Microbiome Of Ecotoxicity Assays

Microorganisms are ubiquitous and present in animal microbiomes, particulates, and colonizable surfaces of test systems. From an ecotoxicological perspective, they are metabolically active biological compartments that respond to test conditions, including test substances. In exposure experiments, microorganisms can both alleviate toxicity via, for example, biotransformation, and reinforce the adverse effects via, for example, disrupted microbiome-host interactions. Acknowledging these interactions is essential for a mechanistic understanding of results in effect studies and developing assays towards more ecologically relevant hazard assessment. Therefore, there is increasing attention toward “microbiome aware ecotoxicology” in recent years, focusing mostly on test organism microbiomes.  I studied how microorganisms present in systems designed for acute and chronic toxicity assays with Daphnia magna affect the test outcome. The experimental studies showed that bacteria introduced in the system intentionally (as a part of the experimental design; Papers I, II, and III) or unintentionally (with the microbiome of the test animals; Paper IV) responded to the test substances and mediated the exposure for the target species. In these studies, we employed the emerging contaminants ciprofloxacin (an antibiotic drug; Paper I) and various fossil-based polymers (microplastic; Papers II, III, and IV), representing a microbiome disrupting and a biofilm promoting type of substance respectively.  In Paper I, we hypothesized that exposure to antibiotics would primarily target the daphnid microbiome with downstream effects on the host fitness. To test this hypothesis, we chronically exposed daphnids to ciprofloxacin, which resulted in decreased microbiome diversity. However, contrary to our hypothesis, there were significant stimulatory effects on the host fitness and antioxidant production due to the direct pro-oxidative ciprofloxacin effects on the host. Although the microbiome was not directly involved in the growth-related responses to the ciprofloxacin exposure, the microbiome’s alterations suggest that exposure to any antimicrobials, which – unlike ciprofloxacin – do not stimulate antioxidant production, would result in gut dysbiosis with possible adverse effects on the host.  Further, we hypothesized that in assays with particulate test materials, such as microplastic, bacterial biofilms increase particle aggregation, affecting exposure levels. This hypothesis was tested using D. magna exposed to a mixture of kaolin clay and polystyrene with and without biofilm (Paper II). We found that biofilm significantly decreased the adverse effects exerted by particulates directly, most likely, by providing nutrition for the daphnids, and indirectly, by inducing particle aggregation. In Paper III, we compared biofilm communities established on the plastic (polyethylene, polypropylene, and polystyrene) vs. non-plastic (cellulose and glass) substrates. The biofilm communities on the plastic were significantly different from those on the non-plastic materials;  hence, microplastic contribution to the suspended solids in the exposure can drive the biofilm community composition in the system. Finally, in Paper IV, we found that in a closed system designed to evaluate microplastic effects on D. magna, bacteria originated from the daphnid microbiome colonize particulates and affect their aggregation and animal survival. Together, these findings suggest that chemical exposure (Paper I), the microbiome of the test animal (Paper IV), the composition of the suspended solids (SS) (Papers II and IV), and their surface properties (Paper III) contribute to the diversity and abundance of the biofilm in the test system, which can affect the test outcome. Thus, the microbiome reacts to and interacts with contaminants within a test system, which calls for the appreciation of these interactions when interpreting the results as well as new developments toward standardization of the bacterial component in (eco)toxicity assays with eukaryotic test species

Ecotoxicological assessment of suspended solids: the importance of biofilm and particle aggregation

Assessment of microplastic impacts in biota is challenging due to the complex behavior of the test particles and their interactions with other particulates, including microorganisms, in the environment. To disentangle responses to microplastic exposure from those to other suspended solids, both microplastic and natural particles must be present in the test system. We evaluated how microplastic, non-plastic particles, and biofilms interacted in their effects on survivorship using acute toxicity assay with Daphnia magna. The animals were exposed to microplastic and kaolin at different concentrations of suspended solids (SS; 10, 100, and 1000 mg/L) with a varying microplastic contribution (%MP; 0–80%) and biofilm (presence/absence) associated with the solids. Also, we examined how these exposure parameters (SS, %MP, and Biofilm) affected aggregate formation that was analyzed using particle size distribution data. Under the exposure conditions, Daphnia mortality was primarily driven by SS concentration but ameliorated by both microplastic and biofilm. The ameliorating effects were related to increased particle aggregation in the presence of biofilm and high %MP. In addition, a weak yet significant positive effect of the biofilm on the survivorship was observed, presumably, due to microbial food supply to the daphniids in the exposure system; the bacteria were utilized at the absence of other food. Therefore, the effects of both natural and anthropogenic particulates depend on the particle behavior and aggregation in the water governed by microbial communities and physicochemical properties of the particles, which must be taken into account in the hazard assessment of plastic litter

Bacicyclin, a new antibacterial cyclic hexapeptide from Bacillus sp. strain BC028 isolated from Mytilus edulis

A new cyclic hexapeptide, cyclo-(Gly-Leu-Val-IIe-Ala-Phe), named bacicyclin (1), was isolated from a marine Bacillus sp. strain associated with Mytilus edulis. The sequences of the amino acid building blocks of the cyclic peptide and its structure were determined by 1D- and 2D-NMR techniques. Marfey's analysis showed that the amino acid building blocks had L-configuration in all cases except for alanine and phenylalanine, which had D-configuration. Bacicyclin (1) exhibited antibacterial activity against the clinically relevant strains Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentration values of 8 and 12 μM, respectively. These results demonstrate the potential of marine bacteria as a promising source for the discovery of new antibiotics

Disparate effects of antibiotic-induced microbiome change and enhanced fitness in Daphnia magna

It is a common view that an organism’s microbiota has a profound influence on host fitness; however, supporting evidence is lacking in many organisms. We manipulated the gut microbiome of Daphnia magna by chronic exposure to different concentrations of the antibiotic Ciprofloxacin (0.01–1 mg L-1), and evaluated whether this affected the animals fitness and antioxidant capacity. In line with our expectations, antibiotic exposure altered the microbiome in a concentration-dependent manner. However, contrary to these expectations, the reduced diversity of gut bacteria was not associated with any fitness detriment. Moreover, the growth-related parameters correlated negatively with microbial diversity; and, in the daphnids exposed to the lowest Ciprofloxacin concentrations, the antioxidant capacity, growth, and fecundity were even higher than in control animals. These findings suggest that Ciprofloxacin exerts direct stimulatory effects on growth and reproduction in the host, while microbiome- mediated effects are of lesser importance. Thus, although microbiome profiling of Daphnia may be a sensitive tool to identify early effects of antibiotic exposure, disentangling direct and microbiome-mediated effects on the host fitness is not straightforward

Disparate effects of antibiotic-induced microbiome change and enhanced fitness in Daphnia magna.

It is a common view that an organism's microbiota has a profound influence on host fitness; however, supporting evidence is lacking in many organisms. We manipulated the gut microbiome of Daphnia magna by chronic exposure to different concentrations of the antibiotic Ciprofloxacin (0.01-1 mg L-1), and evaluated whether this affected the animals fitness and antioxidant capacity. In line with our expectations, antibiotic exposure altered the microbiome in a concentration-dependent manner. However, contrary to these expectations, the reduced diversity of gut bacteria was not associated with any fitness detriment. Moreover, the growth-related parameters correlated negatively with microbial diversity; and, in the daphnids exposed to the lowest Ciprofloxacin concentrations, the antioxidant capacity, growth, and fecundity were even higher than in control animals. These findings suggest that Ciprofloxacin exerts direct stimulatory effects on growth and reproduction in the host, while microbiome- mediated effects are of lesser importance. Thus, although microbiome profiling of Daphnia may be a sensitive tool to identify early effects of antibiotic exposure, disentangling direct and microbiome-mediated effects on the host fitness is not straightforward