Figure 4. IBCs offer a semi-protective niche that delays clearance of bacteria by antibiotics.

(A1–A5) Bacteria persist and grow in IBCs despite antibiotic treatment. Snapshots show the growth of four IBCs with variable growth rates (A1–A2) indicated by yellow arrowheads. Ampicillin treatment eliminates some but not all of the bacteria within each IBC (A3). Growth resumes at all sites in (A4, A5), formation of new IBCs in the second growth cycle is indicated by white arrowheads. (B) Plots of logarithm of bacterial volume within five separate IBCs demonstrates exponential bacterial growth. IBCs are seeded dynamically; growth can occur either in the first or second growth cycle, or prior to the administration of antibiotic, or in few cases continues in the presence of the antibiotic. (C) Plots of the logarithm of bacterial volume vs. time for n=8 IBCs tracked across two growth phases with an intermediate period of ampicillin treatment. In the growth phases, growth is exponential and bacterial volume continues to increase for up to ca. 120 min after administration of antibiotic before declining due to loss of bacteria. In each case, growth resumes after the antibiotic is removed. (D) Scatter plots of the doubling time of bacterial volume in IBCs as measured in the first growth cycle (n=102) and the second growth cycle (n=59) from n = 3 bladder-chips. Growth in the second cycle is significantly slower (p=4.7E-7), red line represents the median value. (E) Doubling time of bacterial volume in IBCs in the first and second growth cycle for some of the IBCs in (D) that survived the antibiotic treatment. In n=16 out of 18 instances, growth is slower in the second growth cycle. (F1–F5) High-resolution time-series that highlights bacterial growth within an IBC prior to (F1, F2) and after (F3–F5) administration of ampicillin. Some bacteria within this IBC are subsequently eliminated (F5). (G1–G5) High-resolution time-series that highlights bacterial growth within an IBC prior to (G1, G2) and after (G3–G5) administration of ampicillin. The bacterial volume within this IBC is not diminished by antibiotic treatment (G5). (H) Plots of logarithm of bacterial volume within n=11 IBCs before, during, and after the second round of ampicillin treatment. (I) Plots of logarithm of bacterial volume within n=9 IBCs before, during, and after the second round of fosfomycin treatment. (J) Plot of the volume of extracellular bacteria upon antibiotic administration from n=103 fields of view, each of which was 206 x 206 μm² in size, from n=3 bladder-chips. The bacterial volumes are normalized to the volume immediately prior to the antibiotic administration. (K) Scatter plot of the extracellular bacterial volume (n=105) and bacterial volume within IBCs (n=22) from n = 3 bladder-chips after ampicillin treatment and extracellular bacterial volume (n=101) and bacterial volume within IBCs (n=23) from n=1 bladder-chip after fosfomycin treatment as a fraction of the maximum bacterial volume prior to antibiotic treatment. Red line represents the median value, p=2.8E-4 (extracellular amp vs IBC amp), p=3.0E-5 (IBC amp vs. IBC fosfomycin) calculated using Kruskal-Wallis ANOVA Test, ‘ns’ represents p>0.05 (L, M) Representative images from the epithelial face of the infected bladder-chips with or without duty cycle, fixed at ca. 13.5 hr after UPEC infection and 6 hr into the first growth cycle. (N) Scatter plots of the logarithm of the total bacterial area across n=14 fields of view from infected bladder-chips with (n=2) and without (n=2) the application of the duty cycle, p = 8.6E-6. p-values calculated using a Mann-Whitney test. Yellow rectangular area in B, C, H and I represent the 1st and 2nd growth cycles. Gray rectangular area in B, C, H, I and J represent the rounds of ampicillin treatment. Scale bars, 10 μm in panels A1–A5, F1–F5, and G1–G5.

Figure 4—figure supplement 1. UPEC regrowth on-chip despite two consecutive periods of fosfomycin treatment.

(A, B) Examples of UPEC growth in infected bladder chips reconstituted with primary epithelial cells. Regrowth and IBC formation after two rounds of antibiotic treatment are observable in A5, B5. (C) Growth statistics for IBCs in chips reconstituted with 5637 cells after the two periods of fosfomycin treatment.

Figure 4—figure supplement 2. UPEC growth in IBCs during ampicillin treatment.

(A1–A5) Additional examples of UPEC growth during ampicillin treatment. (A1) Two IBCs (marked 1 and 2) at the start of ampicillin treatment. The IBC marked one exfoliates (A2), whereas the remaining IBC-2 continues to grow (A1–A3). Toward the end of this period, the bacteria filament (A4) before clearance due to the antibiotic is observed (A5). (B1–B5) High-resolution time-series that highlights bacterial growth within an IBC prior to (B1) and during (B2–B5) administration of ampicillin. The bacterial volume within this IBC is not diminished by antibiotic treatment (B5). Scale bars, 10 μm in all the panels.

Figure 4—figure supplement 3. UPEC infection leads to a higher bacterial burden in bladder-chips perturbed with duty cycle.

Additional examples of UPEC growth in infected bladder chips with (A) and without (B) duty cycle.

Figure 4—video 1. Bacteria within an IBC can persist and grow within an IBC despite the antibiotic treatment (Figure 4A1–A5).

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Proliferation of UPEC (green) within four epithelial cells (magenta, stained with Cell Mask Orange) during the 1^st growth cycle (578–1054 min, ca. 8 hr.) leads to the formation of IBCs. The bacteria within the four IBCs persist during the (~40x MIC) ampicillin treatment (1071–1258 min, ca. 3 hr.) In each case, bacterial clearance was observed during the ampicillin treatment, but all four IBCs persisted over the course of the treatment. Bacterial growth subsequently resumed within all four IBCs post ampicillin washout during the second growth cycle (1275 min onwards). Two new IBCs were formed during the second growth cycle (1275 min onwards). IBC fluxing and filamentation could be observed toward the end of time series.

Figure 4—video 2. Ampicillin-mediated bacterial clearance is delayed within an IBC (Figure 4F1–F5).

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Proliferation of UPEC (green) within an epithelial cell (magenta, stained with Cell Mask Orange) during the 1st growth cycle (578–1054 min, ca. 8 hr.) to form an IBC. The bacteria within the IBC persisted during the (~40x MIC) ampicillin treatment (1071–1258 min, ca. 3 hr.). Bacterial proliferation continued during the first two hours of the ampicillin treatment (1071–1190 min, ca. 2 hr.). Bacterial killing was subsequently observed later during the ampicillin treatment (1190–1258, ca. 1 hr.). Some bacteria within the IBC persisted throughout the ampicillin treatment and resumed proliferation during the second growth cycle (1275 min onwards).

Figure 4—video 3. Ampicillin-mediated bacterial clearance is delayed within an IBC (Figure 4—figure supplement 1A1-A5).

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Proliferation of UPEC (green) within two epithelial cells prior to the ampicillin treatment during the 1st growth cycle (578–1054 min, ca. 8 hr.). IBC#1 exfoliates from the epithelial layer (ca. 1105 min). Bacteria within IBC#2 continued to proliferate during the first 2 hr of the ampicillin treatment (1071–1190 min, ca. 2 hr.). Bacterial clearance was subsequently observed later during the ampicillin treatment (1190–1258, ca. 1 hr.). IBC#2 eventually exfoliated from the epithelial layer toward the end of the time series (1292 min).

Figure 4—video 4. Bacteria can continue growing within an IBC for the entire duration of ampicillin treatment (Figure 4G1–G5).

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Proliferation of UPEC (green) within an epithelial cell (magenta, stained with Cell Mask Orange) during the 1st growth cycle (578–1054 min, ca. 8 hr.) to form an IBC. Bacterial growth within the intact IBC continued both during the ampicillin treatment (~40x MIC, 1071–1258 min, ca. 3 hr.) as well as after the ampicillin was washed out during the 2nd growth cycle (1275 min onwards). Eventually, the IBCs shed bacteria (1700–1751 min, ca. 1 hr) towards the end of time series.

Figure 4—video 5. Bacteria can continue growing within an IBC during the ampicillin treatment (Figure 4—figure supplement 1B1-B5).

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Proliferation of UPEC (green) within an epithelial cell (magenta, stained with Cell Mask Orange) during the 1st growth cycle (578–1054 min, ca. 8 hr.) to form an IBC. Bacterial growth within the intact IBC continued both during the ampicillin treatment (~40x MIC, 1071–1258 min, ca. 3 hr) as well as after the ampicillin was washed out during the 2nd growth cycle (1275 min onwards). The late-stage IBC eventually shed bacteria and filamentous bacterial growth (1632–1717 min, ca. 1.5 hr) was also observed toward the end of time series.