58009915
Diagnosis of Bacterial Pathogens in the Urine of Urinary-Tract-Infection Patients Using Surface-Enhanced Raman Spectroscopy
(1) Background: 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. (2) Methods: 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). (3) Results: 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. (4) Conclusions: SERS can be used in the diagnosis of urinary tract infection with the aid of the recognition software and PCA.
39706914
Multidrug-Resistant Urinary Tract Isolates ofEscherichia coli: Prevalence and Patient Demographics in the United States in 2000
ABSTRACT Concurrent resistance to antimicrobials of different structural classes has arisen in a multitude of bacterial species and may complicate the therapeutic management of infections, including those of the urinary tract. To assess the current breadth of multidrug resistance among urinary isolates of Escherichia coli, the most prevalent pathogen contributing to these infections, all pertinent results in The Surveillance Network Database—USA from 1 January to 30 September 2000 were analyzed. Results were available for 38,835 urinary isolates of E. coli that had been tested against ampicillin, cephalothin, ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. Of these isolates, 7.1% (2,763 of 38,835) were resistant to three or more agents and considered multidrug resistant. Among the multidrug-resistant isolates, 97.8% were resistant to ampicillin, 92.8% were resistant to trimethoprim-sulfamethoxazole, 86.6% were resistant to cephalothin, 38.8% were resistant to ciprofloxacin, and 7.7% were resistant to nitrofurantoin. The predominant phenotype among multidrug-resistant isolates (57.9%; 1,600 of 2,793) included resistance to ampicillin, cephalothin, and trimethoprim-sulfamethoxazole. This was the most common phenotype regardless of patient age, gender, or inpatient-outpatient status and in eight of the nine U.S. Bureau of the Census regions. Rates of multidrug resistance were demonstrated to be higher among males (10.4%) than females (6.6%), among patients >65 years of age (8.7%) than patients ≤17 (6.8%) and 18 to 65 (6.1%) years of age, and among inpatients (7.6%) than outpatients (6.9%). Regionally, the rates ranged from 4.3% in the West North Central region to 9.2% in the West South Central region. Given the current prevalence of multidrug resistance among urinary tract isolates ofE. coli in the United States (7.1%), continued local, regional, and national surveillance is warranted.
26834221
Bacteriuria in representative population samples of persons aged 72-79 years.
Screening for bacteriuria was performed between 1984 and 1988 in persons aged 72-79 years representative of the general population in Göteborg, Sweden. The frequency of bacteriuria (greater than or equal to 10(5)/ml) at a single screening was 6% and 16% at age 72 years and 6% and 14% at age 79 years for the screened men (n = 235 and 259) and women (n = 259 and 297), respectively. By repeated screening after one month and 30 months of those previously negative at age 72 years, an additional 4% and 3% of men and 3% and 7% of women with bacteriuria were detected. Bacteriuric persons were excluded from further screening and controlled by frequent cultures during several years, with careful monitoring of clinical interventions. The persistence of untreated bacteriuria was analyzed in relation to bacterial species and number in the untreated subgroup of bacteriuric individuals. Nine of 10 Escherichia coli (E. coli) with less than 10(6)/ml and 22/22 non-E. coli strains disappeared spontaneously. In contrast, 20/26 (77%, p less than 0.01) with greater than or equal to 10(6) E. coli/ml persisted. Of 17 persons with bacteriuria persisting at least 12 months, 16 were women and 16 had E. coli. Of 201 E. coli cultures obtained from this group, 94% had greater than or equal to 10(6)/ml, and 99% had greater than or equal to 5 x 10(5)/ml. The results indicate that screening for high counts (greater than 10(6)/ml) of E. coli most effectively detects persisting bacteriuria in the general elderly population.
46702756
WIDESPREAD DISTRIBUTION OF URINARY TRACT INFECTIONS CAUSED BY A MULTIDRUG-RESISTANT ESCHERICHIA COLI CLONAL GROUP
A BSTRACT Background The management of urinary tract infections is complicated by the increasing prevalence of antibiotic-resistant strains of Escherichia coli. We studied the clonal composition of E. coli isolates that were resistant to trimethoprim–sulfamethoxazole from women with community-acquired urinary tract infections. Methods Prospectively collected E. coli isolates from women with urinary tract infections in a university community in California were evaluated for antibiotic susceptibility, O:H serotype, DNA fingerprinting, pulsed-field gel electrophoretic pattern, and virulence factors. The prevalence and characteristics of an antibiotic-resistant clone were evaluated in this group of isolates and in those from comparison cohorts in Michigan and Minnesota. Results Fifty-five of the 255 E. coli isolates (22 percent) from the California cohort were resistant to trimethoprim–sulfamethoxazole as well as other antibiotics. There was a common pattern of DNA fingerprinting, suggesting that the isolates belonged to the same clonal group (clonal group A), in 28 of 55 isolates with trimethoprim–sulfamethoxazole resistance (51 percent) and in 2 of 50 randomly selected isolates that were susceptible to trimethoprim–sulfamethoxazole (4 percent, P<0.001). In addition, 11 of 29 resistant isolates (38 percent) from the Michigan cohort and 7 of 18 (39 percent) from the Minnesota cohort belonged to clonal group A. Most of the clonal group A isolates were serotype O11:H(nt) or O77:H(nt), with similar patterns of virulence factors, antibiotic susceptibility, and electrophoretic features. Conclusions In three geographically diverse communities, a single clonal group accounted for nearly half of community-acquired urinary tract infections in women that were caused by E. coli strains with resistance to trimethoprim–sulfamethoxazole. The widespread distribution and high prevalence of E. coli clonal group A have major public health implications. (N Engl J Med 2001;345:1007-13.)
79024721
The laboratory diagnosis of urinary tract infection
Urinary tract infection is common, and it is not surprising that urine specimens make up a large proportion of those samples submitted to the routine diagnostic laboratory. Many of these specimens will show no evidence of infection and several methods can be used to screen out negative samples. Those that grow bacteria need to be carefully assessed to quantify the degree of bacteriuria and hence clinical relevance. To influence treatment, a final report should be produced within 24 hours of specimen receipt, with turnaround times continuously monitored. Much work needs to be done to determine the cost effectiveness involved in processing urine specimens and the evidence base for the final report provided.
45994494
Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
ABSTRACT During the past 5 years, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.
12229134
Sensitive cylindrical SERS substrate array for rapid microanalysis of nucleobases.
In this work, a cylindrical-substrate array for surface-enhanced Raman scattering (SERS) measurements was developed to enable analysis of nucleobases in a few microliters of liquid. To eliminate uncertainties associated with SERS detection of aqueous samples, a new type of cylindrical SERS substrate was designed to confine the aqueous sample at the tip of the SERS probe. Poly(methyl methacrylate) (PMMA) optical fibers in a series of different diameters were used as the basic substrate. A solution of poly(vinylidene fluoride)/dimethylformamide (PVDF/DMF) was used to coat the tip of each fiber to increase the surface roughness and facilitate adsorption of silver nanoparticles (AgNPs) for enhancing Raman signals. A chemical reduction method was used to form AgNPs in and on the PVDF coating layer. The reagents and reaction conditions were systematically examined with the aim of estimating the optimum parameters. Unlike the spreading of aqueous sample on most SERS substrates, particularly flat ones, the new SERS substrates showed enough hydrophobicity to restrict aqueous sample to the tip area, thus enabling quantitative analysis. The required volume of sample could be as low as 1 μL with no need for a drying step in the procedure. By aligning the cylindrical SERS substrates into a solid holder, an array of cylindrical substrates was produced for mass analysis of aqueous samples. This new substrate improves both reproducibility and sensitivity for detection in aqueous samples. The enhancement factor approaches 7 orders in magnitude with a relative standard error close to 8%. Using the optimized conditions, nucleobases of adenine, cytosine, thymine, and uracil could be detected with limits approaching a few hundreds nanomolar in only a few microliters of solution.
36643503
Raman's “Effect” on Molecular Imaging
Raman spectroscopy is an optical technique that offers unsurpassed sensitivity and multiplexing capabilities to the field of molecular imaging. In the past, Raman spectroscopy had predominantly been used as an analytic tool for routine chemical analysis, but more recently, researchers have been able to harness its unique properties for imaging and spectral analysis of molecular interactions in cell populations and preclinical animal models. Additionally, researchers have already begun to translate this optical technique into a novel clinical diagnostic tool using various endoscopic strategies.
33777781
Controlled plasmonic nanostructures for surface-enhanced spectroscopy and sensing.
After its discovery more than 30 years ago, surface-enhanced Raman spectroscopy (SERS) was expected to have major impact as a sensitive analytical technique and tool for fundamental studies of surface species. Unfortunately, the lack of reliable and reproducible fabrication methods limited its applicability. In recent years, SERS has enjoyed a renaissance, and there is renewed interest in both the fundamentals and applications of SERS. New techniques for nanofabrication, the design of substrates that maximize the electromagnetic enhancement, and the discovery of single-molecule SERS are driving the resurgence of this field. This Account highlights our group's recent work on SERS. Initially, we discuss SERS substrates that have shown proven reproducibility, stability, and large field enhancement. These substrates enable many analytical applications, such as anthrax detection, chemical warfare agent stimulant detection, and in vitro and in vivo glucose sensing. We then turn to a detailed study of the wavelength and distance dependence of SERS, which further illustrate predictions obtained from the electromagnetic enhancement mechanism. Last, an isotopic labeling technique applied to the rhodamine 6G (R6G)/silver system serves as an additional proof of the existence of single-molecule SERS and explores the dynamical features of this process. This work, in conjunction with theoretical calculations, allows us to comment on the possible role of charge transfer in the R6G/silver system.
31157493
Diagnosis of bacterial pathogens in the dialysate of peritoneal dialysis patients with peritonitis using surface-enhanced Raman spectroscopy.
BACKGROUND
Bacterial peritonitis is the most common cause of peritoneal dialysis (PD) therapy drop-out. A quick and accurate diagnosis of the bacterial pathogen can reduce the PD drop-out rate. Surface-enhanced Raman spectroscopy (SERS) can rapidly identify bacteria using chips coated with nano-sized metal particles.


METHODS
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.


RESULTS
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.


CONCLUSIONS
SERS techniques can rapidly identify bacterial pathogens in the dialysate of PD peritonitis patients.
16256382
Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers
Rapid bacterial antibiotic susceptibility test (AST) and minimum inhibitory concentration (MIC) measurement are important to help reduce the widespread misuse of antibiotics and alleviate the growing drug-resistance problem. We discovered that, when a susceptible strain of Staphylococcus aureus or Escherichia coli is exposed to an antibiotic, the intensity of specific biomarkers in its surface-enhanced Raman scattering (SERS) spectra drops evidently in two hours. The discovery has been exploited for rapid AST and MIC determination of methicillin-susceptible S. aureus and wild-type E. coli as well as clinical isolates. The results obtained by this SERS-AST method were consistent with that by the standard incubation-based method, indicating its high potential to supplement or replace existing time-consuming methods and help mitigate the challenge of drug resistance in clinical microbiology.
3332015
A novel method for discrimination of beef and horsemeat using Raman spectroscopy.
A new approach, based on the usage of Raman spectroscopy in combination with chemometrics, was developed for the rapid determination of beef adulteration with horsemeat. The data mining process of collected Raman spectra was performed with principal component analysis (PCA). Pure fat samples, extracted from forty-nine meat beef and horsemeat samples, were analysed using the Raman spectroscopy. All meat samples were classified successfully according to their origins. The presence of different concentrations (25%, 50%, 75%, w/w) of horsemeat in beef was also differentiated using the developed model system. This study offers a rapid assay for determination of meat adulteration by discriminating beef and horsemeat with high accuracy, a short analysis time (30s) and no requirement for time-consuming sample preparation procedures.
5252172
Single-molecule surface-enhanced Raman spectroscopy of nonresonant molecules.
Single-molecule surface-enhanced Raman scattering (SERS) detection of nonresonant molecules is demonstrated experimentally using the bianalyte SERS method. To this end, bianalyte SERS is performed at 633 nm excitation using the nonresonant molecule 1,2-di-(4-pyridyl)-ethylene (BPE) in combination with a benzotriazole derivative as a partner. The results are then extended to the even more challenging case of a small nonresonant molecule, adenine, using an isotopically substituted adenine as bianalyte SERS partners. In addition, SERS cross sections of single-molecule events are quantified, thus providing estimates of the enhancement factors needed to see them. It turns out that an enhancement factor on the order of approximately 5 x 10(9) was sufficient for single-molecule detection of BPE, while maximum enhancement factors of approximately 5 x 10(10) were observed in extreme cases. In the case of adenine, single-molecule detection was only possible in the rare cases with enhancement factors of approximately 10(11). This study constitutes a quantitative fundamental test into the lowest detection limits (in terms of differential cross sections) for single-molecule SERS.
137353585
Functionalized arrays of raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood
Detecting bacteria in clinical samples without the time-consuming culture process is most desired for rapid diagnosis. Such a culture-free detection needs to capture and analyze bacteria from a body fluid usually containing complicate constituents. Here we show that vancomycin (Van) coating of a special substrate with arrays of Ag-nanoparticles, which can provide label-free analysis of bacteria via surface enhanced Raman spectroscopy (SERS), leads to 1000 folds increase in its capability to capture bacteria without introducing significant spectral interference. Bacteria spiked in human blood can be concentrated onto a microscopic Van-coated area while blood cells are excluded. Furthermore, A Van-coated substrate provides distinctly different SERS spectra of Van-susceptible and Van-resistant Enterococcus, indicating its potential use for drug-resistance test. Our results represent a critical step towards the creation of SERS-based multifunctional biochips for rapid culture/label-free detection and drug-resistant testing of microorganisms in clinical samples.
206360637
Combined dielectrophoresis-Raman setup for the classification of pathogens recovered from the urinary tract.
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.
22369040
Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis
As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants.
4323103
Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring
SERS spectra excited at 785 nm of the bacteria Chlamydia trahomatis (elementary bodies, EB) and Neisseria gonorrheoae, the causative pathogens for the two most common sexually transmitted diseases (STD), chlamydia and gonorrhea, respectively, are reported. Although both are Gram-negative bacteria, the SERS signatures of C. trachomatis and N. gonorrheoae are completely different. N. gonorrheoae SERS spectra are due to the starvation induced nucleotide metabolites adenine and guanine, and the surface associated co-enzyme nicotinamide adenine dinucleotide and are very similar on Au and Ag although the spectrum appears more rapidly on Ag. The C. trachomatis SERS spectrum is dominated by the vibrational features of cell surface proteins. While features attributable to specific residues and the amide backbone characterize the C. trachomatis spectrum on Ag, the corresponding SERS spectrum on Au substrates displays vibrational characteristics of aggregated proteins. The prospects for the development of a SERS based platform for rapid (<one hour), low-cost bacterial STD diagnostics are promising based on these initial studies. Furthermore, this biomedical application demonstrates the potential for SERS to be a sensitive real time probe of the dynamics of biochemical activity in the cell wall and extracellular regions of microorganisms.
27786171
Nanotools and molecular techniques to rapidly identify and fight bacterial infections.
Reducing the emergence and spread of antibiotic-resistant bacteria is one of the major healthcare issues of our century. In addition to the increased mortality, infections caused by multi-resistant bacteria drastically enhance the healthcare costs, mainly because of the longer duration of illness and treatment. While in the last 20years, bacterial identification has been revolutionized by the introduction of new molecular techniques, the current phenotypic techniques to determine the susceptibilities of common Gram-positive and Gram-negative bacteria require at least two days from collection of clinical samples. Therefore, there is an urgent need for the development of new technologies to determine rapidly drug susceptibility in bacteria and to achieve faster diagnoses. These techniques would also lead to a better understanding of the mechanisms that lead to the insurgence of the resistance, greatly helping the quest for new antibacterial systems and drugs. In this review, we describe some of the tools most currently used in clinical and microbiological research to study bacteria and to address the challenge of infections. We discuss the most interesting advancements in the molecular susceptibility testing systems, with a particular focus on the many applications of the MALDI-TOF MS system. In the field of the phenotypic characterization protocols, we detail some of the most promising semi-automated commercial systems and we focus on some emerging developments in the field of nanomechanical sensors, which constitute a step towards the development of rapid and affordable point-of-care testing devices and techniques. While there is still no innovative technique that is capable of completely substituting for the conventional protocols and clinical practices, many exciting new experimental setups and tools could constitute the basis of the standard testing package of future microbiological tests.
21964491
Label-free highly sensitive detection of proteins in aqueous solutions using surface-enhanced Raman scattering.
We detected concentration-dependent surface-enhanced Raman scattering (SERS) spectra of several label-free proteins (lysozyme, ribonuclease B, avidin, catalase, and hemoglobin) for the first time in aqueous solutions. Acidified sulfate was used as an aggregation agent to induce high electromagnetic enhancement in SERS. Strong SERS spectra of simple and conjugated protein samples could easily be accessed after the pretreatment with the aggregation agent. The detection limits of the proposed method for lysozyme and catalase were as low as 5 microg/mL and 50 ng/mL, respectively. This detection protocol for label-free proteins has combined simplicity, sensitivity, and reproducibility and allows routine qualitative and relatively quantitative detections. Thus, it has great potential in practical high-throughput protein detections.
44849967
Detection of bacteria by surface-enhanced Raman spectroscopy
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
206427811
Extended spectrum beta lactamase (ESBL) producing bacteria urinary tract infections and complex pediatric urology.
AIM OF THE STUDY
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.


METHODS
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.


MAIN RESULTS
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 (ANOVA P=0.1).


CONCLUSION
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.


LEVEL OF EVIDENCE
Level IIc.
11465062
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.
51889837
Risk for subsequent infection and mortality after hospitalization among patients with multidrug-resistant gram-negative bacteria colonization or infection
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.
5953699
Rapid (<5 min) Identification of Pathogen in Human Blood by Electrokinetic Concentration and Surface-Enhanced Raman Spectroscopy
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.
33976438
Isolation and identification of bacteria by means of Raman spectroscopy.
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.
6439350
Increasing local density and purity of molecules/bacteria on a sensing surface from diluted blood using 3D hybrid electrokinetics.
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.
38346184
Microfluidic concentration of bacteria by on-chip electrophoresis.
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.
25094229
Correlation of virulence genes to clinical manifestations and outcome in patients with Streptococcus dysgalactiae subspecies equisimilis bacteremia.
BACKGROUND/PURPOSE
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.


METHODS
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.


RESULTS
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.


CONCLUSION
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.
37830227
Sepsis Pathogen Identification
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.
24028800
Determination of minimum inhibitory concentrations.
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.