Common dCas9-based CRISPR interference (CRISPRi) systems for manipulating bacterial gene expression require antibiotic selection and exogenous inducer molecules, limiting their applicability in infection models. For Staphylococcus aureus, we have developed a programmable, selection-free CRISPRi system leveraging the pCM29 plasmid, which is stable without antibiotic selection. In this system, dCas9 expression is regulated by an endogenous promoter, and sgRNA expression is driven by a constitutive promoter, eliminating the need for exogenous inducer molecules. We programmed the system to silence the expression of the coagulase or autolysin genes whenever their respective endogenous promoters were activated. We confirmed selection-free interference with target gene expression for ≥27 generations by qPCR and protein target-dependent in vitro or in vivo phenotypic assays (plasma coagulation, THP-1 cell, and Galleria mellonella infection). The system is suitable for interrogating gene function in long-term studies of S. aureus pathogenesis and represents a blueprint for similar CRISPRi systems in other species.

Biofilms present a great challenge in antimicrobial therapy due to their inherent tolerance to conventional antibiotics, promoting the need for advanced drug delivery strategies that improve therapy. While various nanoparticles (NPs) have been reported for this purpose, DNA-based NPs remain a largely unexploited resource against biofilm-associated infections. To fill this gap and to lay the groundwork for their potential therapeutic exploitation, we investigated the diffusion, penetration, and retention behaviors of three DNA-based nanocarriers -plain or modified-within P. aeruginosa biofilms. Watson-Crick base pairing or hydrophobic interactions mediated the formation of the plain NPs whilst electrostatic interaction enabled optimization of coated NPs via microfluidic mixing. We assessed the interactions of the nanocarriers with biofilm structures via Single Plane Illumination Microscopy - Fluorescence Correlation Spectroscopy (SPIM-FCS) and Confocal Laser Scanning Microscopy (CLSM). We demonstrate the impact of microfluidic parameters on the physicochemical properties of the modified DNA NPs and their subsequent distinct behaviors in the biofilm. Our results show that single stranded DNA micelles (ssDNA micelle) and tetrahedral DNA nanostructures (TDN) had similar diffusion and penetration profiles, whereas chitosan-coated TDN (TDN-Chit) showed reduced diffusion and increased biofilm retention. This is attributable to the relatively larger size and positive surface charge of the TDN-Chit NPs. The study shows first and foremost that DNA can be used as building block in drug delivery for antibiofilm therapeutics. Moreover, the overall behavioral findings are pivotal for the strategic selection of therapeutic agents to be encapsulated within these structures, possibly affecting the treatment efficacy. This research not only highlights the underexplored potential of DNA-based NPs in antibiofilm therapy but also advocates for further studies using different optimization strategies to refine these nanocarrier systems for targeted treatments in biofilm-related infections.

In search of new putative antimicrobial drug targets in methicillin-resistant Staphylococcus aureus, we aimed to identify and characterize retaining glycosidase activities in this bacterial pathogen. Using activity-based protein profiling (ABPP), a panel of 7 fluorescent probes was screened to detect activities of diverse retaining glycosidase families. Based on this, a cocktail of 3 biotinylated probes (targeting α-glucosidases, β-galactosidases and α-fucosidases) was used for target enrichment and three glycoside hydrolase family proteins were identified by mass-spectrometry: 6-phospho-β-glucosidase (BglA), α-amylase family protein trehalase C (TreC), and autolysin (Atl). The physiological relevance of previously uncharacterized BglA and TreC was addressed in CRISPRi and inhibitor studies with the putative TreC inhibitor α-cyclophellitol-aziridine. Silencing of treC did not affect bacterial growth in rich media, but reduced biofilm formation in vitro, and attenuated virulence during Galleria mellonella infection, warranting future investigations into the biochemical function of this enzyme.

Natural polyphenols are promising alternatives to antifungals for novel treatments of vulvovaginal candidiasis (VVC) in an era of antimicrobial resistance. However, polyphenols are poorly soluble and prone to degradation. To overcome their limitations, we propose incorporation in liposomes. The study aimed to develop chitosan and liposome comprising delivery systems for epicatechin (EC) or propyl gallate (PG) as treatment of VVC. EC was selected for its antioxidative properties and PG as an ester of antifungal gallic acid. To improve formulation retention at vaginal site, mucoadhesive chitosan was introduced into formulation as liposomal surface coating or hydrogel due to intrinsic antifungal properties. These polyphenol-loaded liposomes exhibited an average size of 125 nm with a 64 % entrapment efficiency (for both polyphenols). A sustained in vitro polyphenol release was seen from liposomes, particularly in chitosan hydrogel (p < 0.01 or lower). Viscosity was evaluated since increased viscosity upon mucin contact indicated adhesive bond formation between chitosan and mucin confirming mucoadhesiveness of formulations. Antifungal activity was evaluated by the broth microdilution method on Candida albicans CRM-10231. Unlike PG, incorporation of EC in liposomes enabled antifungal activity. Fungicidal activity of chitosan was confirmed both when used as liposomal coating material and as hydrogel vehicle.

IntroductionImproved understanding of Staphylococcus aureus throat colonization in the presence of other co-existing microbes is important for mapping S. aureus adaptation to the human throat, and recurrence of infection. Here, we explore the responses triggered by the encounter between two common throat bacteria, S. aureus and Streptococcus anginosus, to identify genes in S. aureus that are important for colonization in the presence of human tonsillar epithelial cells and S. anginosus, and further compare this transcriptome with the genes expressed in S. aureus as only bacterium.MethodsWe performed an in vitro co-culture experiment followed by RNA sequencing to identify interaction-induced transcriptional alterations and differentially expressed genes (DEGs), followed by gene enrichment analysis.Results and discussionA total of 332 and 279 significantly differentially expressed genes with p-value < 0.05 and log2 FoldChange (log2FC) ≥ |2| were identified in S. aureus after 1 h and 3 h co-culturing, respectively. Alterations in expression of various S. aureus survival factors were observed when co-cultured with S. anginosus and tonsillar cells. The serine-aspartate repeat-containing protein D (sdrD) involved in adhesion, was for example highly upregulated in S. aureus during co-culturing with S. anginosus compared to S. aureus grown in the absence of S. anginosus, especially at 3 h. Several virulence genes encoding secreted proteins were also highly upregulated only when S. aureus was co-cultured with S. anginosus and tonsillar cells, and iron does not appear to be a limiting factor in this environment. These findings may be useful for the development of interventions against S. aureus throat colonization and could be further investigated to decipher the roles of the identified genes in the host immune response in context of a throat commensal landscape.

The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus-host cell interactions, shedding light on modulating host immune responses to S. aureus, which could involve developing immunomodulatory therapies. IMPORTANCE:This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Klebsiella pneumoniae is a normal resident of the human gastro-intestinal tract and an opportunistic, critical priority pathogen that can cause a variety of severe systemic infections. Due to emerging multi-drug resistance of this pathogen, the discovery and validation of novel targets for the development of new treatment options is an urgent priority. Here, we explored the family of serine hydrolases, a highly druggable and functionally diverse enzyme family which is uncharacterized in K. pneumoniae . Using functionalized covalent fluorophosphonate inhibitors as activity-based probes we identified 10 serine hydrolases by mass spectrometry-based activity-based protein profiling, 7 of which were previously uncharacterized. Functional validation using transposon mutants deficient in either of the putative lysophospholipase PldB, esterase YjfP and patatin-like phospholipase YchK revealed severe growth defects in human colonic organoid co-culture models and reduced virulence during Galleria mellonella infection. Mutants deficient in the PldB and YjfP, but not YchK show increased susceptibility to killing by complement and the antimicrobial peptide antibiotic polymyxin B, suggesting a role in maintaining cell envelope integrity. Biochemical characterization and structural analysis of recombinant YjfP suggest this protein is a deacetylase. This study gives important insights into the molecular mechanisms underlying virulence and cell physiology of K. pneumoniae at the host-pathogen interface and it positions PldB, YjfP and YchK as potential antimicrobial or anti-virulence target candidates, inhibition of which might synergize with existing antibiotics and human immune defenses.### Competing Interest StatementThe authors have declared no competing interest.

Common dCas9-based CRISPR interference (CRISPRi) system for gene regulation requires antibiotic selection and exogenous inducer molecules, posing significant challenges when applied in in vivo bacterial infection models. Using Staphylococcus aureus as a model organism, we have developed a programmable, plasmid-based, but selection-free (ppsf)-CRISPRi system that is based on the pCM29-plasmid which is stable without antibiotic selection. In this ppsf-CRISPRi system, dCas9 expression is regulated by an endogenous virulence gene promoter, and sgRNA expression is driven by a constitutive promoter eliminating the need for exogenous inducer molecules. The system was programmed to silence the expression of genes encoding the virulence factor coagulase or peptidoglycan hydrolase autolysin, whenever their respective endogenous promoter was activated. The selection-free functionality was confirmed over at least 27 generations and verified by qPCR and phenotypic assays depending on the protein target, including coagulation of rabbit plasma and THP-1 macrophage cell infection in vitro as well as in vivo infection of Galleria mellonella larvae, in each case phenocopying the observations made using transposon mutant strains. The system is suitable for long-term studies of S. aureus pathogenesis in vitro or in vivo and represents a blueprint for the development of similar ppsf-CRISPRi systems in other bacterial species. ### Competing Interest Statement The authors have declared no competing interest.

AbstractIsogenic bacterial cell populations are phenotypically heterogenous and may include subpopulations of antibiotic tolerant or heteroresistant cells. The reversibility of these phenotypes and lack of biomarkers to differentiate functionally different, but morphologically identical cells is a challenge for research and clinical detection. To overcome this, we present ´Cellular Phenotypic Profiling and backTracing (CPPT)´, a fluorescence-activated cell sorting platform that uses fluorescent probes to visualize and quantify cellular traits and connects this phenotypic profile with a cell´s experimentally determined fate in single cell-derived growth and antibiotic susceptibility analysis. By applying CPPT on Staphylococcus aureus we phenotypically characterized dormant cells, exposed bimodal growth patterns in colony-derived cells and revealed different culturability of single cells on solid compared to liquid media. We demonstrate that a fluorescent vancomycin conjugate marks cellular subpopulations of vancomycin-intermediate S. aureus with increased likelihood to survive antibiotic exposure, showcasing the value of CPPT for discovery of clinically relevant biomarkers.

Increasing prevalence of infected and chronic wounds demands improved therapy options. In this work an electrospun nanofiber dressing with liposomes is suggested, focusing on the dressing’s ability to support tissue regeneration and infection control. Chloramphenicol (CAM) was the chosen antibiotic, added to the nanofibers after first embedded in liposomes to maintain a sustained drug release. Nanofibers spun from five different polymer blends were tested, where pectin and polyethylene oxide (PEO) was identified as the most promising polymer blend, showing superior fiber formation and tensile strength. The wire-electrospinning setup (WES) was selected for its pilot-scale features, and water was applied as the only solvent for green electrospinning and to allow direct liposome incorporation. CAM-liposomes were added to Pectin-PEO nanofibers in the next step. Confocal imaging of rhodamine-labelled liposomes indicated intact liposomes in the fibers after electrospinning. This was supported by the observed in vitroCAM-release, showing that Pectin-PEO-nanofibers with CAM-liposomes had a delayed drug release compared to controls. Biological testing confirmed the antimicrobial efficacy of CAM and good biocompatibility of all CAM-nanofibers. The successful fiber formation and green production process with WES gives a promising outlook for industrial upscaling.

Enterococcus faecium (Efm) is a versatile pathogen, responsible for multidrug-resistant infections, especially in hospitalized immunocompromised patients. Its population structure has been characterized by diverse clades (A1, A2, and B (reclassified as E. lactis (Ela)), adapted to different environments, and distinguished by their resistomes and virulomes. These features only partially explain the predominance of clade A1 strains in nosocomial infections. We investigated in vitro interaction of 50 clinical isolates (clade A1 Efm) against 75 commensal faecal isolates from healthy humans (25 clade A2 Efm and 50 Ela). Only 36% of the commensal isolates inhibited clinical isolates, while 76% of the clinical isolates inhibited commensal isolates. The most apparent overall differences in inhibition patterns were presented between clades. The inhibitory activity was mainly mediated by secreted, proteinaceous, heat-stable compounds, likely indicating an involvement of bacteriocins. A custom-made database targeting 76 Bacillota bacteriocins was used to reveal bacteriocins in the genomes. Our systematic screening of the interactions between nosocomial and commensal Efm and Ela on a large scale suggests that, in a clinical setting, nosocomial strains not only have an advantage over commensal strains due to their possession of AMR genes, virulence factors, and resilience but also inhibit the growth of commensal strains.

The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components: a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9-controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus. IMPORTANCE The presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters.

In the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) and their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant methicillin-resistant S. aureus strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs' influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the co-crystallized structure of the DesK-DesR from Bacillus subtilis and the modeled structures of the four NarL TCSs in S. aureus, we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and the coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs.IMPORTANCEGiven the widespread conservation of two-component systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents, especially in the face of rising multi-drug-resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence, and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.

Enterococcus faecium is a leading cause of nosocomial infections, particularly in immunocompromised patients. The rise of multidrug-resistant E. faecium, including Vancomycin-Resistant Enterococci (VRE), is a major concern. Vaccines are promising alternatives to antibiotics, but there is currently no vaccine available against enterococci. In a previous study, we identified six protein vaccine candidates associated with extracellular membrane vesicles (MVs) produced by nosocomial E. faecium. In this study, we immunized rabbits with two different VRE-derived MV preparations and characterized the resulting immune sera. Both anti-MV sera exhibited high immunoreactivity towards the homologous strain, three additional VRE strains, and eight different unrelated E. faecium strains representing different sequence types (STs). Additionally, we demonstrated that the two anti-MV sera were able to mediate opsonophagocytic killing of not only the homologous strain but also three unrelated heterologous VRE strains. Altogether, our results indicate that E. faecium MVs, regardless of the purification method for obtaining them, are promising vaccine candidates against multidrug-resistant E. faecium and suggest that these naturally occurring MVs can be used as a multi-antigen platform to elicit protective immune responses against enterococcal infections.

Nucleic acid-based materials showcase an increasing potential for antimicrobial drug delivery. Although numerous reports on drug-loaded DNA nanoparticles outline their pivotal antibacterial activities, their potential as drug delivery systems against bacterial biofilms awaits further studies. Among different oligonucleotide structures, micellar nanocarriers derived from amphiphilic DNA strands are of particular interest due to their spontaneous self-assembly and high biocompatibility. However, their clinical use is hampered by structural instability upon cation depletion. In this work, we used a cationic amphiphilic antibiotic (polymyxin B) to stabilize DNA micelles destined to penetrate P. aeruginosa biofilms and exhibit antibacterial/antibiofilm properties. Our study highlights how the strong affinity of this antibiotic enhances the stability of the micelles and confirms that antibacterial activity of the novel micelles remains intact. Additionally, we show that PMB micelles can penetrate P. aeruginosa biofilms and impact their metabolic activity. Finally, PMB micelles were highly safe and biocompatible, highlighting their possible application against P. aeruginosa biofilm-colonized skin wounds.

Background The nose and the throat are the most predominant colonizing sites of Staphylococcus aureus , and colonization is a risk factor for infection. Nasal colonization is well described; however, we have limited knowledge about S. aureus throat colonization. The main objective of this study was to explore differentially expressed genes (DEGs) in S. aureus throat isolate TR145 exposed to human tonsil epithelial cells (HTEpiC) by using RNA sequencing (RNA-seq) and pathway analysis. DEGs in S. aureus at 1 or 3 hours (h) interaction with its host were explored. Results S. aureus was co-cultured in absence and presence of tonsillar cells at 1 or 3 h. Over the 3 h time frame, the bacteria multiplied, but still caused only minor cytotoxicity. Upon exposure to tonsillar cell line, S. aureus changed its transcriptomic profile. A total of 508 DEGs were identified including unique (1 h, 160 DEGs and 3 h, 78 DEGs) and commonly shared genes (1 and 3 h, 270 DEGs). Among the DEGs, were genes encoding proteins involved in adhesion and immune evasion, as well as iron acquisition and transport. Reverse transcription qPCR was done on selected genes, and the results correlated with the RNA-seq data. Conclusion We have shown the suitability of using HTEpiC as an in vitro model for investigating key determinants in S. aureus during co-incubation with host cells. Several DEGs were unique after 1 or 3 h exposure to host cells, while others were commonly expressed at both time points. As their expression is induced upon meeting with the host, they might be explored further for future targets for intervention to prevent either colonization or infection in the throat.

AbstractIsogenic bacterial cell populations are phenotypically heterogenous and may include subpopulations of antibiotic tolerant or heteroresistant cells. The reversible nature of these phenotypes and lack of biomarkers to differentiate functionally different, but morphologically identical cells is a challenge for research and clinical detection. To overcome this, we present ‘CellularPhenotypicProfiling and backTracing (CPPT)’, a flexible fluorescence-activated cell sorting platform, that uses optical probes to visualize and quantify cellular traits and connects the resulting phenotypic profile with a cell’s experimentally determined fate in single cell-derived growth and antibiotic susceptibility analysis. By applying CPPT onStaphylococcus aureuspopulations we recorded phenotypic signatures for dormant cells, exposed microanatomy-independent bimodal growth patterns in colony-derived cells, and revealed different culturability of single cells on solid compared to liquid media. We demonstrate that vancomycin-bodipyFL marks cellular subpopulations with increased likelihood to survive antibiotic exposure, showcasing the value of CPPT for discovery of clinically relevant biomarkers.

Infected chronic skin wounds and other skin infections are increasingly putting pressure on the health care providers and patients. The pressure is especially concerning due to the rise of antimicrobial resistance and biofilm-producing bacteria that further impair treatment success. Therefore, innovative strategies for wound healing and bacterial eradication are urgently needed; utilization of materials with inherent biological properties could offer a potential solution. Chitosan is one of the most frequently used polymers in delivery systems. This bioactive polymer is often regarded as an attractive constituent in delivery systems due to its inherent antimicrobial, anti-inflammatory, anti-oxidative, and wound healing properties. However, lipid-based vesicles and liposomes are generally considered more suitable as delivery systems for skin due to their ability to interact with the skin structure and provide prolonged release, protect the antimicrobial compound, and allow high local concentrations at the infected site. To take advantage of the beneficial attributes of the lipid-based vesicles and chitosan, these components can be combined into chitosan-containing liposomes or chitosomes and chitosan-coated liposomes. These systems have previously been investigated for use in wound therapy; however, their potential in infected wounds is not fully investigated. In this study, we aimed to investigate whether both the chitosan-containing and chitosan-coated liposomes tailored for infected wounds could improve the antimicrobial activity of the membrane-active antimicrobial chlorhexidine, while assuring both the anti-inflammatory activity and cell compatibility. Chlorhexidine was incorporated into three different vesicles, namely plain (chitosan-free), chitosan-containing and chitosan-coated liposomes that were optimized for skin wounds. Their release profile, antimicrobial activities, anti-inflammatory properties, and cell compatibility were assessed in vitro . The vesicles comprising chitosan demonstrated slower release rate of chlorhexidine and high cell compatibility. Additionally, the inflammatory responses in murine macrophages treated with these vesicles were reduced by about 60% compared to non-treated cells. Finally, liposomes containing both chitosan and chlorhexidine demonstrated the strongest antibacterial effect against Staphylococcus aureus . Both chitosan-containing and chitosan-coated liposomes comprising chlorhexidine could serve as excellent platforms for the delivery of membrane-active antimicrobials to infected wounds as confirmed by improved antimicrobial performance of chlorhexidine.

To avert the poor bioavailability of antibiotics during S. aureus biofilm infections, a series of zwitterionic nanoparticles containing nucleic acid nanostructures were fabricated for the delivery of vancomycin. The nanoparticles were prepared with three main lipids: (i) neutral (soy phosphatidylcholine; P), (ii) positively charged ionizable (1,2-dioleyloxy-3-dimethylaminopropane; D), and (iii) anionic (1,2-dipalmitoyl-sn-glycero-3-phospho((ethyl-1',2',3'-triazole) triethylene glycolmannose; M) or (cholesteryl hemisuccinate; C) lipids. The ratio of the anionic lipid was tuned between 0 and 10 mol %, and its impact on surface charge, size, stability, toxicity, and biofilm sensitivity was evaluated. Under biofilm mimicking conditions, the enzyme degradability (via dynamic light scattering (DLS)), antitoxin (via DLS and spectrophotometry), and antibiotic release profile was assessed. Additionally, biofilm penetration, prevention (in vitro), and eradication (ex vivo) of the vancomycin loaded formulation was investigated. Compared with the unmodified nanoparticles which exhibited the smallest size (188 nm), all three surface modified formulations showed significantly larger sizes (i.e., 222-277 nm). Under simulations of biofilm pH conditions, the mannose modified nanoparticle (PDM 90/5/5) displayed ideal charge reversal from a neutral (+1.69 ± 1.83 mV) to a cationic surface potential (+17.18 ± 2.16 mV) to improve bacteria binding and biofilm penetration. In the presence of relevant bacterial enzymes, the carrier rapidly released the DNA nanoparticles to function as an antitoxin against α-hemolysin. Controlled release of vancomycin prevented biofilm attachment and significantly reduced early stage biofilm formations within 24 h. Enhanced biocompatibility and significant ex vivo potency of the PDM 90/5/5 formulation was also observed. Taken together, these results emphasize the benefit of these nanocarriers as potential therapies against biofilm infections and fills the gap for multifunctional nanocarriers that prevent biofilm infections.