Repurposing Toremifene for Treatment of Oral Bacterial Infections.

The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 µM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 µM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.

Measuring the Viscosity of the Escherichia coli Plasma Membrane Using Molecular Rotors.

The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5-40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes.

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

Implant functionalization with mesoporous silica: A promising antibacterial strategy, but does such an implant osseointegrate?

OBJECTIVES: New strategies for implant surface functionalization in the prevention of peri-implantitis while not compromising osseointegration are currently explored. The aim of this in vivo study was to assess the osseointegration of a titanium-silica composite implant, previously shown to enable controlled release of therapeutic concentrations of chlorhexidine, in the Göttingen mini-pig oral model. MATERIAL AND METHODS: Three implant groups were designed: macroporous titanium implants (Ti-Porous); macroporous titanium implants infiltrated with mesoporous silica (Ti-Porous + SiO2 ); and conventional titanium implants (Ti-control). Mandibular last premolar and first molar teeth were extracted bilaterally and implants were installed. After 1 month healing, the bone in contact with the implant and the bone regeneration in the peri-implant gap was evaluated histomorphometrically. RESULTS: Bone-to-implant contact and peri-implant bone volume for Ti-Porous versus Ti-Porous + SiO2 implants did not differ significantly, but were significantly higher in the Ti-Control group compared with Ti-Porous + SiO2 implants. Functionalization of titanium implants via infiltration of a SiO2 phase into the titanium macropores does not seem to inhibit implant osseointegration. Yet, the importance of the implant macro-design, in particular the screw thread design in a marginal gap implant surgery set-up, was emphasized by the outstanding results of the Ti-Control implant. CONCLUSIONS: Next-generation implants made of macroporous Ti infiltrated with mesoporous SiO2 do not seem to compromise the osseointegration process. Such implant functionalization may be promising for the prevention and treatment of peri-implantitis given the evidenced potential of mesoporous SiO2 for controlled drug release.

New approaches to combat Porphyromonas gingivalis biofilms.

In nature, bacteria predominantly reside in structured, surface-attached communities embedded in a self-produced, extracellular matrix. These so-called biofilms play an important role in the development and pathogenesis of many infections, as they are difficult to eradicate due to their resistance to antimicrobials and host defense mechanisms. This review focusses on the biofilm-forming periodontal bacterium Porphyromonas gingivalis. Current knowledge on the virulence mechanisms underlying P. gingivalis biofilm formation is presented. In addition, oral infectious diseases in which P. gingivalis plays a key role are described, and an overview of conventional and new therapies for combating P. gingivalis biofilms is given. More insight into this intriguing pathogen might direct the development of better strategies to combat oral infections.

Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogen involved in oral diseases such as periodontitis and peri-implantitis. Management of these diseases typically includes mechanical debridement of the colonized surfaces followed by application of antiseptics or antibiotics. Disadvantages associated with the use of antiseptics and the growing worldwide problem of antibiotic resistance have necessitated the search for alternative agents. In this study, the antibacterial and antibiofilm properties of AM404, an active metabolite of paracetamol, were tested against P. gingivalis and other bacterial pathogens. The activity of AM404 was tested against 10 bacteria, including both oral and nonoral human pathogens. The minimal inhibitory concentration (MIC) of AM404 was determined by measuring optical density (OD) values. The minimum biofilm inhibitory concentration (MBIC) was detected by crystal violet staining. The activity of structural analogs of AM404 was tested by MIC determinations. The effect of AM404 on P. gingivalis biofilms formed on titanium disks as a model for dental implants was evaluated by colony forming unit counting. Potential cytotoxicity of AM404 towards HEK-293 (human embryonic kidney cells), HepG2 (human hepatoma cells), IEC-6 (rat intestinal cells), and Panc-1 cells (pancreatic cancer cells) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. To get more insight in the mode of action of AM404, we used the fluorescent dyes N-phenyl-1-napthylamine and SYTOX green to investigate outer and inner membrane damage of P. gingivalis induced by AM404, respectively. Of all tested pathogens, AM404 only inhibited growth and biofilm formation of P. gingivalis. Moreover, it showed potent activity against P. gingivalis biofilms formed on titanium surfaces. A structure-activity analysis demonstrated that the unsaturated carbon chain is essential for its antibacterial activity. Importantly, AM404 was not toxic towards the tested mammalian cells up to concentrations approaching 4× the MIC. Membrane damage assays using fluorescent probes N-phenyl-1-napthylamine and SYTOX green revealed that membrane permeabilization presumably is the primary antibacterial mode of action of AM404. Collectively, our results suggest that AM404 has the potential to be used for the development of new drugs specifically targeting P. gingivalis-related infections.

In vitro activity of the antiasthmatic drug zafirlukast against the oral pathogens Porphyromonas gingivalis and Streptococcus mutans.

Oral infections are among the most common diseases worldwide. Many protocols for the prevention and treatment of oral infections have been described, yet no golden standard has been developed so far. The antiseptic chlorhexidine and antibiotics are often used in these treatment procedures. However, long-term use of chlorhexidine can lead to side effects and extensive use of antibiotics can promote the development of antibiotic-resistant bacteria, which in turn can compromise the effectiveness of the treatment. Consequently, it remains important to search for new antibacterial agents for the treatment of oral infections. In this study, we report on the antibacterial activity of the antiasthma drug zafirlukast against oral pathogens Porphyromonas gingivalis and Streptococcus mutans. Furthermore, its activity against oral biofilms grown on titanium surfaces was confirmed. In addition, we demonstrated that zafirlukast displays no cytotoxicity against human osteoblasts. Combined, this study paves the way for further research to determine the potential of zafirlukast to be used as a new antibiotic against oral pathogens.

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies.