Percorrer por autor "Duarte, Bárbara"
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- Assessment of antimicrobial resistance spread in surface aquatic systems: a study of four rivers in north PortugalPublication . Matos, Cátia; Duarte, Bárbara; Rebelo, Andreia; Lopes, Diana; Freitas, Ana R.; Valente, Margarida; Tavares, Carolina; Rodrigues, Juliana; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaAntimicrobial resistance (AMR) spread in surface water systems, providing water for drinking or for recreational activities, can have serious implications to human, animal and environmental health. The standard criteria to assess water quality are based in the absence of specific bacteria, and do not include AMR study. Here we evaluate the spread of AMR bacteria in rivers of North Portugal under diverse anthropogenic activities.
- Diversity of metal and antibiotic resistance genes in Enterococcus spp. from the last century reflects multiple pollution and genetic exchange among phyla from overlapping ecosystemsPublication . Rebelo, Andreia; Mourão, Joana; Freitas, Ana R.; Duarte, Bárbara; Silveira, Eduarda; Sanchez-Valenzuela, António; Almeida, Agostinho; Baqueroik, Fernando; Coqueik, Teresa M.; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaArsenic (As), mercury (Hg), and copper (Cu) are among the major historical and contemporary metal pollutants linked to global anthropogenic activities. Enterococcus have been considered indicators of fecal pollution and antibiotic resistance for years, but its largely underexplored metallome precludes understanding their role as metal pollution bioindicators as well. Our goal was to determine the occurrence, diversity, and phenotypes associated with known acquired genes/operons conferring tolerance to As, Hg or Cu among Enterococcus and to identify their genetic context (381 field isolates from diverse epidemiological and genetic backgrounds; 3547 enterococcal genomes available in databases representing a time span during 1900–2019). Genes conferring tolerance to As (arsA), Hg (merA) or Cu (tcrB) were used as biomarkers of widespread metal tolerance operons. Different variants of metal tolerance (MeT) genes (13 arsA, 6 merA, 1 tcrB) were more commonly recovered from the food-chain (arsA, tcrB) or humans (merA), and were shared with 49 other bacterial taxa. Comparative genomics analysis revealed that MeT genes occurred in heterogeneous operons, at least since the 1900s, with an increasing accretion of antibiotic resistance genes since the 1960's, reflecting diverse antimicrobial pollution. Multiple MeT genes were co-located on the chromosome or conjugative plasmids flanked by elements with high potential for recombination, often along with antibiotic resistance genes. Phenotypic analysis of some isolates carrying MeT genes revealed up to 128× fold increase in the minimum inhibitory concentrations to metals. The main distribution of functional MeT genes among Enterococcus faecium and Enterococcus faecalis from different sources, time spans, and clonal lineages, and their ability to acquire diverse genes from multiple taxa bacterial communities places these species as good candidates to be used as model organisms in future projects aiming at the identification and quantification of bioindicators of metal polluted environments by anthropogenic activities.
- Enterococcus spp. from chicken meat collected 20 years apart overcome multiple stresses occurring in the poultry production chain: Antibiotics, copper and acidsPublication . Rebelo, Andreia; Duarte, Bárbara; Ferreira, Carolina; Mourão, Joana; Ribeiro, Sofia; Freitas, Ana R.; Coque, Teresa M.; Willems, Rob; Corander, Jukka; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaPoultry 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, ciprofloxacin, 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 0 % 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.
- Exploring peracetic acid and acidic pH tolerance of antibiotic-resistant non-typhoidal Salmonella and Enterococcus faecium from diverse epidemiological and genetic backgroundsPublication . Rebelo, Andreia; Duarte, Bárbara; Freitas, Ana R.; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaAcid stress poses a common challenge for bacteria in diverse environments by the presence of inorganic (e.g., mammals’ stomach) or organic acids (e.g., feed additives; acid-based disinfectants). Limited knowledge exists regarding acid-tolerant strains of specific serotypes, clonal lineages, or sources in human/animal pathogens: namely, non-typhoidal Salmonella enterica (NTS) and Enterococcus faecium (Efm). This study evaluated the acidic pH (Mueller–Hinton acidified with HCl) and peracetic acid (PAA) susceptibility of Efm (n = 72) and NTS (n = 60) from diverse epidemiological/genetic backgrounds and with multiple antibiotic resistance profiles. Efm minimum growth/survival pH was 4.5–5.0/3.0–4.0, and for NTS it was 4.0–4.5/3.5–4.0. Efm distribution among acidic pH values showed that only isolates of clade-non-A1 (non-hospital associated) or the food chain were more tolerant to acidic pH compared to clade-A1 (hospital-associated clones) or clinical isolates (p < 0.05). In the case of NTS, multidrug-resistant (MDR) isolates survived better in acidic pH (p < 0.05). The PAA MIC/MBC for Efm was 70–120/80–150 mg/L, and for NTS, it was 50–70/60–100 mg/L. The distribution of Efm among PAA concentrations showed that clade-A1 or MDR strains exhibited higher tolerance than clade-non-A1 or non-MDR ones (p < 0.05). NTS distribution also showed higher tolerance to PAA among non-MDR and clinical isolates than food chain ones (p < 0.05) but there were no differences among different serogroups. This unique study identifies specific NTS or Efm populations more tolerant to acidic pH or PAA, emphasizing the need for further research to tailor controlled measures of public health and food safety within a One Health framework.
- Organic and inorganic copper feed formulations have a similar impact in the selection of copper tolerant and multidrug resistant Enterococcus faecium from poultry farms and meat available to consumersPublication . Batista, Carina; Rebelo, Andreia; Duarte, Bárbara; Almeida, Marisa; Monteiro, Eulália; Pereira, Beatriz; Ribeiro, Sofia; Freitas, Ana R.; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaCopper-Cu is used as animal feed additive. Inorganic (ITM) or organic (OTM) trace minerals feed formulations with Cu are available (OTM with lower Cu concentrations), but their effect in the selection of Cu tolerant-CuT and multidrug-resistant bacteria-MDR bacteria is unexplored. The impact of ITM/OTM-feed in the occurrence of MDR-CuT E. faecium-Efm in poultry production chain was studied. Broiler feces [n=34; 18 poultry-houses/flocks (10000-64000 animals each); 7 intensive-farms] were collected in each farm from 2 separated poultry-houses using ITM or OTM, at 2-3 days of broilers life (P1) and before slaughter (P2) (2019-2020/Portugal). Meat (n=18) of the same broilers was also collected after slaughter (P3). Samples were plated in Slanetz-Bartley (SL) agar with/without 1mM-CuSO4 (37°C/48h; anaerobiosis). Efm species, tcrB gene coding for CuT were studied by PCR and antibiotics/Cu susceptibility by disk diffusion/microdilution. Results: 256 Efm were from feces-97% and meat-67% samples. Efm-tcrB+ were mostly found in SL+Cu plates (46% of samples vs 15%-SL), with similar rates between OTM and ITM (54% vs 46%). Efm-tcrB+ had an increasing trend between P1 and P2 (56% vs 81%) samples but significantly decrease at P3 (17%). Efm-tcrB+ had a MICCu>12mM (96%), with most being MDR comparing to Efm-tcrB- (98% vs 57%). Efm-tcrB+ were more resistant to tetracycline, erythromycin, quinupristin/dalfopristin or streptomycin than Efm-tcrB- (100%/100%/85%/62% vs 63%/66%/63%/21%). MDR-Efm-tcrB+, including to the clinical-relevant ampicillin or aminoglycosides, were mostly found in feces (P1+P2:47% vs P3:11%), both in OTM and ITM samples (31% vs 38%). MDR-Efm-tcrB+ were similarly detected independently of OTM/ITM feed at poultryfarms but they significantly decreased in carcasses for the consumer. The Cu role in the selection and persistence of MDR-Efm deserves more studies.
- Peracetic acid tolerance of MDR non-typhoidal Salmonella and Enterococcus faecium with diverse epidemiological and genetic backgroundPublication . Rebelo, Andreia; Duarte, Bárbara; Callejón, Ana; Peixe, Luísa; Novais, Carla; Antunes, PatríciaBacteria face multiple stresses in different contexts and developed diverse mechanisms to overcome them individually or through events of cross-tolerance. Peracetic acid (PAA) is widely used in the food-chain as antiseptic/disinfectant (20-3000 mg/mL) and induces oxidative-stress in bacteria. However, data about bacterial tolerance to PAA (PAAT) and the conditions inducing such tolerance remain scarce. Here we assess PAAT of non-typhoidal Salmonella and Enterococcus faecium from diverse epidemiological and genetic backgrounds and determine if induction with PAA and copper-Cu (also associated with oxidativestress and widely used in food-animal production settings) increase PAAT. We included Salmonella (n=66; 23 serotypes) and E. faecium (n=74; clades A1/A2/B) recovered from human (n=54), food-animal production setting (n=20), food (n=56) and environment (n=10) (1997-2018; 6 countries). Most of the isolates were MDR (E. faecium 76%-n=56/74; Salmonella 67%-n=44/66). The MICPAA was performed by broth-microdilution (ISO20776-1:2006; range: 40-140mg/L) followed by MBCPAA determination (NCCLS:1999) (37ºC/48h; 2 replicas/isolate). Induction assays by PAA and by CuSO4 were performed in 6 Salmonella and 6 E. faecium (human, food-animal production settings and food sources; with/without Cu tolerance genes: 3 Salmonella with pcoD+silA genes and 3 E. faecium with tcrB+cueO genes; diverse MIC/MBCPAA) by exposing bacteria (log-phase: 3-4h) to sub-inhibitory PAA or CuSO4 concentrations (up to 10 and 100 times less the MICPAA/Cu) followed by MICPAA assay. MICPAA= 40-60 mg/L and MBCPAA= 50-80 mg/L (MIC90= 60 mg/L; MBC90= 70 mg/L) were observed in Salmonella, and a MICPAA= 60-100 mg/L and MBCPAA= 80-140 mg/L (MIC90= 90 mg/L; MBC90= 140 mg/L) in E. faecium. No differences in MIC/MBCPAA were observed among serotypes/clades, sources or MDR/non-MDR bacteria. The induction with PAA or CuSO4 did not affected the MIC/MBC of Salmonella and E. faecium. Our data suggest that a high number of MDR Salmonella and E. faecium are able to survive to PAA concentrations used in the food-processing industries. Exposure to sub-inhibitory PAA and CuSO4 concentrations, under the tested conditions, does not affect the ability to survive to PAA, in both bacteria. However, further studies are needed to better understand the environmental conditions that can challenge the efficacy of these and other antimicrobial compounds.
- Raw chicken meat is a vehicle of Ampicillin-multidrug-resistant Enterococcus faecium: is it a on health problem?Publication . Novais, Carla; Freitas, Ana R.; Rebelo, Andreia; Ferreira, Carolina; Duarte, Bárbara; Ribeiro, Sofia; Mourão, Joana; Scholes, Christopher; Antunes, Patrícia; Peixe, LuísaResistance to ampicillin-AmpR is associated with Enterococcus faecium-Efm from hospitalized-humans (clade-A1) and at lesser extent with community-based isolates (clade-A2: human and animal strains; clade-B: human-commensal strains). Recently, AmpR combined with specific putative virulence factors were proposed as molecular markers of Efm linked to human infections, which can have an impact in Efm risk assessment in different public health contexts (PMID:29519512). Here we evaluated the occurrence of AmpR-Efm with potential public health impact in chicken-meat samples.
- Uncovering the effects of copper feed supplementation on the selection of copper-tolerant and antibiotic-resistant Enterococcus in poultry production for sustainable environmental practicesPublication . Rebelo, Andreia; Duarte, Bárbara; Freitas, Ana R.; Almeida, Agostinho; Azevedo, Rui; Pinto, Edgar; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaThe use of antibiotics in animal production is linked to the emergence and spread of antibiotic-resistant bacteria, a threat to animal, environmental and human health. Copper (Cu) is an essential element in poultry diets and an alternative to antibiotics, supplementing inorganic or organic trace mineral feeds (ITMF/OTMF). However, its contribution to select multidrug-resistant (MDR) and Cu tolerant Enterococcus, a bacteria with a human-animal-environment-food interface, remains uncertain. We evaluated whether feeding chickens with Cu-ITMF or Cu-OTMF contributes to the selection of Cu tolerant and MDR Enterococcus from rearing to slaughter. Animal faeces [2–3-days-old (n = 18); pre-slaughter (n = 16)] and their meat (n = 18), drinking-water (n = 14) and feed (n = 18) from seven intensive farms with ITMF and OTMF flocks (10.000–64.000 animals each; 2019–2020; Portugal) were sampled. Enterococcus were studied by cultural, molecular and whole-genome sequencing methods and Cu concentrations by ICP-MS. Enterococcus (n = 477; 60 % MDR) were identified in 80 % of the samples, with >50 % carrying isolates resistant to tetracycline, quinupristin-dalfopristin, erythromycin, streptomycin, ampicillin or ciprofloxacin. Enterococcus with Cu tolerance genes, especially tcrB ± cueO, were mainly found in faeces (85 %; E. faecium/E. lactis) of ITMF/OTMF flocks. Similar occurrence and load of tcrB ± cueO Enterococcus in the faeces was detected throughout the chickens' lifespan in the ITMF/OTMF flocks, decreasing in meat. Most of the polyclonal MDR Enterococcus population carrying tcrB ± cueO or only cueO (67 %) showed a wild-type phenotype (MICCuSO4 ≤ 12 mM) linked to absence of tcrYAZB or truncated variants, also detected in 85 % of Enterococcus public genomes from poultry. Finally, < 65 μg/g Cu was found in all faecal and meat samples. In conclusion, Cu present in ITMF/OTMF is not selecting Cu tolerant and MDR Enterococcus during chickens' lifespan. However, more studies are needed to assess the minimum concentration of Cu required for MDR bacterial selection and horizontal transfer of antibiotic resistance genes, which would support sustainable practices mitigating antibiotic resistance spread in animal production and the environment beyond.