Rapid Identification Of Bacteria Research Articles

Rapid identification of bacteria in water by multi-wavelength transmittance spectroscopy and the artificial neural network

Background: Multi-wavelength transmittance spectroscopy, in combination with the artificial neural network, has been a novel tool used to identify and classify microorganisms in recent years.Methods: In our work, the transmittance spectra in the region from 200 to 900 nm for four bacterial species of interest, Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K.pneumoniae), and Salmonella typhimurium (S. typhi), were recorded using an ultraviolet–visible spectrophotometer. Considering too much redundant data on the full-wave band spectra, the characteristic wavelength variables were selected using the competitive adaptive reweighting sampling (CARS) algorithm. Spectra of the initial training set of these targeted microorganisms were used to create identification models representing the spectral variability of each species using four kinds of neural networks, namely, backpropagation (BP), radial basis function network (RBF), generalized regression neural network (GRNN), and probabilistic neural network (PNN).Results: The blinded isolate spectra of targeted species were identified using the four identification models given above. Compared to fullband modeling, after using CARS to screen the wavelength variables, four identification models are established for the 35 preferred characteristic wavelengths, and the prediction performance of the four models is notably improved. Among them, the CARS–PNN model is the best, and the identification rates of all targeted bacteria were achieved with 100% accuracy; the calculation time is just approximately 0.04 s.Discussion: The use of CARS can effectively remove useless information from the spectra, reduce model complexity, and enhance model prediction performance. Multi-wavelength transmission spectroscopy, combined with the CARS–PNN method, can provide a new method for the rapid detection of bacteria in water and could be readily extended for bacterial microbiological detection in blood and food.

Rapid identification of bacteria by the pattern of redox reactions rate using 2′,7′-dichlorodihydrofluorescein diacetate

Bacterial infection is a life-threatening situation, and its rapid diagnosis is essential for treatment. Apart from medical applications, rapid identification of bacteria is vital in the food industry or the public health system. There are various bacterial identification techniques, including molecular-based methods, immunological approaches, and biosensor-based procedures. The most commonly used methods are culture-based methods, which are time-consuming. The objective of this study is to find a fingerprint of bacteria to identify them.Three strains of bacteria were selected, and seven different concentrations of each bacterium were prepared. The bacteria were then treated with two different molar concentrations of the fluorescent fluorophore, dichlorodihydrofluorescein diacetate for 30 minutes. Then, using the fluorescence mode of a multimode reader, the fluorescence emission of each bacterium is scanned twice during 60 minutes. Plotting the difference between two scans versus the bacteria concentration results in a unique fluorescence pattern for each bacterium.Observation of the redox state of bacteria, during 90 minutes, results in a fluorescence pattern that is clearly a fingerprint of different bacteria. This pattern is independent of fluorophore concentration. Mean Squares Errors (MSE) between the fluorescence patterns of similar bacteria is less than that of different bacteria, which shows the method can properly identify the bacteria.In this study, a new label-free method is developed to detect and identify different species of bacteria by measuring the redox activity and using the fluorescence fluorophore, dichlorodihydrofluorescein diacetate. This robust and low-cost method can properly identify the bacteria, uses only one excitation and emission wavelength, and can be simply implemented with current multimode plate readers.

Rapid identification of bacteria using a multiplex polymerase chain reaction system for acute abdominal infections.

Acute abdominal infections can be fatal if the causative organism (s) are misidentified. The spread of antimicrobial-resistant bacteria has become a serious problem worldwide, making antibiotic selection extremely difficult. Using quantitative metagenomic analysis, we evaluated a commercial multiplex polymerase chain reaction (PCR) system (FilmArray™, bioMérieux, Marcy-l'Étoile, France) for the rapid identification of causative bacteria. The cases of 10 patients with acute abdominal infections were enrolled in this retrospective study. There were six cases of perforated peritonitis and four cases of intraabdominal abscess. Fluid collected from the acute surgical abdominal infections were examined. All specimens tested positive for microorganisms in culture, and six involved two or more microorganisms. Using the multiplex PCR system, nine of ten specimens were found to involve at least one microorganism. One specimen was not included in the multiplex PCR system panel. Nineteen of 21 microorganisms (90.5%) isolated by culture were detected by the multiplex PCR system. Microorganisms with very small numbers of reads (19 reads) were detectable. This multiplex PCR system showed a high detection rate for causative microorganisms in ascites and intraabdominal abscesses. This system may be suitable as an affordable rapid identification system for causative bacteria in these cases.

Rapid identification of bacteria from agricultural environment using MALDI-TOF MS

AbstractIn this study, matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) was used to identify bacteria from environmental matrices. The aim of this work was to determine the efficacy of this rapid technique and the bacterial community of agricultural samples. Environmental samples included the collection of irrigation waters and manures, and bacteria from the surface of vegetables were also investigated. From food safety point of view, the investigation of these microbial communities is inevitable considering their potential hazardous impact on the food production chain. Altogether 235 bacterial isolates were identified with the most frequent genera being Pseudomonas, Bacillus, Acinetobacter and Aeromonas. Our results indicated that MALDI-TOF MS can be used to identify causative agents of foodborne illnesses, food spoilage and common plant pathogens. However, limitations of the rapid identification technique were also encountered as we obtained correct identification at species level for 30.2% and at genus level for 69.8% of the isolates.

Sepsityper® Kit versus In-House Method in Rapid Identification of Bacteria from Positive Blood Cultures by MALDI-TOF Mass Spectrometry.

In order to further accelerate pathogen identification from positive blood cultures (BC), various sample preparation protocols to identify bacteria with MALDI-TOF MS directly from positive BCs have been developed. We evaluated an in-house method in comparison to the Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) as well as the benefit of an on-plate formic acid extraction step following positive signal by the BACTECTM FX system. Confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 113 monomicrobial positive BCs were analyzed. The rates of Gram-positive bacteria correctly identified to the genus level using in-house method and Sepsityper® Kit were 63.3% (38/60) and 81.7% (49/60), respectively (p = 0.025). Identification rates at species level for Gram-positive bacteria with in-house method and Sepsityper® kit were 30.0% (18/60) and 66.7% (40/60), respectively (p < 0.001). Identification rates of Gram-negative bacteria were similar with the in-house method and Sepsityper® Kit. Additional on-plate formic acid extraction demonstrated significant improvement in the identification rate of Gram-positive bacteria at both genus and species level for both in-house (p = 0.001, p < 0.001) and Sepsityper® Kit methods (p = 0.007, p < 0.001). Our in-house method is a candidate for laboratory routines with Sepsityper® Kit as a back-up solution when identification of Gram-positive bacteria is unsuccessful.

Performance of a Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Testing Algorithm for the Rapid Identification of Clinical Filamentous Molds.

One of the most significant challenges in the treatment of fungal infections is the relatively long turnaround time (TAT) required for fungal species identification. The length of TAT to identification can impact patient clinical outcomes by delaying appropriate targeted therapy. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has demonstrated exceptional utility in the rapid identification of bacteria and yeasts in the clinical microbiology laboratory. The capability of MALDI-TOF MS for rapid identification of clinical isolates presents an opportunity for significant advancement in the identification of filamentous molds. In this study, we employed a diagnostic algorithm using MALDI-TOF MS for the rapid identification of filamentous molds in order to assess the impact of this technology on TATs. The majority of isolates included in this study were able to be identified by MALDI-TOF MS (78%). Further, these isolates were identified in less than three days from first detection of colony growth. This study demonstrates the utility of MALDI-TOF MS in the rapid identification of filamentous molds in the clinical mycology laboratory.

A Review of Next Generation Sequencing Methods and its Applications in Laboratory Diagnosis

Next-generation sequencing (NGS) is a new technology used to detect the sequence of DNA and RNA and to detect mutations or variations of significance. NGS generates large quantities of sequence data within a short time duration. The various types of sequencing includes Sanger Sequencing, Pyrosequencing, Sequencing by Synthesis (Illumina), Ligation (SoLID), Single molecule Fluorescent Sequencing (Helicos), Single molecule Real time Sequencing (Pacbio), Semiconductor sequencing (Ion torrent technology), Nanopore sequencing and fourth generation sequencing. These methods of sequencing have been modified and improved over the years such that it has become cost effective and accessible to diagnostic laboratories. Management of Outbreaks, rapid identification of bacteria, molecular case finding, taxonomy, detection of the zoonotic agents and guiding prevention strategies in HIV outbreaks are just a few of the many applications of Next Generation sequencing in clinical microbiology.

Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review

Open AccessCCS ChemistryMINI REVIEW1 Feb 2022Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review Yekkuni L. Balachandran and Xingyu Jiang Yekkuni L. Balachandran Shenzhen Key Laboratory of Smart Healthcare Engineering, Department of Biomedical Engineering, Southern University of Science and Technology, Nanshan District, Shenzhen, Guangdong 518055 Google Scholar More articles by this author and Xingyu Jiang *Corresponding author: E-mail Address: [email protected] Shenzhen Key Laboratory of Smart Healthcare Engineering, Department of Biomedical Engineering, Southern University of Science and Technology, Nanshan District, Shenzhen, Guangdong 518055 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101307 SectionsAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Aggregation-induced emission (AIE) fluorogens produce strong, stable fluorescence upon aggregation, thus enabling a new frontier in bio-detection, cell imaging, and other biomedical applications. In this review, we summarize the recent developments of AIE fluorogen design strategy, bio-detection, cell imaging, tumor imaging, vascular imaging, and image-guided tumor therapy. We highlight a section on the microfluidic approach to synthesize AIE nanoparticles (NPs) and current challenges and potentials of AIE NPs. Download figure Download PowerPoint Introduction Noninvasive fluorescence imaging in biomedicine has undergone rapid growth to obtain dynamic information about living cells or organisms.1,2 Fluorescence imaging provides real-time visualization of live cells, increases image sensitivity, improves temporal–spatial resolution, and so forth.3,4 Several well-defined fluorophores of various origins such as organic molecules, quantum dots (QDs), up-conversion nanoparticles (NPs), and fluorescent proteins are synthesized to emit superior and stable fluorescence.5,6 Among them, organic fluorophores are the most studied because of their strong fluorescence, simple preparation method, and satisfactory biocompatibility.7 However, most organic fluorophores suffer from poor solubility in physiological solutions by forming small aggregates or clusters,8,9 which cause partial or complete aggregation-caused quenching (ACQ) due to the π–π stacking.10,11 The ACQ effects restrict fluorophore transformation into NPs because increasing the doping number can provoke fluorescence quenching. Moreover, the fluorophores for cell imaging should be cautiously used (optimum AIEgen concentration) to minimize the intracellular aggregation to prevent compromising fluorescence.12 Furthermore, using small quantities of organic fluorophores produces unsatisfactory fluorescence, poor photostability, and photobleaching.13 Aggregation-induced emission (AIE) fluorogens are a new class of fluorogens that produce strong and stable fluorescence in the aggregation state. AIE fluorophores are nonemissive as single molecules but produce strong fluorescence in an “abnormal” aggregate or cluster state.14,15 The rotor structure of the AIE fluorogens (AIEgens) activates the nonradiative decay pathway to restrict the intermolecular motion to yield bright and stable emissions in the aggregated state.16 Furthermore, the nonplanar conformation of AIEgens prevents π–π stacking, causing strong emission in the aggregate or solid state.17 The AIEgens exhibits a high signal-to-noise ratio (10–40 times brighter than the organic fluorescent molecules), high sensitivity, increased quantum efficiency, anti-photobleaching capability, good photophysical stability, and excellent cytocompatibility.14,15 Tuning the emission characteristics is crucial for fluorescence-based applications. AIEgens produce emission in the visible spectral range,18 and further deep emission in the near-infrared (NIR) window I (700–1000 nm)19,20 and NIR-II window (1000–1500 nm)21,22 to provide high-quality images, deep penetration, and limitation of the photon absorption and scattering. Recently, AIEgens capable of two-photon fluorescence have been explored considerably due to their advantages of longer excitation wavelength, minimal autofluorescence, poor photobleaching, and higher three-dimensional (3D) resolution with deep tissue imaging.23,24 The AIEgens are used in diverse biological applications to detect various biomolecules (amino acids, carbohydrates, DNA or RNA, and proteins or enzymes),25,26 monitor biological processes (protein fibrillation, cell apoptosis, osteogenic differentiation, autophagy or mitophagy, and bacterial labeling and ablation),27 and label cellular organelle.14,15 Due to their exceptional optical properties, AIEgens serve as potential materials of choice for tumor imaging applications.28 Furthermore, the photosensitization and photothermal conversion efficiency of AIEgens broadens the application in tumor diagnosis and image-guided therapy29 such as photodynamic therapy (PDT), photothermal therapy (PTT), and photoacoustic (PA) imaging.30 Herein, we summarize the AIEgens and advances in their biological applications. First, we discuss the design and synthesis of AIE probes and then focus on the advances in applications, such as detection, imaging, and image-guided therapeutics. Next, we focus on the microfluidics approach to prepare AIEgens. We briefly discuss AIEgen fabrication by microfluidics technology and different microfluidic chips (straight and spiral) to prepare various AIEgen compounds. The microfluidic technique manipulates the reaction in a micrometer scale to continuously produce uniform sized nanomaterials in a straightforward approach without laborious postsynthetic processing steps.31–33 The microfluidic technique enables tasks such as generating size-controlled nanomaterials,31 fabricating core–shell materials,34 encapsulating drug molecules,35 and functionalizing the nanomaterial with ligand molecules.35,36 Furthermore, the microfluidic technique promotes continuous large-scale production of AIE nanomaterials for clinical translation and applications. Finally, we discuss the prospective possibilities of AIE platforms for clinical translation and different biomedical applications. AIEgens Phenomenon and Design Strategies Of the various theories proposed for AIE,37,38 the restriction of intramolecular motion (RIM) is a widely accepted phenomenon.14,37,39,40 The RIM mechanism combines restriction of intramolecular rotation (RIR)40 and restriction of intramolecular vibration (RIV) theories (Scheme 1).41 The AIE single molecules are unrestricted and rotate (Scheme 1a) and vibrate freely (Scheme 1b) to activate the nonradiative decay, thereby becoming nonemissive.38 However, when aggregated, the intramolecular rotation and vibrations are restricted, blocking the nonradiative decay channels, thus enabling the radiative decay processes to induce the emission.38 Scheme 1 | RIM phenomenon of AIE emission. (a) The RIR theory proposes, when the AIEgen is in a single molecular state, the intermolecular rotation (e.g., TPE) activates the nonradiative decay pathway resulting in no fluorescence. However, when aggregated, the restricted intramolecular rotation activates radiative decay in excited states to initiate the fluorescence. (b) The RIV theory proposes the free intramolecular vibration of the AIEgens [e.g., 10,10′,11,11′-tetrahydro-5,5′-bidibenzo[a,d][7]-annulenylidene (THBA)] enables the nonradiative decay of the excited state, resulting in no fluorescence, and upon aggregation, the vibration is completely halted, which initiates fluorescence via the radiative decay process. Comprehensively, the AIE phenomena RIR and RIV merge into the RIM theory (RIM = RIR + RIV). Reproduced with permission from ref 38. Copyright 2018 ACS. Download figure Download PowerPoint The excitation and emission of AIEgens are influenced by the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy gaps of the ground and excited state.42,43 Reducing the HOMO–LUMO gap by elevating the HOMO and lowering the LUMO energy level by expanding the π-conjugation length can bring about the emission.42,43 Constructing the low bandgap AIEgens by conjugating the electron donor (D) (to elevate HOMO level) and acceptor (A) (to lower the LUMO level) produces AIE with negligible ACQ.42,43 By enhancing the D–A conjugation, several AIEgens are constructed. Sometimes the electron donation and acceptance characteristics are improved by inserting spacer or bridging molecules or modifying the D or A molecule.38,39 Scheme 2 shows examples of D–A strengthened AIE probes. Acceptor core units such as benzothiazole ( 1),44 cyano ( 2),45 and 9,10-anthraquinone ( 3)46 are conjugated with different electron donor units to lower the bandgap to realize the AIE. Scheme 2 | AIEgen molecular probes. (1) Benzothiazole, (2) cyano, and (3) 9,10-anthraquinone acceptor centric AIEgens are conjugated with AIE moieties to obtain AIE emission. The TPE central fluorogens such as (4) anthraquinone, (5) benzoselenodiazole, and (6) hemicyanine dye molecule impart the needed stable emission. ACQ converted AIEgens formed by conjugating TPE with IR-780: (7) and (8). Download figure Download PowerPoint In contrast, the electron donor core probe units are conjugated with different acceptor molecules to afford a series of new AIEgens, whose spectra range from visible to infrared.18 Especially, the tetraphenylethene (TPE) AIEgens exhibit excellent AIE characteristics.18 Introducing the anthraquinone acceptor into the TPE produced the emission in the infrared region ( 4).19 The conjugation of TPE with the benzoselenodiazole dye induces the AIE emission by taking advantage of the polarization effect of selenium and a strong D–π–A electronic property ( 5).20 The HOMO and LUMO electron density of the TPE-hemicyanine molecules have charge-transfer characteristics with a low bandgap to shift the optical emission ( 6).18 Occasionally, changing the ACQ to AIE has been realized for stable emission.47 The red emissive fluorophores are converted from ACQ to AIE by conjugating with AIE molecules to induce AIE emission. Introducing the TPE with the IR-780 core dye exhibits a typical D–A molecular structure favoring “off–on” fluorescence ( 7 and 8).47 The design strategies are further improved to fine-tune the emission to an NIR region43,48 and enable two-photon imaging.23,49 Because of low photon scattering and phototoxicity, negligible autofluorescence,22,23,50 and deep tissue penetration, the NIR and two-photon capable AIEgens effectively improve biomedical applications.47,48,50,51 The NIR capable AIE molecules are constructed by improving the D–A–D effect by installing substituents with strong electron donors and acceptors to elevate the HOMO level and lower the LUMO level.43 Furthermore, the quantum efficiency of the molecules are boosted by careful design strategies. As the emission wavelength shifts to the red region, the photoluminescence quantum yield (PLQY) decreases due to vibrational overlap of the ground and excited states. Suppressing the twisted intramolecular charge transfer (TICT) and simultaneously increasing the radiative decay and stimulating the AIE feature by preventing close intermolecular interactions cause higher fluorescence quantum efficiency.43 Two-photon capable materials are characterized by the fluorogens possessing a large two-photon absorption cross-section (σ2PA).52–54 The intramolecular charge transfer (ICT) between the terminal donor group and the π-bridge molecule correlates with the cross-section (δ) of the fluorogen, and the magnitude of δ is influenced by the ICT of the molecular structure.52,54 Modification of the molecular structure through extending the conjugation length of the π-system increases the δ of the fluorogens to enable two-photon characteristics.52,55 Therefore, the improvement of σ2PA relies on the strong electron donor and acceptor groups as well as the length of the π-bridge. The NIR building block, benzo[1,2-c:4,5-c′]bis([1,2,5] thiadiazole) (BBTD)56,57 acceptor molecule is widely employed to design NIR AIEgens. By either modifying the acceptor or donor unit, the emission wavelength can be tuned to the NIR range. For example, replacing the sulfur group in the BBTD acceptor unit with selenium shifts the emission maxima from 810 ( 9) to 897 nm ( 11).51 On the other side, strengthening the electron-donor molecules (triphenylamine, thiophene, and alkoxy chains) lowers the bandgap to achieve NIR emission.58 Introducing a strong electron-donating moiety, for example, N,N-diphenyl-4-(1,2,2-triphenylvinyl)aniline and AIE unit (TPE) with the central BBTD core, yields emission ca. 975 nm ( 11).58 Furthermore, inserting a spacer between the central BBTD and modifying the TPE with diethylamine units contributes to electron-donating capability to further shift the emission to 1034 nm ( 12).59 Scheme 3 shows the examples of NIR AIEgens ( 13 and 14)21,60 developed by substituting the donor and acceptor molecules. Similarly, by rigidifying the D–A geometry, various two-photon capable AIEgens were developed with strong and stable fluorescence ( 15 and 16).24,61 Scheme 3 | Structure of NIR and two-photon capable AIEgens. Replacing the sulfur of electron acceptor BBTD (9) with selenium (10) shifts emission to the NIR-II region. Substitution on the electron donor (AIE molecule) (11 and 12) shifts the absorption-maximum to the NIR-II region. When the electron acceptor and donor are modified, the emission-maximum shifts toward the NIR-II region (13 and 14) and enables two-photon capabilities (15 and 16). Download figure Download PowerPoint Applications of AIEgens The distinctive luminescent features of AIE bio-probes have become essential in biomedicine applications.15,16,27,43 The characteristics of AIEgens have been improved for broader biomedical applications.16,43 For example, understanding the mechanism and optimizing the design strategy improves the fluorescence (bio-imaging), photosensitization, and photothermal characteristics of AIEgens.15,16 Furthermore, introducing responsiveness to external stimuli broadens the scope and specificity of AIEgens in biomedical applications.62,63 In particular, an AIEgen response to the external pH is beneficial for tumor applications because of the difference in the tumor microenvironment pH (pH 6.5–5.5).64 The pH-responsive AIEgen platform (such as drug molecules grafted onto pH-responsive polymers with AIE characteristics65,66) induces fluorescence imaging capabilities and delivers therapeutic molecules to eliminate the tumor upon a trigger by the acidic tumor environment.67,68 Such pH-responsive AIE theranostic systems specifically tag the tumor and deliver drug molecules, thereby avoiding damage to the surrounding normal cells.69 Because of the advancements in bioimaging and image-guided theranostic applications, the characteristics of AIEgens are integrated for multifunction biomedical applications.16,43 In the following section, we summarize the major properties of AIEgens in bio-detection, imaging (cellular, vascular, and tumoral) and image-guided therapy. AIEgens Application in Bio-Detection Fluorescent AIE molecular probes have become important as detection sensors due to their noninvasive and real-time visualization features. AIEgens are used to monitor the biomolecules of interest in solutions or more complex biological cell and tissue environments.70,71 Our group has developed various AIE probes for the rapid and efficient identification of biomolecules [e.g., casein and hydrogen sulfide (H2S)]25,26 and bacteria.27 We have developed the TPE-based AIE probe, sodium 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene (BSPOTPE), for detecting casein in milk to evaluate the quality of milk powder (Figure 1a).25 The hydrophobic caves in the casein micelle captures BSPOTPE molecules and the interaction induces BSPOTPE aggregation, thereby enabling fluorescence enhancement by AIE (Figures 1a and 1b). Figure 1 | AIEgen enabled bio-detection. (a) Casein detection using AIE BSPOTPE probe. (b) Confocal image of casein micelles and casein micelle–BSPOTPE complex inducing fluorescence. (c) TPE-indo AIE probe detection of H2S. The nucleophilic addition between TPE-indo and H2S weakens the aggregation and fluorescence. (d) In vivo detection of H2S in HeLa, HUVEC, and MCF-7 cells using TPE-indo AIEgens. (d1) Cells incubated with TPE-indo for 30 min, (d2) cells incubated with NaHS, (d3) cells incubated with NaHS for 60 min, and (d4) bright-field images of the same field of cells. Reproduced with permission from ref 25. Copyright Royal Society of Chemistry 2012; Reproduced with permission from ref 26. Copyright Springer Nature 2015. Download figure Download PowerPoint Our group developed TPE-indolium (TPE-indo) AIEgens to detect H2S in live cells (Figures 1c and 1d).26 H2S is recognized as the third endogenous gasotransmitter, and abnormal levels of H2S are linked to various physiologic effects in health.72,73 H2S has been reported to regulate blood pressure, inflammation, neurodegenerative diseases, and metabolic disorders, including obesity and diabetes.26,72,73 The H2S in the solution and mitochondria of the cells is monitored using TPE-indo AIEgens comprised of the florescent TPE moiety (fluorogen unit) and a H2S reactive positive-charged indolium moiety.26 The TPE-indo interacts with H2S via a specific nucleophilic reaction with CN− on the indolium group. The aggregated TPE-indo probe changes drastically upon interaction with HS−, thereby weakening aggregation and influencing the fluorescence (Figure 1c). With the fluorescence quenching of TPE-indo NPs, the effective visual detection of H2S in the solution and cells is achieved (Figure 1d). Rapid identification of bacteria has become increasingly important in clinical diagnosis and the food industry to monitor the outbreaks of pathogens, trends in infection, and contamination in food products.74,75 Especially in clinical diagnosis, rapid and sensitive detection of bacteria provide evidence of disease origin and guidance for antibiotic treatment. Furthermore, differentiating the clinical drug resistant bacteria gives an advantage for treating the pathogen with a suitable antibiotic. Our group has developed a rapid AIE-based assay to detect and differentiate multidrug-resistant bacteria from normal bacteria (Figure 2).27 Derivatives of TPE AIEgens with different functional groups (Figure 2a) selectively identify eight different kinds of bacteria, including multidrug-resistant bacteria such as methicillin-resistant Staphyloccocus aureus (MRSA), Staphylococcus epidermidis, and Bacillus subtilis; and multidrug-resistant (MDR) Escherichia coli, Escherichia coli, Klebsiella Pneumonia, and Salmonella choleraesuis. The mixing of bacteria and AIE probes differentiates the bacteria simultaneously by the fluorescent intensity data in automated flow cytometry without any washing step. The TPE AIEgens interact with bacterial protoplasm to form aggregates that produce fluorescence, and such fluorescence was unique for each bacteria (Figure 2b). Figure 2 | Detection and differentiation of bacteria. (a) AIEgen probes used for differentiating various bacteria. (b) The design of a sensor array based on AIE probes to differentiate bacteria, in which A1–A5 denote five different AIE probes and B1–B8 represent eight different types of bacteria. Reproduced with permission from ref 27. Copyright 2014 Wiley-VCH GmbH. Download figure Download PowerPoint Tumor Cell and Mitochondria Imaging Real-time imaging of cellular activity is crucial for precisely assessing cell health, which forms the basic structural and functional unit of any organism.37 The function of cellular organelles is important for the diverse biochemical processes and physiological pathways, and studying the abnormities of cellular organelles is vital because the organelles serve as an important biomarker for cell and tissue status and many diseases.76 NIR AIE probes are designed to target specifically the cellular membrane, mitochondria, lysozyme, and nucleus to induce fluorescence.77 Furthermore, the wash-free capability of AIEgens allows cell visualization for a longer duration with extended photostability and antibleaching.78 Of all cellular organelles, the mitochondria has been extensively studied because abnormalities in mitochondria are closely related to the cell and tissue functions and homeostasis.79,80 Dysfunction in mitochondrial morphology is detected in various human pathologies, such as cancer, neurodegeneration, ageing, diabetes, obesity, and cardiomyopathies.79,80 In tumorigenesis, the mitochondria are involved in alteration of fuel utilization, cell death susceptibility, oxidative stress, and allows survival during starvation and resistance toward treatments during chemotherapeutic or targeted cancer treatments.81,82 Furthermore, dysfunction in the mitochondria-regulated apoptosis pathway accelerates tumorigenesis.82 Understanding the mitochondria effectively favors in designing of novel drug molecules to improve cancer therapeutics.83 Mitochondrial imaging is primarily performed using commercial, small organic fluorescent molecules, and such organic fluorophores have limitations including poor cellular uptake, the ACQ effect, and low photostability. The use of AIEgens overcomes these limitations to provide excellent fluorescence and better photostability. Our group has designed a series of one or two-photon multicolor fluorescent AIEgens to target and image mitochondria.61 TPE-based nitrogen (N)-containing heterocycles are designed and synthesized by coupling pyridine (Py), quinoline (Quino), or acridine (Acr) (Figure 3a). The emission shifts toward the longer region in accordance with the number of the phenyl groups in the heterocyclic conjugates (Figure 3b). Because of cationic moieties, TPE AIEgens can aggregate into nanosized dots of 20–40 nm without any need for polymer or cell-penetrating peptide encapsulation (Figure 3c). The TPE-dots possess both one- and two-photon fluorescent capabilities. The two-photon fluorescence upon NIR excitation enables low photobleaching, low background signal, and low phototoxicity and emits light only when it is in the focus position in bioimaging. Furthermore, TPE-dots are wash-free probes78 favoring long-duration imaging of live cells and making the staining process more convenient and economical. The TPE-dots produced strong fluorescence from the mitochondrial regions of HeLa cells (Figure 3d). The MitoTracker Red verified the fluorescence signal of mitochondria, and co-staining experiments showed perfect overlap between TPE-dot and MitoTracker Red FM (Figure 3d). Furthermore, the TPE-dots spontaneously produced both one- and two-photon fluorescence (Figure 3e). The TPE-dots targeting mitochondria are not limited to HeLa cells; the mitochondria in cell types such as HUVEC (HUVEC = human umbilical vein endothelial cells) and NIH 3T3 (NIH 3T3 = National Institutes of Health Swiss mouse embryo fibroblasts/3-day transfer, inoculum 3 × 105 cells) are also efficiently targeted by TPE-dots, indicating the capability of imaging mitochondria in vast cell types. Figure 3 | TPE-dots targeting mitochondria. (a) Chemical structure of TPE-Py, TPE-Quino and TPE-Acr. (b1) Absorbance and (b2) emission spectra of TPE molecules. (c) TEM image of TPE-Acr, TPE-Py, and TPE-Quino dots. (d) One-photon image of HeLa cells after incubation with TPE-dots or MitoTracker red. TPE-dots and MitoTracker red merged images. (e) One- and two-photon fluorescence images of HeLa cells tracking mitochondria. Reproduced with permission from ref 61. Copyright Royal Society of Chemistry 2017. Download figure Download PowerPoint In Vivo Tumor and Tumor Vascular Imaging of AIEgens Effective real-time monitoring of in vivo tumor is necessary to evaluate and intervene with a therapeutic plan.84 Fluorescence imaging, being an indispensable and versatile tool, allows highly sensitive, non-invasive imaging in real-time and for long-term diagnosis.24,57,85 Short-wavelength light is easily absorbed by water in biological tissues, affording undesirable shallow depth bioimaging, and influenced by Rayleigh scattering effect. In contrast, imaging in the NIR range produces low scattering, deeper penetration depth, high fluorescence quantum yield, and reduced phototoxicity.19,21,22,50 AIEgens with NIR emission distinctly portray the tumor mass over the surrounding normal tissues with high sensitivity, deep tissue penetration, and enhanced contrast resolution. Despite the efforts made, off-targeting is of great concern preventing the needed accumulation of AIE NPs for the contrast-enhanced image of the tumor mass. We have recently developed an AIE QD with a NIR-II system that targets tumor in higher numbers evading the liver.31 The NIR-II AIEgen 4,7-(bis4-(4-octylthiophen-2-yl)-N,N-diphenylaniline)benzo [1,2-c:4,5-c′]bis ([1,2,5]thiadiazole) (TTB) (Figure 4a) is made into quantum-sized dots using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG2k) as a surfactant. The transmission electron microscopy (TEM) determined core size of TTB QDs measured <10 nm (Figure 4b). Due to the quantum size, the TTB QDs can internalize more efficiently in cells without any modification with membrane penetrating peptides. The TTB-exposed in vivo tumor produced a much brighter NIR-II fluorescence (Figure 4c). Polyethylene glycol (PEG) coating of TTB QD extends the circulation time in blood86 and, hence, the possibility of higher tumor targeting for bright NIR-II fluorescence. To calculate the off-targeting efficacy, two different sizes of TTB NPs were compared. The TTB QD evaded liver accumulation more than the larger sized 25 nm TTB dots (>25 nm) (Figure 4c).31,87 The tumor and liver mean NIR-II fluorescence intensity ratio from TTB QDs-treated mice was two to three times that of TTB dots (Figure 4d). Furthermore, conjugating with trans-activator of transcription (TAT) membrane penetrating peptide (RKKRRQRRRC) failed to improve tumor accumulation (Figures 4e and 4f). Together, TTB QD improved tumor accumulation to produce brighter NIR-II fluorescence and reduced the off-target accumulation in the liver more than the bigger sized dots and TAT surface-modified TTB QD. Figure 4 | TTB-QDs NIR-II fluorescence imaging of tumor. (a) TTB NIR-II AIE molecule and DSPE-PEG capped TTB QDs. (b) TEM image of TTB QDs. (c) Left side and ventral views of NIR-II tumor (green circle) and liver (yellow ellipse) fluorescent image acquired at different time intervals after exposure to TTB QDs and TTB dots. (d) The tumor (T) to liver (L) mean ratio of fluorescence intensity. (e) The difference in NIR-II fluorescence of TAT capped and uncapped TTB QDs or TTB dots exposed tumor. The yellow line outlines the mice body, and the green circle highlights tumor. (f) NIR-II fluorescence intensity of the tumor treated with TTB QDs or TTB dots with and without conjugation of TAT peptide. Reproduced with permission from ref 31. Copyright 2020 Wiley-VCH GmbH. Download figure Download PowerPoint Vascular imaging in tumors has gained as much consideration as solid tumor imaging.88 In general, the vascular networks play an important role in regulating the delivery of nutrients and oxygen; meanwhile, the tumor vascular network allows the cancer cells to disseminate and influences the response of the tumor cells to therapy.89 Tumor vascular networks are typically sinusoidal, exhibit discontinuous basement membranes, and lack tight endothelial cell junctions making them highly permeable.89 Imaging the tumor vasculature heterogeneity is indispensable to understand the tumor angiogenesis mechanism that is strongly affected by the microenvironment of cancer tissues.89 The NIR AIEgens, due to their deep tissue penetration, provide a superior image of the blood vessels and branching structures than the other imaging modes.90 Our group designed lipid AIE NPs to image tumor vasculature at deeper regions.24 The two-phot

Rapid identification of bacteria from respiratory samples of patients hospitalized in intensive care units, with FilmArray Pneumonia Panel Plus

ObjectivesThis study aimed to evaluate the performance of FilmArray Pneumonia Panel Plus (FA-PP) for the detection of typical bacterial pathogens in respiratory samples from patients hospitalized in intensive care units (ICUs). MethodsFA-PP was implemented for clinical use in the microbiology laboratory in March 2020. A retrospective analysis on a consecutive cohort of adult patients hospitalized in ICUs between March 2020 and May 2020 was undertaken. The respiratory samples included sputum, blind bronchoalveolar lavage (BBAL) and protected specimen brush (PSB). Conventional culture and FA-PP were performed in parallel. ResultsIn total, 147 samples from 92 patients were analysed; 88% had coronavirus disease 2019 (COVID-19). At least one pathogen was detected in 46% (68/147) of samples by FA-PP and 39% (57/147) of samples by culture. The overall percentage agreement between FA-PP and culture results was 98% (93–100%). Bacteria with semi-quantitative FA-PP results ≥105 copies/mL for PSB samples, ≥106 copies/mL for BBAL samples and ≥107 copies/mL for sputum samples reached clinically significant thresholds for growth in 90%, 100% and 91% of cultures, respectively. FA-PP detected resistance markers, including mecA/C, blaCTX-M and blaVIM. The median turnaround time was significantly shorter for FA-PP than for culture. ConclusionsFA-PP may constitute a faster approach to the diagnosis of bacterial pneumonia in patients hospitalized in ICUs.

Leveraging copper import by yersiniabactin siderophore system for targeted PET imaging of bacteria.

There is an emerging need for accurate and rapid identification of bacteria in the human body to achieve diverse biomedical objectives. Copper homeostasis is vital for the survival of bacterial species owing to the roles of the metal as a nutrient, respiratory enzyme cofactor, and a toxin. Here, we report the development of a copper-64–labeled bacterial metal chelator, yersiniabactin, to exploit a highly conserved metal acquisition pathway for noninvasive and selective imaging of bacteria. Compared with traditional techniques used to manufacture probes, our strategy simplifies the process considerably by combining the function of metal attachment and cell recognition to the same molecule. We demonstrate, for the first time to our knowledge, how a copper-64 PET probe can be used to identify specific bacterial populations, monitor antibiotic treatment outcomes, and track bacteria in diverse niches in vivo.

Detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in Culture Negative Cerebrospinal Fluid Samples from Meningitis Patients Using a Multiplex Polymerase Chain Reaction in Nepal.

The rapid identification of bacteria causing meningitis is crucial as delays in the treatment increase mortality rate. Though considered as the gold standard for the laboratory diagnosis of bacterial meningitis, culture might give false negative results in a case of patients under antibiotics prior to lumbar puncture. This study aimed to detect Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by a multiplex polymerase chain reaction (PCR) in culture-negative cerebrospinal fluid samples collected from clinically suspected meningitis cases attending different hospitals in Kathmandu, Nepal from January 2017 to December 2019. S. pneumoniae, N. meningitidis and H. influenzae were detected in 8.59% (33/384) of the specimens by PCR and 7.55% (29/384) of the specimens by culture. Correlation between culture and PCR of the same sample was good (Spearman’s rho correlation coefficient = 0.932). However, the difference in positivity between culture and PCR was statistically not significant (p value > 0.05). In four specimens, culture could not detect any of the targeted bacteria whereas PCR could detect presence of H. influenzae. PCR increases the diagnostic yield for bacterial meningitis. PCR may be considered as an adjunctive test for establishing the cause of infection in culture negative clinically suspected meningitis cases.

The Optoelectronic Nose.

How does one tell the difference between one molecule or mixture of molecules from another? Chemical sensing seeks to probe physical or chemical properties of molecular or ionic species (i.e., analytes) and transform that information into a useful and distinguishable output. The olfactory system of animals is the prototype of chemical sensing. Even for human beings (who are generally more visual than olfactory creatures), the sense of smell is one of our most basic capabilities, and we can discriminate among many thousands, and possibly even billions, of different odors. The chemical specificity of the olfactory system does not come from specific receptors for specific analytes (i.e., the traditional lock-and-key model of enzyme-substrate interactions), but rather olfaction uses pattern recognition of the combined responses of several hundred olfactory receptors.In analogy to olfaction, colorimetric sensor arrays provide high dimensional data from the color changes of chemically responsive colorants as they are exposed to analytes. These colorants include pH responsive dyes, Lewis acid/base indicators, redox dyes, vapochromics, and surface-modified silver nanoparticles. The color difference maps so created provide chemical sensing with high sensitivity (often down to ppb levels), impressive discrimination among very similar analytes, and exquisite fingerprinting of extremely similar mixtures over a wide range of analyte types, both in the gas and liquid phases. Such colorimetric arrays probe a wide range of the chemical reactivity of analytes, rather than the limited dimensionality of physical properties (e.g., mass) or physisorption (e.g., traditional electronic noses). Our sensor arrays are disposable and simple to produce by either inkjet or robotic dip-pen printing onto the surface of porous polymer membranes or even paper.Design of both sensor arrays and optical readers for their analysis has advanced to a fully self-contained pocket-sized instrument, the optoelectronic nose. Quantitative analysis requires appropriate chemometric methods for pattern recognition of data with inherently high dimensionality, e.g., hierarchical cluster analysis and support vector machines. A wide range of applications for the colorimetric sensor arrays has been developed, including personal dosimetry of toxic industrial chemicals, detection of explosives or fire accelerants, monitoring pollutants for artwork and cultural heritage preservation, quality control of foods and beverages, rapid identification of bacteria and fungi, and detection of disease biomarkers in breath or urine. The development of portable, high-accuracy instrumentation using standard imaging devices with the capability of onboard, real-time analysis has had substantial progress and increasingly meets the expectations for real-world use.

Clinical and economical improvements after introducing rapid identification of bacteria and early antibiotic susceptibility testing in sepsis and bloodstream infections. Results of the PHENOMENON study.

Background: Sepsis and bloodstream infections pose severe challenges in intensive care. Early reliable diagnosis is the key to successful therapy. The objective of the study presented here was to investigate the clinical and economical effects of the new PhenoTM BC test, which allows bacteria identification (ID) and antimicrobial susceptibility testing (AST) in approximately 7 hours after a blood culture becomes positive (BC+).Methods: Historically controlled interventional study. Population: patients with BC+ and ICU admission. Inadequate initial antimicrobial therapy (IAT) is need of therapy change based on result. Prospectively the new test was used in addition. Primary endpoint: time-to-result in hours. Contribution margin (CM) i.e. revenue – costs was computed. All patients formed the intention-to-treat population (ITT). Patients with complete cost data formed the modified ITT group (mITT). CM results were calculated for mITT and PP. Further analyses: length-of-stay (LOS) and mortality.Results: 223 historical and 200 prospective patients were included. Time to result (ITT) was shortened by 51.1 hours (83 vs. 31.9; p<0.001). Overall savings (mITT) were 257,100 € (–301,264 € vs. –44,164 €). 143 of 181 (79%) patients had a test performed, 126 of 143 (88%) having a clinically useable result. 40 (32%) had IAT vs. 65 (29%) in the historic cohort. Median time to AST in PP was shortened by 61.7 hours (89.5 vs. 27.8; p<0.001). LOS was shortened 7 days (28 vs. 19; p=0.226) and mortality was 8% (40.5% vs. 32.5%; p=0.440) lower. Median CM +3,074.80 € per case (–2,350.50 € vs. +724.70 €; p=0.040). Conclusion: The new PhenoTM ID+AST test leads to faster and clinically meaningful results and saves money by shortening LOS on the ICU.

Assessment of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF- MS) for Accurate Bacterial Identification in Clinical Labs

Abstract Introduction/Objective On behalf of the diagnostic Medical Laboratory rapid, and accurate identification of bacteria with their one-to-one anti-microbial susceptibility outlines is of ultimate importance for the management of infected patients. Contemporary microbial identification methods employed in routine clinical diagnostic laboratories relies on the use of conventional phenotypic methods. Phenotypic methods are time consuming with minimum turn-around times of at least 24 hrs and in many occurrences of 48hrs. With the intention of accelerate laboratory processes the MALDI-TOF MS was familiarized. MALDI-TOF MS is established on proteomic profiling and permits for rapid identification of bacteria. This technology has not been widely used in Egypt, but has been regularly used in Europe for the past few years. Methods Two hundred forty three positive non duplicate blood cultures were accrued over a period of six months. Experimental aliquots were taken from excess sample material that was collected as part of routine clinical care. 105 were positive for Gram negative bacilli, 123 were positive for Gram positive cocci, 3 positive for Gram positive bacilli, and 7 were positive for yeast. MALDI-TOF identification was compared to conventional identification. Conventional identification consisted of a combination of MALDI-TOF identification of a subcultures colony by direct smear, biochemical reactions, Vitek 2, and molecular identification. Results Ninety seven of the one hundred and five blood cultures positive for Gram negative bacilli were monomicrobial. The majority of these were identified as Escherichia coli by conventional methods, followed by Klebsiella pneumoniae and Pseudomonas aeruginosa. Eighty-four of these monomicrobial cultures were identified by MALDI-TOF to the species level. Eighty-one of the eighty-four were concordant with the conventional identification (96.4%). Conclusion The MALDI-TOF proved to be useful for the rapid and reliable identification of g-ve bacteria from the clinical specimens. The difference in turnaround time for bacterial identification was significant between MALDI-TOF MS and VITEK 2 with minimal preparation for the blood cultures.

OP36 Investigating the role of bioactives produced by gut bacteria to modulate immune response in inflammatory bowel disease

Abstract Background inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. Although the pathogenesis of IBD is not fully understood, it is believed to result from the interaction between various genetic and environmental factors, including the microbiome, resulting in inappropriate gut inflammation. Under homeostatic conditions, the gut immune system exists in a tolerogenic state with the microbiota. However, microbial dysbiosis and an imbalance of pro- and anti-inflammatory cytokines in the gut characterise IBD and recently faecal microbiota transplantation studies have demonstrated that manipulation of the microbiome can induce remission. In this study, the ability of bioactives produced from single species of anaerobic gut bacteria cultured from healthy human faecal samples to modulate NF-kB activity was investigated, their biochemical properties were assessed and their ability to alleviate colitis was also investigated. Methods The NF-κB suppressive effects of culture supernatants (CSs) from 23 different isolates were tested on colonic epithelial cell lines. Suppressive CSs were also tested on human-derived colonic organoids from IBD patients and healthy controls and IL-8 expression was measured. Furthermore, CS from AHG0001 strain was also tested in spontaneous colitis model Winnie in vivo. The supernatant was further characterised by extraction in multiple solvents and fractionated through reversed-phase analytic HPLC column to test for suppressive fractions and further investigate their chemical characteristics. Results LS174T and Caco-2 reporter cells stimulated with TNFα and IL-1β respectively, resulted in NF-κB activation. Of the 23 isolates screened, CS from five isolates significantly suppressed NF-κB activation. The selected CSs also suppressed IL-8 secretion in PBMCs and gut organoids from both UC and CD patients, as well as healthy controls, with notable individual variation. Rectal gavage of CS from AHG0001 also reduced disease activity, improved histologic inflammation and reduced the pro-inflammatory gene expression in Winnie mice. Furthermore, a potent small molecule (IC50 = 3 nM) produced by AHG0001 was also identified through bioassay-guided solvent extractions and filtrations, followed by UPLC-QTOF and comparative metabolomics. Conclusion Our anaerobic culturing and NF-κB reporter assay system allows for the rapid identification of bacteria producing immunomodulatory bioactives, which could lead to the future development of novel therapeutics. Our in vivo and ex vivo testing utilising spontaneous colitis model and patient-derived organoids demonstrates the potential of precision medicine-based approaches for bacterial based therapeutics.

Parasites in Human Stool: To Ignore or Not To Ignore?

Human stool contains a myriad of microorganisms, of which the vast majority are nonpathogenic and represent an important component of the healthy microbiome. The increasing use of molecular techniques has allowed the rapid identification of bacteria, viruses and parasites in human stool. This review focuses on the 3 main classes of parasite responsible for human disease, helminths, protozoa and ectoparasites, and highlights the importance of differentiating between pathogenic and nonpathogenic parasites.

Rapid Identification of Bacteria by Membrane-Responsive Aggregation of a Pyrene Derivative.

An imidazolium-derived pyrene aggregation was developed to rapidly identify and quantify different bacteria species. When the nonemissive aggregates bound to the anionic bacteria surface, the sensor disassembled to turn on significant fluorescence. At the same time, ratiometric signals between pyrene monomer and excimer emission were controlled by different interactions with various bacteria surfaces. The resulted different fluorescent emission profiles then were obtained as fingerprints for various bacterial species. By converting emission profiles directly into output signals of two channels, fluorescence increase and ratiometric change, a two-dimensional analysis map was generated for bacteria identification. We demonstrated that our sensor rapidly identified 10 species of bacteria and 14 clinical isolated multidrug-resistant bacteria, and we determined their staining properties (Gram-positive or Gram-negative).

Culturing the human microbiota and culturomics.

The gut microbiota has an important role in the maintenance of human health and in disease pathogenesis. This importance was realized through the advent of omics technologies and their application to improve our knowledge of the gut microbial ecosystem. In particular, the use of metagenomics has revealed the diversity of the gut microbiota, but it has also highlighted that the majority of bacteria in the gut remain uncultured. Culturomics was developed to culture and identify unknown bacteria that inhabit the human gut as a part of the rebirth of culture techniques in microbiology. Consisting of multiple culture conditions combined with the rapid identification of bacteria, the culturomic approach has enabled the culture of hundreds of new microorganisms that are associated with humans, providing exciting new perspectives on host-bacteria relationships. In this Review, we discuss why and how culturomics was developed. We describe how culturomics has extended our understanding of bacterial diversity and then explore how culturomics can be applied to the study of the human microbiota and the potential implications for human health.

Evaluation of a micro/nanofluidic chip platform for the high-throughput detection of bacteria and their antibiotic resistance genes in post-neurosurgical meningitis

BackgroundPost-neurosurgical meningitis (PNM) is one of the most severe hospital-acquired infections worldwide, and a large number of pathogens, especially those possessing multi-resistance genes, are related to these infections. Existing methods for detecting bacteria and measuring their response to antibiotics lack sensitivity and stability, and laboratory-based detection methods are inconvenient, requiring at least 24h to complete. Rapid identification of bacteria and the determination of their susceptibility to antibiotics are urgently needed, in order to combat the emergence of multi-resistant bacterial strains. MethodsThis study evaluated a novel, fast, and easy-to-use micro/nanofluidic chip platform (MNCP), which overcomes the difficulties of diagnosing bacterial infections in neurosurgery. This platform can identify 10 genus or species targets and 13 genetic resistance determinants within 1h, and it is very simple to operate.A total of 108 bacterium-containing cerebrospinal fluid (CSF) cultures were tested using the MNCP for the identification of bacteria and determinants of genetic resistance. The results were compared to those obtained with conventional identification and antimicrobial susceptibility testing methods. ResultsFor the 108 CSF cultures, the concordance rate between the MNCP and the conventional identification method was 94.44%; six species attained 100% consistency. For the production of carbapenemase- and extended-spectrum beta-lactamase (ESBL)-related antibiotic resistance genes, both the sensitivity and specificity of the MNCP tests were high (>90.0%) and could fully meet the requirements of clinical diagnosis. ConclusionsThe MNCP is fast, accurate, and easy to use, and has great clinical potential in the treatment of post-neurosurgical meningitis.

Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays

BackgroundRapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system.MethodsC60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy.ResultsThe C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture.ConclusionsThe technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.