Raman Spectroscopy Spots Pseudomonas aeruginosa on Wild Turtle Skin in Minutes

A laser beam, a microscope slide, and a few minutes of scanning may soon be all that separates a clinician from a definitive bacterial diagnosis. In a study published in the journal BioRisk, researchers report that standard Raman spectroscopy—without any of the expensive signal-boosting substrates that the technique usually demands—can reliably identify Pseudomonas aeruginosa, a notorious opportunistic pathogen, in bacteria isolated from the skin of free-living European pond turtles (Emys orbicularis). The work, carried out at Silene Nature Park in Latvia, offers a glimpse of a diagnostic future in which microbial identification is fast, cheap, and free of the multi-day cultivation routines that currently dominate clinical microbiology.

Pseudomonas aeruginosa is a gram-negative rod-shaped bacterium measuring roughly one to five micrometers long and half a micrometer to one micrometer wide. According to the Centers for Disease Control and Prevention, it is the most common disease-causing member of the Pseudomonas group. It thrives in healthcare settings, where it causes severe infections in vulnerable patients, but it also lurks in hot tubs, swimming pools, and on contact surfaces associated with free-living animals. The authors note that the bacterium is a principal etiological factor in more than 65 percent of purulent-septic skin lesions, making rapid and reliable identification a pressing task for modern medicine.

The classical bacteriological method—growing a pure culture and identifying it from the totality of its properties—remains the gold standard for reliability, but it is laborious, costly, and slow, sometimes taking several days to deliver an answer. Automated systems such as VITEK2, Phoenix, MALDI-TOF mass spectrometry, and next-generation sequencing can shave 24 to 48 hours off the process, yet they still require substantial time and financial investment. There is also a logistics problem that is easy to overlook: roughly 30 percent of bacterial strains fail to reach laboratory testing at all, so they can never be identified. Any method that shortens the chain between sample and answer therefore carries real clinical value.

Raman spectroscopy attacks the problem from a different direction. The technique detects the vibrations of molecular bonds in a sample, producing a spectrum that acts like a chemical fingerprint. Because bacteria are built from characteristic proportions of proteins, lipids, carbohydrates, and nucleic acids, each species is thought to produce its own ensemble of Raman lines—what the researchers describe as biomolecule fingerprinting—that can be matched against a spectral library in as little as one to two minutes. Crucially, the method does not require long-term cultivation, special sample preparation, additional expensive reagents, or consumables.

Most Raman-based bacterial identification reported to date has relied on surface-enhanced Raman spectroscopy, or SERS, in which the sample is placed on a substrate coated with metallic gold or silver nanoparticles that dramatically amplify the Raman signal. These substrates are expensive and add a layer of complexity to the workflow. The goal of the new study was to demonstrate that P. aeruginosa could be identified with plain, standard Raman spectroscopy—no enhancement, no nanoparticles—by comparing the spectra of wild isolates against a well-characterized control strain, P. aeruginosa ATCC 27853 from the American Type Culture Collection.

The fieldwork began in Silene Nature Park, a NATURA2000 site in Latvia, where ten free-living European pond turtles were collected and samples were taken from the skin surface using sterile transport swabs. In the laboratory, Pseudomonas spp. were isolated by classical bacteriological methods on CHROMagar Orientation and Trypticasein Soy Lab-Agar. Colonies identified as P. aeruginosa were stored at 4 degrees Celsius, then warmed to room temperature, transferred into Brain Heart Infusion Broth, and incubated at 37 degrees Celsius for 24 hours. The cultures were then swabbed onto fresh nutrient agar and incubated for a further 24 to 48 hours before isolated colonies were emulsified in sterile distilled water, vortexed, and adjusted visually to McFarland 0.5 Standard—approximately 1.5 x 10^8 cells per milliliter. One hundred microliters of each suspension was placed on a sterile microscope slide for analysis.

Spectra were recorded with a Renishaw inVia Raman Microscope coupled to a Leica DM 2500 optical microscope, using a 50x objective and an air-cooled CCD array detector. The excitation source was an argon-ion laser operating at a short wavelength of 514 nanometers, chosen to minimize sample self-fluorescence. Laser power was kept remarkably low—no more than 50 milliwatts at the laser head and only about 0.016 milliwatts at the sample surface. More than 50 scans were accumulated for each sample, with integration times of 2 to 40 seconds per exposure and an optimal spectral resolution of 2 per centimeter. Full-length spectra covered the range from 3200 to 200 inverse centimeters, while short-length spectra focused on the 2000 to 600 inverse centimeter window. All data were collected and processed with Renishaw WiRE 5.5 software, and cosmic-ray artifacts were suppressed with dedicated removal functions.

The resulting spectra of the turtle-skin isolates and the control strain coincided closely in both intensity and the localization of their peaks. Fifteen individual Raman shifts stood out: 624, 760, 808, 1002, 1032, 1145, 1150, 1178, 1207, 1330, 1359, 1445, 1580, 1600, and 1620 inverse centimeters. Each peak could be tentatively assigned to a molecular component of the bacterial cell. The 624 line corresponds to skeletal vibrations of aromatic rings in amino acids; the 1002 and 1032 lines to phenylalanine; the 1178 and 1207 lines to aromatic amino acids such as tyrosine, phenylalanine, and tryptophan and to protein C–H stretching regions; the 1445 and 1330 lines to CH deformations in polysaccharides, lipids, and proteins; and the 1580, 1600, and 1620 lines to in-ring C=C and C–C stretching, Amide II, and Amide I vibrations of proteins and unsaturated lipids. The 1145 line was attributed to sulfonic acid residues, while the 1150 line was associated with symmetric glycosidic linkages in polysaccharides or C–C stretching vibrations, possibly including ATP.

Several practical lessons emerged from the experimental work. Taking spectra directly from colonies growing on nutrient agar in Petri dishes proved problematic: the transparent polystyrene base and the agar itself injected extra noise into the measurements. The researchers solved this by measuring drops of bacterial suspension instead, which neutralized any substrate distortion. They also confirmed that the McFarland 0.5 Standard suspension—1.5 x 10^8 cells per unit volume—is suitable for Raman analysis of this bacterium without any surface signal enhancement. The intensity of the Raman signal varied slightly with the concentration of the bacterial suspension, but measurements across different sample groups revealed no significant differences in the spectral positions. The team further noted that high-quality, reproducible spectra require keeping the exposure from any single point on the object to no more than 60 seconds, which also kept spontaneous luminescence minimal and avoided any noticeable effect of laser radiation on the cells.

The authors conclude that Raman spectroscopy, operated under these parameters, can be considered an express method for identifying microorganisms—one that detects the presence of P. aeruginosa even at the tested concentration, delivers quantitative and qualitative results within one to ten minutes for a full-range spectrum, and theoretically allows a large number of cultures to be identified simultaneously. The laser excitation parameters used did not cause destructive changes in the bacterial cells, which retained their integrity and cellular organelles. With high analytical and diagnostic sensitivity and specificity, no additional cultivation stages, and no special sample preparation, the approach offers analytical reliability at high speed. The researchers emphasize that the technology is promising for further research on other microorganisms, though interpreting spectral changes and extending the method to additional bacteria will require further study. If those follow-up efforts succeed, the humble pond turtle may have helped point the way toward a faster, cheaper era of microbial diagnosis.

Subject of Research: Rapid identification of Pseudomonas aeruginosa from European pond turtle skin using standard Raman spectroscopy

Article Title: Prospects and possibilities of using Raman spectroscopy for the identification of Pseudomonas aeruginosa from turtle Emys orbicularis (Linnaeus, 1758) skin

Article References: Petjukevičs, A., Umbraško, I., & Škute, N. (2023). Prospects and possibilities of using Raman spectroscopy for the identification of Pseudomonas aeruginosa from turtle Emys orbicularis (Linnaeus, 1758) skin. BioRisk, 21, 19-28. https://doi.org/10.3897/biorisk.21.111983

Image Credits: AI Generated

DOI: 10.3897/biorisk.21.111983

Keywords: Raman spectroscopy, Pseudomonas aeruginosa, Emys orbicularis, European pond turtle, bacterial identification, microbiology, diagnostics, SERS, spectral fingerprinting, gram-negative bacteria, Latvia, express method