Detecting Pathogenic Mycoplasmas with Surface-Enhanced Raman Spectroscopy

Surface-enhanced Raman spectroscopy (SERS) with silver nanorod-array substrates has been used in various biological applications, such as detection of proteins in body fluids. Duncan C. Krause, who is a professor in the Department of Microbiology at the University of Georgia, worked with his group to establish a SERS method with those substrates for detecting the pathenogenic mycoplasma that causes bronchitis and pneumonia. We recently spoke with him about this research.

Your group has developed a nanorod-array SERS biosensing method for detecting and distinguishing pathogenic mycoplasma strains, specifically Mycoplasma pneumoniae, in clinical samples (1–3). Why is this pathogenic mycoplasma an important target for this method?

Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20–40% of all community-acquired pneumonia as well as being the leading cause of the disease in older children and young adults. Symptoms are generally flu-like and initially can include sore throat, sinus congestion, and occasional middle-ear inflammation. Later the lungs can become involved, including bronchitis and an atypical or “walking” pneumonia, as it is often called, which is usually accompanied by a chronic cough. In some cases chronic airway disease can result, including chronic obstructive pulmonary disease (COPD) and onset or exacerbation of asthma. Because symptoms are largely nondescript and shared with other common respiratory pathogens, diagnosis by primary care providers can be complicated. M. pneumoniae is a bacterial infection and thus is susceptible to treatment with antibiotics, which would hasten recovery and limit spread to others, and underscores the need for rapid detection.

Several other factors limit definitive diagnosis of M. pneumoniae infections, which contributes to the common misperception that mycoplasma disease is rare. Culturing the organism from a throat swab or sputum sample requires up to 8 weeks and has poor sensitivity, even in experienced diagnostic labs. Testing either blood samples for antibodies specific for M. pneumoniae, or throat swabs for evidence of M. pneumoniae DNA by using polymerase chain reaction (PCR), are the most common approaches currently in use, but both have serious limitations. Failure to diagnose delays treatment, prolongs disease, and increases the likelihood of further spread and serious long-term consequences, increasing overall health-care costs. Thus, a critical need exists for a new platform capable of rapid detection to better diagnose and control M. pneumoniae disease.

Can you please briefly describe the method? What characteristics of Raman spectroscopy, and in particular SERS, make the technique suitable for improved detection and identification of infectious agents?

Silver nanorod arrays are fabricated by conventional electron beam vapor deposition at an oblique angle, which results in silver nanorod cylinders having an irregular shape. The specimens, in our case actual or simulated throat-swab samples, are applied to the top of the nanorod array. The mycoplasmas are small enough to settle between the nanorods, which increases the surface contact between the bacteria and the silver, thus maximizing the enhancement of the Raman signature generated. Features of Raman spectroscopy that are appropriate for biosensing with clinical samples include a narrow band width, good spatial resolution, applicability to aqueous samples, and exceptional specificity. The use of SERS makes it possible to overcome the inherently weak nature of Raman scattering to provide the signal amplification necessary for pathogen detection in complex clinical samples. Finally, the silver nanorod arrays provide improved consistency over previous SERS-active substrates.  

How does the technique compare with traditional detection methods in terms of convenience for point-of-care testing and diagnosis accuracy and sensitivity?

There is no point-of-care testing currently for most respiratory pathogens, with two exceptions being influenza and strep throat. Those tests rely on antibody-based detection of pathogen-specific antigens in throat-swab samples. Although these tests are rapid and easily administered, they have limited sensitivity, which could result in false-negative results. PCR-based detection of the genetic material from pathogens offers higher sensitivity but requires more time and generally requires that samples be sent to a diagnostic lab for analysis, so point-of-care testing is not practical. Handheld Raman spectrometers are already available on the market, and we envision the potential for rapid, point-of-care testing using SERS with such handheld units through an internet interface. Furthermore, in theory the testing with a SERS biosensing platform would not require a separate test for each pathogen, but by comparing the spectral features of a clinical sample with a spectral library, it might be possible to test for the presence of a variety of different viral and bacterial pathogens at one time.

What results have you obtained with the technique for identifying variant strains of M. pneumoniae, and what is the significance of that capability?

M. pneumoniae isolates have traditionally been classified into one of two general genotypes, based on variability in the DNA sequence of two genes encoding major proteins found on the mycoplasma surface. Because this difference in DNA sequence also affects the amino acid sequence of these proteins, and because these proteins are found on the mycoplasma surface, we predicted that our silver nanorod array SERS platform could distinguish between isolates of each genotype. This proved to be the case, with high sensitivity and specificity, for about 30 globally diverse clinical isolates. This capability is important because it supports epidemiological studies into what strains are predominating in a given community, as well as patterns of spread.

What are the challenges and limitations encountered in using the SERS method for mycoplasma detection?

The basic challenge of this approach is to decipher a pathogen-specific spectral signature against the biochemically complex background of a clinical sample. This challenge is amplified further by the fact that this complex background is not constant but will vary from one individual to the next and even within the same individual, depending on such factors as age, gender, presence or absence of inflammation, and variation in the normal flora or microbiome. We believe that variable selection-that is, focusing on the most important spectral components allowing detection of M. pneumoniae (or other infectious agents) in a biochemically complex sample background-will yield more consistent sensitivity and specificity. In addition, we believe that improvements in the SERS platform are needed – a simpler and more stable matrix than nanorod arrays. Lastly, perhaps the greatest challenge will be convincing an industry heavily invested in PCR-based technologies that an alternative approach might yield superior applications and versatility.

References

1.     S.L. Hennigan, J.D. Driskell, R.A. Dluhy, Y. Zhao, R.A. Tripp, K.B. Waites, and D.C. Krause, PLoS One5(10), e13633 doi:10.1371/journal.pone.0013633 (2010).

2.     S.L. Hennigan, J.D. Driskell, N. Ferguson-Noel, R.A. Dluhy, Y. Zhao, R.A. Tripp, and D.C. Krause, Appl. Environ. Microbiol.78(6) 1930, doi:10.1128/AEM.07419-11 (2012).

3.     K.C. Henderson, A.J. Benitez, A.E. Ratliff, D.M. Crabb, E.S. Sheppard, J.M. Winchell, R.A. Dluhy, K.B. Waites, T.P. Atkinson, and D.C. Krause, PLoS One10(6), e0131831 doi: 10.1371/journal.pone.0131831 (2015).