Effect of biofilm physical characteristics on their susceptibility to antibiotics: impacts of low-frequency ultrasound.

Biofilms are highly resistant to antimicrobials, often causing chronic infections. Combining antimicrobials with low-frequency ultrasound (LFU) enhances antimicrobial efficiency, but little is known about the underlying mechanisms. Biofilm physical characteristics, which depend on factors such as growth conditions and age, can have significant effects on inactivation efficiency. In this study, we investigated the susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, with and without LFU treatment. The biofilms were grown under low and high fluid shear to provide different characteristics. Low-shear biofilms exhibited greater thickness, roughness, and porosity and lower density, compared to high-shear biofilms. The biofilm matrix of the high-shear biofilms had a three times higher protein-to-polysaccharide ratio, suggesting greater biofilm stiffness. This was supported by microrheology measurements of biofilm creep compliance. For the low-shear biofilms without LFU, the viability of the biofilms in their inner regions was largely unaffected by the antibiotic after a 2-hour treatment. However, when tobramycin was combined with LFU, the inactivation for the entire biofilm increased to 80% after 2 h. For the high-shear biofilms without LFU, higher LFU intensities were needed to achieve similar inactivation results. Microrheology measurements revealed that changes in biofilm inactivation profiles were closely related to changes in biofilm mechanical properties. Modeling suggests that LFU changes antibiotic diffusivity within the biofilm, probably due to a "decohesion" effect. Overall, this research suggests that biofilm physical characteristics (e.g., compliance, morphology) are linked to antimicrobial efficiency. LFU weakens the biofilm while increasing its diffusivity for antibiotics.

Effects of temperature on nitrifying membrane-aerated biofilms: An experimental and modeling study.

Temperature is known to have an important effect on the morphology and removal fluxes of conventional, co-diffusional biofilms. However, much less is known about the effects of temperature on membrane-aerated biofilm reactors (MABRs). Experiments and modeling were used to determine the effects of temperature on the removal fluxes, biofilm thickness and morphology, and biofilm microbial community structure of nitrifying MABRs. Steady state tests were carried out at 10 °C and 30 °C. MABRs grown at 30 °C had higher ammonium removal fluxes (5.5 ± 0.9 g-N/m2/day at 20 mgN/L) than those grown at 10 °C (3.4 ± 0.2 g-N/m2/day at 20 mgN/L). The 30 °C biofilms were thinner and rougher, with a lower protein to polysaccharides ratio (PN/PS) in their extracellular polymeric substance (EPS) matrix and greater amounts of biofilm detachment. Based on fluorescent in-situ hybridization (FISH), there was a higher relative abundance of nitrifying bacteria at 30 °C than at 10 °C, and the ratio of AOB to total nitrifiers (AOB + NOB) was higher at 30 °C (95.1 ± 2.3%) than at 10 °C (77.2 ± 8.6 %). Anammox bacteria were more abundant at 30 °C (16.6 ± 3.7 %) than at 10 °C (6.5 ± 2.4 %). Modeling suggested that higher temperatures increase ammonium oxidation fluxes when the biofilm is limited by ammonium. However, fluxes decrease when oxygen becomes limited, i.e., when the bulk ammonium concentrations are high, due to decreased oxygen solubility. Consistent with the experimental results, the model predicted that the percentage of AOB to total nitrifiers at 30 °C was higher than at 10 °C. To investigate the effects of temperature on biofilm diffusivity and O2 solubility, without longer-term changes in the microbial community, MABR biofilms were grown to steady state at 20 °C, then the temperature changed to 10 °C or 30 °C overnight. Higher ammonium oxidation fluxes were obtained at higher temperatures: 1.91 ± 0.24 g-N/m2/day at 10 °C and 3.19 ± 0.40 g-N/m2/day at 30 °C. Overall, this work provides detailed insights into the effect of temperature on nitrifying MABRs, which can be used to better understand MABR behavior and manage MABR reactors.

Effects of biofilm heterogeneity on the apparent mechanical properties obtained by shear rheometry.

Rheometry is an experimental technique widely used to determine the mechanical properties of biofilms. However, it characterizes the bulk mechanical behavior of the whole biofilm. The effects of biofilm mechanical heterogeneity on rheometry measurements are not known. We used laboratory experiments and computer modeling to explore the effects of biofilm mechanical heterogeneity on the results obtained by rheometry. A synthetic biofilm with layered mechanical properties was studied, and a viscoelastic biofilm theory was employed using the Kelvin-Voigt model. Agar gels with different concentrations were used to prepare the layered, heterogeneous biofilm, which was characterized for mechanical properties in shear mode with a rheometer. Both experiments and simulations indicated that the biofilm properties from rheometry were strongly biased by the weakest portion of the biofilm. The simulation results using linearly stratified mechanical properties from a previous study also showed that the weaker portions of the biofilm dominated the mechanical properties in creep tests. We note that the model can be used as a predictive tool to explore the mechanical behavior of complex biofilm structures beyond those accessible to experiments. Since most biofilms display some degree of mechanical heterogeneity, our results suggest caution should be used in the interpretation of rheometry data. It does not necessarily provide the "average" mechanical properties of the entire biofilm if the mechanical properties are stratified.