International Journal of Antimicrobial Agents 46(2015)S35–S39
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International Journal of Antimicrobial
Agents
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Review
Raman spectroscopy towards clinical application:drug monitoring and pathogen identification
Ute Neugebauer a ,b ,c ,Petra Rösch c ,d ,Jürgen Popp a ,b ,c ,d ,∗
a
Center for Sepsis Control and Care (CSCC),Jena University Hospital,Erlanger Allee 101,D-07747Jena,Germany b
Leibniz Institute of Photonic Technology,Albert-Einstein-Straße 9,D-07745Jena,Germany c
InfectoGnostics Forschungscampus Jena,Philosophenweg 7,D-07743Jena,Germany d
Institute of Physical Chemistry and Abbe Center of Photonics,Friedrich Schiller University Jena,Helmholtzweg 4,D-07743Jena,Germany
a r t i c l e
i n f o
Keywords:
Raman spectroscopy
Therapeutic drug monitoring Infection detection Antibiotic resistance Lab-on-a-chip (LOC)
Fibre-enhanced Raman spectroscopy
a b s t r a c t
Raman spectroscopy is a label-free method that measures quickly and contactlessly,providing detailed information from the sample,and has proved to be an ideal tool for medical and life science r
earch.In this review,recent advances of the technique towards drug monitoring and pathogen identification by the Jena Rearch Groups are reviewed.Surface-enhanced Raman spectroscopy (SERS)and ultraviolet resonance Raman spectroscopy in hollow-core optical fibres enable the detection of drugs at low con-centrations as shown for the metabolites of the immunosuppressive drug 6-mercaptopurine as well as antimalarial agents.Furthermore,Raman spectroscopy can be ud to characteri pathogenic bacteria in infectious dias directly from body fluids,making time-consuming cultivation process dispensable.Using the example of urinary tract infection,it is shown how bacteria can be identified from patients’urine samples within <1h.The methods cover both single-cell analysis and dielectrophoretic capturing of bacteria in suspension.The latter method could also be ud for fast (<3.5h)identification of antibiotic resistance as shown exemplarily for vancomycin-resistant enterococci.
©2015Elvier B.V.and the International Society of Chemotherapy.All rights rerved.
1.Introduction
Raman spectroscopy has proved to be an ideal tool for medical and life science rearch,as Raman measures without con-tact,providing label-free information on process within living cells without di
sturbing them.Furthermore,Raman spectroscopy measures quickly,overcoming the need for complex and time-consuming laboratory analys in many cas.Raman spectroscopy also measures precily,providing the ultransitive detection capabilities needed for clinical applications.Last but not least,Raman spectroscopy can easily be combined with other optical and non-optical methods to enable convenient sample handling and processing of clinical patient samples.In combination with a micro-scope,high spatial resolution (<1m)can be achieved,enabling the analysis of single bacterial cells.As water yields only a very weak Raman spectrum,it is the ideal solvent for Raman spectroscopic analysis.Thus,analysis of body fluids can be carried out by means of Raman spectroscopy [1–3].
∗Corresponding author.Prent address:Institute of Physical Chemistry,Friedrich Schiller University Jena,Helmholtzweg 4,D-07743Jena,Germany.Tel.:+493641948320;fax:+493641948302.牛圈对联
E-mail address:juergen.popp@uni-jena.de (J.Popp).
In the following,the results prented at the 6th European Con-ference on Bloodstream Infections,on 6–7June 2015in Vravrona,Greece,will be summarid.They cover recent rearch highlights from the Jena Rearch Group dealing with Raman spectroscopy-bad concepts for the detection of dru
gs and the technological developments towards therapeutic drug monitoring,the detection and identification of bacteria from body fluids with a special focus on urine from patients suffering from urinary tract infections (UTIs),as well as spectroscopic approaches for fast antibiotic susceptibility testing.
2.Therapeutic drug monitoring
For the detection of low concentrations of drugs,enhancement methods need to be applied.Here especially,surface-enhanced Raman spectroscopy (SERS)enables monitoring of substances at low concentrations [4].Combining SERS with a lab-on-a-chip (LOC)microfluidic device enables enhancement of the reproducibility of the analysis [5,6].By means of LOC-SERS it is possible to detect and quantify antibiotics at micromolar concentrations,which are in the therapeutically important range [7,8].An alternative approach to detect analytes that cannot be directly monitored by means of LOC-SERS is an indirect detection of a fluorescence dye that can react with the analyte [9].
dx.doi/10.1016/j.ijantimicag.2015.10.014
0924-8579/©2015Elvier B.V.and the International Society of Chemotherapy.All rights rerved.
S36U.Neugebauer et al./International Journal of Antimicrobial Agents46(2015)关爱明天
S35–S39
Fig.1.Schematics of thefibre nsing tup.The analyte is injected into thefibre with a syringe pump.Lar light is coupled into the samefibre to interact with the analyte over a large distance(fibre length)enabling the detection of low concen-trations.The backscattered Raman signal is collected through the same objective lens and is further analyd in a spectrometer with a charge-coupled device(CCD) camera.
Adapted with permission from[11].Copyright(2013)American Chemical Society.
LOC-SERS is not only suitable for identifying and quanti-
fying pharmacologically active substances.It can also be ud
to monitor the therapeutic efficacy of drugs with respect to
enzyme activity,which may inactivate the drug too fast or not
at all.An example is thiopurine methyltransfera(TPMT)activ-
ity in red blood cells.Since the concentration of toxic and active
metabolites of the immunosuppressive drug6-mercaptopurine
is rather high in patients,therapy using this drug can result in
rious toxicity as well as failure of efficacy owing to genetic
differences in metabolising enzymes.A huge variety of TPMT
genotypes exhibiting different activities for the methylation of
thiopurines impede determination of a therapeutic dosage,since
high enzyme activity results in the inactivation of thiopurines,
whereas low activity can lead to toxic effects.Here,LOC-SERS was
ud successfully to determine the TPMT activity in blood samples
[10].
An alternative method to detect therapeutic agents isfibre-
enhanced Raman spectroscopy(FERS).Here,a hollow-core optical
椰蓉饼干
fibre is ud to enhance the Raman signal significantly in order
to detect even lower concentrations.Application of an ultraviolet
excitation wavelength in combination with a hollow-core optical
fibre can even detect chloroquine and mefloquine at concentrations
<100M in an aqueous environment(Fig.1)[11].
Applying visible excitation wavelengths enables direct mon-
itoring of human breath,which is a mixture of different major
compounds including N2,O2,CO2and H2O as well as traces
of volatile organic compounds.In addition,there are important
gaous markers for the detection of different dias such ,
acetone(C3H6O)and methane(CH4)for lung cancer,or NH3and 12CO2for Helicobacter pylori infection.Application of FERS with a hollow-core opticalfibre allows the detection of all sorts of gaous
components in human breath.Even the differentiation between
isotope-labelled substances is possible in routine measurements
[12].
Applying a microstructured hollow-core photonic crystalfibre
for FERS even allows simultaneous monitoring of H2in the pres-
ence of all other gas such ,CH4,N2,O2and CO2.This was
achieved by a combination of rotational Raman spectroscopy for H2
and vibrational Raman spectroscopy for the other gas.With this
approach,it was possible to detect H2down to a limit of detection
of4.7ppm besides other gas(CH4,N2,O2,12CO2and13CO2)[13]
.Fig.2.Mean Raman spectra of major pathogens in urinary tract infections ud to construct a support vector machine classification model for the identification of unknown patient samples.
Adapted with permission from[16].Copyright(2013)American Chemical Society.
3.Diagnosis of infectious dias from bodyfluids
这些英文The prevalent caus of death in non-cardiology intensive care units are pathogen-induced psis and its most extreme form,p-tic shock.Before arriving at the state of ptic shock,the patient has undergone veral stages in the continuum of psis,start-ing with a local infection where the pathogen invades the host and releas toxic products,passing the stage where an over-whelming immune respon is not only targeted towards the pathogen but also induces morphological damage to cells and tissue leading to organ failure[14].A faster and more detailed diag-nosis could help to save lives in the future.However,currently established microbiological diagnosis involves time-consuming cultivation steps.Thus,a detailed microbiological analysis is only available after1day up to veral days.Raman spectroscopy is a non-invasive,label-free optical technology that can record in real time the spectroscopicfingerprint of single bacteria,offering a high potential for faster bacterial characterisation directly from patient samples[15–21].
Promising results have been obtained for the diagnosis of UTIs, which account for>150million infections/year with a spectrum ranging from uncomplicated UTI to life-threatening healthcare-associated psis.They are the most frequent infections in women, with>60%of all women having a UTI during their lives.UTIs are also the most common cau of nosocomial infections causing health-care costs of ca.US$6billion.The current gold-standard method for pathogen identification from urine culture takes longer than24h to give a result[22–24].
To evaluate the potential of Raman spectroscopy for fast and reliable identification of pathogens directly from urine samples, a reference databa has been built including the most common causative pathogens,which are Escherichia coli(50%),Klebsiella spp.(14%),enterococci(10%),staphylococci(6%),Pudomonas aeruginosa(3%)and rarely other bacteria,virus and fungi.In total, 11different species have been measured in more than four inde-pendent batches per species yielding more than200spectra from single cells per species.Fig.2displays the averaged Raman spectra per included species.The resulting2952spectra were ud to train a classification model bad on support vector machines.This model was tested with independently measured Raman spectra(in total 514)from the same11species yielding a prediction accuracy of95% [16].This high accuracy makes the model suitable to be evaluated with re
al-world patient samples.However,when analysing urine samples from patients,veral unknown factors will complicate
U.Neugebauer et al./International Journal of Antimicrobial Agents 46(2015)S35–S39
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Fig.3.Evaluation of patient urine specimens:assignment of measured single-cell Raman spectra bad on the previously built support vector machine model.*Samples with a positive inhibitor test.
Adapted with permission from [16].Copyright (2013)American Chemical Society.
the analysis,including the unknown composition of the urine,the unknown dwell time of the bacteria in the urine and,most likely,the urine will not contain a bacterial strain that is already known to the databa.Two Raman-bad approaches have been tested,one focud on single bacterial analysis [16]and the cond optimid to perform the analysis directly in suspension [25].For single-cell analysis,the urine sample is centrifuged and the washed bacteria are placed on a Nickel foil where they are allowed to dry.Raman spectra are recoded from single bacteria cells of the patient’s sam-ple.For each single-cell measurement,the databa predicts the identity of the pathogen.For each specimen,between 66%and 98%of measured cells were classified as one species.In Fig.3,the first ven patient samples were determined to li and the following three samples to contain Enterococcus faecalis as the pre-dominate species.The results agree with the results of the gold standard of urine culture.When using a threshold of ≥60%abun-dance in the single-cell Raman spectra,a correct assignment of all investigated patient samples can be achieved.Noticeably,five of the ten analyd samples showed a positive inhibitor test.Thus,Raman spectroscopic analysis can id
entify pathogens from urine despite the prence of antibiotics or other growth-inhibitory substances.This can bring up very promising applications for the identification
of non-cultivatable pathogens directly from body fluids of patients [16–18].
The cond approach,in which the Raman spectroscopic anal-ysis is performed directly in suspension,us dielectrophoretic forces to capture bacteria in defined locations in space.Only min-imal sample preparation is required for analysis of a conventional urine sample.One droplet of the filtered and washed urine is placed on a dielectrophoresis chip with four gold electrodes (Fig.4).An alternating electric field between the electrodes forces the bacte-ria into the middle where they are analyd by means of Raman spectroscopy.As in the first approach,with the help of a previ-ously established databa the Raman spectra from the patient’s sample are ud to identify the pathogen in the urine sample.High prediction accuracies have been reported for the differentiation li and E.faecalis [25].Furthermore,it was shown that the elec-tric field does not influence the viability of the captured bacteria,which is important if the method is going to be further devel-oped to be implemented in fast,spectroscopy-bad antibiotic susceptibility testing (e Section 4).Instead of dielectrophoresis,centrifugal forces can also be utilid to directly capture bacte-ria from suspension and subquently analy them by means of Raman spectroscopy [26]
.
Fig.4.Visualisation of the workflow for the spectroscopic analysis of urine samples in suspension bad on results in [25].The patient sample arrives in the laboratory and can be placed after a short pre-treatment step onto the dielectrophoresis chip.An alternating electric field is applied on the gold electrodes (depicted in black)and the bacteria experience a force towards the centre (as indicated with yellow arrows in the middle image).The bacteria collect in a cloud in a well-defined region where they are analyd by means of Raman spectroscopy.With the help of a databa,the bacteria can be
identified using the information contained in the Raman spectra.The whole procedure takes ca.35min.(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of the article.)
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Fig.5.(a)Averaged Raman spectra in the fingerprint region of a vancomycin-nsitive strain (Enterococcus faecalis ATCC 29212)and a vancomycin-resistant strain (Entero-coccus faecalis ATCC 51299)at four different time points,both with and without addition of 10g/mL vancomycin.(b)Statistical analysis of the Raman data shown in (a)(top)and for the model of two unknown E.faecium strains (bottom).After 120min,a clear paration of the resistant and nsitive strains can be achieved using the vancomycin score.Whilst the treated nsitive strains show up with positive vancomycin effect score values,the treated resistant strains can be found together with the untreated controls at negative vancomycin effect scores.
Adapted from [28].
4.Spectroscopic characterisation of antibiotic resistance
In times of rising antibiotic resistance,timely characterisation of antibiotic susceptibility is of utmost importance to administer appropriate tailored antibiotic therapy.An increa in the preva-lence of resistant pathogens is en for many antibiotics.In the following,vancomycin-resistant enterococci (VRE)will be ud as a model to demonstrate the power of Raman spectroscopy as a fast method to differentiate nsitive and resistant strains.Currently,the proportion of vancomycin-resistant Enteroco
ccus faecium iso-lates in European hospitals ranges from <1%in Finland and Sweden to 25–50%in Ireland [27].In the USA,VRE are so widespread that vancomycin is almost removed from the guideline recommenda-tions for the treatment of vere enterococcal infections.Whilst established microbiological analysis requires a minimum of 24h and often even veral days owing to time-consuming cultivation steps,a fast Raman-bad method has been developed that can dis-tinguish VRE from vancomycin-nsitive enterococci within only 3.5h [28].The method is bad on the molecular changes that occur within the bacteria owing to the action of the drug and that are detected in the Raman spectra.In vancomycin-nsitive bacteria,the antibiotic binds to the d -alanyl-d -alanine terminus of peptid-oglycan pentapeptide precursors in the nascent cell wall via five hydrogen bonds and by this prevents further cell wall synthesis,finally leading to cell death [29].In the Raman spectra,drug-induced changes can be detected as early as 30min when using antibiotic concentrations above the minimum inhibitory concen-tration (MIC)of the bacteria.The spectral changes can be ud to train a robust classification model that can reliably distinguish treated and untreated bacteria bad on their Raman spectra [30].For vancomycin-resistant bacteria,veral resistance mecha-nisms are known.Most common nowadays are VanA and VanB resistances,which are inducible.This means that initially the drug binds to the growing cell wall.However,unlike in nsitive bacte-ria,this does not lead to cell death but triggers the activation of a cascade of gene clu
sters that ultimately lead to the synthesis of a d -alanyl-d -lactate terminus instead of the previous d -alanyl-d-alanine.Vancomycin shows a reduced binding to this new group
and the bacteria can continue to grow despite the prence of van-comycin.This inducible resistance is also reflected in the Raman spectra (Fig.5a)and can be clearly visualid when analysing the Raman data with partial least squares (PLS)regression and sub-quent linear discriminant analysis (LDA)(Fig.5b)[28].Fig.5a shows the Raman spectra of a nsitive strain (E.faecalis ATCC 29212)and a resistant strain (E.faecalis ATCC 51299)at different time points of interaction with vancomycin as well as the untreated control sam-ples of both strains for comparison.Immediately after vancomycin addition (0min)all Raman spectra appear very similar.At 30min,already the effect of the drug on the bacteria can be detected and the Raman spectra of the treated bacteria show characteristic dif-ferences compared with the untreated controls.However,at that time point,both the nsitive and resistant strains exhibit similar spectral changes.After 60min,the spectral features of the treated resistant strain start to remble tho of the untreated controls,and after 120min differentiation of the resistant and nsitive E.faecalis strains can be achieved with high nsitivity and specificity.This is visualid in Fig.5b where the Raman data are projected into a PLS-LDA model.A positive vancomycin effect score indicates effi-cient binding of vancomycin to the bacteria,whilst the untreate
孕期抑郁
d controls are found at negative vancomycin effect score values.Raman spectra of treated bacteria with an inducible vancomycin resistance and an MIC in the range of the applied drug concen-tration can be found at positive vancomycin effect score values at short vancomycin–bacteria interaction times (30min;Fig.5b,top).However,as time progress,the resistance fully develops and after 120min the Raman spectra of the treated resistant strain can be found at the same negative vancomycin effect score values as the untreated controls,evidencing unobstructed growth of the resistant strain.For a resistant enterococcal strain with an MIC ca.10-fold higher than the administered drug concentration,the effect of an inducible resistance is not so pronounced (Fig.5b,bottom).Notably,the prented Raman-bad algorithm was developed for two E.faecalis strains and also could successfully differentiate a resistant E.faecium strain from a nsitive one,demonstrating the high potential of the method for the analysis of real-world patient’s samples where each patient is likely to carry a different strain [28].
U.Neugebauer et al./International Journal of Antimicrobial Agents46(2015)S35–S39S39
5.Conclusion
Raman spectroscopy is a powerful tool both for drug monitoring at therapeutically significant concent
rations as well as label-free biophotonic characterisation of infections.It yields highly specific molecularfingerprint information from the obrved pathogens enabling differentiation of various species as well as character-isation of antibiotic interactions and identification of resistant bacteria.The spatial resolution when combined with microscopy enables the analysis of single bacterial cells.However,the major advantage for medical diagnosis lies in the short times after which reliable results can be obtained,such as the identification of pathogens from the urinary tract after only35min and information about antibiotic susceptibility after<3.5h.
Funding:Financial support was received from the German Rearch Foundation(DFG)within FOR1738as well as the collabo-rative rearch centre ChemBioSys[SFB1127],from the European Union via the EU project‘HemoSpec’[CN611682],from the Fed-eral Ministry of Education and Rearch(BMBF),Germany,via the Integrated Rearch and Treatment Center‘Center for Sepsis Con-trol and Care’(CSCC)[FKZ01EO1002and FKZ01EO1502],and from the rearch projects RiMaTH[02WRS1276E]Fast Diagnosis [13N11350]as well as funding from the rearch projects Fast-TB [2013FE9057]and BioInter[13022-15]of the Free State of Thuringia and the European Union(EFRE).
Competing interests:None declared.
Ethical approval:Not required.
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