Native fluorescence changes induced by bactericidal agents (original) (raw)
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The steady-state and decay characteristics of primary fluorescence from live bacteria
Applied Spectroscopy, 1987
The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coil, and Bacillus subtilis have been observed. Excitation spectra were obtained while emission at 430, 455, 487 and 514 nm was being monitored. Emission spectra were obtained with the use of excitation wavelengths of 340, 365, 405, 430 and 460 nm. Fluorescence lifetimes were measured at 430, 487, and 514 nm while selective excitation was caused at 340, 405, and 430 nm. The complex nature of the excitation and emission spectra reflects the presence of a number of different fluorophores. Attempts have been made to describe portions of the bacterial fluorescence in terms of the measured fluorescence properties including lifetimes of molecular components known for their widespread occurrence in bacteria and their relatively high quantum yields. Candidate flnorophores which have been considered include the pteridines, the structurally related flavins, and the pyridine coenzymes. The observation that characteristic sets of lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Results are especially definitive in cases such as Pseudomonas fluorescens, where one marker fluorophore, a pteridine, is produced in large amounts.
Applied Optics, 2003
Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis ͑Bs͒, a spore-forming, and Staphylococcus aureus ͑Sa͒, a nonspore-forming bacteria subjected to conditions of starvation. Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan. Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 nm, from dipicolinic acid, appeared. Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores. The intensity of the 410-nm band continued to increase over the next 3 days. The Sa fluorescence was predominantly from tryptophan and did not change over time. In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 nm and excitation peaks at 250, 270, and 400 nm. The addition of -hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission. The addition of Fe 2ϩ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria. We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time.
Rapid Detection of Three Common Bacteria Based on Fluorescence Spectroscopy
Sensors, 2022
As an important part of environmental water quality monitoring, efficient bacterial detection has attracted widespread attention. Among them, LIF (laser-induced fluorescence) technology has the characteristics of high efficiency and sensitivity for bacterial detection. To simplify the experimental process of bacterial detection, fluorescence emission spectra of E. coli (Escherichia coli) and its deactivated controls, K. pneumoniae (Klebsiella pneumoniae) and S. aureus (Staphylococcus aureus), were analyzed with fluorescence excitation by a 266 nm laser. By analyzing the results, it was found that the dominant fluorescence peaks of bacterial solutions at 335~350 nm were contributed by tryptophan, and the subfluorescence peaks at 515.9 nm were contributed by flavin; besides, K. pneumoniae and S. aureus had their own fluoresces characteristics, such as tyrosine contributing to sub-fluorescence peaks at 300 nm. The three species of bacteria can be differentiated with whole fluorescence ...
Environmental Science & Technology, 2009
Relationships between the formation of disinfection byproduct (DBPs) and changes of the fluorescence of natural organic matter (NOM) in chlorinated water were quantified using two fluorescence indexes. They were defined as the change of the wavelength that corresponds to 50% of the maximum intensity of fluorescence (∆λ em 0.5 ) and the differential ratio of fluorescence intensities measured at 500 and 450 nm (∆(I 500 /I 450 )). Although variations of chlorine doses, reaction times and temperatures affected the kinetics of chlorine consumption and DBPs release, correlations between chlorine consumption, concentrations, and speciation of trihalomethanes, haloacetonitriles, haloacetic acids and, on the other hand, ∆(I 500 /I 450 ) and ∆λ em 0.5 values remained unaffected by chlorination conditions and, to some extent, NOM properties. These results allow developing a fluorescence-based approach to monitor DBPs formation in drinking water. FIGURE 7. Correlations between ∆(I 500 /I 450 ) values and concentrations of TTHM (a), , and THAA (c). Chlorinated Ancipa and Potomac waters at pH 7.0, chlorine to DOC ratios from 0.25 to 2.00 mg/mg, reaction times from 10 to 3 days, and temperatures from 3 to 34°C.
Fluorescence Characterization of Clinically-Important Bacteria
2013
Healthcare-associated infections (HCAI/HAI) represent a substantial threat to patient health during hospitalization and incur billions of dollars additional cost for subsequent treatment. One promising method for the detection of bacterial contamination in a clinical setting before an HAI outbreak occurs is to exploit native fluorescence of cellular molecules for a hand-held, rapid-sweep surveillance instrument. Previous studies have shown fluorescence-based detection to be sensitive and effective for food-borne and environmental microorganisms, and even to be able to distinguish between cell types, but this powerful technique has not yet been deployed on the macroscale for the primary surveillance of contamination in healthcare facilities to prevent HAI. Here we report experimental data for the specification and design of such a fluorescence-based detection instrument. We have characterized the complete fluorescence response of eleven clinically-relevant bacteria by generating excitation-emission matrices (EEMs) over broad wavelength ranges. Furthermore, a number of surfaces and items of equipment commonly present on a ward, and potentially responsible for pathogen transfer, have been analyzed for potential issues of background fluorescence masking the signal from contaminant bacteria. These include bedside handrails, nurse call button, blood pressure cuff and ward computer keyboard, as well as disinfectant cleaning products and microfiber cloth. All examined bacterial strains exhibited a distinctive double-peak fluorescence feature associated with tryptophan with no other cellular fluorophore detected. Thus, this fluorescence survey found that an emission peak of 340nm, from an excitation source at 280nm, was the cellular fluorescence signal to target for detection of bacterial contamination. The majority of materials analysed offer a spectral window through which bacterial contamination could indeed be detected. A few instances were found of potential problems of background fluorescence masking that of bacteria, but in the case of the microfiber cleaning cloth, imaging techniques could morphologically distinguish between stray strands and bacterial contamination.
2021
This study is devoted to the development of a UV fluorimetry sensor capable of realtime monitoring of the decontamination process of microbiological pathogens by hydrogen peroxide vapour (HPV) treatment. The sensor is operating on the autofluorescence signal of tryptophan. The Bacillus atrophaeus (B. atrophaeus) and Geobacillus stearothermophilus (G. stearothermophilus) spores were exposed to HPV and the resulting dynamic change in tryptophan fluorescence intensity as a function of time was recorded and analysed. It was revealed that the introduced HPV atmosphere caused a 4time decrease in the fluorescence intensity of the tryptophan emission due to the interaction of HPV with the spores. It was shown that achieving a persistent minimal level of the autofluorescence signal due to the microorganismbound tryptophan during the defined time period is well correlated with the efficiency of the ongoing decontamination process in the HPV treatment course. Therefore, the progress of the HPV decontamination procedure can be firmly evaluated, using fluorescence data obtained in real time and the validity of the method was demonstrated by comparing the fluorescence data with the reference information obtained by implementing classical microbiology viability tests (incl. time behaviour) for various microorganisms in the HPV atmosphere.
Applied Spectroscopy, 1985
Time-resolved fluorescence spectra have been obtained for Escherichia coli, Pseudomonas fluorescens, Staphylococcus epidermidis, and Enterobacter cloacae. Pseudomonas fluorescens has been shown to have distinctly different time-resolved spectra. Fluorescence excitation spectra for the three other organisms showing similar time-resolved spectra are very nearly alike, while spectra of Pseudomonas fluorescens are markedly different. This suggests that the changes in the average fluorescence lifetime with emission wavelength are due to the separate contributions from fluorophores of distinctly different lifetimes which have emission maxima at different wavelengths.
Bacterial bioluminescence inhibition by Chlorophenols
Applied Biochemistry and Microbiology, 2000
Photobacteria were used as a test object for rapid monitoring of ecotoxicants. Specific inhibitory effects of phenol and its chlorinated derivatives (2-chlorophenol, 2,3-dichlorophenol, pentachlorophenol, 2,4dichlorophenoxyacetic acid, and 2,4,5-tdchlorophenoxyacetic acid) on bioluminescence and respiration of intact cells, as well as on the emission activity of the bioluminescence system and luciferase itself, were studied. The toxic effect on the photobacterial cells was found to increase as the number of chlorine atoms in the chlorophenol molecule increased. However, this trend was not observed in cell-free systems (purified luciferase or the protein fraction of a cell-free extract treated with (NH4)2SO 4 at 40-75% saturation). Bacterial cells have a higher threshold sensitivity to chlorophenols in comparison to the sensitivity of the bioluminescence enzyme sy. stem or luciferase. Neutral phenols inhibit luciferase by competing with decanal, whereas a mixed mechamsm of inhibition with this substrate is typical of phenoxyacetic acids. With respect to FMNH 2, all chlorophenols tested in this work were uncompetitive inhibitors. Oxygen uptake by photobacteria was shown to be insensitive to chlorophenols, at least within the concentration range that was effective in bioluminescence inhibition. The results of this study suggest that the bacterial bioluminescence system is not the primary target of the chlorophenol-induced effect on photobacteria.
Spectroscopic analysis of bacterial photoreactivation
Photochemistry and Photobiology, 2024
With the rise of bacterial infections and antibiotic resistance, spectroscopic devices originally developed for bacterial detection have shown promise to rapidly identify bacterial strains and determine the ratio of live to dead bacteria. However, the detection of the photoreactivated pathogens remains a critical concern. This study utilizes fluorescence and Raman spectroscopy to analyze bacterial responses to UV irradiation and subsequent photoreactivation. Our experimental results reveal limitations in fluorescence spectroscopy for detecting photoreactivated bacteria, as the intense fluorescence of tryptophan and tyrosine amino acids masks the fluorescence emitted by thymine molecules. Conversely, Raman spectroscopy proves more effective, showing a detectable decrease in band intensities of E. coli bacteria at 1248 and 1665 cm-1 after exposure to UVC radiation. Subsequent UVA irradiation results in the partial restoration of these band intensities, indicating DNA repair and bacterial photoreactivation. This enhanced understanding aims to improve the accuracy and effectiveness of these spectroscopic tools in clinical and environmental settings.