SC5005 dissipates the membrane potential to kill Staphylococcus aureus persisters without detectable resistance

Abstract Objectives In the past few decades, multiple-antibiotic-resistant Staphylococcus aureus has emerged and quickly spread in hospitals and communities worldwide. Additionally, the formation of antibiotic-tolerant persisters and biofilms further reduces treatment efficacy. Previously, we identified a sorafenib derivative, SC5005, with bactericidal activity against MRSA in vitro and in vivo. Here, we sought to elucidate the resistance status, mode of action and anti-persister activity of this compound. Methods The propensity of S. aureus to develop SC5005 resistance was evaluated by assessment of spontaneous resistance and by multi-passage selection. The mode of action of SC5005 was investigated using macromolecular synthesis, LIVE/DEAD and ATPlite assays and DiOC2(3) staining. The effect of SC5005 on the mammalian cytoplasmic membrane was measured using haemolytic and lactate dehydrogenase (LDH) assays and flow cytometry. Results SC5005 depolarized and permeabilized the bacterial cytoplasmic membrane, leading to reduced ATP production. Because of this mode of action, no resistance of S. aureus to SC5005 was observed after constant exposure to sub-lethal concentrations for 200 passages. The membrane-perturbing activity of SC5005 was specific to bacteria, as no significant haemolysis or release of LDH from human HT-29 cells was detected. Additionally, compared with other bactericidal antibiotics, SC5005 exhibited superior activity in eradicating both planktonic and biofilm-embedded S. aureus persisters. Conclusions Because of its low propensity for resistance development and potent persister-eradicating activity, SC5005 is a promising lead compound for developing new therapies for biofilm-related infections caused by S. aureus.

Download Full-text

CRISPR-Cas9 modified bacteriophage for treatment of Staphylococcus aureus induced osteomyelitis and soft tissue infection

10.1101/705830 ◽

2019 ◽

Author(s):

Leah K. Horstemeyer ◽

JooYoun Park ◽

Elizabeth A. Swanson ◽

Mary Catherine Beard ◽

Emily M. McCabe ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Soft Tissue ◽

Soft Tissue Infection ◽

Bone Infection ◽

Tissue Infection ◽

Fluorescent Imaging ◽

Alginate Hydrogel ◽

Antibiotic Resistant

AbstractOsteomyelitis, or bone infection, is often induced by antibiotic resistant Staphylococcus aureus strains of bacteria. Although debridement and long-term administration of antibiotics are the gold standard for osteomyelitis treatment, the increase in prevalence of antibiotic resistant bacterial strains limits the ability of clinicians to effectively treat infection. Bacteriophages (phages), viruses that effectively lyse bacteria, have gained recent attention for their high specificity, non-toxicity, and the low likelihood of resistance development by pathogens. Previously, we have shown that CRISPR-Cas9 genomic editing techniques could be utilized to expand bacteriophage host range and enhance bactericidal activity through modification of the tail fiber protein, as well as improve safety with removal of major virulence genes. In a dermal infection study, these CRISPR-Cas9 phages reduced bacterial load relative to unmodified phage. Thus, we hypothesized this bacteriophage would be effective to mitigate infection from a biofilm forming S. aureus strain in vitro and in vivo. In vitro, qualitative fluorescent imaging demonstrated superiority of phage to conventional vancomycin and fosfomycin antibiotics against S. aureus biofilm. Quantitative antibiofilm effects increased over time for fosfomycin, phage, and fosfomycin-phage (dual) therapeutics delivered via alginate hydrogel. We developed an in vivo rat model of osteomyelitis and soft tissue infection that was reproducible and challenging and enabled longitudinal monitoring of infection progression. Using this model, phage (with and without fosfomycin) delivered via alginate hydrogel were successful in reducing soft tissue infection but not bone infection, based on bacteriological, histological, and scanning electron microscopy analyses. Notably, the efficacy of phage at mitigating soft tissue infection was equal to that of high dose fosfomycin. Future research may utilize this model as a platform for evaluation of therapeutic type and dose, and alternate delivery vehicles for osteomyelitis mitigation.

Download Full-text

Mechanism of Action of a Prostaglandin E2 Receptor Antagonist with antimicrobial activity against Staphylococcus aureus

10.1101/2020.01.21.914200 ◽

2020 ◽

Author(s):

Elizabeth A. Lilly ◽

Mélanie A. C. Ikeh ◽

Paul L. Fidel ◽

Mairi C. Noverr

Keyword(s):

Staphylococcus Aureus ◽

Antimicrobial Activity ◽

Receptor Antagonist ◽

Mechanism Of Action ◽

Atp Production ◽

Direct Growth ◽

Prostaglandin E2 Receptor ◽

Intra Abdominal Infection

AbstractOur laboratory recently reported that the EP4 receptor antagonist, L-161,982, had direct growth-inhibitory effects on Staphylococcus aureus in vitro and in vivo, reducing microbial burden and providing significant protection against lethality in models of S. aureus monomicrobial and polymicrobial intra-abdominal infection. This antimicrobial activity was observed with both methicillin-sensitive and methicillin-resistant S. aureus (MRSA), as well as other Gram-positive bacteria. The antimicrobial activity of L-161,982 was independent of EP4 receptor inhibitory activity. In this study, we investigated the mechanism of action (MOA) of L-161,982, which contains a sulfonamide functional group. However, results demonstrate L-161,982 does not affect folate synthesis (sulfonamide MOA), oxidative stress, or membrane permeability. Instead, our results suggest that the inhibitor works via effects on inhibition of the electron transport chain (ETC). Similar to other ETC inhibitors, L-161,982 exposure results in a small colony size variant phenotype and inhibition of pigmentation, as well as significantly reduced hemolytic activity, and ATP production. In addition, L-161,982 potentiated the antimicrobial activity of another ETC inhibitor and inhibition was partially rescued by supplementation with nutrients required for ETC auxotrophs. Taken together, these findings demonstrate that L-161,982 exerts antimicrobial activity against MRSA via inhibition the ETC, representing a new member of a potentially novel antimicrobial drug class.

Download Full-text

Mode of Action,In VitroActivity, andIn VivoEfficacy of AFN-1252, a Selective Antistaphylococcal FabI Inhibitor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01411-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5865-5874 ◽

Cited By ~ 70

Author(s):

Nachum Kaplan ◽

Monique Albert ◽

Donald Awrey ◽

Elias Bardouniotis ◽

Judd Berman ◽

...

Keyword(s):

Mode Of Action ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Pharmacokinetic Studies ◽

Coagulase Negative Staphylococci ◽

Resistance Development ◽

Content Type ◽

Peritoneal Infection

ABSTRACTThe mechanism of action of AFN-1252, a selective inhibitor ofStaphylococcus aureusenoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252–FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistantS. aureus, achieving a ≥2-log10reduction inS. aureuscounts over 24 h, and was extremely potent against clinical isolates ofS. aureus(MIC90, 0.015 μg/ml) and coagulase-negative staphylococci (MIC90, 0.12 μg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection ofS. aureusSmith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potentin vitroactivity, andin vivoefficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.

Download Full-text

Rapid resistance development to three antistaphylococcal therapies in antibiotic-tolerant staphylococcus aureus bacteremia

PLoS ONE ◽

10.1371/journal.pone.0258592 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258592

Author(s):

Christopher R. Miller ◽

Jonathan M. Monk ◽

Richard Szubin ◽

Andrew D. Berti

Keyword(s):

Staphylococcus Aureus ◽

Subsequent Development ◽

Multidrug Resistant ◽

Blood Cultures ◽

Therapeutic Interventions ◽

Prevention Measures ◽

Beta Lactam ◽

Resistance Development

Understating how antibiotic tolerance impacts subsequent resistance development in the clinical setting is important to identifying effective therapeutic interventions and prevention measures. This study describes a patient case of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia which rapidly developed resistance to three primary MRSA therapies and identifies genetic and metabolic changes selected in vivo that are associated with rapid resistance evolution. Index blood cultures displayed susceptibility to all (non-beta-lactam) antibiotics with the exception of trimethoprim/ sulfamethoxazole. One month after initial presentation, during the same encounter, blood cultures were again positive for MRSA, now displaying intermediate resistance to vancomycin and ceftaroline and resistance to daptomycin. Two weeks later, blood cultures were positive for a third time, still intermediate resistant to vancomycin and ceftaroline and resistant to daptomycin. Mutations in mprF and vraT were common to all multidrug resistant isolates whereas mutations in tagH, agrB and saeR and secondary mprF mutation emerged sequentially and transiently resulting in distinct in vitro phenotypes. The baseline mutation rate of the patient isolates was unremarkable ruling out the hypermutator phenotype as a contributor to the rapid emergence of resistance. However, the index isolate demonstrated pronounced tolerance to the antibiotic daptomycin, a phenotype that facilitates the subsequent development of resistance during antibiotic exposure. This study exemplifies the capacity of antibiotic-tolerant pathogens to rapidly develop both stable and transient genetic and phenotypic changes, over the course of a single patient encounter.

Download Full-text

Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00028-06 ◽

2006 ◽

Vol 50 (7) ◽

pp. 2448-2454 ◽

Cited By ~ 26

Author(s):

A. S. Bayer ◽

L. I. Kupferwasser ◽

M. H. Brown ◽

R. A. Skurray ◽

S. Grkovic ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cytoplasmic Membrane ◽

Efflux Pump ◽

Multidrug Transporter ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Pump Activity ◽

The Impact

ABSTRACT Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.

Download Full-text

Heme-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

10.1101/2021.06.29.450315 ◽

2021 ◽

Author(s):

Izabela Z Batko ◽

Ronald S Flannagan ◽

Veronica Guariglia-Oropeza ◽

Jessica R Sheldon ◽

David E Heinrichs

Keyword(s):

Staphylococcus Aureus ◽

Iron Acquisition ◽

Iron Starvation ◽

Minimal Growth ◽

Atp Production ◽

Small Colony ◽

Sense And Respond ◽

Outstanding Question

The ability to acquire iron is essential for Staphylococcus aureus to cause infection. Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they too acquire iron. How SCVs obtain iron remains unknown and so here we addressed this outstanding question by creating a stable hemB mutant in S. aureus USA300 strain LAC. The mutant, auxotrophic for hemin, was assessed for its ability to grow under iron-restriction and with various iron sources. The hemB SCV utilizes exogenously supplied heme but was attenuated for growth under conditions of iron starvation. RNA-seq analyses showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrates limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine, which enabled the SCV to sustain only minimal growth in iron deplete media. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo. Indeed, the administration of Desferal failed to promote hemB bacterial growth in vivo, in contrast to WT, in every organ analyzed except for the murine kidney where growth was enhanced. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased heme availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that heme is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes.

Download Full-text

Killing of Staphylococcus aureus via Magnetic Hyperthermia Mediated by Magnetotactic Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.04103-15 ◽

2016 ◽

Vol 82 (7) ◽

pp. 2219-2226 ◽

Cited By ~ 16

Author(s):

Changyou Chen ◽

Linjie Chen ◽

Yong Yi ◽

Chuanfang Chen ◽

Long-Fei Wu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Polyclonal Antibodies ◽

Magnetotactic Bacteria ◽

Magnetic Hyperthermia ◽

Antibiotic Resistant ◽

Treatment Development ◽

Content Type ◽

Conventional Plate

ABSTRACTStaphylococcus aureusis a common hospital and household pathogen. Given the emergence of antibiotic-resistant derivatives of this pathogen resulting from the use of antibiotics as general treatment, development of alternative therapeutic strategies is urgently needed. Here, we assess the feasibility of killingS. aureuscellsin vitroandin vivothrough magnetic hyperthermia mediated by magnetotactic bacteria that possess magnetic nanocrystals and demonstrate magnetically steered swimming. TheS. aureussuspension was added to magnetotactic MO-1 bacteria either directly or after coating with anti-MO-1 polyclonal antibodies. The suspensions were then subjected to an alternating magnetic field (AMF) for 1 h.S. aureusviability was subsequently assessed through conventional plate counting and flow cytometry. We found that approximately 30% of theS. aureuscells mixed with uncoated MO-1 cells were killed after AMF treatment. Moreover, attachment between the magnetotactic bacteria andS. aureusincreased the killing efficiency of hyperthermia to more than 50%. Using mouse models, we demonstrated that magnetic hyperthermia mediated by antibody-coated magnetotactic MO-1 bacteria significantly improved wound healing. These results collectively demonstrated the effective eradication ofS. aureusbothin vitroandin vivo, indicating the potential of magnetotactic bacterium-mediated magnetic hyperthermia as a treatment forS. aureus-induced skin or wound infections.

Download Full-text

Novel Zinc-Attenuating Compounds as Potent Broad-Spectrum Antifungal Agents withIn VitroandIn VivoEfficacy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02024-17 ◽

2018 ◽

Vol 62 (5) ◽

Cited By ~ 5

Author(s):

Karen A. O'Hanlon Cohrt ◽

Laura Marín ◽

Lasse Kjellerup ◽

Johannes D. Clausen ◽

William Dalby-Brown ◽

...

Keyword(s):

Antifungal Activity ◽

Broad Spectrum ◽

Mode Of Action ◽

Zinc Homeostasis ◽

Growth Media ◽

Uptake System ◽

Resistance Development ◽

Content Type

ABSTRACTAn increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resistance to the currently available antifungal drugs calls for new broad-spectrum antifungals with a novel mechanism of action. Here we report the identification and characterization of two novel zinc-attenuating compounds, ZAC307 and ZAC989, which exhibit broad-spectrumin vitroantifungal activity andin vivoefficacy in a fungal kidney burden candidiasis model. The compounds were identified serendipitously as part of a drug discovery process aimed at finding novel inhibitors of the fungal plasma membrane proton ATPase Pma1. Based on their structure, we hypothesized that they might act as zinc chelators. Indeed, both fluorescence-based affinity determination and potentiometric assays revealed these compounds, subsequently termed zinc-attenuating compounds (ZACs), to have strong affinity for zinc, and their growth inhibitory effects onCandida albicansandAspergillus fumigatuscould be inactivated by the addition of exogenous zinc to fungal growth media. We determined the ZACs to be fungistatic, with a low propensity for resistance development. Gene expression analysis suggested that the ZACs interfere negatively with the expression of genes encoding the major components of theA. fumigatuszinc uptake system, thus supporting perturbance of zinc homeostasis as the likely mode of action. With demonstratedin vitroandin vivoantifungal activity, low propensity for resistance development, and a novel mode of action, the ZACs represent a promising new class of antifungal compounds, and their advancement in a drug development program is therefore warranted.

Download Full-text

In Vitro Activity of Rifabutin and Rifampin against Antibiotic-Resistant Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae

mSphere ◽

10.1128/msphere.00920-21 ◽

2021 ◽

Author(s):

Bosul Lee ◽

Jun Yan ◽

Amber Ulhaq ◽

Sarah Miller ◽

Wonjae Seo ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Klebsiella Pneumoniae ◽

General Effect ◽

In Vitro Activity ◽

Antibiotic Resistant ◽

Content Type

Rifabutin has been recently described as a potential adjunctive therapy for antibiotic-resistant A. baumannii infections due to hypersensitivity in iron-depleted media, which may more closely mimic an in vivo environment. Here, we report that this hyperactivity is specific for A. baumannii , rather than being a general effect for other pathogens.

Download Full-text

An Organoselenium Compound Inhibits Staphylococcus aureus Biofilms on Hemodialysis CathetersIn Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05680-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 972-978 ◽

Cited By ~ 52

Author(s):

Phat L. Tran ◽

Nathan Lowry ◽

Thomas Campbell ◽

Ted W. Reid ◽

Daniel R. Webster ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Bloodstream Infections ◽

Organoselenium Compound ◽

Central Venous ◽

Antibiotic Resistant ◽

Content Type

ABSTRACTColonization of central venous catheters (CVCs) by pathogenic bacteria leads to catheter-related bloodstream infections (CRBSIs). These colonizing bacteria form highly antibiotic-resistant biofilms.Staphylococcus aureusis one of the most frequently isolated pathogens in CRBSIs. Impregnating CVC surfaces with antimicrobial agents has various degrees of effectiveness in reducing the incidence of CRBSIs. We recently showed that organoselenium covalently attached to disks as an antibiofilm agent inhibited the development ofS. aureusbiofilms. In this study, we investigated the ability of an organoselenium coating on hemodialysis catheters (HDCs) to inhibitS. aureusbiofilmsin vitroandin vivo.S. aureusfailed to develop biofilms on HDCs coated with selenocyanatodiacetic acid (SCAA) in either static or flowthrough continuous-culture systems. The SCAA coating also inhibited the development ofS. aureusbiofilms on HDCsin vivofor 3 days. The SCAA coating was stable and nontoxic to cell culture or animals. This new method for coating the internal and external surfaces of HDCs with SCAA has the potential to prevent catheter-related infections due toS. aureus.

Download Full-text