Browsing by Author "Freitas, Ana R."
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- Assess of acid tolerance of non-typhoidal Salmonella and Enterococcus faecium from different epidemiological and genetic backgroundsPublication . Rebelo, Andreia; Mourão, Joana; Freitas, Ana R.; Campos, Joana; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaAcid stress is one of the most frequently encountered hostile conditions that bacteria have to face (e.g. foodproduction-chain/feed/disinfectants/human and animal hosts). Nevertheless, acid tolerance profile of bacteria from diverse epidemiological and genetic backgrounds, including multidrug- resistant (MDR), is still poorly explored. The aim of this study was to assess the susceptibility to acidic-pH of non-typhoidal Salmonella and Enterococcus faecium (Efm) from diverse origins. We included Salmonella (n=66; 23 serotypes) and Efm (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). The minimum-growth-pH (growth-pHmin) was assessed by broth-microdilution using Mueller-Hinton-II adjusted with HCl (pH=2.0-6.5/16h-20h ±2h/37ºC) and the minimum-survival-pH (survival-pHmin) by plating the microdilution wells without visible growth in Brain-HeartInfusion-agar (BHI) (24h-48h±2h/37ºC). An Acid-Tolerance-Response (ATR) assay was performed in 3 isolates of each genera (different growth/survival-pHmin), exposing bacteria in log-phase to an acid-shockchallenge (pH=3.0/15’-Salmonella/60’-Efm) or to a pre- adaptation to acidic-pH (pH=4.5/60’ - for both bacteria) followed by an acid-shock-challenge (pH=3.0/15’-Salmonella/60’-Efm). After that a growth-pHmin and survival-pHmin assays were performed. Most Salmonella showed a growth-pHmin of 4.0 (98%- n=65/66) and a survival-pHmin between 4.0 (52%- n=34/66) and 3.5 (48%-n=32/66). In Efm, the growth-pHmin ranged between 4.5 (65%-n=48/74) and 5.0 (35%-n=26/74) and the survival-pHmin between 3.0 (15%-n=11/74), 3.5 (43%- n=32/74) and 4.0 (42%n=31/74). Only Efm isolates from food (39%-n=11/28), mostly from a poultry-processing-plant using peracetic acid as disinfectant (73%-n=8/11), presented the lowest survival-pHmin 3.0 (73%- MDR/82%-clade A2). Similar survival-pHmin=3.5-4.0 were observed for different Salmonella serotypes and Efm clades. However, a higher percentage of MDR-Salmonella (61%-n=27/44) were able to survive at pH=3.5 contrasting with nonMDR-Salmonella (23%-n=5/22) (p<0,05; Fisher-exact test). The ATR-assay (pre-adaptation acidic pH+acid shock challenge) enhanced survival-pHmin from 3 to 2.5 in 1-Efm (clade A2/MDR/from a poultry processing plant) and from 4 to 3.5 in 1-S. 4,[5],12:i:- (MDR/with mcr-1 gene/from pork meat). Our data suggest that MDR-Salmonella and Efm with diverse epidemiological and genetic backgrounds can survive to low-pH values, although differences among clades/serotypes were not detected. MDR- Salmonella showed a better ability to survive to more acidic pH than non-MDR isolates. ATR-assays revealed strainspecific ability to survive under more acidic-pH after a pre-adaptation to middle acidic- pH.
- 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.
- Dairy cattle and the iconic autochthonous cattle in northern Portugal are reservoirs of multidrug-resistant Escherichia coliPublication . Quinteira, Sandra; Dantas, Rui; Pinho, Luís; Campos, Carla; Freitas, Ana R.; Brito, Nuno V.; Miranda, Carla; Campos, CarlaAnimals destined for human consumption play a key role in potentially transmitting bacteria carrying antibiotic resistance genes. However, there is limited knowledge about the carriage of antibiotic-resistant bacteria in native breeds. We aimed to characterize the phenotypic profiles and antibiotic resistance genes in Escherichia coli isolated from bovines, including three native Portuguese bovine breeds. Forty-nine E. coli isolates were selected from 640 fecal samples pooled by age group (eight adult or eight calf samples) from each farm, representing both dairy cattle raised in intensive systems and meat cattle raised in extensive systems in Northern Portugal. The presumptive E. coli colonies plated onto MacConkey agar were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The antibiotic resistance profiles were screened by antimicrobial susceptibility testing (EUCAST/CLSI guidelines), and the antibiotic resistance genes by PCR. Most isolates showed resistance to ampicillin (69%), tetracycline (57%), gentamicin (55%), and trimethoprim + sulfamethoxazole (53%), with no resistance to imipenem. Resistance to at least one antibiotic was found in 92% of isolates, while 59% exhibited multidrug resistance. Most calf isolates, including those from native breeds, showed a multidrug-resistant phenotype. Among the adults, this was only observed in Holstein-Friesian and Barrosã cattle. None of the Holstein-Friesian isolates were susceptible to all the tested antibiotics. ESBL-producing E. coli was identified in 39% of isolates, including those from Holstein-Friesian calves and adults, Cachena calves and Minhota adults. The sul2 gene was detected in 69% of isolates, followed by blaCTX-M (45%), aac(3′)-IV (41%), and aac(6′)-Ib-cr (31%), with a higher prevalence in adults. This pioneering study highlights the concerning presence of multidrug-resistant E. coli in native Portuguese cattle breeds.
- 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.
- Mapping of key bacterial species for Postmorteminterval calculationPublication . Ferreira, Ana Cláudia; Barbosa, Daniel J.; Campos, Carla; Oliveira, Ricardo Jorge Dinis; Freitas, Ana R.Estimation of the PostmortemInterval (PMI), the time elapsed since death, is one of the most challenging issues in forensic sciences. Most studies focus on extensive bacterial sequencing, but culture-based experiments for higher taxonomic resolution remain scarce. We aimed to analyze total bacterial counts and map Enterococcus faecalis, Staphylococcus aureusand Escherichia coliin different organs and timepoints. Male C57BL/6J SPF mice underwent three independent assays during 11 postmortemtimepoints. Feces and organs (n=10: intestine/stomach/skeletical muscle/liver/spleen/kidney/bladder/lungs/ brain/heart) were collected and resuspended in buffered peptone water, then plated onto enriched nonselective and selective culture media (n=4). Following routine aerobic incubation, Colony Forming Units (CFU) per gram/tissue or per mL/sample were quantified for total/individual bacterial loads. Species were identified by MALDI-TOF MS and statistics were done in GraphPad-Prism v.10.0.1. Species (n=44) from 13 families and 3 phyla were identified, with notable consistency in the presence of Staphylococcus xylosus, E. faecalis, and E. coliacross all experiments. Particular families were consistently identified across all organs, including Enterococcaceae and Enterobacteriaceae mostly in the later stages of decomposition, and Bacillaceae resisting often until the last timepoint, whereas Staphylococcaceae was variably detected. The early and substantial contamination observed in skeletal muscle, stomach, and intestine, makes them unsuitable for PMI calculations. E. faecalisappeared promising as a potential biomarker for kidney, liver, and, possibly, brain invasion at later timepoints, whereas E. faecalisand E. coliin the bladder, and E. coliin the spleen and heart, warrant further investigation for similar biomarker potential. This is one of the first quantitative cultural studies assessing how time elapsing postmortem affects the growth/evolution of key bacterial species, with E. faecalisand E.coliemerging as promising traceable biomarkers in real postmortem contexts. While recognizing the limitations of not considering the complex microbiota network, our pilot study brings an easy species-specific approach and offers a baseline for future human-oriented investigations.
- 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.
- 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.