Photodiagnosis and Photodynamic Therapy 40 (2022) 103145

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Photodiagnosis and Photodynamic Therapy

journal homepage: www.elsevier.com/locate/pdpdt

Surface-enhanced Raman spectroscopy for the characterization of pellets of

biofilm forming bacterial strains of Staphylococcus epidermidis
Muhammad Shakeel a, Muhammad Irfan Majeed a, *, Haq Nawaz a, *, Nosheen Rashid b, *,
Aamir Ali c, Asma Haque d, Muhammad Umair Akbar d, Muhammad Tahir a, Saania Munir a,
Zain Ali a, Muhammad Shahbaz a, Mudassar Saleem a
a
Department of Chemistry, University of Agriculture Faisalabad, Faisalabad 38000, Pakistan
b
Department of Chemistry, University of Education, Faisalabad Campus, Faisalabad 38000, Pakistan
c
National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Jhang Road, Faisalabad
38000, Pakistan
d
Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad 38000, Pakistan

A R T I C L E I N F O A B S T R A C T

Editor: Ron R. Allison Background: Surface-enhanced Raman spectroscopy (SERS) is an effective tool for identifying biofilm forming
bacterial strains. Biofilm forming bacteria are considered a major issue in the health sector because they have
Keywords: strong resistance against antibiotics. Staphylococcus epidermidis is commonly present on intravascular devices and
Staphylococcus epidermidis prosthetic joints, catheters and wounds.
Biofilm formation
Objectives: To identify and characterize biofilm forming and non-biofilm forming bacterial strains, surface-
Surface-enhanced Raman spectroscopy
enhanced Raman spectroscopy with principal component analysis (PCA) and partial least square discriminant
Multivariate data analysis techniques
analysis (PLS-DA) were used.
Methods: Surface-enhanced Raman spectroscopy (SERS) with silver nanoparticles were employed for the analysis
and characterization of biofilm forming bacterial strains. SERS is used to differentiate between non biofilm
forming (five samples), medium biofilm forming (five samples) and strong biofilm forming (five samples) bac­
terial strains by applying silver nanoparticles (AgNPs) as SERS substrate. Principal component analysis (PCA) and
Partial least square discriminant analysis (PLS-DA) were used to discriminate between non, medium and strong
biofilm ability of bacterial strains.
Results: Principal component analysis (PCA) and Partial least square discriminant analysis (PLS-DA) have been
used to identify the biochemical differences in the form of SERS features which can be used to differentiate
between biofilm forming and non-biofilm forming bacterial strains. PLS-DA provides successful differentiation
and classification of these different strains with 94.5% specificity, 96% sensitivity and 89% area under the curve
(AUC).
Conclusions: Surface-enhanced Raman spectroscopy can be utilized to differentiate between non, medium and
strong biofilm forming bacterial strains.

1. Introduction patients under immunosuppressive therapy, patients of Acquired Im­

Gram-positive coccus like Staphylococcus epidermidis is usually pre­
sent on human skin and in environment (untreated water, and
contaminated objects) [1]. and can strongly attach to the surface of
medical equipment and thus invade into the blood and different tissues
[2]. During invasive procedures, danger of infection is especially high in
drug addicts and patients with compromised immune systems like
munodeficiency Syndrome AIDS, and premature newborns [3]. The
S. epidermidis bacteria have emerged as a major pathogen in recent
years, and they are now among the top five most common organisms
causing nosocomial infection due to the formation of biofilm [4].
After attaching on biotic or abiotic surfaces, bacteria start growing
on them and form biofilms which are considered a protected mode of
growth. This allows bacteria to survive in the host body [5]. Different

* Corresponding authors.
E-mail addresses: irfan.majeed@uaf.edu.pk (M.I. Majeed), haqchemist@yahoo.com (H. Nawaz), nosheenrasheed@yahoo.com (N. Rashid).

https://doi.org/10.1016/j.pdpdt.2022.103145
Received 2 September 2022; Received in revised form 20 September 2022; Accepted 4 October 2022
Available online 6 October 2022
1572-1000/© 2022 Elsevier B.V. All rights reserved.
M. Shakeel et al.
factors including dormant cells, extracellular matrix, persister cells and
presence of resistance-relating factors are helpful for S. epidermidis
bacteria to form biofilms which make them extremely antibiotics resis­
tant [6]. The formation of biofilm by S. epidermidis on the surfaces lead
to formation of micro colonies of cells. The channels between the micro
colonies help the transfer of nutrients and waste from the biofilm. The
inherent ability of this bacteria to cause infection is associated with the
biofilm formation [7].
All bacterial cells have polysaccharides, lipids, proteins (amino
acids), DNA and RNA (nucleic acids) as major biochemical components.
Bacteria have attachment ability to link them to any surfaces whether in
any natural (living tissues for example bone or skin, vascular grafts,
ossicular and dental prostheses) or man-made systems (such as medical
devices), also called as biofilm forming bacteria, use this ability as de­
fense mechanism to prevent any antibiotic effect. Their attachment
ability helps them in identifying and avoiding any systemic infection
increasing their resistance against antibiotic hence turning them into
susceptible to most medication and hostile for all kind of hosts [8].
X-ray crystallography [9], Mass spectrometry [10], Infrared (FTIR)
spectroscopy [11] and Nuclear Magnetic Resonance (NMR) [12] spec­
troscopy techniques are used for the characterization of bacteria and
their biofilms. These techniques provide information related to
biochemical composition but have some limitations. The X ray crystal­
lography require samples to be in powder form, in large amount and can
destroy the sample due to its high energy. NMR, IR and FTIR require
rigorous sample preparation, not suitable for aqueous environment and
are time consuming. Notably, some techniques are perfectly suited to
research purposes, whereas routine clinical requirements demand
different methods like PCR [13], Congo red staining [14] and Chris­
tensen’s method [15] are used for the identification and characteriza­
tion of biofilm and microorganism but require a lot of time. There is
critical need for the development of rapid identification method to
distinguish microorganisms and biofilm [16].
Raman spectroscopy is a rapid identification and characterization
technique for bacteria [17] because it has potential to fingerprint the
whole organism but it has challenge of weak Raman signal [18]. This
problem is overcome by Surface enhanced Raman spectroscopy (SERS)
which is considered to have great potential for the identification and
characterization of bacteria and provides unique biochemical informa­
tion [19,20]. Surface Enhance Raman Spectroscopy (SERS) employs
nanoparticles for surface enhancement that increase the Raman Signal
which enable this technique for different bioanalytical applications such
as identification and differentiation of drug sensitive and resistant bac­
terial strains [18,21–22], food processing bacteria [23].
The silver nanoparticles (Ag-NPs) have been used to enhance the
Raman signal in the present work. Silver (Ag) and Gold (Au) NPs have
the strong SERS enhancement factor due to their strong surface plasmon
characteristics [24]. Silver nano particles have been intensively used as
SERS substrate for the analysis of various microorganisms [25,26]. In
Silver NPs surface plasmon resonance increased due to a greater number
of active sites hence can cause SERS enhancement higher than Gold NPs.
These can be easily prepared, have high molar extinction coefficient and
inexpensive [27].
Surface enhanced Raman spectroscopy has been used for character­
ization of biofilm of different microorganism like P. aeruginosa, S. epi­
dermidis, and C. albicans [28,29]. In the present work, SERS along with
chemometric analysis technique, PCA (Principal Component Analysis)
and PLS-DA (Partial Least Square Discriminant Analysis) have been
employed for the characterization of different biofilm forming and
non-biofilm forming bacterial strains of Staphylococcus epidermidis. PCA
and PLS-DA have helped to analyze differentiation between different
biofilm producing bacteria. To date, there is no published literature
found yet on characterization of biofilm forming and non-forming bac­
terial strains by using Surface Enhance Raman Spectroscopy.
2
Photodiagnosis and Photodynamic Therapy 40 (2022) 103145
2. Materials and methods
2.1. Bacterial isolation from culture media
The isolates were allowed to make biofilms in 96 well flat-bottom
polystyrene microtiter plates for 48 h at 37 ◦ C under static conditions.
After incubation, the crystal violet (0.1% w/v) staining was performed
as described previously by Christensen et al. [14] and the optical density
(OD) was measured at 570 nm with a 96 well plate reader. The classi­
fication of biofilm formation was done according to Stepanović et al.
[30]. The cut-off value of optical density (ODc) for this classification is
defined as three standard deviations above the mean OD570nm of the
negative control. The isolates were classified as follows: OD ≤ ODc:
non-biofilm former, ODc < OD ≤ 2 x ODc: weak biofilm former, and 4 x
ODc < OD: strong biofilm former. Fifteen bacterial isolates belonging to
the Staphylococcus epidermidis specie, are taken from the institutional
stock cultures. Five isolates for each of the already known category
including strong, medium and non-biofilm forming bacterial strains
have been revived in tryptic soy broth (TSB) at 37 ◦ C overnight. The
bacterial growth has been streaked on nutrient agar plates for colony
morphology. A separate colony was inoculated in 1 mL of sterile TSB and
kept stirring (180 rpm) at 37 ◦ C for 16 h. At 10,000 rpm the growth has
been centrifuged for consecutive 5 min and the pellets were separated
and stored at 4 ◦ C [31] till further Surface enhanced Raman spectral
analysis is performed.
2.2. Preparation of silver nanoparticles (Ag NPs)
The silver nanoparticles to be used as SERS substrate were prepared
by the chemical reduction method. Briefly, 0.0085 g/500 ml of silver
nitrate in deionized water was heated to attain 100 ◦ C followed by the
addition of 0.1 g of Na3C6H5O7 (tri-sodium citrate) as a capping ligand.
The solution was heated on a hot plate for about one and half hours with
continuous stirring with a magnetic stirrer to obtain gray-colored silver
nanoparticles [32].
2.3. SERS spectral acquisition
For the SERS measurements, 50µL of pellets of bacteria suspended in
deionized water were mixed with 50 µL of Silver NPs in Eppendorf tube
and left for half an hour for incubation time for developing interaction
between NPs and sample. The spectral acquisitions were performed
using a Raman Spectroscopy, Peak Seeker Pro-Agiltron Raman spec­
trometer, (USA) equipped with a 785 nm laser as source delivering 100
mW laser through a 40X objective. About 50 µL sample from the incu­
bation mixture in Eppendorf was placed on an aluminum slide for the
acquisition of SERS analysis. For all the samples of bacterial pellets, 15
Raman spectra were acquired in the spectral range of 300 to 1700 cm− 1
with an integration time of 10 s.
2.4. SERS spectral data pre-processing
The raw SERS spectral data contains information about the sample
but it also contains unwanted components like noise, baseline and
substrate contribution which may interfere with distinctive information
about the sample being studied. Therefore, it is necessary to pre-process
the raw SERS data by eliminating noise removal, baseline correction and
other interference to get useful information. MATLAB 7.8 version was
used to perform the pre-processing of SERS raw data by employing
chemometric codes [33]. Firstly, all SERS raw data was imported in
MATLAB in a single matrix and then pre-processed by algorithms for
baseline correction smoothening, substrate removal and vector
normalization. Savitzky-Golay is an algorithm that was applied for
smoothening purpose while polynomial methods and rubber band
correction methods were used for the baseline correction.
M. Shakeel et al.
Fig. 1. Mean SERS spectra of non-biofilm forming, medium biofilm forming and strong biofilm forming of Staphylococcus epidermidis bacterial strains.
2.5. SERS data analysis
For the analysis of SERS processed data, different chemometric
techniques including Principal Component Analysis (PCA) and Partial
Least Square-Discriminant Analysis (PLS-DA) were utilized for the
analysis of multivariate SERS spectral datasets. The differentiating SERS
spectral features associated with the biochemical changes in medium
and strong biofilm-forming bacteria as compared to non-forming ones
were identified SERS spectral data sets acquired from pellets of different
bacterial strains were subjected to PCA which is a chemometric method
and used to analyze differentiation and variability in the distinct data
sets in the form of PCA scatter plot) and the reasons for this differenti­
ation in the form of PCA loadings. PCA reduces the dimensionality while
variability remains unaffected. It is a qualitative analysis technique
which transforms the correlated variables into uncorrelated variables.
The first principal component (PC-1) explains maximum variability in
the data sets while second maximum variability is explained by the
second principal component (PC-2) and so on.
PLS-DA is a versatile and supervised modeling technique that may be
applied to SERS spectral data sets for calibration and validation of
multivariate discriminant models for classifying the non, medium and
strong biofilm-forming bacterial strains. This model uses previous in­
formation of x-variables against y-variables and builds classification
model. All spectral data is combined in a matrix and randomization
command is used to mix up the data and remove biasedness. Then data is
splitted into 60% and 40% calibration set and validation set, respec­
tively. Cross validation also performed to find out the number of latent
variables through monte carlo cross validation. To build the PLS-DA
model, leave one sample (15 SERS spectra) out cross validation was
performed. Notably, during leave one sample (15 spectra) out CV
method, all the replicates of each sample were kept separate.
The receiver operating characteristic (ROC) curve is mostly used for
evaluating the performance of binary classification algorithms. In binary
classification, there would be four possible outcomes including false
positive, true positive, false negative and true negative. ROC curve is
3
Photodiagnosis and Photodynamic Therapy 40 (2022) 103145
plotted by calculating false positive rate (specificity) and true positive
rate (sensitivity). Sensitivity (True Positive Rate) is calculated as the
fraction of "in-class" samples which are above the given threshold on y-
axis.
True Positive
TPR = Sensitivity =
True Positive + Fasle Negative
Specificity (False Positive Rate) is calculated as the fraction of "not-
in-class" samples which are below the given threshold on x-axis.
It can be calculated as:
False Positive
FPR = Specificity =
False Positive + True Negative
Or
FPR = Specificity = 1 − Sensitivity
3. Result and discussion
3.1. Mean SERS spectra
In Fig. 1, some SERS spectral characteristics are observed in mean
SERS plot which can clearly differentiate bacterial pellets of different
biofilm producing bacteria on the basis of different biomolecular con­
tents. Most of the biochemical contents are associated with proteins,
carbohydrates, lipids, amino acids and DNA/RNA. The differentiating
SERS bands are labelled by solid lines while SERS bands with intensity-
based differences are denoted with dotted lines.
The differentiating SERS bands include 604 cm− 1 (CC bending in
benzene ring of carbohydrates), 657 cm− 1 (Guanine ring breathing), 715
cm− 1 (Adenine), 728 cm− 1 (Adenine), 959 cm− 1 (C–N stretching of
amide lipopolysaccharides), 1033 cm− 1 (CH in-plane deformation,
proline/phenylalanine; CO and CC saccharides stretching), 1097 cm− 1
((PO2− symmetrical stretching); and 1430 cm− 1 (δ(CH2) scissoring
(phospholipids, fatty acid chains polysaccharides).
M. Shakeel et al. Photodiagnosis and Photodynamic Therapy 40 (2022) 103145

Table 1 cm− 1, 1322 cm− 1, 1337cm− 1 and 1588 cm− 1 correspond to the DNA. It
SERS peak assignments of pellets of biofilm forming bacterial strains. can be seen that intensity of these DNA related peaks is higher in biofilm
SERS Peak Assignment Components Refs. forming strains as compared to non-biofilm forming bacteria. The SERS
peaks band at 657 cm− 1, 728 cm− 1, and 1097 cm− 1 correspond to DNA which
(cm− 1) is only present in biofilm forming bacteria. This indicates increase in the
388 Polysaccharides Carbohydrates [34] contents of DNA in biofilm forming bacteria which can be due to the
471 C–C–O bending, C–OH twisting Carbohydrates [35] presence of plasmid used for the production of New Delhi metallo beta-
550 S–S stretch Carbohydrates [36] lactamase (NDM). Plasmid is a double strand chromosomal DNA and has
562 CC skeletal; C–O-C glycosidic ring Carbohydrates [37]
deformation of polysaccharide; COO−
ability to replicate and transfer its genes to other colonies which has an
wagging important role in adhesion of bacterial cells to surface and formation of
604 CC bending in benzene ring Carbohydrates [38] biofilm. Notably, this transfer of genetic information by Plasmid through
657 Guanine DNA/RNA [39] conjugation can enable non biofilm forming bacteria to produce biofilm
715 Adenine DNA/RNA [40]
[55].
728 Adenine DNA/RNA [36]
769 polysaccharides; COO− wagging and C–C Carbohydrates [41] The peaks which are related to proteins include 856 cm− 1, 959 cm− 1,
skeletal 1007 cm− 1, 1033 cm− 1, 1237 cm− 1, 1279 cm− 1, 1394 cm− 1 and 1430
785 Cytosine, Uracil (ring, stretching) DNA/RNA [42] cm− 1. SERS features at 856 cm− 1, 1007 cm− 1, 1237 cm− 1 and 1394
856 COC stretching of glycosidic linkage Protein [43] cm− 1 are present in relativity higher intensity in non-biofilm forming
saccharides; Stretching in Proline (CC)
and CCH deformation in ring breathing of
bacteria as compared to biofilm forming bacteria. The SERS peaks at 959
tyrosine; Gram-positive cell wall cm− 1 (C–N stretching (amide lipopolysaccharides) and 1033 cm− 1
(teichuronic acid) (C–H bond deformation of proteins) are solely observed in biofilm
959 C–N stretching (amide Protein [44] forming strains (medium and high) with increasing peak intensities.
lipopolysaccharides)
Some proteins called cell wall anchor proteins (CWA) help peptido­
1007 CC aromatic ring –phenylalanine, and Protein [45,
skeletal stretching of tryptophan; CCH 46] glycan in cell wall of gram-positive bacteria to form covalent bond with
stretching of carotenoids the surface where bacteria want to attach [56]. CWA surface proteins
1033 phenylalanine/proline (C–H in plane Protein [44] help in adhesion of biofilm forming bacteria to the surface of substrate.
deformation); CO and CC stretching The specie S. epidermidis has thirteen CWA proteins which are important
1097 PO2− symmetrical stretching DNA/RNA [47]
during the synthesis of peptidoglycan reaction and can be helpful in the
1134 CC unsaturated fatty acids Lipids [48]
1159 C–C carotenoids (stretching) Carotenoids [45, covalent bond formation between CWA protein and peptidoglycan
46] leading to biofilm formation [57].
1237 N-H bending and CO stretching (amide III) Protein [49] The SERS peaks which are associated with carbohydrates are
and CN amide (stretching)
observed at 471 cm− 1, 550 cm− 1, 562 cm− 1, 604 cm− 1 and 769 cm− 1.
1279 N–H, C–N, amide III (protein) Protein [50,
51] These raman peaks are present in both biofilm forming and non-biofilm
1322 adenine, guanine, Tyr DNA/RNA [52] forming bacteria. The SERS peak at 471 cm− 1 is associated with C–C–O
1337 Amide III; C–OH, HC– –C deformation of DNA/RNA [39] bending, 550 cm− 1 belongs to S–S stretching and 562 cm− 1 represent
polysaccharide; C–H deformation; glycosidic ring deformation in polysaccharide. The increase in the in­
Adenine
tensities of these peaks indicate increase in the carbohydrate contents in
1394 COO− 1 symmetric stretching Proteins [50]
1430 d(CH2) scissoring (phospolipids and fatty Proteins [37] biofilm forming bacteria as compared to non-biofilm forming strains.
acid) The SERS peak at 604 cm− 1 due to CC bending in benzene ring decrease
1472 CH2 deformation Lipids [53] in intensity in medium and strong biofilm forming bacteria as compared
1510 C = C str carotenoids Carotenoids [54]
to non-forming strain. [58]. Cells of bacteria are built up of poly­
1588 A and G stretching DNA/RNA [21]
saccharides that produce slime or capsule outside the bacterial cell wall
that provide bacteria protection, desiccation and host immune response
Table 1. like serum complement resulting in regulation of the host-pathogen
The SERS bands which are denoted by dotted lines indicating interaction on abiotic surface [59]. To increase resistance of biofilm
intensity-based differences as all the bacterial strains belong to same against mechanical force polysaccharide intercellular adhesion (PIA)
species, S. epidermidis, hence some SERS peaks are common in biofilm fixes Staphylococcus cells into fibrous net that built biofilm mass. PIA
forming and non-forming bacterial strains but there are still differences are present higher in concentration in biofilm forming bacterial strains
among them on the basis of intensity. These SERS features include 388 as they have major role in pathogenicity, rapid immune response and
cm− 1 (polysaccharides), 471 cm− 1 (CCO bending, C–OH twisting), 550 mediated intercellular adhesion [60].
cm− 1 (S–S stretching), 562 cm− 1 (COC glycosidic ring deformation of The SERS peaks which are linked to lipids include 1134 cm− 1
polysaccharide; COO- wagging; C–C skeletal), 769 cm− 1 (poly­ (=C–C= of unsaturated fatty acids in lipids) and 1472 cm− 1 (CH2
saccharides; COO− wagging and CC skeletal), 785 cm− 1 (ring stretching deformation) are. Lipids make up the majority of membranes and bac­
of Cytosine and Uracil), 856 cm− 1 saccharides (COC stretching of teria have their own set of lipids that can trigger or modify the host’s
glycosidic linkage, CC proline stretching and CCH deformation of tyro­ innate immune response. These lipids are also helpful in quorum sensing
sine ring breathing, teichuronic acid of Gram-positive cell wall), 1007 mechanism. Different lipids derivatives and proteins are useful in cell-
cm− 1 (C–C skeletal stretching of aromatic ring in phenylalanine; C–CH to-cell communication in biofilm [61]. SERS spectral features
stretching of carotenoids), 1134 cm− 1 (CC of unsaturated fatty acids in observed at 1510 cm− 1 associated with carotenoids due to C=C
lipids), 1159 cm− 1 (CC stretching of carotenoids), 1237 cm− 1 (C=O stretching. Carotenoids perform many major functions in bacteria like
stretching and N–H bending (amide III) and C–N stretching (amide) pigmentation, harvesting photo energy and neutralization of oxidants
(protein)), 1279 cm− 1 (N–H, C–N, amide III), 1322 cm− 1 (adenine, [62].
guanine, Tyr), 1337 cm− 1 (Adenine, amide III; HCO, HCC deformation From the above discussion, it can be concluded that the differenti­
of polysaccharide; CH deformation), 1394 cm− 1 (COO− 1 symmetric ating SERS peaks at 959 cm− 1 (C–N stretching (amide lipopolysaccha­
stretching), 1510 cm− 1(C=C str carotenoids) and 1588 cm− 1 (Adenine rides) and 1033 cm− 1 (C–H bond deformation of proteins) are solely
and Guanine ring stretching). observed in biofilm forming strains (medium and high) with increasing
The SERS bands at 657 cm− 1, 715 cm− 1, 728 cm− 1, 785 cm− 1, 1095 peak intensities. These SERS features of proteins can be associated with
some proteins called cell wall anchor proteins (CWA) which help

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M. Shakeel et al. Photodiagnosis and Photodynamic Therapy 40 (2022) 103145

Fig. 2. PCA scatter plot of SERS spectral data sets non-biofilm forming, medium biofilm forming and strong biofilm forming Staphylococcus epidermidis bacte­
rial strains.

Fig. 3. Pairwise PCA a) Scatter plot b) loadings of Non biofilm forming and medium biofilm forming Staphylococcus epidermidis bacterial strains.

peptidoglycan in cell wall of gram-positive bacteria to form covalent 728 cm− 1, and 1097 cm− 1 correspond to DNA which are only present in
bond with the surface where bacteria want to attach [56] leading to biofilm forming bacteria. This indicates increase in the contents of DNA
biofilm formation [57]. Other differentiating SERS features at 657 cm− 1, in biofilm forming bacteria which can be due to the presence of plasmid

5
M. Shakeel et al. Photodiagnosis and Photodynamic Therapy 40 (2022) 103145

Fig. 4. Pairwise PCA a) Scatter plot b) loadings of Non biofilm forming and strong biofilm forming Staphylococcus epidermidis bacterial strains.

used for the production of New Delhi metallo beta-lactamase (NDM). stretching of aromatic ring in phenylalanine, C–CH carotenoids
Plasmid has ability to replicate and transfer its genes to other colonies stretching in protein), 1134 cm− 1 (=C–C= of unsaturated fatty acids in
which has an important role in adhesion of bacterial cells to surface and lipids), 1237 cm− 1 (C=O stretching and N–H bending (amide III) and C
formation of biofilm [55]. N stretching in protein) and 1394 cm− 1 (COO− 1 symmetric stretching in
proteins) are related to SERS spectral data of non-biofilm forming bac­
terial strains.
3.2. Principal component analysis (PCA)
The positive loadings including 470 cm-1 (C–C–O bending, C–OH
twisting in carbohydrates), 657 cm-1 (guanine and thymine ring
To differentiate SERS spectral data sets of bacterial pellets of non,
breathing in DNA/RNA), 728 cm-1 (adenine in DNA/RNA), 769 cm-1
medium and strong biofilm forming bacteria, PCA is applied which help
(COO− wagging and C–C skeletal in carbohydrates), 785 cm-1 (cytosine,
to differentiate spectra of different strains as well as same strains of
uracil (ring, stretching) in DNA/RNA), 959 cm-1 (CN stretching of amide
bacteria. Fig. 2 shows all SERS spectral data of bacterial pellets including
lipopolysaccharides in proteins), 1033 cm-1 (C–H in-plane deformation -
non, medium and strong biofilm forming bacteria. The black dots
phenylalanine/proline in proteins), 1097 cm-1 PO2- symmetrical
represent spectra of non-biofilm forming and these are all clustered on
stretching in DNA/RNA) and 1472 cm-1 (CH2 deformation in lipids) are
negative side of PC-1 of scatter plot while other clusters like red and blue
linked with medium biofilm forming bacterial strains which is clustered
dots represent spectral data sets of bacteria which are biofilm forming
on positive axis of PC-1 of the PCA scatter plot.
are present on other positive side of PC-1.
PCA pair wise analysis of SERS spectral data sets of non-biofilms
Fig. 3(a) shows the comparison between non biofilm forming and
forming (black cluster dots) and high biofilm forming (blue cluster
medium biofilm forming bacterial strains. The SERS spectral data
dots) bacteria is shown in Fig. 4(a). Both types of spectra are differen­
associated with non-biofilm forming bacteria is clustered as block dots
tiated from each other which indicates that biofilm positive and negative
while medium biofilm forming bacterial strains are clustered as red
strains have significant biochemical differences among them. PC-1 ex­
color. There are two axis present indicating First Principal Component
plains the major variation as 53.52% while PC-2 explains 25.43%
(PC-1) and Second Principal Component (PCA-2).
variations.
The variability explained by first principal component is 74.99% and
In the Fig. 4(b), the positive loadings such as 471 cm− 1 (C–C–O
second principal component is 11.82%. Fig. 3(b) shows the pairwise
bending and C–OH twisting in carbohydrates), 562 cm− 1 (C–O-C
loadings of PCA of SERS spectral data sets of pellets of non-biofilm
glycosidic ring deformation in carbohydrates), 657 cm− 1(Guanine ring
forming and medium biofilm forming bacterial strains of
breathing in DNA/RNA), 728 cm− 1 (Adenine from flavins, NAG and
S. epidermidis. SERS analysis can clearly differentiate between two types
NAM in DNA/RNA), 959 cm− 1 (C–N stretching (amide lipopolysaccha­
of SERS data sets of bacteria. The negative loadings including 604 cm− 1
rides) in protein), 1003 cm− 1 (C–H in-plane deformation-phenylala­
(C–C bending in benzene ring in carbohydrates), 715 cm− 1 (Adenine,
nine/proline in proteins), 1097 cm− 1 (PO2− symmetrical stretching, CC
acetyl coenzyme-A in DNA/RNA), 1007 cm− 1 (C–C skeletal mode

6
M. Shakeel et al.
Fig. 5. Pairwise PCA a) Scatter plot b) loadings of SERS spectral data sets of strong-biofilm forming and medium biofilm forming bacterial strains.
and COC skeletal stretching, glycosidic linkage of saccharides in RNA/
DNA), 1337 cm− 1 (Adenine, amide III; HCC, OH, HCO, COH deforma­
tion of polysaccharide; CH deformation in DNA/RNA) and 1472 cm− 1
(CH2 deformation in carotenoids) are associated with strong biofilm
forming bacterial strains which are clustered on positive axis as blue
dots.
The negative loadings include 715 cm− 1 (Adeninein DNA/RNA), 856
cm− 1 (COC stretching of glycosidic linkage in saccharides), CC stretch­
ing in proline and CCH deformation ring breathing of tyrosine in pro­
tein), 1007 cm− 1 (C–C skeletal stretching of aromatic ring -
phenylalanine/tyrosine/ tryptophan in protein), 1134 cm− 1 (C–C of
unsaturated fatty acids in lipids), 1237 cm− 1 (Adenine, amide III; OH,
COH, HCO, HCC def polysaccharide; CH deformation in DNA/RNA) and
1394 cm− 1 (COO− 1 symmetric stretching in protein).
Fig. 5(a) shows PCA scatter plot between medium biofilm and strong
biofilm forming bacteria. Both of these clusters are clearly separated
from each other indicating that they have significant differences at
biomolecular level. The blue cluster present on the positive side of PC-2
indicate the strong biofilm forming bacterial strains while red cluster
that are present on the negative axis show SERS spectral data sets of
medium-biofilm forming bacterial strains. Fig. 5 have clear differences,
as PCA of these SERS spectra is performed which belong to those strains
which have ability to form biofilm (medium and strong biofilm forming
strains) and they are present in PC-2 while in Figs. 3 and 4, these features
are present in PC-1 which is result of PCA between SERS spectra of
biofilm forming and non-forming bacterial strains. The SERS spectral
data sets of these samples are clearly differentiated in overall scatter plot
7
Photodiagnosis and Photodynamic Therapy 40 (2022) 103145
shown in Fig. 2. PC-1 explains 51.95% variability while PC-2 explains
22.57% variability in spectral data.
Fig. 5(b) shows the loading of 2nd PC, the positive loadings include
features such as (C–O-C glycosidic ring deformation in carbohydrates),
657 cm− 1, (Guanine and thymine ring breathing in DNA/RNA), 728
cm− 1, (Adenine in DNA/RNA), 1033 cm− 1 (C–H in-plane deformation -
phenylalanine/proline in proteins), 1159 cm− 1 (C–C– stretching in ca­
rotenoids), 1337 cm− 1 (Adenine, CH deformation in DNA/RNA) and
1472 cm− 1 (CH deformation in lipids) that are related to the SERS
spectral data of strong biofilm forming bacteria which are clustered on
the positive axis of PC-2 in scatter plot.
The negative loadings include 715 cm− 1 (Adenine in DNA/RNA),
856 cm− 1 (COC stretching of glycosidic linkage in saccharides), CC
stretching in proline and CCH deformation ring breathing of tyrosine in
protein); teichuronic acid of Gram-positive cell wall in proteins), 1007
cm− 1 (C–C skeletal stretching of aromatic ring - phenylalanine/tyrosine/
tryptophan in proteins), 1279 cm− 1 (N–H, amide III in proteins, C–N)
and 1394 cm− 1 (COO− 1 symmetric stretching in proteins) which are
associated to SERS spectral data sets of the medium biofilm forming
bacteria which are scattered on the negative axis of PC-2 of scatter plot.
3.3. Partial least square-discriminant analysis (PLS-DA)
The PLSDA model is very useful chemometric tool that is used for the
discrimination among SERS spectral data sets of bacterial pellets of non-
biofilm forming, medium biofilm forming and strong biofilm forming
bacterial strains of Staphylococcus epidermidis. PLS-DA model is a
M. Shakeel et al. Photodiagnosis and Photodynamic Therapy 40 (2022) 103145

Fig. 6. Scatter score of PLS-DA model for SERS spectral data sets of bacterial strains of non-biofilm forming, medium biofilm forming and strong biofilm form­
ing strains.

Fig. 7. Receiver operating curve of PLS-DA model for SERS spectral data sets of non-biofilm forming, medium biofilm forming and strong biofilm forming bacte­
rial pellets.

represent SERS spectra of non-biofilm forming bacterial strains, blue

Table 2
dots show strong biofilm forming bacterial strains while red dots
PLS-DA classification of SERS spectral data sets
represent medium biofilm forming bacterial strain. This scatter score
of non-biofilm forming, medium and strong
biofilm forming bacterial pellets. shows clear difference between spectral data sets of these different
strains as biofilm negative strains are present on positive axis while
Parameters Values
spectra of biofilm positive strain are present on positive axis of score
Sensitivity 96% plot. Fig. 7 shows the ROC (receiver operating characteristics) curve of
Specificity 94.5%
PLS-DA analysis of SERS spectral data of biofilm positive and negative
AUC 0.8996
Precision 97% strains and value of ROC curve is found to be 0.8996. This model has
Accuracy 97.5% range from 0 to 1 and if the value is close to 1 then the model will be best
fit and indicates maximum accuracy and validation. Table 2 shows the
value of specificity, sensitivity, AUC, precision and accuracy of PLS-DA
supervised and quantitative chemometric technique. In this PLS-DA model of SERS spectral data sets of different bacterial strains. Using
model, SERS spectral data of non-biofilm forming, medium and strong SERS bacterial data sets, the proposed model shows excellent perfor­
biofilm forming bacterial pallets were independently splitted into two mance for discriminating between non, medium and strong biofilm
sets consisting of 45% calibration set and 55% validation set in order to forming bacterial strains.
avoid biasedness among data. For this, five number of latent variables
were selected to be used to build the PLS-DA model in order to avoid 4. Conclusions
over fitting of data set.
Fig. 6 shows scatter score of PLS-DA model including three different Surface enhanced Raman spectroscopy is very helpful technique to
types of clusters of dots. The black dots are present on positive axis that differentiate different bacterial strains of non-biofilm forming, medium

8
M. Shakeel et al.
and strong biofilm forming ones. The characteristic SERS spectral fea­
tures are identified for each specific bacterial strain of the SERS spectra
acquired from the respective bacterial pellets. PCA and PLS-DA are
employed to classify the SERS spectral data sets of non-biofilms forming,
medium and strong biofilm forming strains. The SERS peaks at 959 cm− 1
(C–N stretching (amide lipopolysaccharides) and 1033 cm− 1 (C–H bond
deformation of proteins) are only found in biofilm forming strains
including medium and high and can be associated with some proteins
called cell wall anchor proteins (CWA) which help in the attachment of
bacteria to surface leading to biofilm formation. Other differentiating
SERS features at 657 cm− 1, 728 cm− 1, and 1097 cm− 1 correspond to
DNA which are only present in biofilm forming bacteria. This indicates
increase in the contents of DNA in biofilm forming bacteria which can be
due to the presence of plasmid which has ability to replicate and transfer
its genes to other colonies which has an important role in adhesion of
bacterial cells to surface and formation of biofilm. PLS-DA is also helpful
in differentiating and identification of different spectral data of biofilm
forming and non-biofilm forming bacterial strains with sensitivity
(96%), specificity (94.5%), AUC (0.8996), precision (97%) and accuracy
(97.5).
Funding information
This research did not receive any specific grant from funding
agencies in the public, commercial and or not for profit purpose.
CRediT authorship contribution statement
Muhammad Shakeel: Writing – original draft. Muhammad Irfan
Majeed: Supervision. Haq Nawaz: Conceptualization. Nosheen
Rashid: Investigation. Aamir Ali: Methodology. Asma Haque: Vali­
dation. Muhammad Umair Akbar: Validation. Muhammad Tahir: .
Saania Munir: Data curation. Zain Ali: Software. Muhammad Shah­
baz: Software. Mudassar Saleem: Validation.
Declaration of Competing Interest
The authors have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in
this paper.
References
[1] J.Y. Lee, et al., Global spread of three multidrug-resistant lineages of
Staphylococcus epidermidis, Nat. Microbiol. 3 (10) (2018) 1175–1185.
[2] J. Zhu, et al., Identification of eltrombopag as a repurposing drug against
staphylococcus epidermidis and its biofilms, Curr. Microbiol. 78 (4) (2021)
1159–1167.
[3] G. Méric, et al., Disease-associated genotypes of the commensal skin bacterium
Staphylococcus epidermidis, Nat. Commun. 9 (1) (2018) 1–11.
[4] D. Mack, et al., Microbial interactions in Staphylococcus epidermidis biofilms,
Anal. Bioanal. Chem. 387 (2) (2007) 399–408.
[5] J.P. O’GARA, H. Humphreys, Staphylococcus epidermidis biofilms: importance and
implications, J. Med. Microbiol. 50 (7) (2001) 582–587.
[6] M.H. Muhammad, et al., Beyond risk: bacterial biofilms and their regulating
approaches, Front. Microbiol. 11 (2020) 928.
[7] L.D. Melo, et al., The protective effect of Staphylococcus epidermidis biofilm
matrix against phage predation, Viruses 12 (10) (2020) 1076.
[8] M. Loza-Correa, S. Ramírez-Arcos, Detection of bacterial adherence and biofilm
formation on medical surfaces. Biofilms and Implantable Medical Devices, Elsevier,
2017, pp. 181–193.
[9] A. Diehl, et al., Structural changes of TasA in biofilm formation of Bacillus subtilis,
Proc. Natl. Acad. Sci. 115 (13) (2018) 3237–3242.
[10] C. Bacon, D. Hinton, T. Mitchell, Screening of Bacillus mojavensis biofilms and
biosurfactants using laser ablation electrospray ionization mass spectroscopy,
J. Appl. Microbiol. 125 (3) (2018) 867–875.
[11] M. Dimopoulou, et al., Assessing the biofilm formation capacity of the wine
spoilage yeast Brettanomyces bruxellensis through FTIR spectroscopy,
Microorganisms 9 (3) (2021) 587.
[12] P.D. Majors, et al., NMR methods for in situ biofilm metabolism studies,
J. Microbiol. Methods 62 (3) (2005) 337–344.
[13] G.D. Christensen, et al., Adherence of slime-producing strains of Staphylococcus
epidermidis to smooth surfaces, Infect. Immun. 37 (1) (1982) 318–326.
9
Photodiagnosis and Photodynamic Therapy 40 (2022) 103145
[14] G.D. Christensen, et al., Adherence of coagulase-negative staphylococci to plastic
tissue culture plates: a quantitative model for the adherence of staphylococci to
medical devices, J. Clin. Microbiol. 22 (6) (1985) 996–1006.
[15] M. Kord, et al., Evaluation of biofilm formation and presence of ica genes in
Staphylococcus epidermidis clinical isolates, Osong Public Health Res. Perspect. 9
(4) (2018) 160.
[16] K. Rebrošová, et al., Identification of ability to form biofilm in Candida parapsilosis
and Staphylococcus epidermidis by Raman spectroscopy, Fut. Microbiol. 14 (6)
(2019) 509–517.
[17] L. Wang, et al., Applications of Raman spectroscopy in bacterial infections:
principles, advantages, and shortcomings, Front. Microbiol. (2021) 12.
[18] S. Bashir, et al., Surface-enhanced Raman spectroscopy for the identification of
tigecycline-resistant E. coli strains, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy 258 (2021), 119831.
[19] Y. Liu, et al., Potential of surface-enhanced Raman spectroscopy for the rapid
identification of Escherichia coli and Listeria monocytogenes cultures on silver
colloidal nanoparticles, Appl. Spectrosc. 61 (8) (2007) 824–831.
[20] A. Sengupta, M. Mujacic, E.J. Davis, Detection of bacteria by surface-enhanced
Raman spectroscopy, Anal. Bioanal. Chem. 386 (5) (2006) 1379–1386.
[21] S. Bashir, et al., Rapid and sensitive discrimination among carbapenem resistant
and susceptible E. coli strains using Surface Enhanced Raman Spectroscopy
combined with chemometric tools, Photodiagn.Photodyn. Ther. 34 (2021),
102280.
[22] A. Mushtaq, et al., Surface-enhanced Raman spectroscopy (SERS) for monitoring
colistin-resistant and susceptible E. coli strains, Spectrochim. Acta Part A 278
(2022), 121315.
[23] M. Kashif, et al., Surface-enhanced Raman spectroscopy for identification of food
processing bacteria, Spectrochim. Acta Part A 261 (2021), 119989.
[24] E. Solati, D. Dorranian, Comparison between silver and gold nanoparticles
prepared by pulsed laser ablation in distilled water, J. Cluster Sci. 26 (3) (2015)
727–742.
[25] M. Saleem, et al., Surface-enhanced Raman spectroscopy for the characterization of
the antibacterial properties of imidazole derivatives against Bacillus subtilis with
principal component analysis and partial least squares–discriminant analysis, Anal.
Lett. (2022) 1–15.
[26] Tahir, M., et al., Surface-Enhanced Raman Spectroscopy for Monitoring the
Antibacterial Activity of Lab Synthesized Imidazole Derivative and Commercially
Available Derivative (Tinidazole).
[27] X.-.F. Zhang, et al., Silver nanoparticles: synthesis, characterization, properties,
applications, and therapeutic approaches, Int. J. Mol. Sci. 17 (9) (2016) 1534.
[28] S. Keleştemur, M. Çulha, Understanding and discrimination of biofilms of clinically
relevant microorganisms using surface-enhanced Raman scattering, Appl.
Spectrosc. 71 (6) (2017) 1180–1188.
[29] S. Keleştemur, E. Avci, M. Çulha, Raman and surface-enhanced Raman scattering
for biofilm characterization, Chemosensors 6 (1) (2018) 5.
[30] S. Stepanović, et al., A modified microtiter-plate test for quantification of
staphylococcal biofilm formation, J. Microbiol. Methods 40 (2) (2000) 175–179.
[31] A. Farid, et al., Molecular detection of antimicrobial resistance in local isolates of
Staphylococcus epidermidis from urinary tract infections in Faisalabad region of
Pakistan, EXCLI J. 14 (2015) 697.
[32] H. Fang, et al., Ultrasensitive and quantitative detection of paraquat on fruits skins
via surface-enhanced Raman spectroscopy, Sens. Actuators B 213 (2015) 452–456.
[33] H. Nawaz, et al., Evaluation of the potential of Raman microspectroscopy for
prediction of chemotherapeutic response to cisplatin in lung adenocarcinoma,
Analyst 135 (12) (2010) 3070–3076.
[34] K. Ilaslan, I.H. Boyaci, A. Topcu, Rapid analysis of glucose, fructose and sucrose
contents of commercial soft drinks using Raman spectroscopy, Food Control 48
(2015) 56–61.
[35] Y. Zhao, et al., Study of succinylated food proteins by Raman spectroscopy,
J. Agric. Food Chem. 52 (7) (2004) 1815–1823.
[36] M. Kahraman, et al., Layer-by-layer coating of bacteria with noble metal
nanoparticles for surface-enhanced Raman scattering, Anal. Bioanal. Chem. 395 (8)
(2009) 2559–2567.
[37] K. De Gussem, et al., Raman spectroscopic study of Lactarius spores (Russulales,
Fungi), Spectrochim. Acta Part A 61 (13–14) (2005) 2896–2908.
[38] S. De Marchi, et al., Surface-enhanced Raman scattering (SERS) imaging of
bioactive metabolites in mixed bacterial populations, Appl. Mater. Today 14
(2019) 207–215.
[39] Y. Chao, T. Zhang, Surface-enhanced Raman scattering (SERS) revealing chemical
variation during biofilm formation: from initial attachment to mature biofilm,
Anal. Bioanal. Chem. 404 (5) (2012) 1465–1475.
[40] J. De Gelder, et al., Reference database of Raman spectra of biological molecules,
J. Raman Spectrosc. 38 (9) (2007) 1133–1147.
[41] S. Ramya, et al., Detection of algae and bacterial biofilms formed on titanium
surfaces using micro-Raman analysis, Appl. Surf. Sci. 256 (16) (2010) 5108–5115.
[42] B.C. Spector, et al., Noninvasive fluorescent identification of bacteria causing acute
otitis media in a chinchilla model, Laryngoscope 110 (7) (2000) 1119–1123.
[43] F.S. de Siqueira e Oliveira, et al., Biochemical characterization of pathogenic
bacterial species using Raman spectroscopy and discrimination model based on
selected spectral features, Lasers Med. Sci. 36 (2021) 289–302.
[44] M.L. Paret, et al., Biochemical characterization of Gram-positive and Gram-
negative plant-associated bacteria with micro-Raman spectroscopy, Appl.
Spectrosc. 64 (4) (2010) 433–441.
[45] W.E. Huang, et al., Shining light on the microbial world: the application of Raman
microspectroscopy, Adv. Appl. Microbiol. 70 (2010) 153–186.
M. Shakeel et al.
[46] J. Jehlička, H.G. Edwards, A. Oren, Raman spectroscopy of microbial pigments,
Appl. Environ. Microbiol. 80 (11) (2014) 3286–3295.
[47] A.C.S. Talari, et al., Raman spectroscopy of biological tissues, Appl. Spectrosc. Rev.
50 (1) (2015) 46–111.
[48] M. Nawaz, et al., Characterization and transfer of antibiotic resistance in lactic acid
bacteria from fermented food products, Curr. Microbiol. 62 (3) (2011) 1081–1089.
[49] D. Kusić, et al., Identification of water pathogens by Raman microspectroscopy,
Water Res. 48 (2014) 179–189.
[50] M.L. Laucks, et al., Comparison of psychro-active arctic marine bacteria and
common mesophillic bacteria using surface-enhanced Raman spectroscopy, Appl.
Spectrosc. 59 (10) (2005) 1222–1228.
[51] N.P. Ivleva, et al., Towards a nondestructive chemical characterization of biofilm
matrix by Raman microscopy, Anal. Bioanal. Chem. 393 (1) (2009) 197–206.
[52] R.M. Jarvis, et al., Surface-enhanced Raman scattering from intracellular and
extracellular bacterial locations, Anal. Chem. 80 (17) (2008) 6741–6746.
[53] P. Wang, et al., Characterization of Lactococcus lactis response to ampicillin and
ciprofloxacin using surface-enhanced Raman spectroscopy, Anal. Bioanal. Chem.
408 (3) (2016) 933–941.
[54] P. Rösch, et al., Chemotaxonomic identification of single bacteria by micro-Raman
spectroscopy: application to clean-room-relevant biological contaminations, Appl.
Environ. Microbiol. 71 (3) (2005) 1626–1637.
10
Photodiagnosis and Photodynamic Therapy 40 (2022) 103145
[55] J. Narenkumar, et al., Role of bacterial plasmid on biofilm formation and its
influence on corrosion of engineering materials, J. Bio-Tribo-Corros. 2 (4) (2016)
1–8.
[56] S. Dramsi, H. Bierne, Spatial organization of cell wall-anchored proteins at the
surface of Gram-positive bacteria, Protein Sugar Export Assembly in Gram-positive
Bacteria (2016) 177–201.
[57] M.G. Bowden, et al., Identification and preliminary characterization of cell-wall-
anchored proteins of Staphylococcus epidermidis, Microbiology 151 (5) (2005)
1453–1464.
[58] N. Sharon, Carbohydrates as future anti-adhesion drugs for infectious diseases,
Biochim. Biophys. Acta (BBA)-General Subjects 1760 (4) (2006) 527–537.
[59] R. Pathak, et al., Comparative efficiency of carbohydrates on the biofilm-forming
ability of enteroaggregative Escherichia coli, J. Food Saf. (2022) e12971.
[60] H. Rohde, et al., Structure, function and contribution of polysaccharide
intercellular adhesin (PIA) to Staphylococcus epidermidis biofilm formation and
pathogenesis of biomaterial-associated infections, Eur. J. Cell Biol. 89 (1) (2010)
103–111.
[61] T. Kawai, S. Akira, The role of pattern-recognition receptors in innate immunity:
update on Toll-like receptors, Nat. Immunol. 11 (5) (2010) 373–384.
[62] L. Nupur, et al., ProCarDB: a database of bacterial carotenoids, BMC Microbiol. 16
(1) (2016) 1–8.