Rapid Detection Of Bacterial Pathogens Research Articles

Disposable electrochemical biosensors for the detection of bacteria in the light of antimicrobial resistance.

Persistent and inappropriate use of antibiotics is causing rife antimicrobial resistance (AMR) worldwide. Common bacterial infections are thus becoming increasingly difficult to treat without the use of last resort antibiotics. This has necessitated a situation where it is imperative to confirm the infection to be bacterial, before treating it with antimicrobial speculatively. Conventional methods of bacteria detection are either culture based which take anywhere between 24 and 96 hor require sophisticated molecular analysis equipment with libraries and trained operators. These are difficult propositions for resource limited community healthcare setups of developing or less developed countries. Customized, inexpensive, point-of-care (PoC) biosensors are thus being researched and developed for rapid detection of bacterial pathogens. The development and optimization of disposable sensor substrates is the first and crucial step in development of such PoC systems. The substrates should facilitate easy charge transfer, a high surface to volume ratio, be tailorable by the various bio-conjugation chemistries, preserve the integrity of the biorecognition element, yet be inexpensive. Such sensor substrates thus need to be thoroughly investigated. Further, if such systems were made disposable, they would attain immunity to biofouling. This article discusses a few potential disposable electrochemical sensor substrates deployed for detection of bacteria for environmental and healthcare applications. The technologies have significant potential in helping reduce bacterial infections and checking AMR. This could help save lives of people succumbing to bacterial infections, as well as improve the overall quality of lives of people in low- and middle-income countries.

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Sulfhydryl-mediated etching suppression of gold nanostars for rapid and sensitive detection of bacterial pathogens

Human infectious diseases caused by bacterial pathogens, such as meningitis and pyaemia, threaten the public health worldwide due to their acute onset and high mortality. Sensitive and rapid detection of bacterial pathogens is urgent for the treatment of infectious diseases clinically. However, current techniques are generally time-consuming and require large-scale equipment. Plasmonic nanosensors built on metallic nanomaterials have emerged as excellent tools for biomedical diagnostics. Herein, we report an etching suppression strategy for the sensitive and rapid detection of bacterial pathogens through gold nanostars. Sulfhydryl-containing ligands suppress the iodide-mediated surface etching of gold nanostars, depending on the type, concentration, and size of sulfhydryl-containing ligands. Using mercaptophenylboronic acid (MBA) and Pseudomonas aeruginosa (P. aeruginosa)-specific aptamer as multifunctional ligands, we specifically identify bacterial pathogens through sulfhydryl-mediated etching suppression with a linear detection range from 10 to 106 CFU/mL and a limit of detection of 2 CFU/mL. This etching suppression-based strategy enables the detection of Neisseria meningitidis in the cerebrospinal fluid and P. aeruginosa in the serum within 15 min, showing great potential as a point-of-care platform for clinical diagnosis.

A single thiolated-phage displayed nanobody-based biosensor for label-free detection of foodborne pathogen.

Rapid and sensitive detection of bacterial pathogens present in food and environmental samples is of crucial importance to ensure human health and safety. Here, we present a one-step label-free colorimetric strategy based on M13 bacteriophage-displayed nanobody (phage-Nb) derived from camelid heavy-chain antibodies specific to Vibrio parahaemolyticus (V. parahaemolyticus). The thiolation of phage-Nb (Phage-Nb-SH) on pVIII shell proteins induces the aggregation of gold nanoparticles (AuNPs), whereas the specific interaction between nanobody and bacteria prevents the aggregation of AuNPs, resulting in visible color change due to alteration of surface plasmon resonance properties. Based on this phenomenon, a simple and sensitive colorimetric immunosensor for V. parahaemolyticus was developed. The assay can be accomplished within 100min, and exhibits a visual detection limit of 104 cfu/mL and a quantitative detection limit of 103 cfu/mL, with no cross-reactivity towards other bacterial species. This strategy takes full advantages of both the high specificity of phage-Nbs and the optical properties of AuNPs, enabling simple and rapid detection of bacterial pathogens.

Sensitive and automated detection of bacteria by CRISPR/Cas12a-assisted amplification with digital microfluidics

The development of new diagnostic platforms for sensitive and rapid detection of bacterial pathogens is of great significance for improving precision medication for infectious disease and food safety control. Currently, many bacterial detection methods are limited by low detection sensitivity, sophisticated protocols, long processing times and labor-intensive procedures. Here, we report a DMF-Cas12a method for rapid, sensitive and automated analysis of bacterial pathogens by integrating the CRISPR/Cas12a system with a digital microfluidic chip (DMF). In the assay, Staphylococcus aureus could be detected with high sensitivity and specificity, and the detection limit of the assay was found to be 32 CFU/mL. With the tiny volume and automation capabilities of DMF, this assay was accomplished in 55 min and consumed only 6.6 μL reagent. This platform was successfully applied to urine and milk samples with excellent sensitivity and reliability. The DMF-Cas12a method shows good potential in automated and highly sensitive pathogen detection for a wide variety of biological applications.

Clinical evaluation of cell-direct polymerase chain reaction-based nucleic acid lateral flow immunoassay for rapid detection of bacterial pathogens in clinically suspected sepsis: A multi-center study in Japan

Blood culture, a method for identifying causative agents of bacterial sepsis, requires several days. The combination of cell-direct polymerase chain reaction and nucleic acid lateral flow immunoassay (cdPCR-NALFIA) is a simple and sensitive detection method for identifying pathogenic bacteria. Furthermore, this assay, when applied directly to blood samples yields results within 4.5 h, without requiring culture. This study was performed at five hospitals in Japan between 2013 and 2016. Blood samples from 73 patients with clinically suspected sepsis yielded 18 positive blood cultures, and the isolated bacterial species were detectable using cdPCR-NALFIA in nine samples. Thirteen samples were positive on cdPCR-NALFIA. In total, 17 samples confirmed to have bacterial species were detectable using cdPCR-NALFIA and/or blood culture with a true positive rate of 76.5% and 64.7%, respectively. The combination of blood culture and cdPCR-NALFIA could improve the rate of detection of bacterial sepsis.

Biosensors for rapid detection of bacterial pathogens in water, food and environment.

Conventional techniques (e.g., culture-based method) for bacterial detection typically require a central laboratory and well-trained technicians, which may take several hours or days. However, recent developments within various disciplines of science and engineering have led to a major paradigm shift in how microorganisms can be detected. The analytical sensors which are widely used for medical applications in the literature are being extended for rapid and on-site monitoring of the bacterial pathogens in food, water and the environment. Especially, within the low-resource settings such as low and middle-income countries, due to the advantages of low cost, rapidness and potential for field-testing, their use is indispensable for sustainable development of the regions. Within this context, this paper discusses analytical methods and biosensors which can be used to ensure food safety, water quality and environmental monitoring. In brief, most of our discussion is focused on various rapid sensors including biosensors and microfluidic chips. The analytical performances such as the sensitivity, specificity and usability of these sensors, as well as a brief comparison with the conventional techniques for bacteria detection, form the core part of the discussion. Furthermore, we provide a holistic viewpoint on how future research should focus on exploring the synergy of different sensing technologies by developing an integrated multiplexed, sensitive and accurate sensors that will enable rapid detection for food safety, water and environmental monitoring.

Smart Wearable Fluorescence Sensing of Bacterial Pathogens and Toxic Contaminants by Eu3+-Induced Sodium Alginate/Ag Nanoparticle Aggregates

Rapid detection of bacterial pathogens and toxic contaminants in the environment is essential for providing real-time results to mitigate potential safety hazards. This study prepared a wearable fabric sensing material based on fluorescent nanomaterials. The nano silver/europium complex is assembled layer by layer with sodium alginate to obtain nanoparticle aggregates with strong fluorescence, and then these are covalently bonded onto the cotton fabric surface to obtain the fluorescence cotton fabric (FCF). Via fluorescence quenching, FCF can sensitively sense Escherichia coli and Staphylococcus aureus and show good antibacterial activity. Besides, the selected transition metal ions can be detected qualitatively through the functional relationship between the fluorescence intensity and metal ion dose. The viability test of NIH3T3 cells showed that FCF had good biocompatibility. This fluorescent fabric sensing material has the characteristics of being wearable, antibacterial, and biocompatible. It realizes the real-time detection of bacteria and toxic metal ions and has great application prospects in environmental and biological monitoring.

Rapid Detection of Bacterial Pathogens and Antimicrobial Resistance Genes in Clinical Urine Samples With Urinary Tract Infection by Metagenomic Nanopore Sequencing.

Urinary tract infections (UTIs) are among the most common acquired bacterial infections in humans. The current gold standard method for identification of uropathogens in clinical laboratories is cultivation. However, culture-based assays have substantial drawbacks, including long turnaround time and limited culturability of many potential pathogens. Nanopore sequencing technology can overcome these limitations and detect pathogens while also providing reliable predictions of drug susceptibility in clinical samples. Here, we optimized a metagenomic nanopore sequencing (mNPS) test for pathogen detection and identification in urine samples of 76 patients with acute uncomplicated UTIs. We first used twenty of these samples to show that library preparation by the PCR Barcoding Kit (PBK) led to the highest agreement of positive results with gold standard clinical culture tests, and enabled antibiotic resistance detection in downstream analyses. We then compared the detection results of mNPS with those of culture-based diagnostics and found that mNPS sensitivity and specificity of detection were 86.7% [95% confidence interval (CI), 73.5–94.1%] and 96.8% (95% CI, 82.4–99.9%), respectively, indicating that the mNPS method is a valid approach for rapid and specific detection of UTI pathogens. The mNPS results also performed well at predicting antibiotic susceptibility phenotypes. These results demonstrate that our workflow can accurately diagnose UTI-causative pathogens and enable successful prediction of drug-resistant phenotypes within 6 h of sample receipt. Rapid mNPS testing is thus a promising clinical diagnostic tool for infectious diseases, based on clinical urine samples from UTI patients, and shows considerable potential for application in other clinical infections.

Development of nano-immunosensor with magnetic separation and electrical detection of Escherichia coli using antibody conjugated Fe3O4@Ppy

Detection of bacterial pathogens is the need of the hour due to the increase in antibiotic resistance and the infusion of multi-drug-resistant parasites. The conventional strategies such as ELISA, PCR, and MNP based tests for the detection are efficient but they are cost, time, lab, and manpower intensive. Thus, warranting a simple and effective technique for rapid detection of bacterial pathogens. Magnetic nanoparticles (NPs) have proved to be better alternatives for separation of bacterial pathogens from a variety of sample sources. However, the use of magnetic NPs has not been successful in the detection of these parasites. The current work involves the coating of magnetic NPs (Fe3O4) with a conducting polymer (polypyrrole; Ppy) to facilitate simultaneous separation and detection. Electrical (conductivity) measurement was the mode of choice due to the sensitivity, accuracy, and ease it offers. To enhance the conductivity, carboxylic groups were expressed on the Fe3O4@Ppy complex and to ensure specificity, E. coli specific antibodies were conjugated. The resulting complex at various process parameters was characterized using FTIR, VSM, and SEM. SEM images were recorded to ensure bacterial separation at optimal process parameters. The impedance analysis and conductivity measurements were carried out for the sample volume of 15 μl. The bacterial suspension from 101–106 CFU ml−1 was successfully detected with a limit of detection of 10 CFU ml−1 within 10 min using a simplistic detection method.

Rapid detection of bacterial pathogens in blood through engineered phages-beads and integrated Real-Time PCR into MicroChip

The invasion of pathogens into the bloodstream can produce a systemic inflammatory response syndrome that, if not promptly treated, rapidly evolves in acute life-threatening dysfunction of remote organs (septic shock). Current conventional diagnostic tests are microbial blood cultures that need days for results. Therefore, it is crucial the availability of rapid and reliable pathogens detection for prompt diagnosis. In this work, we developed a new method for fast and sensitive detection of bacteria in blood based on the integration of pathogen capture through engineered M13 phages and molecular detection via PCR in miniaturized silicon microchip. Engineered M13 phage-clones exposing specific peptides able to bind Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and a novel identified phage clone, 9IIIB5, for Staphylococcus epidermidis, were used to functionalize magnetic beads and capturing/concentrate the pathogens. Due to the stability of beads-phage-bacteria complex, PCR inhibitors present in the blood were easily removed without detachment of captured bacteria. The complex was used as target for direct amplification into miniaturized silicon chip by a multiplex PCR followed by Real-Time PCR. The procedure was very fast (less than 3 hs) and sensitive (10 cells/reaction) enabling molecular detection inside to the critical period for clinical decision in sepsis. It could be applied to detect pathogens present in very few numbers or in a metabolically inactive state. The results here presented pay the way to future development of portable genetic Point-of-Care in the rapid identification of microorganisms present in the blood stream or also in different matrices, such as waters and foods.

Early detection of Pseudomonas aeruginosa and Escherichia coli. using zinc tetraphenylporphyrin

Abstract The sensory characteristics of food are considerably effected by the metabolic processes of various micro-organisms in the food stored in the field or at ambient temperature. Also, this microbial contamination can pose serious health hazards to public health. Chemical analysis of the complex mixture of volatiles produced during bacterial growth and investigation methods of these microorganisms presents a big challenge. There remains major unmet need to shorten and improve detection methods. Therefore, early detection of the microorganisms will open many frontiers for quality control in the foodstuffs industry. Accordingly, the aim of this study was to assess the feasibility and performance of chemoreceptive sensors for the rapid detection of bacterial pathogens, specifically Pseudomonas aeruginosa and Escherichia coli. In Uv/vis study, zinc tetraphenyl porphyrin solutions (in DMF) was tested with various volatile compounds, such as propanal, hexanal and heptanal which are commonly found to be released during the growth of bacteria. These sensors were used to detect the bacterial odours of two pathogenic species (E.coli and P.aeruginosa) during their growth cycle at 4 °C and ambient temperature. Hypochromic shifts in Uv/vis and hydrogen bonding in FT-IR studies confirmed the interaction between the volatiles and porphyrin. The porphyrin used detected the presence of microorganisms after 12 hrs incubation and showed more sensitivity for volatiles released during aerobic activity P. aeruginosa as compared to E. coli at 4 °C and ambient temperature. Zinc tetraphenyl porphyrin based chemoreceptive membranes has been proved successful for the detection of P. aeruginosa. Hence, the present study proves wide scope of improvement over current laboratory techniques for the detection of pathogens in terms of speed, ease of use, and cost. Key messages The developed technique allows rapid detection of spoiled food. Chemoreceptive property of porphyrin has been exploited for the early detection of bacteria.

Rapid detection of Escherichia coli using bacteriophage-induced lysis and image analysis.

Rapid detection of bacterial pathogens is a critical unmet need for both food and environmental samples such as irrigation water. As a part of the Food safety Modernization Act (FSMA), The Produce Safety rule has established several requirements for testing for the presence of generic Escherichia coli in water, but the current method available for testing (EPA M1603) demands specified multiple colony verification and highly trained personnel to perform these tests. The purpose of the study was to assess a phage induced bacterial lysis using quantitative image analysis to achieve rapid detection of E. coli at low concentrations within 8 hours. This study aimed to develop a simple yet highly sensitive and specific approach to detect target bacteria in complex matrices. In the study, E. coli cells were first enriched in tryptic soy broth (TSB), followed by T7 phage induced lysis, concentration, staining and fluorescent imaging. Image analysis was conducted including image pre-processing, image segmentation and quantitatively analysis of cellular morphological features (area, eccentricity and full width at half maximum). Challenge experiments using realistic matrices, including simulated fresh produce wash water, coconut water and spinach wash water, demonstrated the method can be applied for use in situations that occur in food processing facilities. The results indicated E. coli cells that are lysed by T7 phages demonstrated significantly (P < 0.05) higher extracellular DNA release, altered cellular shape (from rod to circular) and diffused fluorescent signal intensity. Using this biosensing strategy, a sensitivity to detect Escherichia coli at 10 CFU/ml within 8 hours was achieved, both in laboratory medium and in complex matrices. The proposed phage based biosensing strategy enables rapid detection of bacteria and is applicable to analysis of food systems. Furthermore, the steps involved in this assay can be automated to enable detection of target bacteria in food facilities without extensive resources.

Phage-based assay for rapid detection of bacterial pathogens in blood by Raman spectroscopy

Sepsis is a systemic inflammatory response ensuing from presence and persistence of microorganisms in the bloodstream. The possibility to identify them at low concentrations may improve the problem of human health and therapeutic outcomes. So, sensitive and rapid diagnostic systems are essential to evaluate bacterial infections during the time, also reducing the cost. In this study, from random M13 phage display libraries, we selected phage clones that specifically bind surface of Staphyloccocus aureus, Pseudomonas aeruginosa and Escherichia coli. Then, commercial magnetic beads were functionalized with phage clones through covalent bond and used as capture and concentrating of pathogens from blood. We found that phage-magnetic beads complex represents a network which enables a cheap, high sensitive and specific detection of the bacteria involved in sepsis by micro-Raman spectroscopy. The enter process required 6 h and has the limit of detection of 10 Colony Forming Units on 7 ml of blood (CFU/7 ml).

Hydrothermal green synthesis of magnetic Fe3O4-carbon dots by lemon and grape fruit extracts and as a photoluminescence sensor for detecting of E. coli bacteria

The aim of this work is preparing of a photoluminescence nanostructures for rapid detection of bacterial pathogens. Firstly, carbon dots (CDs) were synthesized by grape fruit, lemon, turmeric extracts and hydrothermal method. Then Fe3O4 (magnetite) nanoparticles was achieved using these bio-compatible capping agents. Finally, magnetite-carbon dots were synthesized as a novel magnetic and photoluminescence nanocomposite. X-ray diffraction (XRD) confirms the crystallinity and phase of the products, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigate the morphology, shape and size of the magnetite, carbon dot and nanocomposites. Fourier transform infrared (FT-IR) spectroscopy shows the purity of the nanostructures. Ultraviolet-visible (UV–Vis) absorption and photo-luminescence (PL) spectroscopy show suitable photo-luminescence under ultraviolet irradiation. Vibrating sample magnetometer (VSM) shows super paramagnetic property of the product. Interestingly carbon dots were used as a non-toxic photoluminescence sensor for detecting of Escherichia coli (E. coli) bacteria. Results show quenching of photoluminescence of the CDs nanocomposite by increasing amount of E. coli bacteria.

Development and Validation of a Combined Cell-direct Polymerase Chain Reaction-based Nucleic Acid Lateral Flow Assay for the Rapid Detection of Bacterial Pathogens Associated with Sepsis

Development and Validation of a Combined Cell-direct Polymerase Chain Reaction-based Nucleic Acid Lateral Flow Assay for the Rapid Detection of Bacterial Pathogens Associated with Sepsis

Rapid Detection of Bacterial Pathogens on 2D Food

This paper investigates a rapid method of detecting bacterial pathogens on 2D food, such as leafy greens. The method is based on our previous work that uses freestanding phage-coated magnetoelastic (ME) biosensors and a surface-scanning coil detector for direct bacterial detection on the surface of food without commonly used sample preparation steps (washing, concentration, enrichment, etc). The ME biosensors used in this work were ferromagnetic alloy strips coated with E2 phage that specifically binds with Salmonella Typhimurium. First, the biosensors (1 mm × 0.2 mm × 30 µm) were placed in rectangular, indexed cavities (1.1 mm × 0.22 mm × 20 µm) that are etched in a glass wafer. A sheet magnet was added to the back of the wafer to hold the sensors in place. The glass wafer with the biosensors and sheet magnet was then flipped and lowered onto Salmonella-tainted spinach leaves. At this time, a weight was added such that the leaves got flattened to improve the chance for the biosensors to physically contact with Salmonella cells. Various weights ranging from 0 g to 120 g were tested to investigate their effects on the Salmonella binding. Measurement sensors loaded with E2 phage and control sensors devoid of the phage were prepared and used. The control sensors served as the reference to compensate for the environmental effects such as temperature and humidity. The biosensors were placed on the surface of the spinach leaves for 2 mins for the binding to occur. The degree of the binding of Salmonella to the measurement sensors was determined by measuring the resonant frequency shift of the sensors using a surface-scanning detection system and confirmed by optical microscopy. The binding of Salmonella was found to increase as the spiked Salmonella concentration increased (103 cfu/ml to 108 cfu/ml). In addition, significant resonant frequency shifts of the measurement sensors (4,800 ± 160 Hz) were observed when 60 g of weight was added. However, further increases in weight did not improve the Salmonella binding with E2 phage. Rather, heavier weights increased the probability of non-specific attachment of Salmonella cells and other residuals from the leaf surface.

Preparation of highly luminescent nitrogen doped graphene quantum dots and their application as a probe for detection of Staphylococcus aureus and E. coli

The rapid detection of bacterial pathogens is very important for medical diagnostics, biosecurity, transportation and the prevention of global pandemics. Here graphene quantum dots (GQDs) are prepared by using citric acid and ethylenediamine and hydrothermal as starting materials and synthesis method. Product is characterized by X-ray diffraction analysis, FT-IR spectroscopy, Transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and photoluminescence (PL) spectroscopy. In the following, graphene quantum dots are used as photoluminescence sensor for detecting Staphylococcus aureus and E. coli. It is found that graphene quantum dots are sensitive at the range of 0 to 9×107 range for Staphylococcus aureus and E. coli.

Culture-Independent Rapid Detection Methods for Bacterial Pathogens and Toxins in Food Matrices.

Rapid detection of bacterial pathogens and toxins in foods is necessary to provide real-time results to mitigate foodborne illness outbreaks. Cultural enrichment methods, although the most widely used, are time-consuming and therefore inadequate for rapid pathogen detection from food samples. The development of novel "rapid" detection methods has decreased detection time dramatically. This review presents an overview of detection methods for various foodborne pathogens, including Listeria monocytogenes, Salmonella enterica, and shiga toxin-producing Escherichia coli, and bacterial toxins in food matrices, with emphasis on those methods which do not require cultural enrichment. Discussed methods include nucleic acid-, immunological-, and biosensor-based techniques. A summary of each type of detection method is given, including referenced methods from the literature. Since these discussed methods do not require cultural enrichment, there is a higher probability of interference from the food matrices. Therefore, the review also discusses the potential interference of food components on detection methods and addresses preprocessing strategies to overcome matrix associated inhibition and to concentrate low quantities of pathogens and toxins in food. Development of rapid and sensitive detection technologies advances and ensures public health safety and security.

Universal ProbeLibrary based real-time PCR for rapid detection of bacterial pathogens from positive blood culture bottles

A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.