Bicarbonate Alters Bacterial Susceptibility to Antibiotics by Targeting the Proton Motive Force

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 the 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

Entrapment of Living Bacterial Cells in Low-Concentration Silica Materials Preserves Cell Division and Promoter Regulation

The entrapment of bacterial cells within inorganic silica materials was reported almost 20 years ago. However, almost all studies to date have shown that these entrapped cells are unable to divide and thus should be expected to have reduced promoter activity. In view of the importance of bacteria as model systems for both fundamental and applied biological studies, it is crucial that immobilized cells retain solutionlike properties, including the ability to divide and display normal promoter activity. Herein we report on a method to immobilize bacterial cells within low-density inorganic silica-based materials, where the cells retain both cell division and promoter activity. Sol–gel processing was used to entrap Escherichia coli cells carrying a variety of green fluorescent protein-linked promoters into sodium silicate-derived materials that were formed in microwell plates. Using a series of assays, we were able to demonstrate that (1) the entrapped cells can divide within the pores of the silica matrix, (2) cellular pathways are regulated in a similar manner in both solution and the sol–gel-derived materials, and (3) promoters in entrapped cells can be specifically induced with small molecules (e.g., antimicrobial compounds) in a concentration-dependent manner to allow assessment of both potency and mode of action. This solid-phase assay system was tested using multiple antimicrobial pathways and should enable the development of solid-phase assays for the discovery of new small molecules that are active against bacteria

Exploiting the Sensitivity of Nutrient Transporter Deletion Strains in Discovery of Natural Product Antimetabolites

Actinomycete secondary metabolites are a renowned source of antibacterial chemical scaffolds. Herein, we present a target-specific approach that increases the detection of antimetabolites from natural sources by screening actinomycete-derived extracts against nutrient transporter deletion strains. On the basis of the growth rescue patterns of a collection of 22 <i>Escherichia coli</i> (<i>E. coli</i>) auxotrophic deletion strains representative of the major nutrient biosynthetic pathways, we demonstrate that antimetabolite detection from actinomycete-derived extracts prepared using traditional extraction platforms is masked by nutrient supplementation. In particular, we find poor sensitivity for the detection of antimetabolites targeting vitamin biosynthesis. To circumvent this and as a proof of principle, we exploit the differential activity of actinomycete extracts against <i>E. coli ΔyigM</i>, a biotin transporter deletion strain versus wildtype <i>E. coli</i>. We achieve more than a 100-fold increase in antimetabolite sensitivity using this method and demonstrate a successful bioassay-guided purification of the known biotin antimetabolite, amiclenomycin. Our findings provide a unique solution to uncover the full potential of naturally derived antibiotics

MICs, MBCs and MBICs for the top 15 compounds tested against ten Bcc strains^a.

<p>^a The strains tested were <i>B</i>. <i>cepacia</i> CEP509, <i>B</i>. <i>multivorans</i> C5393, <i>B</i>. <i>cenocepacia</i> J2315, <i>B</i>. <i>cepacia</i> C7322, <i>B</i>. <i>vietnamiensis</i> PC259, <i>B</i>. <i>cepacia</i> CEP021, <i>B</i>. <i>ambifaria</i> CEP0996, <i>B</i>. <i>anthina</i> AU1293, <i>B</i>. <i>pyrrocinia</i> C1469 and <i>B</i>. <i>contaminans</i> FFH-2055.</p><p>MICs, MBCs and MBICs for the top 15 compounds tested against ten Bcc strains<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128587#t002fn001" target="_blank">^a</a>.</p

A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against <i>Burkholderia cenocepacia</i>

<div><p>Infections with the bacteria <i>Burkholderia cepacia</i> complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, <i>B</i>. <i>cenocepacia</i> K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using <i>C</i>. <i>elegans</i> was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.</p></div

High throughput screening and compound prioritization process.

<p>Steps are shown as arrows with the name of the step to the left and the selection method to the right.</p

<i>C</i>. <i>elegans</i> rescue assays. <i>C</i>. <i>elegans</i> was allowed to feed on <i>B</i>. <i>cenocepacia</i> K56-2 and OP50 for 16 hours.

<p>The <i>B</i>. <i>cenocepacia</i> infected and OP50-fed worms were subsequently treated with (A) Trimethoprim (TP), Tetracycline (Tet), Meropenem (Mero), Chloramphenicol (Chl) and observed for 6 days every 24 hours and were compared to the non-treated (No Antibiotic) worms for survival. p < 0.0001 for all compounds. (B) The worms were treated with MAC-0013209, (p = 0.2503) with MAC-0151023 and MAC-0036650 at their respective MIC (128 μg/mL, 32 μg/mL and 16 μg/mL); p< 0.0001 for both MAC-0151023 and MAC-0036650 compounds. Trimethoprim was used as a control. p < 0.0001 for all concentrations.</p

Survival ₁₀₀ (Surv₁₀₀) assay, and the Surv₁₀₀/MIC ratio determination.

<p>(A) The Surv₁₀₀ assay was conducted in a 96-well format, where each compound was serially diluted along the rows from its highest soluble concentration or the MIC. The last well for each compound was used as a DMSO control. Approximately, 5 to 10 OP50-fed worms were added to the wells containing LKM for a total assay volume of 100 μl. The number of worms was counted and recorded for each concentration on day 0 and again 24 hours later. Percent survival was determined for each concentration. The Surv100/MIC ratio was calculated. The compounds, which demonstrated a ratio of 1 and greater, were used in the in vivo antibiotic activity. (B) Photograph of the worms from the assay illustrating the difference between 0% survival and 100% survival.</p

Zinc Chelation by a Small-Molecule Adjuvant Potentiates Meropenem Activity in Vivo against NDM-1-Producing Klebsiella pneumoniae

The widespread emergence of antibiotic drug resistance has resulted in a worldwide healthcare crisis. In particular, the extensive use of β-lactams, a highly effective class of antibiotics, has been a driver for pervasive β-lactam resistance. Among the most important resistance determinants are the metallo-β-lactamases (MBL), which are zinc-requiring enzymes that inactivate nearly all classes of β-lactams, including the last-resort carbapenem antibiotics. The urgent need for new compounds targeting MBL resistance mechanisms has been widely acknowledged; however, the development of certain types of compoundsnamely metal chelatorsis actively avoided due to host toxicity concerns. The work herein reports the identification of a series of zinc-selective spiro-indoline-thiadiazole analogues that, in vitro, potentiate β-lactam antibiotics against an MBL-carrying pathogen by withholding zinc availability. This study demonstrates the ability of one such analogue to inhibit NDM-1 in vitro and, using a mouse model of infection, shows that combination treatment of the respective analogue with meropenem results in a significant decrease in bacterial burden in contrast to animals that received antibiotic treatment alone. These results support the therapeutic potential of these chelators in overcoming antibiotic resistance

Inhibition of WTA Synthesis Blocks the Cooperative Action of PBPs and Sensitizes MRSA to β‑Lactams

Rising drug resistance is limiting treatment options for infections by methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Herein we provide new evidence that wall teichoic acid (WTA) biogenesis is a remarkable antibacterial target with the capacity to destabilize the cooperative action of penicillin-binding proteins (PBPs) that underlie β-lactam resistance in MRSA. Deletion of gene <i>tarO</i>, encoding the first step of WTA synthesis, resulted in the restoration of sensitivity of MRSA to a unique profile of β-lactam antibiotics with a known selectivity for penicillin binding protein 2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously approved drugs with a cryptic capacity to potentiate its activity against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly potentiated cefuroxime, and this synergy was abolished in strains lacking <i>tarO</i>. The combination was also effective in a <i>Galleria mellonella</i> model of infection. Using both genetic and biochemical strategies, we determined the molecular target of ticlopidine as the <i>N-</i>acetylglucosamine-1-phosphate transferase encoded in gene <i>tarO</i> and provide evidence that WTA biogenesis represents an Achilles heel supporting the cooperative function of PBP2 and PBP4 in creating highly cross-linked muropeptides in the peptidoglycan of <i>S. aureus</i>. This approach represents a new paradigm to tackle MRSA infection