Browsing by Author "Jones-Dias, Daniela"
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- Accessing the molecular basis of transferable quinolone resistance in Escherichia coli and Salmonella spp from food-producing animals and productsPublication . Caniça, Manuela; Jones-Dias, Daniela; Francisco, Ana Patrícia; Manageiro, Vera; Ferreira, EugéniaBackground: Salmonella and Escherichia coli resistant to quinolones frequently arise in animals, being easily transferred to humans through the food chain, which can ultimately lead to the development of untreatable infectious diseases. The aim of the present study was to investigate the presence of PMQR determinants among Salmonella spp and E. coli from food-producing animals and derivative food products. Methods: Salmonella spp (n=183) and E. coli (n=182) isolates were collected from food-producing animals (n=274) and derivative food products (n=91). Antimicrobial susceptibility testing was performed by standard disk diffusion method, according to the CA-SFM veterinary guidelines. PCR and sequencing were used to detect PMQR- (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, and qepA) and β-lactamase-encoding genes (blaTEM, blaSHV, blaOXA and ampC) and to examine the QRDR of gyrA, gyrB, parC and parE genes in PMQR positive isolates. Plasmid characterization was accessed by conjugation followed by replicon-typing. Genetic relatedness of PMQR positive E. coli was examined by MLST and Salmonella isolates were serotyped according to the Kauffmann-White scheme. Mobile genetic elements were also investigated through PCR mapping assays. Results: Overall, 4.7% (17/365) harbored Qnr-encoding genes from qrnB and qnrS families, specifically qnrB2 (n=3), qrnB19 (n=3), and qnrS1 (n=11). All but one isolate presented at least one mutation in QRDR region of genes gyrA, parC or parE genes. 35.3% of Qnr-producing isolates presented resistance to β-lactam antibiotics that were justified by the presence of β-lactamases from TEM (TEM-1, n=10; and TEM-135, n=1) and SHV (SHV-108, n=1) families in QnrB19- and QnrS1-harbouring isolates. All but one Qnr-producing isolates were positively typed by replicon-typing, varying among IncN (n=2), IncFIB (n=11), IncFIC (n=3), IncI1 (n=2), IncHI2 (n=5), IncY (n=1) and IncL/M (n=3) and were, mostly, genetic unrelated. Qnr genes were detected nearby several mobile elements like ISEcl2, IS26 and ISCR1. Conclusions: This study illustrated the existence of Qnr-producing E. coli and Salmonella from food-producing animals, associated to specific mobile elements that can mediate their transference between species and among distinct settings. Epidemiology of PMQR mechanisms and the dissemination of plasmids carrying Qnr-encoding genes in veterinary isolates can compromise the efficacy of fluroquinolone treatments in both animals and humans.
- Antibiotic-resistant bacteria from air samples collected in nursing homesPublication . Salgueiro, Vanessa; Manageiro, Vera; Jones-Dias, Daniela; Cano, Manuela; Ferreira, Eugénia; Caniça, ManuelaIntroduction: Antibiotic resistance is currently a worldwide public health problem that can include diverse mechanisms and bacterial species. The understanding and monitoring of such phenomena is essential to avoid further dissemination. The main aim of this study was the characterization of antibiotic resistance mechanisms in bacterial strains collected in air samples from nursing homes and compare them with clinical samples isolated from the same geographic area. Materials and methods: Air samples were collected from bedrooms, living rooms and outdoor of 4 nursing homes located in Lisbon, Portugal. Screening of antimicrobial susceptibility of 18 Gram-negative (5 Acinetobacter spp., 1 Klebsiella oxytoca, 4 Pantoea spp., 7 Pseudomonas spp., 1 Sphingomonas paucimobilis) and 12 Gram-positive (3 Micrococcus luteus, 3 Staphylococcus spp.) isolates was performed by disk diffusion method. Different antibiotic resistant genes were searched by PCR. PFGE was used to evaluate clonality between K. oxytoca isolated from the air environment and other strains from clinical origin. Results and discussion: The majority of strains were susceptible to all antibiotics tested. Among beta-lactam antibiotics, reduced susceptibility to cefoxitin was detected in Staphylococcus capitis, through expression of the mecA gene, and to ampicillin, piperacillin and piperacillin-tazobactam in 1 K. oxytoca expressing a blaOXY-5-type beta-lactamase. Nonsusceptibility to meropenem and piperacillin-tazobactam was observed in 2 Pseudomonas putida however no antibiotic resistance gene was detected. Regarding quinolones, non-susceptibility was found in 1 S S. capitis, 2 Staphylococcus haemolyticus, and 1 K. oxytoca. The molecular characterization of the mecA-positive S. capitis from the nursing homes and from a hospital within the same region suggests a potential dissemination of strains between these two environments. The genetic relatedness of K. oxytoca from nursing homes (n=1) and clinical isolates (n=9) recovered within the same region, allowed to conclude that they were not genetically related. Conclusions: Globally, nursing homes environments may act as complementary reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. Thus, a better understanding of the antibiotic resistance mechanisms and dissemination pathways in other reservoirs than human is essential to control its emergence and spread.
- Antimicrobial susceptibility and integron diversity in organic and conventionally grown fruits and vegetablesPublication . Jones-Dias, Daniela; Moura, Inês Barata; Ferreira, Eugénia; Caniça, ManuelaIntroduction: During growth and harvesting fresh produce can become contaminated not only with environmental, but also pathogenic bacteria from animal and human sources. This study aimed to characterize the antibiotic resistant bacteria gathered from organically and conventionally grown fruits and vegetables, and to characterize the diversity of class 1, 2 and 3 integrons detected. Methods: Between 2013 and 2014, one conventionally and one organically produced batch of six fruits and vegetables were purchased at retail stores. Products were processed as follows: 50g of product was selected at random without washing or peeling, diluted and homogenized. The selection of resistant Gram negative bacteria was performed in VRBG plates containing ten different antibiotics separately. The isolates were identified through the amplification of the 16S rDNA. Antimicrobial susceptibility was assessed by disc diffusion method using EUCAST guidelines. All isolates were investigated for the presence of class 1, 2 and 3 integrons, through PCR amplification, and their variable region was explored by a strategy of Next Generation Sequencing. Results: A total of 333 isolates showing nonsusceptibility to, at least, one antibiotic were collected among 144 samples. Among others, Enterobactericeae (n=184), Moraxellaceae (n=88), Pseudomonadaceae (n=36), were detected. Nonsusceptibility was mainly identified among β-lactam antibiotics for Enterobactericeae recovered from conventionally produced samples (45.1%); overall, cefoxitin was the most ineffective antibiotic (19.6 % for organic and 26.6% for conventionally grown produce). The molecular screening for the integrase-encoding gene showed the presence of eight isolates with class 1 (2 Enterobacter, 2 Escherichia, 1 Morganella, 2 Acinetobacter and 1 Pseudomonas spp.), three isolates with class 2 (1 Escherichia and 2 Raoutella spp.), and one isolate with class 3 integrons (Klebsiella spp.). The integrons revealed a variety of gene cassettes conferring nonsusceptibility to different classes of antibiotics. Conclusion: Resistance was more frequent in the products grown in close contact with the soil. Besides environmental bacteria, many pathogenic agents were also detected. Lettuce was significantly associated with the existence of class 1 and 2 integrons regardless of the origin. Both organic and conventional produced fruits and vegetables may constitute potential sources of resistant bacteria and of integrons.
- Antimicrobial susceptibility and oxymino-β-lactam resistance mechanisms in Salmonella enterica and Escherichia coli isolates from different animal sourcesPublication . Clemente, Lurdes; Manageiro, Vera; Jones-Dias, Daniela; Correia, Ivone; Themudo, Patricia; Albuquerque, Teresa; Geraldes, Margarida; Matos, Filipa; Almendra, Cláudia; Ferreira, Eugénia; Caniça, ManuelaThe impact of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (PMAβs) of animal origin has been a public health concern. In this study, 562 Salmonella enterica and 598 Escherichia coli isolates recovered from different animal species and food products were tested for antimicrobial resistance. Detection of ESBL-, PMAβ-, plasmid-mediated quinolone resistance (PMQR)-encoding genes and integrons was performed in isolates showing non-wild-type phenotypes. Susceptibility profiles of Salmonella spp. isolates differed according to serotype and origin of the isolates. The occurrence of cefotaxime non-wild-type isolates was higher in pets than in other groups. In nine Salmonella isolates, blaCTX-M (n = 4), blaSHV-12 (n = 1), blaTEM-1 (n = 2) and blaCMY-2 (n = 2) were identified. No PMQR-encoding genes were found. In 47 E. coli isolates, blaCTX-M (n = 15), blaSHV-12 (n = 2), blaCMY-2 (n = 6), blaTEM-type (n = 28) and PMQR-encoding genes qnrB (n = 2), qnrS (n = 1) and aac(6')-Ib-cr (n = 6) were detected. To the best of our knowledge, this study is the first to describe the presence of blaCMY-2 (n = 2) and blaSHV-12 (n = 1) genes among S. enterica from broilers in Portugal. This study highlights the fact that animals may act as important reservoirs of isolates carrying ESBL-, PMAβ- and PMQR-encoding genes that might be transferred to humans through direct contact or via the food chain.
- Antimicrobial susceptibility of Escherichia coli strains isolated from food-producing, companion and wild animals, in Portugal – Characterization of isolates with reduced susceptibility to third generation cephalosporins and cephamycinsPublication . Clemente, Lurdes; Manageiro, Vera; Jones-Dias, Daniela; Ferreira, Eugénia; Correia, Ivone; Albuquerque, Teresa; Geraldes, Margarida; Matos, Filipa; Almendra, Claúdia; Themudo, Patrícia; Caniça, ManuelaEscherichia coli is a common inhabitant of the gastrointestinal tract of humans and animals, but is also a causative agent of diarrhea and extraintestinal infections. There is ongoing concern about the risks posed to human health by antimicrobial resistant bacteria isolated from animals. This study was conducted on 602 Escherichia coli isolates recovered from food-producing (n=217), companion (n=114) and wild animals (n=271), over the period of 2009-2013, to determine their antimicrobial susceptibility to a panel of ten antimicrobials (ampicillin, cefotaxime, ciprofloxacin, nalidixic acid, trimethoprim, sulfamethoxazole, chloramphenicol, streptomycine, gentamicine and tetracycline), through the determination of Minimum Inhibitory Concentration (MIC), using the agar dilution technique. Susceptibility to cefoxitin was determined through disk diffusion method. Molecular characterization of isolates showing a non-wild type MIC to cefotaxime was performed, to determine extended spectrum β-lactamases (ESBL), plasmid mediated AmpC (PMAβ), plasmid mediated quinolone (PMQR) resistance determinants and mobile genetic elements involved in the dissemination of resistance genes. Overall, isolates recovered from food producing animals showed higher frequencies of resistance towards all antimicrobials tested and multidrug resistance (MDR) (53%), followed by companion (43%) and wild animals (30%). Fifty isolates presented a non-wild phenotype to cefotaxime and resistance or intermediate susceptibility to cefoxitin, being 14 (12.3%) from pets, 19 (8.8%) from food producing animals and 17 (6.3%) from wild animals. Polymerase chain reaction and sequencing of the amplicons confirmed the presence of blaCTX-M-type (n=14), blaSHV-type (n=2), blaTEM-type (n=31), blaOXA-type (n=6) and plasmid-mediated AmpC β-lactamases (PMAβ) genes (n=8). Plasmid-mediated quinolone resistance-encoding genes were detected in ten isolates: aac(6')-Ib-cr (n=6) qnrB17 (n=1), qnrS1(n=1) and qnrB19 (n=2). Twenty five isolates carried class 1 integrons and two carried class 2 integrons. This study agrees that animals may act as important reservoirs of E. coli isolates carrying ESBL and PMAβ-encoding genes, which might be transmissible to humans through direct contact or the food chain and, a potential source for human pathogens to acquire these resistance genes.
- Antimicrobial susceptibility of Salmonella enterica isolated from food-producing animals, animal feed and food products of animal origin, in Portugal - Genetic analysis of isolates with reduced susceptibility/resistance to third generation cephalosporins and cephamycinsPublication . Clemente, Lurdes; Manageiro, Vera; Jones-Dias, Daniela; Ferreira, Eugénia; Correia, Ivone; Themudo, Patrícia; Albuquerque, Teresa; Caniça, ManuelaSalmonella is a widely distributed foodborne pathogen and one of the most common causes of bacterial foodborne illnesses in humans. An epidemiologic study was conducted on 1600 Salmonella spp isolates recovered from poultry, swine, other animal species, animal feed and food products of animal origin, over the period of 2009-2013, to determine their serotype and antimicrobial susceptibility to a panel of ten antimicrobials (ampicillin, cefotaxime, ciprofloxacin, nalidixic acid, trimethoprim, sulfamethoxazole, chloramphenicol, streptomycine, gentamicine and tetracycline), through the determination of Minimum Inhibitory Concentration (MIC), using the agar dilution technique. Molecular characterization of isolates showing a non-wild type MIC to cefotaxime was performed, to determine extended spectrum β-lactamases (ESBL), plasmid mediated AmpC (PMAβ), plasmid mediated quinolone (PMQR) resistance determinants and mobile genetic elements involved in the dissemination of resistance genes. In live poultry (breeders, broilers, layers) of the 843 isolates recovered, 27.9% comprised S. Enteritidis, 23,5% Salmonella Havana and 14.1% Salmonella Mbandaka; in turkeys, Salmonella Derby was the most common serovar isolated (44%), followed by Salmonella I 4,[5],12:i:- (16%). In swine, of 101 isolates 21.8% comprised Salmonella Rissen and Salmonella Typhimurium, 10.9% Salmonella Derby and Salmonella London. In other animal species, Salmonella Typhimurium was the prevalent serovar with 65.6% of the isolates, followed by Salmonella I 4,[5],12:i:- (9.8%). Overall, S. 4,[5],12:i:- was the most common serotype recovered from food products (25.8%), followed by S. Typhimurium (19.2%) and Salmonella Rissen (18.4%). S. Enteritidis was the most frequent serotype in poultry products (36.3%). Susceptibility profiles differed according with the serotype and the origin of the isolates. A higher frequency of multidrug resistant isolates was recovered from food of swine and bovine origin, with 62.6% and 59.4%, respectively. Polymerase chain reaction and sequencing of the amplicons confirmed the presence of blaCTX-M-type (n=8), blaSHV-type (n=2), blaTEM-type (n=2) and plasmid-mediated AmpC β-lactamases (PMAβ) genes (n=2). No plasmid mediated quinolone resistance-encoding genes were detected. Six isolates( three S. I 4,[5],12:i:-, two S. Havana and one S. Enteritidis) carried class 1 integrons and one S. I 4,[5],12:i:- isolate harboured a class 2 integron. In conclusion, the growing concern of the emergence of bacterial strains bearing ESBL in food-producing animals highlights the importance of continuous monitoring.
- Architecture of Class 1, 2, and 3 Integrons from Gram Negative Bacteria Recovered among Fruits and VegetablesPublication . Jones-Dias, Daniela; Manageiro, Vera; Ferreira, Eugénia; Barreiro, Paula; Vieira, Luís; Moura, Inês B; Caniça, ManuelaThe spread of antibiotic resistant bacteria throughout the food chain constitutes a public health concern. To understand the contribution of fresh produce in shaping antibiotic resistance bacteria and integron prevalence in the food chain, 333 antibiotic resistance Gram negative isolates were collected from organic and conventionally produced fruits (pears, apples, and strawberries) and vegetables (lettuces, tomatoes, and carrots). Although low levels of resistance have been detected, the bacterial genera identified in the assessed fresh produce are often described not only as environmental, but mostly as commensals and opportunistic pathogens. The genomic characterization of integron-harboring isolates revealed a high number of mobile genetic elements and clinically relevant antibiotic resistance genes, of which we highlight the presence of as mcr-1, qnrA1, bla GES-11, mphA, and oqxAB. The study of class 1 (n = 8), class 2 (n = 3) and class 3 (n = 1) integrons, harbored by species such as Morganella morganii, Escherichia coli, Klebsiella pneumoniae, led to the identification of different integron promoters (PcW, PcH1, PcS, and PcWTNG-10) and cassette arrays (containing drfA, aadA, cmlA, estX, sat, and bla GES). In fact, the diverse integron backbones were associated with transposable elements (e.g., Tn402, Tn7, ISCR1, Tn2 (*), IS26, IS1326, and IS3) that conferred greater mobility. This is also the first appearance of In1258, In1259, and In3-13, which should be monitored to prevent their establishment as successfully dispersed mobile resistance integrons. These results underscore the growing concern about the dissemination of acquired resistance genes by mobile elements in the food chain.
- Assessing antibiotic resistance in Gram negative bacteria from animals and the wider environmentPublication . Jones-Dias, Daniela; Caniça, Manuela; Nogueira, IsabelThe alarming increase in the levels of antibiotic resistant bacteria in clinical practice launched the call for a broader understanding of this event. This Ph.D. thesis aimed to unravel the main mobile antibiotic resistance determinants circulating in Gram negative bacteria from non-human sources, showing the contribution of mobile genetic elements for the overall process. The susceptibility and molecular epidemiological studies performed on different collections of bacterial isolates showed the predominance of multidrug resistant Escherichia coli in animals of different origins, soil and vegetables. E. coli and other species of Gram negative bacteria were related with carriage of diverse antibiotic resistance genes [e.g. blaCTX-M-1, blaCTX-M-15, blaCMY-2, blaGES-11, qnrS1, aac(6’)-Ib-cr, strAB, tet, drfA, aadA, mcr-1] that were associated with a transferable genetic support. Indeed, an assortment of mobile genetic elements (e.g. IncI1 and IncF plasmids, ISEcp1 insertion sequences, Tn402 and Tn7 transposons and class 1, 2 and 3 integrons) was detected in the genetic proximity of those antibiotic resistance genes, suggesting their profound involvement, not only in interspecies dispersion, but also in the movement of the genes within the cell. Specific genomic investigation of two Enterobactericeae isolates and the proteomic study of a third, underscored the potential of massive omic approaches to study antibiotic resistance as a global process. One of the key findings released from these studies is that antibiotic resistance is not only linked to virulence and pathogenicity, but can also be connected to core bacterial metabolic processes. The results obtained throughout this thesis extend our knowledge on the distribution of mobile antibiotic resistance genes in animals, environment and, ultimately, in the food chain. If the gathered results increased our concerns towards the current distribution of antibiotic resistant bacteria, they can also be an encouragement to address this problem at a global scale.
- Assessing the antibiotic susceptibility of freshwater Cyanobacteria spp.Publication . Dias, Elsa; Oliveira, Micaela; Jones-Dias, Daniela; Vasconcelos, Vitor; Ferreira, Eugénia; Manageiro, Vera; Caniça, ManuelaFreshwater is a vehicle for the emergence and dissemination of Antibiotic resistance. Cyanobacteria are ubiquitous in freshwater, where they are exposed to antibiotics and resistant organisms, but their role on water resistome was never evaluated. Data concerning the effects of antibiotics on cyanobacteria, obtained by distinct methodologies, is often contradictory. This emphasizes the importance of developing procedures to understand the trends of antibiotic susceptibility in cyanobacteria. In this study we aimed to evaluate the susceptibility of four cyanobacterial isolates from different genera (Microcystis aeruginosa, Aphanizomenon gracile, Chrisosporum bergii, Planktothix agradhii), and among them nine isolates from the same specie (M.aeruginosa) to distinct antibiotics (amoxicillin, ceftazidime, ceftriaxone, kanamycine, gentamicine, tetracycline, trimethoprim, nalidixicacid, norfloxacin). We used a method adapted from the bacteria standard broth microdilution. Cyanobacteria were exposed to serial dilution of each antibiotic (0.0015–1.6mg/L) in Z8 medium (20±1◦C; 14/10hL/D cycle; light intensity 16±421μEm−s−). Cell growth was followed overtime (OD450nm/microscopic examination) and the minimum inhibitory concentrations (MICs) were calculated for each antibiotic/ isolate. We found that β-lactams exhibited the lower MICs, aminoglycosides, tetracycline and norfloxacine presented intermediate MICs; none of the isolates were susceptible to trimethoprim and nalidixic acid. The reduced susceptibility of all tested cyanobacteria to some antibiotics suggests that they might be naturally non-susceptible to these compounds, or that they might became non-susceptible due to antibiotic contamination pressure, or to the transfer of genes from resistant bacteria present in the environment.
- CTX-M-15-Producing Escherichia coli in Dolphin, PortugalPublication . Manageiro, Vera; Clemente, Lurdes; Jones-Dias, Daniela; Albuquerque, Teresa; Ferreira, Eugénia; Caniça, ManuelaEscherichia coli among humans and its detection in livestock, companion animals, and wildlife is a major cause for concern. Hence, it is imperative to identify and explore its dissemination traits. If CTX-M-15–producing E. coli continues to spread among different environments, therapeutic options in veterinary and human medicine will be greatly narrowed. E. coli is one of the gram-negative bacteria most frequently isolated from bottlenose dolphins. However, few studies about antimicrobial drug–resistant bacteria in dolphins have been published. We explored dissemination linkages between CTX-M-15–producing E. coli isolated from a marine dolphin (Tursiops truncatus) and clinical isolates collected during the same period from humans all over Portugal. (...)