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Deciphering the role of cyanobacteria in water resistome: Hypothesis justifying the antibiotic resistance (phenotype and genotype) in Planktothrix genus

Publication . Dias, Elsa; Oliveira, Micaela; Manageiro, Vera; Vasconcelos, Vitor; Caniça, Manuela

The 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.

2018-10-12Journal article

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Risk Levels of Toxic Cyanobacteria in Portuguese Recreational Freshwaters

Publication . Menezes, Carina; Churro, Catarina; Dias, Elsa

Portuguese freshwater reservoirs are important socio-economic resources, namely for recreational use. National legislation concerning bathing waters does not include mandatory levels or guidelines for cyanobacteria and cyanotoxins. This is an issue of concern since cyanotoxin-based evidence is insufficient to change the law, and the collection of scientific evidence has been hampered by the lack of regulatory levels for cyanotoxins in bathing waters. In this work, we evaluate the profile of cyanobacteria and microcystins (MC) in eight freshwater reservoirs from the center of Portugal, used for bathing/recreation, in order to determine the risk levels concerning toxic cyanobacteria occurrence. Three of the reservoirs did not pose a risk of MC contamination. However, two reservoirs presented a high risk in 7% of the samples according to the World Health Organization (WHO) guidelines for MC in bathing waters (above 20 µg/L). In the remaining three reservoirs, the risk concerning microcystins occurrence was low. However, they exhibited recurrent blooms and persistent contamination with MC up to 4 µg/L. Thus, the risk of exposure to MC and potential acute and/or chronic health outcomes should not be disregarded in these reservoirs. These results contribute to characterize the cyanobacterial blooms profile and to map the risk of toxic cyanobacteria and microcystins occurrence in Portuguese inland waters.

2017-10-18Journal article

Open access

Freshwater cyanobacteria and antibiotic pollution: The Ecotoxicological and Antibiotic Resistance perspectives

Publication . Dias, Elsa; Vasconcelos, Vitor; Caniça, Manuela

The 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.

2017-06-12Conference object

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Resistência a antibióticos em cianobactérias

Publication . Dias, Elsa; Oliveira, Micaela; Manageiro, Vera; Ferreira, Eugénia; Vasconcelos, Vitor; Caniça, Manuela

Os 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.

2017-07-17Conference object

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Minimum inhibitory concentrations and antibiotic resistant genes in the freshwater cyanobacteria Microcystis aeruginosa

Publication . Dias, Elsa; Oliveira, Micaela; Manageiro, Vera; Vasconcelos, Vitor; Caniça, Manuela

Native 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.

2017-06-26Conference object

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