Sequence tag-based analysis of microbial population dynamics

Abstract

We describe sequence tag-based analysis of microbial populations (STAMP) for characterization of pathogen population dynamics during infection. STAMP analyzes the frequency changes of genetically 'barcoded' organisms to quantify population bottlenecks and infer the founding population size. Analyses of intraintestinal Vibrio cholerae revealed infection-stage and region-specific host barriers to infection and showed unexpected V. cholerae migration counter to intestinal flow. STAMP provides a robust, widely applicable analytical framework for high-confidence characterization of in vivo microbial dissemination.

Figure 1

The barriers to V. cholerae infection are heterogeneous along the intestine. (a) Schematic overview of the experimental setup. (b) In vitro calibration curve. Correlation between experimentally determined bottleneck population size (bacterial load) and estimated bottleneck size (N_b) by STAMP. The red, blue and yellow symbols represent biologically independent samples. The solid line indicates the median; the dashed, black lines indicate the 95 % confidence interval. The dashed, grey lines marks the resolution limit for N_b estimation. (c) Representative example of bottleneck populations corrected with the calibration curve (N_b′, black dots) and bacterial load (cfu, red squares) at 20 h post-infection throughout the gastro-intestinal tract of a single animal after inoculation with 10⁹ cfu barcoded V. cholerae. An additional example is shown in supplementary figure 6. (d) Sampling sites used in this study are indicated in light red or blue; S: stomach content, P1–P10, proximal SI sections used in figure 1, I1: proximal SI section used in figures 2 and 3, I2: middle SI, I3: distal SI, Ce: cecum tissue, Cf: cecal fluid, Co: colon.

Figure 2

Spatial and temporal dynamics of founding V. cholerae populations along the intestine. (a) Corrected bottleneck population size (N_b′, black dots) and bacterial load (cfu, red squares) at different loci during early, middle and late phases (~2 h; ~7 h; ~20 h post-infection) of infection from 19 animals from 12 independent litters. Open symbols represent N_b′ values above the resolution limit or no detected colonies; dotted lines indicate the resolution limit for N_b′ estimation. Sample medians are represented by horizontal lines. Corresponding N_b′ and bacterial load from the same animal are aligned vertically and in the same sequential order. Significance was tested with one-sided Wilcoxon signed rank tests; * (p < 0.05) and ** (p < 0.01). (b) Genetic distance of populations during different phases of the disease. The genetic distance between I1 and I3 in the middle phase of the disease is significantly different from the early (p = 0.026) and the late phase (p = 0.015, both two-sided Wilcoxon rank sum test). (c) Model of the spatio-temporal dynamics of V. cholerae infection in the infant rabbit host. The bacterial burden is represented in the heat map (red: high; yellow and white: low); arrows indicate the direction of migration.