Browsing by Author "Oliveira, Micaela"
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- 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.
- Avaliação da resistência a antibióticos em estirpes de cianobactérias isoladas de ambientes hídricosPublication . Oliveira, Micaela; Dias, Elsa; Dias, Deodália[PT] Os ambientes hídricos constituem veículos para a emergência e disseminação da resistência a antibióticos. As cianobactérias encontram-se amplamente distribuidas nestes ambientes, estando frequentemente expostas a antibióticos, bactérias resistentes e genes de resistência. Contudo, o seu papel no resistoma hídrico nunca foi investigado. Este trabalho pretendeu avaliar a susceptibilidade de oito estirpes de Microcystis aeruginosa, oito estirpes de Planktothrix agardhii e oito estirpes de Planktothrix mougeotii a diferentes antibióticos (amoxicilina, ceftazidima, ceftriaxona, canamicina, gentamicina, tetraciclina, ácido nalidíxico, norfloxacina e trimetoprim). Para tal, foi utilizado um procedimento baseado no método standard Broth Microdilution para bactérias, no qual as cianobactérias foram expostas a diluições sucessivas de cada antibiótico em meio Z8 (0,0015 mg/L – 1,6 mg/L) e mantidas numa câmara de culturas sob ciclos de 14 horas de luz – com uma intensidade de 16 ± 4 μEm-2 s-1 – e 10 horas de escuro, a uma temperatura de 20 ± 1ºC. O crescimento celular foi seguido durante 14 dias (observação macroscópica, microscópica e leitura da DO450nm) e as concentrações inibitórias mínimas de cada antibiótico (CIMs) foram calculadas para cada estirpe. Nenhuma das estirpes foi susceptível a qualquer das concentrações de ácido nalidíxico e de trimetoprim testadas. Adicionalmente, as estirpes de M. aeruginosa apresentaram uma susceptibilidade reduzida à tetraciclina, as estirpes de P. agardhii apresentaram uma susceptibilidade reduzida à norfloxacina e as estirpes de P. mougeotii não foram susceptíveis à amoxicilina nem à norfloxacina e apresentaram uma susceptibilidade reduzida à tetraciclina. Foi também pesquisada a presença de genes e de integrões associados à resistência a antibióticos nas cianobactérias em estudo por PCR e posterior sequenciação dos produtos obtidos. Foram encontrados genes de resistência à estreptomicina numa estirpe de M. aeruginosa, em três estirpes de P. agardhii e em quatro estirpes de P. mougeotii; genes de resistência às sulfonamidas em quatro estirpes de M. aeruginosa, em três estirpes de P. agardhii e em cinco estirpes de P. mougeotii; integrões de classe 1 em duas estirpes de M. aeruginosa, em três estirpes de P. agardhii e em três estirpes de P. mougeotii. Adicionalmente, o gene qacE foi encontrado numa estirpe de M. aeruginosa e numa estirpe de P. agardhii. Os resultados obtidos sugerem que as cianobactérias apresentam resistência intrínseca a alguns antibióticos e que, de acordo com o seu local de origem, podem apresentar resistência adquirida a outros. Assim, tudo indica que, de facto, as cianobactérias desempenham um papel importante no resistoma hídrico.
- Deciphering the role of cyanobacteria in water resistome: Hypothesis justifying the antibiotic resistance (phenotype and genotype) in Planktothrix genusPublication . Dias, Elsa; Oliveira, Micaela; Manageiro, Vera; Vasconcelos, Vitor; Caniça, ManuelaThe importance of environmental microorganisms in the emergence and dissemination of antibiotic resistance is an undeniable fact. However, cyanobacteria are not seen yet as putative players in the dynamic of environmental resistome, despite their ubiquity in water environments, where they are exposed to antibiotic pollution and in straight contact with native and pathogenic bacteria harboring antibiotic resistance genes (ARGs). In this work we evaluated the susceptibility of 8 strains of Planktothrix agardhii (from surface freshwaters reservoirs) and 8 strains of Planktothrix mougeotii (from a wastewater treatment plant) to several classes of antibiotics, using a microplate dilution method previously described by us. We also search for ARGs in those strains by molecular methods. None of the 16 tested strains were susceptible to trimethoprim, nalidixic acid and norfloxacin, from 0.0015–1.6 mg/L, but all were susceptible to streptomycin, gentamicin, kanamycin, ceftazidime and ceftriaxone. The minimum inhibitory concentrations (MICs) ranged between 0.05–0.8 mg/L for the aminoglycosides and 0.4–1.6 mg/L for the two β‑lactams. Major differences were found in the susceptibility to amoxicillin and tetracycline, with P. agardhii being susceptible (MIC of 0.05 mg/L and 0.4 mg/L, respectively) and P. mougeotii not susceptible. These distinct responses might be due to differences between species. However, the lower susceptibility of wastewater strains suggests that antibiotic resistance phenotype of cyanobacteria is related with their habitat. The failure to detect acquired genes conferring resistance to trimethoprim/quinolones, strongly supports the hypothesis that cyanobacteria are intrinsically resistant to these antibiotics. Interestingly, we detected a class-1-type integron and a sul1 gene in 3 strains of both P. agardhii and P. mougeotii, which supports the possibility of cyanobacteria to acquire and transfer antibiotic resistance determinants. In conclusion, the identification of ARGs and related integrons, as well as the reduced susceptibility to some antibiotics, suggests that cyanobacteria may play a role on environmental resistome.
- Minimum inhibitory concentrations and antibiotic resistant genes in the freshwater cyanobacteria Microcystis aeruginosaPublication . Dias, Elsa; Oliveira, Micaela; Manageiro, Vera; Vasconcelos, Vitor; Caniça, ManuelaNative aquatic bacteria have been considered as important players in the emergence/dissemination of antibiotic resistance (AR) in water environments [1,2]. Conversely, the role of cyanobacteria (CB) on water resistome is unknown. We have been hypothesizing that CB may contribute to the spread of AR in the environment, considering that [3]: i) they are ubiquitous prokaryotes in aquatic habitats; ii) they are exposed to antibiotics and AR bacteria; iii) they maintain biological relations with their bacterial neighbors; iv) they can change genetic material by horizontal gene transfer. Microcystis aeruginosa is one of the most common CB in freshwater reservoirs worldwide, often exhibiting long residence time in water surface/column. This work aimed to evaluate the antibiotic susceptibility patterns and resistance mechanisms in M. aeruginosa in order to assess their putative contribution to the global pool of resistance determinants in freshwater environments. The presence of AR genes and integrons, as well as the reduced susceptibility to antibiotics, strongly suggests that CB play a role on freshwater resistome, contributing, eventually, to the dissemination of AR in freshwater environments.
- Minimum inhibitory concentrations and antibiotic resistant genes in the freshwater cyanobacteria Planktothirx agardhiiPublication . Dias, Elsa; Oliveira, Micaela; Dias-Jones, Daniela; Manageiro, Vera; Churro, Catarina; Vasconcelos, Vitor; Ferreira, Eugénia; Caniça, ManuelaPlanktothrix agardhii is one of the most common cyanobacteria species in Portuguese freshwater reservoirs, often exhibiting long residence time in those reservoirs. This work aimed to evaluate the antibiotic susceptibility patterns and resistance genes in P. agardhii in order to assess their putative contribution to the global pool of resistance determinants in freshwater. We investigated 9 strains of P. agardhii, previously isolated from different freshwater reservoirs. Antibiotic susceptibility was evaluated by a microdilution method previously adapted for cyanobacteria against β-lactams, aminoglycosides, quinolones, trimethoprim and tetracycline. Minimum inhibitory concentrations (MIC) were determined according to cyanobacterial cell dentisty (DO 450nm) and microscopic examination of cultures integrity. All strains were subjected to the search and identification of antibiotic resistance genes and class 1, 2 and 3 integrons by PCR and sequencing. Preliminary results showed that P. agardhii is not susceptible to trimethoprim and nalidixic acid within the tested concentration range (0.0015-1.6 mg/L). Moreover, the cell growth was strongly inhibited by amoxicillin for the majority of the strains (0.003 mg/L≤MIC≤0.1). The other β-lactams, kanamycine, gentamicine, tetracycline and norfloxacine presented MICs above 0.1 mg/L. Strain LMECYA 260 harboured the sul1 gene, and a 3-type integron was also detected in LMECYA 260 and 257. These strains were isolated from the same freshwater reservoir, which has a persistent bloom of Planktothrix spp. for several years. This study suggests that the presence of a common antibiotic resistant determinant in P. agardhii might be a result from a selective pressure within this reservoir. The presence of antibiotic resistance genes and integrons, as well as the reduced susceptibility to antibiotics detected in these strains, suggest that specific cyanobacteria may play a role on freshwater resistome and eventually contribute to the dissemination of antibiotic resistance in freshwater environments.
- Minimum inhibitory concentrations and antibiotic resistant genes in the freshwater cyanobacteria Planktothirx agardhiiPublication . Dias, Elsa; Oliveira, Micaela; Jones-Dias, Daniela; Manageiro, Vera; Churro, Catarina; Vasconcelos, Vítor; Ferreira, Eugénia; Caniça, ManuelaObjectives: Cyanobacteria are ubiquitous prokaryotes in aquatic ecosystems and although they can be exposed to antibiotics and antibiotic resistant bacteria, their role on water resistome was never investigated. Planktothrix agardhii is one of the most common cyanobacteria species in Portuguese freshwater reservoirs, often exhibition long residence time in those reservoirs. This work aimed to evaluate the antibiotic susceptibility patterns and resistance mechanisms in P. agardhii in order to assess their putative contribution to the global pool of resistance determinants in freshwater. Methods: We investigated 8 strains of P. agardhii, previously isolated from different freshwater reservoirs. The antibiotic susceptibility was evaluated by a microdilution method previously adapted for cyanobacteria, against beta-lactams, aminoglycosides, quinolones, trimethoprim and tetracycline. Minimum inhibitory concentrations (MIC) were determined according to cell density (DO, 450nm) and microscopic examination of cultures integrity. All strains were searched for antibiotic resistance genes and class 1, 2 and 3 integrons by PCR/sequencing. Results: The results showed that P. agardhii is not susceptible to trimethoprim and quinolones within the tested concentrations (0.0015-1.6 mg/L). However, the cell growth is strongly inhibited by amoxicillin (median MIC value of 0.1mg/L). The other antibiotics presented the following median MICs: ceftazidime, 1.6 mg/L; ceftriaxone, 0.8 mg/L; tetracycline, 0.4 mg/L; kanamycine, 0.2 mg/L and gentamicine, 0.1mg/L. None of the P. agardhii strains exhibited genes associated with trimethoprim and quinolones resistance, which suggests that these cyanobacteria are intrinsically non susceptible to this antibiotics. Conversely, genes associated with streptomycin (strAstrB) and sulfonamide (sul1) resistance, as well as a 1-type integron, were detected in three of the strains. These strains were isolated from the freshwater reservoirs where Planktothrix blooms are frequent and. This suggests that the presence of a common antibiotic resistant determinant in P. agardhii might be a result from a similar selective pressure within those reservoirs. Conclusions: The presence of antibiotic resistance genes and integrons, as well as the reduced susceptibility to antibiotics, suggest that cyanobacteria may play a role on freshwater resistome and eventually contribute to the dissemination of antibiotic resistance in freshwater environments.
- Reduced antibiotic susceptibility in wastewater Planktothrix sp.: evaluation of minimum inhibitory concentrations by a method designed for cyanobacteria – molecular identification of resistance genesPublication . Dias, Elsa; Oliveira, Micaela; Jones-Dias, Daniela; Manageiro, Vera; Churro, Catarina; Vasconcelos, Vítor; Ferreira, Eugénia; Caniça, ManuelaBackground: Cyanobacteria are ubiquitous in aquatic ecosystems, being Planktothrix spp. one of the most common bloom-forming species in freshwaters worldwide. In these environments, cyanobacteria are exposed to antibiotics and antibiotic resistant bacteria, but their role on water resistome was never investigated. One limitation of testing antibiotic resistance in cyanobacteria is the lack of standardized methods and interpretative guidelines. Based on the standard broth microdilution method, we developed a procedure for testing antibiotic susceptibility in cyanobacteria. This work aimed to test the applicability of that method in Planktothrix mougeotti from a wastewater treatment plant, in order to assess their susceptibility patterns and their putative contribution to the global pool of resistance determinants in freshwater. Material/methods: We investigated 8 strains of Planktothrix mougeotti isolated from a wastewater treatment plant (WWTP) located in the north of Portugal (41º07’05.22’’N, 8º34’015.40’’W) and dimensioned for a population of 80,000 habitants. Antibiotic susceptibility was evaluated by a microdilution method previously adapted for cyanobacteria, against beta-lactams, aminoglycosides, quinolones, trimethoprim and tetracycline. Minimum inhibitory concentrations (MIC) were determined according to cyanobacterial cell dentisty (optical density, 450nm) and microscopic examination of cultures integrity. All strains were subjected to the search of antibiotic resistance genes and class 1, 2 and 3 integrons by PCR/sequencing. Results: The MIC values of P. mougeotti strains varied between 0.2-0.8 mg/L for ceftazidime, ceftriaxone, kanamycine and gentamicine. On the other hand, all of the tested strains were not susceptible to trimethoprim, quinolones (norfloxacin and nalidixic acid), tetracycline and amoxicillin, within the tested concentration range (0.0015-1.6 mg/L). None of the strains exhibited any gene conferring resistance to these antibiotics. On the contrary, a pair of strA-strB-type genes, a sul1-type gene and a class 1-type integron (int1) were detected in four, five and three strains, respectively. Conclusions: The results suggests that the presence of antibiotic resistance genes and integrons in Planktothrix mougeotti, as well as their reduced susceptibility to some antibiotics, might be a result of their exposure to antibiotics and antibiotic resistance genes in the WWTP. This reinforces the hypothesis that cyanobacteria may play a role on freshwater resistome and point out the need to implement standard methodologies and guidelines for susceptibility testing in cyanobacteria.
- Resistência a antibióticos em cianobactériasPublication . Dias, Elsa; Oliveira, Micaela; Manageiro, Vera; Ferreira, Eugénia; Vasconcelos, Vitor; Caniça, ManuelaOs recursos hídricos constituem reservatórios de antibióticos e microrganismos resistentes, contribuindo, assim, para a disseminação de resistência a antibióticos entre bactérias ambientais e patogénicas. As cianobactérias (CB) são consideradas organismos sensíveis a antibióticos e, inclusivamente, são usadas como indicadores biológicos na avaliação de contaminantes hídricos. No entanto, a nossa equipa tem vindo a investigar o eventual papel das CB no resistoma hídrico e neste trabalho iremos apresentar as principais conclusões desse estudo. A avaliação do fenótipo de suscetibilidade a antibióticos revelou que todas as CB estudadas (28 estirpes de 5 espécies diferentes) apresentam suscetibilidade reduzida ao trimetoprim (TMP). Porém, não foram detetados genes de resistência a este antibiótico, por PCR/sequenciação. Colocámos a hipótese de que as CB têm resistência intrínseca ao TMP, provavelmente por usarem uma via metabólica alternativa à via da dihidrofolato redutase (folA), o alvo intracelular do TMP. De facto, a análise do genoma completo de uma das estirpes (Microcystis aeruginosa, LMECYA7) revelou a presença do gene que codifica a thymidylato synthase (ThyX), enzima alternativo à folA no metabolismo dos folatos nos procariotas. Por outro lado, o padrão de suscetibilidade a outros antibióticos depende da espécie e/ou do local de origem das CB. Destaca-se a menor suscetibilidade do género Planktothrix, o que sugere que as suas características estruturais, fisiológicas e bioquímicas lhes conferem uma maior resistência natural a alguns antibióticos, comparativamente a outros géneros. Foram detetados genes (strA-strB, sul1; qacΔE) e/ou integrões (int1-tipo) associados à resistência e/ou disseminação de alguns antibióticos em 60% das estirpes. A espécie P. mougeotii revelou uma suscetibilidade reduzida a um maior número de antibióticos e a maior percentagem de estirpes com genes de resistência (75%), comparativamente a M. aeruginosa (63%) e P. agardhii (38%). Estes resultados pressupõem a resistência adquirida no habitat, uma vez que as estirpes P. mougeotii foram isoladas numa Estação de Tratamento de Águas Residuais. Foram também isoladas bactérias ambientais associadas às estirpes de CB com padrão de (multi)resistência a várias classes de antibióticos. Estes resultados demonstram que o impacto da poluição antibiótica pode não só afetar as comunidades cianobacterianas e as suas funções ecológicas como, também, conferir-lhes um papel no resistoma hídrico.
- Susceptibilidade reduzida a antibióticos e genes de resistência em cianobactérias de água doce superficial e residualPublication . Dias, Elsa; Oliveira, Micaela; Jones-Dias, Daniela; Vasconcelos, Vitor; Caniça, ManuelaÉ reconhecido que os recursos hídricos representam reservatórios de poluição por antibióticos e microrganismos resistentes e que os mecanismos de resistência em isolados clínicos têm provavelmente origem no resistoma ambiental, através da transferência horizontal de genes entre bactérias ambientais e patogénicas. A temática da resistência a antibióticos em bactérias aquáticas tem vindo a ganhar particular atenção, mas, pelo contrário, o papel das cianobactérias no resistoma hídrico nunca foi investigado. Num estudo prévio, determinámos as concentrações inibitórias mínimas (CIM) de várias classes de antibióticos em 4 estirpes cianobacterianas de espécies diferentes, provenientes de reservatórios de água doce superficial, com base na análise do crescimento celular e na integridade das culturas. Concluímos que todas apresentam susceptibilidade reduzida ao trimetoprim e ao ácido nalidíxico, sendo mais susceptíveis aos antibióticos beta-lactâmicos. Com o presente trabalho, pretendemos avaliar se o padrão de susceptibilidade a antibióticos em cianobactérias é condicionado por características intrínsecas à estirpe/espécie/género e por aspetos extrínsecos como o seu nicho ecológico. Foram avaliadas 8 estirpes de Microcystis aeruginosa e de Planktothrix agardhii isoladas de diferentes reservatórios de água doce superficial e 8 estirpes de Planktothrix mougeotii isoladas de uma estação de tratamento de águas residuais. Foram pesquisados genes de resistência a vários antibióticos por PCR/sequenciação. Os resultados obtidos confirmam a suscetibilidade reduzida ao ácido nalidíxico e ao trimetoprim em todas as estirpes avaliadas, sugerindo que as cianobactérias apresentam uma resistência natural a estes antibióticos. Adicionalmente, as estirpes de M. aeruginosa apresentaram susceptibilidade reduzida à tetraciclina, as estirpes de P. agardhii à norfloxacina e as estirpes de P. mougeotii à tetraciclina, à norfloxacina e à amoxicilina. Os resultados preliminares indiciam que as estirpes de P. mougeotii apresentam um maior número de genes de resistência. Globalmente, os resultados sugerem que o fenótipo/genótipo de resistência de cianobactérias a antibióticos depende da espécie/género e habitat.