Tissue compartmentalization enables Salmonella persistence during chemotherapy - pnas.org
In difficult-to-treat bacterial infections, including tuberculosis, deep-seated Staphylococcus aureus infections, and invasive salmonellosis, adequate antimicrobial chemotherapy can initially clear most pathogen cells and resolve clinical symptoms. However, even extended therapy often fails to eradicate the pathogen, even in the absence of relevant antimicrobial resistance. The persistence of a small subset of pathogen cells can cause relapsing diseases and accelerates the emergence of antimicrobial resistance (1⇓⇓–4). Eradication of such difficult-to-treat infections represents an urgent medical need.
Development of effective treatments requires a detailed understanding of the underlying mechanisms. Various mechanisms have been proposed to explain pathogen persistence during antimicrobial chemotherapy. First, antimicrobials might not reach all bacterial cells in sufficient amounts because anatomical permeation barriers limit drug access to bacteria in certain tissue areas (5, 6). Second, bacteria might adopt physiological states in host tissues that enable them to tolerate antibiotic exposure. Such tolerant states might be triggered by stresses imposed on the pathogen by the host immune system (7⇓⇓⇓⇓⇓–13). Moreover, limited nutrient supply and stress conditions can slow bacterial proliferation, which increases tolerance to most antibiotics (14⇓⇓⇓–18), in some conditions due to low ATP levels (19). Third, pathogen heterogeneity has been suggested as a major cause of treatment failures (1, 2, 20⇓–22). Some bacteria might stop replication either because of stochastic internal processes or in response to external triggers. Such nonreplicating "persisters" can survive exposure to otherwise lethal antibiotic concentrations. Other forms of heterogeneity might also contribute to treatment failures, including asymmetric cell division (23⇓–25), uneven partitioning of efflux pumps among daughter cells (26), heterogeneous expression of prodrug-activating enzymes (27), transient gene amplification (28), and heterogeneous induction of specific stress responses (29). The relevance of these various mechanisms for pathogen persistence in host tissues remains unclear because supporting data have been obtained almost exclusively using in vitro models (1⇓–3, 20⇓–22, 30). In vivo data are critical because bacterial susceptibility depends on environmental factors, and complex and diverse microenvironments in infected tissues are difficult to mimic in vitro (31).
One suitable small-animal infection model for in vivo studies is systemic salmonellosis in mice as a model for human invasive Salmonella infections. Such infections, including typhoid and paratyphoid fever (enteric fever) as well as nontyphoidal Salmonella (NTS) bacteremia, are a major health problem worldwide (32, 33). Antimicrobial chemotherapy frequently fails to eradicate Salmonella, resulting in relapsing disease, even when the bacterial strain is susceptible to the drug (34⇓⇓–37). Mouse models of invasive salmonellosis recapitulate these eradication challenges (38⇓⇓–41). We showed previously that clinically relevant doses of fluoroquinolone antibiotics clear Salmonella from mouse tissues only slowly because Salmonella replicates slowly in vivo with generation times around 6.5 h (42). Nevertheless, clearance continues with a monophasic exponential decay of colony-forming units (CFUs) for at least 5 d. Here, we continued treatment for longer intervals and observed declining clearance rates and eradication failure at later time points. We aimed at unraveling the mechanisms underlying this antibiotic persistence of Salmonella.
In vivo studies of pathogen cells surviving chemotherapy have been thwarted by difficulties in localizing and characterizing few, sparsely distributed, micrometer-sized pathogen cells in entire centimeter-sized host organs. We addressed these limitations by adopting serial two-photon tomography (STP) (43) for detecting individual Salmonella cells across entire spleens of infected mice. We resolved interfering tissue autofluorescence, developed automated pipelines for identifying Salmonella cells in terabytes (TB) of imaging data, and validated accurate detection of single Salmonella cells at densities as low as one bacterium in 100 mm3 tissue. We used STP to localize Salmonella surviving treatment with two clinically relevant antibiotic classes. We combined STP with laser-capture microdissection, flow cytometry, Salmonella reporter constructs, and adjunctive therapies, to determine the relevance of nonreplicating Salmonella persisters, stress-triggered drug tolerance, uneven drug delivery, and tissue infiltration by neutrophils and inflammatory monocytes. We found that Salmonella colonization of spleen in untreated mice was inhomogeneous. A minor Salmonella subset colonized the white pulp (WP) and triggered only limited local infiltration of inflammatory monocytes and neutrophils. These Salmonella-killing host cells supported Salmonella clearance during chemotherapy, but their density collapsed during chemotherapy in response to receding local Salmonella loads resulting in 70-fold better Salmonella survival in the WP compared to other, initially more colonized and inflamed spleen compartments. By contrast, Salmonella dormancy, stress-induced antimicrobial tolerance, or inefficient antibiotic delivery had all minor relevance. Thus, host tissue architecture and the topology and dynamics of host–Salmonella interactions caused locally divergent antimicrobial activities that ultimately resulted in eradication failure.
Results
Chemotherapy Fails to Eradicate Invasive Salmonellosis.
We infected genetically susceptible BALB/c mice by oral administration of Salmonella enterica serovar Typhimurium. Once clinical symptoms emerged, we started chemotherapy with recommended doses of the fluoroquinolone antibiotic enrofloxacin. This treatment prevented mouse mortality, resolved clinical symptoms, and diminished Salmonella loads in the major target-organ spleen ∼200-fold with slow, monophasic exponential kinetics over 6 d (42) (Fig. 1 A and B). However, clearance decelerated thereafter, even though Salmonella survivor cells retained in vitro susceptibility (minimal inhibitory concentration, MIC, 0.03 to 0.06 mg per liter). After 10 d of treatment, Salmonella survived in the spleen (∼2,000 CFUs), the liver (∼2,000), the mesenteric lymph nodes (∼1,000), and the Peyer's patches (∼200 in the last small-intestinal patch) (SI Appendix, Fig. S1A). Reservoirs of surviving Salmonella in these and other organs have been previously observed after treatment of intravenously infected mice (38) and in a streptomycin-pretreated enteritis model with high initial Salmonella loads in the cecum (44).
Spleen-wide monitoring of Salmonella survival during antimicrobial chemotherapy. (A) Disease scores of orally infected mice. From day 5 to day 14, mice received daily 5 mg/kg enrofloxacin. Arithmetic means and SDs are shown for 3 to 26 mice. (B) Salmonella loads in the spleen of orally infected mice as determined by plating (triangles), flow cytometry (diamonds), or serial two-photon tomography (circles). Plating and flow cytometry results are shown as geometric means and SDs for groups of 3 to 20 mice (yellow labels indicate previously reported values) (42). Each tomography data point represents an individual mouse. The dashed line from days 2 to 5 shows an exponential fit; the line from days 5 to 15 shows an exponential decay with y offset; and the line from days 15 to 18 connects the geometric means. (C) Principle of STP tomography. A vibratome removes the surface of a tissue block. A two-photon microscope scans the blockface, before the vibratome removes the next section. (D) Side and top views of collagen fibers (trabeculae) detected by STP tomography of a 5-mm spleen slice. (E) Detection of GFP-Salmonella by STP tomography in one optical section (Left). Neutrophils were stained by in vivo injection of a phycoerythrin-labeled Ly-6G antibody. Green fluorescent particles were confirmed by staining the corresponding vibratome section with an antibody to Salmonella LPS and imaging by confocal microscopy (Right). (Scale bars, 3 μm.) (F) GFP fluorescence intensity distribution of single Salmonella cells in the spleen at day 5 after oral infection as analyzed by flow cytometry (n = 7,685) or STP tomography (n = 681). Intensities were normalized to the respective median. (G) Examples of single Salmonella cells and microcolonies in the spleen as detected by STP tomography. The numbers on the Right represent estimates for the number of Salmonella cells based on total GFP fluorescence. (Scale bar, 5 μm.) (H) Size distributions of Salmonella objects in orally infected mice before (day 5) and during antimicrobial chemotherapy. The data represent pooled data from three mice per group and 39 to 362 Salmonella objects per mouse. The Inset shows the fraction of single Salmonella among all Salmonella objects with each circle representing an individual mouse.
The Salmonella survivors caused disease relapse upon treatment discontinuation in seven out of eight tested mice (Fig. 1 A and B and SI Appendix, Fig. S1B). A second regimen of enrofloxacin again reduced clinical symptoms to baseline (SI Appendix, Fig. S1B) indicating that Salmonella remained susceptible to treatment in vivo. At the end of the second regimen, we still found surviving Salmonella in the spleen, the liver, and the mesenteric lymph nodes but not in the last Peyer's patch (SI Appendix, Fig. S1A). Administration of enrofloxacin at doses 60- to 120-fold higher than recommended (to avoid adverse effects), still requires 5 wk for Salmonella eradication (39, 45, 46).
Similarly, after 10 d of treatment with the beta-lactam ceftriaxone, 480 ± 190 Salmonella cells survived in the spleen (SI Appendix, Fig. S1C). Relapsing disease upon treatment discontinuation occurred in all four tested mice and could be treated with a second regimen of ceftriaxone (SI Appendix, Fig. S1B). Thus, two major classes of antibiotics that are widely used to treat human invasive salmonellosis, failed to eradicate Salmonella in orally infected mice.
Serial Two-Photon Tomography Detects Sparse Salmonella in the Spleen.
Our goal was to identify survival mechanisms and host microenvironments of the persisting Salmonella. Currently available methods (47⇓⇓⇓–51) are impractical for characterizing scattered micrometer-sized Salmonella cells in centimeter-sized host organs. However, STP tomography provides a robust method for a similar task—the detection of thin neuronal axons with submicron resolution in entire mouse brain (43) (Fig. 1C). We were thus interested to adapt STP for infectious diseases. Our initial attempts showed that standard STP protocols were hampered by substantial tissue autofluorescence in the spleen that became particularly strong during infection (SI Appendix, Fig. S2A) and masked the fluorescence signal of GFP-expressing Salmonella. We were unable to boost GFP levels because this would diminish Salmonella in vivo fitness (52), but we discovered that storage of perfusion-fixed spleen in freeze protectant at −20 °C effectively suppressed the interfering tissue autofluorescence (SI Appendix, Fig. S2A). As another challenge, illumination was uneven and varied between samples (SI Appendix, Fig. S2B), precluding subtraction of a single reference background image as can be done for brain datasets. Applying "corrected intensity distributions using regularized energy minimization" (CIDRE) (53) resolved this issue (SI Appendix, Fig. S2B).
Imaging 5-mm slices of spleen at 0.435-μm horizontal and 10-μm vertical resolution (Fig. 1D) revealed Salmonella-like particles with spectral characteristics of GFP (Fig. 1E). A single three-dimensional (3D) image stack for a 5-mm slice of spleen contained ∼0.5 TB of data, prohibiting manual analysis. Simple threshold-based Salmonella detection was hampered by residual autofluorescence and imaging artifacts. To resolve this issue, we utilized machine learning, including support vector machines and a deep convolutional neural network that discriminated GFP-Salmonella from background with >99% sensitivity and specificity. We validated the identification of GFP-Salmonella by antibody staining of Salmonella lipopolysaccharide in spleen sections retrieved from the tomograph (Fig. 1E) and detected no signals classified as GFP-Salmonella in the spleen of mice infected with yellow fluorescent protein (YFP)-Salmonella indicating specific detection. Collagen second-harmonic signals (54) and in vivo staining of host cell surface antigens with fluorescence-labeled antibodies revealed the tissue context of Salmonella cells (Fig. 1 D and E). We focused on infected spleen in this study but STP also identified GFP-Salmonella in liver, mesenteric lymph nodes, and small intestinal Peyer's patches (SI Appendix, Fig. S3 and Movies S1–S3).
Single Salmonella cells had a narrow GFP fluorescence-intensity distribution consistent with flow cytometry data for the same GFP-Salmonella strain in the spleen (55) (Fig. 1F), indicating reliable fluorescence quantification by STP. For unresolved Salmonella microcolonies, we divided total fluorescence by the median single-cell GFP value to estimate the number of Salmonella cells per microcolony (Fig. 1G). The results showed that ∼40% of Salmonella resided alone with no conspecifics in their immediate (<5 μm) neighborhood consistent with previous findings (55, 56) (Fig. 1H). Based on number and size of microcolonies and the imaged tissue volume, we estimated the total number of Salmonella cells per spleen. These data were consistent with plating results and flow cytometry of independently infected mice (Fig. 1B). Thus, STP tomography reliably localized and quantified individual Salmonella cells at population densities down to one cell in 100 mm3 tissue.
Comparison of Salmonella objects before and after 10 d of chemotherapy with either enrofloxacin or ceftriaxone revealed unaltered microcolony-size distributions. We did not detect a preferential survival of isolated single Salmonella cells during antimicrobial clearance of 99% of all Salmonella, which was inconsistent with the proposed exclusive survival of single nonreplicating Salmonella persister cells (57).
Antimicrobial Clearance of Salmonella Is Ineffective in the Splenic WP.
STP tomography enabled us to localize scarce surviving Salmonella during antimicrobial chemotherapy at the whole-organ level. The spleen has three major compartments with distinct physiological functions (58): the red pulp (RP) that serves as a blood reservoir and removes aged erythrocytes; the WP containing high densities of B and T lymphocytes for inducing adaptive immunity to bloodborne pathogens; and the intermittent marginal zone (MZ) that captures bloodborne particles and pathogens. We identified these three regions by in vivo staining of CD169 (also called Siglec-1 or Sialoadhesin), a marker for a macrophage subtype in the MZ (59) (Fig. 2 A and B). We noticed that Salmonella clearance kinetics in RP resembled those in the MZ. We, therefore, combined data for these two compartments (RP/MZ) and compared them with those for WP.
Compartmentalized Salmonella clearance during antimicrobial chemotherapy. (A) Tomogram of a 5-mm spleen slice stained in vivo with an anti-CD169 antibody. (B) Localization of Salmonella cells (cyan, arrowheads) in spleen compartments (RP; MZ, marginal zone containing CD169-positive macrophages; WP) of an orally infected mouse. (C) Salmonella loads in spleen compartments of orally infected mice during enrofloxacin chemotherapy. Each circle represents STP data of an individual mouse. The lines are fits of monoexponential decays with y offsets. (D) Ratio of Salmonella loads in WP vs. RP/MZ during chemotherapy with enrofloxacin (Enro.; P value for deviation of slope from zero after linear regression of log-transformed data, shown as dashed line). Data for 10 d of treatment with ceftriaxone (Ceftr.) are also shown. Each symbol represents STP data of an individual orally infected mouse calculated from the data shown in C. (E) Localization of GFP-expressing Salmonella in CD68-positive macrophages at the periphery of B220-positive B cell zones in the splenic WP of orally infected mice after 10 d of treatment with enrofloxacin. (Scale bars, 5 μm.)
Before treatment, ∼95% of the ∼200,000 Salmonella cells resided in RP/MZ (60, 61) (Fig. 2C). Enrofloxacin cleared Salmonella from these compartments with monophasic exponential kinetics with a rate of 0.44 ± 0.23 log per day for ∼7 d, after which clearance decelerated markedly. After 10 d, ∼300 Salmonella cells or ∼0.15% of the initial load survived. By contrast, at start of therapy only ∼10,000 (∼5%) Salmonella cells resided in WP. Initial clearance of this minor Salmonella subset was 2-fold slower (0.21 ± 0.12 log per day) than in RP/MZ and decelerated markedly already after 4 d of treatment (day 9 postinfection, p.i.) with Salmonella loads declining less than 3-fold over the next 6 d. After a total of 10 d of treatment, ∼1,000 Salmonella cells or ∼10% of the initial load in the WP survived, a 70-fold larger fraction than in RP/MZ (Fig. 2 C and D).
Ceftriaxone, which differs from enrofloxacin by targeting peptidoglycan transpeptidases instead of DNA topoisomerases, and by lacking activity against nonreplicating bacteria (62⇓⇓–65), had similar inhomogeneous activity against Salmonella in the WP compared to RP/MZ (Fig. 2D), indicating that the diverging Salmonella survival did not depend on a particular antimicrobial mode of action or activity against nonreplicating bacteria.
Immunohistochemistry showed that surviving Salmonella in WP resided intracellularly in CD68+ Ly-6C− F4/80− CD21/35− FDC− ER-TR7− gp38− CD3− CD19− WP macrophages (59) at the boundary between T and B cell zones (Fig. 2E). We did not find Salmonella cells in B or T cells, follicular dendritic cells, dendritic cells, inflammatory monocytes, lymphatic epithelial cells, or reticular fibroblasts. Our data differ from previous studies that attempted to identify host cells of surviving Salmonella cells by analyzing tissue homogenates with flow cytometry. Homogenization disrupts most Salmonella-infected cells, even when done gently (55). Released bacteria can bind to other host cells in the tissue homogenate such as B cells (66), which do not harbor Salmonella in intact tissues (61).
Together, these data indicate that antimicrobial chemotherapy was particularly ineffective against a minor Salmonella population in WP.
Enrofloxacin Reaches the WP in Sufficient Amounts.
The poor antimicrobial clearance of Salmonella from WP might reflect inefficient drug delivery (67). Enrofloxacin effectively penetrates tissues (64); however, the splenic WP receives blood solutes only by diffusion through a conduit system (68), in contrast to the direct blood supply to the RP/MZ (69). In addition, oral Salmonella infection caused extensive thrombosis in spleen veins (70) (Fig. 3A) that reduced blood circulation to and within the spleen as revealed by ultrasound imaging (Fig. 3 B and C).
Role of drug delivery and nonreplicating persisters. (A) Autofluorescence micrograph of a spleen section with a highly autofluorescent thrombus in a large vein. Similar images were obtained for all orally infected mice. (B) Ultrasound 3D power Doppler-mode images of blood flow in the spleen of naive and orally infected mice. (Lower) Corresponding blood flow velocities in the splenic artery as determined by pulsed-wave Doppler ultrasound. (C) Mean velocities in splenic arteries and veins of naive and orally infected mice. Each circle represents one mouse (repeated-measures two-way ANOVA comparing naive and infected mice). (D) Enrofloxacin concentration in different spleen compartments of orally infected mice. Each circle represents one mouse (repeated-measures two-way ANOVA comparing RP and WP). The MIC of enrofloxacin for the Salmonella strain as measured in Mueller–Hinton broth is shown for comparison. (E) Salmonella replication-rate distributions in different spleen compartments. The graphs represent pooled data for three orally infected mice per time point. The few nondividing Salmonella cells included enrofloxacin-killed Salmonella corpses that retain the TIMERbac protein. Previously reported in vivo data for the ssrB mutant, which shows a replication arrest in mouse spleen (42) are shown for comparison (gray). (F) Mean Salmonella division rates with each circle representing an individual orally infected mouse (P value: repeated-measures two-way ANOVA comparing red vs. white pulp).
To assess drug delivery, we isolated WP and RP/MZ using laser-capture microdissection (SI Appendix, Fig. S4A) and determined drug concentration with an enrofloxacin-specific ELISA. Enrofloxacin levels were slightly higher in RP/MZ than in WP but were >1 mg mL−1 at day 9 p.i. in both compartments (Fig. 3D), thus exceeding more than threefold the pharmacokinetic–pharmacodynamic target for effective bacterial killing (peak concentration: MIC ratio >12) (71⇓⇓–74). Killing assays with Salmonella cultures growing in chemostat cultures under in vivo mimicking conditions with generation times around 6 h (42) confirmed that enrofloxacin concentrations above 1 mg mL−1 caused saturated killing (SI Appendix, Fig. S4B) (73). Fluoroquinolones accumulate in phagocytes (75), suggesting even higher enrofloxacin exposure of Salmonella residing intracellularly in macrophages (Fig. 2E) compared to bulk average concentrations in the WP. These data suggested that sufficient drug was locally available for effective killing.
To track Salmonella killing by enrofloxacin in vivo, we treated mice at day 5 postinfection with enrofloxacin. Before administration and various times after, we killed mice and prepared spleen homogenates in detergent-containing buffer, which liberates >90% of all Salmonella from host cells and tissue fragments as a suspension of single bacteria (76). We sorted the Salmonella using flow cytometry and determined their survival by comparing CFUs on plates (live Salmonella) and flow cytometric counts (live and drug-killed Salmonella) (42). To ensure sufficient Salmonella numbers for rapid sorting, we used intravenously infected mice at day 5 postinfection (first enrofloxacin dose). Salmonella have indistinguishable replication rates and enrofloxacin susceptibility after oral or i.v. infection (42). The results show that enrofloxacin killed 90 to 95% of all Salmonella within the first hour (42) consistent with pharmacokinetic–pharmacodynamic target attainment (see above). However, little further killing occurred in the following 3 h (SI Appendix, Fig. S4C), consistent with results for chemostat cultures growing under in vivo mimicking conditions (SI Appendix, Fig. S4D) and reported in vitro data for enrofloxacin and other fluoroquinolones (74, 77). This biphasic killing is probably a consequence of a bacterial SOS response to fluoroquinolone exposure ("drug-induced tolerance") (77).
Overall, killing of slowly replicating Salmonella in mice and chemostats was rather limited compared to rapidly replicating bacteria in rich broth, where similar fluoroquinolone concentrations kill several logs of bacteria within 1 h of exposure (42, 74, 77). Killing activity of antibiotics against invasive Salmonella in human patients is also moderate (78, 79), and Salmonella with slightly reduced susceptibility to fluoroquinolones are associated with longer fever clearance times and increased risk of failure (37, 80), while other Enterobacterales with similarly decreased susceptibility can still be treated effectively (Clinical & Laboratory Standards Institute, CLSI, susceptibility breakpoints for ciprofloxacin: Salmonella, ≤0.06 μg/mL; other Enterobacterales, ≤0.25 μg/mL; http://em100.edaptivedocs.net/Login.aspx).
Enrofloxacin levels declined 5- to 10-fold in both RP/MZ and WP from 1 h to 4 h after drug administration (Fig. 3D), consistent with a terminal elimination half-life of enrofloxacin in mice in the range of 1 h (74, 81, 82). Progressively declining enrofloxacin levels will eventually stop enrofloxacin-mediated killing and even enable some Salmonella regrowth when enrofloxacin levels drop below the MIC (see below). Sub-MIC trough levels are known to occur for recommended doses in mice (74) (and occur also in human patients) (83) but are likely less problematic for treatment of more rapidly growing pathogens that are more efficiently killed by peak enrofloxacin levels early after administration of each dose. Indeed, the Cmax/MIC ratio is a marker of effective treatment of various other pathogens with fluoroquinolones, while the fraction of time above the MIC is not (84). Higher or more frequent doses than recommended might reduce the fraction of time with sub-MIC enrofloxacin levels but would also increase adverse reactions (85).
Together, these results show pharmacokinetic–pharmocodynamic target attainment in both spleen compartments. Thus, insufficient drug delivery did not explain the sluggish Salmonella clearance from day 9 p.i. in WP. However, killing was mostly confined to the first hour after drug administration and overall rather limited because of Salmonella's low replication rates.
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