Gilberto Igrejas - Academia.edu (original) (raw)

Papers by Gilberto Igrejas

Italian Journal of Animal Science, 2006

A total of 36 Escherichia coli and 31 enterococci isolates were recovered from 42 common buzzard ... more A total of 36 Escherichia coli and 31 enterococci isolates were recovered from 42 common buzzard faecal samples. The E. coli isolates showed high levels of resistance to streptomycin and tetracycline. The following resistance genes were detected: bla TEM (20 of 22 ampicillin-resistant isolates), tet(A) and/or tet(B) (16 of 27 tetracycline-resistant isolates), aadA1 (eight of 27 streptomycin-resistant isolates), cmlA (three of 15 chloramphenicol-resistant isolates), aac(3)-II with/without aac(3)-IV (all seven gentamicin-resistant isolates) and sul1 and/or sul2 and/or sul3 [all eight sulfamethoxazole/trimethoprim-resistant (SXT) isolates]. intI1 and intI2 genes were detected in four SXT-resistant isolates. The virulence-associated genes fimA (type 1 fimbriae), papC (P fimbriae) and aer (aerobactin) were detected in 61.1, 13.8 and 11.1 % of the isolates, respectively. The isolates belonged to phylogroups A (47.2 %), B1 (8.3 %), B2 (13.9 %) and D (30.5 %). For the enterococci isolates, Enterococcus faecium was the most prevalent species (48.4 %). High levels of tetracycline and erythromycin resistance were found among our isolates (87 and 81 %, respectively). Most of the tetracycline-resistant strains carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 80 % of erythromycin-resistant isolates. The vat(D) and/or vat(E) genes were found in nine of the 17 quinupristin-dalfopristin-resistant isolates. The enterococcal isolates showing high-level resistance for kanamycin, gentamicin and streptomycin contained the aph(39)-IIIa, aac(69)-aph(20) and ant(6)-Ia genes, respectively. This report reveals that common buzzards seem to represent an important reservoir, or at least a source, of multiresistant E. coli and enterococci isolates, and consequently may represent a considerable hazard to human and animal health by transmission of these isolates to waterways and other environmental sources via their faecal deposits.

Frontiers in Microbiology, 2016

Antimicrobial resistance (AMR) is a worldwide problem with serious health and economic repercussi... more Antimicrobial resistance (AMR) is a worldwide problem with serious health and economic repercussions. Since the 1940s, underuse, overuse, and misuse of antibiotics have had a significant environmental downside. Large amounts of antibiotics not fully metabolized after use in human and veterinary medicine, and other applications, are annually released into the environment. The result has been the development and dissemination of antibiotic-resistant bacteria due to many years of selective pressure. Surveillance of AMR provides important information that helps in monitoring and understanding how resistance mechanisms develop and disseminate within different environments. Surveillance data is needed to inform clinical therapy decisions, to guide policy proposals, and to assess the impact of action plans to fight AMR. The Functional Genomics and Proteomics Unit, based at the University of Trás-os-Montes and Alto Douro in Vila Real, Portugal, has recently completed 10 years of research surveying AMR in bacteria, mainly commensal indicator bacteria such as enterococci and Escherichia coli from the microbiota of different animals. Samples from more than 75 different sources have been accessed, from humans to foodproducing animals, pets, and wild animals. The typical microbiological workflow involved phenotypic studies followed by molecular approaches. Throughout the decade, 4,017 samples were collected and over 5,000 bacterial isolates obtained. High levels of AMR to several antimicrobial classes have been reported, including to β-lactams, glycopeptides, tetracyclines, aminoglycosides, sulphonamides, and quinolones. Multi-resistant strains, some relevant to human and veterinary medicine like extended-spectrum β-lactamaseproducing E. coli and vancomycin-resistant enterococci, have been repeatedly isolated even in non-synanthropic animal species. Of particular relevance are reports of AMR bacteria in wildlife from natural reserves and endangered species. Future work awaits as this threatening yet unsolved problem persists.

Breast cancer research : BCR, 2006

Protein-based breast cancer biomarkers are a promising resource for breast cancer detection at th... more Protein-based breast cancer biomarkers are a promising resource for breast cancer detection at the earliest and most treatable stages of the disease. Plasma is well suited to proteomic-based methods of biomarker discovery because it is easily obtained, is routinely used in the diagnosis of many diseases, and has a rich proteome. However, due to the vast dynamic range in protein concentration and the often uncertain tissue and cellular origin of plasma proteins, proteomic analysis of plasma requires special consideration compared with tissue and cultured cells. This review briefly touches on the search for plasma-based protein biomarkers for the early detection and treatment of breast cancer.

1Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro, Vila Real... more 1Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro, Vila Real, Portugal 2Functional Genomics and Proteomics Unit, University of Tras -os-Montes and Alto Douro, Vila Real, Portugal 3Cytogenetics Laboratory, Genetics Service, Hospitalar Centre of Tras -os-Montes and Alto Douro, Vila Real, Portugal 4Bioscope Group, UCIBIO-REQUIMTE. Faculty of Sciences and Technology, New University of Lisbon, Campus de Caparica, Caparica, Portugal 5ProteoMass Scientific Society, Faculty of Sciences and Technology, Campus de Caparica, Portugal 6UCIBIO-REQUIMTE, Faculty of Science and Technology, University Nova of Lisbon, Caparica, Portugal #These authors contributed equally to this work

Frontiers in Microbiology, 2017

Foodborne Pathogens and Disease, 2015

The use of lactic acid bacteria of aquatic origin as probiotics constitutes an alternative strate... more The use of lactic acid bacteria of aquatic origin as probiotics constitutes an alternative strategy to the antibiotic treatment for disease control in aquaculture. Enterococci are currently used as probiotics in human and animal health. In this study, we evaluated the safety of 64 enterococci isolated from rainbow trout (Oncorhynchus mykiss, Walbaum), feed and rearing environment, and their antimicrobial activity against 9 fish pathogens. The 64 enterococcal isolates were identified to the species level by polymerase chain reaction (PCR), using specific primers for the different enterococcal species, and confirmed by superoxide dismutase gene sequencing. Enterococcus faecium and E. hirae were the most common species (42.2 and 35.9%, respectively). A total of 48 isolates (75%) showed phenotypic resistance to at least 1 antibiotic determined by a disk-diffusion method, and 25 isolates (39.1%) harbored at least 1 antibiotic resistance gene [erm(B), tet(M), tet(S), tet(K), tet(L), tet(T), vanC2, and aad(E)], detected by PCR. One (1.6%) isolate produced gelatinase and none produced hemolysin, using a plate assay. The virulence genes gelE (46.9%), efaAfs (17.2%), agg (1.6%), and hyl (1.6%) were detected by PCR. A total of 48 isolates (75%) exerted antimicrobial activity against 1 or more of the tested fish pathogens, using a stab-on-agar test. From these isolates, 21 (43.8%) harbored at least 1 bacteriocin-encoding gene (entP, entL50A and entL50B, hirJM79, entSE-K4, entQ and entA), detected by PCR. None of the enterococci showed bile deconjugation and mucin degradation abilities. A total of 17 enterococcal isolates (26.6%) that did not harbor any antibiotic resistance or virulence factor were considered safe for application as probiotics, including 6 isolates (35.3%) that showed antimicrobial activity against at least 1 fish pathogen and harbored at least 1 bacteriocin-encoding gene. Rainbow trout, feed, and rearing environment constitute an appropriate source for the isolation of enterococci as potential probiotic for aquaculture.

Veterinary journal (London, England : 1997), 2012

The prevalence of vancomycin resistant-enterococci (VRE) in faecal samples from cattle, sheep and... more The prevalence of vancomycin resistant-enterococci (VRE) in faecal samples from cattle, sheep and pigs slaughtered for human consumption was evaluated. Enterococci containing the vanA gene were detected in 25.3% and 2.7% of the porcine and ovine samples, respectively, and were identified as Enterococcus faecium. No vanA-containing enterococcal strains were detected in bovine samples. Enterococcal strains with intrinsic vancomycin resistance were detected in seven (9.9%) faecal samples from pigs and in two samples from both cattle and sheep (3.7% and 2.7%, respectively). All vanA-positive isolates from pigs were resistant to tetracycline and erythromycin, and the mobile element Tn916/Tn1545-like transposon was detected in 90.5% of the tetracycline-resistant isolates that contained the tet(M) gene. Although gelatinase and haemolytic activity were not detected, the hyl and cylB virulence genes were found within the VRE strains isolated.

Marine Pollution Bulletin, 2011

Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinica... more Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinical setting but also to other environments such as marine species that harbor antibiotic resistant bacteria and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to evaluate antibiotic resistance phenotypes in enterococci isolated from fecal samples of gilthead seabream and the associated mechanisms of resistance. A collection of 118 samples were analyzed and 73 enterococci were recovered. The strains showed high percentages of resistance to erythromycin and tetracycline (58.9% and 17.8%, respectively). Lower level of resistance (<13%) was detected for quinupristin-dalfopristin, ampicillin, high-level-gentamicin, high-level-streptomycin, high-level-kanamycin, ciprofloxacin and chloramphenicol. The erm(B), tet(L) or tet(M),…

Journal of Proteomics, 2012

Proteomic approaches have been considerably improved during the past decade and have been used to... more Proteomic approaches have been considerably improved during the past decade and have been used to investigate the differences in protein expression profiles of cells grown under a broad spectrum of growth conditions and with different stress factors including antibiotics. In Europe, the most significant disease threat remains the presence of microorganisms that have become resistant to antimicrobials and so it is important that different scientific tools are combined to achieve the largest amount of knowledge in this area of expertise. The emergence and spread of the antibiotic-resistant Gram-negative pathogens, such as Escherichia coli, can lead to serious problem public health in humans. E. coli, a very well described prokaryote, has served as a model organism for several biological and biotechnological studies increasingly so since the completion of the E. coli genome-sequencing project. The purpose of this review is to present an overview of the different proteomic approaches to antimicrobial-resistant E. coli that will be helpful to obtain a better knowledge of the antibiotic-resistant mechanism(s). This can also aid to understand the molecular determinants involved with pathogenesis, which is essential for the development of effective strategies to combat infection and to reveal new therapeutic targets. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Journal of Proteomics, 2010

Using Salmonella strains identical to those present in the gastrointestinal tract of different an... more Using Salmonella strains identical to those present in the gastrointestinal tract of different animals we aim to determine and compare the proteome of two serotypes, Salmonella Typhimurium and Enteritidis recovered from faecal samples of wild boars and wild rabbits, respectively. The presence of genes responsible for antibiotic resistance was detected by PCR. Proteomes of the two distinct serotypes were determined using 2-DE in order to identify proteins associated with antibiotic resistance or virulence. Through 2-DE we obtained a total of 229 spots from both strains. All were suitable for MALDI-TOF/TOF and, in correlation with bioinformatic databases, allowed accurate identification and characterization of proteins. S. Enteritidis recovered from wild rabbits was sensitive to all the antibiotics tested in contrast to S. Typhimurium isolated from wild boars which presented a resistance phenotype to ampicillin, streptomycin and chloramphenicol. Nevertheless, despite the different ratio of proteins observed in each proteome according to their biological function, no significant difference was observed in the involvement of these proteins in pathogenicity. Bearing in mind that serotypes are related to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.

Journal of Basic Microbiology, 2008

os-Montes and Alto Douro Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopept... more os-Montes and Alto Douro Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopeptide with activity against gram-positive organisms. We investigated the activity of dalbavancin against faecal enterococci isolates from wild animals, pets, poultry and healthy humans in Portugal. The in vitro activity of dalbavancin was determined by the microbroth dilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines in 589 enterococci of different species and origins. All vancomycin-susceptible Enterococcus spp. were inhibited by ≤0.25 mg/l dalbavancin. Although vancomycin-resistant-enterococci (VRE) showed higher dalbavancin MIC values (16 mg/l), the isolates that exhibited the VanC resistance phenotype were inhibited at dalbavancin concentrations ≤0.125 mg/l. Only vanA isolates were not inhibited by low concentrations of dalbavancin since vanA strains showed higher dalbavancin MIC values (16 mg/l).

Journal of Basic Microbiology, 2009

The presence of vanA-containing E. faecium isolates was demonstrated in three of 77 faecal sample... more The presence of vanA-containing E. faecium isolates was demonstrated in three of 77 faecal samples (3.9%) of wild rabbits recovered in Portugal. Enterococcal strains with intrinsic vancomycin resistance (vanC-1 or vanC-2/3 gene) were found in five (6.5%) and three (3.9%) faecal samples, respectively. The mechanisms of resistance for other antibiotics were studied in these vancomycin-resistant isolates. All vanA strains showed resistance for tetracycline [with the presence of tet(L) gene, associated or not with tet(M) gene] and for erythromycin [with the presence of the erm(B) gene]. Two isolates were resistant to ciprofloxacin and one to ampicillin. Two vanC-1 strains and one vanC-2/3 strain were tetracycline resistant [containing the tet(M) gene associated with tet(L) gene] and erythromycin resistant [with erm(B) gene]. Two vanC-1 and two vanC-2/3 strains were also ciprofloxacin resistant and one vanC-1 strain was, additionally, resistant to quinupristin-dalfopristin. The two remaining isolates (vanC-1, vanC-2/3) did not show resistance for any additional antibiotic. The intestinal tract of wild rabbits could be a reservoir of vanA-containing enterococci.

Journal of Basic Microbiology, 2008

The production of antimicrobial, haemolytic and gelatinase activities was tested in 67 enterococc... more The production of antimicrobial, haemolytic and gelatinase activities was tested in 67 enterococci (39 E. faecium, 24 E. hirae, 2 E. faecalis, and 2 Enterococcus spp.), recovered from faecal samples of wild boars. In addition, the presence of genes encoding bacteriocin and virulence factors was also analysed by PCR and sequencing. Production of antimicrobial activity was checked in all enterococci against 9 indicator bacteria and it was detected in 11 E. faecium isolates (16.5%); eight and two of them harboured the genes encoding enterocin A + enterocin B and enterocin L50A/B, respectively. Sixty-seven per cent of our enterococci harboured different combinations of genes of the cyl operon, but none of them contained the complete cylL L L S ABM operon, necessary for cytolysin expression. The presence of gelE gene, associated with the fsrABC locus, was identified in 4 E. faecium and two E. faecalis isolates, exhibiting all of them gelatinase activity. β-hemolytic activity was not found in our isolates. Both cpd and ace genes, encoding respectively the accessory colonisation factor and pheromone, were detected in two E. faecalis isolates, and the hyl gene, encoding hyalorunidase, in two E. faecium isolates, one of them gelatinase-positive. Genes encoding bacteriocins and virulence factors are widely disseminated among faecal enterococci of wild boars and more studies should be carried out to know the global distribution of these determinants in enterococci of different ecosystems.

Journal of Antimicrobial Chemotherapy, 2010

one bilioma). In addition, in one neighbouring hospital, crosstransmission has also been observed... more one bilioma). In addition, in one neighbouring hospital, crosstransmission has also been observed. All the KPC-Kp isolates were identical to a previously identified K. pneumoniae ST258-type clone that is epidemic in Greece, Israel and the USA, as revealed by PFGE, plasmid analysis, multilocus sequence typing and Tn4401 transposon typing. 2,8 PCR experiments, followed by sequencing, identified additional b-lactamase genes coding for the naturally occurring narrowspectrum SHV-11, the plasmid-encoded narrow-spectrum TEM-1 and extended-spectrum SHV-12. In France, KPC-Kp remain rare and, to date, have always been linked to patient transfer from a country where KPC-Kp are endemic. 2 This is the first KPC-Kp outbreak in France and the first worldwide to be linked to a contaminated endoscope. Although the risk of endoscopy-related infection is low, 6 changes to endoscope reprocessing, by replacing a glutaraldehyde decontamination bath with an automated peracetic acid washer (to prevent Creutzfeldt-Jacob transmission), may have been deleterious to the endoscope. However, careful checking of the endoscope by the instrument's manufacturer did not reveal any obvious signs of degradation. Careful auditing of endoscope reprocessing revealed two possible explanations for the contamination: (i) the pre-wash of the endoscope may have been delayed 24 h, thus resulting in drying of the device; and (ii) after the peracetic wash, the drying procedure was not long enough for the novel automated washer, thus the endoscope was not completely dried. In light of this outbreak, new local guidelines for endoscope reprocessing have been established, taking into account the specificities of the new automated peracetic acid washer. Microbiological testing of endoscopes, performed until now twice a year, will now be undertaken on a more frequent basis.

Journal of Antimicrobial Chemotherapy, 2009

Microbial drug resistance (Larchmont, N.Y.), 2011

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of ... more The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This s...

Foodborne Pathogens and Disease, 2010

Background: Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteria... more Background: Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we characterize a new AmpC variant presenting a broadened substrate activity towards fourth-generation cephalosporins, selected in vivo following cefepime treatment for Enterobacter aerogenes. Methods: Two consecutive clonally related isolates of E. aerogenes were evaluated. Screening for ESAC production was performed using plates containing 200 mg/L cloxacillin. MICs were determined by microdilution (CLSI guidelines). bla AmpC genes were cloned into a pCR-Blunt II-TOPO vector and expressed in Escherichia coli. The ampC genes were cloned into vector pGEX-6P-1 for protein purification. Results: Isolate Ea595 was resistant to two fourth-generation cephalosporins, cefepime and cefpirome; using plates containing cloxacillin, susceptibility to ceftazidime and cefepime was restored, suggesting overproduction of the ESAC b-lactamase. Sequencing identified a new AmpC b-lactamase variant presenting one amino acid substitution, Val291Gly, inside the H-10 helix. Recombinant plasmids harbouring this ESAC b-lactamase conferred a broadened resistance profile to cefepime and cefpirome, with resistance levels increasing from 16-to 32-fold in E. coli. AmpC-Ea595 hydrolysed ceftazidime, cefepime and cefpirome at high levels, presenting a lower K m and enabling us to classify the enzyme as an ESAC. Homology modelling suggested that the size of the active site could have increased. Conclusions: We characterized an ESAC b-lactamase selected in vivo and conferring a high level of resistance to fourth-generation cephalosporins in E. aerogenes. The broadened spectrum was caused by a new modification to the H-10 helix, which modified the active site.

Foodborne Pathogens and Disease, 2011

Foodborne Pathogens and Disease, 2013

We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escheri... more We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy pigs, and to evaluate their clonality and associated resistance. Forty-nine percent of pigs sampled (n = 35/71) in a slaughterhouse in Portugal revealed ESBL-producing E. coli isolates. Most isolates produced CTX-M-1 enzyme (71.4%; n = 25/35), followed by CTX-M-9 (11.4%; n = 4/35), CTX-M-14 (5.7%; n = 2/35), SHV-12 (5.7%; n = 2/35), and CTX-M-32 (5.7%; n = 2/35). Ninety-four percent of the isolates presented a phenotype of multi-resistance. Most isolates belonged to phylogroups B1 (42.8%; n = 15/35) and A (40%; n = 14/35). Multilocus sequence typing (MLST) analysis revealed nine sequence types (STs) under six clonal complexes (CCs) and nine singletons, including overrepresentation of CC10 and three new STs (ST2524, ST2525, ST2528). We observed the frequent presence of CTX-M-producing E. coli in pigs at slaughter level, most of them belonging to CC10, commonly recovered from clinical samples.

Italian Journal of Animal Science, 2006

A total of 36 Escherichia coli and 31 enterococci isolates were recovered from 42 common buzzard ... more A total of 36 Escherichia coli and 31 enterococci isolates were recovered from 42 common buzzard faecal samples. The E. coli isolates showed high levels of resistance to streptomycin and tetracycline. The following resistance genes were detected: bla TEM (20 of 22 ampicillin-resistant isolates), tet(A) and/or tet(B) (16 of 27 tetracycline-resistant isolates), aadA1 (eight of 27 streptomycin-resistant isolates), cmlA (three of 15 chloramphenicol-resistant isolates), aac(3)-II with/without aac(3)-IV (all seven gentamicin-resistant isolates) and sul1 and/or sul2 and/or sul3 [all eight sulfamethoxazole/trimethoprim-resistant (SXT) isolates]. intI1 and intI2 genes were detected in four SXT-resistant isolates. The virulence-associated genes fimA (type 1 fimbriae), papC (P fimbriae) and aer (aerobactin) were detected in 61.1, 13.8 and 11.1 % of the isolates, respectively. The isolates belonged to phylogroups A (47.2 %), B1 (8.3 %), B2 (13.9 %) and D (30.5 %). For the enterococci isolates, Enterococcus faecium was the most prevalent species (48.4 %). High levels of tetracycline and erythromycin resistance were found among our isolates (87 and 81 %, respectively). Most of the tetracycline-resistant strains carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 80 % of erythromycin-resistant isolates. The vat(D) and/or vat(E) genes were found in nine of the 17 quinupristin-dalfopristin-resistant isolates. The enterococcal isolates showing high-level resistance for kanamycin, gentamicin and streptomycin contained the aph(39)-IIIa, aac(69)-aph(20) and ant(6)-Ia genes, respectively. This report reveals that common buzzards seem to represent an important reservoir, or at least a source, of multiresistant E. coli and enterococci isolates, and consequently may represent a considerable hazard to human and animal health by transmission of these isolates to waterways and other environmental sources via their faecal deposits.

Frontiers in Microbiology, 2016

Antimicrobial resistance (AMR) is a worldwide problem with serious health and economic repercussi... more Antimicrobial resistance (AMR) is a worldwide problem with serious health and economic repercussions. Since the 1940s, underuse, overuse, and misuse of antibiotics have had a significant environmental downside. Large amounts of antibiotics not fully metabolized after use in human and veterinary medicine, and other applications, are annually released into the environment. The result has been the development and dissemination of antibiotic-resistant bacteria due to many years of selective pressure. Surveillance of AMR provides important information that helps in monitoring and understanding how resistance mechanisms develop and disseminate within different environments. Surveillance data is needed to inform clinical therapy decisions, to guide policy proposals, and to assess the impact of action plans to fight AMR. The Functional Genomics and Proteomics Unit, based at the University of Trás-os-Montes and Alto Douro in Vila Real, Portugal, has recently completed 10 years of research surveying AMR in bacteria, mainly commensal indicator bacteria such as enterococci and Escherichia coli from the microbiota of different animals. Samples from more than 75 different sources have been accessed, from humans to foodproducing animals, pets, and wild animals. The typical microbiological workflow involved phenotypic studies followed by molecular approaches. Throughout the decade, 4,017 samples were collected and over 5,000 bacterial isolates obtained. High levels of AMR to several antimicrobial classes have been reported, including to β-lactams, glycopeptides, tetracyclines, aminoglycosides, sulphonamides, and quinolones. Multi-resistant strains, some relevant to human and veterinary medicine like extended-spectrum β-lactamaseproducing E. coli and vancomycin-resistant enterococci, have been repeatedly isolated even in non-synanthropic animal species. Of particular relevance are reports of AMR bacteria in wildlife from natural reserves and endangered species. Future work awaits as this threatening yet unsolved problem persists.

Breast cancer research : BCR, 2006

Protein-based breast cancer biomarkers are a promising resource for breast cancer detection at th... more Protein-based breast cancer biomarkers are a promising resource for breast cancer detection at the earliest and most treatable stages of the disease. Plasma is well suited to proteomic-based methods of biomarker discovery because it is easily obtained, is routinely used in the diagnosis of many diseases, and has a rich proteome. However, due to the vast dynamic range in protein concentration and the often uncertain tissue and cellular origin of plasma proteins, proteomic analysis of plasma requires special consideration compared with tissue and cultured cells. This review briefly touches on the search for plasma-based protein biomarkers for the early detection and treatment of breast cancer.

1Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro, Vila Real... more 1Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro, Vila Real, Portugal 2Functional Genomics and Proteomics Unit, University of Tras -os-Montes and Alto Douro, Vila Real, Portugal 3Cytogenetics Laboratory, Genetics Service, Hospitalar Centre of Tras -os-Montes and Alto Douro, Vila Real, Portugal 4Bioscope Group, UCIBIO-REQUIMTE. Faculty of Sciences and Technology, New University of Lisbon, Campus de Caparica, Caparica, Portugal 5ProteoMass Scientific Society, Faculty of Sciences and Technology, Campus de Caparica, Portugal 6UCIBIO-REQUIMTE, Faculty of Science and Technology, University Nova of Lisbon, Caparica, Portugal #These authors contributed equally to this work

Frontiers in Microbiology, 2017

Foodborne Pathogens and Disease, 2015

The use of lactic acid bacteria of aquatic origin as probiotics constitutes an alternative strate... more The use of lactic acid bacteria of aquatic origin as probiotics constitutes an alternative strategy to the antibiotic treatment for disease control in aquaculture. Enterococci are currently used as probiotics in human and animal health. In this study, we evaluated the safety of 64 enterococci isolated from rainbow trout (Oncorhynchus mykiss, Walbaum), feed and rearing environment, and their antimicrobial activity against 9 fish pathogens. The 64 enterococcal isolates were identified to the species level by polymerase chain reaction (PCR), using specific primers for the different enterococcal species, and confirmed by superoxide dismutase gene sequencing. Enterococcus faecium and E. hirae were the most common species (42.2 and 35.9%, respectively). A total of 48 isolates (75%) showed phenotypic resistance to at least 1 antibiotic determined by a disk-diffusion method, and 25 isolates (39.1%) harbored at least 1 antibiotic resistance gene [erm(B), tet(M), tet(S), tet(K), tet(L), tet(T), vanC2, and aad(E)], detected by PCR. One (1.6%) isolate produced gelatinase and none produced hemolysin, using a plate assay. The virulence genes gelE (46.9%), efaAfs (17.2%), agg (1.6%), and hyl (1.6%) were detected by PCR. A total of 48 isolates (75%) exerted antimicrobial activity against 1 or more of the tested fish pathogens, using a stab-on-agar test. From these isolates, 21 (43.8%) harbored at least 1 bacteriocin-encoding gene (entP, entL50A and entL50B, hirJM79, entSE-K4, entQ and entA), detected by PCR. None of the enterococci showed bile deconjugation and mucin degradation abilities. A total of 17 enterococcal isolates (26.6%) that did not harbor any antibiotic resistance or virulence factor were considered safe for application as probiotics, including 6 isolates (35.3%) that showed antimicrobial activity against at least 1 fish pathogen and harbored at least 1 bacteriocin-encoding gene. Rainbow trout, feed, and rearing environment constitute an appropriate source for the isolation of enterococci as potential probiotic for aquaculture.

Veterinary journal (London, England : 1997), 2012

The prevalence of vancomycin resistant-enterococci (VRE) in faecal samples from cattle, sheep and... more The prevalence of vancomycin resistant-enterococci (VRE) in faecal samples from cattle, sheep and pigs slaughtered for human consumption was evaluated. Enterococci containing the vanA gene were detected in 25.3% and 2.7% of the porcine and ovine samples, respectively, and were identified as Enterococcus faecium. No vanA-containing enterococcal strains were detected in bovine samples. Enterococcal strains with intrinsic vancomycin resistance were detected in seven (9.9%) faecal samples from pigs and in two samples from both cattle and sheep (3.7% and 2.7%, respectively). All vanA-positive isolates from pigs were resistant to tetracycline and erythromycin, and the mobile element Tn916/Tn1545-like transposon was detected in 90.5% of the tetracycline-resistant isolates that contained the tet(M) gene. Although gelatinase and haemolytic activity were not detected, the hyl and cylB virulence genes were found within the VRE strains isolated.

Marine Pollution Bulletin, 2011

Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinica... more Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinical setting but also to other environments such as marine species that harbor antibiotic resistant bacteria and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to evaluate antibiotic resistance phenotypes in enterococci isolated from fecal samples of gilthead seabream and the associated mechanisms of resistance. A collection of 118 samples were analyzed and 73 enterococci were recovered. The strains showed high percentages of resistance to erythromycin and tetracycline (58.9% and 17.8%, respectively). Lower level of resistance (<13%) was detected for quinupristin-dalfopristin, ampicillin, high-level-gentamicin, high-level-streptomycin, high-level-kanamycin, ciprofloxacin and chloramphenicol. The erm(B), tet(L) or tet(M),…

Journal of Proteomics, 2012

Proteomic approaches have been considerably improved during the past decade and have been used to... more Proteomic approaches have been considerably improved during the past decade and have been used to investigate the differences in protein expression profiles of cells grown under a broad spectrum of growth conditions and with different stress factors including antibiotics. In Europe, the most significant disease threat remains the presence of microorganisms that have become resistant to antimicrobials and so it is important that different scientific tools are combined to achieve the largest amount of knowledge in this area of expertise. The emergence and spread of the antibiotic-resistant Gram-negative pathogens, such as Escherichia coli, can lead to serious problem public health in humans. E. coli, a very well described prokaryote, has served as a model organism for several biological and biotechnological studies increasingly so since the completion of the E. coli genome-sequencing project. The purpose of this review is to present an overview of the different proteomic approaches to antimicrobial-resistant E. coli that will be helpful to obtain a better knowledge of the antibiotic-resistant mechanism(s). This can also aid to understand the molecular determinants involved with pathogenesis, which is essential for the development of effective strategies to combat infection and to reveal new therapeutic targets. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Journal of Proteomics, 2010

Using Salmonella strains identical to those present in the gastrointestinal tract of different an... more Using Salmonella strains identical to those present in the gastrointestinal tract of different animals we aim to determine and compare the proteome of two serotypes, Salmonella Typhimurium and Enteritidis recovered from faecal samples of wild boars and wild rabbits, respectively. The presence of genes responsible for antibiotic resistance was detected by PCR. Proteomes of the two distinct serotypes were determined using 2-DE in order to identify proteins associated with antibiotic resistance or virulence. Through 2-DE we obtained a total of 229 spots from both strains. All were suitable for MALDI-TOF/TOF and, in correlation with bioinformatic databases, allowed accurate identification and characterization of proteins. S. Enteritidis recovered from wild rabbits was sensitive to all the antibiotics tested in contrast to S. Typhimurium isolated from wild boars which presented a resistance phenotype to ampicillin, streptomycin and chloramphenicol. Nevertheless, despite the different ratio of proteins observed in each proteome according to their biological function, no significant difference was observed in the involvement of these proteins in pathogenicity. Bearing in mind that serotypes are related to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.

Journal of Basic Microbiology, 2008

os-Montes and Alto Douro Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopept... more os-Montes and Alto Douro Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopeptide with activity against gram-positive organisms. We investigated the activity of dalbavancin against faecal enterococci isolates from wild animals, pets, poultry and healthy humans in Portugal. The in vitro activity of dalbavancin was determined by the microbroth dilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines in 589 enterococci of different species and origins. All vancomycin-susceptible Enterococcus spp. were inhibited by ≤0.25 mg/l dalbavancin. Although vancomycin-resistant-enterococci (VRE) showed higher dalbavancin MIC values (16 mg/l), the isolates that exhibited the VanC resistance phenotype were inhibited at dalbavancin concentrations ≤0.125 mg/l. Only vanA isolates were not inhibited by low concentrations of dalbavancin since vanA strains showed higher dalbavancin MIC values (16 mg/l).

Journal of Basic Microbiology, 2009

The presence of vanA-containing E. faecium isolates was demonstrated in three of 77 faecal sample... more The presence of vanA-containing E. faecium isolates was demonstrated in three of 77 faecal samples (3.9%) of wild rabbits recovered in Portugal. Enterococcal strains with intrinsic vancomycin resistance (vanC-1 or vanC-2/3 gene) were found in five (6.5%) and three (3.9%) faecal samples, respectively. The mechanisms of resistance for other antibiotics were studied in these vancomycin-resistant isolates. All vanA strains showed resistance for tetracycline [with the presence of tet(L) gene, associated or not with tet(M) gene] and for erythromycin [with the presence of the erm(B) gene]. Two isolates were resistant to ciprofloxacin and one to ampicillin. Two vanC-1 strains and one vanC-2/3 strain were tetracycline resistant [containing the tet(M) gene associated with tet(L) gene] and erythromycin resistant [with erm(B) gene]. Two vanC-1 and two vanC-2/3 strains were also ciprofloxacin resistant and one vanC-1 strain was, additionally, resistant to quinupristin-dalfopristin. The two remaining isolates (vanC-1, vanC-2/3) did not show resistance for any additional antibiotic. The intestinal tract of wild rabbits could be a reservoir of vanA-containing enterococci.

Journal of Basic Microbiology, 2008

The production of antimicrobial, haemolytic and gelatinase activities was tested in 67 enterococc... more The production of antimicrobial, haemolytic and gelatinase activities was tested in 67 enterococci (39 E. faecium, 24 E. hirae, 2 E. faecalis, and 2 Enterococcus spp.), recovered from faecal samples of wild boars. In addition, the presence of genes encoding bacteriocin and virulence factors was also analysed by PCR and sequencing. Production of antimicrobial activity was checked in all enterococci against 9 indicator bacteria and it was detected in 11 E. faecium isolates (16.5%); eight and two of them harboured the genes encoding enterocin A + enterocin B and enterocin L50A/B, respectively. Sixty-seven per cent of our enterococci harboured different combinations of genes of the cyl operon, but none of them contained the complete cylL L L S ABM operon, necessary for cytolysin expression. The presence of gelE gene, associated with the fsrABC locus, was identified in 4 E. faecium and two E. faecalis isolates, exhibiting all of them gelatinase activity. β-hemolytic activity was not found in our isolates. Both cpd and ace genes, encoding respectively the accessory colonisation factor and pheromone, were detected in two E. faecalis isolates, and the hyl gene, encoding hyalorunidase, in two E. faecium isolates, one of them gelatinase-positive. Genes encoding bacteriocins and virulence factors are widely disseminated among faecal enterococci of wild boars and more studies should be carried out to know the global distribution of these determinants in enterococci of different ecosystems.

Journal of Antimicrobial Chemotherapy, 2010

one bilioma). In addition, in one neighbouring hospital, crosstransmission has also been observed... more one bilioma). In addition, in one neighbouring hospital, crosstransmission has also been observed. All the KPC-Kp isolates were identical to a previously identified K. pneumoniae ST258-type clone that is epidemic in Greece, Israel and the USA, as revealed by PFGE, plasmid analysis, multilocus sequence typing and Tn4401 transposon typing. 2,8 PCR experiments, followed by sequencing, identified additional b-lactamase genes coding for the naturally occurring narrowspectrum SHV-11, the plasmid-encoded narrow-spectrum TEM-1 and extended-spectrum SHV-12. In France, KPC-Kp remain rare and, to date, have always been linked to patient transfer from a country where KPC-Kp are endemic. 2 This is the first KPC-Kp outbreak in France and the first worldwide to be linked to a contaminated endoscope. Although the risk of endoscopy-related infection is low, 6 changes to endoscope reprocessing, by replacing a glutaraldehyde decontamination bath with an automated peracetic acid washer (to prevent Creutzfeldt-Jacob transmission), may have been deleterious to the endoscope. However, careful checking of the endoscope by the instrument's manufacturer did not reveal any obvious signs of degradation. Careful auditing of endoscope reprocessing revealed two possible explanations for the contamination: (i) the pre-wash of the endoscope may have been delayed 24 h, thus resulting in drying of the device; and (ii) after the peracetic wash, the drying procedure was not long enough for the novel automated washer, thus the endoscope was not completely dried. In light of this outbreak, new local guidelines for endoscope reprocessing have been established, taking into account the specificities of the new automated peracetic acid washer. Microbiological testing of endoscopes, performed until now twice a year, will now be undertaken on a more frequent basis.

Journal of Antimicrobial Chemotherapy, 2009

Microbial drug resistance (Larchmont, N.Y.), 2011

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of ... more The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This s...

Foodborne Pathogens and Disease, 2010

Background: Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteria... more Background: Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we characterize a new AmpC variant presenting a broadened substrate activity towards fourth-generation cephalosporins, selected in vivo following cefepime treatment for Enterobacter aerogenes. Methods: Two consecutive clonally related isolates of E. aerogenes were evaluated. Screening for ESAC production was performed using plates containing 200 mg/L cloxacillin. MICs were determined by microdilution (CLSI guidelines). bla AmpC genes were cloned into a pCR-Blunt II-TOPO vector and expressed in Escherichia coli. The ampC genes were cloned into vector pGEX-6P-1 for protein purification. Results: Isolate Ea595 was resistant to two fourth-generation cephalosporins, cefepime and cefpirome; using plates containing cloxacillin, susceptibility to ceftazidime and cefepime was restored, suggesting overproduction of the ESAC b-lactamase. Sequencing identified a new AmpC b-lactamase variant presenting one amino acid substitution, Val291Gly, inside the H-10 helix. Recombinant plasmids harbouring this ESAC b-lactamase conferred a broadened resistance profile to cefepime and cefpirome, with resistance levels increasing from 16-to 32-fold in E. coli. AmpC-Ea595 hydrolysed ceftazidime, cefepime and cefpirome at high levels, presenting a lower K m and enabling us to classify the enzyme as an ESAC. Homology modelling suggested that the size of the active site could have increased. Conclusions: We characterized an ESAC b-lactamase selected in vivo and conferring a high level of resistance to fourth-generation cephalosporins in E. aerogenes. The broadened spectrum was caused by a new modification to the H-10 helix, which modified the active site.

Foodborne Pathogens and Disease, 2011

Foodborne Pathogens and Disease, 2013

We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escheri... more We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy pigs, and to evaluate their clonality and associated resistance. Forty-nine percent of pigs sampled (n = 35/71) in a slaughterhouse in Portugal revealed ESBL-producing E. coli isolates. Most isolates produced CTX-M-1 enzyme (71.4%; n = 25/35), followed by CTX-M-9 (11.4%; n = 4/35), CTX-M-14 (5.7%; n = 2/35), SHV-12 (5.7%; n = 2/35), and CTX-M-32 (5.7%; n = 2/35). Ninety-four percent of the isolates presented a phenotype of multi-resistance. Most isolates belonged to phylogroups B1 (42.8%; n = 15/35) and A (40%; n = 14/35). Multilocus sequence typing (MLST) analysis revealed nine sequence types (STs) under six clonal complexes (CCs) and nine singletons, including overrepresentation of CC10 and three new STs (ST2524, ST2525, ST2528). We observed the frequent presence of CTX-M-producing E. coli in pigs at slaughter level, most of them belonging to CC10, commonly recovered from clinical samples.