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- Escherichia coli Isolated from Potomida littoralis and Margaritifera margaritífera: phylogenetic group determinationPublication . Gomes, Sónia; Piteira, Martinha; Fernandes, Conceição; Varandas, Simone; Saavedra, Maria JoséAntimicrobial resistance (AMR) is a major global health problem. Water use by human activities represent a natural reservoir of AMR, thus a time-integrated approach is required in aquatic ecosystems. Freshwater bivalves are widely applied as accumulation indicators and monitoring tools of contaminant effects on different levels of biological integration since they are suspension feeders that actively filter, retain and concentrate particles from their surrounding water, including free living or particle-bound bacteria. In this study, we aimed to (i) investigate the genetic diversity of E. coli strains isolated from freshwater bivalves, and (ii) determine their virulence factors. Samples were collected in the middle sector of the Tua River. Isolates were retrieved in Chromocult® Coliform Agar plates (Merck, Germany). Antimicrobial susceptibility testing (AST) was done by the Kirby-Bauer disk diffusion method against 21 antimicrobial agents, according to the recommendations issued by the EUCAST and CLSI. Antibiotic resistance rates were ticarcillin (TIC) 87.5%, ticarcillin-clavulanic acid (TIM) 87.5%, piperacillin (PRL) 6.3%, cefoxitin (FOX) 18.8%, tobramycin (TOB) 31.3%, gentamicin (CN) 12.5%, and amikacin (AK) 87.5%. Antibiotics whose antimicrobial resistance was 100% belong to β-lactam, namely penicillins and carbapenems. The antimicrobial susceptibility test revealed that 43.75% of the isolates were MDR. PCR-based assays were designed for phylogenetic E. coli groups evaluation. The E. coli phylogenetic group D or E was the most prevalent (50%), followed by phylogenetic groups E or clade I, B1 and A. Phylogenetic groups D or E and E or clade I should be confirmed using E-specific primers. Our results indicated that bivalves provide an efficient, time-integrating tool for identifying/quantifying faecal indicators, including multidrug resistant bacteria
- Endangered bivalves as antimicrobial-resistant Escherichia coli and other species of the Enterobacteriaceae family.Publication . Saavedra, Maria José; Fernandes, Conceição; Ballem, Andressa; Varandas, SimoneFreshwater unionoids are one of the most threatened animal groups worldwide, suffering dramatic regressions globally. The freshwater pearl mussel Margaritifera margaritifera, currently listed as critically endangered in Europe, and Potomida littoralis, listed as endangered, are both present in the River Tua basin (Portugal). These aquatic long-lived organisms, responsible for important functions and vital ecosystem services [1], are sensitive to environmental changes, making them excellent bioindicators [2]. According to Grizzle & Brunner [3] mussels are capable of establishing symbiotic mutualistic or antagonistic relationships with bacteria. The inappropriate use of antibiotics has led to the emergence and spread of resistant bacteria, recognized today as a serious public health problem. Based in the concept of “One health”, the objective of this study was to examine the antibacterial resistance rates in Escherichia coli and other Enterobacteriaceae isolates obtained from water and the two freshwater bivalve species collected in the River Tua Basin.
- Prevalence of shigatoxigenic Escherichia coli in bovine cattle: a survey in the North Region of PortugalPublication . Ballem, Andressa; Gonçalves, Soraia; Ferreira, A.I.; Fernandes, Conceição; Saavedra, Maria José; Pinto, C.; Oliveira, H.; Almeida, Gonçalo; Almeida, CarinaShiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens that can be transmitted to humans through many different routes, but mainly through consumption of contaminated foods. STEC strains are characterized by the production of Shiga toxins (Stx) that are encoded by two genes stx1 and/or stx2. Each toxin can be subdivided into subtypes and, currently, there are three known subtypes for stx1 (stx1a, stx1c, and stx1d) and seven for stx2 (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g). Others virulence factors, such the production of intimin protein, encoded by eae gene, are associated with severe outcomes of STEC-associated diseases. The most common STEC serotype implicated worldwide is E. coli O157:H7, but many other STEC strains are associated with severe human diseases. Ruminants, especially cattle, are a major reservoir for O157 and non-O157 STEC. Information on STEC prevalence in Portuguese dairy cattle is limited, so we analysed for the presence of STEC 329 faecal specimens collected from the rectum of healthy dairy cattle. The samples were collected from adult lactating cows (n=194) and from heifers (n=135) with ages among 6 to 18 months, between March and June 2019, at milk farms (n=17) in the North region of Portugal. After enrichment, in modified TSB with novobiocin, samples were analysed by real time PCR to detect the presence of stx1, stx2 and eae in accordance to ISO/TS 13136:2012(E). In addition, conventional PCR for the detection of sxt1 and stx2 gene subtypes was performed according to the guidelines of the VTEC European Union Reference
- Antibiotic resistance profile among Shiga toxin-producing Escherichia coli isolated from dairy cattlePublication . Ballem, Andressa; Almeida, Gonçalo; Almeida, Carina; Fernandes, Conceição; Saavedra, Maria JoséShiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that can cause serious diseases in humans, including bloody diarrhoea and kidney failure. Ruminants, such as cattle, are considered the main reservoirs and source of STEC. Human infection can occur through contaminated food and water, or direct contact with infected animals. STEC antimicrobialresistance (AMR) is increasingly frequent in patients with serious disease. It is necessary to understand the epidemiology, the emergence, and the prevalence of AMR in STEC isolated from cattle to investigate how resistance spreads from ruminants to humans. Thus, susceptibility tests were performed on 55 STEC strains belonging to 29 serogroups. The strains were isolated from healthy dairy cattle faeces (cows and heifers) in the North of Portugal. Antibiotic susceptibility testing (AST) was performed by disc diffusion method following European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2020) and Clinical and Laboratory Standards Institute (CLSI, 2020). The antibiotics used included penicillins (ampicillin; amoxicillin-clavulanic acid), cephalosporins (ceftazidime; cefotaxime; cefoxitin; cephalothin), carbapenems (imipenem; meropenem), aminoglycosides (kanamycin), phenicol (chloramphenicol), sulphonamides/ trimethoprim (trimethoprim-sulfamethoxazole), fluoroquinolones (moxifloxacin; levofloxacin) and tetracyclines (tetracycline; tigecycline). Results reveal low level of resistance among the isolates tested. However, five (9%) STEC isolates were resistant to one antibiotic, and three (5,5%) to three or more antibiotic classes (multidrug resistance-MDR). The MDR strains were resistant to trimethoprim-sulfamethoxazole, tetracycline and other antibiotics commonly used to treat gastroenteritis. Two strains MDR belonged to O91 serogroup and were founded in heifers in the same farm. O91 is an important serogroup to public health surveillance, as it is commonly associated with contamination of products from animal origin, and it has been isolated from patients with severe gastrointestinal disease. Overall, the AMR did not seem to be widely spread in STEC isolates from cattle; but serotype O91 might be of special concern as two O91-multidrug resistance profiles have been identified.
- Antibiotic-Resistant Escherichia coli on Asian clam (Bivalvia, Corbiculidea): Case Study in the Tua River, Portugal.Publication . Saavedra, Maria José; Gomes, Sónia; Fernandes, Conceição; Teixeira, Amílcar; Varandas, SimoneAntimicrobial resistance (AMR) is a major global health problem. Water use by human activities represent a natural reservoir of AMR, thus a time-integrated approach is required in aquatic ecosystems. Freshwater bivalves are widely applied as accumulation indicators and monitors of contaminant effects on different levels of biological integration once they are suspension feeders that actively filter, retain and concentrates particles from their surrounding water, including free living or particle-bound bacteria. In this study, we aimed to (i) investigate the genetic diversity of E. coli, (ii) determine the virulence factors of E. coli isolates, and (iii) detect antibiotic resistant E. coli. Samples were collected in the middle sector of the Tua River. Isolates were retrieved in Chromocult® Coliform Agar plates (Merck, Germany). Susceptibility testing was performed by the disc diffusion method, according to the Clinical Laboratory Standards Institute (CLSI), and the screening of phenotypic ESBL production was carried out by the double disk synergy test using cefotaxime (30μg), ceftazidime (30μg), and amoxicillin/clavulanate (20/10μg) acid discs. PCR-based assays were designed for phylogenetic E. coli groups evaluation, resistance and detection of ESBL genes. The E. coli phylogenetic group B1 was the most prevalent (44 %), followed by phylogenetic groups A and D. Phylogenetic groups A and B1 comprised more than half (70 %) of the total E. coli isolated. Our results show that Corbicula spp. provide an efficient, time-integrating tool for identifying/quantifying faecal indicators, including multidrug resistant bacteria.
- Antibiotic resistance profile among Shiga toxin-producing Escherichia coli isolated from dairy cattlePublication . Ballem, Andressa; Almeida, Gonçalo; Almeida, Carina; Fernandes, Conceição; Saavedra, Maria JoséShiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that can cause serious diseases in humans, including bloody diarrhoea and kidney failure. Ruminants, such as cattle, are considered the main reservoirs and source of STEC. Human infection can occur through contaminated food and water, or direct contact with infected animals.
- Endangered mussels as antimicrobial-resistant E. coli and other species (Enterobacteriaceae family) reservoirsPublication . Saavedra, Maria José; Fernandes, Conceição; Ballem, Andressa; Varandas, SimoneFreshwater unionoids are one of the most threatened animal groups worldwide, suffering dramatic regressions globally. The freshwater pearl mussel Margaritifera margaritifera, currently listed as critically endangered in Europe, and Potomida littoralis, listed as endangered, are both present in the River Tua basin (Portugal). The inappropriate use of antibiotics has led to the emergence and spread of resistant bacteria, recognized today as a serious public health problem. Based in the concept of “One health”, the objective of this study was to examine the antibacterial resistance rates in Escherichia coli and other Enterobacteriaceae isolates obtained from both bivalves collected in the River Tua Basin during the summer of 2018. Activity of 22 antibiotics was studied using disc diffusion (Kirby–Bauer) method. Multidrug resistant in E. coli and Enterobacteriaceae isolates was observed only from P. littoralis. Overall, results show that M. margaritifera inhabits more pristine sectors of river not subject to the development of multidrug resistance, unlike P. littoralis which, although does not tolerate high levels of contamination, is naturally found in lower stretches is subject to greater pressure and, as such, a greater probability of the appearance of multi-resistant organisms. The presence of E. coli in freshwater bivalves can represents serious public health problems once bacteria are one the most important biological contaminants in foodborne diseases. Given the conservation status of both studied species their protection against this silent/invisible enemy should be a priority, although work done with UnionidMussels fromNorthern Portugal showed that they had a great antimicrobial and antibiofilm activity.