Unveiling the fungal color palette: pigment analysis of Fusarium solani species complex and Curvularia verruculosa clinical isolates.

Fungal species in the Nectriaceae, such as Fusarium spp. (Hypocreales: Nectriaceae), are etiologic agents of hyalohyphomycosis capable of producing violaceous or yellowish pigments under certain conditions, while Curvularia spp. (Pleosporales: Pleosporaceae) are agents of phaeohyphomycosis and typically produce melanin in their cell walls. In nectriaceous and pleosporaceous fungi, these pigments are mainly constituted by polyketides (e.g., azaphilones, naphthoquinones, and hydroxyanthraquinones). Considering the importance of pigments synthesized by these genera, this work focused on the selective extraction of pigments produced by eight Fusarium solani species complex and one Curvularia verruculosa isolate recovered from dermatomycosis specimens, their separation, purification, and posterior chemical analysis. The pigments were characterized through spectral and acid-base analysis, and their maximum production time was determined. Moreover, spectral identification of isolates was carried out to approach the taxonomic specificity of pigment production. Herein we describe the isolation and characterization of three acidic pigments, yellowish and pinkish azaphilones (i.e., coaherin A and sclerotiorin), and a purplish xanthone, reported for the first time in the Nectriaceae and Pleosporaceae, which appear to be synthesized in a species-independent manner, in the case of fusaria.

Determination of streptomycin and doxycycline using LC/MS towards an effective treatment against an experimental Brucella abortus infection in mice.

Herein we described a versatile liquid chromatographic method for detection and quantification of the total levels of two antimicrobials [i.e., streptomycin (STM) and doxycycline (DOX)], in mice plasma and selected tissues, with the aid of a single quadrupole as a detection method. The method included a few sample preparation steps, including freeze-drying and in situ triphasic solvent-assisted defatting, precipitation, and extraction, allowing easy and fast tissue sample processing and avoiding analyte loss. Using a murine model, we demonstrated that mass spectrometry detects simultaneously and with high specificity two of the most widespread antimicrobials used against Brucellosis. An accurate [recoveries varied from 75.23 (bone marrow) to 101.33% (liver)] and sensitive (LoD in the ng g-1 range) method to assess STM and DOX in murine tissue, including subtherapeutic and therapeutic doses of the antimicrobials, was achieved. This validated method can be successfully used to monitor the depletion of STM and DOX in several mice tissues and plasma during metabolism after administration.

Nitrogen/protein and one-step moisture and ash examination in foodstuffs: Validation case analysis using automated combustion and thermogravimetry determination under ISO/IEC 17025 guidelines.

Method validation within food science is a not only paramount to assess method certainty and ensure the quality of the results, but a pennant in analytical chemistry. Proximate analysis is an indispensable requirement for food characterization. To improve proximate analysis, automated protein and thermogravimetric methods were validated according to international guidelines (including ISO 17025) and acceptance criteria of results based on certified reference materials and participation within international recognized proficiency schemes. Common food groups (e.g., meat, dairy, and grain products) were included and at the end of validation, we obtained three rugged and accurate methods with adequate z scores (-2 ≥ x ≤ 2) and recoveries (92-105%). During optimization, variables such as gas flows, subsample masses, and temperatures were varied and specific conditions (those that rendered the best results) were selected for each food group. For each validated method, a comparison (technical and economic) among the data obtained and the data extracted for its traditional counterpart were included: assays validated demonstrate to be more cost-effective labor-wise (ca. 9 and 16-fold) than their traditional alternatives. Specifically for combustion assay regression analysis (y = 0.9361x, y = 1.1001x, and y = 0.9739x, for meat, dairy and grain products, respectively) were performed to assess the factor, if any, which must be applied to the results to effectively match those obtained for Kjeldahl method. Finally, in the case of protein, samples can be analyzed under 5 min with no residue and a subsample mass below 400 mg. Moisture and ash analysis can be performed simultaneously using the same subsample. Data herein will also help harmonize and advance food analysis toward more efficient greener methods for proximate analysis.

Microbiological Safety and Presence of Major Mycotoxins in Animal Feed for Laboratory Animals in a Developing Country: The Case of Costa Rica.

Safety and quality of compound feed for experimental animals in Costa Rica is unknown. Some contaminants, such as Salmonella spp. and mycotoxins, could elicit confounding effects in laboratory animals used for biomedical research. In this study, different batches of extruded animal feed, intended for laboratory rodents in Costa Rica, were analyzed to determine mycotoxin and microbiological contamination (i.e., Salmonella spp., Escherichia coli, total coliform bacteria, and total yeast and molds enumeration). Two methods for Salmonella decontamination (UV light and thermal treatment) were assessed. Only n = 2 of the samples were negative (representing 12.50%) for the 26 mycotoxins tested. Enniatins and fumonisins were among the most frequent toxins found (with n = 4+ hits), but the level of contamination and the type of mycotoxins depended on the supplier. None of the indicator microorganisms, nor Salmonella, were found in any of the tested batches, and no mold contamination, nor Salmonella growth, occurs during storage (i.e., 2-6 months under laboratory conditions). However, mycotoxins, such as enniatins and fumonisins tend to decrease after the fourth month of storage, and Salmonella exhibited a lifespan of 64 days at 17 °C even in the presence of UV light. The D-values for Salmonella were between 65.58 ± 2.95 (65 °C) and 6.21 ± 0.11 (80 °C) min, and the thermal destruction time (z-value) was calculated at 15.62 °C. Results from this study suggest that laboratory rodents may be at risk of contamination from animal feed that could significantly affect the outcomes of biomedical experiments. Thus, improved quality controls and handling protocols for the product are suggested.

Selected Instrumental Techniques Applied in Food and Feed: Quality, Safety and Adulteration Analysis.

This review presents an overall glance at selected instrumental analytical techniques and methods used in food analysis, focusing on their primary food science research applications. The methods described represent approaches that have already been developed or are currently being implemented in our laboratories. Some techniques are widespread and well known and hence we will focus only in very specific examples, whilst the relatively less common techniques applied in food science are covered in a wider fashion. We made a particular emphasis on the works published on this topic in the last five years. When appropriate, we referred the reader to specialized reports highlighting each technique's principle and focused on said technologies' applications in the food analysis field. Each example forwarded will consider the advantages and limitations of the application. Certain study cases will typify that several of the techniques mentioned are used simultaneously to resolve an issue, support novel data, or gather further information from the food sample.

Total and resistant starch from foodstuff for animal and human consumption in Costa Rica.

Starchy ingredients are a key source of carbohydrates and have an essential role in a healthy diet. Starch amount in foodstuffs is paramount as it allows diet professionals to base their formulations on scientific data. Herein, the total (TS) and resistant starch (RS) content, in a selection of typical starchy foods available on the Costa Rican market, for both human and animal consumption, is reported. The major types of starch, including physically encapsulated starch, were determined using in vitro methods AOAC OMASM methods 996.11, 2014.10, 996.11, 2002.02 and AACC 76-13.01 and 32-40.01. Samples were collected during 5 years as part of national surveillance plans. For feedstuffs, n = 252 feed ingredients (e.g., cornmeal and wheat products), n = 103 feeds (e.g., dairy and beef cattle), and n = 150 feed ingredient samples (selected based on their usage in feed formulations) were assessed for RS. In food commodities, sample numbers ascended to n = 287 and n = 371 for TS and RS, respectively (e.g. bananas). Feed ingredients with higher TS values were cassava meal, bakery by-products, rice/broken, sweet potato, and cornmeal (93.37, 81.67, 72.33, 66.66, and 61.43 g/100 g, respectively). TS for beef and dairy cattle, pig, and calf feeds, ranged from 30.26 to 34.46 g/100 g. Plantain/green banana flour, as a feed ingredient, exhibited RS absolute and relative contributions of 37.04 g/100 g and 53.89%, respectively. Products with a higher TS content included banana flour, green plantain flour, japonica rice, and cassava flour (62.87, 63.10, 72.90, 83.37 g/100 g). The primary RS sources in the Costa Rican diet are, in absolute terms, green plantain and malanga (50.41 and 56.59 g/100 g). Depending on a person's food habits, these sources may contribute in the range of 20-30 grams of RS per day. TS and RS intake may vary considerably among ingredients, and the contribution of RS may be of nutritional importance for specific individuals.

Effect of the veterinary ionophore monensin on the structure and activity of a tropical soil bacterial community.

Monensin (MON) is a coccidiostat used as a growth promoter that can reach the environment through fertilization with manure from farm animals. To verify whether field-relevant concentrations of this drug negatively influence the structure and activity of tropical soil bacteria, plate counts, CO2 efflux measurements, phospholipid fatty acids (PLFA) and community-level physiological profiling (CLPP) profiles were obtained for soil microcosms exposed to 1 or 10 mg kg-1 of MON across 11 days. Although 53% (1 mg kg-1) to 40% (10 mg kg-1) of the MON concentrations added to the microcosms dissipated within 5 days, a subtle concentration-dependent decrease in the number of culturable bacteria (<1 log CFU g-1), reduced (-20 to -30%) or exacerbated (+25%) soil CO2 effluxes, a marked shift of non-bacterial fatty acids, and altered respiration of amines (1.22-fold decrease) and polymers (1.70-fold increase) were noted in some of the treatments. These results suggest that MON quickly killed some microorganisms and that the surviving populations were selected and metabolically stimulated. Consequently, MON should be monitored in agronomic and environmental systems as part of One Health efforts.

Simultaneous LC/MS Analysis of Carotenoids and Fat-Soluble Vitamins in Costa Rican Avocados (Persea americana Mill.).

Avocado (a fruit that represents a billion-dollar industry) has become a relevant crop in global trade. The benefits of eating avocados have also been thoroughly described as they contain important nutrients needed to ensure biological functions. For example, avocados contain considerable amounts of vitamins and other phytonutrients, such as carotenoids (e.g., ß-carotene), which are fat-soluble. Hence, there is a need to assess accurately these types of compounds. Herein we describe a method that chromatographically separates commercial standard solutions containing both fat-soluble vitamins (vitamin A acetate and palmitate, Vitamin D2 and D3, vitamin K1, α-, δ-, and γ-vitamin E isomers) and carotenoids (ß-cryptoxanthin, zeaxanthin, lutein, ß-carotene, and lycopene) effectively (i.e., analytical recoveries ranging from 80.43% to 117.02%, for vitamins, and from 43.80% to 108.63%). We optimized saponification conditions and settled at 80 °C using 1 mmol KOH L-1 ethanol during 1 h. We used a non-aqueous gradient that included methanol and methyl tert-butyl ether (starting at an 80:20 ratio) and a C30 chromatographic column to achieve analyte separation (in less than 40 min) and applied this method to avocado, a fruit that characteristically contains both types of compounds. We obtained a method with good linearity at the mid to low range of the mg L-1 (determination coefficients 0.9006-0.9964). To determine both types of compounds in avocado, we developed and validated for the simultaneous analysis of carotenoids and fat-soluble vitamins based on liquid chromatography and single quadrupole mass detection (LC/MS). From actual avocado samples, we found relevant concentrations for cholecalciferol (ranging from 103.5 to 119.5), δ-tocopherol (ranging from 6.16 to 42.48), and lutein (ranging from 6.41 to 15.13 mg/100 g dry weight basis). Simmonds cultivar demonstrated the higher values for all analytes (ranging from 0.03 (zeaxanthin) to 119.5 (cholecalciferol) mg/100 g dry weight basis).

Pet Food Quality Assurance and Safety and Quality Assurance Survey within the Costa Rican Pet Food Industry.

Costa Rican animal feed production is continually growing, with approximately 1,238,243 metric tons produced in 2018. Production-wise, pet cat and dog food are in fifth place (about 41,635 metric tons per year) amongst animal feeds, and it supplies up to 90% of the national market. Pet food production has increased as a response to the increase in the population of dogs and cats in Costa Rica, where 50.5% of households own at least one dog and indicates more responsible ownership in terms of feeding pets. Part of the process of making dry pet food involves a thermal process called extrusion, which is capable of eliminating the microbial load. However, extrusion can compromise nutritional quality to some extent by denaturing proteins, oxidizing lipids, and reducing digestibility. The objective of this study was to evaluate the quality and safety of dry pet food and to assess the effect of the extrusion process on digestibility and the quality of proteins, amino acids, and fatty acids. Pet food samples were collected before and after extrusion and were used to evaluate Good Manufacturing Practices (GMP), based on Central American Technical Regulation (RTCA 65.05.63:11). In general terms, weaknesses in infrastructure, documentary evidence, and post-process practices were observed in two Costa Rican feed manufactories. Feed safety was surveyed through the analysis of Salmonella spp., Escherichia coli, Listeria spp., Staphylococcus aureus, aerobic mesophilic microorganisms, fungi, and yeasts counts. The extrusion process effectively reduced pathogenic microorganisms, and showed no effect on the digestibility of dog food (p = 0.347), however, it could reduce the availability of some nutrients (e.g., amino acids, fatty acids). Furthermore, a retrospective diagnosis was made for puppy food (n = 68), dog food (n = 158), and cat food (n = 25), to evaluate the history of nutritional quality and safety. Finally, it can be confirmed that the correct implementation of GMP allows feed manufacturers to deliver a product of optimum texture, smell, nutritional composition, and safety.

Mycotoxins at the Start of the Food Chain in Costa Rica: Analysis of Six Fusarium Toxins and Ochratoxin A between 2013 and 2017 in Animal Feed and Aflatoxin M1 in Dairy Products.

Mycotoxins are secondary metabolites, produced by fungi of genera Aspergillus, Penicillium and Fusarium (among others), which produce adverse health effects on humans and animals (carcinogenic, teratogenic and immunosuppressive). In addition, mycotoxins negatively affect the productive parameters of livestock (e.g., weight, food consumption, and food conversion). Epidemiological studies are considered necessary to assist stakeholders with the process of decision-making regarding the control of mycotoxins in processing environments. This study addressed the prevalence in feed ingredients and compound feed of eight different types of toxins, including metabolites produced by Fusarium spp. (Deoxynivalenol/3-acetyldeoxynivalenol, T-2/HT-2 toxins, zearalenone and fumonisins) and two additional toxins (i.e., ochratoxin A (OTA) and aflatoxin M1 (AFM1)) from different fungal species, for over a period of five years. On the subject of Fusarium toxins, higher prevalences were observed for fumonisins (n = 80/113, 70.8%) and DON (n = 212/363, 58.4%), whereas, for OTA, a prevalence of 40.56% was found (n = 146/360). In the case of raw material, mycotoxin contamination exceeding recommended values were observed in cornmeal for HT-2 toxin (n = 3/24, 12.5%), T-2 toxin (n = 3/61, 4.9%), and ZEA (n = 2/45, 4.4%). In contrast, many compound feed samples exceeded recommended values; in dairy cattle feed toxins such as DON (n = 5/147, 3.4%), ZEA (n = 6/150, 4.0%), T-2 toxin (n = 10/171, 5.9%), and HT-2 toxin (n = 13/132, 9.8%) were observed in high amounts. OTA was the most common compound accompanying Fusarium toxins (i.e., 16.67% of co-occurrence with ZEA). This study also provided epidemiological data for AFM1 in liquid milk. The outcomes unveiled a high prevalence of contamination (i.e., 29.6-71.1%) and several samples exceeding the regulatory threshold. Statistical analysis exposed no significant climate effect connected to the prevalence of diverse types of mycotoxins.

Liquid Chromatography Analysis of Common Nutritional Components, in Feed and Food.

Food and feed laboratories share several similarities when facing the implementation of liquid-chromatographic analysis. Using the experience acquired over the years, through application chemistry in food and feed research, selected analytes of relevance for both areas were discussed. This review focused on the common obstacles and peculiarities that each analyte offers (during the sample treatment or the chromatographic separation) throughout the implementation of said methods. A brief description of the techniques which we considered to be more pertinent, commonly used to assay such analytes is provided, including approaches using commonly available detectors (especially in starter labs) as well as mass detection. This manuscript consists of three sections: feed analysis (as the start of the food chain); food destined for human consumption determinations (the end of the food chain); and finally, assays shared by either matrices or laboratories. Analytes discussed consist of both those considered undesirable substances, contaminants, additives, and those related to nutritional quality. Our review is comprised of the examination of polyphenols, capsaicinoids, theobromine and caffeine, cholesterol, mycotoxins, antibiotics, amino acids, triphenylmethane dyes, nitrates/nitrites, ethanol soluble carbohydrates/sugars, organic acids, carotenoids, hydro and liposoluble vitamins. All analytes are currently assayed in our laboratories.

Two alternative chromatography methods assisted by the sulfonic acid moeity for the determination of furosine in milk.

N6-(2-(2-Furanyl-2-oxoethyl))-l-lysine (furosine) is a deteriorative reaction product that is produced during heat treatment and storage of milk. This compound affects the quality of commercial dairy products. Accurate determination of furosine is necessary as it may serve as a measure of the degree of protein degradation in dairy products. In this article, two HPLC based methods (1. a novel ion-pairing reagent 2. a strong cation exchange column) are proposed to quantify furosine. These methods were optimized and validated for their application to analyze fluid milk and dried milk powder. •Two methods that can be used for routine milk quality control, including heat damage and adulteration, were developed.•Compared to previous methods, the modified procedures herein using aromatic sulfonic acids (a pairing agent or covalently bound to a matrix on a strong cation exchange column) provide less expensive and more sensitive determinations.•The identification and quantification of the furosine chromatographic signal was successfully achieved during analysis of commercial and spiked samples.

Total starch in animal feeds and silages based on the chromatographic determination of glucose.

Starch is an important nutrient in animal feed, and so its analysis is of considerable concern as it is one of the most relevant energy containing fractions. Method AOAC 996.11 was modified to exchange the enzymometric and colorimetric step full approach to a simpler HPLC amine-based column one. The method was optimized and validated for its application in animal feeds and silages. •We demonstrated that the method could be used for quality control for animal feeds and silages•We modified the final incubation time, the initial sample mass, the quantity of enzyme added and buffered, to pH 6.2, the medium to which α-amylase is added.•We applied a chromatographic analysis of the glucose that resulted from starch enzymatic hydrolysis, via a refractive index detector and amine-based chromatographic column.

A straightforward LC approach using an amine column and single quad mass detector to determine choline chloride in feed additives and feeds.

Considering choline (ChCl) as an essential ingredient for animals and that it is administered through feed, we developed an easy, accurate, and sensitive method for its analysis. The method is straightforward, derivatization-free and has no secondary chromatographic interactions. •We demonstrated that the method can be used for quality control for feeds and feed additives containing choline chloride•We report a simple chromatographic method which takes advantage of the hydroxyl moiety present in ChCl and a MS detector.•We demonstrated that a single quadrupole detector is an effective option for the quantification of ChCl in feeds as an alternative for the more expensive tandem MS system.

Tetracyclines in Food and Feedingstuffs: From Regulation to Analytical Methods, Bacterial Resistance, and Environmental and Health Implications.

Antibiotics are widely used as growth promoters in animal husbandry; among them, the tetracyclines are a chemical group of relevance, due to their wide use in agriculture, surpassing in quantities applied almost every other antibiotic family. Seeing the considerable amounts of tetracyclines used worldwide, monitoring of these antibiotics is paramount. Advances must be made in the analysis of antibiotics to assess correct usage and dosage of tetracyclines in food and feedstuffs and possible residues in pertinent environmental samples. The tetracyclines are still considered a clinically relevant group of antibiotics, though dissemination of tolerance and resistance determinants have limited their use. This review focuses on four different aspects: (i) tetracyclines, usage, dosages, and regulatory issues that govern their food-related application, with particular attention to the prohibitions and restrictions that several countries have enforced in recent years by agencies from both the United States and the European Union, (ii) analytical methods for tetracyclines, determination, and residues thereof in feedstuffs and related matrices with an emphasis on the most relevant and novel techniques, including both screening and confirmatory methods, (iii) tetracycline resistance and tetracycline-resistant bacteria in feedstuff, and (iv) environmental and health risks accompanying the use of tetracyclines in animal nutrition. In the last two cases, we discuss the more relevant undesirable effects that tetracyclines exert over bacterial communities and nontarget species including unwanted effects in farmers.

Vigilance for Salmonella in Feedstuffs Available in Costa Rica: Prevalence, Serotyping and Tetracycline Resistance of Isolates Obtained from 2009 to 2014.

Relevant epidemiological information is provided in this report for Salmonella based on data obtained from a Costa Rican surveillance program for animal feeds. In addition to prevalence, a description in terms of serotypes and tetracycline (TET) resistance of the isolates is included. A total of 1725 feed and feed ingredients samples were analyzed during 2009 and 2014, from which 110 Salmonella strains were recovered (76 from poultry, 23 from meat and bone meal [MBM], 3 from pet foods, and 8 from other feed). Retrieved isolates were serotyped and tested for minimum inhibitory concentration (MIC) against TET. Salmonella strains were found mainly from poultry feed (different growth stages, n = 76/110; 69.1%) and MBM (n = 23/109; 21.1%). The rest of the isolates were recovered from feather meal, pet food, fish meal (n = 3/110; 2.3% each) and swine feed (n = 1/110; 0.9%). From the different serotypes recovered (n = 21), the most common were Salmonella Give (n = 18; 13.8%) and Salmonella Rissen (n = 6; 4.6%) for MBM and Salmonella Havana (n = 14; 10.8%), Salmonella Rissen, Salmonella Soerenga, and Salmonella Schwarzengrund (n = 8; 6.2% each) in poultry feed. Recovered strains were regarded to be sensitive or have an intermediate resistance to TET as evidenced by their MIC50 and MIC90 concentrations of 4 and 8 µg/mL for MBM and poultry feed, respectively. Compound feed and MBM samples exhibited strains characterized by 86.8 and 88.9% of the isolates classified (according to CLSI, 2015 ) as sensitive, 7.7 and 3.7% as intermediate, and 5.5% (with >256 µg/mL as the highest concentration) and 7.4% (with 64 µg/mL as the highest concentration) as resistant to TET, respectively. Salmonella serovars Anatum and Havana exhibited the highest resistance profile >256 and 128 µg/mL, respectively. Hence, MBM and poultry feed seem to be a target of interest if Salmonella incidence is to be controlled. Serotypes recovered have in the past demonstrated pathogenic capability; therefore, hereafter a stricter surveillance program may be in order.

Detection of aflatoxin M1 in milk, cheese and sour cream samples from Costa Rica using enzyme-assisted extraction and HPLC.

Aflatoxins are toxic fungal metabolites, which can be found in feed. Aflatoxin M1 (AFM1) is excreted into milk when ruminants ingest aflatoxin B1 contaminated feedstuffs. Due to its carcinogenic potential, contamination of milk and dairy products with AFM1 may pose a risk for consumers. Hence, it is considered a public health concern. In this survey, the level of AFM1 contamination of dairy products marketed in Costa Rica was determined by enzyme-assisted extraction, immunoaffinity clean-up and high-performance liquid chromatography coupled with a fluorescent detector (HPLC-FLD) in fluid milk (n = 70), fresh cheese (n = 70) and sour cream (n = 70) collected at local convenience stores and supermarkets. AFM1 concentrations in milk and fresh cheese ranged from 19 to 629 ng/L and from 31 to 276 ng/L, with mean values of 136 ng/L and 74 ng/L, respectively, whereas none of the sour cream samples analysed tested positive for this aflatoxin. In 30 milk samples, and 10 cheese samples, AFM1 concentrations surpassed threshold concentrations as established by the European Commission. Thus, sour cream and - to a lesser extent - cheese manufacturing seems to reduce the amount of AFM1 present in milk, possibly due to fraction redistribution or microbiological degradation. The survey results reveal improper quality control procedures in the Costa Rican dairy industry. Therefore, a surveillance programme for dairy products in our country is recommended.

Unravelling a vicious circle: animal feed marketed in Costa Rica contains irregular concentrations of tetracyclines and abundant oxytetracycline-resistant Gram-positive bacteria.

Diverse tetracyclines are used to prevent and control bacterial infections in livestock and farmed fish. These drugs are administered through the diet, but farmers seldom check whether feed contains antibiotic-resistant bacteria that may colonise their crops or transfer their resistance traits to species of veterinary relevance. To examine whether antibiotic dosage defines the abundance of antibiotic-resistant bacteria in animal feed, we determined the concentration of parental compounds and epimers of oxytetracycline (OTC), doxycycline, tetracycline and chlortetracycline, as well as the abundance and resistance level of OTC-resistant bacteria in samples of fish (n = 21), poultry (n = 21), swine (n = 21), and shrimp feed (n = 21) marketed in Costa Rica. Fish feed contained the highest amounts of tetracyclines (119-8365 mg kg(-1)) and the largest proportion of bacteria resistant to 10 µg ml(-1) (1.8-92.4%) or 100 µg ml(-1) of OTC (12.5-63.8%). Poultry (78-438 mg kg(-1)) and swine (41-1076 mg kg(-1)) feed had intermediate concentrations of tetracyclines and OTC-resistant bacteria (0.2-66% and 0.3-49%, respectively), whereas shrimp feed showed the lowest amounts of tetracyclines (21.5-50.3 mg kg(-1)), no OTC and no culturable OTC-resistant bacteria. In line with these results, the MIC50 of OTC for 150 isolates from fish and poultry feed was > 256 µg ml(-1), while that of 150 bacteria isolated from swine feed was 192 µg ml(-1). Phenotypic tests, fatty acid profiles and proteotypic analyses by matrix-assisted laser desorption/ionisation-time of flight mass-spectroscopy revealed that most OTC-resistant isolates were Gram-positive bacteria of low G+C% content from the genera Staphylococcus and Bacillus. Clear correlations between OTC dosage and feed colonisation with OTC-resistant bacteria were seen in medicated feed for fish (r = 0.179-0.651). Nonetheless, some unmedicated feed for fish, swine and poultry contained large populations of OTC-resistant bacteria, suggesting that raw materials and manufacturing processes may also influence carriage of OTC-resistant bacteria in animal feed.

Tetracycline and 4-epitetracycline modified the in vitro catabolic activity and structure of a sediment microbial community from a tropical tilapia farm idiosyncratically.

Aquaculture farmers commonly add tetracycline to fish feed or to their ponds to prevent or treat bacterial infections in their crops. To assess the short-term effect of tetracycline (TET) and of one of its reversible epimers, 4-epitetracycline (ETC), on the function and structure of a sediment microbial community from a tropical tilapia farm, we contrasted community-level physiological profiles (CLPP) and phospholipid fatty acid profiles (PLFA) obtained from microcosms exposed for 12 days to 5, 10, 50, or 75 mg kg(-1)of these antibiotics. Notwithstanding that the concentration of the antibiotics during the experiment decreased between 13-100% (TET) or 16-61% (ETC), both compounds provoked opposing metabolic responses that did not revert. TET displayed a tendency to inhibit respiration at concentrations < 50 mg kg(-1), whereas ETC showed the opposite effect. As revealed by the finding of the fatty acids 11:0 iso 3OH, 16:1w6c, and 18:1w6c, the sediment analyzed was predominantly colonized by Gram-negative bacteria. A marked decrease in fatty acid diversity accompanied the aforementioned metabolic responses, with TET concentrations > 50 mg kg(-1)leading to an enrichment of yeast and fungal biomarkers and both antibiotics at concentrations < 10 mg kg(-1)selecting for microorganisms with 11:0 iso 3OH. In agreement with CLPP data, differences between the PLFA profiles of control and treated microcosms were more pronounced for TET than for ETC. We conclude that high, yet field-relevant, concentrations of TET and ETC have the potential to modify the composition, and to a lesser extent, the functioning of a sediment microbial community. This study highlights the importance of considering antibiotic degradation products in ecotoxicological research.

A novel green chemistry method for nonaqueous extraction and high-performance liquid chromatography detection of first-, second-, and third-generation tetracyclines, 4-epitetracycline, and tylosin in animal feeds.

Although tetracyclines and macrolides are common additives for animal nutrition, methods for their simultaneous determination in animal feeds are nonexistent. By coupling an organic extraction and solid-phase extraction cleanup to a high-performance liquid chromatography separation and a nonaqueous postcolumn derivatization, we succeeded in detecting from 0.2 to 24.0 µg kg(-1) of tetracycline, oxytetracycline, chlortetracycline, doxycycline, tigecycline, and 4-epitetracycline in this complex and heterogeneous matrix. Minocycline and tylosin could also be detected with our procedure, but using UV spectrophotometry (1.5 ≤ LOD ≤ 1.9 mg kg(-1)). Linear responses with correlation coefficients between 0.996 and 0.999 were obtained for all analytes in the 0.5-10 mg kg(-1) concentration range. Average recoveries between 59 and 97% and between 98 and 102% were obtained for the tetracyclines and tylosin, respectively. Replicate standard deviations were typically below 5%. When this method was applied to 20 feeds marketed in Costa Rica, we detected labeling inconsistencies, banned mixtures of tetracyclines, and tetracycline concentrations that contravene international regulation.