Sensitivity of phytoplankton, zooplankton and macroinvertebrates to hydrogen peroxide treatments of cyanobacterial blooms.

Addition of hydrogen peroxide (H2O2) is a promising method to acutely suppress cyanobacterial blooms in lakes. However, a reliable H2O2 risk assessment to identify potential effects on non-target species is currently hampered by a lack of appropriate ecotoxicity data. The aim of the present study was therefore to quantify the responses of a wide diversity of freshwater phytoplankton, zooplankton and macroinvertebrates to H2O2 treatments of cyanobacterial blooms. To this end, we applied a multifaceted approach. First, we investigated the 24-h toxicity of H2O2 to three cyanobacteria (Planktothrix agardhii, Microcystis aeruginosa, Anabaena sp.) and 23 non-target species (six green algae, eight zooplankton and nine macroinvertebrate taxa), using EC50 values based on photosynthetic yield for phytoplankton and LC50 values based on mortality for the other organisms. The most sensitive species included all three cyanobacterial taxa, but also the rotifer Brachionus calyciflores and the cladocerans Ceriodaphnia dubia and Daphnia pulex. Next, the EC50 and LC50 values obtained from the laboratory toxicity tests were used to construct a species sensitivity distribution (SSD) for H2O2. Finally, the species predicted to be at risk by the SSD were compared with the responses of phytoplankton, zooplankton and macroinvertebrates to two whole-lake treatments with H2O2. The predictions of the laboratory-based SSD matched well with the responses of the different taxa to H2O2 in the lake. The first lake treatment, with a relatively low H2O2 concentration and short residence time, successfully suppressed cyanobacteria without major effects on non-target species. The second lake treatment had a higher H2O2 concentration with a longer residence time, which resulted in partial suppression of cyanobacteria, but also in a major collapse of rotifers and decreased abundance of small cladocerans. Our results thus revealed a trade-off between the successful suppression of cyanobacteria at the expense of adverse effects on part of the zooplankton community. This delicate balance strongly depends on the applied H2O2 dosage and may affect the decision whether to treat a lake or not.

Dispersing and Sonoporating Biofilm-Associated Bacteria with Sonobactericide.

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 µg/mL). Biofilms were exposed to ultrasound (2 MHz, 100-400 kPa, 100-1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy.

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms.

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0-12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.