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A Fluorescence Based-Proliferation Assay for the Identification of Replicating Bacteria Within Host Cells
摘要： Understanding host pathogen interactions is paramount to the development of novel antimicrobials. An important facet of this pursuit is the accurate characterization of pathogen replication within infected host cells. Here we describe the use of a fluorescence-based proliferation assay to identify intracellular populations of replicating bacteria at the subcellular level. Using Staphylococcus aureus as a model Gram-positive bacterial pathogen and macrophages as a model host phagocyte, we demonstrate this assay can be used to reliably identify individual phagocytes that contain replicating bacteria. Furthermore, we demonstrate this assay is compatible with additional cellular probes that enable characterization of cellular compartments in which replicating bacteria reside. Finally, we demonstrate that this assay facilitates the investigation of both Gram-negative and Gram-positive bacteria within host cells.
关键词： microscopy,Staphylococcus,phagolysosome,fluorescence,phagocytosis,macrophage
更新于2025-09-23 15:22:29
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Changes of Intracellular Porphyrin, Reactive Oxygen Species, and Fatty Acids Profiles During Inactivation of Methicillin-Resistant Staphylococcus aureus by Antimicrobial Blue Light
摘要： Antimicrobial blue light (aBL) has attracted increasing interest for its antimicrobial properties. However, the underlying bactericidal mechanism has not yet been verified. One hypothesis is that aBL causes the excitation of intracellular chromophores; leading to the generation of reactive oxygen species (ROS) and the resultant oxidization of various biomolecules. Thus, monitoring the levels of redox-sensitive intracellular biomolecules such as coproporphyrins, as well as singlet oxygen and various ROS may help to uncover the physiological changes induced by aBL and aid in establishing the underlying mechanism of action. Furthermore, the identification of novel targets of ROS, such as fatty acids, is of potential significance from a therapeutic perspective. In this study, we sought to investigate the molecular impact of aBL treatment on methicillin-resistant Staphylococcus aureus (MRSA). The results showed that aBL (5–80 J/cm2) exhibited a bactericidal effect on MRSA, and almost no bacteria survived when 80 J/cm2 had been delivered. Further studies revealed that the concentrations of certain intracellular molecules varied in response to aBL irradiation. Coproporphyrin levels were found to decrease gradually, while ROS levels increased rapidly. Moreover, imaging revealed the emergence and increase of singlet oxygen molecules. Concomitantly, the lipid peroxidation product malondialdehyde (MDA) increased in abundance and intracellular K+ leakage was observed, indicating permeability of the cell membrane. Atomic force microscopy showed that the cell surface exhibited a coarse appearance. Finally, fatty acid profiles at different illumination levels were monitored by GC-MS. The relative amounts of three unsaturated fatty acids (C16:1, C20:1, and C20:4) were decreased in response to aBL irradiation, which likely played a key role in the aforementioned membrane injuries. Collectively, these data suggest that the cell membrane is a major target of ROS during aBL irradiation, causing alterations to membrane lipid profiles, and in particular to the unsaturated fatty acid component.
关键词： membrane injuries,coproporphyrin,lipids,methicillin-resistant Staphylococcus aureus,antimicrobial blue light,unsaturated fatty acids
更新于2025-09-23 15:22:29
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[IEEE 2018 IEEE 8th International Conference Nanomaterials: Application & Properties (NAP) - Zatoka, Ukraine (2018.9.9-2018.9.14)] 2018 IEEE 8th International Conference Nanomaterials: Application & Properties (NAP) - The Study of the Activation of Antibacterial Activity of Silver Nanoparticles by Laser Radiation
摘要： Silver nanoparticles are known to have inhibitory and bactericidal effects and they play a significant role in the development of new antimicrobial substances against pathogenic microorganisms. In this article the minimum bactericidal concentration of the silver nanoparticles on two different bacteria cultures (Escherichia coli and Staphylococcus aureus) as well as the possibility to enhance their antimicrobial activity using laser irradiation was investigated. It was found, that solutions of silver nanoparticles at concentrations ≥ 3120 μg/ml cause death of both types of studied microorganisms. Dispersion of nanoparticles after laser irradiation caused the inhibition of Staphylococcus aureus at lower concentrations.
关键词： surface plasmon resonance,Escherichia coli,Staphylococcus aureus,bactericidal properties,silver nanoparticles
更新于2025-09-23 15:21:01
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Blood–Retinal Barrier Compromise and Endogenous <i>Staphylococcus aureus</i> Endophthalmitis
摘要： PURPOSE. To test the hypothesis that blood–retinal barrier compromise is associated with the development of endogenous Staphylococcus aureus endophthalmitis. METHODS. To compromise the blood–retinal barrier in vivo, streptozotocin-induced diabetes was induced in C57BL/6J mice for 1, 3, or 5 months. Diabetic and age-matched nondiabetic mice were intravenously injected with 108 colony-forming units (cfu) of S. aureus, a common cause of endogenous endophthalmitis in diabetics. After 4 days post infection, electroretinography, histology, and bacterial counts were performed. Staphylococcus aureus–induced alterations in in vitro retinal pigment epithelial (RPE) cell barrier structure and function were assessed by anti–ZO-1 immunohistochemistry, FITC-dextran conjugate diffusion, and bacterial transmigration assays. RESULTS. We observed one bilateral infection in a control, nondiabetic animal (mean ? 1.54 3 103 6 1.78 3 102 cfu/eye, 7% incidence). Among the 1-month diabetic mice, we observed culture-con?rmed unilateral infections in two animals (mean ? 5.54 3 102 6 7.09 3 102 cfu/ eye, 12% incidence). Among the 3-month diabetic mice, infections were observed in 11 animals, three with bilateral infections (mean ? 2.67 3 102 6 2.49 3 102 cfu/eye, 58% incidence). Among the 5-month diabetic mice, we observed infections in ?ve animals (mean ? 7.88 3 102 6 1.08 3 103 cfu/eye, 33% incidence). In vitro, S. aureus infection reduced ZO-1 immunostaining and disrupted the barrier function of cultured RPE cells, resulting in diffusion of ?uorophore-conjugated dextrans and transmigration of live bacteria across a permeabilized RPE barrier. CONCLUSIONS. Taken together, these results indicated that S. aureus is capable of inducing blood–retinal barrier permeability and causing endogenous bacterial endophthalmitis in normal and diabetic animals.
关键词： Staphylococcus aureus,endogenous endophthalmitis,blood–retinal barrier,bacteria,diabetes,retinal pigment epithelium
更新于2025-09-23 15:21:01
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Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for accurate discrimination of Staphylococcus schleiferi subspecies
摘要： S. schleiferi is one of the main species isolated from canine otitis externa, pyoderma and from apparently healthy dogs. The species is divided into two subspecies, S. schleiferi schleiferi and S. schleiferi coagulans. MALDI-TOF MS does not distinguish correctly these two subspecies. This study aimed to identify biomarkers that could possibly discriminate Staphylococcus schleiferi subspecies by MALDI-TOF MS. Twelve strains (eight S. schleiferi schleiferi and four S. schleiferi coagulans) were firstly identified. Each isolate was submitted to a protein extraction protocol and used for improved subspecies identification in clinical samples.
关键词： MALDI-TOF MS,Staphylococcus schleiferi,biomarkers
更新于2025-09-23 15:19:57
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Incorporating Statistical Test and Machine Intelligence Into Strain Typing of Staphylococcus haemolyticus Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
摘要： Staphylococcus haemolyticus is one of the most significant coagulase-negative staphylococci, and it often causes severe infections. Rapid strain typing of pathogenic S. haemolyticus is indispensable in modern public health infectious disease control, infections to prevent further infectious facilitating the identification of the origin of outbreak. Rapid identification enables the effective control of pathogenic infections, which is tremendously beneficial to critically ill patients. However, the existing strain typing methods, such as multi-locus sequencing, are of relatively high cost and comparatively time-consuming. A practical method for the rapid strain typing of pathogens, suitable for routine use in clinics and hospitals, is still not available. Matrix-assisted laser desorption ionization-time of flight mass spectrometry combined with machine learning approaches is a promising method to carry out rapid strain typing. In this study, we developed a statistical test-based method to determine the reference spectrum when dealing with alignment of mass spectra datasets, and constructed machine learning-based classifiers for categorizing different strains of S. haemolyticus. The area under the receiver operating characteristic curve and accuracy of multi-class predictions were 0.848 and 0.866, respectively. Additionally, we employed a variety of statistical tests and feature-selection strategies to identify the discriminative peaks that can substantially contribute to strain typing. This study not only incorporates statistical test-based methods to manage the alignment of mass spectra datasets but also provides a practical means to accomplish rapid strain typing of S. haemolyticus.
关键词： MALDI-TOF MS,strain typing,Fisher’s exact test,Staphylococcus haemolyticus,machine learning
更新于2025-09-16 10:30:52
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[IEEE 2019 18th International Conference on Optical Communications and Networks (ICOCN) - Huangshan, China (2019.8.5-2019.8.8)] 2019 18th International Conference on Optical Communications and Networks (ICOCN) - High sensitivity biosensor for Staphylococcus Aureus detection based on tapered a singlemode-no core-singlemode fiber structure
摘要： A fiber biosensor based on tapered singlemode-no core-singlemode (SNS) fiber structure was investigated for the detection of staphylococcus aureus. The fibre sensor was functionalised by depositing the pig IgG antibodies onto tapered biosensor surface, which has specific binding with staphylococcus aureus. The experimental results showed that transmission spectrum of functionalized sensor undergone wavelength blue shift when it was immersed into Staphylococcus aureus solution, and 1.1 nm wavelength shift was observed with a concentration of 101colony-forming units (CFU)/mL.
关键词： optical fiber sensor,biosensor,staphylococcus aureus,foodborne pathogens,the pig IgG
更新于2025-09-16 10:30:52
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2125. Staphylococcus Species Identification by Fourier Transform Infrared (FTIR) Spectroscopic Techniques: A Cross-Lab Study
摘要： Staphylococcus aureus is well known to be associated with atopic dermatitis. Recent studies also report S. aureus presence in lesional skin of squamous cell carcinoma (SCC) and its precursor lesion, actinic keratosis (AK). Therefore, it is of potential clinical interest to monitor skin S. aureus colonization on AK lesions. Fourier transform infrared (FTIR) spectroscopy is a cost-effective, nondestructive, and reagent-free technique for rapid microbial identification. It is based on the use of spectral databases developed with well-characterized strains in conjunction with the application of multivariate statistical analysis to elaborate classification models. In the present cross-lab study, spectral databases containing FTIR spectra of over 1000 staphylococcal isolates obtained from reference and clinical microbiology laboratories across Canada were employed in the FTIR spectroscopic identification of Staphylococcus spp. isolated from AK, SCC and perilesional skin of patients at the Princess Alexandra Hospital Dermatology Clinic in Brisbane, Australia.
关键词： atopic dermatitis,actinic keratosis,squamous cell carcinoma,microbial identification,Staphylococcus aureus,Fourier transform infrared spectroscopy
更新于2025-09-16 10:30:52
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Realisation and assessment of a low-cost LED device for contact lens disinfection by visible violet light
摘要： This study presents a device for efficient, low-cost and eye-friendly overnight disinfection of contact lenses by visible violet light as an alternative to disinfection with biocide-containing solutions. Bacterial solutions with one Pseudomonas and one Staphylococcus strain each were irradiated for up to 8 h in commercial transparent contact lens cases by the presented light-emitting diode (LED) device. Samples were taken at different intervals and distributed on agar plates. The surviving bacteria were determined by counting of colony-forming units and compared to the specific requirements of the stand-alone test for contact lens disinfection of the hygiene standard ISO 14729. The concentration of both microorganisms was reduced by three orders of magnitude after less than 4 h of irradiation. The LED current and intensity have not yet been at maximum and could be further increased if necessary for other microorganisms. The presented device fulfils the requirement of the stand-alone test of the contact lens hygienic standard ISO 14729 for the tested Pseudomonas and Staphylococcus strains. According to literature data, the inactivation of Serratia marcescens, Candida albicans and Fusarium solani seems also possible, but may require increased LED current and intensity.
关键词： Staphylococcus,LED device,contact lens,visible light,photoinactivation,Pseudomonas,disinfection
更新于2025-09-12 10:27:22
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Flexible organic light-emitting diodes for antimicrobial photodynamic therapy
摘要： Bacterial infection and the growth of antibiotic resistance is a serious problem that leads to patient suffering, death and increased costs of healthcare. To address this problem, we propose using flexible organic light-emitting diodes (OLEDs) as light sources for photodynamic therapy (PDT) to kill bacteria. PDT involves the use of light and a photosensitizer to generate reactive oxygen species that kill neighbouring cells. We have developed flexible top-emitting OLEDs with the ability to tune the emission peak from 669 to 737 nm to match the photosensitizer, together with high irradiance, low driving voltage, long operational lifetime and adequate shelf-life. These features enable OLEDs to be the ideal candidate for ambulatory PDT light sources. A detailed study of OLED–PDT for killing Staphylococcus aureus was performed. The results show that our OLEDs in combination with the photosensitizer methylene blue, can kill more than 99% of bacteria. This indicates a huge potential for using OLEDs to treat bacterial infections.
关键词： Photodynamic therapy,Flexible organic light-emitting diodes,Staphylococcus aureus,Antimicrobial,Methylene blue
更新于2025-09-12 10:27:22