Elsa Dias

PRODUCTION OF PARALYTIC SHELLFISH TOXINS BY APHANIZOMENON SP. LMECYA 31 (CYANOBACTERIA)1

We examined intracellular and extracellular paralytic shellfish toxins (PST) in a strain of Aphanizomenon sp. (LMECYA31) isolated from a Portuguese freshwater reservoir throughout the growth cycle and under different conditions affected by temperature and nitrate and phosphate availability. PST concentrations and compositions were greatly influenced by cell density, growth stage, and temperature and nutrients conditions. On a per‐cell basis results showed (1) the enhancement of PST cell quota after the end of exponential growth phase in nutrient replete batch cultures, (2) the absence of a PST increment at late growth stages under phosphate limitation, (3) a rise in PST maximum cell quota under nitrate depletion, and (4) the enhancement of toxin production at higher temperatures. The relative proportion of the four toxins detected, neoSTX, dcSTX, STX and GTX5, also changed within and between culture settings. While growing under phosphate rich media cells produced mainly GTX5 and neoSTX, whereas under phosphate limitation the proportion of STX and dcSTX increased substantially with culture age. Large amounts of extracellular toxins were found in the culture medium, increasing during culture time. Extracellular toxin composition in each culture was fairly constant and always similar to the intracellular composition found at late stages of growth. This further supported other research that indicates that PSTs are released to the water through cell lysis, and a significant concentration of PST may be expected to remain in the water upon the collapse of a toxic bloom or after cells removal by water treatment.

Taxonomy and production of paralytic shellfish toxins by the freshwater cyanobacterium Aphanizomenon gracile LMECYA40

Detection of Paralytic Shellfish Toxins (PST) in an algal bloom in a Portuguese drinking water reservoir (Lake Crato) was followed by isolation of a strain of Aphanizomenon gracile. The strain, coded as LMECYA40, was cultured and identified by combining a morphological study with 16S rRNA gene sequencing. The toxin profile of this isolate, as revealed by HPLC-FLD analysis, was similar to that of other Aphanizomenon strains (e.g. NH-5) isolated from North America, consisting of two PST analogues: neoSTX (0.27 fmol cell−1) and STX (0.05 fmol cell−1). Based on these toxin levels, we estimated that a culture with a cell density of 107 cells ml−1 should contain approximately 910 μg of STX equivalents per litre. Small volumes of water containing PST concentrations similar to those estimated from the cultured A. gracile would therefore contain PST amounts comparable to those used as limit enforcement for harvesting and consumption of shellfish in marine environments (80 μg of STX equivalents per 100 g of shellfish).

Morphological and ultrastructural effects of microcystin-LR from Microcystis aeruginosa extract on a kidney cell line.

The aim of this study was to examine the toxic effects of a microcystin-LR (MCLR)-containing cyanobacteria extract on the subcellular organization of a kidney cell line (Vero-E6). Cells were exposed to different MCLR concentrations (1.3-150 microM) for 24, 48 and 72h and two cytotoxicity assays were performed. This information was combined with the analysis of lysosomal, mitochondrial and cytoskeleton integrity and with an ultrastructural study. Biochemical and microscopic data revealed a good agreement and demonstrated that cellular response to MCLR is dependent on the dose/exposure time. Cell viability decayed markedly after 24h of exposure to toxin concentrations greater than 30 microM. Furthermore, it was demonstrated that lysosome destabilization precedes mitochondria dysfunction. The ultrastructural analysis showed that mild toxin incubation conditions induce endoplasmic reticulum (ER) vacuolization and assembly of large autophagic vacuoles, suggesting that autophagy is an early cellular response to the toxin. After exposure to higher MCLR doses, the number of apoptotic cells increased, as identified by microscopic observations and confirmed with TUNEL assay. Additionally, drastic exposure conditions induced the increase of necrotic cells. These results suggest that the ER is the primary microcystin target in Vero cells and that autophagy, apoptosis and necrosis are induced in a dose- and time-dependent manner.

Microcystin-LR activates the ERK1/2 kinases and stimulates the proliferation of the monkey kidney-derived cell line Vero-E6.

Microcystin-LR (MCLR) is a peptide produced by freshwater cyanobacteria that induces severe hepatotoxicity in humans and animals. MCLR is also a potent tumour promoter and it has been proposed that this activity is mediated by the inhibition of protein phosphatases PP1/PP2A, possibly through the activation of proto-oncogenes c-jun, c-fos and c-myc. However, the mechanisms underlying MCLR-induced tumour promotion are still largely unknown, particularly in non-liver cells. In previous studies we have demonstrated that micromolar concentrations of MCLR induce cytotoxic effects in the kidney Vero-E6 cell line. The purpose of the present work was to evaluate whether the exposure to subcytotoxic concentrations of MCLR was sufficient to induce the proliferation of Vero-E6 cells. Through BrdU incorporation assay we show that at nanomolar concentrations MCLR stimulates cell cycle progression in Vero-E6 kidney cell line. Moreover, the analysis of mitogen-activated protein kinases p38, JNK and ERK1/2 activity revealed that the proliferative effect of MCLR is associated with the activation of the pro-proliferative ERK1/2 pathway. These results emphasise the importance to confirm in vivo the impact of MCLR on tumour promotion at kidney level.

Involvement of endoplasmic reticulum and autophagy in microcystin-LR toxicity in Vero-E6 and HepG2 cell lines

This work investigates the involvement of the endoplasmic reticulum (ER) and autophagy in microcystin-LR (MCLR) toxicity in Vero-E6 and HepG2 cell lines. Additionally, morphological alterations induced by MCLR in lysosomes and mitochondria were studied. Cytotoxicity evaluation showed that pure MCLR and MCLR from LMECYA110 extract induce concentration dependent viability decays after 24h exposure. HepG2 cells showed an increased sensitivity to MCLR than Vero cells, with lower cytotoxic thresholds and EC(50) values. Conversely, LC3B immunofluorescence showed that autophagy is triggered in both cell lines as a survival response to low MCLR concentrations. Furthermore, MCLR induced a MCLR concentration-dependent decrease of GRP94 expression in HepG2 cells while in Vero cells no alteration was observed. This suggests the involvement of the ER in HepG2 apoptosis elicited by MCLR, while in Vero cells ER destructuration could be a consequence of cytoskeleton inflicted damages. Additionally, in both cell lines, lysosomal destabilization preceded mitochondrial impairment which occurred at high toxin concentrations. Although not an early cellular target of MCLR, mitochondria appears to serve as central mediators of different signaling pathways elicited by the organelles involved in MCLR toxicity. As a result, kidney and hepatic cell lines exhibit cell type and dose-dependent mechanisms to overcome MCLR toxicity.

Genotoxicity of Microcystin-LR in In Vitro and In Vivo Experimental Models

Microcystin-LR (MCLR) is a cyanobacterial toxin known for its acute hepatotoxicity. Despite being recognized as tumour promoter, its genotoxicity is far from being completely clarified, particularly in organs other than liver. In this work, we used the comet and/or the micronucleus (MN) assays to study the genotoxicity of MCLR in kidney- (Vero-E6) and liver-derived (HepG2) cell lines and in blood cells from MCLR-exposed mice. MCLR treatment (5 and 20 μM) caused a significant induction in the MN frequency in both cell lines and, interestingly, a similar positive effect was observed in mouse reticulocytes (37.5 μg MCLR/kg, i.p. route). Moreover, the FISH-based analysis of the MN content (HepG2 cells) suggested that MCLR induces both chromosome breaks and loss. On the other hand, the comet assay results were negative in Vero-E6 cells and in mouse leukocytes, with the exception of a transient increase in the level of DNA damage 30 minutes after mice exposure. Overall, the present findings contributed to increase the weight of evidence in favour of MCLR genotoxicity, based on its capacity to induce permanent genetic damage either in vitro or in vivo. Moreover, they suggest a clastogenic and aneugenic mode of action that might underlie a carcinogenic effect.

Current perspectives on the dynamics of antibiotic resistance in different reservoirs

Antibiotic resistance consists of a dynamic web. In this review, we describe the path by which different antibiotic residues and antibiotic resistance genes disseminate among relevant reservoirs (human, animal, and environmental settings), evaluating how these events contribute to the current scenario of antibiotic resistance. The relationship between the spread of resistance and the contribution of different genetic elements and events is revisited, exploring examples of the processes by which successful mobile resistance genes spread across different niches. The importance of classic and next generation molecular approaches, as well as action plans and policies which might aid in the fight against antibiotic resistance, are also reviewed.

The Kidney Vero-E6 Cell Line: a Suitable Model to Study the Toxicity of Microcystins

Microcystins (MCs) are toxins produced by cyanobacteria from water environments that can induce acute and chronic effects on humans and animals, after ingestion/contact with conta‐ minated water [1]. This group of cyclic heptapeptides comprises approximately 80 variants, being microcystin-LR (MCLR) the most frequent and toxic variant [1]. MCs are mainly known for their hepatotoxicity due to their inhibitory activity of serine/threonine phosphatases PP1 and PP2A [2]. This inhibition interferes with hepatocyte homeostasis and structure, leading to the collapse of liver tissue organization, liver necrosis and hemorrhage (Figure 1), which can culminate, in severe cases, in the death of the intoxicated individuals [3, 4].

Assessing the antibiotic susceptibility of freshwater Cyanobacteria spp.

Freshwater 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, nalidixic acid, norfloxacin). We used a method adapted from the bacteria standard broth microdilution. Cyanobacteria were exposed to serial dilution of each antibiotic (0.0015–1.6 mg/L) in Z8 medium (20 ± 1°C; 14/10 h L/D cycle; light intensity 16 ± 4 μEm−2s−1). 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.

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

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.