The structure and composition of bacterial communities can compromise antibiotic efficacy. For example, the secretion of β-lactamase by individual bacteria provides passive resistance for all residents within a polymicrobial environment. Here, we uncover that collective resistance can also develop via intracellular antibiotic deactivation. Real-time luminescence measurements and single-cell analysis demonstrate that the opportunistic human pathogen Streptococcus pneumoniae grows in medium supplemented with chloramphenicol (Cm) when resistant bacteria expressing Cm acetyltransferase (CAT) are present. We show that CAT processes Cm intracellularly but not extracellularly. In a mouse pneumonia model, more susceptible pneumococci survive Cm treatment when coinfected with a CAT-expressing strain. Mathematical modeling predicts that stable coexistence is only possible when antibiotic resistance comes at a fitness cost. Strikingly, CAT-expressing pneumococci in mouse lungs were outcompeted by susceptible cells even during Cm treatment. Our results highlight the importance of the microbial context during infectious disease as a potential complicating factor to antibiotic therapy.

Antibiotic-resistant bacterial infections are on the rise and pose a serious threat to society. The influence of genetic resistance mechanisms on antibiotic therapy is well described. However, other factors, such as epigenetic resistance or the impact of the environment on antibiotic therapy, are less well understood. Here, we describe and characterize a mechanism of noninherited antibiotic resistance that enables the survival and outgrowth of genetically susceptible bacteria during antibiotic therapy. We show that bacteria expressing the resistance factor chloramphenicol (Cm) acetyltransferase (CAT) can potently deactivate Cm in their immediate environment. The reduced Cm concentration then allows for the outgrowth of genetically susceptible bacteria in the same environment. Mathematical modeling demonstrates the presence of a parameter space in which stable coexistence between Cm-susceptible and -resistant bacteria is possible during antibiotic therapy, which we validated using single-cell analyses. Strikingly, mixed culture experiments in which mice were infected with both Cm-susceptible and -resistant pneumococci revealed that Cm-sensitive “freeloader” bacteria even outcompeted resistant bacteria during antibiotic therapy. Together, we show that the microbial context during infection is a potential complicating factor to antibiotic treatment outcomes.

Funding: NIH (grant number U54-HD071600).Received by VN. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. European Research Council Starting grant (grant number 337399-PneumoCell). Received by JWV. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Netherlands Organisation for Scientific Research (grant number VIDI 864.12.001). Received by JWV. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. European Research Council Starting grant (grant number 309555). Received by GSvD. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH (grant number U01 AI124316). Received by VN. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Netherlands Organisation for Scientific Research (grant number VIDI 864.11.012). Received by GSvD. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Here, we describe another mechanism by which bacteria survive antibiotic therapy without obtaining genetic resistance, with the example of the bacteriostatic antibiotic chloramphenicol (Cm) and the opportunistic human pathogen S. pneumoniae. We show that Cm-resistant pneumococci expressing the resistance factor Cm acetyltransferase (CAT) can provide passive resistance for Cm-susceptible pneumococci by intracellular antibiotic deactivation. CAT covalently attaches an acetyl group from acetyl coenzyme A (acetyl-CoA) to Cm [ 26 , 27 ] and thus prevents the drug from binding to bacterial ribosomes [ 28 ]. Intracellular CAT in resistant bacteria can potently detoxify an entire environment in growth culture, semisolid surfaces of microscopy slides, or in a mouse infection model, supporting the survival and growth of genetically susceptible bacteria in the presence of initially effective Cm concentrations. Our results expand recent findings on the basis of E. coli growth cultures and indicate a potential clinical relevance of passive Cm resistance [ 29 , 30 ].

As an alternative to reduced drug susceptibility, bacteria can also clear lethal doses of antibiotics from their environment. High cell densities and thus the presence of many drug target sites may be sufficient to lower the concentration of active compound by titration of free drug molecules [ 19 ]. Furthermore, antibiotic degradation via β-lactamase enables growth not only of resistant cells but also of susceptible cells in their vicinity [ 20 – 22 ], even across species, as demonstrated for amoxicillin-resistant H. influenzae and susceptible S. pneumoniae [ 23 , 24 ]. This mechanism is of direct relevance to clinical medicine and is alternatively referred to as passive or indirect resistance (from the perspective of susceptible cells) or collective resistance (from the perspective of mixed populations) [ 25 ].

While an individual pneumococcal cell competes for limited resources with all other bacteria present in the niche, it may also benefit from a community setting. In a collective effort, bacteria become recalcitrant to antibiotics when forming biofilms that represent a physical constraint for drug accessibility [ 7 , 8 ]. Additional population-based survival strategies involve the phenotypic diversification of an isogenic population, either to preadapt for environmental changes (bet-hedging) or to enable division of labor [ 9 ]. Because the impact of most antibiotics is growth rate dependent [ 10 – 12 ], a bifurcation into growing and nongrowing cells increases the drug tolerance for the latter fraction, commonly referred to as persisters [ 13 , 14 ]. Cell-to-cell communication represents another way to react to antibiotic inhibition by allowing bacteria to coordinate a common response; S. pneumoniae, for example, activates the developmental process of competence whereupon it may acquire resistance [ 15 – 17 ]. A quorum-sensing mechanism that compromises antibiotic effectiveness was also found in evolved Escherichia coli cultures, in which cells of increased resistance induce drug efflux pumps in susceptible cells via the signaling molecule indole [ 18 ].

Antibiotics are indispensable for fighting bacterial infections. Yet the rapid emergence of resistance during the last decades renders current drugs increasingly ineffective and poses a serious threat to human health [ 1 ]. Drug action and bacterial resistance mechanisms are well understood in population assays of isogenic cultures in vitro. However, ecological factors and cell physiological parameters in natural environments influence the impact of antibiotics [ 2 , 3 ]. Streptococcus pneumoniae (pneumococcus) is an important human pathogen that resides in complex and dynamic host environments. The bacterium primarily populates the nasopharynx of healthy individuals, together with numerous commensal microbiota, and often alongside disease-associated species, including Staphylococcus aureus, Moraxella catarrhalis, and Haemophilus influenzae [ 4 – 6 ].

Results

Antibiotic Resistance of the Pneumococcus Resistances to all currently prescribed antibiotics have been identified in clinical isolate strains of S. pneumoniae [31]. Genes that transfer antibiotic resistance can be classified according to their mode of action [32]. One class keeps the cytoplasmic drug level low by preventing drug entry or by exporting drug molecules. Another class alters the targeted enzymes by modifying their drug binding sites or by replacing the entire functional unit. A third class alters the drug molecules themselves. Only members of the latter group are potential candidates for establishing passive resistance. In the pneumococcus, resistance genes that deactivate antibiotics include aminoglycoside phosphor- or acetyltransferases and cat. To date, β-lactam antibiotic-degrading enzymes have not been reported in S. pneumoniae genomes or plasmids [33]. Standard therapy of pneumococcal infections does not include aminoglycosides because of the relatively high intrinsic resistance of S. pneumoniae to members of this antibiotic family. In contrast, Cm, a member of the World Health Organization Model List of Essential Medicines [34], is regularly prescribed throughout low-income countries for infections with S. pneumoniae and other Gram-positive pathogens due to its broad spectrum, oral availability, and excellent tissue distribution, including the central nervous system. Recently, the antibiotic was also discussed as candidate for a comeback in developed nations due to spreading resistances against first-line agents [35–37]. To test whether passive resistance emerges from antibiotic-deactivating resistance markers with S. pneumoniae, we used the drug-susceptible clinical isolate D39 [38]. We constructed an antibiotic-susceptible reporter strain expressing firefly luciferase (luc) and antibiotic-resistant strains expressing single-copy genomic integrated kanamycin 3′-phosphotransferase (aphA1), gentamicin 3′-acetyltransferase (aacC1), and chloramphenicol acetyltransferase (cat). Resistant and susceptible cells were grown at a one-to-one ratio, and optical density (both strains) and bioluminescence (emitted by susceptible cells only) were measured (Fig 1). Expression of cat, but not aphA1 or aacCI, conferred passive resistance to susceptible cells (as observed by increased luminescence in mixed populations compared with assays of susceptible cells only; S1 Fig), mirroring prior investigations of antibiotic deactivation by resistant isolates of S. pneumoniae [39]. Aminoglycosides permeate the bacterial cell only at low frequency [40]; high permeability, however, was recently shown to represent an important precondition for the establishment of passive resistance, explaining why the phenomenon could not be observed with aphA1 and aacCI expression [29]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Experimental setup to determine passive resistance. Antibiotic-susceptible cells (AbS) constitutively expressing luc are grown together with antibiotic-resistant cells (AbR, which do not express luc). Only when the concentration of the antibiotic in the medium is reduced by enzymatic deactivation of resistant cells will the genetically antibiotic-susceptible cells be able to grow and produce light. https://doi.org/10.1371/journal.pbio.2000631.g001

Collective Resistance to Cm In Vitro To characterize the observed Cm collective resistance in more detail, we used the Cm-susceptible strain D-PEP2K1 (from here on CmS), which constitutively expresses luc and the kanamycin resistance marker aphA1 [41], and the Cm-resistant strain D-PEP1-pJS5 (from here on CmR), which expresses cat from plasmid pJS5 [42] (see Methods). Luminescence allowed for the real-time estimation of growth (or inhibition) of the CmS population, and kanamycin resistance allowed for the monitoring of their viable cell count by plating assays in the presence of kanamycin. Cm represses the growth of susceptible pneumococci at a minimal inhibitory concentration (MIC) of 2.2 μg ml−1, and during Cm exposure, luminescence from luc expression of susceptible pneumococci was previously shown to decrease at a rate that depends on the applied Cm concentration [12]. However, when CmS was co-inoculated with CAT-expressing CmR, luminescence (indicative for growth or inhibition of the CmS cell fraction) recovered, both for a Cm concentration slightly above the MIC (3 μg ml−1; Fig 2A) and even for a Cm concentration of more than two times the MIC (5 μg ml−1; S2 Fig). Luminescence recovery in mixed population assays (CmR + CmS) exceeded the values measured with CmS monoculture by up to 10-fold (Fig 2A and S2 Fig), and plating assays (with kanamycin) revealed that the difference in viable cell count was 1,000-fold greater after 8 h of cocultivation (Fig 2B and S2 Fig). Although Cm is commonly regarded as bacteriostatic, bactericidal activity has also been demonstrated against S. pneumoniae [43], explaining the observed decrease in viability of CmS monoculture (Fig 2B and S2 Fig). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Cm deactivation during mixed population assays. (a) Plate reader assay sets in quadruplicate (average and standard error of the mean [s.e.m.]) measuring luminescence (symbols with color outline) and cell density (corresponding grey symbols) of S. pneumoniae CmS growing in the presence of 3 μg ml−1 Cm, in presence (+) or absence (−) of CmR cells. (b) Development of the count of viable CmS cells (colony-forming units per ml [CFUs ml−1]) during the cultivation assay presented in a, determined via plating in the presence of kanamycin; average values of duplicates are shown. (c) Culture supernatant (S) samples after 0, 1, 2, and 4 h of CmR cultivation (inoculation at optical density OD 0.001) in the presence of 5 μg ml−1 Cm, analyzed for Cm content by high-performance liquid chromatography (HPLC) separation and ultraviolet (UV) detection at 278 nm. (d) Luminescence and cell density profiles of CmS cells treated with 3 μg ml−1 Cm (inoculation at OD 0.001) in dependency of the inoculum size of CmR cells. (e, f), CmS luminescence and growth analysis (e) in Cm-supplemented medium (3 μg ml−1) that was pretreated with CmR cell pellet (P), S, and culture lysate (L), and controls without (C−) and with Cm (C+); (f) schematic overview of the assay (see also Methods and S1 Data). https://doi.org/10.1371/journal.pbio.2000631.g002 To confirm that CmR cells actually deactivate Cm in the growth medium, we analyzed culture supernatant (S) by high-performance liquid chromatography (HPLC) [44]. As shown in Fig 2C, within 4 h of growth, CmR cells entirely converted an initial Cm concentration of 5 μg ml−1, as evidenced by the disappearance of the corresponding Cm peak at wavelength 278 nm. New peaks (at later elution times) appeared and gradually increased in HPLC profiles of S collected after 1, 2, and 4 h of cultivation; these peaks were previously shown to correspond to acetylated Cm derivates (1- and 3-acetylchloramphenicol) [44]. Next, we focused on whether the initial amount of CAT-expressing CmR cells was important for the survival and growth of CmS cells during drug treatment. To test this, we inoculated microtiter plate wells with a fixed number of CmS cells (inoculation at optical density [OD] 0.001, corresponding to ~1.5 × 106 colony-forming units per ml [CFUs ml−1]) while varying the number of CmR cells (Fig 2D). High inoculation densities of CmR cells (OD 0.01) resulted in a fast recovery of luminescence activity of CmS cells; however, the peak of luminescence was lower compared to intermediate CmR inoculation densities. This difference can be explained by cells reaching the carrying capacity of the growth medium before the pool of Cm is completely deactivated; luciferase expression activity was previously shown to slow down when cultures reach high cell densities (above ~OD 0.05) [41]. Relatively low CmR inoculation densities (OD 0.0001) also limited luminescence recovery of CmS cells during cocultivation. This finding likely reflects fewer CmR cells requiring more time to deactivate Cm, resulting in increased time spans of CmS drug exposure. Prolonged drug exposure of susceptible pneumococci was previously shown to result in increasing lag periods after drug removal, indicating a more severe perturbation of cell homeostasis [12]. The time span before outgrowth of CmS cells consequently consists of both the period required for drug clearance (by CmR cells) and the period required to reestablish intracellular conditions allowing for cell division.

Intracellular Deactivation of Cm To test whether Cm processing by CAT is an intracellular process, or if it takes place after secretion or cell lysis, we examined the potential of the S and the cytosolic content of CmR cells to deactivate Cm (assay scheme in Fig 2F). Precultured CmR cells were diluted to OD 0.02 and translation activity was blocked by adding 1 μg ml−1 tetracycline ([Tc]; S. pneumoniae D39 MIC: 0.26 μg ml−1) [12] for 1 h at 37°C to prevent ongoing protein synthesis and thus CAT expression. Next, the Tc-treated culture was split into three fractions: cell pellet (P) and S, separated via centrifugation, and cell culture lysate (L), obtained by sonication. The P was resuspended in C+Y medium containing 3 μg ml−1 Cm (and 1 μg ml−1 Tc), and 3 μg ml−1 Cm was added to the S and the L, followed by incubation at 37°C. After 2 h, the remaining cells and cell debris were removed by centrifugation and filtration, and the treated medium was used to test cell growth of a Tc-resistant variant of the CmS strain. Neither the S nor the L could support growth of CmS, whereas medium preincubated with the P did (Fig 2E). Together, these experiments indicate that CAT is only active inside living cells, in which acetyl-CoA is present [26,27].

Single-Cell Observations of Collective Resistance Because the abovementioned experiments were performed in bulk assays, we wondered whether CAT-expressing bacteria would also efficiently deactivate Cm, and thus support the growth of susceptible cells, in a more complex environment, such as on semi-solid surfaces. To do so, we spotted CmR cells together with Cm-susceptible D-PEP33 cells expressing green fluorescent protein (GFP) on a matrix of 10% polyacrylamide C+Y medium containing 3 μg ml−1 Cm. Indeed Cm-susceptible D-PEP33 cells were able to grow and divide under these conditions (S3 Fig). S. pneumoniae cohabitates the human nasopharynx with other bacteria, such as S. aureus [6]. Therefore, we investigated whether CAT-expressing S. aureus could also support growth of Cm-susceptible S. pneumoniae in environments containing Cm. As shown in Fig 3 and S1 Movie, all S. aureus cells grew and divided from the starting point of the experiment, whereas S. pneumoniae CmS cells did not grow initially. However, after 8 h, a fraction of CmS cells grew out to form microcolonies. Note that CmS cells spotted in the absence of S. aureus did not grow under these conditions (S2 Movie). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Interspecies collective resistance. Still images (overlay of phase contrast and fluorescence microscopy) of a time-lapse experiment of S. pneumoniae CmS, cocultivated with a strain of the pneumococcal niche competitor S. aureus (strain LAC pCM29) that expresses CAT and GFP, growing on a semi-solid surface supplemented with 3 μg ml−1 Cm. Scale bar 10 μm. https://doi.org/10.1371/journal.pbio.2000631.g003

Requirements for Stable Coexistence The observation that CmS cells grow only when Cm-deactivating cells are present in their close vicinity (Fig 3) suggests that the establishment of collective resistance requires CmS and CmR bacteria to be present in the same niche. However, such coexistence is subject to ecological constraints (e.g., the competitive exclusion principle) [45], particularly if susceptible and resistant strains compete for the same limiting resource. We therefore developed an ecological model to assess the scope for coexistence between CAT-producing bacteria and an antibiotic-susceptible strain (S1 Text). Consistent with this objective, we employed a minimalist modeling strategy and disentangled the qualitative effects of different factors (antibiotic stress, relative cost of Cm degradation and density regulation by ecological resource competition) from the interaction between CmS and CmR bacteria rather than aiming for a precise quantitative reconstruction of the experimental conditions. In fact, in contrast to natural environments (such as the human nasopharynx) that provide ample opportunities for coexistence because of spatial structure and concentration gradients of multiple resources, the model considers a worst-case scenario for coexistence: the two populations are assumed to grow in a well-mixed, homogeneous chemostat environment and are limited by the same resource. Nonetheless, we found that coexistence between CmR and CmS bacteria was feasible (Fig 4A and 4B), albeit under a restricted range of conditions (Fig 4C and S4 Fig). A mathematical analysis of the model (S1 Text) indicates that resistant and susceptible bacteria can establish a stable coexistence when CAT expression has a modest fitness cost. Without such a cost, the CmR strain is predicted to outcompete the CmS strain in the presence of antibiotics. Conversely, if the cost of expressing resistance is too high, the CmS strain will be the superior competitor. Interestingly, the model furthermore predicts parameter ranges that result in the extinction of mixed populations during drug treatment, while CmR populations on their own could survive (S4 and S5 Figs). A second condition for coexistence demands that the CmR population has a significant impact on the extracellular Cm concentration in its ecological niche. This requires that the population density reached at steady state must be high, so that coexistence can be stabilized by frequency-dependent selection, generated by a negative feedback loop between the relative abundance of drug-deactivating cells and the level of antibiotic stress in the environment. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Population dynamics of bacterial communities. (a) Simulated growth trajectories for CmR and CmS populations subject to antibiotic stress and resource competition. (b) Dynamic of intracellular Cm (y r and y s ) and growth-limiting resource (z). Simulation time is scaled relative to the mean residence time of cells in a chemostat, which is equal to the generation time at steady state. At low population densities, the CmR strain can grow, whereas CmS cannot, due to a high concentration of Cm. However, the invasion of CmR lowers antibiotic stress, generating permissive conditions for the growth of CmS cells. The chemostat is then rapidly colonized by both strains (shortly after t = 180) until the resource becomes limiting. From that moment onwards, total cell density changes little, while the relative frequencies of the two strains continue to shift. Eventually, a stable equilibrium is reached, at which the cost and benefit of CAT expression (i.e., reduced growth rate efficiency for CmR cells versus their lower intracellular Cm concentration) balance out. Inset (c), The dark-red dot pinpoints the parameter set used in the simulation shown in a and b: r = 20.0, η = 0.9, k z = 4.0, c = 1.0, p = 50.0, h Y = 0.25/Y 0 , k Y = 2.5/Y 0 , d = 30.0/Y 0 and Y 0 = 0.8. These parameters were selected to lie in a restricted area of parameter space (highlighted in red) where stable coexistence between CmS and CmR cells is observed Alternative model outcomes, which were identified by a numerical bifurcation analysis (see S1 Text and S4 Fig), include establishment of CmS only (area S), establishment of CmR only (area R), no bacterial growth (area N), and competition-induced extinction (area E, where CmS bacteria first outcompete CmR bacteria and subsequently are cleared by the antibiotic; see S5 Fig). https://doi.org/10.1371/journal.pbio.2000631.g004 We note that competitive exclusion acts at a local scale in structured environments, where the presence of spatial gradients in Cm and resources may help to create refuges in which either strain can escape competition from the other. In addition, we expect that coexistence between resistant and susceptible bacteria would be promoted in vivo by previously evolved ecological niche partitioning between co-occurring species.