Browsing by Author "Manageiro, Vera"
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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.
- Alkyl deoxyglycoside-polymyxin combinations against critical priority carbapenem-resistant gram-negative bacteriaPublication . Matos, Ana M. de; Calado, Patrícia; Miranda, Mónica; Almeida, Rita; Rauter, Amélia P.; Oliveira, M. Conceição; Manageiro, Vera; Caniça, ManuelaThe escalating antimicrobial resistance crisis urges the development of new antibacterial treatments with innovative mechanisms of action, particularly against the critical priority carbapenem-resistant Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA) and Enterobacteriaceae (CRE). Membrane-disrupting dodecyl deoxyglycosides have been reported for their interesting phosphatidylethanolamine-associated bactericidal activity against Gram-positive strains; however, their inability to penetrate the Gram-negative outer membrane (OM) renders them useless against the most challenging pathogens. Aiming to repurpose alkyl deoxyglycosides against Gram-negative bacteria, this study investigates the antimicrobial effects of five reference compounds with different deoxygenation patterns or anomeric configurations in combination with polymyxins as adjuvants for enhanced OM permeability. The generation of the lead 4,6-dideoxy scaffold was optimized through a simultaneous dideoxygenation step and applied to the synthesis of a novel alkyl 4,6-dideoxy C-glycoside 5, herein reported for the first time. When combined with subtherapeutic colistin concentrations, most glycosides demonstrated potent antimicrobial activity against several multidrug-resistant clinical isolates of CRAB, CRE and CRPA exhibiting distinct carbapenem resistance mechanisms, together with acceptable cytotoxicity against human HEK-293T and Caco-2 cells. The novel 4,6-dideoxy C-glycoside 5 emerged as the most promising prototype structure for further development (MIC 3.1 μg/mL when combined with colistin 0.5 μg/mL against CRPA or 0.25 μg/mL against several CRE and CRAB strains), highlighting the potential of C-glycosylation for an improved bioactive profile. This study is the first to show the potential of IM-targeting carbohydrate-based compounds for the treatment of infections caused by MDR Gram-negative pathogens of clinical importance.
- Antibiotic resistance in foodborne bacteriaPublication . Caniça, Manuela; Manageiro, Vera; Abriouel, Hikmate; Moran-Gilad, Jacob; Franz, Charles M.A.P.Background: Antibiotic resistance, mainly due to imprudent use of antibiotics in agriculture, environment, animal and human medicine, has been widely recognized as one of the main global health concerns, threatening food security, and human and animal health, causing considerable economic losses. Scope and approach: We summarize the state of the art in antibiotic resistant foodborne bacteria and related reservoirs, some actions to overcome this threat, and the future perspectives in the field. Key findings and conclusions: Food and food production may be a vehicle of antibiotic resistant bacteria and antibiotic resistance genes to humans that have a public health impact. Mobile genetic elements such as plasmids, transposons, have the ability to form hybrid elements interplaying with or from the environment and foodborne bacteria. These genetic structures are able to encode for resistance for many antibiotics, namely those that are last resort treatments for patients infected with multidrug resistant bacteria. Information, education and training, surveillance, monitoring, record-keeping, reduction of infection, legislation, optimization and reduced antibiotic use, and sustainable investment for alternatives, are important actions to bring antibiotic resistant foodborne bacteria under control. Omics technologies such as genomics, metagenomics and transcriptomics, are valuable tools for surveillance and control of antibiotic resistance in different One Health settings, notably with respect to the selection, dissemination, and distribution of antibiotic resistant bacteria in food, as well as to unravel the antibiotic resistance mechanism involved. In the future metatranscriptomics, proteomics and metabolomics are expected to enlarge next-generation-sequencing tools to strengthen control of antibiotic resistance in food.
- Antibiotic resistance in freshwater cyanobacteria and associated bacteriaPublication . Dias, Elsa; Dias, Daniela; Ferreira, Eugénia; Manageiro, Vera; Vasconcelos, Vitor; Pereira, Paulo; Caniça, ManuelaObjectives: Cyanobacteria are ubiquitous prokaryotes in aquatic ecosystems and although they can be exposed to antibiotics, their role on water resistome was never investigated. Thus, this work aimed to evaluate the antibiotic susceptibility patterns and resistance mechanisms of cyanobacteria and co-occurring bacteria in order to assess their contribution to the global pool of resistance determinants in freshwater. Methods: We investigated 4 cyanobacterial genera (Microcystis, Aphanizomenon, Anabaena and Planktothrix), previously isolated from freshwater reservoirs, and several bacteria isolated from those cyanobacterial cultures. Antibiotic susceptibility of cyanobacteria was evaluated by microdilution method, under specific culturing conditions, against beta-lactams, aminoglycosides, quinolones, sulfonamides and tetracyclines. Minimum inhibitory concentrations (MIC) were determined according to cyanobacterial cell dentisty (DO, 450nm) and microscopic examination of cultures integrity. Bacteria were identified by 16S sequencing and their susceptibility patterns were determined by disk diffusion, according to SFM 2012 non-specific breakpoints, against the same antibiotics. All strains were subjected to the search of class 1, 2 and 3 integrons and antibiotic resistance genes according to the phenotype. Results: Overall, we observed a great diversity of susceptibility to the tested antibiotics, among the distinct strains. Microcystis showed the lowest susceptibility regarding beta-lactams. Conversely, Microcystis was more susceptible to quinolones, while Planktothrix showed higher MIC values. Bacteria from cyanobacterial cultures were identified as Hydrogenophaga atypica, Limnobacter thioxidans, Rhizobium radiobacter, Sphingobium sp. and Brevundimonas lenta. Even though no known antibiotic resistance genes were yet identified, bacteria from different species and showing distinct phenotypes exhibited class 1 and 2 integrons. L. thioxidans, for example, revealed to be resistant to aminoglycosides and harbored a class 2 integron. Conclusions: Although no known antibiotic resistance genes were found in cyanobacteria and co-occurring bacteria, the presence of integrons and the susceptibility to antibiotics, suggest that they may play a role on freshwater resistome and eventually contribute to the dissemination of antibiotic resistance. These results may also be helpful to define guidelines and breakpoints to access cyanobacteria antibiotic susceptibility.
- Antibiotic resistance: where are we and where are we going?Publication . Manageiro, VeraAntibiotic resistance is an emerging problem, becoming a serious threat to global public health. Indeed, we are seeing across Europe, namely in Portugal, an increasing number of patients with multidrug-resistant (MDR), extensively-drug resistant (XDR) and pandrug-resistant (PDR) infections, meaning there is no effective treatment available. New resistance mechanisms are disseminating worldwide, and the situation is aggravated by a low investment in this area, and specifically a substantial decline in the research and development of antibacterial agents, among others. Hence, very few new antibacterial classes are brought to market when older classes lose their efficacy. Antibiotic resistance constitutes a global health priority with severe consequences to humans, animals and to the environment, and should be tackled on all fronts in view to the actual concept of “One World - One Medicine - One Health”.
- 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 activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteriaPublication . Gonçalves, Tiago; Marques, Andreia T.; Manageiro, Vera; Tanoeiro, Luis; Vital, Joana S.; Duarte, Aida; Vítor, Jorge M.B.; Caniça, Manuela; Gaspar, Maria Manuela; Vale, Filipa F.Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.
- Antimicrobial Resistance - Human Health: Portugal experience – focused on human health issuesPublication . Manageiro, VeraThe EU initiative on AMR, aims at promoting a strong collaboration and sharing experiences in preventing and containing AMR. Aspects of human and animal health, general prevention, such as hospital infection control, antibiotic stewardship programs in hospitals, antibiotic free farming practices, research and innovation and trade will be addressed during a series of 7 events in different countries in South America. The EU will be delighted to count on the support and expertise of Argentina, Brazil, Chile, Colombia Paraguay, Peru and Uruguay and will welcome representatives by the relevant National Authorities working on the National Action Plan (NAP) on AMR in the different events. Following the opening conference in Brasilia in March, the first seminar that focused on Animal Health in Buenos Aires in June and the second workshop in Santiago in October, this workshop aims at raising awareness of antimicrobial resistance and the relevant studies and their latest results in the human health related aspects; sharing best practices that will facilitate drawing-up and implementing national AMR action plans; making synergies and aligning policies and practices between partner countries, as well as focusing on implementation of commitments made at the UN high-level event on AMR.
- Antimicrobial Resistance and Molecular Epidemiology of Staphylococcus aureus from Hunters and Hunting DogsPublication . Silva, Vanessa; Caniça, Manuela; Manageiro, Vera; Vieira-Pinto, Madalena; Pereira, José Eduardo; Maltez, Luís; Poeta, Patrícia; Igrejas, GilbertoSeveral studies have showed that a dog-to-human transmission of Staphylococcus aureus occurs. Hunting dogs do not have as much contact with their owners as dogs that live in the same household as the owners; however, these dogs have contact with their owners during hunting activities as well as when hunting game; therefore, we aimed to isolate S. aureus from hunters and their hunting dogs to investigate a possible S. aureus transmission. Nose and mouth samples were collected from 30 hunters and their 78 hunting dogs for staphylococcal isolation. The species identification was performed using MALDI-TOF. The antimicrobial susceptibility profiles were accessed using the Kirby-Bauer method and respective antimicrobial resistance genes were investigated by PCR. Multilocus sequence typing (MLST) and spa- and agr-typing was performed in all S. aureus isolates. S. aureus were detected in 10 (30%) human samples and in 11 (15.4%) dog samples of which 11 and 5 were methicillin-resistant S. aureus (MRSA). Other staphylococci were identified, particularly, S. pseudintermedius. Most S. aureus isolates were resistant to penicillin, erythromycin, and tetracycline. Evidence of a possible transmission of S. aureus between human and dogs was detected in three hunters and their dogs. S. aureus isolates were ascribed to 10 STs and 9 spa-types. A moderate colonization of S. aureus in hunting dogs and their owners was detected in this study. A few dog-to-dog and dog-to-human possible transmissions were identified.
- 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.