Betty San Martín

Genetic Characterization of Antibiotic Resistance Genes Linked to Class 1 and Class 2 Integrons in Commensal Strains of Escherichia coli Isolated from Poultry and Swine

The aim of this research was to identify the presence of integrons among Escherichia coli strains isolated from poultry and swine and to characterize the topological association of these integrons with resistance genes and assess their potential ability to transfer these elements by conjugation. One hundred and seventy-two strains of E. coli were isolated. Their resistance to tetracycline, streptomycin, sulfamethoxazole-trimethoprim, ciprofloxacin, and enrofloxacin was studied by plate dilution. In resistant strains the presence of integrons and resistance genes was assessed by PCR. In the variable region, genes aadA1, dfrA1, and qnr were analyzed. Also, presence of tetA, tetB, and sul1 was assessed. Transference of these genes and integrons in vitro was evaluated by conjugation assays, using E. coli J53 Az(r) as recipient strain. Seventy-eight percent and 83% of the poultry and swine strains, respectively, were resistant to at least one of the studied antimicrobials. Of the isolated strains 91 presented integrons. Resistance genes detected within the integrons were aadA1, dfrA1, and sat1. Gene qnr was not detected. Genes tet and sul1 were identified in 105 and 53 strains, respectively. Seven strains transferred their resistance determinants by conjugation. The results verify the high percentage of antibiotic resistance in the E. coli strains isolated, and these represent a reservoir of resistance genes and integrons.

Increased urinary glucocorticoid metabolites are associated with metabolic syndrome, hypoadiponectinemia, insulin resistance and β cell dysfunction.

Metabolic syndrome (MetS) may have increased cortisol (F) production caused by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in liver and adipose tissue and/or by HPA axis dysregulation. F is then mainly metabolized by liver reductases into inactive tetrahydrometabolites (THMs). We measured THM levels in patients with or without MetS and evaluate the correlation between THMs and anthropometric and biochemical parameters. We recruited 221 subjects, of whom 130 had MetS by ATP III. We evaluated F, cortisone (E), adipokines, glucose, insulin and lipid profiles as well as urinary (24h) F, E and THM levels. β Cell function was estimated by the HOMA Calculator. We observed that patients with MetS showed higher levels of THMs, HOMA-IR and leptin and lower levels of adiponectin and HOMA-β but no differences in F and E in plasma or urine. THM was associated with weight (r = +0.44, p<0.001), waist circumference (r = +0.38, p<0.01), glycemia (r = +0.37, p<0.01), and triglycerides (r = +0.18, p=0.06) and negatively correlated with adiponectin (r = -0.36, p<0.001), HOMA-β (r = -0.21, p<0.001) and HDL (r = -0.29, p<0.01). In a logistic regression model, THM levels were associated with hypertension, hyperglycemia and dyslipidemia. We conclude that MetS is associated with increased urinary THMs but not with F and E levels in plasma or urine. Increased levels of THM, reflecting the daily cortisol production subsequently metabolized, are correlated with hypoadiponectinemia, hypertension, dyslipidemia, insulin resistance and β cell dysfunction. A subtle increased in glucocorticoid production may further account for the phenotypic and biochemical similarities observed in central obesity and Cushings syndrome.

Withdrawal Times of Oxytetracycline and Tylosin in Eggs of Laying Hens after Oral Administration

Antimicrobials administered to laying hens may be distributed into egg white or yolk, indicating the importance of evaluating withdrawal times (WDTs) of the pharmaceutical formulations. In the present study, oxytetracycline and tylosins WDTs were estimated. The concentration and depletion of these molecules in eggs were linked to their pharmacokinetic and physicochemical properties. Twenty-seven Leghorn hens were used: 12 treated with oxytetracycline, 12 treated with tylosin, and 3 remained as an untreated control group. After completion of therapies, eggs were collected daily and drug concentrations in egg white and yolk were assessed. The yolk was used as the target tissue to evaluate the WDT; the results were 9 and 3 days for oxytetracycline and tylosin, respectively. In particular, oxytetracycline has a good oral bioavailability, a moderate apparent volume of distribution, a molecular weight of 460 g/mol, and is lightly liposoluble. Tylosin, a hydrosoluble compound, with a molecular weight of 916 g/mol, has a low oral bioavailability and a low apparent volume of distribution, too. Present results suggest that the WDTs of the studied antimicrobials are strongly influenced by their oral bioavailability, the distribution, and the molecular weight and solubility, and that these properties also influence the distribution between the egg yolk and white.

Determination of Oxytetracycline and 4-Epi-Oxytetracycline Residues in Feathers and Edible Tissues of Broiler Chickens Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Antibiotics have been widely used in poultry production for the treatment of bacterial diseases. However, drug residues can remain in products derived from animals after the cessation of the drug therapies. Feathers, in particular, have shown an affinity for antibiotics such as tetracycline, suggesting the persistence of these drugs in nonedible tissue. After the birds are slaughtered, feathers are ground into feather meals, which are used as organic fertilizer or an ingredient in animal diets, thereby entering into the food chain and becoming a potential risk for public health. To evaluate the depletion of oxytetracycline (OTC) and its metabolite 4-epi-oxytetracycline (4-epi-OTC) in the muscles, liver, and feathers, 64 broiler chickens, bred under controlled conditions, were treated orally with a commercial formulation of 10% OTC for 7 days. The analytes were quantified using liquid chromatography-tandem mass spectrometry. OTC and 4-epi-OTC were found in the feathers for 46 days, whereas they were found in the muscle and liver for only 12 and 6 days, respectively. These results prove that the analytes remain in feathers in higher concentrations than they do in edible tissues after treatment with tetracyclines. Thus, feather meals represent a potential source of antimicrobial residue contamination in the food chain.

In-House validation of HPLC-MS/MS methods for detection and quantification of tetracyclines in edible tissues and feathers of broiler chickens

For the detection of tetraciclines in feathers, muscle and liver, an internal protocol was designed for in-house validation of two methods, through high performance liquid chromatography with tandem mass spectrometric (HPLC-MS/MS) techniques. This protocol was based on the recommendation of the decision 2002/657/EC from the European Community as well as the food and drug administration (FDA) VICHGL49. Limit of detection (LOD) was set at 20 μg kg and the limit of quantification (LOQ) ranged from 21.5 to 24.2, 21.2 to 21.6, and 25.0 to 27.7 μg kg in feahers, muscle and liver samples, respectively, for all analytes. The calibrations curves show a coefficient of determination (R) above 0.98, 0.99 and 0.96 for feathers, muscle and liver samples, respectively. Analyte recovery ranged from 92 to 108%. In conclusion, these methods can be deemed accurate and reliable, and their validation is a fundamental step to be performed in depletion studies on these matrices.

Determination of sulfachloropyridazine residue levels in feathers from broiler chickens after oral administration using liquid chromatography coupled to tandem mass spectrometry

Several antimicrobials are routinely used by the poultry farming industry on their daily operations, however, researchers have found for some antimicrobials that their residues persist for longer periods in feathers than they do in edible tissues, and at higher concentrations, as well. But this information is not known for other classes of antimicrobials, such as the sulfonamides. Therefore, this work presents an accurate and reliable analytical method for the detection of sulfachloropyridazine (SCP) in feathers and edible tissues from broiler chickens. This method was also validated in-house and then used to study the depletion of sulfachloropyridazine in those matrices. The experimental group comprised 54 broiler chickens, who were raised under controlled conditions and then treated with a commercial formulation of 10% sulfachloropyridazine for 5 days. Samples were analyzed via LC-MS/MS, using 13C6-sulfamethazine (SMZ-13C6) as an internal standard. Aromatic sulfonic acid solid phase extraction (SPE) cartridges were used to clean up the samples. The Limit of Detection (LOD) for this method was set at 10 μg kg-1 on feathers and liver; and at 5 μg kg-1 on muscle. Within the range of 10–100 μg kg-1, the calibration curves for all matrices presented a determination coefficient greater than 0.96. Our results show, with a 95% confidence level, that sulfachloropyridazine persisted in feathers for up to 55 days after ceasing treatment, and its concentrations were higher than in edible tissues. In consequence, to avoid re-entry of antimicrobial residues into the food-chain, we recommend monitoring and inspecting animal diets that contain feather derivatives, such as feathers meals, because they could be sourced from birds that might have been medicated with sulfachloropyridazine.

Depletion of tylosin residues in feathers, muscle and liver from broiler chickens after completion of antimicrobial therapy

ABSTRACT Tylosin is one of the most commonly used antimicrobial drugs from the macrolide family and in broiler chickens it is used specially for the treatment of infectious pathologies. The poultry industry produces several by-products, among which feathers account for up to 7% of a chicken’s live weight, thus they amount to a substantial mass across the whole industry. Feathers have been repurposed as an animal feed ingredient by making them feather meal. Therefore, the presence of high concentrations of residues from antimicrobial drugs in feathers might pose a risk to global public health, due to re-entry of these residues into the food chain. This work aimed to characterise the depletion behaviour of tylosin in feather samples, while considering its depletion in muscle and liver tissue samples as a reference point. To achieve this goal, we have implemented and validated an analytical methodology suitable for detecting and quantifying tylosin in these matrices. Sixty broiler chickens, raised under controlled conditions, received an oral dose of 32 mg kg-1 of tylosin for 5 days. Tylosin was quantified in muscle, liver and feathers by liquid chromatography coupled with a photodiode array detector (HPLC-DAD). High concentrations of tylosin were detected in feather samples over the whole experimental period after completing both the therapy and the recommended withdrawal time (WDT). On the other hand, tylosin concentrations in muscle and liver tissue samples fell below the limit of detection of this method on the first sampling day. Our results indicate that the WDT for feather samples is 27 days, hence using feather meal for the formulation of animal diets or for other agricultural purposes could contaminate with antimicrobial residues either other livestock species or the environment. In consequence, we recommend monitoring this matrix when birds have been treated with tylosin, within the context of poultry farming.

Determination of Chlortetracycline Residues, Antimicrobial Activity and Presence of Resistance Genes in Droppings of Experimentally Treated Broiler Chickens

Tetracyclines are important antimicrobial drugs for poultry farming that are actively excreted via feces and urine. Droppings are one of the main components in broiler bedding, which is commonly used as an organic fertilizer. Therefore, bedding becomes an unintended carrier of antimicrobial residues into the environment and may pose a highly significant threat to public health. For this depletion study, 60 broiler chickens were treated with 20% chlortetracycline (CTC) under therapeutic conditions. Concentrations of CTC and 4-epi-CTC were then determined in their droppings. Additionally, this work also aimed to detect the antimicrobial activity of these droppings and the phenotypic susceptibility to tetracycline in E. coli isolates, as well as the presence of tet(A), tet(B), and tet(G) resistance genes. CTC and 4-epi-CTC concentrations that were found ranged from 179.5 to 665.8 µg/kg. Based on these data, the depletion time for chicken droppings was calculated and set at 69 days. All samples presented antimicrobial activity, and a resistance to tetracyclines was found in bacterial strains that were isolated from these samples. Resistance genes tet(A) and tet(B) were also found in these samples.

Residue Depletion of Florfenicol and Florfenicol Amine in Broiler Chicken Claws and a Comparison of Their Concentrations in Edible Tissues Using LC–MS/MS

Antimicrobial residues might persist in products and by-products destined for human or animal consumption. Studies exploring the depletion behavior of florfenicol residues in broiler chicken claws are scarce, even though claws can enter the food chain directly or indirectly. Hence, this study intended to assess the concentrations of florfenicol (FF) and florfenicol amine (FFA)—its active metabolite—in chicken claws from birds that were treated with a therapeutic dose of florfenicol. Furthermore, concentrations of these analytes in this matrix were compared with their concentrations in edible tissues at each sampling point. A group of 70 broiler chickens were raised under controlled conditions and used to assess residue depletion. Sampling points were on days 5, 10, 20, 25, 30, 35, and 40 after ceasing treatment, thus extending beyond the withdrawal period established for muscle tissue (30 days). Analytes were extracted using HPLC-grade water and acetone, and dichloromethane was used for the clean-up stage. Liquid chromatography coupled to mass spectroscopy detection (LC–MS/MS) was used to detect and quantify the analytes. The analytical methodology developed in this study was validated in-house and based on the recommendations described in the Commission Decision 2002/657/EC from the European Union. Analyte concentrations were calculated by linear regression analysis of calibration curves that were fortified using an internal standard of chloramphenicol-d5 (CAF-d5). The depletion time of FF and FFA was set at 74 days in claws, based on a 95% confidence level and using the limit of detection (LOD) as the cut-off point. Our findings show that FF and FFA can be found in chicken claws at higher concentrations than in muscle and liver samples at each sampling point.