Jodi L. Connell

3D printing of microscopic bacterial communities

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. 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.

Probing Prokaryotic Social Behaviors with Bacterial “Lobster Traps”

ABSTRACT Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 108 bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 101 to 105 cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial “lobster traps”) defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (1012 cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations. IMPORTANCE Prokaryotes are social organisms capable of coordinated group behaviors, including the abilities to construct antibiotic-resistant biofilms and to communicate with small signaling molecules (quorum sensing [QS]). While there has been significant effort devoted to understanding biofilm formation and QS, few studies have examined these processes in high-density/low-cell-number populations. Such studies have clinical significance, as many infections are initiated by small bacterial populations (<105) that are organized into dense clusters. Here, we describe a technology for studying such bacterial populations in picoliter-sized porous cavities (referred to as bacterial “lobster traps”) capable of capturing a single bacterium and tracking growth and behavior in real time. We provide evidence that small changes in the size of the bacterial cluster as well as flow rate of the surrounding medium modulate QS in Pseudomonas aeruginosa. We also demonstrate that as few as ~150 confined bacteria are needed to exhibit an antibiotic-resistant phenotype similar to biofilm bacteria. Prokaryotes are social organisms capable of coordinated group behaviors, including the abilities to construct antibiotic-resistant biofilms and to communicate with small signaling molecules (quorum sensing [QS]). While there has been significant effort devoted to understanding biofilm formation and QS, few studies have examined these processes in high-density/low-cell-number populations. Such studies have clinical significance, as many infections are initiated by small bacterial populations (<105) that are organized into dense clusters. Here, we describe a technology for studying such bacterial populations in picoliter-sized porous cavities (referred to as bacterial “lobster traps”) capable of capturing a single bacterium and tracking growth and behavior in real time. We provide evidence that small changes in the size of the bacterial cluster as well as flow rate of the surrounding medium modulate QS in Pseudomonas aeruginosa. We also demonstrate that as few as ~150 confined bacteria are needed to exhibit an antibiotic-resistant phenotype similar to biofilm bacteria.

Mechanisms of synergy in polymicrobial infections.

Communities of microbes can live almost anywhere and contain many different species. Interactions between members of these communities often determine the state of the habitat in which they live. When these habitats include sites on the human body, these interactions can affect health and disease. Polymicrobial synergy can occur during infection, in which the combined effect of two or more microbes on disease is worse than seen with any of the individuals alone. Powerful genomic methods are increasingly used to study microbial communities, including metagenomics to reveal the members and genetic content of a community and metatranscriptomics to describe the activities of community members. Recent efforts focused toward a mechanistic understanding of these interactions have led to a better appreciation of the precise bases of polymicrobial synergy in communities containing bacteria, eukaryotic microbes, and/or viruses. These studies have benefited from advances in the development of in vivo models of polymicrobial infection and modern techniques to profile the spatial and chemical bases of intermicrobial communication. This review describes the breadth of mechanisms microbes use to interact in ways that impact pathogenesis and techniques to study polymicrobial communities.

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

Significance Bacteria commonly reside in vivo as communities comprised of small, densely packed aggregates. Aggregates display important phenotypes, including enhanced antibiotic resistance, and recent evidence suggests that chemical interactions between aggregates are critical in human-associated microbial communities. However, studying aggregates is challenging because of the inability to confine and spatially organize small microbial populations. Here, we interface two analytical technologies, micro-3D printing and scanning electrochemical microscopy, to develop an in vitro platform with the capacity to manipulate the size and spatial arrangement of bacterial aggregates and quantify chemical interactions between aggregates in real time. We show that a quorum-sensing metabolite is produced by Pseudomonas aeruginosa aggregates containing as few as 500 cells and determine how spatial structure impacts communication between neighboring aggregates. Microbes frequently live in nature as small, densely packed aggregates containing ∼101–105 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.

Oxygen Limitation within a Bacterial Aggregate

ABSTRACT Cells within biofilms exhibit physiological heterogeneity, in part because of chemical gradients existing within these spatially structured communities. Previous work has examined how chemical gradients develop in large biofilms containing >108 cells. However, many bacterial communities in nature are composed of small, densely packed aggregates of cells (≤105 bacteria). Using a gelatin-based three-dimensional (3D) printing strategy, we confined the bacterium Pseudomonas aeruginosa within picoliter-sized 3D “microtraps” that are permeable to nutrients, waste products, and other bioactive small molecules. We show that as a single bacterium grows into a maximally dense (1012 cells ml−1) clonal population, a localized depletion of oxygen develops when it reaches a critical aggregate size of ~55 pl. Collectively, these data demonstrate that chemical and phenotypic heterogeneity exists on the micrometer scale within small aggregate populations. IMPORTANCE Before developing into large, complex communities, microbes initially cluster into aggregates, and it is unclear if chemical heterogeneity exists in these ubiquitous micrometer-scale aggregates. We chose to examine oxygen availability within an aggregate since oxygen concentration impacts a number of important bacterial processes, including metabolism, social behaviors, virulence, and antibiotic resistance. By determining that oxygen availability can vary within aggregates containing ≤105 bacteria, we establish that physiological heterogeneity exists within P. aeruginosa aggregates, suggesting that such heterogeneity frequently exists in many naturally occurring small populations. Before developing into large, complex communities, microbes initially cluster into aggregates, and it is unclear if chemical heterogeneity exists in these ubiquitous micrometer-scale aggregates. We chose to examine oxygen availability within an aggregate since oxygen concentration impacts a number of important bacterial processes, including metabolism, social behaviors, virulence, and antibiotic resistance. By determining that oxygen availability can vary within aggregates containing ≤105 bacteria, we establish that physiological heterogeneity exists within P. aeruginosa aggregates, suggesting that such heterogeneity frequently exists in many naturally occurring small populations.

Sociomicrobiology in engineered landscapes

A growing body of evidence points to the importance of microcolonies in the dissemination of bacteria, yet there is a dearth of tools for systematically assessing the behavior of cells within such communities. New strategies for landscaping three-dimensional culture environments on microscopic scales may have a critical role in revealing how bacteria orchestrate antibiotic resistance and other social behaviors within small, dense aggregates.

Development of a versatile in vitro platform for studying biological systems using micro-3D printing and scanning electrochemical microscopy.

We report a novel strategy for studying a broad range of cellular behaviors in real time by combining two powerful analytical techniques, micro-3D printing and scanning electrochemical microscopy (SECM). This allows one, in microbiological studies, to isolate a known number of cells in a micrometer-sized chamber with a roof and walls that are permeable to small molecules and observe metabolic products. In such studies, the size and spatial organization of a population play a crucial role in cellular group behaviors, such as intercellular interactions and communication. Micro-3D printing, a photolithographic method for constructing cross-linked protein microstructures, permits one to compartmentalize a small population of microbes by forming a porous roof and walls around cells in situ. Since the roof and walls defining the microchamber are porous, any small molecules can freely diffuse from the chamber to be detected and quantified using SECM. The size of the chamber and the roof permeability can be obtained by SECM using a small probe molecule, ferrocenemethanol (FcMeOH). The chamber permeability to FcMeOH can be tuned by varying printing parameters that influence the cross-linking density of the proteinaceous material. These analyses establish a versatile strategy as a sensitive platform to quantitatively monitor small molecules produced by microbes.

Automated colorimetric gadolinium assay for verification of clearance and estimation of glomerular filtration rate

BACKGROUND NSF (nephrogenic systemic fibrosis) is a potentially serious adverse effect for renal patients undergoing MRI (magnetic resonance imaging) procedures using gadolinium-containing contrast agents. There is therefore a need to verify clearance of these agents and to confirm appropriate renal status of patients treated with these drugs. METHODS Serum samples from canine and feline subjects dosed with 0.1 mmol/kg of gadolinium agent, or from spiked samples were assayed for gadopentetate (Magnevist), gadodiamide (Omniscan) or gadoversetamide (OptiMARK) using a new dye reagent on the Olympus AU400. Accuracy was verified by ICP-MS. RESULTS The reportable dynamic range is 3-600 micromol/l Gd. Split serum samples from animals dosed with 0.1 mmol/kg of gadopentetate ranged from 7-458 micromol/l Gd: y=1.121x+0.267, r=0.996, for the Olympus method as a function of Gd measurement by ICP-MS. Between-day imprecision was 1.3% CV-3.6% CV for samples ranging from 12-400 micromol/l Gd. CONCLUSIONS The assay is useful to verify the clearance of gadolinium and for evaluation of renal status by estimation of GFR using gadopentetate.

Analyzing secondary metabolite production by 3D printed bacterial populations using scanning electrochemical microscopy

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 [1]. 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.

In Situ Imprinting of Topographic Landscapes at the Cell–Substrate Interface

In their native environments, adherent cells encounter dynamic topographical cues involved in promoting differentiation, orientation, and migration. Ideally, such processes would be amenable to study in cell culture using tools capable of imposing dynamic, arbitrary, and reversible topographic features without perturbing environmental conditions or causing chemical and/or structural disruptions to the substrate surface. To address this need, we report here development of an in vitro strategy for challenging cells with dynamic topographical experiences in which protein-based hydrogel substrate surfaces are modified in real time by positioning a pulsed, near-infrared laser focus within the hydrogel, promoting chemical cross-linking which results in local contraction of the protein matrix. Scanning the laser focus through arbitrary patterns directed by a dynamic reflective mask creates an internal contraction pattern that is projected onto the hydrogel surface as features such as rings, pegs, and grooves. By subjecting substrates to a sequence of scan patterns, we show that topographic features can be created, then eliminated or even reversed. Because laser-induced shrinkage can be confined to 3D voxels isolated from the cell-substrate interface, hydrogel modifications are made without damaging cells or disrupting the chemical or structural integrity of the surface.