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

Abstract

Therapeutic failures and relapses are critical challenges in Staphylococcus aureus bone and joint infections. These issues may stem, in part, from the incomplete eradication of S. aureus residing within osteoblasts, the bone-forming cells, despite recommended antibiotic treatment. However, the mechanisms underlying intraosteoblastic S. aureus survival remain poorly understood. Here, we used automated real-time fluorescence microscopy at the single-host-cell level to monitor the intracellular replication dynamics of clinical S. aureus strains and their survivors of rifampicin treatment in MG-63 osteoblast cell line. S. aureus replication dynamics was heterogeneous both within and across strains, while survival to rifampicin treatment was uniformly characterized by a non-replicative phenotype. Surprisingly, rifampicin killed less than 0.3 log of intraosteoblastic S. aureus, and only during the early phase of infection. The majority of S. aureus that survived rifampicin treatment remained non-replicative intracellularly after rifampicin withdrawal, yet they retained the capacity to regrow on agar following release from host cells. This high proportion of non-replicative antibiotic-tolerant S. aureus inside osteoblasts may contribute to the high rates of therapeutic failures in bone and joint infections.

Fig 1. Intracellular Staphylococcus aureus replication dynamics is heterogeneous over time.

(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.

Fig 2. Intracellular Staphylococcus aureus replication dynamics is heterogeneous among clinical isolates.

(A-F) MG63 cells (red) were seeded, labeled and infected as previously described in Fig. 1 with a range of S. aureus strains and clinical isolates expressing GFP (green) and pre-labeled by eFluor-450 (blue), (S1 Table). Time-lapse imaging was conducted over 24 hours with hourly acquisitions using automated confocal microscopy. (A) Quantification of infected cells based on intracellular S. aureus replication dynamics (RD) over an infection period of 24 hours. (Classification: profile mainly non-replicative (blue); intermediate replicative profile (yellow); mainly replicative profile (green)). (B) Quantification of the classical fast replicative phase intensities, that represent the increase in S. aureus population size, through the ratio of total green (GFP) pixel count from replication onset to host cell lysis. (C-F) Representative confocal images and corresponding quantification of green (GFP) and blue (eFluor-450) fluorescence intensities over time. Images show single osteoblastic cells infected by S. aureus experiencing a classical fast replicative phase either in an irregular shape (C, D: strain HG001), or constrained shape (D, E: strain HG001), (scale bar = 10 µm). (A, B) Results were presented as mean ± SD representing 9 individual values (A) from 3 independent experiments (A: N = 1631 infected cells, B).

Fig 3. Fast replicative phases are heterogeneous.

(A-F) MG63 cells (red) were seeded, labeled and infected as previously described in Fig 1 with a range of S. aureus strains and clinical isolates expressing GFP (green) and pre-labeled by eFluor-450 (blue), (S1 Table). Time-lapse imaging was conducted over 24 hours with hourly acquisitions using automated confocal microscopy. (A-D) Representative confocal images and corresponding quantification of green (GFP) and blue (eFluor-450) fluorescence intensities over time. Images show single osteoblastic cells infected by S. aureus experiencing either: an aborted fast replicative phase (A, B: strain BJI001), or an arrested fast replicative phase (C, D: BJI019) (scale bar = 10 µm). (E, F) Quantification of the proportion of aborted (E) and arrested (F) fast replicative phases relative to the total fast replicative events measured. (E, F) Results were presented as mean ± SD representing 9 individual values from 3 independent experiments.

Fig 4. Intracellular Staphylococcus aureus survivors to rifampicin are non-replicative, regardless of strain.

(A-E) MG63 cells (red) were seeded, labeled and infected as previously described in Fig 1 with a range of S. aureus strains and clinical isolates expressing GFP (green) and pre-labeled by eFluor-450 (blue), (S1 Table, BJI035: rifampicin resistant). 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. Time-lapse imaging was conducted over 24 hours with hourly acquisitions using automated confocal microscopy. (A) Quantification of infected cells based on intracellular S. aureus replication dynamics (RD) over an infection period of 24 hours. (B-E) Representative confocal images and corresponding quantification of green (GFP) and blue (eFluor-450) fluorescence intensities of control (B, C) and rifampicin-treated conditions (D, E) over time. Images show population of osteoblastic cells infected by S. aureus BJI076 experiencing either quiescent and replicative states (B) or only quiescence (D), (scale bar = 40 µm). (A) Results were presented as mean ± SD representing 9 individual values from 3 independent experiments (N = 1848 infected cells).

Fig 5. Rifampicin treatment rescue host cells from lysis and S. aureus survivors regrow on agar plate with a global reduce colony size.

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 Fig 4 (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.

Fig 6. Arrested fast replicative phases are induced by ciprofloxacin treatment.

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.

Fig 7. Model of the intracellular S. aureus replication dynamics and corresponding antibiotics impact.

After S. aureus internalization by osteoblastic cells, the entire intracellular bacterial population can remain quiescent, forming the exclusively non-replicative host cell subset. It is the most represented over time for all the tested strains. Within the host cells, at least one S. aureus can transition from quiescence to slow or fast replication. This transition occurs gradually over the time course of the infection. Host cells are classified into 3 groups depending on the fast replicative phase pattern: (i) “classical” leading to host cell lysis and the subsequent release of intracellular S. aureus; (ii) “aborted” where the S. aureus signal disappears without impacting the host cell phenotype; (iii) “arrested” where S. aureus experience a growth arrest inside phenotypically unaltered host cell. The rates of transitioning as well as the patterns and intensities of fast replicative phases are strain-dependent. Survival to rifampicin treatment exhibits a population-wide strain-independent predominant or exclusive non-replicative profile. In contrast, ciprofloxacin treatment induces arrested fast replicative phase in a concentration-dependent manner. The combination of both antibiotics does not show superiority compared to rifampicin alone.