Browsing by Author "Amador, Paula"
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- Antibiotic resistance in enterobacteriaceae isolated from Portuguese deli meatsPublication . Amador, Paula; Fernandes, Rúben; Brito, Luísa; Prudêncio, CristinaThis study aimed to identify the presence of β-lactam-resistant bacteria in different types of Portuguese deli meats. The numbers of ampicillin resistant bacteria varied from negative in 25 g to 1.0 × 108colony-forming units/g. Within 78 randomly selected β-lactam-resistant bacteria, 24 different resistant phenotypes were found and 35.9% were multidrug resistant (MDR). The majority (87.2%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the blaTEM gene was detected in 23 out of 67 isolates (34.3%) and 16 of them presented MDR phenotypes. Four Klebsiella oxytoca isolates (6%) harbored a gene for the CTX-M/OXY-type enzyme. The direct sequencing of their purified amplicons confirmed the presence of three types of blaOXYgenes (blaOXY-1, blaOXY-2 and blaOXY-5). These results suggest that without good hygienic practices, deli meats may act as a vehicle of transfer of β-lactam-resistant bacteria to the gastrointestinal tract of consumers.
- Characterization of antibiotic resistance in enterobacteriaceae from agricultural manure and soil in PortugalPublication . Amador, Paula; Duarte, Isabel M.; Costa, R.P. Roberto da; Fernandes, Rúben; Prudêncio, CristinaIntensive livestock production uses antibiotics to treat and prevent infectious diseases. Antibiotics are expelled unaltered in animal manure, which is often used as a fertilizer. This practice spreads antibiotic residues and antibiotic-resistant bacteria and their genes into the soil. These resistance determinants affect soil microbial communities, raising concerns about the potential environmental and public health risks. This study aims to characterize soil contaminated with antibiotic resistant bacteria and the putative role played by manure as a vector for these resistance determinants. Six poultry and dairy cattle farms in the central region of Portugal were sampled. Ampicillin-resistant Enterobacteriaceae isolates from manure and manured-soil samples were tested for their resistance profile to 13 antibiotics and the presence of tetracycline and sulfonamide resistance genes. High multidrug resistance rates (>70%) were observed in both manure and soil samples. This resistance was higher in the poultry samples; manured-soil isolates were more resistant to cefoxitin (91.7%), sulfamethoxazole/trimethoprim (79.2%), chloramphenicol (79.2%), and to a lesser extent tetracycline (12.5%). Manure isolates have high resistance frequency to tetracycline (73.9%), amoxicillin with clavulanic acid (66.0%), chloramphenicol (65.3%), and sulfamethoxazole with trimethoprim (36.5%). Manure isolates revealed high frequency of tet(A), sul1, and sul3 genes. The multiple antibiotic resistance index reveals a putative risk of soil contamination due to antibiotic exposure. The results obtained are important for soil management regarding resistance determinants spread through agricultural practices. To our knowledge, this is the first study of tetracycline and sulfonamide resistance in Portuguese manure and soil.
- ESBL and AmpC β-Lactamases in Clinical Strains of Escherichia coli from Serra da Estrela, PortugalPublication . Oliveira, Cátia; Amador, Paula; Prudêncio, Cristina; Tomaz, Cândida T; Tavares-Ratado, Paulo; Fernandes, RúbenBackground and Objectives: Given the considerable spatial, temporal, and ecological factors, heterogeneity, which affects emergency response, persistence, and dissemination of genetic determinants that confer microorganisms their resistance to antibiotics, several authors claim that antibiotics' resistance must be perceived as an ecological problem. The aim of this study was to determine the prevalence of broad-spectrum bla genes, not only Extended-spectrum β-lactamases (ESBL) but also AmpC-types, in clinical strains of Escherichia coli isolated from Portugal (in the highest region of the country, Serra da Estrela) to disclose susceptibility profiles among different genotypes, and to compare the distribution of bla genes expressing broad-spectrum enzymes. Materials and Methods: Clinical strains of Escherichia coli presenting resistance to third generation (3G) cephalosporins and susceptibility to inhibition by clavulanic acid were studied by means of phenotypic and molecular profiling techniques for encoding β-lactamases genes. Results: Strains were mainly isolated from hospital populations (97%). Molecular analysis enabled the detection of 49 bla genes, in which 55% (27/49) were identified as blaOXA-1-like, 33% (16/49) as blaCTX-M-group-1, 10% (5/49) as blaTEM, and 2% (1/49) were identified as genes blaCIT (AmpC). Among all blaOXA-1-like detected, about 59% of strains expressed at least another bla gene. Co-production of β-lactamases was observed in 40% of strains, with the co-production of CTX-M group 1 and OXA-1-like occurring as the most frequent. Conclusions: This is the first study using microorganisms isolated from native people from the highest Portuguese mountain regions, showing an unprecedent high prevalence of genes blaOXA-1-like in this country.
- Extended-spectrum β-lactamases from the North of Portugal in the boundaries to Spain: emergence of high resistance to 4th generation cephalosporinsPublication . Fernandes, Rúben; Ferraz, Ricardo; Vieira, Mónica; Amador, Paula; Brito, Luísa; Coelho, Pedro; Silva, Nuno; Oliveira, C.; Prudêncio, CristinaDuring the past 15 years, emergence and dissemination of third-generation cephalosporins resistance in nosocomial Enterobacteriaceae became a serious problem worldwide, due to the production of extended-spectrum-β-lactamases (ESBLs). The aim of this study was to investigate among the presence of ESBL-producing enterobacteria among Portuguese clinical isolates nearby Spain, to investigate the antimicrobial susceptibility patterns and to compare the two countries. The β-lactamases genes, blaTEM, blaSHV and blaCTX-M were detected by molecular methods. Among the ESBL-producing isolates it was found extraordinary levels (98.9%) of resistance to the fourth-generation cephalosporin Cefepime. These findings point to the need of reevaluate the definition of ESBL.
- In vitro transference and molecular characterization of bla TEM genes in bacteria isolated from Portuguese ready-to-eat foodsPublication . Amador, Paula; Fernandes, Rúben; Duarte, I.; Brito, Luísa; Prudêncio, CristinaThe principal aim of this study was to investigate the possibility of transference to Escherichia coli of β-lactam resistance genes found in bacteria isolated from ready-to-eat (RTE) Portuguese traditional food. From previous screenings, 128 β-lactam resistant isolates (from different types of cheese and of delicatessen meats), largely from the Enterobacteriaceae family were selected and 31.3% of them proved to transfer resistance determinants in transconjugation assays. Multiplex PCR in donor and transconjugant isolates did not detect bla CTX, bla SHV and bla OXY, but bla TEM was present in 85% of them, while two new TEMs (TEM-179 and TEM-180) were identified in two isolates. The sequencing of these amplicons showed identity between donor and transconjugant genes indicating in vitro plasmid DNA transfer. These results suggest that if there is an exchange of genes in natural conditions, the consumption of RTE foods, particularly with high levels of Enterobacteriaceae, can contribute to the spread of antibiotic resistance.
- Molecular Characterization of ESBL-Producing Enterobacteriaceae in Northern PortugalPublication . Fernandes, Rúben; Amador, Paula; Oliveira, Carla; Prudêncio, CristinaExtended-spectrum β-lactamases (ESBLs) prevalence was studied in the north of Portugal, among 193 clinical isolates belonging to citizens in a district in the boundaries between this country and Spain from a total of 7529 clinical strains. In the present study we recovered some members of Enterobacteriaceae family, producing ESBL enzymes, including Escherichia coli (67.9%), Klebsiella pneumoniae (30.6%), Klebsiella oxytoca (0.5%), Enterobacter aerogenes (0.5%), and Citrobacter freundii (0.5%). β -lactamases genes blaTEM, blaSHV, and blaCTX-M were screened by polymerase chain reaction (PCR) and sequencing approaches. TEM enzymes were among the most prevalent types (40.9%) followed by CTX-M (37.3%) and SHV (23.3%). Among our sample of 193 ESBL-producing strains 99.0% were resistant to the fourth-generation cephalosporin cefepime. Of the 193 isolates 81.3% presented transferable plasmids harboring bla ESBL genes. Clonal studies were performed by PCR for the enterobacterial repetitive intragenic consensus (ERIC) sequences. This study reports a high diversity of genetic patterns. Ten clusters were found for E. coli isolates and five clusters for K. pneumoniae strains by means of ERIC analysis. In conclusion, in this country, the most prevalent type is still the TEM-type, but CTX-M is growing rapidly.
- Prevalence of antibiotic resistance genes in multidrug-resistant enterobacteriaceae on Portuguese Livestock ManurePublication . Amador, Paula; Fernandes, Rúben; Prudêncio, Cristina; Duarte, IsabelThe exposure of both crop fields and humans to antibiotic-resistant bacteria in animal excreta is an emergent concern of the One Health initiative. This study assessed the contamination of livestock manure from poultry, pig, dairy farms and slaughterhouses in Portugal with resistance determinants. The resistance profiles of 331 Enterobacteriaceae isolates to eight β-lactam (amoxicillin, cefoxitin, cefotaxime, cefpirome, aztreonam, ceftazidime, imipenem and meropenem) and to five non-β-lactam antibiotics (tetracycline (TET), trimethoprim/sulfamethoxazole (SXT), ciprofloxacin (CIP), chloramphenicol (CHL) and gentamicin) was investigated. Forty-nine integron and non-β-lactam resistance genes were also screened for. Rates of resistance to the 13 antibiotics ranged from 80.8% to 0.6%. Multidrug resistance (MDR) rates were highest in pig farm samples (79%). Thirty different integron and resistance genes were identified. These were mainly associated with resistance to CHL (catI and catII), CIP (mainly, qnrS, qnrB and oqx), TET (mainly tet(A) and tet(M)) and SXT (mostly dfrIa group and sul3). In MDR isolates, integron presence and non-β-lactam resistance to TET, SXT and CHL were positively correlated. Overall, a high prevalence of MDR Enterobacteriaceae was found in livestock manure. The high gene diversity for antibiotic resistance identified in this study highlights the risk of MDR spread within the environment through manure use.
- Resistance to β-lactams in bacteria isolated from different types of Portuguese cheesePublication . Amador, Paula; Fernandes, Rúben; Prudêncio, Cristina; Brito, LuísaThe purpose of this study was to investigate the presence of beta-lactam-resistant bacteria in six different types of Portuguese cheese. The numbers of ampicillin resistant (AMP(r)) bacteria varied from 4.7 x 10(2) to 1.5 x 10(7) CFU/g. Within 172 randomly selected beta-lactam-resistant bacteria, 44 resistant phenotypes were found and 31.4% were multidrug resistant. The majority (85%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the bla(TEM) gene was detected in 80.9% of the tested isolates. The results suggest that without thermal processing of the milk and good hygienic practices, cheese may act as a vehicle of transfer of beta-lactam-resistant bacteria to the gastrointestinal tract of consumers.
- ß-lactamases in the biochemistry and molecular biology laboratoryPublication . Amador, Paula; Prudêncio, Cristina; Vieira, Mónica; Ferraz, Ricardo; Fonte, Rosália; Silva, Nuno; Coelho, Pedro; Fernandes, Rúbenβ-lactamases are hydrolytic enzymes that inactivate the β-lactam ring of antibiotics such as penicillins and cephalosporins. The major diversity of studies carried out until now have mainly focused on the characterization of β-lactamases recovered among clinical isolates of Gram-positive staphylococci and Gram-negative enterobacteria, amongst others. However, only some studies refer to the detection and development of β-lactamases carriers in healthy humans, sick animals, or even in strains isolated from environmental stocks such as food, water, or soils. Considering this, we proposed a 10-week laboratory programme for the Biochemistry and Molecular Biology laboratory for majors in the health, environmental, and agronomical sciences. During those weeks, students would be dealing with some basic techniques such as DNA extraction, bacterial transformation, polymerase chain reaction (PCR), gel electrophoresis, and the use of several bioinformatics tools. These laboratory exercises would be conducted as a mini research project in which all the classes would be connected with the previous ones. This curriculum was compared in an experiment involving two groups of students from two different majors. The new curriculum, with classes linked together as a mini research project, was taught to a major in Pharmacy and an old curriculum was taught to students from environmental health. The results showed that students who were enrolled in the new curriculum obtained better results in the final exam than the students who were enrolled in the former curriculum. Likewise, these students were found to be more enthusiastic during the laboratory classes than those from the former curriculum.
- ß-Lactams: chemical structure, mode of action and mechanisms of resistancePublication . Fernandes, Rúben; Amador, Paula; Prudêncio, CristinaThis synopsis summarizes the key chemical and bacteriological characteristics of β-lactams, penicillins, cephalosporins, carbanpenems, monobactams and others. Particular notice is given to first-generation to fifth-generation cephalosporins. This review also summarizes the main resistance mechanism to antibiotics, focusing particular attention to those conferring resistance to broad-spectrum cephalosporins by means of production of emerging cephalosporinases (extended-spectrum β-lactamases and AmpC β-lactamases), target alteration (penicillin-binding proteins from methicillin-resistant Staphylococcus aureus) and membrane transporters that pump β-lactams out of the bacterial cell.