Mobile resistome of microbial communities and antimicrobial residues from drinking water supply systems in Rio de Janeiro, Brazil.

Antibiotic resistance genes (ARGs) are widespread in the environment due to the overuse of antibiotics and other pollutants, posing a threat to human and animal health. In this study, we evaluated antimicrobial residues, bacterial diversity and ARGs in two important watersheds, Guandu and São João, that supply drinking water to Rio de Janeiro city, Brazil. In addition, tap water samples were collected from three different cities in Rio de Janeiro State, including the metropolitan area of Rio de Janeiro city. Clarithromycin, sulfamethoxazole and azithromycin were found in untreated water and drinking water in all samples. A greater abundance of Proteobacteria was observed in Guandu and São João watersheds, with most of the sequences belonging to the Gammaproteobacteria class. A plasmidome-focused metagenomics approach revealed 4881 (Guandu), 3705 (São João) and 3385 (drinking water) ARGs mainly associated with efflux systems. The genes encoding metallo-ß-lactamase enzymes (blaAIM, blaGIM, blaIMP, and blaVIM) were detected in the two watersheds and in drinking water samples. Moreover, we demonstrated the presence of the colistin resistance genes mcr-3 and mcr-4 (both watersheds) and mcr-9 (drinking water and Guandu) for the first time in Brazil. Our data emphasize the importance of introducing measures to reduce the disposal of antibiotics and other pollutants capable of promoting the occurrence and spread of the microbial resistome on aquatic environments and predicting possible negative impacts on human health.

Tetracyclines in pasteurized milk commercialized in São Paulo, Brazil: occurrence and dietary risk assessment based on single and combined exposure with polyether ionophores.

Tetracyclines and polyether ionophores are veterinary drugs frequently used in animals reared for food production. Despite the benefits, residues can be harmful for human health if levels are higher than those considered safe. The aim of this study was to evaluate, by LC-MS/MS, tetracyclines residues in pasteurised milk samples marketed in São Paulo state, Brazil and estimate the exposure of the adult/adolescent/children population. Within the eight antibiotics monitored two were detected, tetracycline and 4-epitetracycline, found in 22.5% and 12.5% of the samples, respectively. Since the same samples had been previously tested for polyether ionophore residues, risk assessment based on dietary exposure to tetracyclines when considered individually and in combination with the ionophores was performed. The daily intakes were below the acceptable daily intake (ADI) for tetracycline. Considering the levels of monensin reported in a previous study, the combined exposure with tetracycline and 4-epi-tetracycline also indicated low potential health concerns.

Dietary exposure assessment to macrolide antimicrobial residues through infant formulas marketed in Brazil.

Macrolides are critically important antimicrobials for both human and animal health and should be prioritized in risk assessments, as inappropriate use may result in antimicrobial resistance. The antimicrobials erythromycin, oleandomycin, spiramycin, tilmicosin and tylosin were analysed in infant formula samples by HPLC-MS/MS using a validated analytical method based on a modified QuEChERS extraction. The results of the occurrence study were employed to perform a dietary exposure assessment of infants to residues. In a total of 30 analysed samples, 73% contained spiramycin residues and 27%, tilmicosin residues. Calculated daily intakes ranged from 1.47 × 10-6 to 2.71 × 10-4 mg kg-1 body weight considering all analytes, representing 0.01-0.59% of acceptable daily intakes. The results of the dietary exposure assessment were all below acceptable daily intakes, indicating low potential health concerns. However, according to Brazilian regulations, infant formulas containing residues of one or more of the investigated analytes were deemed as non-compliant.

Polyether ionophores residues in pasteurized milk marketed in the state of São Paulo, Brazil: Occurrence and exposure assessment.

This paper aimed to survey the occurrence of six polyether ionophores (lasalocid, maduramycin, monensin, narasin, salinomycin and semduramycin) in pasteurized milk using a validated method by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). For that, 40 samples of pasteurized milk, from seven brands, collected in six locations (supermarkets, grocery stores and greengrocers) in the metropolitan region of São Paulo and Campinas, São Paulo State, Brazil, were evaluated. Sampling included whole (18), skimmed (8) and semi-skimmed (10) milk as well as lactose-free (4) products. Only monensin antibiotic residues were found in the analyzed samples. This ionophore was confirmed in 45% of the samples and quantified in 32.5% at concentrations ranging from 0.1 to 0.27 µg kg-1. The positive samples were whole or semi-skimmed milk. Considering the levels of monensin reported in the present study and data on milk consumption in Brazil, an exposure assessment indicated that the intake of this antibiotic does not represent a risk to health.

Monensin residues in the production of Minas Frescal cheese: Stability, effects on fermentation, fate and physicochemical characteristics of the cheese.

Considering the widespread use of the antibiotic monensin (MON) in the Brazilian livestock and the possibility of residues in milk, this paper aimed to study the stability and fate of this drug during the production of Brazilian Minas Frescal cheese, its effects on milk fermentation and on the physicochemical characteristics of this product. For that, samples of raw milk were fortified with MON at three different nominal concentrations (1.0, 2.0 and 8.0 µg/kg), passed through heat treatment and used to produce Minas Frescal cheese. Pasteurization efficiency was certified by alkaline phosphatase and peroxidase enzyme tests and cheese samples were evaluated for pH, moisture and total protein and fat content. MON residues were determined by LC-MS/MS in the following steps: raw milk, heat-treated milk, whey and cheese. No significant degradation of MON due to heat treatment was observed, suggesting that the drug is resistant to high temperatures. Moreover, the residue levels quantified in cheese and whey demonstrated a concentration of this antibiotic in the curd by about 5-fold, with a small amount of MON being lost during draining. There were no significant differences (p > 0.05) considering the physicochemical parameters evaluated in cheese samples. Fermentation was also not affected by the presence of the drug. The results showed that residues of MON in milk are stable during cheese production and may be concentrated in the final product, as well as indicate the need to establish a MON safe residue level for this food commodity.

A preliminary study of simultaneous veterinary drug and pesticide residues in eggs produced in organic and cage-free alternative systems using LC-MS/MS.

In this study, a preliminary food quality and safety assessment was performed on organic and cage-free egg samples marketed in the state of Rio de Janeiro, Brazil, that were analyzed concerning veterinary drug and pesticide residues using high performance and ultra performance liquid chromatography coupled to tandem mass spectrometry. The polyether ionophore salinomycin was detected in two organic egg samples (25% of the organic samples), one with an estimated concentration even higher than the maximum permissible amount of 3 µg kg-1 established for conventional eggs by the European Commission. The other sample presented a concentration higher than the limit of detection of 0.3 µg kg-1, but lower than the lowest calibration level of 1.5 µg kg-1. Regarding pesticide residues, spiroxamine, pirimiphos, mephosfolan and pyraclostrobin were identified at residual levels below the lowest calibration level of 4.5 µg kg-1, except for one organic egg sample, presenting 8.3 µg kg-1 of spiroxamine. Spiroxamine was identified in 62% of the assessed samples. These findings indicate that non-conformities were found even with a limited number of samples, impacting the confidence in the quality of organic and cage-free alternative systems in egg production. The hazard index (HI) approach demonstrated that chemical food safety might be at risk, since a mixture of the detected analytes may pose a risk for children up to 27 kg, through egg consumption.

A feasibility study for producing an egg matrix candidate reference material for the polyether ionophore salinomycin.

The aim of this work was to study the feasibility of producing an egg matrix candidate reference material for salinomycin. Preservation techniques investigated were freeze-drying and spray drying dehydration. Homogeneity and stability studies of the produced batches were conducted according to ISO Guides 34 and 35. The results showed that all produced batches were homogeneous and both freeze-drying and spray drying techniques were suitable for matrix dehydrating, ensuring the material stability. In order to preserve the material integrity, it must be transported within the temperature range of -20 up to 25°C. The results constitute an important step towards the development of an egg matrix reference material for salinomycin is possible.

Validation of a liquid chromatography-electrospray ionization tandem mass spectrometric method to determine six polyether ionophores in raw, UHT, pasteurized and powdered milk.

This study aimed to validate a method developed for the determination of six antibiotics from the polyether ionophore class (lasalocid, maduramicin, monensin, narasin, salinomycin and semduramicin) at residue levels in raw, UHT, pasteurized and powdered milk using QuEChERS extraction and high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The validation was conducted under an in-house laboratory protocol that is primarily based on 2002/657/EC Decision, but takes in account the variability of matrix sources. Overall recoveries between 93% and 113% with relative standard deviations up to 16% were obtained under intermediate precision conditions. CCα calculated values did not exceed 20% the Maximum Residue Limit for monensin and 25% the Maximum Levels for all other substances. The method showed to be simple, fast and suitable for verifying the compliance of raw and processed milk samples regarding the limits recommended by Codex Alimentarius and those adopted in European Community for polyether ionophores.

Innovative mixture of salts in the quick, easy, cheap, effective, rugged, and safe method for the extraction of residual macrolides in milk followed by analysis with liquid chromatography and tandem mass spectrometry.

A simple extraction technique has been developed for seven macrolide antibiotics in milk. The procedure involves a modified quick, easy, cheap, effective, rugged, and safe method based on acetonitrile extraction, followed by the addition of a mixture of salts (sodium sulfate, sodium chloride, and potassium carbonate) not yet reported in literature. The method was validated for tylosin and was selective, free of matrix effect, and linear in the range of 0.78-18.75 ng/mL in the final extract, corresponding to 0.125-3 times the maximum residue limit. The limit of detection, limit of quantification, decision limit, and detection capability were, respectively, 0.84, 2.79, 58.4, and 71.7 µg/kg. The overall average recovery at 25, 50, and 75 µg/kg ranged from 89-97%. Repeatability and intermediate precision expressed by relative standard deviations were below 10.5 and 12%, respectively. The extension of the validation for spiramycin, throleandomycin, oleandomycin, roxithromycin, erythromycin, and clarithromycin is under consideration since the procedure proved to be able to efficiently extract all studied macrolides, with recoveries from 74-104% at 50 µg/kg for tylosin, erythromycin, spiramycin, and oleandomycin and 20 µg/kg for throleandomycin, roxithromycin, and clarithromycin.

Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography-electrospray ionization tandem mass spectrometry using a simple solvent extraction.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the determination of residues of 6 polyether ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin, semduramicin), 3 macrolides (erythromycin, tylosin, clarithromycin) and 1 lincosamide (lincomycin) in eggs. Nigericin was used as qualitative internal standard. Samples were deproteinizated/extracted with acetonitrile without pH adjustments. Aliquots of the extracts were evaporated and reconstituted for injection in the instrument operated in positive multiple reaction monitoring (MRM) mode. The stability of the antibiotics and the intensity of the formed ions were considered in order to select a suitable solvent for the reconstitution of the obtained dry extracts. No clean-up steps were required and matrix effects were controlled by sample dilution, selection of appropriate chromatographic conditions and reduced injection volume. Good within-laboratory reproducibility was obtained, with relative standard deviations (RSD(R)) from 4.0 (semduramicin at 5 µgkg(-1)) to 18.6 (erythromycin at 25 µgkg(-1)) for the ionophores and macrolides. Lincomycin showed the least precise results, with a maximum RSD(R) of 20.2% at 75 µgkg(-1)). Satisfactory decision limits (CCα) and detection capabilities (CCß) were also attained. Method limits of detection (LODs) from 0.04 (salinomycin) to 1.6 µgkg(-1) (lincomycin) were achieved. Method limits of quantification (LOQs) were from 0.14 to 5.3 µgkg(-1) for the same drugs, respectively. All the LOQs, except that obtained for maduramicin were remarkably below the lowest validation level. The proposed method is suitable for routine application in commercial egg samples.

Pilot survey of hen eggs consumed in the metropolitan area of Rio de Janeiro, Brazil, for polyether ionophores, macrolides and lincosamides residues.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which has recently been developed and validated, was used for the identification and quantification of polyether ionophore, macrolide and lincosamide residues in commercial eggs sold in the metropolitan area of Rio de Janeiro, Brazil. The method was applied to 100 samples and the results showed a high incidence of polyether ionophore residues (25%). Salinomycin was detected in 21% of samples, but only two non-compliant results (5.3 and 53 µg kg(-1)) were found if maximum limits (tolerances) established by European Union were adopted in Brazil and if a method decision limit (CCα) of 3.4 µg kg(-1) was considered. In 8% of analyzed samples, more than one studied coccidiostat was found. The lincosamide, lincomycin, and the macrolide, tylosin, were detected at trace levels in 4 and 1% of the samples, respectively. Lasalocid, clarithromycin and erythromycin were not found.

Pilot survey of commercial pasteurized milk consumed in the metropolitan area of Rio de Janeiro, Brazil, for tetracyclines residues, including the 4-epimers of oxytetracycline, tetracycline and chlortetracycline.

This pilot survey aimed to assess the occurrence of tetracyclines and the 4-epimers of oxytetracycline, tetracycline and chlortetracycline in commercial pasteurized milks sold in the metropolitan area of Rio de Janeiro, Brazil, between October 2009 and March 2010. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, developed and validated in our laboratory, was used. All 100 analyzed samples were compliant, but 14 contained oxytetracycline in concentrations ranging from the method limit of detection (3.7 µg l(-1)) to the method limit of quantification (12.2 µg l(-1)). One sample contained oxytetracycline and tetracycline simultaneously (at a concentration slightly higher than method limit of quantification, 7.0 µg l(-1)). The presence of 4-epioxytetracycline and 4-epitetracycline in contaminated samples with the parent drugs could not be confirmed as traces were detected only in the quantification MRM transition. No other tetracyclines were detected.

A liquid chromatography-tandem mass spectrometry confirmatory assay for the simultaneous determination of several tetracyclines in milk considering keto-enol tautomerism and epimerization phenomena.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method for the analysis of several tetracyclines residues in bovine milk has been developed. Milk deproteinization/extraction of samples was performed with acidified acetonitrile. After diluting and purification by solid-phase extraction (SPE), the extracts were injected into the instrument operated in Multiple Reaction Monitoring (MRM) acquisition mode. The reversible epimerization at C-4 of oxytetracycline, tetracycline and chlortetracycline and the keto-enol tautomerism of chlortetracycline between C-11a and C-12 were considered for reliable quantification. Degradation was also taken in account and minimized for the same purpose. A central composite (response surface) design with desirability function was employed for the optimization of extraction and clean-up steps. The optimization improved the extraction efficiency of the more polar analytes reaching 93.9% for 4-epioxytetracycline and 95.8% for oxytetracycline at 100 microg L(-1). The validation was performed following the criteria established by Commission Decision 2002/657/EC.