Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets

Abstract

Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail. To overcome these limitations, we have developed a droplet microfluidic system for isolating individual episodes of bacterial transformation between two confined cells of pneumococcus. By encapsulating the cells in a 10 μm diameter aqueous droplet, we provide an improved experimental model of genetic transformation, as both participating cells can be identified, and the released DNA is spatially restricted near the attacking strain. Specifically, the bacterial cells, one rifampicin (R) resistant, the other novobiocin (N) and spectinomycin (S) resistant were encapsulated in droplets carried by the fluorinated oil FC-40 with 5% surfactant and allowed to carry out competence-specific attack and DNA uptake (and consequently gain antibiotic resistances) within the droplets. The droplets were then broken, and recombinants were recovered by selective plating with antibiotics. The new droplet system encapsulated 2 or more cells in a droplet with a probability up to 71%, supporting gene transfer rates comparable to standard mixtures of unconfined cells. Thus, confinement in droplets allows characterization of natural genetic transformation during a strictly defined interaction between two confined cells.

Figure 1.. Summary of phenotypes of pneumococcal strains used for transformation in droplets and schematic of cell encapsulation for confined gene transfer between the strains.

CP2204, inducible with competence-stimulating peptide (CSP), is designated as attacker or DNA recipient; CP2215, genetically incompetent, is the natural donor of DNA. In an environment with CSP, CP2204 can attack and lyse CP2215 and take up liberated fragments of DNA, gaining resistance to novobiocin (N) or spectinomycin (S).

Figure 2.. Microfluidic device for encapsulation, observation, and recovery of bacterial cells.

a) Droplet microfluidic device with droplet generator and imaging chamber. Droplets are generated by flow-focusing from three inlets; one oil inlet for Pico-Surf (FC-40 fluorinated oil with 5% surfactant) and two aqueous inlets, for cell mixture and inducer cocktail, respectively. b) Detailed design of the droplet generating device and the focusing nozzle. In-line 10-μm-gap filters are placed at the aqueous inlets to trap debris. Two in-line 6-μm-gap filters are also placed at oil inlets to prevent debris from clogging the droplet generating narrowed neck. c) Detailed design of the imaging chamber, which stores droplets for imaging and cell count in characterization experiments.

Figure 3.. Experimental design for pneumococcus encapsulation and genetic transformation in droplets.

Two strains of pneumococcus were grown and re-suspended in fresh medium so that each strain had an OD of 4. The strains were mixed with a volume ratio of 1:1 and stored on ice. In the cold room, oil, inducer, and cell mixture were loaded into 1-mL syringes with 23G x 1’’ blunt needles and controlled by syringe pumps. Droplet-collecting vials, prepared with 300 μL of CAT medium to prevent droplet evaporation, were maintained on ice. For a specific experiment, when droplets were stably formed, two parallel unconfined culture control experiments, positive control and Pico-exposed control, proceeded by mixing an equal volume of cell mixture and inducer in each control so that the total volume of control would be similar to the total volume of collected aqueous droplets. The droplet-collecting vials and control vials were maintained on ice during the droplet generating process, then incubated at 37°C to activate competence and allow cell-cell attack. After opening of droplets with Pico-Break, cells were diluted, incubated again at 37°C for 1 hour for gene integration and expression, and plated for selection.

Figure 4.. Quantitative image analysis of encapsulation of pneumococci in droplets.

a) Droplet occupancy distribution of the system operated with cell feedstocks at OD 4.0 for each strain: 100 representative droplets were collected and imaged at each trial and manually counted during live video imaging for number of cells in each droplet; 18 trials total. b) Image of pneumococci in ~10 μm diameter droplets.

Figure 5.. Dependence of gene transfer on droplet incubation at 37°C.

Comparison of number of transformants and yield of recombinants in 25-minute incubated droplets (Droplets, 25 mins, 37°C) and non-incubated droplets (Droplets, 0 min, 37°C) at post-droplet dilution factors of 10, 100 or 1000. Yield of recombinants is calculated as a percentage of Nov^RRif^R transformants among initial input of CP2204 (Rif^R) recipients. The error bar shown in the figure is the standard deviation for number of CFU/mL.

Figure 6.. Comparison of kinetics of competence development within droplets vs. in unconfined planktonic cultures.

Seven equivalent droplet samples were collected sequentially for every 10 minutes in the cold room and then incubated at 37°C for different amount of time (0, 10, 15, 20, 25, 30, 35 minutes). Competence development is expressed as the number of Nov^RRif^R transformants per mL and yield of recombinants, as a function of incubation time. Yield of recombinants is calculated as a percentage of Nov^RRif^R transformants among initial input of CP2204 (Rif^R) recipients. The error bar shown in the figure is the standard deviation for number of CFU/mL.

Figure 7.. Nov^R and Spc^R gene transfer in micro-droplets and in unconfined cultures.

Nov^R and Spc^R gene transfer in micro-droplets and in unconfined cultures are shown as total transformants and yield of recombinants in droplets, in a positive (+) control, and in a Pico-exposed control (Pico Control) experiment. Yield of recombinants is calculated as percentage of total transformants for each antibiotic resistance marker among initial input CP2204 (Rif^R) recipients. a) Number of transformants and yield of recombinants for Nov^R marker. b) Number of transformants and yield of recombinants for Spc^R marker. c) Recovery of viable cells of each strain. CP2204 recipient determined as Rif^R colonies; CP2215 donor determined independently as Nov^R or Spc^R colonies. The error bar shown in the figure is the standard deviation for number of CFU/mL.