Metabolites potentiate low gentamicin doses against Gram-negative pathogens

To test whether metabolic stimulation could reduce effective aminoglycoside concentrations, we supplemented aminoglycoside treatment with specific metabolites (glucose, mannitol, or fructose) in three prevalent Gram-negative pathogens: E. coli, S. typhimurium, and K. pneumoniae (see Materials and Methods). All cultures were grown to stationary phase, where bacteria enter a tolerant, non-dividing state after exhausting their nutrients, and were then treated for 2 hours, corresponding to the pharmacokinetic properties of aminoglycosides in patients [34].

We first tested gentamicin, the most commonly administered aminoglycoside [35]. Gentamicin alone demonstrated poor efficacy against E. coli, the primary cause of hospital-acquired Gram-negative infections [36], producing only 80% killing at the peak clinical concentration of 10 μg/mL (Fig 1C). Conversely, gentamicin-metabolite combinations produced 95% reduction even at a gentamicin concentration of 2.5 μg/mL, and approximately 4 orders of magnitude at peak concentrations. No killing was observed with 2.5 μg/mL gentamicin alone. Similar results were obtained for S. typhimurium, a pathogen closely related to E. coli and cause of gastrointestinal disease, urinary tract infections, and bacteremia [37]. Consistent with E. coli, all metabolites boosted elimination of S. typhimurium up to 4 orders of magnitude at peak doses, while gentamicin alone produced no killing (Fig 1D). As with E. coli, adding metabolite improved treatment by orders of magnitude, even at one-fourth the concentration of gentamicin.

This approach was most effective for K. pneumoniae, a pathogen more distantly related to E. coli and a major threat due to its development of resistance to many antibiotics and pernicious ability to spread in hospitals [38]. All three metabolites potentiated killing at gentamicin concentrations of 10 μg/mL by four and a half orders of magnitude (Fig 1E). Mannitol was the only metabolite that significantly enhanced killing at 1 μg/mL, producing 90% reduction. Treatment times extended to 8 hours nearly sterilized E. coli, but had no additional effect on S. typhimurium or K. pneumoniae (S1a–S1g Fig in S1 File). Moreover, we found this approach was also effective at killing bacterial persisters within the stationary phase population (S2 Fig in S1 File). Collectively, these data suggest that aminoglycoside-metabolite combinations are effective in treating Gram-negative bacteria at lower concentrations than the peak serum concentrations targeted by clinicians [39,40].

Having demonstrated potentiation using specific metabolites, we sought to determine whether their effects were due to catabolism, rather than an osmotic effect, by testing gentamicin combined with sorbitol (S3a–S3c Fig in S1 File), which resembled gentamicin treatment without metabolite. Though sorbitol can serve as a carbon source, starved bacteria preferentially consume glucose present in the inoculum before consuming sorbitol [41]. Additionally, we confirmed that metabolic potentiation occurs for the aminoglycoside class more broadly by testing two additional antibiotics, tobramycin and amikacin. In both cases, metabolites greatly improved killing by multiple orders of magnitude, except in the case of tobramycin in S. typhimurium (S4a–S4f Fig in S1 File). Only glucose potentiated tobramycin in K. pneumoniae and amikacin in E. coli. Metabolites did not potentiate aminoglycoside killing in rapidly growing cells, which are already highly susceptible to antibiotics, nor did they promote growth or antibiotic tolerance (S5a–S5c Fig in S1 File).

We considered this metabolite-potentiation might be applied to “rescue” neomycin, an aminoglycoside no longer used for intravenous therapy due to its severe toxic side effects [42]. We found that mannitol reduced the effective dose to treat K. pneumoniae below the 5 μg/mL serum threshold associated with human toxicity [43] (Fig 1F), while potentiating doses of 20 μg/mL or greater for both E. coli and S. typhimurium (S6a–S6b Fig in S1 File).

Metabolites potentiate low aminoglycoside doses against bacterial biofilms

Bacteria commonly exist in structured communities known as biofilms, which can form on both abiotic and biotic surfaces, play important roles as reservoirs of infection, and exhibit high tolerance to antibiotics [32,44]. The tolerance of biofilms is a type of phenotypic resistance arising from the heterogeneity of cells [26,32,45], though, in some cases, tolerance results from poor penetrance of antimicrobials, particularly for β-lactam antibiotics [46]. Thus, we sought to determine the ability of metabolites to reduce the concentration of aminoglycosides required to eliminate biofilm cells. Biofilms of E. coli, S. typhimurium, and K. pneumoniae were grown for 48 hours and were then treated with antibiotics and mannitol. We found that gentamicin-mannitol treatments were highly effective at eradicating Gram-negative biofilm cells, whereas gentamicin alone had very little impact on biofilm cell viability. Specifically, for E. coli (Fig 2A) and S. typhimurium (Fig 2B), concentrations of gentamicin could be reduced ten-fold while still eliminating biofilm cells when mannitol was added. Low gentamicin plus mannitol treatment was less effective for K. pneumoniae (Fig 2C), achieving only 90% reduction compared to the 99% reduction with high gentamicin and mannitol. Ampicillin did not have any effect on the viability of any species tested, while norfloxacin only achieved killing in S. typhimurium by less than one order of magnitude.

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Fig 2. Metabolite-aminoglycoside combination improves treatment of Gram-negative biofilms.

a, Survival of E. coli biofilms after treatment with norfloxacin (5 μg/mL), ampicillin (100 μg/mL), mannitol (10 mM), low gentamicin (1 μg/mL), high gentamicin (10 μg/mL), low gentamicin plus mannitol, and high gentamicin plus mannitol. b, Survival of S. typhimurium biofilms after treatment with norfloxacin, ampicillin, mannitol, low gentamicin, high gentamicin, low gentamicin plus mannitol, and high gentamicin plus mannitol. c, Survival of K. pneumoniae biofilms after treatment with norfloxacin, ampicillin, mannitol, low gentamicin, high gentamicin, low gentamicin plus mannitol, and high gentamicin plus mannitol. d, Crystal violet (CV) (0.1%) stain of E. coli biofilm (grey), S. typhimurium (blue), and K. pneumoniae (green) cells after treatment with norfloxacin, ampicillin, mannitol, low gentamicin, high gentamicin, low gentamicin plus mannitol, and high gentamicin plus mannitol. Absorbance values were recorded at 550 nm and were normalized by no-treatment CV stains for each strain. Plots represent mean +/- standard deviation for three or more replicates.

https://doi.org/10.1371/journal.pone.0237948.g002

We used crystal violet staining to determine if treatments were liberating cells from biofilms rather than killing them, a possible confounding factor with implications for treatment in patients. We found that low gentamicin treatments that included metabolite, despite potentiating killing, actually caused a mild increase in biofilm volume for E. coli and K. pneumoniae, although a small decrease for S. typhimurium was observed (Fig 2D). This suggests that combination treatments were in fact killing biofilm cells rather than dispersing them. Such a phenotype might be explained by the production of exopolysaccharide typically associated with aminoglycoside treatment [47]. Intermediate doses of gentamicin, when supplemented with mannitol, were similarly effective (S7a–S7c Fig in S1 File). To ensure cells were not detaching and growing planktonically, we tested the cell viability from the biofilm supernatants by flow cytometry and agar plating. For E. coli and K. pneumoniae, gentamicin-mannitol treatment resulted in a viability of 0.1% and 1%, respectively (S8a–S8b Fig in S1 File) (Materials and Methods). These findings suggest aminoglycoside-metabolite combinations may be useful for the treatment or prevention of infections resulting from Gram-negative biofilms.

Metabolite supplementation does not increase aminoglycoside resistance

Antibiotic dosing regimens can affect the development of antibiotic resistance. To study the possible emergence of aminoglycoside-resistant mutants, we investigated whether prolonged aminoglycoside-metabolite treatment might affect minimum inhibitory concentration (MIC), the standard measure of resistance. Stationary-phase cultures were treated on two consecutive days for two hours in the presence of gentamicin and mannitol, and MICs were determined (Materials and Methods). No significant difference was observed between untreated and gentamicin-mannitol cases, while a slight increase of 0.05 μg/mL occurred between gentamicin-mannitol and gentamicin-only treatments (Fig 3). Nonetheless, the MIC following gentamicin-mannitol treatment was twenty-fold lower than levels denoting genetic resistance [48].

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Fig 3. Metabolite potentiation does not accelerate development of aminoglycoside resistance.

Minimum inhibitory concentrations following one- or two-day treatment of stationary phase E. coli with gentamicin (5 μg/mL) with or without mannitol (10 mM). Dashed line indicates minimum inhibitory concentration threshold for low-level gentamicin resistance in E. coli, as defined by the European Committee on Antimicrobial Susceptibility Testing [48]. Plots represent mean +/- standard deviation for three or more replicates.

https://doi.org/10.1371/journal.pone.0237948.g003

Role of metabolites in aminoglycoside potentiation

Past studies uncovered the importance of proton-motive force (PMF) in potentiating aminoglycoside against E. coli [6,49] and have indicated that metabolites do not return cells to active growth given that other classes of antibiotics are not potentiated [6,49]. We sought to test the specificity of potentiation in these Gram-negative species and treated each with norfloxacin or ampicillin in presence of metabolite (Fig 4A). Neither norfloxacin nor ampicillin were potentiated by mannitol in any species tested, suggesting that the processes of cell-wall synthesis and DNA replication targeted by these antibiotics remained inactive. Additionally, using carbonyl cyanide m-chlorophenyl hydrazone (CCCP), we showed that PMF was a requirement for potentiation in these species (Fig 4A), as was seen in E. coli persisters previously [6,49].

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Fig 4. Metabolite-aminoglycoside killing is specific to aminoglycoside antibiotics, requires PMF, and does not require cell growth.

a, Survival of stationary-phase E. coli (grey), S. typhimurium (blue), and K. pneumoniae (green) cells after treatment with gentamicin (10 μg/mL), gentamicin plus mannitol (10 mM), norfloxacin (5 μg/mL), norfloxacin plus mannitol, ampicillin (100 μg/mL), ampicillin plus mannitol, and gentamicin plus mannitol and CCCP (2.5 μg/mL). b, Growth of stationary-phase E. coli cultures after addition of 10 mM of the following metabolites: mannitol, glucose, fructose, and sorbitol. Plots represent mean +/- standard deviation for three or more replicates. c, Illustrative examples of stationary-phase E. coli cells (~400 observed) containing constitutively-expressed scarlet (red) and inducible GFP (green) tracked by microscopy at 37˚C after addition of 20 nM anhydrotetracycline and 10 mM mannitol (top) or no metabolite (bottom). Scale bar represents 6 μm. d, Illustrative examples of rare “cheater” cells (~3%) from the same experiments.

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Curious of the inactivity of norfloxacin and ampicillin, we investigated if any of the metabolites used stimulated cellular growth over longer time scales. Similar to potentiation experiments, metabolites were added to stationary phase cultures, but no antibiotic was added and colony forming data was collected over an 8 hours rather than 2 hours (Fig 4B). We found that little-to-no growth was induced by any metabolite during this time frame, though all could serve as carbon sources if cultures were diluted in minimal media with extended growth periods. We reasoned that the bulk-scale nature of these experiment might mask more complicated dynamics occurring at the cellular level. To further explore the physiological effects of metabolite supplementation on stationary-phase cells, we performed single-cell imaging with an E. coli strain harboring inducible green fluorescent protein. Cells were grown to stationary phase without inducer then were added to agarose pads made from spent media (see Materials and Methods) with inducer in the presence or absence of mannitol. This experiment allowed us to simultaneously observe division and nascent protein synthesis resulting from metabolite supplementation. Dynamically tracking ~400 cells, we found that mannitol supplementation did not cause significant cell division (Fig 4C). Cells in both mannitol (+) and mannitol (-) conditions remained non-dividing for up to 12 hours, supporting the bulk-culture data (Fig 4B). Intriguingly, new protein synthesis was observed to a similar degree in both mannitol (+) and mannitol (-) samples. This suggests that, despite nutrient limitation, stationary-phase cells are capable of translation and adapting in response to their environment, adding to past findings in bacterial persisters [50]. Given that these cells tolerate aminoglycosides (which target translation) in the absence of mannitol, this result further suggests that the metabolite’s role in potentiation is to generate PMF, thereby facilitating antibiotic uptake.

Surprisingly, growth heterogeneity was observed in the late stages of these experiments: rare cells (~3%) in both mannitol (+) and mannitol (-) conditions began dividing around 8 hours and had formed microcolonies by 12 hours (Fig 4D). This is not due to heterogeneous availability of nutrients as dividing cells are present in them same environment as, and with little distance from, non-dividing cells. Instead, these cells appear to be phenotypic “cheaters” escaping from a regulatory blockade to utilize available nutrients. The behavior of these cells further indicates that the population majority is not arrested because of a lack of nutrients (also supported by the small difference between mannitol (+) and mannitol (-)), and instead may be stuck at a growth-phase checkpoint. In light of the observed translation (Fig 4C), these findings (Fig 4D) suggest that stationary-phase cells actively maintain a growth-arrested state.

Mannitol protects kidney cells from aminoglycoside toxicity

Aminoglycoside nephrotoxicity, a primary limitation of aminoglycoside treatment, results from the accumulation of aminoglycosides in the kidneys, where they can induce apoptosis and necrosis by causing mitochondrial dysfunction and oxidative damage [51]. Mannitol is often used to protect against kidney toxicity during cancer chemotherapy with cisplatin, though the protective mechanism remains unclear. We hypothesized that mannitol, which is not a human metabolite and is commonly used clinically, might also protect against aminoglycoside-induced nephrotoxicity. To investigate this possibility, we cultured normal human primary renal epithelial cells (Material and Methods) and treated with gentamicin and mannitol, in combination and individually, for 72 hours before quantifying apoptosis and cell death by staining with Annexin-V FITC (indicating apoptosis) and propidium iodide (indicating necrosis). A gentamicin concentration of 2 mM (equivalent to 1033 μg/mL) was used, consistent with previous in vitro tissue culture studies in embryonic rat fibroblasts and porcine and canine renal cells [52]. The higher concentrations required for in vitro nephrotoxicity studies (~100 greater than ideal serum drug concentrations) may be due to aminoglycoside renal tubule concentrations reaching levels 10-times greater than serum concentrations [53].

We observed significant renal cytotoxicity in gentamicin-treated cells (12.0%) compared to untreated cells (4.1%) (Fig 5B), similar to previous studies [52]. However, adding mannitol greatly reduced cytotoxicity in gentamicin-treated cultures (4.5%). Annexin V and propidium iodide levels in gentamicin/mannitol samples were indistinguishable from untreated cells, suggesting that mannitol had blocked gentamicin toxicity. Polymyxin B, a macrolide antibiotic also used as a second-line treatment for serious Gram-negative infections, was used as a positive control given its severe nephrotoxicity (82.9%). The observed protective effect of mannitol was further evidenced by differences in cellular morphology (Fig 5A). The morphology of samples treated with gentamicin alone resembled samples treated with polymyxin B, as cells took on a spherical shape and appeared ready to detach from the plates. Alternatively, cells treated with gentamicin plus mannitol were morphologically indistinguishable from cells that did not receive treatment. These results indicate that mannitol may serve as a cytoprotectant against the kidney toxicity induced by aminoglycoside antibiotics. These findings reveal that specific metabolites can exploit bacterial metabolism while protecting human cells from antibiotic toxicity.

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Fig 5. Mannitol reduces aminoglycoside-induced renal cytotoxicity.

a, Bright field and fluorescent micrographs of normal primary human renal cells showing no treatment, gentamicin (2 mM or 1033 μg/mL), gentamicin plus mannitol (10 mM), or polymyxin B (0.5 mM). Annexin-V FITC (green) binds to phosphatidylserine, a lipid that presents extracellularly during cellular apoptosis, while propidium iodide (PI) (red) binds to DNA and indicates cell death. Images are representative examples. Scale bar represents 100 μm. b, Percent cytotoxicity (Annexin-V FITC or PI staining) of treatments. Plot represents mean +/- standard deviation for three biological replicates. Over 1,000 cells were quantified for each replicate.

https://doi.org/10.1371/journal.pone.0237948.g005

Bacterial strains and culture conditions

All experiments were performed with the following Gram-negative bacterial strains: Escherichia coli MG1665, Klebsiella pneumoniae subspecies pneumoniae (Schroeter) Trevisan, and Salmonella enterica serovar typhimurium LT2. K. pneumoniae (ATCC^® 43816^™) and S. typhimurium (ATCC^® 700720^™) were both purchased from the American Tissue Type Culture Collection. Escherichia coli MG1665Pro (F-, λ-, Sp^R, lacR, tetR) with genomic-integrated PRO1-scarlet containing pEZ21-GFP was used in microscopy experiments.

All bacteria were cultured in 25 mL of Neidhardt supplemented MOPS defined medium (Teknova M2105, in which glucose is the primary carbon source) in 250 mL flasks at 37°C at 300 RPM. This media was specifically developed for the study of Gram-negative Enterobacteriaceae [74]. Cultures were grown for 16 hours or until OD₆₀₀ 0.2–0.3, for stationary-phase (non-dividing) or exponential-phase (growing) experiments, respectively. For treatment, bacterial cultures were added in 1 mL volumes to 14 mL Falcon tubes already containing antibiotics and/or metabolites, then incubated at 37°C at 300 RPM for two hours. Two-hour treatment time was chosen to correspond to human pharmacokinetic characteristics of the antibiotics, representing the approximate half-life of aminoglycosides in normal patients [35], although some experiments were also performed using four- and eight-hour treatment times. Following treatment, cultures were serially diluted in phosphate-buffered solution (PBS) and plated on LB plates to determine colony-forming units.

Antibiotics and chemicals

Metabolites were purchased from Fisher Scientific and Sigma-Aldrich. For all experiments, metabolites were diluted from concentrated stocks to 10 mM. Antibiotics and Carbonyl-cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma-Aldrich. Concentrations of gentamicin, tobramycin, and amikacin were consistent with target serum peak and trough concentrations used clinically [39,40,75,76].

Microtiter plate biofilm assay

Biofilms were grown and quantified as previously described [77], with slight modifications. Briefly, overnight cultures of E. coli, S. typhimurium, and K. pneumoniae were diluted 1:100 into 100 μL of MOPS defined medium in 96-well plates (Corning) and incubated with gas permeable membranes (Breath Easy, RPI) at 37°C with no agitation for 48 hours. After washing twice with PBS, biofilms were treated with antibiotics and metabolites in fresh media for 2 hours at 37°C with gentle shaking at 150 RPM. After incubation, cells were washed twice with PBS. To quantify cellular viability, biofilm cells were liberated by sonication, serial diluted, and then plated on LB plates.

Biofilm production was quantified by crystal violet (CV) staining. After treatment, biofilms were washed twice with distilled water, incubated with 0.1% CV at room temperature for 15 minutes, and rinsed with distilled water four times to remove loosely associated bacteria. Plates were placed upside down for three hours to dry. Next, 30% acetic acid was added to each well to solubilize bound CV and plates were incubated for 15 minutes at room temperature. The solubilized CV were then transferred to a black-wall, flat-bottomed, 96-well plate, and absorbance was measured spectrophotometrically at 550 nm on a Biotech Synergy (Biotek).

To quantify the viability of cells in the biofilm supernatant, biofilms were grown as previously described, and then treated with gentamicin (5 μg/mL) or gentamicin and mannitol (10 mM) for 2 hours at 37°C with gentle shaking at 150 RPM. Next, 100 μL of supernatant was transferred into 0.5 mL 1X PBS and at least 500 cells were sorted by BD FACSAria II into 0.5 mL 1X PBS. Cells were spun down at 10 G for 2 minutes, then cells were resuspended in 50 μL of PBS and plated on LB agar plates. Cell viability was then calculated based on the number of colonies formed and theoretical number plated. These experiments were not performed for S. typhimurium after determining cells were made unviable by the flow cytometry apparatus, possibly as a result of the high-pressure fluidics or lasers that the machine uses to count cells.

Acquisition of resistance via determination of minimum inhibitory concentrations

E. coli was treated with either gentamicin (5 ug/mL) or gentamicin plus mannitol (10 mM) for 2 hours as previously described. Cultures were then diluted 1:1000 and grown to OD₆₀₀ 0.2–0.3 in 15 mL culture tubes, at which point cells were again diluted 1:1000 and grown overnight for 16 hours in 125 mL flasks. Cultures were then diluted 1:10,000 in the presence of a range of gentamicin concentrations in 96-well flat-bottomed plates and were incubated at 37°C for 18–20 hours, and MICs were determined corresponding to one-day treatment. Second-day treatments were then performed and subsequent MICs were measured in the same manner.

Bacterial microscopy

Stationary phase cultures were diluted and added to 1% agarose pads made from spent medium prepared by filtering Neidhardt supplemented MOPS from stationary cultures. Images were obtained using a Leica DMi8 microscope equipped with a DIC HCPL APO 63X oil immersion objective, Hamamatsu ORCA-Flash 4.0 camera, and Lumencor Spectra-X light engine. Multi-channel, large-field, tiled-image time courses were collected using the Leica X software. Temperature was maintained at 37ᵒC throughout image acquisition using a stage-top incubator (Tokai-Hit). Excitation and emission for fluorescence microscopy was performed at 470 nm and 500–550 nm for green fluorescence and 510 nm and 592–668 nm for red fluorescence, respectively. Fluorescent exposures were 10 ms at 15% intensity and 20 ms at 20% intensity for green and red, respectively. Image scaling, processing, and cropping were performed uniformly across raw data using Leica Application Suite X (LAS X) software.

Renal cell growth

Normal Human Renal Proximal Tubule Epithelial Cells (ATCC^®PCS-400-010^™) were cultured in renal growth medium (ATCC) with the supplements of fetal bovine serum (0.5%), triiodothyronine (10 nM), rh EGF (10 ng/mL), hydrocortisone hemisuccinate (100 ng/mL), rh insulin (5 mg/mL), epinephrine (1 mM), transferrin (5 mg/mL), and L-alanyl-L-glutamine (2.4 mM). Treatment media was supplemented with 0.001% Tween-20 to circumvent the established loss of aminoglycoside permeability typically observed in cultured human renal cells [78,79]. Cells were cultured at 37°C under 5% CO₂ in monolayers in culture flasks or culture plates until 80% confluence before passage at a ratio of 1:3.

Renal cell viability and cytotoxicity

Renal cytotoxicity was quantified with the Dead Cell Apoptosis Kit (ThermoFisher Scientific), which includes Annexin-V FITC and PI to quantify markers of apoptosis and cell death, respectively. Briefly, cells were grown in monolayers in 6-well plates as described above. After treatment with metabolite and antibiotic (at concentrations specified in Fig 5) for 72 hours, cells were stained in 1 mL PBS with Annexin V (5 μL), propidium iodide (1 μL), and Annexin-V binding buffer (360 μL) (see Dead Cell Apoptosis Kit) for 15 minutes in the dark. Stained cells were then fixed with 1% paraformaldehyde for 15 minutes on ice. Treatment with 0.5 mM Polymyxin B for 24 hours was used as a positive control. Cytotoxicity was determined by fluorescence microscopy on a Zeiss Apotome 2 (Carl Zeiss) based on Annexin V and PI staining. One hundred images were taken of each treatment condition per plate (totaling three wells per condition). Identical intensity and contrast scaling was applied across samples for fluorescent images. Annexin V- and PI-stained cells were counted for each condition and normalized by the number of cells to compute a percentage of cells experiencing cytotoxicity. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc; La Jolla, CA). When comparing the means of three independent groups, a one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons tests was performed.

Renal cell microscopy

All images were collected using a Zeiss Apotome.2 microscope, using an EC Plan-Neofluar 10x/0.30 M27 objective and an Axiocam 503 camera. DIC, green fluorescent, and red fluorescent images were taken using exposure times of 2 ms, 750 ms, and 750 ms respectively. Excitation wavelengths for green fluorescence and red fluorescence were 495 nm and 592 nm, respectively, while emission wavelengths were 519 and 614, respectively. One hundred images were taken of each treatment condition per plate (totaling three wells per condition). Identical intensity and contrast scaling was applied across samples for fluorescent images. Annexin V- and propidium iodide-stained cells were counted for each condition and normalized by the number of cells to compute a percentage of apoptotic or necrotic cells.