Bicarbonate Alters Bacterial Susceptibility to Antibiotics by Targeting

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Bicarbonate alters bacterial susceptibility to antibiotics by targeting the proton motive force Maya A. Farha, Shawn French, Jonathan Stokes, and Eric D Brown ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00194 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 25, 2017

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ACS Infectious Diseases

Bicarbonate alters bacterial susceptibility to antibiotics by targeting the proton motive force

Maya A. Farhaa, Shawn Frencha, Jonathan M. Stokesa and Eric D. Browna,*

Affiliations: a

Michael G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry

and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5, Canada

*Correspondence: [email protected]

ACS Paragon Plus Environment

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Abstract The antibacterial properties of sodium bicarbonate have been known for years, yet the molecular understanding of its mechanism of action is still lacking. Utilizing chemical-chemical combinations, we first explored the effect of bicarbonate on the activity of conventional antibiotics to infer on mechanism. Remarkably, the activity of 8 classes of antibiotics differed in the presence of this ubiquitous buffer. These interactions and a study of mechanism of action revealed that, at physiological concentrations, bicarbonate is a selective dissipater of the pH gradient of the proton motive force across the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Further, while components that make up innate immunity have been extensively studied, a link to bicarbonate, the dominant buffer in the extracellular fluid, has never been made. Here, we also explored the effects of bicarbonate on components of innate immunity. Although the immune response and the buffering system have distinct functions in the body, we posit there is interplay between these, as the antimicrobial properties of several components of innate immunity were enhanced by a physiological concentration of bicarbonate. Our findings implicate bicarbonate as an overlooked potentiator of host immunity in the defense against pathogens. Overall, the unique mechanism of action of bicarbonate has far-reaching and predictable effects on the activity of innate immune components and antibiotics. We conclude that bicarbonate has remarkable power as an antibiotic adjuvant and suggest that there is great potential to exploit this activity in the discovery and development of new antibacterial drugs by leveraging testing paradigms that better reflect the physiological concentration of bicarbonate.

Keywords Bicarbonate buffer system; antibiotic activity; drug discovery; mechanism of action; proton motive force; innate immunity


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Bacterial infection remains one of the leading causes of death worldwide and the spread of drug-resistant bacteria continues to erode the effectiveness of existing antibiotics. At the same time, we live in an era where not a single new antibacterial drug has been discovered in more than thirty years. Innovations to improve the success of modern antibacterial drug discovery and development are urgently needed. To this end, screens for antibacterial agents are increasingly being developed with the goal of mimicking the environment of the sites of infection1. The impetus for this stems from the growing appreciation that conventional laboratory media does not accurately represent conditions encountered by bacterial pathogens in the host2. Further, chemical collections used to find leads for new antibacterial drugs have been exhaustively screened via the traditional route and chances for new discoveries are low. Recent innovations include screens for growth inhibitory chemicals in host-like conditions that are recapitulated with serum-based3, nutrient-limited4-5 and eukaryotic cell culture6 growth media. Most recently, an antibacterial susceptibility testing study showed that bicarbonate, a ubiquitous buffer found in humans, influenced the potencies of a wide number of antibiotics and concluded that bicarbonate should be included in routine testing of bacterial pathogens in the clinical microbiology laboratory7. No reports to date however have addressed the mechanism of action of bicarbonate in this context and none have sought to explore a role for this ubiquitous host buffer in innate immunity. Sodium bicarbonate has been celebrated for years as a multi-use household item and additive in food and dental products, but has not been exploited for its antibacterial properties. While it has been reported to inhibit the growth of Escherichia coli8, fungi9 and cariogenic Streptococcus mutans10, the antibacterial activity of sodium bicarbonate has not been extensively studied and certainly, a molecular understanding of its mechanism of action is still lacking. Herein, to begin exploring the mechanism of action of bicarbonate, we characterized a variety of interactions – synergistic, antagonistic and neutral – of bicarbonate with assorted chemical classes of antibiotics using systematic analyses of growth inhibition in a matrix of



compound concentrations. Indeed, chemical-chemical combinations hold great power in revealing insights into the mechanism of action for uncharacterized molecules11-12. This work, along with genome-scale studies of gene expression and enhancement of bicarbonate activity by gene deletion, led to hypothesis testing using physiological and biochemical approaches. In all, these experiments were consistent with a mode of action for bicarbonate as a selective dissipater of the pH gradient of the bacterial proton motive force, a key bioenergetics process in all bacterial cells. Further, we found that bicarbonate exerts antibacterial activity against various disease-causing bacteria and works together with components of innate immunity to inhibit the growth of bacterial pathogens. Thus, we conclude that bicarbonate, as the dominant buffering system in the human body, is an overlooked antimicrobial component of the host defense system. This work provides a model for the action of bicarbonate as an antibacterial adjuvant that impacts the potency of conventional antibiotics and components of the innate immunity in a manner that is consistent and predictable based on mechanism of action and/or mode of uptake of these compounds. We conclude that bicarbonate is an overlooked component of host defense with strong potential to be leveraged for its adjuvant properties in the design of novel antibacterial schemes.

Results Effects of bicarbonate on various classes of antibiotics and deduction of its mechanism of action We first analyzed the interaction of sodium bicarbonate (pH 7.4) with conventional antibiotics at the sub-minimum inhibitory concentration (MIC) but physiological concentration of 25 mM. Our goal was to establish the nature of the interaction with various chemical classes of antibiotics as a first step to understanding the mechanism of action of bicarbonate. Remarkably, eight classes of antibiotics investigated had appreciably altered activities in the presence of a physiological concentration sodium bicarbonate. The fold enhancement in MIC for a variety of


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antibiotics in standard microbiological media relative to media supplemented with sodium bicarbonate is shown for E. coli and S. aureus in Figure 1 and Table S1. With a few exceptions, these Gram-negative and Gram-positive bacteria, behaved similarly. Of the antibiotics tested, the activity of some fluoroquinolones, macrolides, and aminoglycosides was enhanced. The activity of polymyxin B was enhanced strictly in S. aureus. In contrast, the activity of other fluoroquinolones, various cell wall active drugs, tetracyclines, fosfomycin and novobiocin was suppressed in the presence of bicarbonate. The effect on other classes such as chloramphenicol, linezolid, the antifolate drugs, trimethoprim and sulfamethoxazole, remained largely unchanged. Where antibiotics were potentiated or suppressed, follow-up studies using systematic microbroth checkerboard techniques were completed to assess the dosedependence of the interaction (Figure S1). Indeed, in all cases, enhancement or suppression was further pronounced with increasing concentrations of sodium bicarbonate. As a test case, we further explored the enhancement of various antibiotics by sodium bicarbonate for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa and methicillin-resistant S. aureus (Figure S2). In all cases, bicarbonate was a strong potentiator of the action of these antibiotics. Importantly, the chemical bicarbonate was uniquely responsible for the enhancements observed. The activity was not due to a trivial effect on pH. Test media were pH-adjusted upon addition of sodium bicarbonate for all studies reported herein. Of note, sodium bicarbonate at physiological concentration (25 mM) produces media with a pH typical of standard susceptibility testing conditions. Further, using dirithromycin as a test case, we tested many equimolar organic salts, with differing ionic strengths and steric properties, and none had impact on antibacterial activity, ruling out osmotic-mediated mechanisms (Table 1). Lastly, sodium did not contribute to the potentiation of dirithromycin, as equally potent synergy was observed with other salts of bicarbonate (Table 1). Idiosyncratic interaction patterns – synergistic, indifferent or antagonistic – of



antibacterial compounds of unknown mechanism with known antibiotics can provide strong hypotheses for the mode of action of an antibacterial compound11, 13. Here, a potential role for bicarbonate in perturbing the proton motive force (PMF) of the bacterial cytoplasmic membrane became evident on examination of the interactions depicted in Figure 1. The product of cellular respiration, PMF, describes the electrochemical potential at the cytoplasmic membrane that is composed of an electrical potential (∆ψ, negative inside) and a proton gradient (∆pH, acidic outside)14. This electrochemical potential crucially underpins energy production so that bacterial cells work to maintain a constant PMF15. Agents that selectively perturb either ∆ψ or ∆pH are known to prompt a compensatory increase in the other component in order to maintain PMF. In this context, the suppression and enhancement by bicarbonate of the growth inhibition by tetracyclines and aminoglycosides, respectively, suggested that bicarbonate might target the ∆pH component of PMF. Tetracyclines penetrate bacterial cells in a ∆pH-dependent fashion16, while positively charged aminoglycosides exploit the ∆ψ component for transport17. A direct test of the cellular uptake of tetracycline revealed that that suppression observed was indeed due to inhibition of tetracycline uptake on addition of bicarbonate (Figure 2A). Consistent with the observed increase in aminoglycoside activity, selective dissipation of ∆pH by sodium bicarbonate would be compensated for by an increase in ∆ψ that would in turn drive uptake of aminoglycosides. Accordingly, treatment of E. coli with 25 mM bicarbonate led to a higher transmembrane distribution of 3,3'-dipropylthiadicarbocyanine iodide (DiSC3(5)), a fluorescent probe that exhibits ∆ψ-dependent membrane accumulation (Figure S3). Further, pre-incubation of E. coli with the proton ionophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP; selectively targets the pH gradient of cells) prior to treatment with sodium bicarbonate and gentamicin, reversed the potentiation observed (Figure 2B). Other antibiotics that rely on cellular energetics for entry include fosfomycin and novobiocin. The former is actively transported via a glycerol-3-phosphate permease where transport activity has been shown to be dependent on ∆pH18. Uptake of novobiocin is similarly an active transport mechanism supported


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by ∆pH such that uncouplers and inhibitors of respiration have been shown to reduce its cellular accumulation19. Accordingly, sodium bicarbonate suppressed the activity of fosfomycin and novobiocin (Figure S1). So too did nigericin, an ionophore that selectively dissipates the pH gradient (Figure S4). Overall, sodium bicarbonate influences the entry of antibacterial agents that are driven by PMF, suppressing those that require an intact pH gradient across the cytoplasmic membrane and enhancing those that are driven by ∆ψ, such as the polycationic aminoglycosides. Fluoroquinolone (FQ) antibiotics showed a variety of responses in the presence of 25 mM sodium bicarbonate depending on their physicochemical properties and the organism in question. While convention holds that FQ uptake is a passive process, previous studies have noted that the addition of the protonophore CCCP results in increased uptake of some FQs20-21, suggesting a role for the ∆ψ component of the proton motive force. Consistent with this, we saw a potentiation of various FQs by bicarbonate. The activity of FQs in the presence of bicarbonate against E. coli correlated with the nature of the substituents at the C-7 position of the quinolone nucleus (Table S2). We observed that the activities of FQs containing more basic substituents at C-7 (e.g. ciprofloxacin and besifloxacin) increased in the presence of bicarbonate, while those with more acidic substituents (e.g. nalidixic acid and pefloxacin) were suppressed (Figure 1, Table S1). These results are consistent with the idea that the electrochemical component (∆ψ) of the proton motive force has a role in FQ uptake. Compensatory increases in ∆ψ associated with dissipation of ∆pH by bicarbonate would favour the uptake of positively charged species22. Interestingly, in S. aureus, there was no enhancement by bicarbonate of FQs. Instead, a small suppression was observed for the antibacterial activity of this chemical class. Indeed, antibiotic uptake is a complex function of permeability and efflux23. Below, we note that the impact of bicarbonate on the pH gradient likely also impacts drug efflux, particularly in Gram-negative bacteria. Many multidrug efflux pumps depend on the PMF, where energy from the proton



gradient is harnessed to expel drugs from the cell, such as the Resistance-Nodulation-Division (RND)-system AcrAB-TolC in E. coli. Consistent with a role in dissipating ∆pH, we reasoned that bicarbonate should reduce efflux activity. The potentiation of dirithromycin, for example, by bicarbonate was lost in a strain lacking the outer membrane channel of this tripartite efflux system (∆tolC) (Figure 2C), suggesting it was inhibition of efflux by sodium bicarbonate that led to its enhanced activity. Notably, macrolide antibiotics are thought to be of little value for the treatment of Gram-negative bacteria due to their diminished accumulation24. Nevertheless, our experiments suggest that in the bicarbonate-rich environment of the host, energy-dependent efflux systems may be less effective than predicted by increases in MICs in conventional in vitro MIC determinations, often assumed to be the result of efflux pumps. Interestingly, macrolide antibiotics have been previously shown to be potentiated by normal human serum, but the serum factor responsible for the effect has remained elusive25. Our studies suggest bicarbonate is the causative agent. The antibacterial activity of inhibitors of cell wall synthesis was attenuated in the presence of sodium bicarbonate in E. coli only 2-4 fold on average, but this suppression was more pronounced in S. aureus, which is generally more susceptible to cell wall synthesis inhibitors than Gram-negative bacteria. Where cell wall-active compounds are most effective on actively dividing bacteria, suppression of the action of the cidal antibiotics, β-lactams and cephalosporins, for example, may be due to reduced respiratory energy production to fuel growth in the presence of PMF-altering concentrations of bicarbonate26. Accordingly, we observed a profound effect on cellular respiration (70% reduction) in E. coli treated with 25 mM sodium bicarbonate (Figure S5A). Consistent with this finding, E. coli grown in high concentrations of sodium bicarbonate exhibited an extended lag phase, implying lowered metabolic resources (Figure S5B). Further, intracellular ATP levels, which are produced via the F0F1-ATPase utilizing PMF, were reduced by ~30% in sodium bicarbonate-treated E. coli compared to the untreated control (Figure S5C). Overall, these experiments suggest that


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bicarbonate is a bacteriostatic compound that perturbs cellular respiration and reduces the activity of cell wall-active antibiotics that require actively growing bacteria for activity. Chemical genomics studies further reveal action of bicarbonate on the proton motive force To further explore the mode of action of bicarbonate on E. coli physiology, we assessed the impact of 25 mM sodium bicarbonate on an ordered E. coli gene-deletion collection of ∼4,000 strains27. Sodium bicarbonate profoundly reduced the growth of 28 deletion strains. Most significantly the missing genes encoded proteins involved in redox reactions and oxidative stress responses (Figure 3A, Table S3). Among them was dsbB, whose gene product is required to maintain disulfide bonds in periplasmic enzymes at extreme pHs28, and the gene encoding the sigma factor RpoS that regulates several components of resistance to both acid and base28. Deletion in the gene cydX, coding for a cytochrome oxidase, caused sensitization to bicarbonate. Cytochrome oxidases are, of course, key components of the bacterial respiratory chain. Additionally, deletion in the gene nhaA, which encodes a Na+:H+ antiporter that has a prominent role in sodium ion and alkaline pH homeostasis in E. coli and many enterobacteria29, sensitized cells to bicarbonate. Thus, a defect in proton expulsion enhanced the growth inhibition by bicarbonate. Deletion of cya was also sensitized to bicarbonate. Interestingly, this mutation is known to reduce ∆ψ30. Overall, gene deletions sensitized to bicarbonate involved pH-related processes, through proton expulsion or stress responses, that when deleted amplify bicarbonate’s action on the pH gradient across the inner membrane. We further explored the action of sodium bicarbonate on E. coli, by analyzing promoter activity in response to 25 mM sodium bicarbonate using a genome-scale, promoter-reporter library where nearly all of the promoters in E. coli have been transcriptionally fused to gfp31 (Figure 3B, Table S3). Overall, we observed changes in promoter activity that reflected adaptive strategies by the bacterium to maintain pH homeostasis. For instance, promoter activity for a large number of substrate/proton



antiporters was repressed in the presence of bicarbonate, presumably a practical response to a loss in the utility of these transporters as a result of reduction in ∆pH by bicarbonate. Promoter activity for nhaA was enhanced in the presence of bicarbonate. Stimulation of Na+ efflux by this electrogenic antiporter may facilitate a compensatory increase in ∆ψ, and survival under the dissipated ∆pH imposed by bicarbonate. Further, the activation of nhaA may help to restore a transmembrane pH gradient through cytoplasmic alkalinization, as previously reported32. Indeed, upon treatment with bicarbonate, cytoplasmic pH, which began at ~7.5 endured a rapid cytoplasmic alkalinization, as measured by BCECF-AM (Figure S5D). The expression of a number of inner membrane protein/transporters was differentially regulated in the presence of bicarbonate. While their functions are poorly understood, these transporters might make available or restrict different kinds of substrates to aid in pH homeostasis. Finally, decreased promoter activity was also observed for many ATP-dependent processes, likely as an adaptive effort to conserve energy. Overall, E. coli adaptation to bicarbonate involved strategies to respond to periplasmic pH changes, increase membrane potential and preserve energy. Bicarbonate selectively dissipates the transmembrane pH gradient We have previously shown that agents that selectively dissipate ∆ψ and ∆pH, respectively, are synergistic inhibitors of bacterial growth33. Accordingly, we reasoned that bicarbonate should enhance the activity of molecules that selectively perturb ∆ψ. To test this, we combined the following ∆ψ dissipaters with sodium bicarbonate: valinomycin, a selective potassium ionophore, as well as compounds we have previously characterized as dissipaters of ∆ψ, namely I1, I2 and I333 and loperamide34. All combinations yielded synergistic interactions, consistent with the role of bicarbonate as a selective dissipater of ∆pH (Figure S6). Importantly, the finding suggests that membrane active agents that target ∆ψ, while frequently eschewed in drug discovery efforts for potential cytotoxicity, may have superior activity in the bicarbonate-rich environment of the host. PMF is driven in part by a transmembrane gradient where the periplasmic side of the


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membrane has a greater concentration of protons. Thus, the addition of buffering agents to alter the external pH can have a drastic effect on PMF35. To test the impact of such a perturbation, we added trisodium phosphate (Na3PO4), which increased the pH of the media by 3 units and noted that this also potentiated the activity of dirithromycin. Adjusting the pH back to neutrality, however, led to a loss of synergy (Figure S7A). Importantly, this is was not the case with sodium bicarbonate. So, what makes bicarbonate special? Scheme 1 illustrates the bicarbonate buffer system that equilibrates carbonic acid (H2CO3), bicarbonate (HCO3-) and CO2 in order to maintain pH in human blood and tissues. The system is a complex equilibrium where carbonic acid exists in balance with its conjugate base bicarbonate, and carbonic anhydrases (human or bacterial) speed up the interconversion of carbonic acid with carbon dioxide and water. With a pKa of 6.1 for carbonic acid, bicarbonate is the dominant species at physiological pH (7.4) and has particular capacity to buffer protons. We speculate that because CO2 can escape the system, whether from a petri dish or the human body, where the latter is facilitated by the respiratory apparatus, the bicarbonate buffer system is uniquely driven by Le Chatelier’s Principle. The latter pushes this reaction to the left, liberating CO2 and consuming protons until excess acid is removed. We posit that the bicarbonate buffer system is one-of-a-kind and especially capable of consuming excess protons that comprise the pH gradient of the PMF. Bicarbonate and its conjugate acid carbonic acid are poorly lipid soluble and must be transported to enter bacterial cells. Consistent with the idea that bicarbonate is acting extracytoplasmic, presumably in the periplasmic space, we saw no difference in bicarbonate’s ability to potentiate dirithromycin in a bicarbonate transporter-deficient strain (∆ychM) and in a wildtype strain (Figure S7B). Effect of bicarbonate on the antibacterial activity of components of innate immunity The notion that bicarbonate is the dominant buffer in the human body that acts on a universal bacterial target led us to investigate the hypothesis that bicarbonate is an overlooked



component of antimicrobial host defense that may alter the antibacterial activity of other components of innate immunity. To this end, we investigated the influence of sodium bicarbonate (pH 7.4) on the in vitro antibacterial activity of various secretory molecules and cellular components that make up innate immunity against bacterial pathogens. Sodium bicarbonate itself exhibited antibacterial activity against many clinically relevant pathogens, with MIC values ranging from 50-100 mM (Table S4). We assessed the ability of sodium bicarbonate, at the sub-MIC but physiological concentration of 25 mM, to potentiate the activity of various mediators of host defense, including defensins and cathelicidins, whose family members make up the principal components of innate immunity in vertebrates36. The antimicrobial activity of alpha-defensin and LL-37 were enhanced 4 to 8-fold against E. coli (Figure 4A,E) and S. aureus (Table S5). Other antimicrobial peptides, such as indolicidin and bactenesin, were also highly potentiated in the presence of bicarbonate, 128- and 256-fold, respectively against E. coli (Figure 4B,C), and 16- and 256-fold, respectively against S. aureus (Table S5). Also enhanced in the presence of bicarbonate, was the activity of the porcrine leukocyte protegrin (8-fold in both E. coli and S. aureus) (Figure 4D, Table S5). Additionally, a physiological concentration of sodium bicarbonate enhanced the inhibitory activity of other innate immunity chemical factors such as lysozyme and bile salts against E. coli (Figure 4F,G). Interestingly, the innate immunity chemical barrier, hyaluronic acid, which is ubiquitously expressed in the extracellular matrix of all vertebrate tissues was also highly potentiated in the presence of sodium bicarbonate, 64-fold in both E. coli and S. aureus (Figure 4H, Table S5). Common among these components of innate immunity is their ultimate action on the cytoplasmic membrane causing membrane depolarization, suggesting for the first time, a role for bicarbonate as a mediator of membrane attack. Indeed, the majority of innate immunity constituents tested herein are ultimately perturbants of membrane potential37-40, suggesting a concerted attack by the host on bacterial PMF using these factors in bicarbonate-rich environment. Overall, our studies of innate immune components suggested that sodium


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bicarbonate is intrinsically antibacterial and an integral player in immunity, working in synergy with physical and chemical barriers to eliminate pathogens that cause infection. Discussion In the work described here, we explored the unique interactions of bicarbonate with antibiotics and components of the innate immune system. Combinatorial analyses with conventional antibiotics and chemical genomic studies led to hypothesis testing using physiological and biochemical methods to reveal that bicarbonate is intrinsically antibacterial and has a distinct mechanism of action. It acts as a selective dissipater of the transmembrane pH gradient that, together with the membrane potential, make up the proton motive force (PMF) of bacteria. We showed that with this unique mode of action is a powerful modulator of the activity of many components of innate immunity and of a variety of antibiotics, as these depend on the PMF for activity and/or uptake. We also demonstrated that, among many other organic salts, bicarbonate is unique in its effects, reinforcing the power of this buffer system. Indeed, the efficiency of bicarbonate buffer system in consuming excess protons provides an elegant mechanism to short-circuit the bacterial transmembrane proton gradient. The widespread impact, both enhancement and suppression, of the activity of conventional antibiotics in the presence of physiological concentrations of bicarbonate raises important clinical implications. For instance, our work suggests potentially unrecognized in vivo activities for aminoglycosides, macrolides and some fluoroquinolones, while other antibiotics, such as tetracyclines, fosfomycin, novobiocin, cell wall actives and other fluoroquinolones may not be as effective in the bicarbonate-rich environment of the host. Importantly, a deep understanding of the mode of action of bicarbonate can enable predictions to enrich for in vivoeffective molecules. Overall, the activities of antibacterial agents that are cationic in nature, that utilize the bacterial membrane potential for entry, that are substrates to PMF-dependent efflux systems and/or that selectively disrupt the membrane potential should be potentiated by bicarbonate.



Where physiological barriers that make up innate immunity are generally thought to consist of various body secretions such as antimicrobial peptides, bile and lysozyme, our work suggests that the natural bicarbonate buffering system of the human body is an overlooked component of innate immunity that not only prevents the growth of microbes but also works in synergy with other components to disrupt bacterial PMF. Indeed, the ubiquitous distribution of sodium bicarbonate throughout the bloodstream and various bodily tissues raises intriguing possibilities regarding its potential role in innate immunity against infection. In all, the mechanistic work described herein suggests that tests of antibacterial potency in conventional microbiological media may fail to predict in vivo susceptibility of antibacterial compounds that are impacted by physiological concentrations of bicarbonate. Most interestingly, such compounds include many chemical components of the innate immune system that we show are potentiated by this ubiquitous buffer system. We conclude that bicarbonate has remarkable power as an antibiotic adjuvant and suggest that there is great potential to exploit this activity in the discovery and development of new antibacterial drugs.

Methods Materials and bacterial strains. All antibiotics and chemicals used in the study were purchased from Sigma Aldrich. Defensin was purchased from Innovagen. CCCP concentration of 20 µM was used. The main strains used in this study were E. coli (K-12 BW25113) and S. aureus (Strain Newman). Knockout strains (∆tolcC and ∆ychM) were used from the Keio knockout collection 27. In all experiments, bacterial cells were cultured in 96-well microtiter plates in cation-adjusted Mueller-Hinton Broth (MHB). In all experiments, where necessary, pH was adjusted with HCl upon addition of the higher concentrations of sodium bicarbonate. MIC determinations and checkerboard analyses. Protocol for MIC determinations was based on CLSI guidelines. Plates were incubated at 37°C for 18 hours (stationary incubator for E. coli and 250 r.p.m for S. aureus) and optical density read at 600 nm. At least 3 replicates


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were done for each query compound. The MIC was the lowest concentration showing

Bicarbonate Alters Bacterial Susceptibility to Antibiotics by Targeting

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