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Chapter 20 : Raman Spectroscopy for Bacterial Strain Typing
Category: Clinical Microbiology; Bacterial Pathogenesis
In the past 20 years, phenotypic typing methods have been largely replaced by typing methods based on the comparison of genomic DNA (molecular typing), such as PCR fingerprinting, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). An alternative approach to bacterial typing is based on applying Raman spectroscopy to test subtle differences in the molecular composition of the biomass, reflecting differences in the genomic DNA. The most important advantages of Raman spectroscopy compared to established molecular typing methods are speed, high sample throughput, and ease of use. In a Raman scattering event, an incident photon transfers some of its energy to the molecule, which leads to a lower energy in the scattered photon than in the incident photon. The approach can be as simple as a visual assessment of clearly identifiable spectral features that can only correlate to the biochemical component of interest. A well-known example of a microorganism causing hospital-acquired infections (HAI) is methicillin-resistant Staphylococcus aureus (MRSA). Therefore, the authors used an MRSA reference collection to demonstrate the capabilities of Raman spectroscopy. This reference collection contained 20 well-characterized MRSA isolates that had previously been analyzed by multiple typing techniques. Using Raman spectroscopy as a bacterial typing tool, infection control teams will have a tool for the continuous monitoring of isolates in their hospital, and they will be aware of the need for corrective action earlier, all leading to an accurate, real-time rather than retrospective surveillance approach in combating HAI.
Examples of Raman spectra obtained by measuring a single bacterial cell (Staphylococcus aureus), yeast cell (Candida krusei), and spore (Bacillus megaterium). a.u., arbitrary units.
(A) Most of the incident light will be scattered from a sample with the identical wavelength (λ), the so-called Rayleigh scattering. A fraction of the incident light will be scattered with a slightly higher wavelength (λ + Δ) due to the interaction with the molecules in the sample. (B) Raman spectrum of chloroform. This small molecule produces a relatively simple Raman spectrum. The peaks in the spectrum can be attributed to specific vibrations within the molecule. (C) Raman spectrum of Staphylococats aureus. Due to the complex molecular composition of the sample involved, a complex Raman spectrum is obtained. Based on the Raman spectra of purified compounds, spectral features can be assigned to specific molecular moieties in the bacterial cell. Most of the time, the whole spectrum is seen as a spectroscopic fingerprint and used for bacterial typing, a.u., arbitrary units.
(A) Every instrument for Raman spectroscopy consists of four basic parts: 1, a laser as an excitation source; 2, a sample stage, where the light is focused on the sample and Raman scattered light is collected; 3, a spectrometer, in which the scattered light is detected; and 4, a computer to analyze the collected spectra. (B) Renishaw Raman instrument using a microscope. (C) River Diagnostics Raman module coupled to an inverted microscope. (D) Spectracell™ Raman analyzer developed by River Diagnostics as a dedicated instrument for microbiological analyses.
(A) In automated analyses, an electrophoresis band pattern is transformed into a densitogram. Raman spectra resemble such a complex electrophoresis profile. (B) Difference between a spectrum of Stahylococcus aureus (solid line) and a spectrum of Staphylococcus epidermidis (dotted line). This difference spectrum shows many similarities to a typical carotene spectrum.
Raman classification of five MRSA reference isolates. Each isolate was cultured and measured in five independent sessions, and spectra were obtained after 18 h, 20 h, 22 h, and 24 h of incubation time. Raman clusters are found based on the PFGE patterns of the isolates. a.u., arbitrary units.
To demonstrate the capabilities of Raman spectroscopy, an MRSA reference collection was used. The reproducibility of the Raman procedure is high, since the multiple independent measurements on the same isolate result in identical Raman clustering. The Raman clustering has a high concordance with PFGE, MLST, and variable-number tandem repeat analyses. Superscripts: 1, PFGE results obtained previously ( 43 ); 2, sequence type as analyzed by MLST ( 44 ); 3, results of a multilocus variable-number tandem repeat method ( 14 ); 4, results obtained by using random amplification of polymorphic DNA analysis using three different primers (ERIC-2, AP1, and AP7) ( 44 ).
Example of a local MRSA outbreak analysis. On day 1, a contact screening was started in the General Surgery ward. In total, 11 S. aureus isolates were found. All three isolates from a staff member were found to be MRSA and analyzed. On day 3, the Raman results showed that the two MRSA isolates were not identical. This was confirmed by PFGE 4 days later.
Comparing outbreak and surveillance isolates. While MLVA finds mixed clusters, both AFLP and Raman find single-type clusters and distinguish between outbreak and unrelated isolates. na, not available.
The role of Raman-based strain typing in an infection prevention strategy. Using this technology will lead to actionable typing results at an early stage, leading to rapid intervention possibilities, limited further transmission, and a reduction in HAI.