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Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near-exclusively performing redox reactions. We present an unexpected discovery of non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-materials, and non-metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan-glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as "nanozyme prodrug therapy" to mediate antibacterial measures.
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Nanoestruturas/química , Pró-Fármacos/química , Catálise , HumanosRESUMO
The development of bacteria-specific infection radiotracers is of considerable interest to improve diagnostic accuracy and enabling therapy monitoring. The aim of this study was to determine if the previously reported radiolabelled 1,4,7,10-tetraazacyclododecane-N,N',Nâ³,Nâ´-tetraacetic acid (DOTA) conjugated peptide [68 Ga]Ga-DOTA-K-A9 could detect a staphylococcal infection in vivo and distinguish it from aseptic inflammation. An optimized [68 Ga]Ga-DOTA-K-A9 synthesis omitting the use of acetone was developed, yielding 93 ± 0.9% radiochemical purity. The in vivo infection binding specificity of [68 Ga]Ga-DOTA-K-A9 was evaluated by micro positron emission tomography/magnetic resonance imaging of 15 mice with either subcutaneous Staphylococcus aureus infection or turpentine-induced inflammation and compared with 2-deoxy-2-[18 F]fluoro-D-glucose ([18 F]FDG). The scans showed that [68 Ga]Ga-DOTA-K-A9 accumulated in all the infected mice at injected doses ≥3.6 MBq. However, the tracer was not found to be selective towards infection, since the [68 Ga]Ga-DOTA-K-A9 also accumulated in mice with inflammation. In a concurrent in vitro binding evaluation performed with a 5-carboxytetramethylrhodamine (TAMRA) fluorescence analogue of the peptide, TAMRA-K-A9, the microscopy results suggested that TAMRA-K-A9 bound to an intracellular epitope and therefore preferentially targeted dead bacteria. Thus, the [68 Ga]Ga-DOTA-K-A9 uptake observed in vivo is presumably a combination of local hyperemia, vascular leakiness and/or binding to an epitope present in dead bacteria.
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Extracellular DNA (eDNA) is a major matrix component of many bacterial biofilms. While the presence of eDNA and its role in biofilm stability have been demonstrated for several laboratory biofilms of oral bacteria, there is no data available on the presence and function of eDNA in in vivo grown dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated with either DNase or heat-inactivated DNase for 1 h. The bacterial biovolume was determined with digital image analysis. Staining with TOTO®-1 allowed visualization of eDNA both on bacterial cell surfaces and, with a cloud-like appearance, in the intercellular space. DNase treatment strongly reduced the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover the interplay of eDNA and other matrix components and to explore the therapeutic potential of DNase treatment for biofilm control.
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Biofilmes , DNA Bacteriano/fisiologia , Adulto , DNA Bacteriano/análise , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Caries is caused by acid production in biofilms on dental surfaces. Preventing caries therefore involves control of microorganisms and/or the acid produced. Here, calcium-phosphate-osteopontin particles are presented as a new approach to caries control. The particles are made by co-precipitation and designed to bind to bacteria in biofilms, impede biofilm build-up without killing the microflora, and release phosphate ions to buffer bacterial acid production if the pH decreases below 6. Analysis of biofilm formation and pH in a five-species biofilm model for dental caries showed that treatment with particles or pure osteopontin led to less biofilm formation compared to untreated controls or biofilms treated with osteopontin-free particles. The anti-biofilm effect can thus be ascribed to osteopontin. The particles also led to a slower acidification of the biofilm after exposure to glucose, and the pH always remained above 5.5. Hence, calcium-phosphate-osteopontin particles show potential for applications in caries control.
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Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Biofilmes , Fosfatos de Cálcio/farmacologia , Cárie Dentária/prevenção & controle , Osteopontina/farmacologia , Desequilíbrio Ácido-Base/metabolismo , Desequilíbrio Ácido-Base/prevenção & controle , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cárie Dentária/metabolismo , Cárie Dentária/microbiologia , Combinação de Medicamentos , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacosRESUMO
The ability of bacteria to bind different compounds and to adhere to biotic and abiotic surfaces provides them with a range of advantages, such as colonization of various tissues, internalization, avoidance of an immune response, and survival and persistence in the environment. A variety of bacterial surface structures are involved in this process and these promote bacterial adhesion in a more or less specific manner. In this review, we will focus on those surface adhesins and exopolymers in selected foodborne pathogens that are involved mainly in primary adhesion. Their role in biofilm development will also be considered when appropriate. Both the clinical impact and the implications for food safety of such adhesion will be discussed.
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Adesinas Bacterianas/análise , Bactérias/metabolismo , Aderência Bacteriana , Fenômenos Fisiológicos Bacterianos , Biopolímeros/metabolismo , Doenças Transmitidas por Alimentos/microbiologia , Biofilmes/crescimento & desenvolvimentoRESUMO
Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles.
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Antibacterianos/metabolismo , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Listeria/metabolismo , Polilisina/metabolismo , Transporte Biológico , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli/química , Listeria/química , Listeria/efeitos dos fármacos , Polilisina/farmacologiaRESUMO
Bringing the study of bacterial adhesion down to a single-cell level is critical for understanding the molecular mechanisms involved in initial bacterial attachment. We have developed a simple and versatile method for making single-cell bacterial probes to study the adhesion of single bacterial cells by atomic force microscopy (AFM). A single-cell probe was made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated with a commercial cell adhesive Cell-Tak. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, and mica). Attachment to the cantilever was stable during the AFM force measurements that were conducted for 2 h, and viability was confirmed by Live/Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and contact time. The single-cell probe offers control of cell immobilization and thus holds advantages over the commonly used multicell probes with which random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported method provides a general platform for investigating single-cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding of the mechanisms of bacterial attachment.
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Proteínas de Bactérias/química , Escherichia coli/citologia , Microscopia de Força Atômica , Pseudomonas fluorescens/citologia , Análise de Célula Única , Staphylococcus/citologia , Adesão Celular , Escherichia coli/crescimento & desenvolvimento , Pseudomonas fluorescens/crescimento & desenvolvimento , Staphylococcus/crescimento & desenvolvimentoRESUMO
OBJECTIVES: To evaluate the effect of daily use of a multiple-enzyme lozenge on de novo plaque formation, on gingivitis development, and on the oral microbiome composition. METHODS: This trial with two parallel arms included 24 healthy adults allocated to the Active (n = 12) or Placebo (n = 12) group. Subjects consumed one lozenge three times daily for seven days, and no oral hygiene procedures were allowed. Differences in de novo plaque accumulation between a baseline period, and one and seven days of intervention were assessed by the Turesky-modification of the Quigley-and-Hein-Plaque-Index (TM-QHPI). The development of gingivitis after seven days of intervention was assessed by the Gingival Index (GI). Plaque and saliva samples were collected at baseline and after seven days of intervention, and evaluated by 16S rRNA gene sequencing. RESULTS: All subjects completed the study, and no adverse events were reported. After one day, the average TM-QHPI was significantly lower in the Active than in the Placebo group, as compared to baseline (p = 0.012). After 7 days, average TM-QHPI values did not differ significantly between groups (p = 0.37). GI values did not increase during the intervention period, with no difference between groups (p = 0.62). Bacterial richness increased in both plaque and saliva samples over a seven-day oral hygiene-free period, with a statistically significant difference for the saliva samples (p = 0.0495) between groups. CONCLUSIONS: A multiple-enzymes lozenge decreased the build-up of de novo plaque after one day and slowed down the process of species increment in saliva. The lozenge may be an adjunct to regular mechanical plaque removal. CLINICAL SIGNIFICANCE: Dental plaque is the main cause of caries, gingivitis, and periodontitis. The search for therapeutic adjuncts to mechanical plaque removal that have no harmful effects on the oral microbiome is important. Treatment with multiple plaque-matrix degrading enzymes is a promising non-biocidal approach to plaque control.
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Biofilmes , Índice de Placa Dentária , Placa Dentária , Gengivite , Índice Periodontal , Saliva , Humanos , Placa Dentária/microbiologia , Feminino , Gengivite/microbiologia , Masculino , Biofilmes/efeitos dos fármacos , Adulto , Saliva/microbiologia , Projetos Piloto , Adulto Jovem , RNA Ribossômico 16S , Microbiota/efeitos dos fármacos , Método Duplo-Cego , Higiene Bucal , Resultado do Tratamento , Hidrolases/uso terapêutico , Pessoa de Meia-IdadeRESUMO
Polyether ionophores are complex natural products known to transport various cations across biological membranes. While several members of this family are used in agriculture (e.g., as anti-coccidiostats) and have potent antibacterial activity, they are not currently being pursued as antibiotics for human use. Polyether ionophores are typically grouped as having similar functions, despite the fact that they significantly differ in structure; for this reason, how their structure and activity are related remains unclear. To determine whether certain members of the family constitute particularly interesting springboards for in-depth investigations and future synthetic optimization, we conducted a systematic comparative study of eight different polyether ionophores for their potential as antibiotics. This includes clinical isolates from bloodstream infections and studies of the compounds' effects on bacterial biofilms and persister cells. We uncover distinct differences within the compound class and identify the compounds lasalocid, calcimycin, and nanchangmycin as having particularly interesting activity profiles for further development. IMPORTANCE Polyether ionophores are complex natural products used in agriculture as anti-coccidiostats in poultry and as growth promoters in cattle, although their precise mechanism is not understood. They are widely regarded as antimicrobials against Gram-positive bacteria and protozoa, but fear of toxicity has so far prevented their use in humans. We show that ionophores generally have very different effects on Staphylococcus aureus, both in standard assays and in more complex systems such as bacterial biofilms and persister cell populations. This will allow us to focus on the most interesting compounds for future in-depth investigations and synthetic optimizations.
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Antibacterianos , Anti-Infecciosos , Humanos , Animais , Bovinos , Ionóforos/farmacologia , Ionóforos/química , Antibacterianos/uso terapêutico , Anti-Infecciosos/farmacologia , Bactérias Gram-Positivas , Biofilmes , Testes de Sensibilidade MicrobianaRESUMO
Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluorescent protein reporters in their native environment, because they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.
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The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms.
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Anti-Infecciosos , Imunoconjugados , Camundongos , Animais , Liberação Controlada de Fármacos , Bactérias , BiofilmesRESUMO
Bacterial biofilms are involved in numerous infections of the human body, including dental caries. While conventional therapy of biofilm diseases aims at eradication and mechanical removal of the biofilms, recent therapeutic approaches target the mechanisms of biofilm formation and bacterial adhesion in particular. The effect of bovine milk osteopontin, a highly phosphorylated whey protein, on adhesion of Streptococcus mitis, Streptococcus sanguinis, and Actinomyces naeslundii, three prominent colonizers in dental biofilms, to saliva-coated surfaces was investigated. While adhesion of A. naeslundii was not affected by osteopontin, a strong, dose-dependent reduction in the number of adhering S. mitis was shown. No difference in bacterial adhesion was observed for caseinoglycomacropeptide, another phosphorylated milk protein. Osteopontin did not affect bacterial viability, but changed bacterial surface hydrophobicity, and may be suggested to prevent the adhesins of S. mitis from interacting with their salivary receptors. The antiadhesive effect of osteopontin may be useful for caries prevention.
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Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Osteopontina/farmacologia , Dente/microbiologia , Actinomyces , Adesinas Bacterianas/efeitos dos fármacos , Animais , Bovinos , Cárie Dentária/microbiologia , Cárie Dentária/prevenção & controle , Película Dentária/microbiologia , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Leite/química , Saliva/microbiologia , Streptococcus mitis/efeitos dos fármacos , Streptococcus sanguis/efeitos dos fármacosRESUMO
Treatment of Staphylococcus aureus biofilm infections using conventional antibiotic therapy is challenging as only doses that are sublethal to the biofilm can be administered safely to patients. A potential solution to this challenge is targeted drug delivery. In this study, we tailored an aptamer-targeted liposomal drug delivery system for accumulation and delivery of antibiotics locally in S. aureus biofilm. In our search for a suitable targeting ligand, we identified six DNA aptamers that bound to S. aureus cells in biofilms, and we demonstrated that one of these aptamers could facilitate accumulation of liposomes around S. aureus cells inside the biofilm. Aptamer-targeted liposomes encapsulating a combination of vancomycin and rifampicin were able to eradicate S. aureus biofilm upon 24 h of treatment in vitro. Our results point to that aptamer-targeted drug delivery of antibiotics is a potential new strategy for treatment of S. aureus biofilm infections.
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Infecções Estafilocócicas , Staphylococcus aureus , Antibacterianos/uso terapêutico , Biofilmes , Sistemas de Liberação de Medicamentos , Humanos , Lipossomos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/genéticaRESUMO
Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at â¼7 nm localization precision and with a surface density of 38-46 µm-2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.
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Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/metabolismo , Fibronectinas/metabolismo , Aderência Bacteriana , Integrina alfa5beta1/metabolismo , DNA/metabolismo , Adesinas Bacterianas/metabolismoRESUMO
Introduction. Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp).Hypothesis/Gap Statement. We hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo.Aim. We address the importance of PIA and Embp in biofilm grown in 'humanized' media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix.Methodology. S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis.Results. S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility.Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell-cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.
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Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/fisiologia , Aderência Bacteriana , Regulação Bacteriana da Expressão Gênica , HumanosRESUMO
The increasing demand and limited natural resources for industrially important platinum-group metal (PGM) catalysts render the recovery from secondary sources such as industrial waste economically interesting. In the process of palladium (Pd) recovery, microorganisms have revealed a strong potential. Hitherto, bacteria with the property of dissimilatory metal reduction have been in focus, although the biochemical reactions linking enzymatic Pd(II) reduction and Pd(0) deposition have not yet been identified. In this study we investigated Pd(II) reduction with formate as the electron donor in the presence of Gram-negative bacteria with no documented capacity for reducing metals for energy production: Cupriavidus necator, Pseudomonas putida, and Paracoccus denitrificans. Only large and close-packed Pd(0) aggregates were formed in cell-free buffer solutions. Pd(II) reduction in the presence of bacteria resulted in smaller, well-suspended Pd(0) particles that were associated with the cells (called "bioPd(0)" in the following). Nanosize Pd(0) particles (3-30 nm) were only observed in the presence of bacteria, and particles in this size range were located in the periplasmic space. Pd(0) nanoparticles were still deposited on autoclaved cells of C. necator that had no hydrogenase activity, suggesting a hydrogenase-independent formation mechanism. The catalytic properties of Pd(0) and bioPd(0) were determined by the amount of hydrogen released in a reaction with hypophosphite. Generally, bioPd(0) demonstrated a lower level of activity than the Pd(0) control, possibly due to the inaccessibility of the Pd(0) fraction embedded in the cell envelope. Our results demonstrate the suitability of bacterial cells for the recovery of Pd(0), and formation and immobilization of Pd(0) nanoparticles inside the cell envelope. However, procedures to make periplasmic Pd(0) catalytically accessible need to be developed for future nanobiotechnological applications.
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Cupriavidus necator/metabolismo , Nanopartículas , Paládio/metabolismo , Paracoccus denitrificans/metabolismo , Pseudomonas putida/metabolismo , Cupriavidus necator/química , Formiatos/metabolismo , Hidrogênio/metabolismo , Oxirredução , Paracoccus denitrificans/química , Periplasma/química , Pseudomonas putida/químicaRESUMO
The risk of foodborne diseases has increased over the last years. We have developed a simple, portable, and label-free optical sensor via aptamer recognition of Staphylococcus aureus at nanostructured plasmonic elements. The developed aptamers conjugated to a localized surface plasmon resonance (LSPR) sensing device were applied in both pure culture and artificially contaminated milk samples enabling a limit of detection of 103 CFU/mL for S. aureus in milk. There was no need for a pre-enrichment step, and the total analysis time decreased from 30 min to 120 s. Finite-difference time-domain was used to simulate the experimentally measured optical responses for a range of different sensor designs (100 and 200 nm disks), addressing the role of the near field and intrinsic refractive index sensitivity. A comparison of the aptamer to antibody-based recognition approaches showed that the thickness of the sensing layer was critical with a significantly larger response for the thinner aptamer layer. Comparison of differently sized metal nanostructures showed a significantly higher sensitivity for 200 nm diameter compared to 100 nm diameter disk structures resulting from both increases in bulk refractive index sensitivity and the extent to which the local field extends out from the metal surface. These findings confirmed that the developed gold nanodisk-based LSPR sensing chips could facilitate sensitive detection of S. aureus in food samples.
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Nanomaterials have emerged as antimicrobial agents due to their unique physical and chemical properties. The development of nanoparticles (NPs) composed of natural biopolymers and biosurfactants have sparked interest, as they can be obtained without the use of complex chemical synthesis and toxic materials. In this study, we develop antimicrobial nanoparticles combining the biopolymer chitosan with the biosurfactant rhamnolipid. Addition of rhamnolipid reduced the size and polydispersity index of chitosan nanoparticles showing a more positive surface charge with improved stability, suggesting that chitosan-free amino groups are predominantly present on the surface of nanoparticles. Antimicrobial activity of chitosan/rhamnolipid nanoparticles (C/RL-NPs) against Staphylococcus strains surpassed that of either single rhamnolipid or chitosan, both in planktonic bacteria and biofilms. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C/RL-NPs were determined considering the concentration of each individual molecule in NPs. MIC values of 14/19 µg mL-1 and MBC of 29/37 µg mL-1 were observed for S. aureus DSM 1104 and MIC and MBC of 29/37 and 58/75 µg mL-1 were observed against S. aureus ATCC 29213, respectively. For S. epidermidis, MIC and MBC of 7/9 and 14/19 µg mL-1 were noticed. Chitosan and chitosan nanoparticles eliminate the bacteria present in the upper parts of biofilms, while C/RL-NPs were more effective, eradicating most sessile bacteria and reducing the number of viable cells below the detection limit, when NPs concentration of 58/75 µg mL-1 was applied for both S. aureus DSM 1104 and S. epidermidis biofilms. The improved antibacterial efficacy of C/RL-NPs was linked to the increased local delivery of chitosan and rhamnolipid at the cell surface and, consequently, to their targets in Gram-positive bacteria. The combination of chitosan and rhamnolipid offers a promising strategy to the design of novel nanoparticles with low cytotoxicity, which can be exploited in pharmaceutical and food industries.
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Anti-Infecciosos , Bactérias/efeitos dos fármacos , Quitosana , Glicolipídeos , Nanopartículas/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/química , Quitosana/farmacologia , Glicolipídeos/química , Glicolipídeos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Testes de Sensibilidade MicrobianaRESUMO
We utilize an aqueous extract of fish proteins (FPs) as a coating for minimizing the adsorption of fibrinogen (Fg) and human serum albumin (HSA). The surfaces include stainless steel (SS), gold (Au), silicon dioxide (SiO(2)), and poly(styrene) (PS). The adsorption processes (kinetics and adsorbed mass) are followed by quartz crystal microbalance with dissipation (QCM-D). Complementary surface information is provided by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). QCM-D shows no mass increases to any of the FP-coated surfaces upon treating with Fg or HSA. Also, when Fg- or HSA-coated surfaces are exposed to the FPs, a significant increase in adsorbed mass occurs because the FPs are highly surface-active displacing Fg. Additionally, fluorescence microscopy confirms that very little Fg adsorbs to the FP-coated surfaces. We propose that FP coatings prevent protein adsorption by steric stabilization and could be an alternative method for preventing unwanted bioadhesion on medical materials.
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Proteínas/química , Adsorção , Animais , Peixes , Cinética , Microscopia de Força Atômica , Microscopia de Fluorescência , Propriedades de SuperfícieRESUMO
Treatment of polymicrobial infections requires combination therapy with drugs that have different antimicrobial spectra and possibly work in synergy. However, the different pharmacokinetics and adverse side effects challenge the simultaneous delivery of multiple drugs at the appropriate concentrations to the site of infection. Formulation of multiple drugs in nano-carrier systems may improve therapeutic efficacy by increasing the local concentration and lowering the systemic concentration, leading to fewer side effects. In this study, we loaded polymyxin B and vancomycin on bare and carboxyl-modified mesoporous silica nanoparticles (B-MSNs and C-MSNs, respectively) to achieve simulataneous local delivery of antibiotics against Gram-positive and -negative bacteria. Polymyxin B adsorbed preferentially to nanoparticles compared to vancomycin. The total antibiotic loading was 563 µg and 453 µg per mg B-MSNs or C-MSNs, respectively. Both B-MSNs and C-MSNs loaded with antibiotics were effective against Gram-negative and Gram-positive bacteria. The antibiotics had synergistic interactions against Gram-negative bacteria, and the antimicrobial efficacy was higher for antibiotic-loaded C-MSNs compared to free antibiotics at the same concentration even though the cytotoxicity was lower. Our study shows that formulations of existing antibiotics in nanocarrier systems can improve their therapeutic efficiency, indicating that combination therapy with drug-loaded silica nanoparticles may provide a better treatment outcome for infections that require high concentrations of multiple drugs.