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Raman spectroscopy as a new tool for early detection of bacteria in patients

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IOP PUBLISHING LASER PHYSICS LETTERS
Laser Phys. Lett. 10 (2013) 075603 (7pp) doi:10.1088/1612-2011/10/7/075603

LETTER

Raman spectroscopy as a new tool for

early detection of bacteria in patients with
cystic fibrosis
Giulia Rusciano1 , Paola Capriglione1 , Giuseppe Pesce1 , Pasquale Abete2 ,
Vincenzo Carnovale3 and Antonio Sasso1
1
Department of Physics, University of Naples ‘Federico II’, Complesso Universitario Monte S Angelo,
Via Cinthia, I-80126 Napoli, Italy
2
Department of Clinical Medicine, Cardiovascular and Immunological Sciences, University of Naples
‘Federico II’, I-80131 Naples, Italy
3
Cystic Fibrosis Center, University of Naples ‘Federico II’, Italy
E-mail: giulia.rusciano@unina.it

Received 3 October 2012, in final form 8 March 2013

Accepted for publication 9 March 2013
Published 14 May 2013
Online at stacks.iop.org/LPL/10/075603
Abstract
Respiratory infections represent a major threat for people affected by cystic fibrosis, leading to
pulmonary deterioration and lung transplantation as a therapeutic option for end-stage
patients. A fast and correct identification of pathogens in airway fluid of these patients is
crucial to establish appropriate therapies, to prevent cross-infections and, ultimately, to
preserve lung function. In this study, we used Raman spectroscopy to reveal bacteria in the
sputa of patients such as Pseudomonas aeruginosa and Staphylococcus aureus, which are
among the earliest and the most frequent bacteria affecting cystic fibrosis patients. We found
that Raman analysis, combined with principal component analysis, is able to provide a correct
identification of these bacteria, with a global accuracy higher than 95%. Interestingly, bacterial
identification is performed by analysing patients’ sputa as a whole, avoiding, therefore,
time-consuming procedures involving bacterial isolation or even bacterial cultures. This study
suggests that Raman spectroscopy could be a suitable candidate for the development of
innovative and non-invasive procedures for a fast and reliable identification of respiratory
infections in cystic fibrosis patients.
(Some figures may appear in colour only in the online journal)

1. Introduction impaired mucociliary clearance of inhaled microbes [4]. As

Cystic fibrosis (CF) is a multiorgan, worldwide diffuse
disease, whose incidence reaches one in 2500 newborns in
Caucasian populations [1]. In the world, it is possible to
estimate that around 70 000 people are affected by CF [2].
Most of the clinical manifestations of CF are related
to the mutations of the cystic fibrosis transmembrane
regulator gene (CFTR) on chromosome 7, responsible for
the normal function of the chloride channel [3]. This leads
to the production of abnormally viscous mucus and to
a result, from early childhood, CF patients are affected
by respiratory infections and airway inflammations, which
represent the most serious threat during the disease, leading
often to respiratory failure and even to the option of lung
transplantation [1, 5]. Hence, it is clear that a fast and correct
identification of bacteria involved in these infections is critical
for the therapeutic management of patients.
Different approaches are actually used to identify bacteria
from cystic fibrosis sputum samples, including morphological
and biochemical tests such as Gram staining, enzyme activity

1612-2011/13/075603+07$33.00 1 c 2013 Astro Ltd Printed in the UK & the USA

Laser Phys. Lett. 10 (2013) 075603
tests and antibiotic susceptibility profiles [6]. The gold
standard for bacterial identification remains 16S ribosomal
deoxyribonucleic acid (DNA) or 16S ribosomal ribonucleic
acid (RNA) analysis.
All these tests can be applied only on pure, isolated
bacteria, deriving from culture plates [7]. However, culturing
bacteria is a time-consuming procedure that can hamper the
proper management of patients. Complete identification is
routinely achieved within two days, but may be longer for
atypical bacteria. For this reason, it is highly desirable to
explore the potentiality of alternative techniques for bacterial
detection and identification. With this purpose, during the
last few years, several innovative biophysical techniques
for characterizing and typing microorganisms have been
introduced; the most prominent ones are those based on
mass spectrometry [8] and Raman spectroscopy (RS) [9]. In
particular, RS is becoming more and more a well consolidated
tool for characterizing bio-materials, from tissues to single
cells, since Raman spectra constitute a sort of chemical
fingerprint, which enables their identification. Very recent
works by several groups have demonstrated that the Raman
spectrum of a bacterium allows bacterial classification at a
species level or even at strain level [10–14]. In addition, the
information carried by a Raman spectrum can be obtained
even from a single bacterium, avoiding therefore the need for
a culture step.
Unfortunately, there are some important cases in which
this analysis cannot be ordinarily applied. As a matter of
fact, the localization of microorganisms in very complex
and optically thick samples introduces another level of
difficulty [15]. This is the case of airway fluid in CF patients,
where bacterial identification is also hindered by the presence
in the sample of many particles with sizes and shapes often
similar to bacteria. More importantly, the CF sputum is rich in
the residue of lysed bacteria while the concentration of intact
cells is relatively low [16]. Getting rid of this step would be an
undoubted breakthrough for a prompt clinical management of
bacterial infections. With this in mind, we decided to test what
kind of information on the bacterial content can be provided
by Raman analysis when the CF sputum is analysed. Notably,
we found that even this bulk phase carries information on
the content of bacteria. In particular, we show that Raman
analysis, combined with principal component analysis, reveals
infection from Pseudomonas aeruginosa and Staphylococcus
aureus, which are among the earliest and the most frequent
bacteria detected in CF patients. Furthermore, we aimed to
find any correlations of our experimental data with clinical
parameters such as the forced expiratory volume in 1 s, which
represents a key outcome of lung measure in CF [17].
2. Material and methods
2.1. Samples
Sputum was expectorated spontaneously, and in some cases
with the help of a physiotherapist, from 38 adults attending
the University clinics (21 males and 17 females; mean age
29 years; range 18–56 years). Anonymity was maintained for
2
G Rusciano et al
all the samples collected, according to an ethical protocol
approved by the University of Naples Federico II, Italy.
Samples were collected by direct expectoration into a sterile
cup. Pulmonary function was evaluated for all the patients by
spirometry assays.
Sample processing was initiated approximately 1 h after
donation. The samples were divided in two aliquots. One
aliquot was seeded into or smeared onto bacterial culture
media in accordance with the techniques routinely employed
at Microbiology Laboratory of Azienda Ospedaliera Univer-
sitaria of the University of Naples Federico II, as follows: by
means of a 1:100 calibrated loop and the streaking technique,
the material was seeded onto MacConkey agar (10:4 and
10:6 dilutions), mannitol salt agar (MSA) (10:6 dilution),
and B. cepacia selective agar (BCSA) (10:6 dilution) plates.
The plates were incubated at 35–37 ◦ C for up to 48 h (on
MacConkey agar and MSA) or for up to 72 h (on BCSA) in
room air. All of the colonies obtained were identified [18]. The
second aliquot from samples was moved to the Department of
Physics for Raman analysis, making care to keep the samples
at controlled temperature (∼0 ◦ C).
The donors analysed in this study belong to three different
classes. The first two classes class include CF patients affected
by P. aeruginosa (class 1, 26 donors) or S. aureus (class 2,
9 donors) chronic infection. The third class includes three
negative control donors (NC), i.e. people for which the search
for both P. aeruginosa and S. aureus in microbial assays
was negative. In particular, these donors were selected among
chronic obstructive pulmonary disease (COPD) patients.
From a clinical point of view, COPD patients are quite similar
to CF patients: in both cases, patients suffer from an excess
of mucus stasis, associated with increased mortality and a
more rapid decline in pulmonary function [19]. However,
while microbial infections are usually fully eradicated from
respiratory tree of COPD patients under proper antibiotic
therapies, they tend to chronicize in CF patients.
2.2. Spectroscopic measurements
The spectroscopic analysis was performed by using a
micro-Raman system described previously [20, 21]. Briefly, it
is based on an inverted microscope equipped with a Raman
probe at 532 nm (Laser Quantum, Opus 532). This laser
was focused into the sample by an Olympus oil-immersion
infinity corrected objective lens (100×, 1.4 NA). Raman
scattering generated at the focus was collected by the same
objective, passed through a notch filter, and then focused
through a 50 µm pinhole for background signal rejection.
The Raman photons were then dispersed by a spectrometer
(Jobin Yvon, Triax 180) equipped with a 1800 grooves mm−1
grating and, finally, projected on a thermo-electrically cooled
CCD (PIXIS 100, Princeton Instruments). The final spectral
resolution was 2 cm−1 . The sample under investigation was
sealed between two 150 µm microscope cover slips to avoid
drying. Measurements were made at room temperature. The
acquisition time was 20 s while the power impinging on the
sample was 300 µW. For each sample, 30 Raman spectra were
acquired.
Laser Phys. Lett. 10 (2013) 075603
Figure 1. (a) Raman spectra showing the effect of photo-induced
bleaching of a sputum sample from a CF patient affected by
P. aeruginosa. (b) Fluorescence background level (estimated at
1800 cm−1 ) versus bleaching time for the same sample. The
continuous line is a fit of data with an exponential curve.
2.3. Sample photo-bleaching
For nearly all samples analysed in this work, the characteristic
Raman peaks were initially almost completely embedded
in a large fluorescent background. The origin of this
background can be mostly ascribed to bacterial pigmentations,
which are also responsible for sputum colour [22]. For
instance, P. aeruginosa secretes a variety of pigments,
including pyocyanin (blue–green), pyorubin (red–brown)
and pyoverdine (yellow–green). To avoid this problem,
we performed an extended sample photo-bleaching. This
technique has been previously suggested to reduce fluorescent
background in the analysis of bacterial strains [23]. A
typical situation is reported in figure 1. In particular, part
(a) shows three Raman spectra acquired after different times
of continuous sample irradiation. At t = 0 min, the Raman
features are almost completely masked by a large fluorescence
background. As laser exposure time passes, Raman peaks
begin to emerge (see spectra at t = 15 and 30 min).
In part (b) of figure 1 we also report the signal
background level, estimated at 1800 cm−1 , as a function of
the exposure time. Data were normalized to the highest value
observed at t = 0 and fitted by an exponential decay. A decay
time τ equal to 86 s was found, which is quite close to the
value τ ∼ 100 s found by Scholtes-Timmerman et al [23]
to extinguish most pigment fluorescence in the spectra of P.
aeruginosa.
2.4. Data analysis
To highlight differences in the Raman spectra from different
donors, we applied principal component analysis (PCA)
to the recorded spectra. This allowed us to reduce the
dimensionality of spectral variables, retaining, at the same
time, the variables of the original data set that contribute most
3
G Rusciano et al
Figure 2. Comparison between the mean Raman spectra relative to
two CF patients. Spectra (a) and (b) come from donors infected by
P. aeruginosa and S. aureus, respectively.
to its variance (principal components). In our analysis, PCA
was performed by using MATLAB software subroutines. The
Raman raw data were pretreated by using a custom-made
routine, developed in order to eliminate spurious cosmic
ray contributions and to automatically subtract the residual
background with a fourth order polynomial curve. Therefore,
the data were normalized, mean centred and, finally, analysed
by PCA. The results of PCA were given in terms of
component scores and loadings. The scores for the first three
components were used to create a three-dimensional plot
(score plot) in order to verify clustering and/or separation
of data. To test the reliability of our results, we used
the leave-one-out cross-validation procedure (LOOCV) [24].
With this purpose, we verified that the first five PCs had a
relevance of at least 95% for all the subgroups considered in
the leave-one-out procedure. The results of this analysis were
used to calculate the confusion matrix [25], in which all the
correct guesses are located on the diagonal of the matrix (true
positive, TP and true negative, TN), while misclassified data
(false positive, FP and false negative, FN) are represented by
the off-diagonal elements. From this matrix, the sensitivity
TP/(TP+FN), the specificity TN/(TN+FP) and the accuracy
(TP + TN)/(TP + TN + FP + FN) in the leave-one-out
procedure were estimated.
3. Results and discussion
3.1. Raman analysis on CF patients’ sputum
Sputum is the (spontaneous or induced) expectorated fluid
from the airway. In healthy people, it mainly contains mucins,
the normal secretion from mucous cells and submucosal
glands. However, in CF patients sputum contains very little
mucin, while there is a large amount of cellular debris,
including actin, DNA, bacteria and bacterial breakdown
products [16]. This complex composition is translated into
complex Raman spectra, with relatively broad features
(see figure 2). They contain major peaks at 1005 cm−1
Laser Phys. Lett. 10 (2013) 075603 G Rusciano et al

Table 1. Tentative assignments for the main Raman bands observed in the spectra of sputa, according to the references indicated in the
fourth column. Abbreviations: G, guanine; A, adenine; P, Phe, phenylalanine; Tyr, tyrosine.
Wavenumber (cm−1 ) Component Assignment References
668 DNA Ring breathing (G) [27, 28]
725 DNA Ring breathing (A) [27, 28]
1004 Protein Ring breathing (Phe) [28, 29]
1032 Protein Phe [28]
1157 Carotenoids Carotene –C–H– stretch [23, 30]
1245 Protein Amide III (β strand) [28, 29, 31]
1319 Protein Amide III [28, 29]
1447 Protein/lipids CH2 –CH3 bend [28, 31]
1480 DNA Ring mode (G, A) [27, 28]
1525 Carotenoids Carotene –C=H– stretch [23, 30]
1584 Protein Phe [28]
1617 Protein Tyr [28, 31]
1667 Protein Amide I [28]

(phenylalanine ring vibration), 1445 (CH2 and CH3 lipid and

protein) and 1650 cm−1 (amide I protein band). Moreover,
many peaks due to carotenoids, at 1157 and 1525 cm−1 can
be identified in the spectra. A tentative assignment of the
observed Raman features is reported in table 1.
For this study, we performed an experiment on 38
patients, selected by the medical staff of the Cystic Fibrosis
Center at the University of Naples Federico II as described
before. Researchers involved in Raman measurements were
prevented from knowing donors’ clinical conditions. Samples
were collected in seven different sessions. Surprisingly, very
similar spectra were obtained for all the samples analysed. A
typical example is shown figure 2, where we report the spectra
of two CF patients, affected by different bacterial infections.
Minimal differences can only be seen in the intensity of
bands due to amide bands and carotenoids around 1650
and 1150 cm−1 , respectively. However, a reliable evaluation
of these differences is an hard task, due to the presence
of a residual background signal. Taken from the viewpoint
of clinicians and medical analysts, this completely impairs
their effective utility for diagnostic purposes. Therefore,
as it was impossible to distinguish samples in a direct
way, we implemented a classification model based on PCA. Figure 3. Scatter plots relative to PCA analysis of samples
As a matter of fact, in all the measurement sessions, obtained in two different experimental sessions. Points of the same
we found that PCA allowed separation of samples into colour represent spectra from the same patient. Dots correspond to
two or three subgroups (see figure 3, part (a) and (b) patients affected by P. aeruginosa, triangles correspond to patients
affected by S. aureus while squares correspond to NCs. In both
respectively). The significance of this separation (obtained cases, the first three PCA components contribute over 98% of the
under blind conditions) became clear when clinicians revealed total relevant information on the system. As it is possible to see,
the patients’ clinical data to the researchers involved in data reported in (a) collapse in two different subgroups,
Raman analysis. This a posteriori knowledge allowed us to corresponding to CF patients affected by different infections, while
identify the subgroups as corresponding to patients belonging in (b) a third subgroup appears, corresponding to NC donors.
to different classes (P. aeruginosa-infected, S. aureus-infected
or negative control donors). For instance, in figure 3(a),
dots correspond to patients affected by P. aeruginosa, while patients’ sputum? The answer to this question can be found
triangles correspond to patients affected by S. aureus. Similar by analysing the PCA loadings. These last indicate the specific
results were obtained for all the patients analysed in the contribution of each wavenumber in the calculation of the total
present study. In all examined cases, our analysis showed variance of the spectral data. The interpretation of the loading
significant clustering of data deriving from homogeneous plots is often difficult since they did not directly represent
donors and, at the same time, separation between the three a real Raman spectrum, presenting, in general, both positive
previously mentioned subgroups of patients. and negative features. However, sometimes it is possible to
What makes the sputum samples different? Or, in other give a reasonable interpretation of loadings [26], as in our
words, what kind of signature do bacteria leave in CF case. Figure 4 shows the loading relative to the first principal

4
Laser Phys. Lett. 10 (2013) 075603 G Rusciano et al

Figure 5. PCA score plot relative to eight donors with different

FEV1 values. All the donors were affected by the same bacterial
Figure 4. PC1 loading plot relative to the analysis of sputum infection (P. aeruginosa). For each data set (represented by dots of
samples from six CF patients. The corresponding scatter plot is the same colour), the 95% confidence level ellipsoid is also shown.
shown in figure 3, part (b).

component for data of figure 3. Interestingly, this loading

presents some sharp features, easily assigned to known Raman
peaks, which are completely hidden in the original Raman
spectra (see figure 2). More importantly, there is an interesting
similarity between the PC1 loading and typical bacteria
spectra (see, for instance [23], for a comparison with the P.
aeruginosa Raman spectrum). This outcome confirms that
what makes the sample distinguishable in our analysis is their
bacterial content, rather than any other biochemical feature.
This can be ascribed to the use of a Raman probe at 532 nm,
a wavelength at which the contribution from bacteria (and
bacterial lysis products) is resonantly enhanced [15].

3.2. Correlation of data with the FEV1 values

As shown in section 3.1, our analysis enables us to distinguish
groups of CF patients infected by different bacterial strains.
However, some more information can be gained by analysing
CF patients presenting the same bacterial infection but
exhibiting different clinical conditions. In fact, we noticed that
there is a correlation between the spread of data in the PCA
scatter plot and the patient respiratory function, quantified
by the FEV1 values obtained in spirometry assays. FEV1
corresponds to the forced expiratory volume at 1 s. It is the
clinical parameter most commonly used to evaluate the upper
airway obstructions and in assessing patient conditions in
lung related disease such as cystic fibrosis [17]. In figure 5,
we show the PCA score plot relative to eight CF patients
infected by P. aeruginosa but presenting different FEV1
values measured just before sputum expectoration. The data
corresponding to CF patients presenting a moderate (FEV1
between 40% and 70%) or severe (FEV1 lower than 40%)
respiratory manifestation tend to occupy a smaller region of
the PCA scatter plot, while data from patients with only
a slight lung manifestation of the disease (FEV1 higher
than 70%) are scattered in a much wider region. This is
quantitatively illustrated in figure 6, where we report the
volume corresponding to the 95% confidence level ellipsoid
Figure 6. Volume of the 95% confidence level ellipsoid in the
PC-score space for CF patients presenting different FEV1 values
before sputum expectoration.
for each of the data sets of figure 5. In a sense, we can say
that the lower the FEV1, the clearer is the bacterial fingerprint
in the Raman spectrum, so that the data tend to cluster in the
PCA plot.
3.3. Application of the LOOCV procedure
To test the global reliability of our approach in discriminating
patients affected by a different bacterial infections, we used
the LOOCV procedure. According to this, denoting by N the
total number of data sets relating to the N different donors,
PCA was calculated using N − 1 data sets (used as the training
set), and the scores of the left-out data set (relating to the Nth
donor) were calculated by projecting it on the PC vectors of
the training data. The attribution of each of the 30 spectra
belonging to the left-out set into the proper class was done by
evaluating its Mahalanobis distance [25] from the training set
(whose classification was considered as known) in the space
of the first five scores. Finally, donor assignment to a given
patient class was performed on the basis of the majority of

5
Laser Phys. Lett. 10 (2013) 075603
Table 2. Confusion matrix giving the classification for
P. aeruginosa- and S. aureus-infected CF patients, as well as for NC
donors.
Predicted
P. aeruginosa S. aureus NC
True P. aeruginosa 25 1 0
S. aureus 1 8 0 a correlation between an important clinical parameter, the
NC 0 0 3 FEV1 value, and spectroscopy-derived parameters, such as
Table 3. Sensitivity, specificity and accuracy estimated by LOOCV
procedure for the three donor classes.
P. aeruginosa S. aureus NC
Sensitivity 96 89 100
Specificity 92 97 100
Accuracy 95 95 100
spectra attribution. This procedure was iteratively repeated
until all the data sets were left out. For almost all the patients,
more than 95% of spectra were attributed to the same donor
class; in only one case, the majority of spectra corresponded
only to 17 spectra out of 30. The results of LOOCV analysis
are summarized by the confusion matrix shown in table 2.
From the elements of this matrix, we evaluated the
sensitivity, specificity and accuracy reached in discriminating
between the three different classes. These values are reported
in table 3. It is worth noticing that our methodology is
able to identify correctly all the NC donors with a 100%
accuracy. Moreover, P. aeruginosa in patients’ sputum is
identified with both high sensitivity (percentage of infected
people who are correctly identified as having the infection)
and high specificity (percentage of uninfected people who are
correctly identified as not having the infection). A slightly
worse result was obtained in revealing infection by S. aureus.
However, this is probably due to the lower number of
S. aureus-infected patients here analysed, which severely
reduces the obtained sensitivity even if only one sample was
misclassified. Although the study presented herein has still a
preliminary character, it holds promise for the discrimination
of bacterial infections in CF patients by analysing directly
patients’ sputa. Prospectively, this could pave the way for
the development of new protocols for a reliable and very fast
identification of these bacteria, which are among the earliest
and the most frequent bacteria detected in people with CF.
4. Conclusions
Our experimental outcomes demonstrate that Raman analysis,
combined with a multivariate statistical data analysis,
is able to reveal different species of bacteria through
the lysed material they leave in the patients’ sputa. In
particular, we found a global accuracy higher than 95% for
differentiating between bacterial infections in CF patients
due to P. aeruginosa or S. aureus. This means that there
is actually a relatively small chance of misclassification of
the patients according to their bacterial infection. To the
best of our knowledge, the current study represents the
6
G Rusciano et al
ultimate limit of Raman spectroscopy in bacterial detection
and identification in a highly heterogeneous environment
such as CF patient sputum. Notably, our analysis can be
carried out directly on CF patient sputum, avoiding the
necessity of time-consuming procedures involving bacteria
isolation or even bacteria cultures. Interestingly, we also found
the spread of points in the score plot representation. Although
the study presented herein has still a preliminary character,
it holds promise for the use of Raman analysis for the
development of new clinical protocols for bacteria in the
airway fluid of CF patients.
Acknowledgments
The authors thank Professors G Miele and V Raia for their
critical reading of the manuscript.
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