Diagnosis of Bacterial Pathogens in the Urine of Urinary-Tract-Infection Patients Using Surface-Enhanced Raman Spectroscopy

surface-enhanced Raman spectroscopy (SERS) is a novel method for bacteria identification. However, reported applications of SERS in clinical diagnosis are limited. In this study, we used cylindrical SERS chips to detect urine pathogens in urinary tract infection (UTI) patients.  Urine samples were retrieved from 108 UTI patients. A 10 mL urine sample was sent to conventional bacterial culture as a reference. Another 10 mL urine sample was loaded on a SERS chip for bacteria identification and antibiotic susceptibility. We concentrated the urine specimen if the intensity of the Raman spectrum required enhancement. The resulting Raman spectrum was analyzed by a recognition software to compare with spectrum-form reference bacteria and was further confirmed by principal component analysis (PCA).  There were 97 samples with single bacteria species identified by conventional urine culture and, among them, 93 can be successfully identified by using SERS without sample concentration. There were four samples that needed concentration for bacteria identification. Antibiotic susceptibility can also be found by SERS. There were seven mixed flora infections found by conventional culture, which can only be identified by the PCA method.  SERS can be used in the diagnosis of urinary tract infection with the aid of the recognition software and PCA.

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Known bacteria were loaded in the SERS-chips and illuminated with laser light to establish a reference Raman spectra library. Dialysate from PD peritonitis patients was concentrated by centrifuge and examined with the same SERS, and the resulting Raman spectra were compared with library spectra for bacteria identification. Principal component analysis was used for further confirmation. The same batches of dialysate were sent to routine culture as a reference bacteria identification method. The results of the 2 identification methods were compared.



A total of 43 paired-samples were sent for study. There were 37 samples with bacteria identified but 6 were culture-negative by the reference method. 31 bacteria were identified in paired-samples by SERS, among which, 29 bacteria were exactly the same as those identified by the reference method. Bacteria not included in the reference library spectra cannot be identified.



SERS techniques can rapidly identify bacterial pathogens in the dialysate of PD peritonitis patients.

title: Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers
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title: Functionalized arrays of raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood
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title: Combined dielectrophoresis-Raman setup for the classification of pathogens recovered from the urinary tract.
abstract: Rapid and effective methods of pathogen identifications are of major interest in clinical microbiological analysis to administer timely tailored antibiotic therapy. Raman spectroscopy as a label-free, culture-independent optical method is suitable to identify even single bacteria. However, the low bacteria concentration in body fluids makes it difficult to detect their characteristic molecular fingerprint directly in suspension. Therefore, in this study, Raman spectroscopy is combined with dielectrophoresis, which enables the direct translational manipulation of bacteria in suspensions with spatial nonuniform electrical fields so as to perform specific Raman spectroscopic characterization. A quadrupole electrode design is used to capture bacteria directly from fluids in well-defined microsized regions. With live/dead fluorescence viability staining, it is verified, that the bacteria survive this procedure for the relevant range of field strengths. The dielectrophoretic enrichment of bacteria allows for obtaining high quality Raman spectra in dilute suspensions with an integration time of only one second. As proof-of-principle study, the setup was tested with Escherichia coli and Enterococcus faecalis, two bacterial strains that are commonly encountered in urinary tract infections. Furthermore, to verify the potential for dealing with real world samples, pathogens from patients' urine have been analyzed. With the additional help of multivariate statistical analysis, a robust classification model could be built and allowed the classification of those two strains within a few minutes. In contrast, the standard microbiological diagnostics are based on very time-consuming cultivation tests. This setup holds the potential to reduce the crucial parameter diagnosis time by orders of magnitude.

title: Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis
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title: Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring
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title: Nanotools and molecular techniques to rapidly identify and fight bacterial infections.
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title: Label-free highly sensitive detection of proteins in aqueous solutions using surface-enhanced Raman scattering.
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title: Detection of bacteria by surface-enhanced Raman spectroscopy
abstract: AbstractThe detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 103 cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D2O are also reported.
 FigureLight scattering apparatus used to detect bacteria

title: Extended spectrum beta lactamase (ESBL) producing bacteria urinary tract infections and complex pediatric urology.
abstract: Extended spectrum beta lactamase (ESBL) producing bacteria are resistant to most beta-lactam antibiotics including third-generation cephalosporins, quinolones and aminoglycosides. This resistance is plasmid-borne and can spread between species. Management of ESBL is challenging in children with recurrent urinary tract infections (UTIs) and complex urological abnormalities. We aim to quantify the risk in children and specifically in urological patients.



Retrospective review of a microbiology database (April 2014 to November 2015). This identified urine isolates, pyuria, ESBL growth and patient demographics. Data analysis was by Chi square, Mann-Whitney U-test and ANOVA. A P value of <0.05 was taken as significant.



Analysis of 9418 urine samples showed 2619 with pure isolates, of which 1577 had pyuria (>10×106 WC/L). 136 urine cultures (n=79 patients) grew purely ESBL. Overall, 5.2% of urine isolates were ESBL and 9.5% isolates with pyuria (>100×106 WC/L) had ESBL, whereas only 22/1032 (2.1%) with no pyuria, (P<0.0001). Urology patients had 86/136 (63%) ESBL positive cultures. These represented 86/315 (27%) of all positive cultures for urology patients vs. 50/2267 (2.2%) for all other specialties (P<0.0001). Potential ESBL transmission between organisms occurred in 3 (all on prophylactic antibiotics). Over the study period, there was no significant rise of the monthly incidence between 2014 and 2015 (.



This study is the first to document the incidence of ESBL in children (5%), and estimate the frequency of possible plasmid transmission between bacterial species in children. This quantifies the risk of ESBL, especially to urology patients, and mandates better antibiotic stewardship.

title: Bacteremic Urinary Tract Infection Caused by Multidrug-Resistant Enterobacteriaceae Are Associated With Severe Sepsis at Admission
abstract: The purpose of this study is to compare the clinical features and treatment outcomes among patients with bacteremic urinary tract infection (UTI) caused by multidrug-resistant (MDR) and non-MDR Enterobacteriaceae and to identify whether MDR pathogens were independently associated with severe sepsis or septic shock at presentation. The clinical data of adult patients visiting and being treated at Chia-Yi Christian Hospital due to bacteremic UTI caused by Enterobacteriaceae from January 2006 to August 2015 were retrospectively analyzed. A total of 585 patients were enrolled. Among them, 220 (37.6%) were caused by the MDR Enterobacteriaceae. A total of 206 patients (35.2%) developed severe sepsis or septic shock at presentation. Patients in the MDR group tend to be male and have a past history of gout, recurrent UTI, prior hospitalization, hydronephrosis, renal stone, ureteral stone, indwelling urinary catheter, newly development of renal dysfunction, severe sepsis or septic shock, intensive care unit (ICU) admission, receipt of ineffective empirical therapy, longer hospital stay, and higher in-hospital mortality (2.7% vs 1.9%, P = 0.569). Using multivariate logistic regression analysis, it is revealed that independent predictors associated with severe sepsis or septic shock at presentation were liver cirrhosis (OR 2.868; 95% CI 1.439–5.716; P = 0.003), indwelling urinary catheter (OR 1.936; 95% CI 1.238–3.027; P = 0.004), and MDR Enterobacteriaceae (OR 1.447; 95% CI 1.002–2.090; P = 0.049). Multidrug resistance was associated with the development of severe sepsis or septic shock upon presentation among patients with bacteremic UTI caused by Enterobacteriaceae. Therefore, empirical antibiotics therapy for patients with UTI presented with severe sepsis and/or septic shock should be more broad-spectrum to effectively cover MDR Enterobacteriaceae.

title: Risk for subsequent infection and mortality after hospitalization among patients with multidrug-resistant gram-negative bacteria colonization or infection
abstract: BackgroundRisks for subsequent multidrug-resistant gram-negative bacteria (MDRGNB) infection and long-term outcome after hospitalization among patients with MDRGNB colonization remain unknown.MethodsThis observational study enrolled 817 patients who were hospitalized in the study hospital in 2009. We defined MDRGNB as a GNB resistant to at least three different antimicrobial classes. Patients were classified into MDRGNB culture-positive (MDRGNB-CP; 125 patients) and culture-negative (MDRGNB-CN; 692 patients) groups based on the presence or absence of any MDRGNB identified from either active surveillance or clinical cultures during index hospitalization. Subsequent MDRGNB infection and mortality within 12 months after index hospitalization were recorded. We determined the frequency and risk factors for subsequent MDRGNB infection and mortality associated with previous MDRGNB culture status.ResultsIn total, 129 patients had at least one subsequent MDRGNB infection (MDRGNB-CP, 48.0%; MDRGNB-CN, 10.0%), and 148 patients died (MDRGNB-CP, 31.2%; MDRGNB-CN, 15.9%) during the follow-up period. MDR Escherichia coli and Acinetobacter baumannii were the predominant colonization microorganisms; patients with Proteus mirabilis and Pseudomonas aeruginosa had the highest hazard risk for developing subsequent infection. After controlling for other confounders, MDRGNB-CP during hospitalization independently predicted subsequent MDRGNB infection (hazard ratio [HR], 5.35; 95% confidence interval [CI], 3.72–7.71), all-cause mortality (HR, 2.42; 95% CI, 1.67–3.50), and subsequent MDRGNB infection-associated mortality (HR, 4.88; 95% CI, 2.79–8.52) after hospitalization.ConclusionsHarboring MDRGNB significantly increases patients’ risk for subsequent MDRGNB infection and mortality after hospitalization, justifying the urgent need for developing effective strategies to prevent and eradicate MDRGNB colonization.

title: Rapid (<5 min) Identification of Pathogen in Human Blood by Electrokinetic Concentration and Surface-Enhanced Raman Spectroscopy
abstract: This study reports a novel microfluidic platform for rapid and long-ranged concentration of rare-pathogen from human blood for subsequent on-chip surface-enhanced Raman spectroscopy (SERS) identification/discrimination of bacteria based on their detected fingerprints. Using a hybrid electrokinetic mechanism, bacteria can be concentrated at the stagnation area on the SERS-active roughened electrode, while blood cells were excluded away from this region at the center of concentric circular electrodes. This electrokinetic approach performs isolation and concentration of bacteria in about three minutes; the density factor is increased approximately a thousand fold in a local area of ~5000 μm2 from a low bacteria concentration of 5 × 103 CFU/ml. Besides, three genera of bacteria, S. aureus, E. coli, and P. aeruginosa that are found in most of the isolated infections in bacteremia were successfully identified in less than one minute on-chip without the use of any antibody/chemical immobilization and reaction processes.

title: Isolation and identification of bacteria by means of Raman spectroscopy.
abstract: Bacterial detection is a highly topical research area, because various fields of application will benefit from the progress being made. Consequently, new and innovative strategies which enable the investigation of complex samples, like body fluids or food stuff, and improvements regarding the limit of detection are of general interest. Within this review the prospects of Raman spectroscopy as a reliable tool for identifying bacteria in complex samples are discussed. The main emphasis of this work is on important aspects of applying Raman spectroscopy for the detection of bacteria like sample preparation and the identification process. Several approaches for a Raman compatible isolation of bacterial cells have been developed and applied to different matrices. Here, an overview of the limitations and possibilities of these methods is provided. Furthermore, the utilization of Raman spectroscopy for diagnostic purposes, food safety and environmental issues is discussed under a critical view.

title: Increasing local density and purity of molecules/bacteria on a sensing surface from diluted blood using 3D hybrid electrokinetics.
abstract: We present a long-range and selective nanocolloid/molecular/bacteria concentrator based on 3D hybrid AC electrokinetics (ACEK) that includes AC dielectrophoresis (DEP) and biased AC electroosmosis (ACEO). Through a convergency comb-shaped electrode design, this long-range ACEO allows the effective transport of a high number of targets into the centre of the detection zone. In the proposed 3D hybrid electrokinetics model, 3D ACEO provides long-range transportation, and the 3D DEP provides an effective separation mechanism. Thus, detection targets ranging from nanoscale to micrometers could be selectively concentrated long-range from diluted blood. The proposed design was used for selectively concentrating nanocolloids and bacteria in the diluted blood sample, respectively. Compared to a 3D short-range dipolar electrode configuration, the detection limit of long-range 3D convergency tripolar electrode configuration is one order of magnitude higher. The result also shows that the 3D hybrid ACEK demonstrated a higher purity of any plane above the electrode, which compared positively to the same design of a 2D hybrid ACEK. The concentration factor of the proposed 3D hybrid electrokinetics device increased by several orders of local density and raised the local purity at least 6 orders (from 0.05% to greater than 99.9%). The chip is capable of making a DNA/protein/bacterial aggregate characterized by high local density and purity for further molecular and bacteria detection/analysis.

title: Microfluidic concentration of bacteria by on-chip electrophoresis.
abstract: In this contribution, we present a system for efficient preconcentration of pathogens without affecting their viability. Development of miniaturized molecular diagnostic kits requires concentration of the sample, molecule extraction, amplification, and detection. In consequence of low analyte concentrations in real-world samples, preconcentration is a critical step within this workflow. Bacteria and viruses exhibit a negative surface charge and thus can be electrophoretically captured from a continuous flow. The concept of phaseguides was applied to define gel membranes, which enable effective and reversible collection of the target species. E. coli of the strains XL1-blue and K12 were used to evaluate the performance of the device. By suppression of the electroosmotic flow both strains were captured with efficiencies of up to 99%. At a continuous flow of 15 μl/min concentration factors of 50.17 ± 2.23 and 47.36 ± 1.72 were achieved in less than 27 min for XL1-blue and K12, respectively. These results indicate that free flow electrophoresis enables efficient concentration of bacteria and the presented device can contribute to rapid analyses of swab-derived samples.

title: Correlation of virulence genes to clinical manifestations and outcome in patients with Streptococcus dysgalactiae subspecies equisimilis bacteremia.
abstract: Streptococcus dysgalactiae subsp. equisimilis (SDSE) is increasingly recognized as a human pathogen responsible for invasive infection and streptococcal toxic shock syndrome (STSS). The pathogen possesses virulence genes that resemble those found in Streptococcus pyogenes (GAS). We analyzed the association between these specific toxic genes, clinical presentations, and outcome in patients with SDSE infections.



Patients (older than 18 years) with community-acquired invasive bacteremia caused by SDSE bacteremia who were undergoing treatment at China Medical University Hospital from June 2007 to December 2010 were included in this study. Multiplex polymerase chain reaction was performed to identify virulence genes of the SDSE isolates. Demographic data, clinical presentations, and outcome in patients with SDSE infections were reviewed and analyzed.



Forty patients with 41 episodes of SDSE bacteremia were reviewed. The median age of the patients with SDSE infection was 69.7 years; 55% were female and 78% had underlying diseases. Malignancy (13, 33%) and diabetes mellitus (13, 33%) were the most common comorbidities. The 30-day mortality rate was 12%. Compared with the survivors, the non-survivors had a higher rate of diabetes mellitus (80% vs. 26%), liver cirrhosis (60% vs.11%), shock (60% vs.17%), STSS (60% vs. 8%), and a high Pittsburgh bacteremia score >4 (40% vs. 6%). Most isolates had scpA, ska, saga, and slo genes, whereas speC, speG, speH, speI, speK, smez, and ssa genes were not detected. speA gene was identified only in one patient with STSS (1/6, 17%). All isolates were susceptible to penicillin, cefotaxime, levofloxacin, moxifloxacin, vancomycin, and linezolid.



In invasive SDSE infections, most isolates carry putative virulence genes, such as scpA, ska, saga, and slo. Clinical SDSE isolates in Taiwan remain susceptible to penicillin cefotaxime, and levofloxacin.

title: Sepsis Pathogen Identification
abstract: Sepsis is a rapidly progressing, severe inflammatory response to infection, causing more than 200 000 deaths per year. Rapid, specific pathogen identification is important to guide sepsis treatment. In this review, we describe and compare currently available commercial products for sepsis diagnosis and pathogen identification, based on microbiological, molecular, and mass spectrometric technologies. Microbiological techniques, the current “gold standard” in sepsis pathogen identification, include blood culture followed by subculturing and pathogen identification via biochemical or microscopic means. These methods have been automated but nevertheless require several days to generate results. Alternative technologies, including highly multiplexed PCR-based methods and mass spectrometric approaches, can decrease the required turnaround time. Matrix-assisted laser-desorption ionization time-of-flight–based systems have recently become an attractive option to rapidly identify a broad spectrum of sepsis pathogens with good sensitivity and specificity. Effectively integrating rapid sepsis pathogen identification into the hospital workflow can improve patient outcomes and can reduce the length of hospitalization and cost per patient.

title: Determination of minimum inhibitory concentrations.
abstract: Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation, and minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media. MICs are used by diagnostic laboratories mainly to confirm resistance, but most often as a research tool to determine the in vitro activity of new antimicrobials, and data from such studies have been used to determine MIC breakpoints. MBC determinations are undertaken less frequently and their major use has been reserved for isolates from the blood of patients with endocarditis. Standardized methods for determining MICs and MBCs are described in this paper. Like all standardized procedures, the method must be adhered to and may not be adapted by the user. The method gives information on the storage of standard antibiotic powder, preparation of stock antibiotic solutions, media, preparation of inocula, incubation conditions, and reading and interpretation of results. Tables giving expected MIC ranges for control NCTC and ATCC strains are also supplied.