Repositório Colecção: Sat, 25 Oct 2014 22:09:00 GMT 2014-10-25T22:09:00Z REVIVE a surveillance programme for ticks and tick-borne diseases: molecular detection of B. burgdorferi s.l. and Rickettsia spp. Título: REVIVE a surveillance programme for ticks and tick-borne diseases: molecular detection of B. burgdorferi s.l. and Rickettsia spp. Autor: Lopes de Carvalho, I.; Santos, A.S.; Santos-Silva, M.M.; de Sousa, R.; Núncio, M.S. Resumo: Objectives: To assess the risk posed by ticks and tick-borne pathogens to Public Health is essential to have reliable data on tick population, namely main species, status, distribution and the changing trends in tick distribution and abundance. Since 2011 the Ministry of Health requested the National Institute of Health to implement and coordinate a national surveillance programme for ticks and tick-borne diseases. Methods: During 2012 and 2013, ticks were collected from hosts and on the vegetation by flagging method. Results: Through the engagement of several health agencies, more than > 18 000 ticks from 119 municipalities across mainland Portugal have been submitted, representing 13 tick species Dermacentor marginatus; D. reticulatus; Haemaphysalis punctata; Hyalomma lusitanicum; H. marginatum; Ixodes canisuga; I. hexagonus; I. ricinus; I. ventalloi; Rhipicephalus annulatus; R. bursa; R. pusillus and R. sanguineus. The majority of ticks sent to the laboratory were R. sanguineus (69%), followed by R. pusillus (16.4%) and H. marginatum (9.7%). The other tick species were collected in occasionally, such as I. ricinus with only 0.3%. Two hundred and thirty three ticks were collected on humans and R. sanguineus was the most frequent (41%) followed by I. ricinus (20%). All the human ticks and about 5% of the ticks collected from vegetation or in hosts were analyzed for the presence of Borrelia spp and Rickettsia spp. B. lusitaniae and seven rickettsial species were detected, R. aeschlimannii, R. conorii Malish, R. helvetica, R. massilae, R. raoulti and R. slovaca. Some ticks species was found only infected with a bacterial agent, as for example H. marginatum with R. aeschlimannii, and D. reticulatus with R. slovaca, when others as R. sanguineus and I. ricinus were infected with three different agents (R. conorii Malish, R. massilae and B. lusitaniae and R. helvetica, R. monacensis, B. lusitaniae, respectively). Due to the high density of Hyalomma spp. present in Portugal and the recent detection of crimean congo hemorrhagic fever virus (CCHF) in Spain, the surveillance of this virus is being implemented during this year. Conclusions: REVIVE program has already produced valuable results and will be maintained for at least the next three years that will allow to observe the trends in the Portuguese tick fauna and pathogens. Another important output of this network is to raise awareness about tick-borne diseases among the populations and the healthcare providers as medical doctors and nurses. Keywords: surveillance programme, ticks, tick-borne diseases Sat, 10 May 2014 00:00:00 GMT 2014-05-10T00:00:00Z Resistencia a antibióticos em cianobactérias e bactérias de água doce Título: Resistencia a antibióticos em cianobactérias e bactérias de água doce Autor: Dias, Elsa; Jones-Dias, Daniela; Manageiro, Vera; Vasconcelos, Vitor; Pereira, Paulo; Ferreira, Eugénia; Caniça, Manuela Resumo: Os recursos hídricos são reservatórios de antibióticos e de bactérias de origem humana e animal resistentes a antibióticos. Embora as cianobactérias estejam expostas a este tipo de contaminantes, o seu papel no resistoma hídrico nunca foi investigado. Neste trabalho estudámos os padrões de suscetibilidade de cianobactérias (e bactérias associadas) a diferentes classes de antibióticos e pesquisámos mecanismos de resistência de forma a avaliar o contributo das cianobactérias e bactérias-associadas para o pool de determinantes de resistência a antibióticos em água doce. Analisámos a suscetibilidade de 4 estirpes de cianobacterianas das espécies Microcystis aeruginosa, Aphanizomenon gracile, Anabaena bergii e Planktothrix agardhii a vários tipos de antibióticos (beta-lactâmicos, aminoglicosídeos, quinolonas, sulfonamidas e tetraciclina) pelo método da microdiluição, adaptado às condições de cultura in vitro de cianobactérias. A concentração inibitória mínima (MIC) para cada antibiótico foi determinada com base no crescimento celular (OD450nm) e na integridade das culturas (observação microscópica). As bactérias isoladas das culturas cianobacterianas foram identificadas por sequenciação do gene 16S rRNA e o seu padrão de suscetibilidade a antibióticos foi avaliado pelo método da difusão em disco, de acordo com os breakpoints não específicos da Sociedade Francesa de Microbiologia. Foram pesquisados por PCR integrões de classe 1, 2 e 3 bem como genes de resistência a antibióticos beta-lactâmicos (blaTEM, blaSHV, blaOXA) e à sulfonamida (sul1, sul2) em todas as estirpes. Dos resultados obtidos com as cianobactérias, destaca-se o seguinte: 1) as estirpes não são suscetíveis ao ácido nalidíxico e ao trimetoprim nas concentrações testadas; 2) Microcystis aeruginosa (LMECYA 7) foi a estirpe menos suscetível aos beta-lactâmicos; 3) Planktothrix agardii (LMECYA 260) a estirpe menos suscetível à gentamicina e à norfloxacina; 4) Anabaena bergii (LMECYA 246) a menos suscetível à canamicina, norfloxacina e tetraciclina; 5) Aphanizomenon gracile (LMECYA 40) suscetível a um maior número de antibióticos; 6) A estirpe Planktothrix agardii (LMECYA 260) exibiu um integrão de classe 3-tipo e o gene sul1. As 27 bactérias isoladas apresentaram padrões de suscetibilidade diferentes entre si e das suas cianobactérias de origem. As bactérias isoladas da Anabaena bergii revelaram-se resistentes a um maior número de antibióticos, destacando-se, de entre estas, algumas estirpes de Novosphingobium panipatens positivas para o integrão de classe 1. O fenótipo de resistência a antibióticos exibido por cianobactérias e bactérias, bem como a presença de integrões de classe 1 e 3-tipo e do gene de resistência sul 1 nalgumas estirpes, sugerem que as cianobactérias e bactérias associadas podem ter um papel na emergência e disseminação de genes de resistência em reservatórios de água doce superficial. A inclusão de um maior número de estirpes de cianobactérias e bactérias associadas neste estudo permitirá avaliar se a resistência a antibióticos em cianobactérias é condicionada por características intrínsecas como o género, espécie e toxicidade (produção de cianotoxinas) e por aspetos extrínsecos como o seu nicho ecológico. Wed, 10 Jul 2013 00:00:00 GMT 2013-07-10T00:00:00Z Antibiotic resistance in freshwater cyanobacteria and associated bacteria Título: Antibiotic resistance in freshwater cyanobacteria and associated bacteria Autor: Dias, Elsa; Dias, Daniela; Ferreira, Eugénia; Manageiro, Vera; Vasconcelos, Vitor; Pereira, Paulo; Caniça, Manuela Resumo: Objectives: 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. Sat, 27 Apr 2013 00:00:00 GMT 2013-04-27T00:00:00Z Screening of cryptic species among clinical Aspergillus isolates collected during one year period in a Portuguese reference laboratory Título: Screening of cryptic species among clinical Aspergillus isolates collected during one year period in a Portuguese reference laboratory Autor: Sabino, R.; Veríssimo, C.; Parada, H.; Clemons, K.V.; Stevens, D.A. Resumo: Objectives: Correct identification of Aspergillus species is important given that sibling species may show variable susceptibilities to multiple antifungal drugs and also because sharper definition of species may facilitate epidemiological studies. Thus, we screened Aspergillus clinical isolates from Portuguese hospitals to determine which, if any, of the cryptic species of Aspergillus were involved in patient infections. Methods: Over a one year period, Aspergillus isolates from Portuguese health institutions were collected. These isolates were identified on the basis of microscopic morphology and through the use of molecular tools. Genomic DNA was prepared from each isolate and the sequencing of the Internal Transcribed Spacers (ITS) regions, specifically the ITS1 and ITS2 non-coding regions flanking the 5.8S rDNA was used to determine the species complex, whereas β-tubulin and calmodulin sequencing was done to achieve the correct species identification. Results: Over the study period, 57 Aspergillus isolates from clinical samples were collected from 10 Portuguese health institutions. According to the morphological observations, 29 isolates were identified as Aspergillus fumigatus, 11 A. flavus, 8 A. niger, 3 A. nidulans, 2 A. terreus, 2 A. candidus and 2 Aspergillus sp. Among those isolates, six species-complexes were detected by ITS sequencing, and were distributed as follows: fumigati (50.1%), flavi (21.0%), nigri (15.8%), terrei (5.3%), nidulantes (3.6%) and versicolores (3.6%). β-tubulin and calmodulin sequencing resulted in ten (17.5%) cryptic species being identified among the 57 isolates. Six of those isolates belonged to the nigri complex (A. brasiliensis, A. awamorii and A. tubigensis), two to the versicolores complex (A. sidowii and A. fructus), one to the fumigati complex (A. lentulus) and one to the nidulantes complex (Emmericella echinulata). Conclusion: With rigorous application of molecular tools, cryptic species of Aspergillus are not uncommon in the clinic. The identification of cryptic species among the collected clinical isolates of Aspergillus alerts the clinician to isolates with reduced susceptibilities to antifungal drugs and emphasizes a correct identification to species level. Tue, 01 Oct 2013 00:00:00 GMT 2013-10-01T00:00:00Z