Compositions and methods for measuring bacterial growth
AssigneeDuke University
InventorsLawrence David, Rachael Bloom
Life of this patent
Estimating the term…
Abstract
The invention disclosed herein relates generally to the fields of microbiology, ecology and microfluidics. Particularly, the invention disclosed herein provides compositions and methods for isolating bacteria from complex microbial communities and measuring growth rates of the isolated bacteria in a given environmental condition.
Claims25
A method for measuring the growth rate of a bacterial strain from a mixed microbial community, comprising:
- (a) isolating a single bacterium by encapsulating the bacterium in an aqueous droplet surrounded by an oil-phase to obtain an encapsulated bacterium;
- (b) incubating the encapsulated bacterium under conditions appropriate for growth to obtain a single encapsulated bacterial strain;
- (c) extracting DNA from the encapsulated bacterial strain at various time points during incubation;
- (d) measuring:
- (i) a quantity of total DNA extracted from each data point using quantitative Polymerase Chain Reaction (“qPCR”) via primers that target a variable region within a conserved gene sequence;
- and
- (ii) a relative abundance of the encapsulated bacterial strain through sequencing of the same variable region within a conserved gene sequence as in (i) using the same primers as in (i);
- and
- (e) generating a growth curve based on the measurements obtained in (d) at each time point.
A method for assessing the potential for a bacterial strain from a mixed microbial community to develop resistance to an antibiotic drug, comprising:
- (a) isolating a single bacterium by encapsulating the bacterium in an aqueous droplet surrounded by an oil-phase to obtain an encapsulated bacterium;
- (b) incubating the encapsulated bacterium in the presence of an antibiotic drug, under conditions appropriate for growth to obtain a single encapsulated bacterial strain;
- (c) extracting DNA from the encapsulated bacterial strain at various time points during incubation;
- (d) measuring:
- (i) a quantity of total DNA extracted from each data point using quantitative Polymerase Chain Reaction (“qPCR”) via primers that target a variable region within a conserved gene sequence;
- and
- (ii) a relative abundance of the encapsulated bacterial strain through sequencing of the same variable region within a conserved gene sequence as in (i) using the same primers as in (i);
- and
- (e) generating a growth curve based on the measurements obtained in (d) at each time point.
A method for characterizing the microflora in a patient's gut, comprising:
- (a) isolating each bacterium within a patient's stool sample by individually encapsulating the bacterium in an aqueous droplet surrounded by an oil-phase to obtain individually encapsulated bacteria;
- (b) incubating each encapsulated bacterium under conditions appropriate for growth to obtain encapsulated bacterial strains;
- (c) extracting DNA from each encapsulated bacterial strain at various time points during incubation;
- (d) measuring:
- (i) a quantity of total DNA extracted from each data point using quantitative Polymerase Chain Reaction (“qPCR”) via primers that target a variable region within a conserved gene sequence;
- and
- (ii) a relative abundance of the encapsulated bacterial strain through sequencing of the same variable region within a conserved gene sequence as in (i) using the same primers as in (i);
- and
- (e) generating a growth curve based on the measurements obtained in (d) at each time point.
(canceled)
(canceled)
(canceled)
A method for identifying bacteria from a mixed microbial community which have an inhibitory effect on a target bacteria, comprising:
- (a) adding a fluorescent-labeled target bacterial strain to a suspension containing a mixed microbial community;
- (b) isolating a bacterial cell from a mixed microbial community and a fluorescent-labeled target bacterial cell together by encapsulating the two bacterial cells in an aqueous droplet surrounded by an oil-phase to obtain an encapsulated co-culture;
- (b) incubating the encapsulated co-culture under conditions appropriate for growth to stationary phase;
- (c) sorting the co-cultured droplets using Fluorescence Activated Cell Sorting (FACS);
- and
- (d) detecting co-cultured droplets exhibiting bacterial growth of a mixed community bacterial strain, but lacking fluorescence.
(canceled)
Dependencies are inferred from the claim text.