Antibiotic therapy often fails to eliminate a fraction of transiently refractory bacteria, causing relapses and chronic infections. Multiple mechanisms can induce such persisters with high antimicrobial tolerance in vitro, but their in vivo relevance remains unclear. Using a fluorescent growth rate reporter, we detected extensive phenotypic variation of Salmonella in host tissues. This included slow-growing subsets as well as well-nourished fast-growing subsets driving disease progression. Monitoring of Salmonella growth and survival during chemotherapy revealed that antibiotic killing correlated with single-cell division rates. Nondividing Salmonella survived best but were rare, limiting their impact. Instead, most survivors originated from abundant moderately growing, partially tolerant Salmonella. These data demonstrate that host tissues diversify pathogen physiology, with major consequences for disease progression and control.

To determine pathogen growth variation in vivo, we devised a single-cell growth reporter and applied it to a mouse typhoid fever model. The results revealed distinct Salmonella subsets with divergent division rates in spleen and other tissues. Analysis of ex vivo purified subsets suggested that differential host nutrient supply contributed to this heterogeneity. To assess antimicrobial tolerance of the various subsets, we monitored Salmonella division and killing during fluoroquinolone therapy under clinically relevant conditions. Slow-growing Salmonella survived best after each dose but, surprisingly, overall eradication was delayed primarily by abundant subsets of moderately growing Salmonella with partial tolerance. These results provide a new paradigm for pathogen variation in host tissues and its impact on antimicrobial chemotherapy.

Only a few studies have analyzed mechanisms that cause pathogen antimicrobial tolerance in infected host tissues. Subsets of Mycobacterium marinum in infected fish larvae express high levels of drug efflux pumps conferring antimicrobial tolerance (), whereas minor nondividing Salmonella subsets can persist in infected mice during early-onset/high-dose antimicrobial chemotherapy (). The extent of pathogen phenotypic variation and its impact on treatment efficacy under clinically relevant conditions is still unclear ().

Treatment of infections with appropriate antibiotics rapidly reduces bacterial burden, but often fails to eliminate a fraction of refractory cells that can cause relapses and chronic infections (). Recalcitrant cells are phenotypic variants that transiently tolerate extraordinary levels of antibiotics but remain genetically drug sensitive. Such so-called persisters can be induced in vitro by diverse mechanisms (). Persisters may originate from rare stochastic nondividing subpopulations (), but high tolerance can also occur in actively growing pathogen subsets ().

This orange Salmonella subset survived ciprofloxacin treatment best ( Figure 7 F) and, because of its abundance before treatment, also dominated among survivors ( Figure 7 F, inset). One day later, most Salmonella had not yet resumed growth, based on the low proportion of green Salmonella subsets ( Figures 7 G and 7H). This might be the consequence of growth arrest due to continuously high ciprofloxacin levels (). Together, these data support the proposed major role of nondividing Salmonella in this streptomycin-pretreatment model ().

Before ciprofloxacin treatment, a surprisingly large proportion of TIMER-Salmonella had low green/orange ratios, indicative of poor growth in mesenteric lymph nodes (but not in spleen; Figure S2 ). This subset might reflect initial growth arrest of hundreds of Salmonella that continuously arrive from the densely colonized gut lumen (). New arrivers might require extensive adaptations to tissue microenvironments () before starting replication.

In another model (), mice are pretreated with streptomycin to partially deplete endogenous gut microbiota and then orally infected with Salmonella. From day 1 p.i., mice are treated twice daily with 62 mg/kg ciprofloxacin. This therapy eradicates Salmonella from most organs, but some Salmonella with low proliferation rates persist in mesenteric lymph nodes over many days of treatment. In this case, we successfully recovered viable colony-forming Salmonella from mesenteric lymph nodes before and after ciprofloxacin treatment, as expected.

For comparison, we investigated two other Salmonella infection/treatment models. In one model (), mice are orally infected with 2 × 10CFUs Salmonella and continuously treated with 2 g lenrofloxacin in drinking water from day 1 p.i. (mice drink 1–1.5 ml/10 g body weight per day). After 5 days of treatment, a few fluorescent Salmonella persist in mesenteric lymph nodes (). We detected fluorescent Salmonella in this model, but our attempts to demonstrate their viability/colony-forming capability were unsuccessful in three independent experiments, even when using preenrichment in liquid lysogeny broth medium and prolonged plate incubation. Poor recovery of live Salmonella under similar treatment conditions has previously been reported ().

After each enrofloxacin dose, initially fast-growing Salmonella survived poorly, whereas nondividing/slow-growing Salmonella subsets (<0.04 hr; less than 1 division per day) survived best ( Figure 7 D). However, the largest number of survivors originated from an abundant Salmonella subset with moderate growth rates (1–3.4 divisions per day) and partial tolerance ( Figures 7 D and 7E). Survivors again generated a dominant subset of moderately growing Salmonella at the time of the next enrofloxacin dose 1 day later ( Figure 7 E). Repeating cycles of extensive killing/partial tolerance and resumption of growth resulted in only slow Salmonella clearance and dominance of moderately growing Salmonella subsets throughout therapy.

In spleen, each enrofloxacin dose killed 88%–92% of all Salmonella, whereas TIMERcontent and color remained again stable for at least 1 hr ( Figure S6 B). One day after the first enrofloxacin dose, many Salmonella had moderate-to-high division rates, suggesting that most survivors had resumed growth ( Figure 7 B), consistent with enrofloxacin tissue concentrations dropping below the minimal inhibitory concentration between doses ( http://www.animalhealth.bayer.com/5176.0.html ). In addition, a new subset emerged with high orange fluorescence, low green/orange ratios ( Figure 7 C, red box), and low colony-forming capability (87% ± 3% dead), which might represent Salmonella with blocked division but residual expression of accumulating TIMER

TIMERcolors in spleen, mesenteric lymph nodes, and Peyer’s patches at onset of treatment resembled data for spleen of i.v. infected mice ( Figure S2 ). This was consistent with similar net growth (about 1 log per day) in spleen regardless of oral or i.v. inoculation (). Data were more surprising for Peyer’s patches and mesenteric lymph nodes, in which net growth stops a few days postoral infection ( Figure S7 ; data were derived from). In these organs, active growth (as suggested by high green/orange ratios) might be counterbalanced by equally fast killing/dissemination to other organs. Differential oxygen availability might also affect TIMERcolor in these organs. More work is required to clarify this issue.

Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two-colour flow cytometry.

Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two-colour flow cytometry.

Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two-colour flow cytometry.

To investigate antimicrobial efficacy under more clinically relevant conditions, we infected mice via the natural oral route and waited until disease signs appeared before starting treatment according to the recommended schedule (daily intraperitoneal [i.p.] doses of 0.1 mg enrofloxacin equivalent to about 5 mg/kg body weight; http://www.animalhealth.bayer.com/5176.0.html ). CFU counts in spleen, mesenteric lymph nodes, and Peyer’s patches declined during treatment and disease signs improved ( Figure 7 A) but, even after 5 days of treatment, some mice still harbored residual Salmonella and/or showed weak disease signs, reproducing effective but slow fluoroquinolone therapy of severe human cases of invasive salmonellosis ().

(G) Fluorescence of Salmonella in mesenteric lymph nodes 1 hr or 1 day after an oral dose of 1.3 mg ciprofloxacin.

(F) Survival of Salmonella with various TIMER bac colors 1 hr after an oral dose of 1.3 mg ciprofloxacin in mesenteric lymph nodes in the streptomycin-pretreatment model (averages ± SD for three mice from two independent experiments; one-way repeated-measures ANOVA with posttest for linear trend). The inset shows the proportions of Salmonella with log(green/orange) <−0.055 among survivors in our typhoid fever/enrofloxacin model (En) and the streptomycin/ciprofloxacin model (CIP).

(E) Proportions of live slow (s; <0.04 hr −1 ), moderate (m), and fast (f; >0.14 hr −1 ) Salmonella cells before or 1 hr after (“survivors”) daily enrofloxacin doses (averages ± SD of two to five mice per time point). At doses 4 and 5, low Salmonella loads prevented sorting-based analysis of survivor distributions.

(D) Salmonella survival (CFUs per sorted TIMER bac -Salmonella cell) in subsets with different division rates from mice treated with one to three doses of enrofloxacin (averages ± SD for two to five mice from two independent experiments; one-way repeated-measures ANOVA with posttest for linear trend).

(C) TIMER fluorescence 1 hr or 1 day after the first enrofloxacin dose. The red box indicates an antibiotic-induced new subset. Similar observations were made for 14 mice in five independent experiments.

(B) Division rate distributions at various time points during multidose therapy. Pooled data for two to five mice per time point are shown.

(A) Salmonella tissue loads during therapy with daily doses of 0.1 mg enrofloxacin. Spleen colonization levels before start of therapy (b; gray) were estimated based on flow cytometry counts. Each symbol represents one mouse. To avoid delays in TIMER flow cytometry, we prepared only the most distal small intestinal Peyer’s patch (Last PP) (mLN, mesenteric lymph nodes). Average disease scores ± SEM of between 20 (dose 1) and 3 (dose 5) mice from three independent experiments are also shown.

In i.v. infected mice, treatment with 0.2 mg enrofloxacin reduced spleen CFUs by 93% ± 3%, but fluorescent counts and TIMERcolor remained stable for at least 1 hr ( Figure S6 B), as in vitro. Slowly responding TIMERthus still reports on pretreatment growth rates of both live and dead Salmonella, without confounding effects by later antibiotic-induced growth alterations. This enabled us to investigate the impact of Salmonella growth heterogeneity at the onset of treatment on antimicrobial efficacy. Specifically, we sorted Salmonella subsets with different colors at 1 hr after treatment and determined their viability by comparing plating and flow cytometry counts (CFUs per sorted fluorescent Salmonella). There was a strict correlation between killing and color/pretreatment growth rate ( Figures S6 C–S6E), consistent with in vitro data (). Fast-growing subsets were extensively killed, whereas slow-growing/nondividing Salmonella tolerated treatment better, resulting in their overrepresentation among survivors. Nevertheless, most survivors originated from abundant Salmonella with moderate pretreatment growth rates and intermediate antimicrobial tolerance.

The rate of killing of Escherichia coli by β-lactam antibiotics is strictly proportional to the rate of bacterial growth.

Treatment of TIMER-Salmonella chemostat cultures with 5 mg lenrofloxacin caused some 90% drop in CFUs within 1 hr but fluorescent cell counts remained stable ( Figure S6 A), indicating that dead Salmonella initially retained fluorescent TIMER. This was consistent with fluoroquinolone-mediated killing by double-strand DNA breaks () without immediate lysis. Green/orange ratios of (mostly dead) Salmonella did not change during 1 hr treatment ( Figure S6 A, inset), consistent with slow TIMERresponse times ( Figure 1 G).

(E) Proportions of Salmonella subsets among survivors 1 hr after an i.p. dose of 0.2 mg enrofloxacin (averages ± SD of three mice from three independent experiments).

(D) Salmonella survival (CFU per sorted TIMER bac -Salmonella cell) in subsets with different division rates in spleen of i.v. infected mice at day 4, 1 hr after an i.p. dose of 0.2 mg enrofloxacin. Each line represents data for one mouse from three independent experiments. Salmonella survival correlated with division rate (one-way repeated-measures ANOVA with posttest for linear trend).

(C) Proportions of Salmonella subsets with different division rate at day 4 after i.v. infection (averages ± SD of three mice from three independent experiments).

(B) Salmonella CFU and fluorescent particle counts 1 hr after an i.p. dose of 0.2 mg enrofloxacin in spleen of i.v. infected mice (open red circles), an i.p. dose of 0.1 mg enrofloxacin in spleen of orally infected mice with clear disease signs during multi-dose therapy (filled red circles), or vehicle in i.v. infected mice (open black circles). Similar to in vitro conditions, Salmonella loose colony-forming capability but retain fluorescence. The inset shows median green/orange ratios in orally infected mice without treatment (black) or 1 hr after the first 0.1 mg enrofloxacin dose. Each symbol represents an individual mouse.

(A) Colony-forming units and fluorescent particle counts in chemostat cultures growing at 0.13 h -1 , 1 hr after addition of 5 mg l -1 (final concentration) enrofloxacin or vehicle. Most treated Salmonella are killed but remain detectable as fluorescent particles with unaltered green/orange ratios (inset) at least for 1 hr after treatment. Each symbol represents one reactor from one experiment.

To investigate the impact of antimicrobial chemotherapy on differentially growing Salmonella, we used the fluoroquinolone enrofloxacin that is effective in the mouse typhoid fever model (). Fluoroquinolones are the antibiotic class of choice for human typhoid fever ().

Green Salmonella contained slightly less enzymes for purine nucleoside biosynthesis but more enzymes for purine nucleoside degradation ( Figure 6 D), suggesting a diverging purine supply. Indeed, Salmonella purH, which depends on purine supplementation, had a striking bimodal TIMER color distribution ( Figures 6 B and 6C), with both growth-arrested and normally growing subsets. Fluorescence dilution data confirmed increased growth rate diversity in Salmonella purH ( Figure S5 B). This increased cell-to-cell variation in purH single-cell growth rates, and the high proportion of nongrowing cells, differs from previous assumptions that mutant attenuation at the population level reflects more-or-less uniformly attenuated growth at the single-cell level. Instead, Salmonella purH appeared to partially reside in microenvironments with insufficient purine availability, resulting in growth arrest, whereas other subsets maintained normal growth, suggesting sufficient host purine supply.

Green Salmonella contained more transporters and enzymes for utilization of sialic acid and galactose ( Figure 6 D) but Salmonella galP mglB and Salmonella nanT had indistinguishable color distributions ( Figure 6 C), consistent with full virulence of these mutants (). Green subsets might be exposed to levels above the catabolism induction threshold for these carbohydrates, but other nutrients are apparently more relevant for growth.

Histidine biosynthesis enzymes were more abundant in orange Salmonella, suggesting a potentially limiting host histidine supply. Histidine biosynthesis is inactive in strain SL1344 due to a dysfunctional hisGallele (), but restoring functional hisGhad no significant effect on spleen colonization (spleen CFU increase in 4 days: SL1344, 2.8 ± 0.2 log; SL1344 hisG, 3.1 ± 0.3 log; p = 0.55) or green/orange ratios ( Figure 6 C). These data indicate sufficient histidine supply for all subsets of auxotrophic SL1344, although orange cells might encounter lower levels that induce biosynthesis enzymes.

Members of oxygen-dependent FNR () and ArcA () regulons were similarly abundant (p = 0.42 and p = 0.49, respectively), suggesting similar oxygen tension around green and orange Salmonella. This is important, because inhomogeneous oxygenation could confound TIMER as a growth rate reporter (see Discussion ). Many other metabolic enzymes were differentially abundant ( Figure 6 A; Figure S5 A; Table S1 ; an interactive map with descriptions of all associated enzymatic reactions is available at http://www.biozentrum.unibas.ch/personal/bumann/Claudi_et_al/TIMER.html ). A small number of pathways showed consistent differences for several enzymes ( Figure 6 D).

(B) Analysis of in vivo division numbers of SL1344 and its purine-auxotrophic derivate purH using the Fluorescence Dilution technique. The gray areas show GFP fluorescence of arabinose-induced S1344/pFcGi and S1344 purH/pFcGi cultures used as inocula. The black lines show Salmonella GFP levels in spleen at day 2 postinfection. In both strains, most Salmonella diluted their GFP contents more than 20-fold (equivalent to > 4 divisions) during this infection time interval. However, a distinct subset in purH (but not SL1344) retained high GFP levels indicative of slow growth. The proportion of this subset is small compared to nongrowing subsets observed for purH using the TIMERapproach ( Figure 6 B). This could reflect that in the Fluorescence Dilution approach only Salmonella with minimal growth throughout the entire infection period retain distinct high GFP levels. Such arrested Salmonella would be rapidly overgrown by normally proliferating Salmonella. Salmonella that initially divided a few times and diluted their GFP, but then entered microenvironments with poor purine supply and slowed down, might be indistinguishable from the majority of continuously growing Salmonella based on Fluorescence Dilution, whereas they would acquire detectably distinct orange color ratios in the TIMERapproach.

(A) Abundance ratios for metabolic enzymes based on proteome comparisons of green and orange TIMER-Salmonella subsets (see Table S1 for the full data set). Symbols represent metabolites (squares, carbohydrates; triangles, amino acids; circles, other metabolites; filled symbols, phosphorylated metabolites) and proteins (diamonds). The connecting lines present enzymes catalyzing the corresponding conversions. The brown lines represent the inner and outer membranes. An interactive version of this map with detailed descriptions for all reactions is available at http://www.biozentrum.unibas.ch/personal/bumann/Claudi_et_al/TIMER.html

Green cells contained more IgaA (internal growth attenuator A), which inhibits detrimental activation of the RcsD/RcsC/RcsB signaling system () ( Figure 6 A). Orange Salmonella might contain inadequate IgaA amounts, resulting in elevated RcsC activity and slow growth. However, Salmonella rcsC had slightly orange-shifted color distributions ( Figure 6 B), consistent with minor attenuation of such mutants (). This is the opposite of what one would expect if insufficient IgaA-RcsC interactions impaired growth of orange Salmonella.

Repression of the RcsC-YojN-RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence.

Repression of the RcsC-YojN-RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence.

Orange cells contained more entericidin B, the toxin component of the ecnAB toxin/antitoxin (TA) module () ( Figure 6 A). Toxins can arrest bacterial growth, in particular when antitoxin degradation is stimulated by elevated (p)ppGpp levels (), but Salmonella ecnB had wild-type green/orange ratios ( Figure 6 B), arguing against a major impact of entericidin B. Salmonella ecnB shpAB phD-doc (TA Δ3) defective for two additional TA modules influencing Salmonella growth and persistence in macrophage cell cultures () had unaffected TIMERcolors ( Figures 6 B and 6C) and almost normal virulence (competitive index versus wild-type at day 5 postoral infection: 0.8 ± 0.4, p = 0.03). We did not detect other Salmonella toxins () ( Table S1 ). Efflux pumps AcrAB and MacAB had similar abundance in both subsets (ratios 0.96–1.19), whereas other efflux systems including IceT (the ortholog of a mycobacterial system involved in antimicrobial tolerance;) were not detected.

Proteome analysis of sorted subsets ( Figure 6 A; Table S1 ) revealed higher levels of ribosomal proteins in green Salmonella, consistent with faster growth (), but more starvation sigma factor (σ)-dependent proteins in orange Salmonella, implying slower growth/carbon starvation/ATP shortage (). The transcription factor CRP appeared to be more active in the orange subset (p = 0.011), suggesting carbon limitation (). Enzymes RelA, SpoT, and GppA involved in (p)ppGpp metabolism, and (p)ppGpp-activated proteins (), were also more abundant in the orange Salmonella subset (p = 0.0036), suggesting elevated (p)ppGpp levels in response to amino acid starvation, other nutrient limitations, and/or heat and oxidative stress (). Nutrient limitation regulating Salmonella growth would be consistent with our previous observations ().

(D) Abundance ratios of enzymes involved in biosynthesis of histidine (His) or purines (Pur), or degradation of Pur, sialic acid (Sial), or galactose (Gal) ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; two-tailed t test on log-transformed data).

(B) Color distributions of parental strain SL1344 and various mutants in spleen. Similar data were obtained for another rcsC, another TA Δ3, two ssrB, four purH, and four SL1344 replicates.

(A) Proteome comparison of sorted green and orange TIMER-Salmonella subsets (see Table S1 for the full data set). Ribosomal proteins were more abundant in green subpopulations (p < 0.0001; two-tailed Wilcoxon signed-rank test), whereas σ-dependent proteins were more abundant in orange subpopulations (p < 0.0001). Many metabolic enzymes had differential abundance ratios.

The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression.

Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium.

Calculated TIMER-based division rates for sorted fractions a–d correlated with respective plasmid dilution data ( Figure 5 B, inset). The lower TIMER-based values for slow-to-medium fractions a–c suggest that some Salmonella initially proliferated with substantial plasmid dilution but subsequently slowed down, resulting in low green/orange TIMER ratios. Indeed, Salmonella with division rates above the median were predicted to generate some 90% of the daily new Salmonella daughter cells ( Figure 5 B), but many of these new, initially green daughter cells likely slowed down and turned more orange, because overall color distributions remained stable at increasing loads over several days of infection ( Figure S2 ).

Similar color and division rate distributions were obtained for low-level TIMERexpression from pSC101_TIMER ( Figure S4 ; FHWM, 0.05–0.20 hr), confirming that the approach is robust against differences in TIMER protein quantities. TIMER-Salmonella in genetically resistant 129/Sv mice had an even broader distribution (FHWM, 0.01–0.13 hr) and lower division rates (median rate, 0.063 ± 004 hrversus 0.133 ± 0.001 hrfor BALB/c mice, p < 0.0001; Figure S4 B). This was consistent with slower Salmonella growth as the primary cause of dramatically lower tissue loads ( Figure S4 B, inset) in resistant compared to genetically susceptible mice ().

(B) Calculated division rate distributions based on data in (A) (averages ± SEMs for four mice from one experiment). The inset shows Salmonella loads in spleen of individual mice.

(A) Color distributions for SL1344/pSC101_TIMER in spleen of 129/Sv and BALB/c mice 4 days after i.v. infection with 700 CFU. For comparison, data for SL1344/pBR322_TIMER in BALB/c mice at the same time point are also shown (shaded area). Data were pooled for four mice from one experiment in each group. Median log(green/orange) ratios for SL1344/pSC101_TIMER in individual mice are shown in the inset.

The primary effect of the Ity locus is on the rate of growth of Salmonella typhimurium that are relatively protected from killing.

We calculated division rate distributions based on in vivo TIMER colors and calibration data from chemostats at 5% O Figure 1 F) (that yielded reasonable average growth rate estimates; see above). The results suggested extensive Salmonella division rate heterogeneity with a full width at half maximum (FWHM) spanning a 5-fold range (0.04–0.19 hr Figure 5 B, blue line), compared to commonly studied in vitro conditions (our chemostat cultures, FWHM 1.3- to 1.5-fold range; microfluidic devices, FWHM <2-fold ranges for Escherichia coli and Mycobacterium smegmatis;).

The broad color distribution could indicate divergent Salmonella growth rates. We tested this hypothesis with the thermosensitive plasmid pVE6007 conferring resistance to chloramphenicol (CAM) (). pVE6007 cannot replicate at 37°C and is thus transmitted in vivo to only one of the two daughter cells at each division, resulting in progressive dilution of plasmid-harboring CAMcells. Comparison of the TIMER-Salmonella/pVE6007 inoculum and spleen homogenates at 48 p.i. revealed a pVE6007 dilution factor (input frequency/output frequency of CAMclones) of 145 ± 17, equivalent to log(145) = 7.2 ± 0.2 Salmonella divisions within 48 hr. This is equivalent to an average growth rate of 0.15 ± 0.005 hr, consistent with our previous data (0.16 ± 0.03 hr). Importantly, TIMER-Salmonella subsets sorted according to their green/orange ratios contained diverging proportions of CAMclones, indicative of differential plasmid dilution and division numbers ( Figure 5 A), consistent with TIMERcolor reporting on growth rate. These results demonstrate that (1) Salmonella subsets with distinct growth rates coexist in infected spleen, and (2) TIMERcan be used to detect and purify these subsets.

(C) Estimated daughter cell generation per day for Salmonella cells growing at various division rates (averages and SDs of four independently infected mice; based on data shown in B). Median and mean division rates are also shown.

(B) In vivo division rate distribution calculated from in vivo colors and in vitro color-division rate relations at 5% O 2 (averages ± SD of four independently infected mice). The blue line represents the FWHM, an expression of the extent of a distribution. Division rate estimates were compared to data from plasmid dilution for various Salmonella subsets (inset; averages ± SD of four mice).

(A) In vivo dilution of the nonreplicating plasmid pVE6007 (CAM R ) in Salmonella subsets. Mice infected with TIMER bac -Salmonella/pVE6007 had a typical color distribution at 48 hr p.i. (upper panel; pooled data from four mice infected in two independent experiments). Salmonella subsets with different green/orange ratios were sorted and analyzed for the fraction of pVE6007-bearing CAM R clones (lower left panel; each data point represents a specific subset in one individual mouse; Exp, experiment). From these data, the number of divisions was calculated (lower right panel). Significance was tested with one-way repeated-measures ANOVA with posttest for linear trend.

TIMER-Salmonella had broad color distributions in infected spleen ( Figure 3 B, left) compared to chemostat cultures ( Figure 1 E). Ex vivo sorting and reanalysis confirmed the presence of Salmonella subsets with genuine color differences ( Figure 4 A). Reinjection of sorted subsets into naive mice yielded superimposable color distributions in spleen 4 days later ( Figure 4 A), suggesting phenotypic rather than heritable variation. TIMER-Salmonella resided in spleen red pulp, as expected (), and showed divergent colors in confocal microscopy, consistent with flow cytometry data. Salmonella within the same infected host cell had similar colors whereas neighboring infected cells often contained Salmonella with different colors ( Figures 4 B and S3 A), suggesting a potential impact of individual host cells rather than regional factors. Salmonella colors did not correlate with Salmonella load ( Figure S3 B) or host cell type ( Figure S3 C). Further work might explore additional, potentially relevant, host cell properties.

(A) Analysis of Salmonella colors in single infected host phagocytes. Green/orange ratios of individual Salmonella are compared with averages colors of all Salmonella in the same cell. Most Salmonella had individual colors close to the average for the respective phagocyte.

(B) Confocal micrograph of an infected spleen cryosection. The insets show regions of interest with an overlay of F4/80 antibody staining (gray) recognizing resident red pulp macrophages. Additional micrographs show infected CD11b hi infiltrating cells (blue). The scale bars represent 5 μm. Similar observations were made in multiple sections from five independently infected mice.

(A) Flow cytometry sorting, reanalysis, and reinfection of TIMER bac -Salmonella subpopulations with different colors. Mice infected with sorted orange or green subsets were analyzed at day 4 p.i. (lower panel). This experiment was done once.

Comparison of in vivo TIMERfluorescence ( Figure 3 B, left) with chemostat cultures under in vivo-like conditions (5% oxygen, acidic pH, limiting nutrients) ( Figure 1 F) suggested an average in vivo division rate of 0.14 ± 0.02 hr, in excellent agreement with plasmid dilution data (0.15 ± 0.005 hr; see below) and generation times of 6–8 hr in mouse spleen (). In contrast, the SPI-2 mutant Salmonella ssrB poorly colonized spleen and had low green/orange ratios at day 4 p.i. ( Figure 3 B, right), consistent with marginal growth of SPI-2 mutants from day 2 p.i. (). Other Salmonella mutants had intermediate TIMERcolors ( Figure 3 C). Color-based growth rate estimates correlated with data from competitive infections () ( Figure 3 D). This is remarkable, because competitive infections reflect Salmonella growth and killing over several days of infection, whereas TIMERreports on current growth and provides no information on killed nonfluorescent Salmonella ().

During initial adaptation to host conditions, Salmonella show substantial nondividing subsets () before reaching optimal growth rates at 24–48 hr postinfection (p.i.) (). Consistent with these observations, TIMER-Salmonella subsets with low green/orange ratios were present at day 1 p.i. but declined to stable low levels thereafter ( Figure 3 B, left; Figure S2 ). Color distributions of adapted Salmonella resembled macrophage cell cultures infected with exponentially grown Salmonella ( Figures 2 B and S2 A).

(A) SL1344/pBR322_TIMER colors 1 or 4 days after i.v. infection (top panel), in various organs after appearance of disease signs in orally infected mice (middle panel), and in mesenteric lymph nodes (mLN) and spleen of streptomycin-pretreated mice at day 3 p.i. (bottom panel). The data represent pooled data for 3 to 4 mice from 2 to 3 experiments. The proportion of orange Salmonella with log(green/orange) < −0.055 (dashed lines) in individual mice is shown in (B).

To evaluate TIMERin vivo, we infected genetically susceptible BALB/c mice, a model for human typhoid fever (). TIMER-Salmonella retained full virulence (spleen colony-forming units [CFUs] 4 days after intravenous [i.v.] infection with some 1,000 CFUs: SL1344, [1.3 ± 0.3] × 10; Salmonella/pBR322_TIMER, [1.6 ± 0.2] × 10; Salmonella/pSC101_TIMER, [1.2 ± 0.4] × 10). Salmonella stably maintained functional episomal TIMERexpression cassettes (all colonies recovered on nonselective medium were orange). Flow cytometry of detergent-treated spleen homogenates revealed released green/orange Salmonella with baseline separation from autofluorescent host debris for episomal, but not single-copy, chromosomal expression cassettes ( Figures 3 A and Figure S1 ). We used pBR322_TIMER, yielding bright fluorescence in most subsequent experiments.

(C) Comparison of green fluorescence intensities for various strains. The single-copy chromosomal construct SL1344 virKp::timer bac emitted detectable fluorescence under in vitro inducing conditions, but the intensities were too low for in vivo detection.

(B) Gating strategy for Salmonella with moderate TIMER content (pSC101 replicon). Due to considerably overlap in the green channels, we use in addition the spectral properties of orange TIMER molecules to further separate TIMER signals from host autofluorescence.

(A) Gating strategy for Salmonella with high TIMER content (pBR322 replicon). A tissue homogenate from mice infected with Salmonella SL1344 not expressing any fluorescent protein (right panel) confirms absence of any host background particle with similar fluorescence thus enabling to detect even few Salmonella in entire organ homogenates (Ex, excitation; Em, emission).

All TIMER-expressing Salmonella contain green emitting molecules that are spectrally distinct from host tissue autofluorescence. We exploit this fact for gating.

(D) Comparison of TIMER-based growth rate estimates with data from competitive infections (CIs, competitive indices). Data represent averages ± SD for one to five TIMER replicates and three to six CI values from individual mice (r, Spearman’s rank-order correlation coefficient).

(C) Fluorescence colors of various attenuated TIMER-Salmonella strains in infected spleen at day 4 postinfection. Median green/orange ratios for replicates from individual mice are shown in Figure 6 C.

(B) Fluorescence of wild-type (WT) TIMER-Salmonella (left panel) and TIMER-Salmonella ssrB deficient for expression of the SPI-2-associated type III secretion system. Green/orange fluorescence ratios for Salmonella in vitro cultures at 5% Oand various division rates ( Figure 1 E) are shown in blue for comparison. Median green/orange ratios for replicates from individual mice are shown in Figure 6 C.

(A) Flow cytometry of an infected mouse spleen homogenate (for gating, see Figure S1 ). Similar observations were made for more than 25 infected mice.

In cell-culture macrophage infections, Salmonella have diverse intracellular growth rates, including a large nondividing subset () ( Figure 2 A). Consistent with this finding, intracellular TIMER-Salmonella showed varying green/orange ratios including a prominent subset with low green/orange ratios, indicative of poor replication ( Figure 2 B, Stat. phase). Live-cell imaging confirmed green fluorescence of growing Salmonella and orange fluorescence of nondividing Salmonella ( Movies S1 and S2 available online). Interestingly, growth heterogeneity depended on the inoculum. Large nondividing subsets occurred after infection with Salmonella from stationary cultures (as used in previous studies), possibly reflecting the extensive cell-to-cell variation in stress sensitivity in such cultures (). In contrast, exponentially grown Salmonella showed more homogeneous intracellular growth, with few nondividing bacteria both in the fluorescence dilution () and TIMERapproaches ( Figures 2 A and 2B; Movies S3 and S4 ). The close agreement with fluorescence dilution and video microscopy confirms the utility of TIMERas a reporter for intracellular Salmonella growth.

(B) Fluorescence colors of TIMER bac -Salmonella 16 hr after infection of Maf-DKO macrophages with stationary (red) or exponentially grown (blue) in vitro cultures. Similar results were obtained for TIMER bac -SL1344 orgA as well as bone-marrow-derived macrophages in nine independent experiments.

(A) Fluorescence dilution analysis of GFP-loaded Salmonella 30 min (shaded areas) or 16 hr (red/blue lines) after infection of bone-marrow-derived macrophages with stationary (red; left panel) or exponentially grown (blue; right panel) in vitro cultures (under conditions suppressing SPI-1-mediated macrophage pyroptosis). Growth results in progressive GFP dilution and diminishing fluorescence intensities. Similar observations were made for the SPI-1 mutant SL1344 orgA and Maf-DKO macrophages in a total of eight independent experiments.

During continuous growth in chemostats, TIMER-Salmonella had green/orange fluorescence ratios that correlated with division rates and were robust against cell-to-cell variations in protein content and cell size ( Figure 1 E). Green/orange ratios depended on oxygen tension ( Figure 1 F), as expected based on maturation kinetics ( Figure 1 D). After switching cultures from one division rate to another, color changed with response times of several hours ( Figure 1 G), consistent with slow TIMERmaturation and dilution as decisive processes determining color.

To test this hypothesis, we exchanged serine 197 for threonine in a DsRed variant with high yields in bacteria () and expressed the resulting TIMERin Salmonella enterica serovar Typhimurium SL1344. Salmonella with constitutive TIMERexpression grew as orange colonies ( Figure 1 B). Freshly induced TIMERformed green fluorophores (emission peak at 503 nm), followed by orange fluorophores (peak at 587 nm) ( Figure 1 C). Orange TIMERmolecules had a bimodal excitation spectrum with peaks at 483 and 561 nm ( Figure 1 C), consistent with FRET within mixed green/orange TIMER tetramers. Maturation kinetics depended on oxygen partial pressure ( Figure 1 D), as expected (). This oxygen dependency represents an important caveat for using TIMER as a growth rate reporter in environments with inhomogeneous oxygenation (see Discussion ).

DsRed variants are very stable against proteolysis (). In nonproliferating cells, both fast green and slowly maturing orange TIMER molecules should thus accumulate over time, yielding green/orange fluorescence ( Figure 1 A). In contrast, growing cells dilute both forms with each cell division. Fast-maturing green TIMER molecules should emerge earlier, at a more concentrated stage, compared to slowly maturing orange TIMER molecules, resulting in a dominant green fluorescence ( Figure 1 A). TIMER color might thus serve as a growth rate reporter.

(G) TIMER bac -Salmonella fluorescence dynamics after switching division rates in chemostats (averages ± SD for two or three reactors from one experiment).

(F) TIMER bac -Salmonella fluorescence colors at different division rates and oxygen concentrations. Combined data from four experiments are shown (averages ± SD of two to four reactors for each data point).

(E) Flow cytometry of Salmonella with constitutive expression of TIMER bac at defined division rates in chemostats maintained at 5% O 2 (n.g., nongrowing culture). Similar data were obtained in three independent experiments.

(D) Fluorescence maturation kinetics in TIMER bac -Salmonella at two different oxygen concentrations calculated from spectral data such as shown in (C).

(C) Fluorescence spectra of TIMER bac -Salmonella 18 hr after induction of TIMER bac expression (excitation spectra; left) or at various time intervals during maturation at 37°C, 5% O 2 , and pH 5.0 (emission spectra; right) (numbers show time points in hours). Similar data were obtained in two independent experiments.

(A) Schematic representation of TIMER fluorescence in nongrowing (upper panel) or actively growing (lower panel) cells. In nongrowing cells, both rapidly maturing green and slowly maturing orange TIMER molecules can accumulate. In dividing cells, rapidly maturing green molecules dominate over orange molecules that are diluted by cell division before maturation.

The DsRed S197T variant called TIMER spontaneously changes fluorescence color from green to green/orange (). This results from a branched maturation pathway with rapid emergence of green fluorophores and delayed formation of orange fluorophores. Fluorescence resonance energy transfer (FRET) from green to orange fluorophores in mixed green/orange TIMER tetramers increasingly quenches green fluorescence and further enhances orange fluorescence ().

Discussion

Grant et al., 2008 Grant A.J.

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Tenson T. Cell division in Escherichia coli cultures monitored at single cell resolution. Successful antimicrobial chemotherapy of infections might depend on pathogen growth patterns. Testing this hypothesis requires monitoring of pathogen growth and survival during disease and treatment. Sequence tags and nonreplicating plasmids/phages () reveal pathogen growth, clearance, and spreading between organs at the population level but lack single-cell resolution. Fluorescence dilution () is the first single-cell method for assessing pathogen growth, but is mostly suitable for early infection stages because of decreasing signal to background and does not necessarily reveal current growth (cells that stopped dividing after initial rapid growth, or that started to proliferate after an initial lag phase, are indistinguishable).

We developed a complementary approach based on the TIMER protein that changes fluorescence color over time. TIMER reports on single-cell growth rates at various stages of disease. TIMER is blind to short-term fluctuations, but can resolve growth changes occurring over several hours (about one in vivo generation time). TIMER color also depends on oxygen tension. This is an important caveat in environments with inhomogeneous oxygenation, where green TIMER fluorescence might reflect fast growth, poor oxygenation, or a combination of both parameters. Further developments decreasing TIMER oxygen sensitivity are thus highly desirable.

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Bumann D. Disparate impact of oxidative host defenses determines the fate of Salmonella during systemic infection in mice. However, TIMER can be used as a growth rate reporter in rather homogeneously oxygenated tissues such as spleen red pulp. Oxygen tension data for mouse spleen range from 2% to 9% (), averaging over white pulp with limited blood flow and red pulp with strong circulation of arterial blood and low oxygen consumption (). Live Salmonella reside exclusively in red pulp in close vicinity to erythrocytes (), suggesting similar oxygen availability for various subsets, consistent with homogeneous levels of Salmonella oxygen-regulated proteins. Most importantly, TIMER-based Salmonella growth estimates closely correlated with an independent approach, plasmid dilution, confirming the utility of TIMER as an in vivo division rate reporter under these conditions.

TIMER revealed extensive phenotypic variation in Salmonella single-cell growth rates. Rapidly growing subsets dominated overall Salmonella proliferation and disease progression. These Salmonella might have superior access to nutrients such as purines, carbohydrates, and amino acids, but other yet-unidentified factors likely influence growth as well.

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et al. Antibiotic resistance—the need for global solutions. To assess the impact of Salmonella growth on therapy, we treated infected mice with a potent fluoroquinolone following recommended dosing regimens. Resolution of disease signs and Salmonella eradication required many doses, similar to severe cases of human typhoid fever. Delayed eradication can cause serious problems in critically ill patients and promote resistance development, a major concern in medicine today ().

In our model, slow eradication was the consequence of Salmonella antimicrobial tolerance, with a strong dependency on pretreatment single-cell growth rates. Fast-growing Salmonella survived poorly, whereas nondividing/slow-growing Salmonella survived best but were rare, limiting their impact. Instead, Salmonella with moderate growth rates (one to four divisions per day) and intermediate levels of tolerance dominated throughout therapy. More effective targeting of this large subset could substantially accelerate therapy. Pathogen physiology and antimicrobial action at moderate growth rates might thus deserve more research efforts in addition to the current focus on rich in vitro cultures with vigorously growing, drug-sensitive cells and rare nondividing, highly tolerant cells.

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Hardt W.D. Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment. In our disease/treatment model, we tried to mimic common clinical settings where patients with established infections and clear disease signs are treated with safe antibiotic doses. In other models (), mice are treated with high antibiotic doses already at early infection stages. Under these conditions, tissues harbor many maladapted, nondividing Salmonella with high antimicrobial tolerance at the onset of therapy, and surviving Salmonella cannot resume growth because of steady high antibiotic levels. As a consequence, long-term persisters are mostly nondividing Salmonella, in contrast to our conditions.

For many infectious diseases, pathogen eradication with antibiotics is slow compared to standard in vitro cultures. Pathogen subsets with moderate growth and partial antimicrobial tolerance might be involved in some cases, as shown here for Salmonella. In other cases, nondividing/dormant subpopulations with high tolerance or a growth-independent tolerance mechanism might be more relevant. Single-cell approaches as developed in this study might help to clarify these important issues as a basis for more efficacious therapies.