Real-time mapping of a hydrogen peroxide concentration profile across a polymicrobial bacterial biofilm using scanning electrochemical microscopy

Abstract

Quantitative detection of hydrogen peroxide in solution above a Streptococcus gordonii (Sg) bacterial biofilm was studied in real time by scanning electrochemical microscopy (SECM). The concentration of hydrogen peroxide was determined to be 0.7 mM to 1.6 mM in the presence of 10 mM glucose over a period of 2 to 8 h. The hydrogen peroxide production measured was higher near the biofilm surface in comparison to Sg grown planktonically. Differential hydrogen peroxide production was observed both by fluorometric as well as by SECM measurements. The interaction between two different species in a bacterial biofilm of Sg and Aggregatibacter actinomycetemcomitans (Aa) in terms of hydrogen peroxide production was also studied by SECM. One-directional y-scan SECM measurements showed the unique spatial mapping of hydrogen peroxide concentration across a mixed species biofilm and revealed that hydrogen peroxide concentration varies greatly dependent upon local species composition.real-time (detection) | metabolite efflux | local concentration | oral flora | Au UME S treptococcus gordonii (Sg) is a member of the viridans group streptococci-Gram-positive oral microbes that are known to ferment sugars into lactic acid and produce hydrogen peroxide in the presence of oxygen (1). The presence of these beneficial oral streptococci has been shown to improve oral health, by either competition with pathogens for nutrients in the oral cavity or by the production of inhibitory concentrations of hydrogen peroxide. Populations of viridans group streptococci negatively correlate with the presence of many notable oral pathogens (2, 3). However, recent work has demonstrated that in vitro Sg can grow in coculture with the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans (Aa) (4). In co-culture Aa preferentially utilizes Sg-produced lactic acid (5) and detoxifies Sg-produced hydrogen peroxide using the KatA enzyme (6). Recent work has demonstrated that hydrogen peroxide induces katA expression as well as apiA, which encodes an immunoprotective factor that renders Aa more resistant to killing by host innate immunity (5). These studies demonstrated induction of gene expression in mixed species biofilms by Sg-produced hydrogen peroxide. Because hydrogen peroxide is rapidly degraded by catalase and can also react with other biological materials, we sought to quantify local hydrogen peroxide concentrations in real time to be utilized for future polymicrobial experiments between Sg, Aa, and other oral bacteria.Previous measurements of hydrogen peroxide have been performed using fluorescence, spectroscopy and other methods (1, 7-10). However, current techniques lack the ability to quantify local hydrogen peroxide concentrations at the surface of a biofilm. In this study, scanning electrochemical microscopy (SECM) was used to address this problem. SECM has the unique ability to set the exact distance from a sensing tip [an ultramicroelectrode (UME) of size ∼10 to 25 μm diameter] to a substrate through a feedback approach curve (11)...

Discovery of a biofilm electrocline using real-time 3D metabolite analysis

“…Our laboratories previously adapted SECM to acquire quantitative data detailing metabolite concentration heterogeneity surrounding a biofilm (24). These initial studies led us to expand the utility of SECM not only to quantify the concentration of the signal/cue PYO, but also to noninvasively determine the redox status of biofilm-produced small molecules in 3D space.…”

“…The primary challenge to these experiments is the ability to quantify and spatially resolve small molecules immediately surrounding biofilms in real-time. In a previous study (24), our laboratories have begun to address the challenge of spatial metabolite profiling through the use of scanning electrochemical microscopy (SECM) to quantify a primary metabolite produced by a bacterial biofilm in real-time. SECM has the unique ability to set the exact distance from a ultramicroelectrode sensing tip to a biological substrate through a feedback approach curve (25) and, thus, is able to measure the local concentration of redox active small molecules over a biofilm.…”

Bacterial fight-and-flight responses enhance virulence in a polymicrobial infection

“…Prior in vitro studies pointed to the likely importance of catalase. Catalase is strongly up-regulated during coculture (28) and responsible for locally depleting H 2 O 2 levels in mixed Aa-Sg biofilms (24). In light of these previous findings, we anticipated that catalase would be an essential Aa fitness determinant during coinfections with Sg.…”

“…Many streptococci are known to produce H 2 O 2 at amounts sufficient to inhibit the growth of surrounding microbes (24). For instance, H 2 O 2 production by Streptococcus pneumoniae reduces the prevalence of other pathogens within the upper respiratory tract, including Haemophilus influenzae (52).…”

“…In the oral cavity, Aa and Sg inhabit the gingival crevice (20), a niche defined as the narrow pocket between the gum and the tooth; however, these organisms can also spread systemically and contribute to extraoral infections, including polymicrobial abscesses (21,22). Similar to other oral streptococci, Sg rapidly consumes carbohydrates and excretes two primary metabolic end products, L-lactate and hydrogen peroxide (H 2 O 2 ), which can accumulate to high (millimolar) concentrations within mixed-species biofilms (23,24). Rapid sugar consumption and H 2 O 2 production render the streptococci extremely competitive by respectively limiting carbon source availability and causing oxidative stress to surrounding microbes.…”