Browsing by Author "Novais, Carla"
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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.
- Atypical Non-H2S-producing monophasic salmonella typhimurium ST3478 strains from chicken meat at processing stage are adapted to diverse stressesPublication . Mourão, Joana; Rebelo, Andreia; Ribeiro, Sofia; Peixe, Luísa; Novais, Carla; Antunes, PatríciaPoultry products are still an important cause of Salmonella infections worldwide, with an increasingly reported expansion of less-frequent serotypes or atypical strains that are frequently multidrug-resistant. Nevertheless, the ability of Salmonella to survive antimicrobials promoted in the context of antibiotic reducing/replacing and farming rethinking (e.g., organic acids and copper in feed/biocides) has been scarcely explored. We investigated Salmonella occurrence (conventional and molecular assays) among chicken meat at the processing stage (n = 53 batches/29 farms) and characterized their tolerance to diverse stress factors (antibiotics, copper, acid pH, and peracetic acid).
- Comprehensive assessment of the ability of non-typhoidal Salmonella and Enterococcus faecium to survive acidic environmentsPublication . Rebelo, Andreia; Mourão, Joana Vanessa Cordeiro Melro; Freitas, Ana Raquel; Campos, Joana; Peixe, Luísa Maria Vieira; Antunes, Patrícia; Novais, CarlaAcidic-pH resulting from acid-products use or naturally occurring in different environments (e.g.food-production-chain-feed/disinfectants/human/animal hosts) potentially selects multidrug-resistant (MDR)-bacterial strains. Remains unknown if particular MDR-strains of pathogenic bacteria are more prone to overcome acid-stress.
- Decoding Klebsiella pneumoniae in poultry chain: Unveiling genetic landscape, antibiotic resistance, and biocide tolerance in non-clinical reservoirsPublication . Mourão, Joana; Magalhães, Mafalda; Ribeiro-Almeida, Marisa; Rebelo, Andreia; Novais, Carla; Peixe, Luísa; Novais, Ângela; Antunes, PatríciaThe rise of antibiotic resistance in the food chain is influenced by the use of antimicrobial agents, such as antibiotics, metals, and biocides, throughout the entire farm-to-fork continuum. Besides, non-clinical reservoirs potentially contribute to the transmission of critical pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae. However, limited knowledge exists about the population structure and genomic diversity of K. pneumoniae circulating in conventional poultry production. We conducted a comprehensive characterization of K. pneumoniae across the whole chicken production chain (flocks/environment/meat, 2019-2022), exploring factors beyond antibiotics, like copper and quaternary ammonium compounds (QACs). Clonal diversity and adaptive features of K. pneumoniae were characterized through cultural, molecular (FT-IR), and whole-genome-sequencing (WGS) approaches. All except one flock were positive for K. pneumoniae with a significant increase (p < 0.05) from early to pre-slaughter stages, most persisting in chicken meat batches. Colistin-resistant K. pneumoniae rates were low (4%), while most samples carried MDR strains (67%) and copper-tolerant isolates (63%; sil+pco clusters; MICCuSO4≥16mM), particularly at pre-slaughter. Benzalkonium chloride consistently exhibited activity in K. pneumoniae (MIC/MBC range=4-64mg/L) from diverse and representative strains independently of the presence/absence of genes linked to QACs tolerance. A polyclonal K. pneumoniae population, discriminated by FT-IR and WGS, included various lineages dispersed throughout the chicken’s lifecycle at the farm (ST29-KL124, ST11-KL106, ST15-KL19, ST1228-KL38), until the meat (ST1-KL19, ST11-KL111, ST6405-KL109, and ST6406-CG147-KL111), or over years (ST631-49 KL109, ST6651-KL107, ST6406-CG147-KL111). Notably, some lineages were identical to those from human clinical isolates. WGS also revealed F-type multireplicon plasmids carrying sil+pco (copper) co-located with qacEΔ1±qacF (QACs) and antibiotic resistance genes like those disseminated in humans. In conclusion, chicken farms and their derived meat are significant reservoirs for diverse K. pneumoniae clones enriched in antibiotic resistance and metal tolerance genes, some exhibiting genetic similarities with human clinical strains. Further research is imperative to unravel the factors influencing K. pneumoniae persistence and dissemination within poultry production, contributing to improved food safety risk management. This study underscores the significance of understanding the interplay between antimicrobial control strategies and non-clinical sources to effectively address the spread of antimicrobial resistance.
- Detection of Salmonella serotypes adapted to diverse stresses in poultry meat at processing level in PortugalPublication . Ribeiro, Sofia; Mourão, Joana; Rebelo, Andreia; Novais, Carla; Peixe, Luísa; Antunes, PatríciaNon-typhoidal Salmonella causes frequently foodborneinfections mainly associated with the consumption of eggs/poultry products. A decreasing trend in human salmonellosis, particularly of S.Enteritidis, has been observed in EU dueto successful control programs at theavian production level. Nevertheless, expansion of less frequent serotypes and/or certain well-adapted clones has been reported.Theeffectiveness of control practices (e.g. organicacids in feed/biocides) on theelimination of Salmonella,and particularly of EU targeted serotypes, in poultry has been scarcely explored. Here, weinvestigated the presence of Salmonella, using conventional and molecular approaches,and characterizetheir clinically-relevant serotypes,among fresh chickenmeat samples at poultry processing level in Portugal.
- Distribution of Arsenic Tolerance Genes (arsA) among Enterococcus spp. from different sources, continents and timeframes (<1906-2015).Publication . Rebelo, Andreia; Mourão, Joana; Freitas, Ana; Coque, Teresa; Almeida, Agostinho; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaSuccessful bacteria accumulate different genetic features shaping their evolution and fitness to diverse environments/hosts. Arsenic-compounds are widespread in nature and possibly contribute to selection of particular strains. The aim of this study was to evaluate the dispersion of arsA genes among Enterococcus from different sources, continents and timeframes. The arsA genes (coding for arsenical-pump-driving-ATPases) were searched in GenBank Enterococcus genomes and used to construct a maximum-likelihood phylogenetic-tree. A PCR scheme+sequencing was developed to detect arsA alleles from all phylogenetic subgroups identified among 333 isolates (Portugal; human/animal/environment/food; 1996-2012)1. Na2HAsO4 susceptibility was evaluated by agar dilution (0,25 to 128mM; n=143 isolates).
- 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.
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