DDI - Apresentações orais em encontros internacionais
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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.
- Ampicillin Resistance Mechanisms in Clinical Haemophilus influenzae: What is Happening in Portugal?Publication . Bajanca-Lavado, Maria PaulaHaemophilus influenzae (Hi) remains a key etiological agent of upper and lower respiratory tract infections. Two major mechanisms are involved in ampicillin (AMP) resistance: β-lactam hydrolysis due to β-lactamase production (TEM-1 or ROB-1) and decreased affinity of penicillin-binding protein 3 (PBP3) for β-lactam antibiotics as a result of ftsI gene mutations encoding PBP3. Isolates exhibiting this latter resistant mechanism are termed β-lactamase-non-producing ampicillin-resistant (BLNAR), while isolates with both resistant mechanisms are defined as β-lactamase-positive amoxicillin-clavulanic acid-resistant (BLPACR). A variety of amino acid (AA) substitutions within the transpeptidase domain of PBP3 are mainly responsible for resistance. According to specific substitutions, these isolates have been classified in one of three mutational groups: I-III. Group II was further divided into subgroups IIa-IId. More recently, a new group was described, “III-like”, with additionally AA substitutions to the ones described in group III. Decreased ampicillin susceptibility have been associated to group I and II, while group III is normally associated with high resistance levels to ampicillin. Isolates with the non enzymatic resistance mechanisms have been described and emerging worldwide. In this context, we aimed to characterize ampicillin resistance mechanisms in clinical Hi strains isolated in Portugal. Amplification and sequencing of ftsI gene was performed in 568 clinical Hi isolates. Analysis of mutations characterized 61% of isolates as gBLNAR or gBLPACR. Most of the strains were included in group II (85%), with predominance of IIb (61%). Rare isolates were of group I and no isolate was classified in group III, although few strains were of group III-like. Our results are indicative of wide dissemination of a non-enzymatic resistance mechanism to β-lactams among H. influenzae isolates circulating in our country, probably due to inappropriate use of oral antibiotics, which is a matter of concern. A better understanding of this issue may help to establish adequate therapeutic and preventive measures to avoid selection or dissemination of such strains.
- Antimicrobial drug resistance of Campylobacter spp and Salmonella enterica: national data in food producing animals and food of animal originPublication . Clemente, L.; Correia, I.; Ferreira, E.; Manageiro, V.; Jones-Dias, D.; Albuquerque, T.; Themudo, P.; Rocha, T.; Tavares, A.; Geraldes, M.; Barahona, M.J.; Caniça, M.Campylobacter spp and Salmonella enterica are the two most common causes of bacterial foodborne illnesses in humans in developed countries, being food producing animals one the main reservoirs. Antimicrobial susceptibility testing was determined through Minimum Inhibitory Concentration, in 448 isolates of Campylobacter spp recovered from broiler ceca at slaughter (n=351) and broiler carcasses (n=97); and 1600 isolates of S. enterica feed (n=43) and food products of animal origin (n=527). Screening and identification of beta-lactamase and plasmid-mediated quinolone resistance (PMQR) genes were performed by PCR and sequencing. The highest level of resistance in Campylobacter spp isolates recovered from broilers and carcasses was recorded to ciprofloxacin, followed by tetracycline, erythromycin and streptomycin. Four isolates of Campylobacter coli were resistant to gentamicin.
- 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.
- Assessment of Francisella Tularensis in PortugalPublication . Lopes de Carvalho, I.; Carvalho, C.L.; Kingry, L.R.; Zé-Zé, Líbia; Petersen, J.M.; Núncio, M.S.
- Bacterial biofilms: a story of persistence and invasionPublication . Sousa, Sara; Bandeira, Maria; Carvalho, Patricia ALmeida; Duarte, Aida; Jordão, LuísaBiofilms are described as colonies of microorganisms that are attached to each other and to a surface, in an irreversible mode. These structures are virtualy everywhere: natural and humanized environments, as well as within living beings (humans and animals). For a long time biofilms where regarded as a bacterial survival strategie. Nowadays, in the industrialized world, the impact of acute bacterial infections caused by rapidly proliferating planktonic cells have gradually decreased in comparison to chronic infections owing to environmental organisms growing as biofilms. The major concern in this field is the healthcare-associated infections (HAIs). In 2012, HAIs estimated cases reached 6.7 million either in long-term care facilities or acute-care hospitals from which result 37,000 deaths configuring a serious public health problem [1]. The etiological agents are diverse being often resistant to antimicrobials and able to assemble biofilms both in abiotic and biotic surfaces. Here we evaluated the ability of different bacteria to assemble biofilms on a model surface and materials mimicking surfaces present either on healthcare units or medical devices. All bacteria were able to assemble biofilmls although following different kinetics and exhibiting different structural features assessed by electron microscopy. Additionally a link was established between bacteria ability to assemble biofilms and increased antibiotic resistance [2]. 1. ECDC Europe; (2012), Annual epidemiological report 2011 2. Bandeira M; (2014), Pathogens (doi:10.3390/pathogens3030720)
- Biofilm Formation by ST17 and ST19 Strains of Streptococcus agalactiaePublication . Silvestre, Inês; Borrego, Maria José; Jordão, LuísaBacterial biofilms are an important virulence factor with a vital role in evasion from the host immune system, colonization and infection. The aim of the present study was to evaluate in vitro the effects of three environmental factors (H+ , glucose and human plasma) in biofilm formation, by carrier and invasive S. agalactiae strains of ST17 and ST19 sequence types, including DNase producers and nonproducers. Bacteria ability to assemble biofilms was classified based on crystal violet assay. Biofilm formation was also monitored by scanning electron microscopy. Depending on the growth medium used, each bacterial isolate could fit in different biofilm production categories. Our data showed that optimal conditions for S. agalactiae biofilm assembly were reached after 48 h incubation at pH 7.6 in the presence of glucose and inactivated human plasma. In the presence of inactivated human plasma, the biofilm biomass of ST19 strains experienced a higher increase than ST17 strains. The composition of the extracellular polymeric matrix of the three strongest biofilm producers (all from ST17) was accessed by enzymatic digestion of mature biofilms and proteins were shown to be the predominant component. The detailed identification of the extracellular protein components should contribute to the development of new therapeutic strategies to fight S. agalactiae infections.
- Biofilms and catheter related bloodstream infection: a tale of two kigdomsPublication . Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel; Faria, Isabel; Pessanha, Maria Ana; Verissimo, Cristina; Sabino, Raquel; Rodrigues, Joao; Matias, Rui; Martins, Filomena; Carvalho, Patricia; Gomes, Joao Paulo; Jordão, LuísaBackground: Biofilm-associated infections are a public health concern in the context of healthcare-associated infections (HAI) such as catheter-related bloodstream infections (CRBSI). Here, we studied two top ten CRBS etiological agents, Enterobacter cloacae and Candida parapsilosis, isolated from a patient with CRBSI in order to understand the role played by biofilms on this HAI. Materials/methods: E.cloacae and C.parapsilosis were isolated from CVC and peripheral blood by standard procedures. EUCAST guidelines were followed for antimicrobial susceptibility evaluation. Single and/or mixed biofilms were assembled on different materials in Mueller-Hinton broth with 2% glucose. Biofilm assembly was assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification and to assess microorganisms distribution within the biofilm (3D reconstruction). In addition, Focus Ion Beam (FIB)-SEM was used to assess biofilms assembled on inner and outer surfaces of CVCs and construct tomograms. CVC and hemoculture (HC) isolates were subjected to whole-genome sequencing (WGS). Results: All Enterobacter and Candida isolates were antimicrobial resistant. Of note, E. cloacae-CVC revealed an additional resistance (ceftolozame-tazobactam) in comparison to the HC- isolate. Both microorganisms assembled biofilms on glass, polystyrene and polyurethane. Mixed biofilms were denser when both microorganisms were present from the beginning. Biofilm phenotype was not dependent of biofilm initiation by E.cloacae or C.parapsilosis. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Environmental isolates of the same species were also tested, showing that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), we observed denser mixed biofilms richer in EPS. FIB-SEM preliminary results suggest that biofilms assembled on inner and outer catheter surface might differ on microorganisms’ distribution. WGS confirmed the genetic identity of the CVC/HC pairs while corroborating the virulence potential and antimicrobial resistant character of the CRBSI-driving pathogens. Conclusions: The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Borrelia wingmen: dispersal and maintenance of Borrelia burgdorferi s.l. by birdsPublication . Norte, A. C.; Ramos, J.A.; Araújo, P.M.; da Silva, L.P.; Heylen, D.; Costantini, D.; Eens, M.; Núncio, M.S.; Lopes de Carvalho, I.Lyme borreliosis, caused by Borrelia burgdorferi s.l., is the most prevalent vector-borne disease of moderate climates of the northern hemisfere. In Portugal, several Borrelia genospecies are present in questing ticks, which have different associations with vertebrate reservoir hosts and Lyme borreliosis etiology. To better understand disease risk it is necessary to evaluate the relationships among Borrelia genospecies, their tick vectors and vertebrate reservoir hosts.