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BioRisk 21: 19-28 (2023)
DOI: 10.3897/biorisk.21.111983

Research Article

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

Aleksandrs Petjukeviés'®, Inta UmbraSko™®, Natalja Skute’©

1 Institute of Life Sciences and Technology, Daugavpils University, Parades Str. 1A, 121, Daugavpils, Latvia
Corresponding author: Aleksandrs Petjukevics (aleksandrs.petjukevics@du.|v)

OPEN Qaceess

Academic editor: Pavel Stoev
Received: 1 September 2023
Accepted: 6 November 2023
Published: 17 November 2023

Citation: Petjukevics A, Umbrasko

|, Skute 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

Copyright: © Aleksandrs Petjukevics et al.

This is an open access article distributed under
terms of the Creative Commons Attribution
License (Attribution 4.0 International -

CC BY 4.0).

Abstract

This study describes an express method for identifying microorganisms: Pseudomonas
aeruginosa by standard Raman spectroscopy, without surface-enhanced Raman spec-
troscopy (SERS). The short-wavelength 514 nm Ar-lon laser was used for P aeruginosa
spectral identification in the Raman shift range from 3200 cm" to 200 cm". The research
results showed a high analytical and diagnostic sensitivity of the technology to the ex-
press identification of P aeruginosa and can be used as one of the reliable methods. The
proven technology is promising for further research of other microorganisms due to sev-
eral significant advantages of the method. It does not require long-term cultivation of bac-
teria and special sample preparation, additional expensive reagents or consumables.

Key words: Bacteria, identification of microorganisms, Pseudomonas aeruginosa,
Raman spectroscopy, reptiles, turtle

Introduction

Nowadays, microbiological research is an important and relevant activity in bi-
ology and medicine since these studies confirm or deny the presence of certain
bacteria with high accuracy and reliability. The classical bacteriological research
method and Automated bacteriological diagnostics by different identification sys-
tems, VITEK2, Phoenix, MALDI-TOF (matrix-assisted laser description/ionisation
time-of-flight mass spectroscopy), Next-generation sequencing, solves the prob-
lem of isolating a pure culture of the pathogen with its subsequent identification,
but it requires much time and financial investments. Thanks to microbiological
research methods, it is possible to establish the causative agents of certain infec-
tious diseases and choose a rational treatment for these diseases (Ramalho et al.
2002). Therefore, developing faster and cheaper routine methods for diagnosing
pathogenic microorganisms is a priority area in modern microbiology. Pseudo-
monas spp. is a group of bacteria that can cause several infections. P aeruginosa
is the most common disease-causing form of these bacteria, according to the
Centers for Disease Control and Prevention (CDC). P aeruginosa belongs to the
group of gram-negative bacteria and it is an opportunistic pathogen. Colonies
of P aeruginosa consist of rod-shaped bacterium sizes about 1-5 um long and

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

0.5-1.0 um wide (Pauw et al. 2008). They can infect different organs and tissues;
the infection is usually severe. Serious infections from P. aeruginosa primarily oc-
cur in healthcare settings, but people can also become infected from hot tubs
and swimming pools or after contact with free-living animals. P aeruginosa is one
of the main etiological factors in more than 65% of cases of purulent-septic skin
lesions and the fight against these diseases remains an urgent task of modern
medicine (Katuzna et al. 2014; Lee et al. 2021; Umbrasko et al. 2022, 2023).

The study of microorganisms that are the causative agents of many infectious
diseases is an urgent and important task and progress in solving this can only
be achieved if various research methods are used, combining them to obtain the
fastest, most reliable and economically justified results (Terrones-Fernandez et
al. 2023). The method for detecting microorganisms should not require complex
stages of sample preparation while providing rapid identification of the bacte-
ria and be relatively inexpensive and automated. An important criterion for set-
ting up a microbiological study, based on determining the type of bacteria, is its
speed. However, the classical bacteriological diagnostic method, based on the
isolation and identification of a pure culture of bacteria based on the totality of
their specific properties, with all its reliability and information content, is very la-
borious, financially costly and, most importantly, it is lengthy (it may take several
days). In addition to the microbiological method, other laboratory studies allow
for obtaining objective information about the microbial composition of the ma-
terial under study. The use of automated systems with multi-wavelength lasers
for the identification of bacteria can speed up the process of microorganism
identification by 24-48 hours and obtain reliable information about microorgan-
isms faster (Huang et al. 2004, 2010; Zhu et al. 2014; Pezzotti 2021).

One of the possible methods for the identification of microorganisms is Raman
spectroscopy. This method is based on the detection of the molecular structure

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Figure 1. Sampling site: Silene Nature Park NATURA2000 (Latvia: 55.690835°N, 26.788760°E).

BioRisk 21: 19-28 (2023), DOI: 10.3897/biorisk.21.111983 20

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

vibrations of the object and has established itself as a reliable analytical tool in var-
ious fields of science (Petjukevics and Skute 2017; Vaitiekiinaité and Snitka 2021;
VaitiekUnaité et al. 2022). It is assumed that bacteria are also characterised by in-
dividual spectral lines of Raman scattering (like biomolecule fingerprinting), which
make it possible to identify them in a short time (1-2 min) with a very high degree
of reliability of the information obtained. In turn, a library of microorganisms is cre-
ated, based on the spectral information of Raman spectra. Raman spectroscopy
makes it possible to apply this method to rapidly identify microorganisms in more
comprehensive applications (Patel et al. 2008; Stegelmeier et al. 2019; Kumar et
al. 2020; Vaitieknaité et al. 2022). One of the existing problems is the timely de-
livery of bio-material, as about 30% of bacterial strains do not reach laboratory
testing and, thus, cannot be identified. Modern achievements in microbiology us-
ing the Raman spectroscopy method and its limitations were considered, as well
as the most important world trends in the use of this diagnostic technology for the
study and indication of the causative agents of bacterial pathogenic flora and viral
infections (Lee et al. 2020; Nakar et al. 2022; Umbrasko et al. 2022).

Based on the preceding, the purpose of this study is to develop an express
method for detecting and identifying the bacterium, P. aeruginosa, without the use
of expensive SERS substrates with metallic gold or silver nanoparticles, based on
Raman spectroscopy by analysing and comparing the obtained spectra of anal-
ysed samples with the test-control strain: P aeruginosa ATCC 27853 (American
Type Culture Collection), as well as optimising the conditions for obtaining spec-
tra and also developing an algorithm for processing primary spectral information.

Materials and methods
Study site, sampling, microbiological identification

For the classic bacteriological method, ten free-living turtles, Emys orbicularis
(L.) (European pond turtle), were collected and biological material was taken
from the skin surface. The sampling site was Silene Nature Park NATURA2000
(Latvia) (55.690835°N, 26.788760°E) (Fig. 1). CliniswabTS Sterile Transport
Swabs (Italy) were used to collect samples in wild nature and preserve the mi-
crobiological flora for further analysis in the laboratory. Subsequently, Pseudo-
monas spp. was isolated and identified by the classical bacteriological method.
Solid nutrient media were used for cultivation and primary microbiological anal-
ysis: CHROMagar Orientation and Trypticasein Soy Lab-Agar (BioMaxima S.A.,
Poland) (Merlino et al. 1996; Garcia and Isenberg 2010). As an objective, daily
cultures of the control strain (culture number: P aeruginosa ATCC 27853) and
P aeruginosa were taken, which were identified from the skin surface of turtles.
Identified colonies from the surface of the skin of turtles were stored in a refrig-
erator at a temperature of 4+ 1 °C. Then for the study, the material was warmed
up to room temperature 20 + 1 °C. Bacterial colonies were transferred into 3
ml BHI (Brain Heart Infusion Broth, BioMaxima S.A., Poland) with a disposable
sterile loop (COPAN). In the next step, colonies were incubated in a thermostat
at 37 + 1 °C for 24 hours. BHI is a nutrient-rich liquid medium suitable for the in-
oculation of P aeruginosa. After 24 hours of incubation, the samples were trans-
ferred with a sterile disposable viscose swab with a plastic stick (APTACA) to a
nutrient medium (Trypticasein Soy Lab-Agar) and incubated in a thermostat at

BioRisk 21: 19-28 (2023), DOI: 10.3897/biorisk.21.111983 21

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

37 +1 °C for 24-48 hours. Isolated colonies were placed in sterile distilled wa-
ter, the obtained emulsion vortexed = 10 s by Vortex V-1 plus Biosan (Latvia) and
visually compared with McFarland 0.5 Standard, 1.5 x 10® cells per volume unit
(approximate bacterial suspension/ml) and after that, 100 ul of bacterial sus-
pension transferred on sterile microscope 1 - 1.2 mm thick slide (ChannelMED).

Raman spectroscopy of P. aeruginosa

Raman scattering spectra were recorded using Renishaw inVia Raman Micro-
scope (United Kingdom), equipped with an optical microscope, Leica DM 2500
(Germany). Raman scattered light from the sample collected through a micro-
scope with a short-distance objective, Leica L 50x/0.50 (eyepiece: HC PLAN
10x/20) and analysed by an inVia Spectrometer. Scattered light focused on
a Renishaw air-cooled Ren Cam CCD array detector with insertion/retraction
speed > 20 mm s', repeatability < 0.5 um, laser spot size < 2 um FWHM, spa-
tial resolution 2 um and a field of view > 25um. During Raman spectroscopy,
> 50 scans were accumulated for each sample. To improve the noise/signal
quality ratio, the laser power was minimised (reduced sample self-fluorescence)
and an excitation source Renishaw Stellar-Ren Ar-lon laser with 514.0 nm wave-
length (VIS: 2400 I/mm grating and back-illuminated CCD camera) was used.
Lens-focused laser radiation on a quartz glass slide with the sample and Raman
spectra were collected by the Raman inVia Reflex microspectroscopy system
in the range from 3200 cm" to 200 cm” for the full-length spectrum (Fig. 4b)
and from 2000 cm" to 600 cm” for the short-length spectrum (Fig. 4a). The Ar-
lon laser power was < 50 mW at the laser head and < 0.016 mW at the sample
surface (Nova II PD300-3W-V1, Ophir Photonics, Israel). All spectra were read
out three times for each bacterial suspension of ten and the results of Raman
scattering signals were averaged. All collected data are unsmoothed averages
of exposures obtained with an integration time of 2-40 s/exposure. The optimal
spectral resolution from the sample surface was reached at 2 cm’. Data collec-
tion was accomplished with Renishaw WiRE 5.5 Raman software, scans, pro-
cessing (including decomposition of the complex band-shapes analyses) and
spectral analysis was controlled by a personal computer. Raman spectral shifts
were compared with the Raman spectrum of the control strain (P aeruginosa
ATCC 27853) and the S.T. Japan spectral databases, Thermo scientific software,
Grams Spectral ID library, Version 9.0.7 and the data from scientific articles.

Results

The results of the research are presented in the form of graphs of the spectral
characteristics of P aeruginosa (Fig. 2a) and (Fig. 2b). Ten spectra of P. aerugino-
sa bacterial substances (bacterial colonies suspension, concentration measured
according to McFarland 0.5 Standard), systematised and analysed by Raman
spectroscopy. In the Raman spectra of control samples, low-intensity lines were
detected that were not related to compounds characteristic of bacteria and were
taken into account as side noise since the range of detection of Raman shifts and
the ratio of line intensities did not correspond to lipids, proteins or nucleic acids.
However, it was known that lipids, which are inherent in bacterial cells increasing
the reflectivity, also play an important role, probably influencing the intensity level

BioRisk 21: 19-28 (2023), DOI: 10.3897/biorisk.21.111983 22

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

a

counts

500

\

increase in spectra. It was noted that spontaneous luminescence was absent or
was minimal during short-term (up to 60 s) focusing on the sample, which indi-
cates the absence of a noticeable effect of laser radiation on them. Cosmic rays’
peaks or unusually high and unstable Raman shifts on the abscissa axis — x was
also not considered and was suppressed with cosmic ray removing functions.

The specific Raman spectra observed in this research represent an ensemble
of Raman signals that arise from the different molecular vibrations of individual
cell components, integrating over nucleic acids, lipids, carbohydrates and pro-
teins. The resulting Raman spectra had a significant number of peaks, for which it
was difficult to unambiguously assign to the type of vibrations of groups of atoms
in analyte molecules. As can be seen from the graph, the scattered light intensity
peaks of this bacterium species coincide in intensity and localisation in the spec-
tral region of Raman scattering in the short and extended spectra for P. aerugino-
sa. The individual Raman shifts were localised at 624 cm"', 760 cm”, 808 cm",
1082-cm:*,.1032-cm"*, 1145-0m=,, 11-58 -enr 171178: em> |, 1207 €m- 1330 em,
1359 cm, 1445 cm"', 1580 cm", 1600 cm” and 1620 cm’; the values and char-
acteristics of the principal obtained components are detailed in table (Table 1).

All obtained spectra from P aeruginosa bacteria had similar characteristics
of the values in the studied region 1700-600 cm” and slightly differ only in the
intensity of the peaks. Recorded spectra previously filtered from high-frequen-
cy noise (software suppression of cosmic rays) are presented in the paper for
further analysis and interpretation. The specific Raman spectra observed here
represent an ensemble of signals that arise from the molecular vibrations of
individual cell components of gram-negative bacteria, integrating over proteins,
lipids and carbohydrates (Fig. 3).

|

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| A | vi i
Nnagei” Varna " } i Ka a \semunpll

wey
bit en on
sy War

1000 1500 2000 2500 3000
Raman shift/ em!

Figure 2. Averaged short-length spectrum: 2000 cm“ - 600 cm" and extended full-length Raman spectrum: 3200 cm"
- 200 cm” of P aeruginosa bacteria isolated from turtle skin (a) and control strain: P aeruginosa culture number: ATCC
27853 (b). On the abscissa axis x—- Raman shift (cm) along the y-axis is the scattered light intensity (a.u).

BioRisk 21: 19-28 (2023), DOI: 10.3897/biorisk.21.111983 23

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

Table 1. Raman shifts and tentative assignments of bacteria cells of P aeruginosa isolated from turtle skin.

Raman shifts, (em)
624

760

808

1002

1032

1145

1150

1178
1207

1330

1359

1445

1580 (1600-1585)
1600 (1645-1540)
1620 (1680-1640)
650-600
1280-1160
1333-1313
1440-1360
1460-1440
1645-1545
2000-1665
3100-2800

Tentative assignment #
Skeletal vibrations of aromatics rings of amino acid
Carbohydrates COO-def, CH, rocking
Nonpolar amino acids: proline, valine; Polar, uncharged amino acid: u (CN) tyrosine
amino acid: Phe
C-H in plane, Phe

sulfonic acid residues

Could be associated with the stretching vibration from symmetric glycosidic linkages (C-O-C) and rbr of polysaccharides or C—C

str vibrations. ATP
Aromatic amino acids: 6 (C—H), Tyr, Phe; Proteins: C-H str Region
Proteins: Amide Ill, C-C,H, str. Phe, Trp
CH deformations can be assigned to polysaccharides and lipids, as well as to protein
u(COO-), 6(C—H) proteins
CH deformations can be assigned to polysaccharides and lipids, as well as to protein
C=C str, C-C str (in-ring)
C-C str (in-ring), Amide II, u(CN), y(NH), unsaturated lipids
Amide |
Proteins
B-sheet (proteins)
CH def stretch band
u(COO-) sym
5(CH,) fatty acid molecules without double bonds
Amide II, u(CN), y(NH)
Overtones of fundamental or compound vibrations, weak vibrations

C-H str region

@ Abbreviations: Phe — phenylalanine; Tyr — tyrosine; Trp — tryptophan; rbr — ring breathing; def — deformation; str — stretching; sym — symmetric.

Discussion

The possibility of taking spectra of P aeruginosa directly from the surface of
Petri dishes with nutrient agar was initially studied. However, this technology
added extra noise to the spectrum due to the agar on which the colonies were
grown. The plastic base of the Petri dishes (transparent polystyrene) with nu-
trient agar on which bacterial colonies were grown (Fig. 4). Raman spectra of
P aeruginosa wild type were acquired between 3200 cm"! and 200 cm"'. How-
ever, the studies reported here concentrate on vibrational bands found in the fin-
gerprint region 1700 — 600 cm’. Outside this range, C—H stretching vibrations in
the 3050 — 2750 cm" range dominate spectra. This information is unimportant
in this paper, although these Raman bands carry important information about
cell membrane fluidity (Huang et al. 2010; Kumar et al. 2020; Pezzotti 2021).

Previously, before Raman scattering, the quality of preparations of the nu-
trient medium to luminescence capability was checked out. Accordingly, the
possibility of the substrate distorting the obtained Raman spectra was neu-
tralised using bacterial suspension drops against direct spectra from the Petri
dish surface. The spectra of bacterial suspensions were measured one by one.

Measurements of the bacterial suspension of samples from different groups
did not reveal significant differences; however, there was a slight change in the
peak height (the intensity of the Raman signal varied depending on the concen-
tration of bacterial suspension in the test sample).

BioRisk 21: 19-28 (2023), DOI: 10.3897/biorisk.21.111983 24

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

O-specific poly-
saccharide chain
(3-8 units)

Lipopoly-
saccharides |
| Core poly-
saccharide
OUTER
MEMBRANE

QOODQDOOOOOCOOO0O0C
PERIPLASMIC
Peptidoglycan — SPACE

Murein Lipoprotein

INNER

CYTOSOL

Membrane
Proteins

Figure 3. Cell wall components of Gram-negative bacteria (Kagle 2023).

Figure 4. Microscopic image of Pseudomonas aeruginosa bacterial cells distribution in agar: (Trypticasein Soy Lab-Agar,
BioMaxima).

BioRisk 21: 19-28 (2023), DOI: 10.3897/biorisk.21.111983 25

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

It was also experimentally confirmed that this bacterial test-suspension by
McFarland 0.5 Standard method, 1.5 x 10° cells per volume unit, is suitable for
Raman spectroscopy during this type of bacterium P aeruginosa analysis and
is Suitable for Raman spectroscopy without the use of surface signal enhance-
ment technique (SERS).

Each sample of studied bacteria for P aeruginosa is characterised by individ-
ual spectral shifts of Raman scattering, which make it possible to identify them
in a short time (total time for whole full-range spectrum 1-10 min) and, theoret-
ically, makes it possible to identify a large number of cultures simultaneously.
This method is characterised by high sensitivity (100 ul of the prepared suspen-
sion according to the McFarland Standard method, 1.5 x 108 cells per volume
unit) and rapid microorganism identification. The spectra recorded for the same
sample remain almost unchanged in the spectrum over a short time, while they
could only slightly differ in signal intensity and resolution of the leading bands.
It was noted that, to take high-quality, optimally-reproducible spectra, the spec-
trum from an object should not exceed 60 seconds from one point

Conclusions

The study showed the possibility of obtaining fast and high-quality Raman spec-
tra of P aeruginosa bacterial cells. The used parameters of laser excitation did not
cause pronounced destructive changes in bacterial cells. Bacterial cells retained
their integrity and cellular organelles with decreased laser beam power. However,
the self-luminescence of the samples was reduced to a minimum background ef-
fect, which did not significantly affect the quality of the obtained Raman spectra.
Laser diagnostics, based on Raman spectroscopy, can be considered an express
method for identifying microorganisms and allows detection of the presence of
a microorganism even at this concentration (1.5 x 108 cells per volume unit). The
application of Raman spectroscopy is characterised by high analytical and diag-
nostic sensitivity and specificity, which is necessary for accurately identifying mi-
croorganisms. We can also note the high speed of obtaining results (quantitative
and qualitative). The method does not require additional stages of bacterial culti-
vation or special sample preparation, which are important characteristics for a re-
liable study and provide analytical reliability and high speed for obtaining results.
These advantages of the method give reason to consider it a promising universal
express method for microbiological diagnosis of diseases of microbial etiology.
The study's results indicate the information content of using Raman spectrosco-
py to identify microorganisms. However, interpreting these changes and possibly
using this technology to study other bacteria requires additional research.

Acknowledgements

We thank the two anonymous reviewers for helpful criticisms that improved the
final manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

BioRisk 21: 19-28 (2023), DOI: 10.3897/biorisk.21.111983 26

Aleksandrs Petjukevics et al.: Raman spectroscopy for the identification of Pseudomonas aeruginosa from skin

Ethical statement

No ethical statement was reported.

Funding

No funding was reported.

Author contributions

Formal analysis: AP. Investigation: IU. Methodology: IU, AP. Project administration: NS. Soft-
ware: AP. Visualization: AP. Writing — original draft: IU, AP. Writing — review and editing: NS.

Author ORCIDs

Aleksandrs Petjukeviés © https://orcid.org/0000-0001-691 7-2677
Inta Umbrasko © https://orcid.org/0009-0009-5792-9033
Natalja Skute © https://orcid.org/0000-0002-3584-0347

Data availability

All of the data that support the findings of this study are available in the main text.

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