DDI - Apresentações orais em encontros internacionais
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Browsing DDI - Apresentações orais em encontros internacionais by Subject "Antibiotic Resistance"
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- 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.
- Characterization of Arcobacter butzleri isolates from poultry and slaughterhouse EnvironmentPublication . Ferreira, Susana; Fraqueza, Maria João; Queiroz, João; Domingues, Fernanda; Oleastro, MónicaThe genus Arcobacter is an emerging pathogen associated with several clinical symptoms, namely diarrhoea, abdominal pain or bacteraemia. This genus is widely distributed and has been isolated from environmental, animal, food and human samples, being poultry considered the major reservoir. In this study, forty three Arcobacter butzleri strains were isolated from poultry of three flocks from different farms and environment samples at a Portuguese slaughterhouse, also three reference strains were included. All isolates were confirmed at species level by multiplex PCR and genomic DNA fingerprints of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) after enzymatic digestion with SmaI and the isolates with undistinguishable SmaI patterns were further analyzed with a second restriction enzyme, SacII. Phenotypic resistance profiles to nine antibiotics were assessed by broth microdilution method. Biofilm formation assays were performed in the 36 out of the 43 A. butzleri isolates, either presenting different pulsetypes or similar PFGE patterns but different origin or even different resistance patterns. PFGE patterns obtained using restriction enzymes SmaI and SacII revealed genetic diversity, with 32 distinct PFGE patterns. A high percentage of A. butzleri isolates was found to be resistant, recording four different resistance profiles. Twenty four of the 43 isolates presented a phenotypic resistance to ciprofloxacin, in contrast to the great susceptibility against gentamicin and chloramphenicol. Among the 36 selected A. butzleri isolates, 13.9% were categorized as moderately adherent, while 58.3% were defined as weakly adherent. Overall, the results showed a high degree of genetic heterogeneity among A. butzleri isolates, along with high levels of resistance to several antibiotics. Biofilm formation ability of A. butzleri can possibly favour dispersion and cross-contamination along the slaughterhouse processing line. These findings may represent a contribution to get insight the survival and even persistence mechanisms of this organism in the environment and on its relevance as a potential hazard for foodborne infections.
- Concerns on antibiotic resistance: Cross-talk between different environments.Publication . Caniça, ManuelaThe arisal of antibiotic resistance is a major public health problem, at national and at international level. It has serious consequences in the control of infections, in hospital and in the community, as some classes of antibiotics are often the last resort to treat bacterial infections. Among others, in last years, it has been seen an explosion of extended-spectrum beta-lactamases, namely those from CTX-M lineage, which have become particularly widespread, as well as beta-lactamases from non-ESBL families, such as carbapemenases (KPC and metallo-beta-lactamases). We also emphasize the problem of methicillin-resistant Staphylococcus aureus isolates in different environments, and the appearance of vancomycin-resistantce in Staphylococcus aureus, a resistance mechanism recently identified in Portugal. The factors involved in the emergence of antibiotic resistance are numerous, and the constant adaptation of microorganisms to the selective pressure exerted by antibiotics is impressive. However, the success of spread of certain resistant clones remains sometimes difficult to determine. Thus, it is manifest that action must be taken, and research in this area should be enlarged, not only to better understand the dynamics of spread of resistance between different bacteria and different ecosystems, but also to enlarge the pharmaceutical pipeline of antibacterials against multidrug resistant pathogens.
- Exploring a link between healthcare associated infections, biofilms and antimicrobial resistancePublication . Bandeira, Maria; Borges, Vítor; Nascimento, Maria; Duarte, Aida; Gomes, Joao Paulo; Jordão, LuísaHAIs are a major public health problem affecting 4 million patients per year in Europe. Here we present results of a retrospective study in wich the ability of K. pneumoniae isolates (collected during a period of 31 years) to assemble biofilms was evaluated; and an ongoing study of multispecies biofilms. In the first study, we found major differences in biofilm assembly kinetics, extracellular polymeric substances (EPS) content, which were further corroborated by in-depth EPS composition analysis. WGS analysis revealed a high nucleotide similarity within the core-genome, but relevant differences in the accessory genome that may account for the detected biofilm phenotypic dissimilarities, such as genes already associated with biofilm formation in other pathogenic bacteria (e.g., genes coding haemogglutinins and haemolysins). These data reinforce that the research efforts to defeat bacterial biofilms should take into account that their dynamics may be contingent on the medical devices-associated materials. The second study is ongoing and we aim at understanding the basis of multispecies biofilm assembly on different surfaces. Bacterial and fungi isolates have been used as models and until now all results suggest that the two microorganisms play different roles on biofilm onset.
- Exploring the aquatic resistomePublication . Rosado, Tânia; Manageiro, Vera; Balata, Duarte; Menezes, Carina; Ferreira, Eugénia; Paulo, Octávio; Caniça, Manuela; Dias, ElsaAntibiotic resistance is one of the major problems in public health today since the failure of antibiotherapy has dramatic clinical implications such as the increase of mobility/mortality and of health costs. Antibiotic resistance is a dynamic process since antibiotic-resistant microorganisms, and the corresponding genetic material, flows and disseminates among several settings simultaneously: humans, animals and natural habitats. Water environments are recognized as important pools of antibiotic pollution and antibiotic resistance genes, but the water resistome (collection of all genes responsible for antibiotic resistance in water environments) is far from being characterized. It is well known that antibiotic pollution affects the structure/functioning/diversity of aquatic ecosystems, namely the cyanobacteria community. However, the role of cyanobacteria in the context of antibiotic resistance was never characterized. In this presentation, the aims, the team, the methodologies and the preliminary results of the national project EXPLORAR – Exploring the Aquatic Resistome (PTDC/BIA-BMA/31451/2017), founded by the Portuguese Foundation for Science and Technology (FCT, PT) will be discussed. In this project, we are investigating the contribution of indigenous freshwater organisms (cyanobacteria and bacteria) to water resistome. We expect to identify and characterize ecological niches associated with antibiotic resistance in freshwater environments. This may contribute to define a monitoring strategy to map the antibiotic resistance profiles of national freshwater resources. We expect to contribute to the definition of preventive measures against the dissemination of antibiotic resistance in the environment.
- Freshwater cyanobacteria and antibiotic pollution: The Ecotoxicological and Antibiotic Resistance perspectivesPublication . Dias, Elsa; Vasconcelos, Vitor; Caniça, ManuelaThe release of antibiotics in natural ecosystems led to the development of antibiotic resistant (AR) microorganisms and alters the diversity/functioning of natural microbial communities. Freshwater cyanobacteria (CB) have been considered sensitive to antibiotics and they have been used as biologic indicators in environmental risk assessment of water contaminants, such as antibiotics. However, we have shown that CB strains from water environments exhibits reduced susceptibility to some antibiotics (nalidixic acid, trimethoprim), irrespective of the tested specie (Microcystis aeruginosa, Aphanizomenon gracile; Anabaena berghii, Planktothrix agardhii, Planktothrix mougeotti). The failure to detect genes conferring resistance to these antibiotics by PCR does not exclude the possibility of CB harbor AR determinants. NGS sequencing will help us to understand if unknown AR genes are present in CB genomes. Besides, we may also hypothesize that CB are intrinsically resistant to those compounds. On the other hand, the susceptibility to other antibiotics depended of the specie and/or the specie origin (freshwater reservoirs, wastewater treatment plants). Moreover, we also detect genes associated with resistance to other antibiotics (streptomycin, sulfonamides) and a class-1-type integron, in some strains. This suggests that CB might acquire resistant determinants from AR microbiota sharing the same habitats. Thus, the impact of antibiotic pollution in natural occurring CB is far from being elucidated. CB seems to have a role on water resistome but CB communities and their important ecological roles (primary and O2 production, CO2 fixation) may be also hampered by antibiotic exposure.
- NDM-1-producing Providencia stuartii isolates in a Portuguese HospitalPublication . Manageiro, Vera; Ferreira, Eugénia; Rodrigues, João; Sampaio, Daniel A; Vieira, Luís; Pereira, Patrícia; Rodrigues, Paulo; Palos, Carlos; Caniça, ManuelaObjective: Providencia stuartii is an opportunistic pathogen typically associated with urinary infections, and is intrinsically resistance to a wide range of antibiotics. The main aim of this study was to characterize five carbapenemase (CA) NDM-1-producing P. stuartii isolates obtained during an outbreak detected in a Hospital. Methods: MICs were obtained by the reference microdilution broth method, according to EUCAST guidelines. PCR amplification and DNA sequencing were applied to identify the presence of CA genes from class A, B and D. Direct transfer of the CA resistance phenotype was attempted by mating-out assays. Genetic relatedness was examined by PFGE. One isolate, INSRA21868, recovered from the urine of an 88-year-old male patient admitted to the intensive care unit, was selected for genetic characterization using whole-genome sequencing (WGS), performed using 150 bp paired-end reads on a MiSeq (Illumina). A set of bioinformatic web tools were used to estimate the presence of pathogenicity determinants, antibiotic resistance (AR) genes, and clinically relevant mobile genetic elements. Results: All isolates, genetically indistinguishable by PFGE, presented multidrug-resistance with non-susceptibility to all carbapenems tested. Transconjugants had AR profiles similar to those of their parental clinical isolates. All NDM-1 determinants tested were found to be carried on conjugative plasmids. In silico AR analyses using ResFinder-v2.1 revealed genes conferring resistance to β-lactams [blaNDM-1, blaCMY-4 and ΔblaDHA-1), aminoglycosides (aac(2’)-Ia, armA), tetracycline (tetB), macrolides (mphE and msrE), chloramphenicol (catB3), and sulfonamides (sul1). PlasmidFinder-v1.2 analyses revealed the presence of an IncA/C2, which has been associated with wide dissemination of blaNDM-1. In the 3’ region, the blaNDM-1 gene was adjacent to a bleomycin resistance-encoding gene (bleMBL), followed by a trpF and part of the blaDHA-1-ampR region. The ISAba125 element upstream of blaNDM-1 was interrupted by an IS26 element. Conclusion: This study emphasizes the elements involved in dissemination of nosocomial infections and the potential of WGS in epidemiological investigations in the prevention of CA dissemination among hospitals as well as to other bacterial genera.
- Next Generation Sequencing as a tool to detect antibiotic resistance mechanismsPublication . Manageiro, Vera; Caniça, ManuelaAntibiotic resistance is an emerging problem, becoming a serious threat to global public health.The causes of its spread are complex, as are the strategies to combat this threat. Following recent improvements in sequencing technologies, whole-genome sequencing (WGS) provides a comprehensive alternative in the evaluation and detection of antibiotic resistance mechanisms. In the scope of the analysis of nonsusceptibility of Gram-negative isolates recovered from human, veterinary and environment samples, we identified the presence of a high diversity of resistance mechanisms, with emphasis in the recently described plasmid-mediated mcr gene, conferring resistance to colistin. Therefore, the objective of this study was to characterize the phenotype and genotype of those isolates using conventional microbiological methods and WGS. Whenever appropriate, genetic relatedness of isolates was also investigated by pulsed-field gel electrophoresis (PFGE). To understand the genetic background of those resistance mechanisms, which included oxyimino-β-lactam, fluoroquinolone and colistin resistance-encoding genes, we performed whole genome and plasmid sequencing using a 454 (Roche) and/or MiSeq (Illumina) sequencing strategy. A set of bioinformatic web tools were used to estimate the presence of pathogenicity determinants, antibiotic resistance (AR) genes, and clinically relevant mobile genetic elements. Indeed, the efficient gene capture and spread of resistance determinants by mobile genetic elements are factors to be taken into account, due to their contribution for the co-selection of multidrug resistant strains in the different settings and environment. Furthermore, WGS might be used with great benefit in combination with phenotypic methods for surveillance purposes.
- The use of Whole-Genome Sequencing to predict antibiotic resistance: practical casesPublication . Manageiro, Vera; Caniça, ManuelaAntibiotic resistance is an emerging problem, becoming a serious threat to global public health. The causes of its spread are complex, as are the strategies to combat this threat. Following recent improvements in sequencing technologies, whole-genome sequencing (WGS) provides a comprehensive alternative in the evaluation and detection of antibiotic resistance mechanisms. In the scope of the analysis of nonsusceptibility of Gram-negative isolates recovered from human, veterinary and environment samples, we identified the presence of a high diversity of resistance mechanisms. To understand the genetic background of those resistance mechanisms, which included oxyimino-β-lactam, fluoroquinolone and colistin resistance-encoding genes, we performed whole genome and plasmid sequencing using a 454 (Roche) and/or MiSeq (Illumina) sequencing strategy. A set of bioinformatic web tools were used to estimate the presence of pathogenicity determinants, antibiotic resistance (AR) genes, and clinically relevant mobile genetic elements. Indeed, the efficient gene capture and spread of resistance determinants by mobile genetic elements are factors to be taken into account, due to their contribution for the co-selection of multidrug resistant strains in the different settings and environment. Furthermore, WGS might be used with great benefit in combination with phenotypic methods for surveillance purposes.