Publications

Abstract Poultry meat has been a vehicle of antibiotic resistant bacteria and genes. Yet, the diversity of selective pressures associated with their maintenance in the poultry-production chain remains poorly explored. We evaluated the susceptibility of Enterococcus spp. from chicken meat collected 20 years apart to antibiotics, metals, acidic pH and peracetic acid-PAA. Contemporary chicken-meat samples (n = 53 batches, each including a pool of neck skin from 10 single carcasses) were collected in a slaughterhouse facility using PAA as disinfectant (March-August 2018, North of Portugal). Broilers were raised in intensive farms (n = 29) using CuSO4 and organic acids as feed additives. Data were compared with that of 67 samples recovered in the same region during 1999-2001. All 2018 samples had multidrug resistant-MDR isolates, with >45 % carrying Enterococcus faecalis, Enterococcus faecium or Enterococcus gallinarum resistant to tetracycline, erythromycin, ampicillin, quinupristin-dalfopristin, ciprofloxa-cin, chloramphenicol or aminoglycosides. Resistance rates were similar (P > 0.05) to those of 1999-2001 samples for all but five antibiotics. The decrease of samples carrying vancomycin-resistant isolates from 46 % to % between 1999-2001 and 2018 was the most striking difference. Isolates from both periods were similarly susceptible to acid pH [minimum-growth pH (4.5-5.0), minimum-survival pH (3.0-4.0)] and to PAA (MIC90 = 100-120 mg/L/MBC90 = 140-160 mg/L; below concentrations used in slaughterhouse). Copper tolerance genes (tcrB and/or cueO) were respectively detected in 21 % and 4 % of 2018 and 1999-2001 samples. The tcrB gene was only detected in E. faecalis (MICCuSO4 > 12 mM), and their genomes were compared with other international ones of chicken origin (PATRIC database), revealing a polyclonal population and a plasmid or chromosomal location for tcrB. The tcrB plasmids shared diverse genetic modules, including multiple antimicrobial resistance genes (e.g. to tetracyclines, chloramphenicol, macrolide-lincosamide-streptogramin B-MLSB, aminoglycosides, bacitracin, coccidiostats). When in chromosome, the tcrB gene was co-located closely to merA (mercury) genes. Chicken meat remains an important vehicle of MDR Enterococcus spp. able to survive under diverse stresses (e.g. copper, acid) potentially contributing to these bacteria maintenance and flux among animal-environment -humans.

Abstract The expansion of mcr-carrying bacteria is a well-recognized public health problem. Measures to contain mcr spread have mainly been focused on the food-animal production sector. Nevertheless, the spread of MCR producers at the environmental interface particularly driven by the increasing population of gulls in coastal cities has been less explored. Occurrence of mcr-carrying Escherichia coli in gull's colonies faeces on a Portuguese beach was screened over 7 months. Cultural, molecular and genomic approaches were used to characterize their diversity, mcr plasmids and adaptive features. Multidrug-resistant mcr-1-carrying E. coli were detected for 3 consecutive months. Over time, multiple strains were recovered, including zoonotic-related pathogenic E. coli clones (e.g. B2-ST131-H22, A-ST10 and B1-ST162). Diverse mcr-1 genetic environments were mainly associated with ST2/ST4-HI2 (ST10, ST131, ST162, ST354 and ST4204) but also IncI2 (ST12990) plasmids or in the chromosome (ST656). Whole-genome sequencing revealed enrichment of these strains on antibiotic resistance, virulence and metal tolerance genes. Our results underscore gulls as important spreaders of high-priority bacteria and genes that may affect the environment, food-animals and/or humans, potentially undermining One-Health strategies to reduce colistin resistance.

Abstract This work reports the development of a simple and rapid electrochemical immunosensor for the determination of breast cancer biomarker Cancer Antigen 15-3 (CA 15-3). Disposable and cost-effective chips, consisting of gold screen-printed electrodes (AuSPEs), were used to develop the portable electrochemical devices for monitoring the biomarker in point-of-care (PoC), under clinical context. The biosensor preparation consisted of two simple steps. First, a self-assembled monolayer (SAM) of mercaptosuccinic acid (MSA) was formed at the AuSPE surface. Then, the CA 15-3 antibody was covalently bound to the carboxylic groups standing at the electrode surface using EDC/NHS chemistry. The performance of the developed immunosensor was evaluated by assessing the sensor sensitivity, linear response interval, selectivity and detection limit (LOD). The developed immunosensor provided a wide linear concentration range (from 1.0 to 1000 U mL(-1)) and low detection levels were achieved (LOD of 0.95 U mL(-1)), enabling the sensitive detection of the cancer biomarker at clinically relevant levels, using square wave voltammetry (SWV) as electroanalytical technique. Moreover, selectivity studies performed against other cancer biomarkers (CA 125 and CA 19-9) revealed that the antibody has high selectivity for CA 15-3 antigen. The immunosensor was applied to the quantification of CA 15-3 in artificial serum samples with satisfactory results.

Abstract Emergence and expansion of frequent multidrug-resistant (MDR) major Salmonella clones/serotypes has been a significant threat in the last years. Metal compounds, such as copper, commonly used in animal-production settings, have been pointed out as possible contributors for the selection of such strains/clones. However, the scarcity of studies limits our understanding of the impact of other metal environmental contaminants as arsenic (used in insecticides/herbicides/coccidiostats). We analysed arsenic tolerance (AsT) dispersion by phenotypic and genotypic (PCR/sequencing/I-CeuI/S1/XbaI-PFGE/hybridization) assays among Salmonella with diverse epidemiological and genetic backgrounds. Then, to better understand ars operon genetic contexts, the whole genome of five representative strains was sequenced. We found a high dispersion of ars operons conferring AsT, especially among copper-tolerant and relevant serotypes/clones related to pig-production setting. The acr3-type was found dispersed in the chromosome of diverse serotypes, including the emergent S. Rissen. Conversely, arsBII was almost confined to the MDR ST34 European clone of S. Typhimurium/S. 4,[5],12:i:-, always along with copper/silver tolerance sil + pco clusters in an integrative conjugative element. These data suggest that AsT is an essential adaptive feature for the ecological success of these epidemic clones/serotypes and alerts for global strategies to reduce arsenic-based compounds' impact thus preventing environmental/food contamination with frequent MDR foodborne pathogens.

Abstract Considering the high incidence level and mortality rate of ovarian cancer, particularly among the European female population, the carbohydrate antigen 125 (CA-125) was selected as the protein target for this study for the development of a MIP-based biosensor. This work presents the development of molecular imprinting polymers (MIPs) on gold electrode surface for CA-125 biomarker recognition. The preparation of the CA-125 imprinting was obtained by electropolymerization of pyrrole (Py) monomer in a gold electrode using cyclic voltammetry (CV) in order to obtain highly selective materials with great molecular recognition capability. The quantification of CA-125 biomarker was made through the comparison of two methods: electrochemical (square wave voltammetry -SWV) and optical transduction (surface plasmon resonance -SPR). SWV has been widely used in biological molecules analysis since it is a fast and sensitive technique. In turn, SPR is a non-destructive optical technique that provides high-quality analytical data of CA-125 biomarker interactions with MIP. Several analytical parameters, such as sensitivity, linear response interval, and detection limit were determined to proceed to the performance evaluation of the electrochemical and optical transduction used in the development of the CA-125 biosensor. The biosensor based in the electrochemical transduction was the one that presented the best analytical parameters, yielding a good selectivity and a detection limit (LOD) of 0.01 U/mL, providing a linear concentration range between 0.01 and 500 U/mL. This electrochemical biosensor was selected for the study and it was successfully applied in the CA-125 analysis in artificial serum samples with recovery rates ranging from 91 to 105% with an average relative error of 5.8%.

Abstract In the past few years a large effort is being made aiming at the development of fast and reliable tests for cancer biomarkers. Protein imprinted sensors can be a fast and reliable strategy to develop tailor made sensors for a large number of relevant molecules. This work aims to produce, optimize and use in biological samples a biosensor for microseminoproteinbeta (MSMB). Caffeic acid (CAF) electropolimerization was performed in the presence of microseminoprotein-beta (MSMB) creating target protein specific cavities on the surface of a screen-printed carbon. Dopamine was introduced as charged monomer labelling the binding site and was allowed to self-organize around the protein. The subsequent electropolimerization was made by applying a constant potential of +2.0 V, for 30s, on a carbon screen-printed electrode, immersed in a solution of protein and CAF prepared in phosphate buffer. The sensor with charged monomers showed a more sensitive response, with an average slope of -7.59 mu A/decade, linear concentration range of 0.5-100 ng/mL and a detection limit of 0.12 ng/mL. The corresponding non-imprinted sensor displayed an inconsistent response over the range of the calibration curve. The biosensor was successfully applied to the analysis of MSMB in serum and urine samples.

Abstract The development of fast and reliable methods for protein determination are of great relevance to a diversity of areas from industry to diagnostics. Molecular Imprinted Materials (MIM) has proved to be an interesting methodology for protein analysis however further studies of the effect of the experimental parameters and starting materials in the performance of the MIM are still required. Caffeic acid (CAF) is employed for the first time as a monomer to tailor a synthetic receptor for a protein target. This was done by bulk-electropolymerization, applying a constant potential of +2.0 V, for 30 s, on a carbon screen printed electrode, immersed in a solution of protein and CAF prepared in phosphate buffer. Annexin A3 (ANXA3) was selected as protein target due to the fact that this is an emerging biomarker in prostate cancer. The assembly of the protein imprinted material (PIM) was followed by Electrochemical Impedance Spectroscopy (EIS) and Raman Spectroscopy. A non-imprinted material (NIM) was prepared in parallel as control. Square wave voltammetry (SWV) was used to monitor the electrochemical signal of the [Fe(CN)(6)](3-)/[Fe(CN)(6)](4-) redox for the quantification of ANXA3. The optimized PIM-based device showed average detection limits (LOD) of 0.095 ng/mL, a linear behavior against log (concentration) between 0.10, and 200 ng/mL and good selectivity. The NIM-based device showed random behavior against protein concentration. Finally, the PIM-sensor was successfully applied to the analysis of ANXA3 in spiked urine samples.

Abstract Prostate Specific Antigen (PSA) is widely used as a biomarker for prostate cancer. Recently, an electrochemical biosensor for PSA detection by means of molecularly imprinted polymers (MIPs) was developed. This work evaluated the performance and the effectiveness of that PSA biosensor in screening the biomarker PSA in biological media with complex composition, collected from different human prostate cell line cultures. For that, the prostate cancer LNCaP and PC3 cells, and the non-cancerous prostate cell line PNT2 were cultured for 2, 7 and 14 days in either alpha-MEM or RPMI in the presence of 10% or 30% fetal bovine serum. Human gingival fibroblasts were used as a non-cancerous non-prostatic control. The different culture conditions modulated cellular proliferation and the expression of several prostate markers, including PSA. The electrochemical biosensor was able to specifically detect PSA in the culture media and values obtained were similar to those achieved by a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit, the most commonly used method for PSA quantification in prostate cancer diagnosis. Thus, the tested biosensor may represent a useful alternative as a diagnostic tool for PSA determination in biological samples.

Abstract As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H2O2 (at 0.6 V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16 nM, using a linear concentration range between 10 and 100 nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.

Abstract This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 x 10(-11) mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9 to 106.1% and relative errors of 6.8%.