Journal: PLOS Pathogens

Article Title: Monitoring intracellular replication dynamics unveils high proportion of non-replicating antibiotic-tolerant Staphylococcus aureus inside osteoblasts

doi: 10.1371/journal.ppat.1013525

Figure Lengend Snippet: (A) Experimental design for assessing S. aureus replication dynamics in MG63 osteoblastic cells. (B-K) MG63 cells, seeded at sparse density and labeled with CellTracker Red CMTPX (red), were infected at MOI 8 with S. aureus SH1000 expressing GFP (green) pre-labeled by eFluor-450 (blue). Following 2 hours of co-incubation, lysostaphin at 10 µg/mL was added to eliminate extracellular S. aureus . Time-lapse imaging was conducted over 24 hours with hourly acquisitions using automated confocal microscopy. (B-G) Representative confocal images and corresponding quantification of green (GFP) and blue (eFluor-450) fluorescence intensities over time. Images show single osteoblastic cells infected by: exclusively non-replicative S. aureus (B, C ), or at least one S. aureus transitioning from quiescence to slow replication (D, E) or fast replication leading to host cell lysis (F, G) (scale bar = 10 µm). (H) Hourly quantification of infected cells based on intracellular S. aureus replication dynamics (RD) across the 24-hour infection period. (I) Initiation and duration of the fast replicative phases. (J) Hourly quantification of infected cells experiencing either an ongoing or an ended fast replicative phase. (K) Global quantification of infected cells based on intracellular S. aureus replication dynamics over an infection period of 24 hours (ENR: Exclusively non-replicative; SR: slow replicative; FR: fast replicative). (H-K) Results were presented as mean ± SD (H, J, K) or median and quartiles (I) , representing 36 individual values (H, J, K) from 12 independent experiments ( H , K : N = 1005 infected cells, I , J : N = 474 infected cells). Mann-Whitney test: **** p < 0.0001.

Article Snippet: Infection was performed at MOI 8 (or 2 or 4 where indicated) using sparse CellTracker Red CMTPX-labeled cells in 24-well plates (4TITUDE, 4TI-0241) or 96-well plates (Greiner bio-one, 655090), as described above.

Techniques: Labeling, Infection, Expressing, Incubation, Imaging, Confocal Microscopy, Fluorescence, Lysis, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Monitoring intracellular replication dynamics unveils high proportion of non-replicating antibiotic-tolerant Staphylococcus aureus inside osteoblasts

doi: 10.1371/journal.ppat.1013525

Figure Lengend Snippet: MG63 cells labeled (B , D) or not ( A , C , E ) with CellTracker Red CMTPX (red) were seeded at sparse (B , D) or confluent (A , C , E) density and infected and treated as previously described in (BJI035: rifampicin resistant). (A) At 2 hpi propidium iodide (PI) was added at 2 µg/mL. At 24 hpi PI fluorescence intensity was measured with a plate reader. PI fluorescence intensity is normalized to uninfected cells in untreated and rifampicin-treated conditions. (B) Time-lapse imaging was conducted over 24 hours with hourly acquisitions using automated confocal microscopy. Quantification of the S. aureus SH1000 population size per cell over time represented by the total green (GFP) pixel count normalized per cell. (C, E) Intracellular S. aureus SH1000 were collected at 1 hpi and 24 hpi, and the total number of S. aureus forming colonies on agar plate was investigated (C) as well as their area distribution frequency at 24 hpi (E) and the small colony variants rates (F) . (D) At 6 hpi, rifampicin treatment was either withdrawn by washing or maintained, and incubation continued. Time-lapse imaging was conducted over 18 hours post-withdrawal with hourly acquisitions using automated confocal microscopy. Quantification of infected cells based on intracellular S. aureus replication dynamics (RD). Results were presented either as single value and mean ± SD from 3 independent experiments (B, E) or as mean ± SD representing 9 individual values from 3 independent experiments (A , D : N = 165 infected cells, F) or 12 individual values from 4 independent experiments (C) . One-way ANOVA with Dunnett’s correction for multiple post hoc comparisons with the control (A) and Mann-Whitney test (C , D, F) : *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Infection was performed at MOI 8 (or 2 or 4 where indicated) using sparse CellTracker Red CMTPX-labeled cells in 24-well plates (4TITUDE, 4TI-0241) or 96-well plates (Greiner bio-one, 655090), as described above.

Techniques: Labeling, Infection, Fluorescence, Imaging, Confocal Microscopy, Incubation, Control, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Monitoring intracellular replication dynamics unveils high proportion of non-replicating antibiotic-tolerant Staphylococcus aureus inside osteoblasts

doi: 10.1371/journal.ppat.1013525

Figure Lengend Snippet: MG63 cells labeled (A , C - G) or not (B) with CellTracker Red CMTPX (red) were seeded at sparse density and infected at MOI 8 with S. aureus SH1000 expressing GFP (green) pre-labeled by eFluor-450 (blue). Following 2 hours of co-incubation, lysostaphin at 10 µg/mL was added to eliminate extracellular S. aureus . Concomitantly, cells were treated with rifampicin at 6 µg/mL and/or ciprofloxacin at 2, 5, or 10 µg/mL or left untreated. Time-lapse imaging was conducted over 24 hours with hourly acquisitions using automated confocal microscopy (A , C - G) or intracellular S. aureus were collected at 1 hpi and 24 hpi, and total number of S. aureus forming colonies on agar plate was investigated (B) . (A , C) Quantification of infected cells based on intracellular S. aureus replication dynamics (RD) over an infection period of 24 hours. (B) Total number of intracellular S. aureus SH1000 forming colonies on plates. (D) Quantification of the S. aureus population size per cell over time represented by the total green (GFP) pixel count normalized per cell. Represent 1 experiment of 3 for easier readability. (E , F) Representative confocal images (E) and corresponding quantification of green (GFP) and blue (eFluor-450) fluorescence intensities over time (F) . Images show a single osteoblastic cell infected by S. aureus SH1000 experiencing an arrested fast replicative phase under ciprofloxacin treatment at 2 µg/mL (scale bar = 10 µm). (G) Quantification of the proportion of arrested fast replicative phases relative to the total fast replicative events measured. Results were presented either as mean ± SD representing 9 individual values from 3 independent experiments ( A : N = 1288, C : N = 745, G : N = 258 infected cells) or as 12 individual values from 4 independent experiments (B) . One-way ANOVA with Dunnett’s correction for multiple post hoc comparisons with the control (A , C , G) or Two-way ANOVA test with Sidak’s correction for multiple comparisons post hoc test ( B ; p < 0.05: treatment, p < 0.0001: time): * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Infection was performed at MOI 8 (or 2 or 4 where indicated) using sparse CellTracker Red CMTPX-labeled cells in 24-well plates (4TITUDE, 4TI-0241) or 96-well plates (Greiner bio-one, 655090), as described above.

Techniques: Labeling, Infection, Expressing, Incubation, Imaging, Confocal Microscopy, Fluorescence, Control