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.