Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation

Alimova, Alexandra; Katz, Alvin; Savage, Howard E.; Shah, Mahendra; Minko, Glenn; Will, D.V.; Rosen, Richard B.; McCormick, Steven A.; Alfano, R. R.

doi:10.1364/ao.42.004080

Cited by 58 publications

(37 citation statements)

References 33 publications

(31 reference statements)

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“…Very few studies on the use of autofluorescence for the detection of bacteria have been reported (2,14). Some bacteria exhibit spontaneous emission spectra following excitation at specific wavelengths.…”

Section: Discussionmentioning

confidence: 99%

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Patino¹,

Alamo²,

Cimino³

et al. 2008

Journal of Clinical Microbiology

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The diagnosis of tuberculosis in developing countries still relies on direct sputum examination by light microscopy, a method that is easy to perform and that is widely applied. However, because of its poor sensitivity and requirement for significant labor and training, light microscopy examination detects the bacilli in only 45 to 60% of all people whose specimens are culture positive for Mycobacterium tuberculosis. Therefore, new diagnostic methods that would enable the detection of the undiagnosed infected population and allow the early commencement of antituberculosis treatment are needed. In this work, the potential use of mycobacterial cyan autofluorescence for the detection of Mycobacterium tuberculosis was explored. The tubercle bacilli were easily visualized as brilliant fluorescent bacilli by microscopy and were easily tracked ex vivo during macrophage infection. Assays with seeded sputum and a 96-well microplate reader fluorimeter indicated that <10 6 bacilli ml ؊1 of sputum could be detected. Moreover, the use of microplates allowed the examination of only 200 l of sputum per sample without a loss of sensitivity. Treatment with heat or decontaminating chemical agents did not interfere with the autofluorescence assay; on the contrary, they improved the level of bacterial detection. Autofluorescence for the detection of bacilli is rapid and easy to perform compared to other methodologies and can be performed with minimal training, making this method suitable for implementation in developing countries.The diagnosis of tuberculosis (TB) in low-and middle-income countries, where more than 90% of TB cases occur, is mainly performed by microscopy examination of stained sputum smears for acid-fast bacilli (25). The International Standards for Tuberculosis Care considers the microscopic analysis of two or three specimens per patient to be a cornerstone for the diagnosis of TB (13). Although light microscopy is inexpensive, easy to perform, and highly specific in areas where there is a high prevalence of TB, it is relatively insensitive, requiring Ն10 4 bacilli ml Ϫ1 of sputum to achieve a positive result (15,35). In addition, the procedure requires the observation of from 100 to 300 fields in order to obtain accurate results (31).The sensitivity of microscopy is influenced by numerous factors, such as the prevalence and severity of disease, the quality of specimen collection, the type of specimen, the method of processing (direct or concentrated, centrifugation, liquefaction), staining, and finally, the quality of the examination (20,23,26,29). In well-trained hands, the test detects tubercle bacilli in 75% of all people who have active pulmonary TB, but the sensitivity may fall to 45 to 60%, depending on the training, eye, and motivation of the laboratory technician (1). The basis of conventional light microscopy was developed more than 100 years ago, and, at present, conventional light microscopy uses carbolfuchsin Ziehl-Neelsen or Kinyoun acid-fast stains. Fluorescence microscopy with fluorochrome dyes suc...

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Section: Discussionmentioning

confidence: 99%

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Patino¹,

Alamo²,

Cimino³

et al. 2008

Journal of Clinical Microbiology

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“…But this experimental challenge cannot account for the two orders of magnitude differences seen. It has been noted, however, that the procedures used to prepare biological species strongly influence the fluorescence spectra and fluorescence quantum efficiencies -the growth medium, and the number of times the sample has been washed, are known to greatly influence fluorescence profiles; large differences in behavior have also been observed between wet and dry samples; and illumination laser intensities and local concentrations of different fluorophores contained within the biological sample also impact not only the measured fluorescence intensities, but also the wavelength-resolved spectral profiles (Farris et al 1997;Fell et al 1998;Johnson et al 1999;Hill et al 2001;Alimova et al 2003;Kunnil et al 2005;Sarasanandarajah et al 2005b;Atkins et al 2007). …”

Section: Uv Fluorescence Lidarmentioning

confidence: 99%

Modeling LIDAR Detection of Biological Aerosols to Determine Optimum Implementation Strategy

Sheen¹,

Aker²

2007

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SummaryThis report summarizes work performed for a larger multi-laboratory project named the Background Interferent Measurement and Standards project. While PNNL was originally tasked to develop algorithms to optimize biological warfare agent detection using UV fluorescence LIDAR, the current uncertainties in the reported fluorescence profiles and cross sections preclude the development of any meaningful models. It was decided that a better approach would be to model the wavelength-dependent elastic backscattering from a number of ambient background aerosol types, and compare this with that generated from representative sporulated and vegetative bacterial systems. Calculations in this report show that a 266, 355, 532, and 1064 nm elastic-backscatter LIDAR experiment will allow an operator to immediately recognize when sulfate, VOC-based, or road dust (silicate) aerosols are approaching, independent of humidity changes. It will be more difficult to distinguish soot aerosols from biological aerosols, or vegetative bacteria from sporulated bacteria. In these latter cases, the elastic scattering data will most likely have to be combined with UV fluorescence data to enable a more robust categorization.iii iv Acknowledgments

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“…This molecule is prevalent in bacterial spores [17,18] and has been used as a marker molecule in various spectroscopic detection schemes [17,[19][20][21][22]. This molecule is an ideal test molecule for our parameters as it contains the atoms oxygen and nitrogen yet consists of a parent ring structurally similar to benzene, a molecule we have previously investigated with SERID [5,23].…”

Section: Dpa Equilibrium Propertiesmentioning

confidence: 99%

Semiquantitative model for response of biological molecules containing C, N, O and H to laser pulses, with initial application to dipicolinic acid

Sauer

Allen

2006

Journal of Modern Optics

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We have constructed a semiquantitative model which can be used to treat the response of molecules containing carbon, nitrogen, oxygen and hydrogen to ultrafast laser pulses. The parameters for the pairwise interaction of both nitrogen and oxygen are obtained from a simple scaling of carbon and hydrogen parameters calculated by Frauenheim and co-workers. For the initial application of this model, we have chosen dipicolinic acid (DPA), an important constituent of biological spores. The equilibrium bond lengths of all of the test molecules, including DPA, are within 5% of the experimental bond lengths. The calculated molecular orbital structure of DPA near the HOMO-LUMO gap region shows three nearly degenerate occupied molecular orbitals and two nearly degenerate unoccupied orbitals. Photoexcitation with an ultrashort 5 fs FWHM laser pulse matched to the HOMO-LUMO gap energy produced transitions from the lowest of the the three nearly degenerate occupied orbitals to both of the nearly degenerate unoccupied molecular orbitals. The precise nature of the transition depends on the polarization of the laser pulse. These results are shown to be in qualitative agreement with fully ab initio methods.

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Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation

Cited by 58 publications

References 33 publications

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Modeling LIDAR Detection of Biological Aerosols to Determine Optimum Implementation Strategy

Semiquantitative model for response of biological molecules containing C, N, O and H to laser pulses, with initial application to dipicolinic acid

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