Eradication of Bacterial Persister Cells By Leveraging Their Low Metabolic Activity Using Adenosine Triphosphate Coated Gold Nanoclusters.

Bacteria first develop tolerance after antibiotic exposure; later genetic resistance emerges through the population of tolerant bacteria. Bacterial persister cells are the multidrug-tolerant subpopulation within an isogenic bacteria culture that maintains genetic susceptibility to antibiotics. Because of this link between antibiotic tolerance and resistance and the rise of antibiotic resistance, there is a pressing need to develop treatments to eradicate persister cells. Current anti persister cell strategies are based on the paradigm of "awakening" them from their low metabolic state before attempting eradication with traditional antibiotics. Herein, we demonstrate that the low metabolic activity of persister cells can be exploited for eradication over their metabolically active counterparts. We engineered gold nanoclusters coated with adenosine triphosphate (AuNC@ATP) as a benchmark nanocluster that kills persister cells over exponential growth bacterial cells and prove the feasibility of this new concept. Finally, using AuNC@ATP as a new research tool, we demonstrated that it is possible to prevent the emergence of antibiotic-resistant superbugs with an anti-persister compound. Eradicating persister cells with AuNC@ATP in an isogenic culture of bacteria stops the emergence of superbug bacteria mediated by the sub-lethal dose of conventional antibiotics. Our findings lay the groundwork for developing novel nano-antibiotics targeting persister cells, which promise to prevent the emergence of superbugs and prolong the lifespan of currently available antibiotics.

Povidone-Iodine Fails to Eradicate Chronic Suppurative Otitis Media and Demonstrates Ototoxic Risk in Mice.

HYPOTHESIS: Commercially available povidone-iodine solution can eliminate biofilms and persister cells rapidly in in vivo achievable concentrations without inducing ototoxicity. BACKGROUND: Chronic suppurative otitis media (CSOM) is a substantial global problem. Current treatment options often induce a temporary remission without leading to a permanent cessation of symptoms secondary to the treatments' inability to eliminate persister cells. Povidone-iodine has been shown to be able to clear biofilm and planktonic cells in in vitro assays, but there are reports of ototoxic effects limiting its clinical utility. METHODS: Bacterial and biofilm growth with quantification by spectrophotomer, murine auditory brainstem response (ABR), and distortion product otoacoustic emissions, immunohistochemistry, in vivo povidone-iodine treatment of murine CSOM, persister cell assay. RESULTS: Commercially available 10% povidone-iodine solution is able to completely eradicate multiple clinical strains of Pseudomonas aeruginosa and Staphylococcus aureus in vitro with 10 minutes of exposure. Mice that have received a transtympanic injection of 1% povidone-iodine solution did not have significantly different auditory brainstem response or distortion product otoacoustic emission results compared with the control. Mice that received a povidone-iodine scrub or 10% povidone-iodine solution had significantly worsened hearing (25- and 13-dB increase in threshold, respectively; p < 0.05). In vivo CSOM infection recurred in all mice after the completion of treatment with 10% povidone-iodine solution, and there was no improvement in the bacterial load after treatment, indicating in vivo failure of therapy. CONCLUSION: Povidone-iodine solution is effective at eliminating biofilm and persister cells in vitro at in vivo achievable concentrations but fails in vivo most likely because of kinetics of distribution in vivo. Even if drug distribution could be improved, the therapeutic window is likely to be too small given that the diluted solution does not have ototoxic potential, whereas while the scrub variant, which contains detergents, and the undiluted solution are ototoxic after a single treatment.

Chronic suppurative otitis media causes macrophage-associated sensorineural hearing loss.

BACKGROUND: Chronic suppurative otitis media (CSOM) is the most common cause of permanent hearing loss in children in the developing world. A large component of the permanent hearing loss is sensory in nature and our understanding of the mechanism of this has so far been limited to post-mortem human specimens or acute infection models that are not representative of human CSOM. In this report, we assess cochlear injury in a validated Pseudomonas aeruginosa (PA) CSOM mouse model. METHODS: We generated persisters (PCs) and inoculated them into the mouse middle ear cavity. We tracked infection with IVIS and detected PA using RT-PCR. We assessed cochlear damage and innate immunity by Immunohistochemistry. Finally, we evaluated cytokines with multiplex assay and quantitative real-time PCR. RESULTS: We observed outer hair cell (OHC) loss predominantly in the basal turn of the cochlear at 14 days after bacterial inoculation. Macrophages, not neutrophils are the major immune cells in the cochlea in CSOM displaying increased numbers and a distribution correlated with the observed cochlear injury. The progression of the morphological changes suggests a transition from monocytes into tissue macrophages following infection. We also show that PA do not enter the cochlea and live bacteria are required for cochlear injury. We characterized cytokine activity in the CSOM cochlea. CONCLUSIONS: Taken together, this data shows a critical role for macrophages in CSOM-mediated sensorineural hearing loss (SNHL).

Anti-persister and Anti-biofilm Activity of Self-Assembled Antimicrobial Peptoid Ellipsoidal Micelles.

Although persister cells are the root cause of resistance development and relapse of chronic infections, more attention has been focused on developing antimicrobial agents against resistant bacterial strains than on developing anti-persister agents. Frustratingly, the global preclinical antibacterial pipeline does not include any anti-persister drug. Therefore, the central point of this work is to explore antimicrobial peptidomimetics called peptoids (sequence-specific oligo-N-substituted glycines) as a new class of anti-persister drugs. In this study, we demonstrate that one particular antimicrobial peptoid, the sequence-specific pentamer TM5, is active against planktonic persister cells and sterilizes biofilms formed by both Gram-negative and Gram-positive bacteria. Moreover, we demonstrate the potential of TM5 to inhibit cytokine production induced by lipopolysaccharides from Gram-negative bacteria. We anticipate that this work can pave the way to the development of new anti-persister agents based on antimicrobial peptoids of this class to simultaneously help address the crisis of bacterial resistance and reduce the occurrence of the relapse of chronic infections.

Gold nanocluster adjuvant enables the eradication of persister cells by antibiotics and abolishes the emergence of resistance.

Persister cells are responsible for relapses of infections common in cystic fibrosis and chronic suppurative otitis media (CSOM). Yet, there are no Food and Drug Administration (FDA) approved antibiotics to eradicate persister cells. Frustratingly, the global preclinical bacterial pipeline does not contain antibacterial agents targeting persister cells. Therefore, we report a nontraditional antimicrobial chemotherapy strategy based on gold nanoclusters adjuvant to eradicate persister cells by existing antibiotics belonging to that different class. Compared to killing with antibiotics alone, combining antibiotics and AuNC@CPP sterilizes persister cells and biofilms. Enhanced killing of up to 4 orders of magnitude in a validated mouse model of CSOM with Pseudomonas aeruginosa infection was observed when combining antibiotics and AuNC@CPP, informing a potential approach to improve the treatment of CSOM. We established that the mechanism of action of AuNC@CPP is due to disruption of the proton gradient and membrane hyperpolarization. The method presented here could compensate for the lack of new antibiotics to combat persister cells. This method could also benefit the current effort to slow resistance development because AuNC@CPP abolished the emergence of drug-resistant strains induced by antibiotics.

Topical Therapy Failure in Chronic Suppurative Otitis Media is Due to Persister Cells in Biofilms.

OBJECTIVE: Chronic suppurative otitis media (CSOM) is characterized by a chronically draining middle ear. CSOM is typically treated with multiple courses of antibiotics or antiseptics which are successful in achieving quiescence; however, the disease is prone to relapse. Understanding why these treatment failures occur is essential. STUDY DESIGN: The minimum inhibitory concentration (MIC), minimal biofilm eradication concentration, and the inhibitory zone were determined for ototopicals and ofloxacin for the laboratory strains and CSOM-derived isolates. The percentage of persister cells and bacterial biofilm formation were measured. Disease eradication was tested in a validated in-vivo model of CSOM after treatment with ofloxacin. SETTING: Microbiology Laboratory. METHODS: Basic science experiments were performed to measure the effectiveness of a number of compounds against CSOM bacteria in a number of distinct settings. RESULTS: The minimal biofilm eradication concentration is higher than is physiologically achievable with commercial preparations, except for povo-iodine. Clincial isolates of CSOM have equivalent biofilm-forming ability but increased proportions of persister cells. Ofloxacin can convert to inactive disease temporarily but fails to eradicate disease in an in-vivo model. CONCLUSIONS: Higher percentages of persister cells in clinical CSOM isolates are associated with resistance to ototopicals. Current ototopicals, except povo-iodine, have limited clinical effectiveness; however, it is unknown what the maximum achievable concentration is and there are ototoxicity concerns. Fluoroquinolones, while successful in producing inactive disease in the short term, have the potential to encourage antimicrobial resistance and disease recalcitrance and do not achieve a permanent remission. Given these limitations, clinicians should consider surgery earlier or use of clinically safe concentrations of povo-iodine earlier into the treatment algorithm.

Locally administered heparin-binding epidermal growth factor-like growth factor reduces radiation-induced oral mucositis in mice.

Oral mucositis refers to lesions of the oral mucosa observed in patients with cancer being treated with radiation with or without chemotherapy, and can significantly affect quality of life. There is a large unmet medical need to prevent oral mucositis that can occur with radiation either alone or in combination with chemotherapy. We investigated the efficacy of locally administered heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent epithelial proliferation and migration stimulator of the oral mucosa as a potential therapy to prevent radiation induced oral mucositis. Using a single dose (20 Gy) of radiation to the oral cavity of female C57BL/6 J mice, we evaluated the efficacy of HB-EGF treatment (5 µl of 10 µg/ml) solution. The results show that HB-EGF delivered post radiation, significantly increased the area of epithelial thickness on the tongue (dorsal tongue (42,106 vs 53,493 µm2, p < 0.01), ventral tongue (30,793 vs 39,095 µm2, *p < 0.05)) compared to vehicle control, enhanced new epithelial cell division, and increased the quality and quantity of desmosomes in the oral mucosa measured in the tongue and buccal mucosa. This data provides the proof of concept that local administration of HB-EGF has the potential to be developed as a topical treatment to mitigate oral mucositis following radiation.

Antimicrobial Gold Nanoclusters Eradicate Escherichia coli Biofilms and Are Nontoxic by Oral Administration.

Escherichia coli biofilms are a major causative agent of many intestinal infections, and there is ongoing research aimed at E. coli biofilm eradication. Gold nanoclusters (AuNCs) conjugated with various surface ligands have been extensively investigated for antimicrobial properties and provide a potential solution. There is little known about their in vivo safety because current standards of nanosafety research involve incubation of AuNCs with cells in vitro to confirm biocompatibility. In addition to systemic administration, nanosafety research on AuNC-based antimicrobials designed to treat gastrointestinal infections must also consider the potential for inducing gastrointestinal disorders. We report the design and application of two AuNCs coated with either hydroxyl (AuNC@PEG-OH)- or amine (AuNC@PEG-NH2)-functionalized poly(ethylene glycol), which enables the eradication of E. coli biofilms. Gastrointestinal safety of AuNC@PEG-OH and AuNC@PEG-NH2 was evaluated in healthy mice up to 35 days after administration by oral gavage at a dose of 10 mg/kg (or 1 mg/mL) daily for 14 days. No changes were detected in the histopathology of major organs, serum chemistry, hematology, and feces. Thus, oral administration of AuNCs is unlikely to be of concern for systemic toxicity or in the induction of gastrointestinal illnesses. Further studies on increasing time exposure and doses are necessary to determine whether toxicity occurs at higher doses or whether there is no adverse effect limit.