of chitosan application in the levels of phenolic compounds of Vitis vinifera L. red grapes berry skin (cv. Tinto Cão) during veraison. Grapevines were treated with chitosan (0.01% in 0.01% acetic acid) while control grapevines were sprayed with 0.01% acetic acid. Results showed that several monomeric anthocyanins increased significantly in berry skins after treatment with chitosan. Additionally, Catechin, Rutin and Querecetin-3-O-galactoside were also recorded in higher amount upon chitosan treatment. Besides modulating the phenolic content, chitosan treatment also induced modifications in several target genes encoding key enzymes and transporters involved in secondary metabolic pathways. For instance, the genes PAL, CHS, F3H, ANR, UFGT, ABCC1, GST, MATE1 were upregulated in leaves and berry skins at veraison cessation in response to chitosan treatment. Overall, the results demonstrated that chitosan has a stimulatory effect on the accumulation of phenolic compounds, including anthocyanins, mediated by modifications in the transcription of key genes involved in their biosynthesis and transport in grape berries.

Evaluation of the Phenolic Profile of Castanea sativa Mill. By-Products and Their Antioxidant and Antimicrobial Activity against Multiresistant Bacteria.

The chestnut industry generates a large amount of by-products. These agro-industrial wastes have been described as potential sources of phenolic compounds with high bioactive potential. Therefore, we aimed to extract the phenolic compounds from chestnut by-products and assess their antioxidant potential and evaluate their antimicrobial activity against multidrug resistant bacteria. The individual phenolic compounds in the ethanolic extracts of chestnut shell, inner shell, bur, and leaves were characterized by HPLC-DAD/electrospray ionization (ESI)-MS. The antioxidant properties were determined by DPPH and ABTS assays. The minimum inhibitory concentration (MIC) and the antimicrobial susceptibility was performed using the Kirby-Bauer disc diffusion method against 10 bacterial strains. The major phenolic compounds identified in the extracts were trigalloyl-HHDP-glucose, gallic acid, quercetin, and myricetin glycoside derivatives. All chestnut by-products presented promising antioxidant activity in both assays, with leaf samples the ones presenting the highest antioxidant capacity. The inner shell's extract was effective against all Gram-positive and two Gram-negative bacteria; nevertheless, all extracts showed antibacterial activity. Staphylococcus epidermidis showed susceptibility to all extracts while none of the extracts was able to suppress the growth of Escherichia coli and Salmonella enteritidis. Chestnut by-products are a source of phenolic compounds with prominent antioxidant and antimicrobial activities. Nevertheless, further studies should be conducted to assess the correlation between phenolic compounds and the bioactivities obtained.

Chitosan Upregulates the Genes of the ROS Pathway and Enhances the Antioxidant Potential of Grape (Vitis vinifera L. 'Touriga Franca' and 'Tinto Cão') Tissues.

Chitosan is an environmentally-friendly active molecule that has been explored for numerous agricultural uses. Its use in crop protection is well-known, however, other properties, such as bioactivity, deserve attention. Moreover, the modes of actions of chitosan remain to be elucidated. The present study assessed the levels of total phenolic compounds, the antioxidant potential, and the expression of reactive oxygen species (ROS) scavenging genes in the berries (skins and seeds), leaves, cluster stems, and shoots upon chitosan application on two red grapevine varieties (Touriga Franca and Tinto Cão). The application of chitosan on the whole vine before and after veraison led to the increased levels of polyphenols, anthocyanins, and tannins in Tinto Cão berries, and polyphenols and tannins in Touriga Franca berries, respectively. CUPric Reducing Antioxidant Capacity (CUPRAC) and Ferric Reducing Antioxidant Power (FRAP) assays indicated an increase in the antioxidant potential of berries. With the exception of ascorbate peroxidase (APX), all the ROS pathway genes tested, i.e., iron-superoxide dismutase (Fe-SOD), copper-zinc-superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione reductase (GR), glutaredoxin (Grx), respiratory burst oxidase (Rboh), amine oxidase (AO), peroxidase (POD) and polyphenol oxidase (PPO), were found up-regulated in chitosan-treated berries. Results from the analyses of leaves, stems, and shoots revealed that chitosan not only induced the synthesis of phenolic compounds but also acted as a facilitator for the transfer of polyphenols from the leaves to the berries.

Influence of Food Characteristics and Food Additives on the Antimicrobial Effect of Garlic and Oregano Essential Oils.

Utilization of essential oils (EOs) as antimicrobial agents against foodborne disease has gained importance, for their use as natural preservatives. Since potential interactions between EOs and food characteristics may affect their antimicrobial properties, the present work studies the influence of fat, protein, pH, aw and food additives on the antimicrobial effect of oregano and garlic EOs against Salmonella spp. and Listeria monocytogenes. Results showed that protein, pH, aw, presence of beef extract, sodium lactate and nitrates did not influence their antimicrobial effect. In contrast, the presence of pork fat had a negative effect against both EOs associated with their dilution of the lipid content. The addition of food phosphates also exerts a negative effect against EOs probably associated with their emulsification properties as observed with the addition of fat. The results may help the food industry to select more appropriate challenges to guarantee the food safety of foodstuffs.

Spontaneous variation regarding grape berry skin color: A comprehensive study of berry development by means of biochemical and molecular markers.

Understanding grape berry development and the metabolism of different classes of compounds responsible for traits like berry's color is imperative to control and improve quality aspects of grapes. A colorimetric, biochemical and molecular characterization allowed the comprehensive description of the pigment-related characteristics of nine berry skin color somatic variants, belonging to four different varieties. Although the observed berry skin color variability was not fully explained by MybA locus, the phenolic profiles allowed inferring about specific interferences among the biosynthetic pathways. Data were consistent concerning that grapes showing cyanidin-3-O-glucoside as the major anthocyanin and flavonols with two substituent groups in the lateral B-ring are generally originated by a white ancestor. After retro-mutation, these grapes seem to keep the dysfunction on flavonoid hydroxylases enzymes, which negatively affect the synthesis of both flavonols and anthocyanins with three substituent groups in the lateral B-ring. Overall, the obtained results indicate that the color differences observed between somatic variants are not solely the result of the total amount of compounds synthesized, but rather reflect a different dynamics of the phenolic pathway among the different color variants of the same variety. CHEMICAL COMPOUNDS: Gallic acid (PubChem CID: 370); Caftaric acid (PubChem CID: 6,440,397); Catechin (PubChem CID: 73,160); Epigallocatechin gallate (PubChem CID: 65,064); Quercetin-3-O-galactoside (PubChem CID: 5,281,643); Quercetin-3-O-glucoside (PubChem CID: 25,203,368); Malvidin-3-O-glucoside (PubChem CID: 443,652); Peonidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,849); Malvidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,988); Resveratrol-3-O-glucoside (PubChem CID: 25,579,167).

Synergistic Activity of Essential Oils from Herbs and Spices Used on Meat Products against Food Borne Pathogens.

Essential oils (EOs) could be utilized as natural agents to improve the safety of meat products. However, the high concentration required to achieve an antimicrobial effect in foods might be incompatible with their sensory acceptance. To avoid this problem, combinations of EOs provide an effective approach reducing the odds of sensory rejection. In our study, 13 EOs of herbs and spices commonly used in the seasoning of meat products were assessed for their antimicrobial activity against Salmonella spp., Listeria monocytogenes, Escherichia coli and Staphylococcus aureus. However, only 7 of them were selected to study their synergistic effect based on their antimicrobial activity and minimum inhibitory concentration (MIC) against foodborne pathogens. EOs of thyme and cinnamon presented the largest antibacterial activity against foodborne pathogens. Combinations of selected EOs displayed a synergic effect against foodborne pathogens and also an important decrease in their individual MIC. Thyme EO presented the lowest individual MIC, but its utilization in combination decreased the MIC of the other EOs. Utilization of cinnamon EO also improved the reduction of the individual MICs of the EOs of cumin and parsley. Our results suggest the potential use of EO mixtures to control foodborne pathogens in meat products. Although the individual MIC values of selected EOs decreased, the sensory impact on meat products needs to be assessed.

Identification of Vitis vinifera L. grape berry skin color mutants and polyphenolic profile.

A germplasm set of twenty-five grapevine accessions, forming eleven groups of possible berry skin color mutants, were genotyped with twelve microsatellite loci, being eleven of them identified as true color mutants. The polyphenolic profiling of the confirmed mutant cultivars revealed a total of twenty-four polyphenols, comprising non-colored compounds (phenolic acids, flavan-3-ols, flavonols and a stilbene) and anthocyanins. Results showed differences in the contribution of malvidin-3-O-glucoside to the characteristic Pinot Noir anthocyanins profile. Regarding the two Pique-Poul colored variants, the lighter variant was richer than the darker one in all classes of compounds, excepting anthocyanins. In Moscatel Galego Roxo the F3'H pathway seems to be more active than F3'5'H, resulting in higher amounts of cyanidin, precursor of the cyanidin derivatives. As far as we are aware, this is the first time that a relationship between the content of polyphenolic compounds is established in groups of grape berry skin color mutant cultivars.

Nitrogen addition influences formation of aroma compounds, volatile acidity and ethanol in nitrogen deficient media fermented by Saccharomyces cerevisiae wine strains.

The effects of nitrogen addition into nitrogen deficient/depleted media on the release of aroma compounds post-fermentation were investigated in three commercial yeast strains of Saccharomyces cerevisiae which highlight the yeast strain effect as well as nitrogen effects. By comparing the two timings of nitrogen addition, prior to fermentation or later at stationary phase (72 h), it was shown that nitrogen addition at stationary phase significantly decreases ethanol and acetic acid formation and significantly increases the following compounds: 2-phenylethanol, ethyl isobutyrate, 2-phenylethyl acetate, ethyl 2-methylbutyrate and ethyl propionate in the three strains, and also isovaleric acid, isoamyl alcohol and ethyl isovalerate in both PYCC4072 and UCD522. The strain EC1118 produced significantly less medium chain fatty acids, hexanoic, octanoic and decanoic acids and their respective esters after nitrogen addition. Therefore, timing of nitrogen addition to a ferment media can vary the concentration of certain aroma compound and might provide a means for varying wine composition.

The production of hydrogen sulphide and other aroma compounds by wine strains of Saccharomyces cerevisiae in synthetic media with different nitrogen concentrations.

The aim of this study was to evaluate the combined effect of initial nitrogen content on the production of hydrogen sulphide and other volatile compounds during alcoholic fermentation. For that propose, three commercial wine strains of Saccharomyces cerevisiae were used to ferment synthetic grape juice media under different nitrogen concentrations. H(2)S was measured throughout fermentations and other aroma compounds were analyzed at the end of the experiments. The trigger levels at which an inverse relationship between the initial nitrogen present in media and total H(2)S production varied among the three strains tested. For UCD522 and PYCC4072, the highest H(2)S levels were produced in media with 267 mg N l(-1) of initial nitrogen, whereas the lowest levels were detected with nitrogen limitation/starvation conditions (66 mg N l(-1)). Moreover, 21 other aroma compounds belonging to different chemical classes were identified and quantified by solid phase micro-extraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). The initial nitrogen concentration more than yeast strain had a decisive effect on the final aroma composition, suggesting that modulation of nutrients emerges as a useful tool for producing desired flavour and odour compounds.

Effects of elevated CO2 on grapevine (Vitis vinifera L.): volatile composition, phenolic content, and in vitro antioxidant activity of red wine.

The impact of elevated carbon dioxide concentration ([CO2]) on the quality of berries, must, and red wine (with special reference to volatile composition, phenolic content, and antioxidant activity) made from Touriga Franca, a native grape variety of Vitis vinifera L. for Port and Douro wine manufacturing grown in the Demarcated Region of Douro, was investigated during 2005 and 2006. Grapevines were grown either in open-top chambers (OTC) with ambient (365 +/- 10 ppm) or elevated (500 +/-16 ppm) [CO2] or in an outside plot. In general, the increase of [CO2] did not affect berry characteristics, especially the total anthocyan and tannin concentrations. However, the total anthocyan and polyphenol concentrations of the red wine were inhibited under elevated [CO2]. The antioxidant capacity of the wines was determined by DPPH, ABTS, and TBARS assays and, despite the low concentrations of phenolics, the elevated [CO2] did not significantly change the total antioxidant capacity of the red wines. Thirty-five volatile compounds belonging to seven chemical groups were identified: C6 alcohols, higher alcohols, esters, terpenols, carbonyl compounds, acids, volatile phenols, and C13 norisoprenoids. Generally, the same volatile compounds were present in all of the wines, but the relative levels varied among the treatments. The effect of elevated [CO2] was significant because it was detected as an increase in ethyl 2-methylbutyrate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, butyric acid, and isovaleric acid concentrations and a decrease in ethyl acetate concentration when compared to wines produced in ambient [CO2] in 2005. In elevated [CO2], wines from 2006 had lower methionol, 1-octanol, and 4-ethylguaiacol and higher ethyl lactate and linalool concentrations. The increase in [CO2] did not significantly affect C6 alcohols, citronellol, carbonyl compounds, and beta-damascenone concentrations. This study showed that the predicted rise in [CO2] did not produce negative effects on the quality of grapes and red wine. Although some of the compounds were slightly affected, the red wine quality remained almost unaffected.