Microbial exposure during early human development primes fetal immune cells.

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth.

Screening of the PA14NR Transposon Mutant Library Identifies Genes Involved in Resistance to Bacteriophage Infection in Pseudomomas aeruginosa.

Multidrug-resistant P. aeruginosa infections pose a serious public health threat due to the rise in antimicrobial resistance. Phage therapy has emerged as a promising alternative. However, P. aeruginosa has evolved various mechanisms to thwart phage attacks, making it crucial to decipher these resistance mechanisms to develop effective therapeutic strategies. In this study, we conducted a forward-genetic screen of the P. aeruginosa PA14 non-redundant transposon library (PA14NR) to identify dominant-negative mutants displaying phage-resistant phenotypes. Our screening process revealed 78 mutants capable of thriving in the presence of phages, with 23 of them carrying insertions in genes associated with membrane composition. Six mutants exhibited total resistance to phage infection. Transposon insertions were found in genes known to be linked to phage-resistance such as galU and a glycosyl transferase gene, as well as novel genes such as mexB, lasB, and two hypothetical proteins. Functional experiments demonstrated that these genes played pivotal roles in phage adsorption and biofilm formation, indicating that altering the bacterial membrane composition commonly leads to phage resistance in P. aeruginosa. Importantly, these mutants displayed phenotypic trade-offs, as their resistance to phages inversely affected antibiotic resistance and hindered biofilm formation, shedding light on the complex interplay between phage susceptibility and bacterial fitness. This study highlights the potential of transposon mutant libraries and forward-genetic screens in identifying key genes involved in phage-host interactions and resistance mechanisms. These findings support the development of innovative strategies for combating antibiotic-resistant pathogens.

Experimental colonization with Blastocystis ST4 is associated with protective immune responses and modulation of gut microbiome in a DSS-induced colitis mouse model.

BACKGROUND: Blastocystis is a common gut protistan parasite in humans and animals worldwide, but its interrelationship with the host gut microbiota and mucosal immune responses remains poorly understood. Different murine models of Blastocystis colonization were used to examine the effect of a common Blastocystis subtype (ST4) on host gut microbial community and adaptive immune system. RESULTS: Blastocystis ST4-colonized normal healthy mice and Rag1-/- mice asymptomatically and was able to alter the microbial community composition, mainly leading to increases in the proportion of Clostridia vadinBB60 group and Lachnospiraceae NK4A136 group, respectively. Blastocystis ST4 colonization promoted T helper 2 (Th2) response defined by interleukin (IL)-5 and IL-13 cytokine production, and T regulatory (Treg) induction from colonic lamina propria in normal healthy mice. Additionally, we observed that Blastocystis ST4 colonization can maintain the stability of bacterial community composition and induce Th2 and Treg immune responses to promote faster recovery from experimentally induced colitis. Furthermore, fecal microbiota transplantation of Blastocystis ST4-altered gut microbiome to colitis mice reduced the severity of colitis, which was associated with increased production of short-chain fat acids (SCFAs) and anti-inflammatory cytokine IL-10. CONCLUSIONS: The data confirm our hypothesis that Blastocystis ST4 is a beneficial commensal, and the beneficial effects of Blastocystis ST4 colonization is mediated through modulating of the host gut bacterial composition, SCFAs production, and Th2 and Treg responses in different murine colonization models.

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Rabbit Fungal Keratitis Model of Fusarium solani Tested Against Three Commercially Available Antifungal Drugs.

OBJECTIVES: The purpose of this study was to develop a reproducible preclinical Fusarium solani keratitis model, which would allow comparative testing of currently available antifungals (NATACYN [Alcon, Fort Worth, TX], voriconazole 1%, and amphotericin B 0.1%) as well as efficacy testing of new antifungals for translation into clinical practice in the future. METHODS: The rabbit F. solani keratitis model was developed in New Zealand white rabbits using local and systemic immunosuppression. Infection was introduced by intrastromal injection of F. solani spores into one of the immunosuppressed rabbit eyes while the contralateral eye was a control. Progress of the infection was assessed by the clinical features, histopathology, and viable fungal counts. In this study, the efficacy of currently available antifungals (NATACYN [Alcon], voriconazole 1%, and amphotericin B 0.1%) was compared. Rabbits were randomly divided (n=4 in each group), and the respective antifungal was instilled topically 5 times/day for 7 days. Treatment effects were analyzed by evaluating the anterior segment with the help of slit-lamp, histopathological findings and viable fungal culture at the end of the experiment. RESULTS: We report the development of a reproducible and progressive rabbit F. solani keratitis model as shown by the substantial viable fungal counts (3 log CFU), the presence of large patchy lesions and substantial hypopyon in the 12-day model correlated with specific histopathological analysis for fungus (extended F. solani hyphae from midcorneal stroma into the anterior chamber and traverse Descemet membrane with anterior chamber suppurative plaque). Voriconazole 1% and NATACYN revealed significant reduction of the fungal wound area (P=0.02 and 0.021), respectively, while amphotericin B 0.1% exhibited P value of 0.083 compared with their infected nontreated controls. Voriconazole 1% and amphotericin B 0.1% showed significant viable fungal count differences (P=0.004 and 0.01), respectively, whereas P value of NATACYN was 0.337 compared with control infected corneas. CONCLUSION: The reported rabbit fungal keratitis model can be used for screening new antifungals and evaluating currently available antifungals to facilitate better clinical outcomes. Voriconazole 1% showed the best efficacy among the three tested currently available antifungals by showing the significant differences in both wound size and viable fungal count comparisons in our F. solani rabbit keratitis model.

Repurposing the anticancer drug cisplatin with the aim of developing novel Pseudomonas aeruginosa infection control agents.

Antibiotic resistance threatens effective treatment of microbial infections globally. This situation has spurred the hunt for new antimicrobial compounds in both academia and the pharmaceutical industry. Here, we report how the widely used antitumor drug cisplatin may be repurposed as an effective antimicrobial against the nosocomial pathogen Pseudomonas aeruginosa. Cisplatin was found to effectively kill strains of P. aeruginosa. In such experiments, transcriptomic profiling showed upregulation of the recA gene, which is known to be important for DNA repair, implicating that cisplatin could interfere with DNA replication in P. aeruginosa. Cisplatin treatment significantly repressed the type III secretion system (T3SS), which is important for the secretion of exotoxins. Furthermore, cisplatin was also demonstrated to eradicate in vitro biofilms and in vivo biofilms in a murine keratitis model. This showed that cisplatin could be effectively used to eradicate biofilm infections which were otherwise difficult to be treated by conventional antibiotics. Although cisplatin is highly toxic for humans upon systemic exposure, a low toxicity was demonstrated with topical treatment. This indicated that higher-than-minimal inhibitory concentration (MIC) doses of cisplatin could be topically applied to treat persistent and recalcitrant P. aeruginosa infections.

Antimicrobial Activity and Cell Selectivity of Synthetic and Biosynthetic Cationic Polymers.

The mammalian and microbial cell selectivity of synthetic and biosynthetic cationic polymers has been investigated. Among the polymers with peptide backbones, polymers containing amino side chains display greater antimicrobial activity than those with guanidine side chains, whereas ethylenimines display superior activity over allylamines. The biosynthetic polymer ε-polylysine (εPL) is noncytotoxic to primary human dermal fibroblasts at concentrations of up to 2,000 µg/ml, suggesting that the presence of an isopeptide backbone has greater cell selectivity than the presence of α-peptide backbones. Both εPL and linear polyethylenimine (LPEI) exhibit bactericidal properties by depolarizing the cytoplasmic membrane and disrupt preformed biofilms. εPL displays broad-spectrum antimicrobial properties against antibiotic-resistant Gram-negative and Gram-positive strains and fungi. εPL elicits rapid bactericidal activity against both Gram-negative and Gram-positive bacteria, and its biocompatibility index is superior to those of cationic antiseptic agents and LPEI. εPL does not interfere with the wound closure of injured rabbit corneas. In a rabbit model of bacterial keratitis, the topical application of εPL (0.3%, wt/vol) decreases the bacterial burden and severity of infections caused by Pseudomonas aeruginosa and Staphylococcus aureus strains. In vivo imaging studies confirm that εPL-treated corneas appeared transparent and nonedematous compared to untreated infected corneas. Taken together, our results highlight the potential of εPL in resolving topical microbial infections.

Biofilms of Pathogenic Nontuberculous Mycobacteria Targeted by New Therapeutic Approaches.

Microbial infections of the cornea are potentially devastating and can result in permanent visual loss or require vision-rescuing surgery. In recent years, there has been an increasing number of reports on nontuberculous mycobacterial infections of the cornea. Challenges to the management of nontuberculous mycobacterial keratitis include delayed laboratory detection, low index of clinical suspicion, poor drug penetration, slow response to therapy, and prolonged use of antibiotic combinations. The ability of nontuberculous mycobacteria to evade the host immune response and the ability to adhere and to form biofilms on biological and synthetic substrates contribute to the issue. Therefore, there is an urgent need for new antimicrobial compounds that can overcome these problems. In this study, we evaluated the biofilm architectures for Mycobacterium chelonae and Mycobacterium fortuitum in dynamic flow cell chamber and 8-well chamber slide models. Our results showed that mycobacterial biofilms were quite resistant to conventional antibiotics. However, DNase treatment could be used to overcome biofilm resistance. Moreover, we successfully evaluated a new antimicrobial compound (AM-228) that was effective not only for planktonic mycobacterial cells but also for biofilm treatment and was compared favorably with the most successful "fourth-generation" fluoroquinolone, gatifloxacin. Finally, a new treatment strategy emerged: a combination of DNase with an antibiotic was more effective than an antibiotic alone.

Colonization with two different Blastocystis subtypes in DSS-induced colitis mice is associated with strikingly different microbiome and pathological features.

Rationale: The gut microbiota plays a significant role in the pathogenesis of inflammatory bowel disease (IBD). However, the role of Blastocystis infection and Blastocystis-altered gut microbiota in the development of inflammatory diseases and their underlying mechanisms are not well understood. Methods: We investigated the effect of Blastocystis ST4 and ST7 infection on the intestinal microbiota, metabolism, and host immune responses, and then explored the role of Blastocystis-altered gut microbiome in the development of dextran sulfate sodium (DSS)-induced colitis in mice. Results: This study showed that prior colonization with ST4 conferred protection from DSS-induced colitis through elevating the abundance of beneficial bacteria, short-chain fatty acid (SCFA) production and the proportion of Foxp3+ and IL-10-producing CD4+ T cells. Conversely, prior ST7 infection exacerbated the severity of colitis by increasing the proportion of pathogenic bacteria and inducing pro-inflammatory IL-17A and TNF-α-producing CD4+ T cells. Furthermore, transplantation of ST4- and ST7-altered microbiota resulted in similar phenotypes. Conclusions: Our data showed that ST4 and ST7 infection exert strikingly differential effects on the gut microbiota, and these could influence the susceptibility to colitis. ST4 colonization prevented DSS-induced colitis in mice and may be considered as a novel therapeutic strategy against immunological diseases in the future, while ST7 infection is a potential risk factor for the development of experimentally induced colitis that warrants attention.

Propranolol Ameliorates the Antifungal Activity of Azoles in Invasive Candidiasis.

The effectiveness of current antifungal therapies is hampered by the emergence of drug resistance strains, highlighting an urgent need for new alternatives such as adjuvant antifungal treatments. This study aims to examine the synergism between propranolol and antifungal drugs, based on the premise that propranolol is known to inhibit fungal hyphae. In vitro studies demonstrate that propranolol potentiates the antifungal activity of azoles and that the effect is more pronounced for propranolol-itraconazole combination. Using an in vivo murine systemic candidemia model, we show that propranolol-itraconazole combination treatment resulted in a lower rate of body weight loss, decreased kidney fungal bioburden and renal inflammation when compared to propranolol and azole treatment alone or untreated control. Altogether, our findings suggest that propranolol increases the efficacy of azoles against C. albicans, offering a new therapeutic strategy against invasive fungal infections.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Hybrid derivative of cathelicidin and human beta defensin-2 against Gram-positive bacteria: A novel approach for the treatment of bacterial keratitis.

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5-25.0 µg/ml (5.2-10.4 µM)] and S. epidermidis [MIC = 12.5 µg/ml (5.2 µM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 µg/ml (10.4-20.8 µM)]. CaD23 (at 25 µg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 µg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

Multifunctional Antimicrobial Nanofiber Dressings Containing ε-Polylysine for the Eradication of Bacterial Bioburden and Promotion of Wound Healing in Critically Colonized Wounds.

Bacterial colonization of acute and chronic wounds is often associated with delayed wound healing and prolonged hospitalization. The rise of multi-drug resistant bacteria and the poor biocompatibility of topical antimicrobials warrant safe and effective antimicrobials. Antimicrobial agents that target microbial membranes without interfering with the mammalian cell proliferation and migration hold great promise in the treatment of traumatic wounds. This article reports the utility of superhydrophilic electrospun gelatin nanofiber dressings (NFDs) containing a broad-spectrum antimicrobial polymer, ε-polylysine (εPL), crosslinked by polydopamine (pDA) for treating second-degree burns. In a porcine model of partial thickness burns, NFDs promoted wound closure and reduced hypertrophic scarring compared to untreated burns. Analysis of NFDs in contact with the burns indicated that the dressings trap early colonizers and elicit bactericidal activity, thus creating a sterile wound bed for fibroblasts migration and re-epithelialization. In support of these observations, in porcine models of Pseudomonas aeruginosa and Staphylococcus aureus colonized partial thickness burns, NFDs decreased bacterial bioburden and promoted wound closure and re-epithelialization. NFDs displayed superior clinical outcome than standard-of-care silver dressings. The excellent biocompatibility and antimicrobial efficacy of the newly developed dressings in pre-clinical models demonstrate its potential for clinical use to manage infected wounds without compromising tissue regeneration.

Antimicrobial activity profiles of Amphiphilic Xanthone derivatives are a function of their molecular Oligomerization.

Currently, membrane-targeting small antimicrobial peptidomimetics (SAP) are important in antibiotic development because bacteria appear to develop resistance to these surface-active compounds less readily. However, the molecular membrane-targeting action of SAPs has received little attention. In this study, we investigated the effect of oligomerization of amphiphilic xanthone, a model SAP, on its antimicrobial properties against both Gram-positive and Gram-negative bacteria. First, oligomer formation by an amphiphilic xanthone, compound 2 (also coded as AM052), was investigated via solution-state nuclear magnetic resonance (NMR) spectroscopy. Then, the effects of oligomerization on membrane disruption were further studied via biophysical approaches. The results showed that the antimicrobial activities of SAPs develop in several stages: oligomer formation in aqueous solution, initial binding of oligomers to the membrane-water interface followed by insertion into the membrane bilayer, aggregation of antimicrobial oligomers in the membrane, and induced membrane leakage. Ultimately, the presence of the oligomers in the bacterial membrane leads to decreased membrane fluidity and bacterial cell death. Interestingly, the early formation of large oligomers leads to stronger membrane disruption and more rapid bacterial killing. However, reduced antimicrobial activities against Gram-negative bacteria were observed for compounds that formed larger oligomers because the LPS layer acts as a barrier to large complexes. Taken together, our results suggest that oligomerization of SAPs has a strong impact on their antimicrobial properties.

Discovery of novel antimycobacterial drug therapy in biofilm of pathogenic nontuberculous mycobacterial keratitis.

PURPOSE: The potential of slow-growing mycobacteria to form biofilms in human tissues contributes to the problem of establishing an effective treatment strategy. The purpose of this study was to examine new antibiotic strategies to enhance current treatment options for these infections. METHODS: Sensitivities of Mycobacterium fortuitum ATCC 49404 and Mycobacterium chelonae ATCC 35752 were evaluated for different antimicrobials singly and in combination using broth microdilution and FICI (Fractional Inhibitory Concentration Index) synergy screening. Anti-biofilm effects were evaluated in an 8-well chamber slide biofilm model. The efficacy of a new treatment strategy was validated using the novel neutropenic mouse keratitis model and monitored by slit-lamp microscopy, confocal microscopy, and colony forming unit measurements. RESULTS: We reported the very first evidence that these organisms develop corneal biofilms by the accumulation of extracellular DNA (eDNA) and the presence of microcolonies using a novel mycobacterial neutropenic mouse keratitis model. The combination of amikacin and gatifloxacin or besifloxacin was more effective than the current gold-standard drug, amikacin, and we developed a novel treatment strategy (amikacin + gatifloxacin + DNase), the destruction of biofilm matrix component, eDNA, which increased the efficacy of the new antibiotic combination for treating mycobacterial infection in in vitro (P = 0.002) and in vivo (P = 0.001) compared to its respective control. CONCLUSION: Biofilms have a role in mycobacterial keratitis leading to poor treatment outcomes in clinical practice and the use of combination therapy (amikacin + gatifloxacin + DNase) could be a useful new treatment option.

Symmetrically Substituted Xanthone Amphiphiles Combat Gram-Positive Bacterial Resistance with Enhanced Membrane Selectivity.

This is the first report of the design of a new series of symmetric xanthone derivatives that mimic antimicrobial peptides using a total synthesis approach. This novel design is advantageous because of its low cost, synthetic simplicity and versatility, and easy tuning of amphiphilicity by controlling the incorporated cationic and hydrophobic moieties. Two water-soluble optimized compounds, 6 and 18, showed potent activities against Gram-positive bacteria, including MRSA and VRE (MICs = 0.78-6.25 µg/mL) with a rapid bactericidal effect, low toxicity, and no emergence of drug resistance. Both compounds demonstrated enhanced membrane selectivity that was higher than those of most membrane-active antimicrobials in clinical trials or previous reports. The compounds appear to kill bacteria by disrupting their membranes. Significantly, 6 was effective in vivo using a mouse model of corneal infection. These results provide compelling evidence that these compounds have therapeutic potential as novel antimicrobials for multidrug-resistant Gram-positive infections.

Semisynthetic Flavone-Derived Antimicrobials with Therapeutic Potential against Methicillin-Resistant Staphylococcus aureus (MRSA).

A new series of semisynthetic flavone-based small molecules mimicking antimicrobial peptides has been designed from natural icaritin to combat drug-resistant Gram-positive bacterial infections. Compound 6 containing two arginine residues exhibited excellent antibacterial activity against Gram-positive bacteria, including MRSA, and very low toxicity to mammalian cells, resulting in a high selectivity of more than 511, comparable to that of several membrane-active antibiotics in clinical trials. Our data show for the first time that icaritin derivatives effectively kill bacteria. Meanwhile, this is the first study deploying a biomimicking strategy to design potent flavone-based membrane targeting antimicrobials. 6 showed rapid bactericidal activity by disrupting the bacterial membrane and can circumvent the development of bacterial resistance. Importantly, 6 was highly efficacious in a mouse model of corneal infection caused by MRSA and Staphylococcus aureus.

Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria.

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.

N-Lipidated Peptide Dimers: Effective Antibacterial Agents against Gram-Negative Pathogens through Lipopolysaccharide Permeabilization.

Treating infections caused by multidrug-resistant Gram-negative pathogens is challenging, and there is concern regarding the toxicity of the most effective antimicrobials for Gram-negative pathogens. We hypothesized that conjugating a fatty acid moiety onto a peptide dimer could maximize the interaction with lipopolysaccharide (LPS) and facilitate the permeabilization of the LPS barrier, thereby improving potency against Gram-negative pathogens. We systematically designed a series of N-lipidated peptide dimers that are active against Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE). The optimized lipid length was 6-10 carbons. At these lipid lengths, the N-lipidated peptide dimers exhibited strong LPS permeabilization. Compound 23 exhibited synergy with select antibiotics in most of the combinations tested. 23 and 32 also displayed rapid bactericidal activity. Importantly, 23 and 32 were nonhemolytic at 10 mg/mL, with no cellular or in vivo toxicity. These characteristics suggest that these compounds can overcome the limitations of current Gram-negative-targeted antimicrobials such as polymyxin B.