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Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection.

Henderson KC, Benitez AJ, Ratliff AE, Crabb DM, Sheppard ES, Winchell JM, Dluhy RA, Waites KB, Atkinson TP, Krause DC - PLoS ONE (2015)

Bottom Line: At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use.Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy.Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Georgia, Athens, GA, United States of America.

ABSTRACT
Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP). At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl) and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

No MeSH data available.


Related in: MedlinePlus

Averaged, baseline-corrected, and normalized SERS spectra for the nanorod substrate, growth medium control, and M. pneumoniae reference strain controls and clinical isolates, as indicated.Raw spectra of the three sample classes were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). For the nanorod substrate background class, n = 20; for the growth medium control class, n = 20; and for the M. pneumoniae class, n = 350.
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pone.0131831.g001: Averaged, baseline-corrected, and normalized SERS spectra for the nanorod substrate, growth medium control, and M. pneumoniae reference strain controls and clinical isolates, as indicated.Raw spectra of the three sample classes were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). For the nanorod substrate background class, n = 20; for the growth medium control class, n = 20; and for the M. pneumoniae class, n = 350.

Mentions: We analyzed 32 clinical isolates, including reference strains M129 (type 1) and FH (type 2), alongside a growth medium control prepared in parallel with the M. pneumoniae samples. Full details regarding isolate origin and year, P1 genotype, macrolide susceptibility, protein and DNA content, and genome equivalents for M. pneumoniae strains are given in Table 1. CFU values were determined for both reference strains and six randomly chosen additional isolates to assess cell viability at time of fixation and ranged from 1x105 to 1x107 CFU/ml. Due to the propensity for mycoplasma cells to clump, a confounding factor in using CFU values as a metric for sample content is the potential discrepancy between CFU value and actual cell number, which can differ by as much as 103-fold [39]. Therefore, protein content and genome equivalents were determined in order to better define the content of the samples at the concentration analyzed by SERS. These values fell within comparable ranges and were consistent with published values for bacterial cells [40]. Protein concentration per cell was higher for M. pneumoniae isolates harvested during stationary phase relative to those harvested during log phase (growth phase based on the color of the pH indicator in the SP4 medium), but no notable differences in genome equivalents or SERS spectra were observed between M. pneumoniae samples relative to growth phase at time of harvest (data not shown). Average SERS spectra of the nanorod substrate background, growth medium control, and M. pneumoniae samples are shown in Fig 1, with each class exhibiting a distinct band pattern, as expected.


Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection.

Henderson KC, Benitez AJ, Ratliff AE, Crabb DM, Sheppard ES, Winchell JM, Dluhy RA, Waites KB, Atkinson TP, Krause DC - PLoS ONE (2015)

Averaged, baseline-corrected, and normalized SERS spectra for the nanorod substrate, growth medium control, and M. pneumoniae reference strain controls and clinical isolates, as indicated.Raw spectra of the three sample classes were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). For the nanorod substrate background class, n = 20; for the growth medium control class, n = 20; and for the M. pneumoniae class, n = 350.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487258&req=5

pone.0131831.g001: Averaged, baseline-corrected, and normalized SERS spectra for the nanorod substrate, growth medium control, and M. pneumoniae reference strain controls and clinical isolates, as indicated.Raw spectra of the three sample classes were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). For the nanorod substrate background class, n = 20; for the growth medium control class, n = 20; and for the M. pneumoniae class, n = 350.
Mentions: We analyzed 32 clinical isolates, including reference strains M129 (type 1) and FH (type 2), alongside a growth medium control prepared in parallel with the M. pneumoniae samples. Full details regarding isolate origin and year, P1 genotype, macrolide susceptibility, protein and DNA content, and genome equivalents for M. pneumoniae strains are given in Table 1. CFU values were determined for both reference strains and six randomly chosen additional isolates to assess cell viability at time of fixation and ranged from 1x105 to 1x107 CFU/ml. Due to the propensity for mycoplasma cells to clump, a confounding factor in using CFU values as a metric for sample content is the potential discrepancy between CFU value and actual cell number, which can differ by as much as 103-fold [39]. Therefore, protein content and genome equivalents were determined in order to better define the content of the samples at the concentration analyzed by SERS. These values fell within comparable ranges and were consistent with published values for bacterial cells [40]. Protein concentration per cell was higher for M. pneumoniae isolates harvested during stationary phase relative to those harvested during log phase (growth phase based on the color of the pH indicator in the SP4 medium), but no notable differences in genome equivalents or SERS spectra were observed between M. pneumoniae samples relative to growth phase at time of harvest (data not shown). Average SERS spectra of the nanorod substrate background, growth medium control, and M. pneumoniae samples are shown in Fig 1, with each class exhibiting a distinct band pattern, as expected.

Bottom Line: At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use.Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy.Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Georgia, Athens, GA, United States of America.

ABSTRACT
Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP). At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl) and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

No MeSH data available.


Related in: MedlinePlus