Revision as of 16:36, 22 September 2018

Citation

Connell, J. L., Ritschdorff, E. T., Whiteley, M., & Shear, J. B. (2013). 3D printing of microscopic bacterial communities. Proceedings of the National Academy of Sciences, 110(46), 18380-18385.

Links

• http://www.pnas.org/content/110/46/18380
• http://hawaiireedlab.com/pdf/c/connelletal2013.pdf (internal lab link only)

Published 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 three-dimensional (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 cross-linked 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.

Published Significance

Bacteria within the human body commonly thrive within structured three-dimensional (3D) communities composed of multiple bacterial species. Organization of individuals and populations within bacterial aggregates is believed to play key roles in mediating community attributes, affecting, for example, the virulence of infections within the cystic fibrosis lung and oral cavity. To gain detailed insights into how geometry may influence pathogenicity, we describe a strategy for 3D printing bacterial communities in which physically distinct but chemically interactive populations of defined size, shape, and density can be organized into essentially any arrangement. Using this approach, we show that resistance of one pathogenic species to an antibiotic can enhance the resistance of a second species by virtue of their 3D relationship.

Notes

The importance of local interactions, while perhaps overlooked, should not be too surprising. Satellite colonies on antibiotic selective media are a good example. Cells that incorporate a plasmid that gives antibiotic resistance are soon locally surrounded by "satellite" colonies without the plasmid. The antibiotic, which spreads through the media by diffusion, is being broken down in the immediate area on the agar plate, allowing nearby colonies to grow. However, this work is focused on even smaller scales on the order of tens of micrometers and volumes as small as 1 pL (1 picoliter or one trillionth of a liter).

The scale bars used in the figures are 10 or 20 μm. It is useful to recall that an average bacteria like E. coli can be ~2μm long and ~0.5μm wide.

Staphylococcus aureus and Pseudomonas aeruginosa are often found in coinfections when they infect humans. The authors showed that S. aureus in a container nested within a P. aeruginosa container are shielded from ampicillin antibiotic by the P. aeruginosa.

They found that some P. aeruginosa cells could not be removed from the area outside the printed containers by washing. Did single cells adhere to the glass surface similar to biofilm adhesion?

Could this type of approach be applied to sponge (Porifera) cells? Sponge cells can be isolated from each other, survive, then regroup to form a new multicellular sponge. Perhaps different 3D filtering designs for artificial bioremediation vessels could be constructed and tested?

The polypeptides of the gelatin matrix are cross linked with a laser focused on the point of the desired solid voxel. There is a threshold light intensity ("nonlinear dependence") so that the cross-linking can be spatially restricted. The laser light is at 740 nm or 0.74 µm. The solid structures created have smallest constituent "wall" cross sections on the order of micrometers, which is about the size of typical bacterial cells. This is probably at or near the limit of smallest construction allowed by this particular technology. The lower bound focus of a light source is limited by its wavelength (this is the reason for using an Electron Microscope for imaging objects smaller than what light microscopy allows).

Terms

• Sociomicrobiology - Studying microbe interactions and social behaviors.
• Optical tweezers - Physically manipulating microscopic objects with a laser. https://en.wikipedia.org/wiki/Optical_tweezers
• Confocal microscopy - A method that uses fine scale focusing to image a narrow depth and block out of focus parts of the image from above and below that depth. Planes of the image at a range of depths can then be assembled to reconstruct the imaged object in three dimensions. for https://en.wikipedia.org/wiki/Confocal_microscopy
• Lithography - The term is used here in a different way from what I think of. Originally lithography meant etching a pattern into a flat surface but here is seems to be used to refer to making a solid pattern in three dimensions. https://en.wikipedia.org/wiki/Lithography https://en.wikipedia.org/wiki/Photolithography What is done here might be better referred to as laser sintering or sterolithography. https://en.wikipedia.org/wiki/Selective_laser_sintering https://en.wikipedia.org/wiki/Stereolithography
• in situ - Some students in class were unsure about this term. Here it is used to refer to building the laser crosslinked gelatin containers in place around the bacteria.

Related Publications

On the to do list to read:

• Schaffner, M., Rühs, P. A., Coulter, F., Kilcher, S., & Studart, A. R. (2017). 3D printing of bacteria into functional complex materials. Science Advances, 3(12), eaao6804. http://advances.sciencemag.org/content/3/12/eaao6804.short