҉ Mycoplasma is a genus of a bacteria that is characterized by the absence of a cell wall, a limited genome and biosynthetic capabilities that necessitate the need for a symbiotic life ҉ Mycoplasma pneumoniae is a respiratory tract pathogen that commonly causes pneumonia, particularly in immunocompromised persons, children, and the elderly ҉ Current methods of M. pneumoniae detection are not very effective, costly, and/or not simple; common methods involve serology/PCR, but these methods are flawed in that they require time for host to develop antibodies, too much loss of sample during processing, or results producing false negatives ҉ There is a need for a more simple, rapid detection method for M. pneumoniae that has high sensitivity, specificity so in our study we examined the Layer-by-layer (LBL) method along with surface enhanced Raman spectroscopy (SERS) for M. pneumoniae detection    

Richard A. Dluhy: dluhy@uga.edu Omar E. Rivera: omar6683@uga.edu Duncan C. Krause: dkrause@uga.edu Seyed R. Taghavi: srtaghav@uark.edu

Contact Information

Layer-by-layer polyelectrolyte encapsulation of Mycoplasma pneumoniae for enhanced Raman detection Omar E. Rivera1, Seyed R. Taghavi3, Duncan C. Krause2, Richard A. Dluhy1

1Department of Chemistry, University of Georgia; 2Department of Microbiology, University of Georgia; 3University of Arkansas Results

Conclusions and Future Work

Abstract

Methodology

Introduction

Mycoplasma pneumonia is a major cause of respiratory disease in humans and accounts for as much as 20% of all community-acquired pneumonia. Existing mycoplasma diagnosis is primarily limited by the poor success rate at culturing the bacteria from clinical samples. There is a critical need to develop a new platform for mycoplasma detection that has high sensitivity, specificity, and expediency. Here we report the layer-by-layer (LBL) encapsulation of M. pneumoniae and mycoplasma commensal cells with Ag nanoparticles in two different polyelectrolyte matrixes; one matrix is the combination poly(allylamine hydrochloride) (PAH) with poly(styrene sulfonate) (PSS) and the other is poly(diallyldimethylammonium chloride) (PDADMAC) with PSS. We evaluated nanoparticle encapsulated mycoplasma cells as a platform for the differentiation of M. pneumoniae from mycoplasma commensal strains using surface enhanced Raman scattering (SERS) combined with multivariate statistical analysis. Pathogenic strains such as M. pneumoniae (M129) and M. genitalium, along with a series of commensal mycoplasma strains, were studied. Scanning electron microscopy, fluorescence imaging, and AFM showed that the Ag nanoparticles were incorporated between the oppositely charged polyelectrolyte layers. SERS spectra showed that LBL encapsulation provides excellent spectral reproducibility. Multivariate statistical analysis of the Raman spectra differentiated the pathogenic strains from the commensal strains with near 90-100% specificity and sensitivity, and low root mean square error. The technique shows promise for adaptation to sample preparation of M. pneumoniae infections in clinical specimens and represents a valuable alternative to current bacterial diagnostic techniques.

  Bacterial cells are regarded as being slightly negatively charged   Strong bases PAH and PDADMAC used as anchoring mechanism for layers   PSS used as negative layer   Differing molecular weights of PDADMAC used to measure effect of sturdiness of encapsulated cell   HCl/NaCl used to increase ionic strength   Approach employs consecutively placed polyelectrolytes of opposite charge onto surface of bacteria to form layers   Bacterial samples used include pathogenic wild type M129 and 12 commensals, including the commensal M. genitalium that is also considered pathogenic   Raman spectroscopy performed on a copper foil substrate with dried liquid phase sample

•  M. pneumoniae whole cells were encapsulated layer-wise with consecutive polyelectrolyte thin films along with silver nanoparticles incorporated between these films; this was proven through the use of various microscopic techniques.

•  The platform described here detected and differentiated M.pneumoniae separate from other species with excellent specificity and sensitivity when analyzing spectra taken with SERS in conjunction with chemometric methods

•  The technique shows great promise in its potential to improve diagnosis of M. pneumoniae infections

•  The model dependent PLS-DA provided quantitative statistical measurements of the sensitivity and specificity of the LBL encapsulation methods for discrimination between the wild type M129 and the twelve commensal mycoplasma strains.

•  Since both the sensitivity and specificity yielded inconsistent results when all the strains in this work were modeled, statistical methods such spectral variability and feature selection will be employed to analyze this data.

Acknowledgements

Research reported in this publication was supported by the National Science Foundation with the project title REU Site: Interdisciplinary Research Experiences in Nanotechnology and Biomedicine, under award number EEC-1359095.

This work supported by the NIH under grant number AI096364

References • O.E. Rivera-Betancourt, E.S. Sheppard, D.C. Krause and R.A. Dluhy, Analyst, 2014, DOI: 10.1039/C4AN00596A. • R. F. Fakhrullin, A. I. Zamaleeva, M. V. Morozov, D. I. Tazetdinova, F. K. Alimova, A. K. Hilmutdinov, R. I. Zhdanov, M. Kahraman and M. Culha, Langmuir, 2009, 25, 4628–4634. • S. L. Hennigan, J. D. Driskell, R. A. Dluhy, Y. Zhao, R. A. Tripp, K. B. Waites and D. C. Krause, PLoS One, 2010, 5, e13633.

Spectroscopy

SEMs of encapsulated cells

SEM of encapsulated FH/PAH/PSS/PAH/AgNP

SEM of encapsulated FH/PDADMAC/PSS/PDADMAC/AgNP:

Results

Ϟ Spectral processing of results included analysis of 10 raw spectra in the 1650-700cm-1 region and preprocessing, which includes baseline correction and normalizing

M. pneumoniae M129 uncoated whole cells

M. pneumoniae FH/PDADMAC/PSS/PDADMAC/AgNP encapsulated cell when using PDADMAC-HMW

M. pneumoniae FH/PDADMAC/PSS/PDADMAC/AgNP encapsulated cell when using PDADMAC-LMW

SERS spectra of the bacteria with LBL PDADMAC of LMW