3D printing of microscopic bacterial communities

Abstract

Bacteria communicate via short-range physical and chemical signals, interactions known to mediate quorum sensing, sporulation, and other adaptive phenotypes. Although most in vitro studies examine bacterial properties averaged over large populations, the levels of key molecular determinants of bacterial fitness and pathogenicity (e.g., oxygen, quorum-sensing signals) may vary over micrometer scales within small, dense cellular aggregates believed to play key roles in disease transmission. A detailed understanding of how cell-cell interactions contribute to pathogenicity in natural, complex environments will require a new level of control in constructing more relevant cellular models for assessing bacterial phenotypes. Here, we describe a microscopic threedimensional (3D) printing strategy that enables multiple populations of bacteria to be organized within essentially any 3D geometry, including adjacent, nested, and free-floating colonies. In this laser-based lithographic technique, microscopic containers are formed around selected bacteria suspended in gelatin via focal cross-linking of polypeptide molecules. After excess reagent is removed, trapped bacteria are localized within sealed cavities formed by the crosslinked gelatin, a highly porous material that supports rapid growth of fully enclosed cellular populations and readily transmits numerous biologically active species, including polypeptides, antibiotics, and quorum-sensing signals. Using this approach, we show that a picoliter-volume aggregate of Staphylococcus aureus can display substantial resistance to β-lactam antibiotics by enclosure within a shell composed of Pseudomonas aeruginosa.multiphoton lithography | microfabrication | antibiotic resistance | polymicrobial U ncovering relationships between structure and function remains a central goal of biology. At molecular dimensions, protein structure can be modified to systematically evaluate how conformation gives rise to ligand binding, catalysis, and other functional properties. On the far larger scale of ecological habitats, organization plays a similarly vital role in mediating "function," where the social behavior of organisms-including their reproduction rate, mobility, and involvement in cooperative and predatory relationships-depends on the spatial arrangement of the community. As with molecular function, a detailed understanding of how organization affects behavior of communities would benefit from technologies for creating variants of defined structure.Nowhere is the potential value of geometrical control more evident than in the study of microbial ecosystems. The burgeoning field of sociomicrobiology has revealed a richness in the mechanisms by which bacteria engage in cooperative and adversarial relationships, affecting nearby individuals through physical contact and modifications to the chemical composition of their shared microenvironment. Spatially dependent interactions can result from perturbations to the nutritional state of the local habitat, but also may be caused by release of di...

Synergistic Interactions of Pseudomonas aeruginosa and Staphylococcus aureus in an In Vitro Wound Model

“…In addition, Hoffman et al have demonstrated that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) could protect S. aureus from killing by the aminoglycoside tobramycin in S. aureus-P. aeruginosa cocultures by suppressing S. aureus respiration (24). This type of "bystander" protection was also seen in the production of ␤-lactamases by P. aeruginosa, which were thought to protect S. aureus from ampicillin treatment in 3-dimensionally (3D) printed S. aureus-P. aeruginosa bacterial communities (46). Accumulation of environmental DNA (eDNA) in cocultures represents another possibility that could be examined further.…”

Microfluidic Studies of Biofilm Formation in Dynamic Environments

“…Alternatives have been proposed, such as integration of an electrochemical sensor for real-time analysis (56), yet they are not widespread and make fabrication and operation more complex. A second limitation is that most microfluidic devices used in microbial studies to date are essentially two-dimensional, with very few exceptions (57). To attain a closer representation of natural habitats, it will be important in the future to introduce a three-dimensional component, as was done in tissue engineering (58,59).…”

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

“…The protein-based walls and roof of the microtrap define aggregate size and shape in three dimensions and are permeable to many small molecules. Cells confined within microtraps divide at normal rates and reach maximum cell density (10 12 cells mL −1 ) while maintaining cell numbers typical of natural aggregates (14,28,42).Using microtraps, our group showed that as few as 2,600 P. aeruginosa cells engage in QS-mediated behaviors when present at maximum density (42). This work used a cell-based biosensor, in which production of GFP served as a proxy for QS-mediated communication (14,42).…”