Fabio Granados-Chinchilla

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.

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.

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.

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−1of 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−1leading to an enrichment of yeast and fungal biomarkers and both antibiotics at concentrations < 10 mg kg−1selecting 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 single exposure of sediment sulphate-reducing bacteria to oxytetracycline concentrations relevant to aquaculture enduringly disturbed their activity, abundance and community structure

Although feed medicated with antibiotics is widely used in animal production to prevent and treat bacterial infections, the effect of these drugs on nontarget anaerobic bacteria is unknown. We aimed to clarify whether a single exposure of sulphate‐reducing bacteria (SRB) from a tilapia pond to oxytetracycline (OTC) concentrations relevant to aquaculture impacts their function, abundance and community structure.

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. Graphical Abstract

Inorganic contaminants and composition analysis of commercial feed grade mineral compounds available in Costa Rica

BackgroundHeavy metals such as arsenic (As), mercury (Hg), lead (Pb) and cadmium (Cd), are potential toxic substances that may incorporate in productive systems in multiple ways including contaminated feedstuff. In this regard, we hypothesize that the main input of heavy metal contamination include mineral feed ingredients which, in turn, are included in compound feed to meet animals’ nutritional requirements. Hence, we offer a comprehensive heavy metal determination on imported feed grade mineral supplement samples (n = 435), comprised of 27 different sources including calcium/phosphorus, iron, cobalt, copper, cobalt, manganese, iodide, sulfur, potassium, sodium, selenium and magnesium were collected from eight different local feedingstuff manufactures, mineral and heavy metal as cadmium (Cd), lead (Pb), arsenic (As) and mercury (Hg) analyses were performed using atomic absorption spectroscopy and microwave assisted digestion. Based on this premise, the main goals of the study were to determine mineral and trace mineral content and contrast these values with those advertised by the manufacturers and to determine heavy metal concentrations and compare these levels with the current regulation in commercially available mineral sources which are used in premixes and downstream formulation of compound feeds; a matter which remains undocumented.ResultsOur results show that occasionally mineral values for these supplements were lower than those declared. Additionally, several samples contravene, in at least one heavy metal, current regulations; samples in this condition correspond to 0.5 (n = 2), 13.8 (n = 60), 4.1 (n = 18) and 2.5 % (n = 11) for As, Hg, Pb and Cd, respectively with mercury as the most frequent mineral to surpass, in the majority of cases, current thresholds. Overall, 21.1 % (n = 92) of the samples exhibited concentrations of heavy metals above those stipulated by European guidelines. Meanwhile potassium chloride, (n = 17), exhibited the lower overall concentrations of heavy metals.ConclusionSamples of mineral origin may surpass, in some cases with elevated concentrations, permitted levels of undesirable substances, therefore, a monitoring programme for mineral ingredients in our country is recommended.Graphical abstractApproach for mineral and heavy metal analysis in feed grade minerals.

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

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

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

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