Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy

Abstract

Microbes frequently live in nature as small, densely packed aggregates containing ∼10 1 -10 5 cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.Pseudomonas aeruginosa | scanning electrochemical microscopy | quorum sensing | 3D printing | pyocyanin B acterial populations are often found in nature as small, densely packed aggregates containing ∼10 1 -10 5 cells (1-5). These aggregates serve as building blocks for larger biofilm communities as well as a primary mode of transmission for pathogenic microbes (5-8). Similar to biofilm communities, aggregates develop microscale physical and chemical heterogeneity and display clinically relevant phenotypes, including enhanced antibiotic resistance (2,(8)(9)(10)(11)(12)(13)(14)(15)(16). Moreover, aggregate sizes containing as few as 10 3 bacteria have been shown to engage in quorum sensing (QS)-mediated behaviors (17-21). In its simplest form, QS is a communication strategy that allows bacteria to effectively monitor their population density through the secretion and sensing of extracellular signals (7,(22)(23)(24). When the population reaches a specific density, activation of the QS regulatory cascade results in enhanced transcription of a defined set of genes. These genes control distinct behaviors, including virulence, in the opportunistic pathogen Pseudomonas aeruginosa (25). In addition to displaying QS-mediated behaviors, bacterial aggregates have been shown to interact with neighboring aggregates both in vitro and in vivo (9,(26)(27)(28). Indeed, these interactions have a profound impact on virulence and are often mediated by small diffusible molecules (8-10, 22, 29-31).Despite the prevalence of aggregates in nature, understanding the mechanisms controlling their behavior and intera...

The biogeography of polymicrobial infection

“…96) can spatially regulate biofilm formation 97 and dispersion 98 , and can also stimulate other virulence properties, such as antibiotic resistance 94,99 and per-sistence 96 . As these signals are often restricted to very short-range (<10 µm) effects 100 , they have the potential to generate fine-scale spatial structure. However, signalling is restricted to only those species with an appropriate receptor, suggesting that non-discriminatory metabolic interactions may be a more widespread force that generates spatial structure.…”

Benefits and limitations of three-dimensional printing technology for ecological research

“…In one application, designer bacterial ecosystems that varied in geometry and spatial structure were printed using a gelatin-based material in order to study cell-to-cell interactions ([47, 48]; Table 1). In a second application, nano-scale 3D printing technology was used to print replicas of abdominal scales from rainbow peacock spiders ( Maratus robinsoni and M. chrysomelas ) and specialized hairs from blue tarantulas ( Poecilotheria metallica and Lampropelma violaceopes ) with comparable visual properties to the actual structures [49, 50].…”

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Bacterial Chat: Intestinal Metabolites and Signals in Host-Microbiota-Pathogen Interactions

“…Bacteria employ chemical signaling to communicate among themselves upon the availability of favorable or detrimental compounds. Different aspects of this signaling have been shown in the bacterial cell-to-cell communication field (25,(122)(123)(124). The advent of novel technologies will help to obtain more detailed information on the intricate microbiotapathogen-host relationships to directly identify single molecules or more complex compounds present during in vivo infections.…”