Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors

Abstract

Biofilms are communities of microorganisms enclosed in a self-generated matrix of extracellular polymeric substances. While biofilm recalcitrance and persistence are caused by several factors, a reduction in antimicrobial susceptibility has been closely associated with the generation of pH gradients within the biofilm structure. Cells embedded within the biofilm create a localized acidic microenvironment, which is unaffected by the external pH. Therefore, pH monitoring is a promising approach for understanding the complexities of a three-dimensional heterogeneous biofilm. A fluorescent pH nanosensor was designed through the synthesis of mesoporous silica nanoparticles (47 ± 5 nm diameter) conjugated to a pH-sensitive dye (fluorescein) and a pH-insensitive dye (rhodamine B) as an internal standard (dye-MSNs). The fluorescence intensity of fluorescein (I F ) reduced significantly as the pH was decreased from 8.5 to 3.5. In contrast, the fluorescence intensity of rhodamine B (I R ) remained constant at any pH. The ratio of I F /I R produced a sigmoidal curve with respect to the pH, in a working pH range between 4.5 and 7.5. Dye-MSNs enabled the measurement of pH gradients within Pseudomonas fluorescens WCS 365 biofilm microcolonies. The biofilms showed spatially distinct low-pH regions that were enclosed into large clusters corresponding to high-cell-density areas. Also present were small low-pH areas that spread indistinctly throughout the microcolony caused by the mass transfer effect. The lowest detected pH within the inner core of the microcolonies was 5.1, gradually increasing to a neutral pH toward the exterior of the microcolonies. The dye-MSNs were able to fully penetrate the biofilm matrix and allowed a quantitative ratiometric analysis of pH gradients and distribution throughout the biofilm, which was independent of the nanoparticle concentration.

<p>Tailoring Nanoparticle-Biofilm Interactions to Increase the Efficacy of Antimicrobial Agents Against <em>Staphylococcus aureus</em></p>

“…The synthesis of MSNs was performed using an adapted Stöber method as previously described. 14 The functionalization of MSNs was adapted from Gounani et al (2018); 40 50 mg of bare-MSNs (MSN-B) were dispersed in 4.6 mL of absolute ethanol in an ultrasonic bath, then 200 µL of deionized water, 100 µL of acetic acid and 100 µL of DETA were added, and the reaction mixture stirred at room temperature for 1 h. The obtained aminated product (MSN-D) was washed three times with ethanol by centrifugation (9,000 rpm, 15 min). In order to obtain the carboxyl functionalized MSNs, a suspension of dried MSN-D in DMF (6 mL of 2 mg mL −1 ) was added to a solution of succinic anhydride in DMF (50 mL of 10 mg mL −1 ).…”

“…11,12 The complex interlink of the EPS components leads to poor antibiotic penetration due to diffusion limitation. 13 The EPS components and acidic microenvironments 14 within the matrix can also interact with and inactivate drug molecules reducing the local concentration around the bacterial cells. 15 Among all drug-resistant infections, methicillinresistant Staphylococcus aureus (MRSA) is the most frequent, with a high incidence worldwide.…”

Engineering Inorganic Nanoflares with Elaborate Enzymatic Specificity and Efficiency for Versatile Biofilm Eradication

“…[38] And the endogenous H 2 O 2 content of infection sites was estimated to be ≈90.2 × 10 −6 m (Table S2, Supporting Information), which was consistent with the previous reports (100 × 10 −6 m). [39] Besides endogenous H 2 O 2 , these biofilminfected tissues are also encoded with acidic environment due to the bacterial metabolism-mediated production of acidic by-products. [40] Thus, the endogenous H 2 O 2 and the pathological acidic environment of infected tissue could facilitate the robust CPPcatalyzed gene ration of •OH, which then subsequently leads to the highly efficient biofilms disinfection (Scheme 1B).…”

How Microbial Biofilms Control the Environmental Fate of Engineered Nanoparticles?

“…(Hunter and Beveridge, 2005;Hidalgo et al, 2009). For instance, Pseudomonas fluorescens WCS 365 biofilms were reported to exhibit low pH regions (down to pH 5.1) in cells clusters located at the inner-core of microcolonies, which gradually evolved to neutral pH in vicinity of the bulk solution at the biofilm surface (Fulaz et al, 2019a). These pH variations within relatively short spatial scales could be explained by the fast production of metabolic residues combined with a limited solute transfer through the EPS matrix, resulting in a local accumulation of acidic by-products (Fulaz et al, 2019a).…”

“…For instance, Pseudomonas fluorescens WCS 365 biofilms were reported to exhibit low pH regions (down to pH 5.1) in cells clusters located at the inner-core of microcolonies, which gradually evolved to neutral pH in vicinity of the bulk solution at the biofilm surface (Fulaz et al, 2019a). These pH variations within relatively short spatial scales could be explained by the fast production of metabolic residues combined with a limited solute transfer through the EPS matrix, resulting in a local accumulation of acidic by-products (Fulaz et al, 2019a). Multispecies biofilms, presenting an array of different metabolisms, could generate more acidic by-products under oxygen-limiting conditions compared to single-species biofilms, resulting in lower minimal pH values, reported between 4 and 5 for multi-species oral biofilms (Schlafer et al, 2018).…”