A membrane basis for bacterial identification and discrimination using laser-induced breakdown spectroscopy

Abstract

Follow this and additional works at: https://scholar.uwindsor.ca/physicspub Recommended Citation Recommended Citation Rehse, Steven J.; Jeyasingham, N.; and Palchaudhuri, S.. (2009). A membrane basis for bacterial identification and discrimination using laser-induced breakdown spectroscopy. Journal of Applied Physics, 105 (10).Nanosecond single-pulse laser-induced breakdown spectroscopy ͑LIBS͒ has been used to discriminate between two different genera of Gram-negative bacteria and between several strains of the Escherichia coli bacterium based on the relative concentration of trace inorganic elements in the bacteria. Of particular importance in all such studies to date has been the role of divalent cations, specifically Ca 2+ and Mg 2+ , which are present in the membranes of Gram-negative bacteria and act to aggregate the highly polar lipopolysaccharide molecules. We have demonstrated that the source of emission from Ca and Mg atoms observed in LIBS plasmas from bacteria is at least partially located at the outer membrane by intentionally altering membrane biochemistry and correlating these changes with the observed changes in the LIBS spectra. The definitive assignment of some fraction of the LIBS emission to the outer membrane composition establishes a potential serological, or surface-antigen, basis for the laser-based identification. E. coli and Pseudomonas aeruginosa were cultured in three nutrient media: trypticase soy agar as a control, a MacConkey agar with a 0.01% concentration of bile salts including sodium deoxycholate, and a trypticase soy agar with a 0.4% deoxycholate concentration. The higher concentration of deoxycholate is known to disrupt bacterial outer membrane integrity and was expected to induce changes in the observed LIBS spectra. Altered LIBS emission was observed for bacteria cultured in this 0.4% medium and laser ablated in an all-argon environment. These spectra evidenced a reduced calcium emission and in the case of one species, a reduced magnesium emission. Culturing on the lower ͑0.01%͒ concentration of bile salts altered the LIBS spectra for both the P. aeruginosa and two strains of E. coli in a highly reproducible way, although not nearly as significantly as culturing in the higher concentration of deoxycholate did. This was possibly due to the accumulation of divalent cations around the bacteria by the formation of an extracellular polysaccharide capsule. Lastly, a discriminant function analysis demonstrated that in spite of alterations in the LIBS spectrum induced by growth in the three different media, the analysis could correctly identify all samples better than 90% of the time. This encouraging result illustrates the potential utility of LIBS as a rapid bacteriological identification technology.

Towards the clinical application of laser-induced breakdown spectroscopy for rapid pathogen diagnosis: the effect of mixed cultures and sample dilution on bacterial identification

“…Introduction Laser-induced breakdown spectroscopy (LIBS) is a sensitive spectrochemical technique that has recently begun to be used for characterizing biological samples such as microorganisms (i.e., bacteria) and tissues [1][2][3][4][5][6][7][8][9][10][11][12]. The speed, portability, and robustness of the technique suggest that LIBS may be applicable as rapid point-of-care medical diagnostic technology.…”

The Effect of Bacterial Environmental and Metabolic Stresses on a Laser-Induced Breakdown Spectroscopy (LIBS) Based Identification of <I>Escherichia coli</I> and <I>Streptococcus viridans</I>

“…As well, it is likely that the bacteria cannot survive if their elemental composition is significantly altered beyond a narrow range, particularly given the important role that the divalent cations of Ca and Mg play in regulating cell function and membrane porosity. 27 Undoubtedly media could be obtained or created that would significantly alter bacterial elemental concentrations while still encouraging growth. However, because we are attempting to develop a biomedical diagnostic, we are primarily interested in testing bacteria in conditions that they are likely to experience in vivo, and not in arbitrary or unrealistic chemical environments.…”

“…The specimens of E. coli C cultured on the two different media (TSA and MacConkey) were still closely grouped despite possible membrane alteration due to the detergent action of the deoxycholate on the lipid bilayer outer membrane of the E. coli. 27 In this DFA, 100% of the bacteria were correctly classified by strain, regardless of nutritional environmental conditions. This analysis therefore suggests that clinical specimens obtained from infected persons could be identified on the basis of their LIBS spectra independent of the chemical environment present in the host.…”

A comparison of multivariate analysis techniques and variable selection strategies in a laser-induced breakdown spectroscopy bacterial classification

“…This has been discussed by us in depth previously. [9] The remaining nineteen variables were composed of ratios of these sums (ten independent variables) and also unique combinations of the summed intensities forming complex ratios (nine independent variables). This approach has been utilized with success by Gottfried et al to discriminate LIBS spectra obtained from explosives residues.…”

“…[5][6][7][8] The intensities of strong specific elemental atomic emission lines normalized by the total observed spectral power have been utilized as independent variables in this multivariate analysis. [9] The selection of specific spectral lines to serve as independent variables in the multivariate analysis is known as variable down-selection. [10] However it is not yet known whether the use of down-selected variables or the entire LIBS spectrum provides optimal discrimination and classification of unknown LIBS spectra, and this is an ongoing area of investigation.…”