Detection Of Pathogenic Bacteria Research Articles

An Amplification-Free Digital Assay Based on Primer Exchange Reaction-Mediated Botryoidal-Like Fluorescent Polystyrene Dots to Detect Multiple Pathogenic Bacteria.

Multiple and ultrasensitive detection of pathogenic bacteria is critical but remains a challenge. Here, we introduce a digital assay for multiplexed and target DNA amplification-free detection of pathogenic bacteria using botryoidal-like fluorescent polystyrene dots (PS-dots), which were first prepared through the hybridization reaction between primer exchange reaction chains and polystyrene nanospheres that encapsulated polymer dots for signal preamplification. The pathogenic bacteria's DNA was cleavaged by the argonaute (Ago) protein-mediated multiple and precise cleavage reactions, where the obtained target sequences bridged the magnetic beads (MBs) and botryoidal-like PS-dots via a hybridization reaction, and the fluorescent MB-botryoidal PS-dot complexes were utilized as digital probes based on colors and sizes for digital encoding. An artificial-intelligence-fluorescent microsphere counting algorithm was applied to identify and count the fluorescent MBs for digital readout. This digital assay combined the ultrabright botryoidal-like PS-dots with Clostridium butyricum Ago's precise enzyme cleavage properties, achieving simultaneous detection of three pathogenic bacteria with a linearity range from 102 to 106 CFU/mL without target DNA amplification within 1.5 h. This digital assay has also been applied to detect aquatic and clinical samples with accepted accuracy (98%), which offers an avenue for a next-generation multiplexed digital platform for pathogenic bacteria analysis.

“Dandelion” magnetic bead-driven CDs@CeO2 based-multifunctional nanoplatform for tri-modal ultrasensitive detection and efficient eradication of pathogenic bacteria

To address the challenge of foodborne pathogens, we have developed an ultra-sensitive tri-modal detection and effective pathogen eradication strategy utilizing a “dandelion” magnetic bead-driven cerium dioxide doped with carbon dots (CDs@CeO2) multifunctional nanoplatform. Aptamer (Apt) modification CDs@CeO2 specifically recognizes and binds to the outer membrane proteins of Salmonella typhimurium (S. typhimurium). Concurrently, mannose-functionalized dandelion-like magnetic beads selectively recognize adhesins of S. typhimurium, avoiding competition with Apt for binding sites and leveraging the strong bacterial capture capability for high-sensitivity detection. The prepared CDs@CeO2 has been demonstrated to exhibit favourable fluorescence characteristics. In the presence of H2O2, CDs@CeO2 is capable of oxidising colourless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxide, displaying exceptional photothermal properties. CDs@CeO2 have the potential to generate •OH through a Fenton-like reaction, which could directly damage cells. Notably, coupling the photosensitizer Ce6 to CDs@CeO2 and subsequent illumination generates 1O2, which synergistically eradicates bacteria with •OH. Detection limits of the fluorescence, colorimetry, and photothermal methods were 3.85 cfu/mL, 9.44 cfu/mL, and 5.00 cfu/mL, respectively. The platform successfully achieves precise detection of S. typhimurium in actual pre-prepared food samples through fluorescence, colorimetry, and photothermal tri-modal signals, and effectively kills bacteria, achieving trace detection and source control.

Graphene oxide-based fluorescent biosensors for pathogenic bacteria detection: a review

Graphene oxide-based fluorescent biosensors for pathogenic bacteria detection: a review

Functional Nucleic Acids as Bacterial Biosensors: A Focus on Clostridioides difficile Infection

Clostridioides difficile (CD) is a Gram‐positive, anaerobic, and spore‐forming bacillus that colonizes the human gut and causes a range of diseases, such as pseudomembranous colitis and antibiotic‐associated diarrhea, that are generally known as CD infection (CDI). Rapid and accurate detection of CDI with high sensitivity and specificity is crucial for patient treatment, infection control, and epidemiological monitoring. Current diagnostic methods for CDI have several limitations, such as high cost, long turnaround time, suboptimal sensitivity, and the need for specialized equipment. Hence, novel detection methods that can overcome these limitations are needed. Functional nucleic acids (FNAs) are a promising class of molecular recognition element (MRE) that can be incorporated into biosensors for detecting infectious pathogens. Several FNAs have been developed for detecting CD. In this review, an overview of CD, CDI, and current diagnostic methods for CDI and their drawbacks are provided. Furthermore, the design principles and working mechanisms of FNAs as well as their applications for the detection of pathogenic bacteria, including CD, are discussed. The potential for developing point‐of‐care paper sensors using currently available CD‐selective FNAs is also highlighted.

Dual-electrode signal amplification self-powered biosensing platform based on nanozyme boosting target-induced DNA nanospace array for ultrasensitive detection of sugarcane Pokkah Boeng disease pathogenic bacteria

Sugarcane is a crop with significant economic importance worldwide. However, pokkah boeng disease poses a serious threat to its production and the sustainable development. There is a pressing necessity for precise and portable detection methods. We develop a dual-electrode signal amplification biosensing platform, for highly sensitive detection of sugarcane pokkah boeng disease pathogenic bacteria. This innovative platform integrates highly catalytic AuNPs/Mn3O4 nanozymes with N-GDY, along with a target-induced development of DNA nanostructure arrays. AuNPs/N-GDY serves as dual electrode substrates, and AuNPs/Mn3O4 nanozymes are surface-loaded as the bioanode. The biocathode is constructed by introducing DNA nanospace arrays onto the electrode through target-induced methods. [Ru(NH3)6]3+ is embedded into the nucleic acid double-helix scaffold via electrostatic adsorption, generating an EOCV signal that is strongly correlated with the target concentration. To further enhance sensitivity, the detection platform is combined with a capacitor to amplify the detection signal, utilizing its high power density, which results in a 22.5-fold increase in sensitivity. The method offers a linear detection range of 0.0001 to 10,000 pM and an detection limit of 32.5 aM (S/N = 3). This method supplies a novel approach for real-time monitoring and competent oversight of pokkah boeng disease pathogenic bacteria.

CRISPR-Cas12a-based nanoparticle biosensor for detection of pathogenic bacteria in food

Pathogenic bacterial contamination of food is a major challenge in food safety and public health management. To monitor pathogenic bacteria in food, a rapid, sensitive, and specific pathogen detection method is needed. CRISPR-Cas12a has been developed as a sensitive method for detecting pathogenic bacteria, but its output signal is fluorescent, which need equipment to detect. Here, the nanoparticle probe was designed to detect the activity of Cas12a and were able to visualize the assay results by observing the color change by naked eyes. The target sequences of pathogenic bacteria were amplified by recombinase polymerase, then recognized and bound by the crRNA and Cas12a complex. The activated Cas12a cleaved the linker-ssDNA connecting the nanoparticles, causing the nanoparticles to transition from aggregation to dispersion, and the color showed as purple. The sensitivity analysis showed that the limits of detection (LOD) reached 1 × 10°CFU/mL, 1 × 104 CFU/mL, 1 × 102 CFU/mL, 1 × 103 CFU/mL, 1 × 101 CFU/mL for Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes, which were all specific for five bacteria in the milk samples. Therefore, this method is a dependable, fast, sensitive and specific method, which make the CRIPSR-Cas12a detection method more useful in bacterial pathogenic detection.

Enzyme-accelerated catalytic DNA circuits enable rapid and one-pot detection of bacterial pathogens

Catalytic DNA circuits, serving as signal amplification strategies, can enable simple and accurate detection of pathogenic bacteria in complex matrices but suffer from low reaction rates and depths. Herein, we design an enzyme-accelerated catalytic hairpin assembly (EACHA) in which duplex DNA products are converted into hairpin reactants to continue participating in the next circuit reaction with the assistance of RNase H. Profiting from the high recyclability of the reactants, EACHA exhibits an approximately 37.6-fold enhancement in the rate constant and a two-order-of-magnitude improvement in sensitivity compared to conventional catalytic hairpin assembly (CHA). By integrating an allosteric probe with EACHA, a one-pot method is developed for rapid and direct detection of S. enterica Enteritidis (S. Enteritidis). This method is capable of detecting 15 CFU mL−1 of S. Enteritidis within 20 min, which is superior to that of real-time PCR. By testing 60 milk samples, we demonstrate this method's high accuracy in discriminating contaminated samples, with an area under the curve (AUC) of 0.997. Moreover, this method can be employed to accurately diagnose early-stage infected mice, with an AUC of 1.00 for feces samples and 0.986 for serum samples. Therefore, this study offers a simple and feasible method for identifying pathogens in complex matrices.

Bioreceptor modified electrochemical biosensors for the detection of life threating pathogenic bacteria: a review.

The lack of reliable and efficient techniques for early monitoring to stop long-term effects on human health is an increasing problem as the pathogenesis effect of infectious bacteria is growing continuously. Therefore, developing an effective early detection technique coupled with efficient and continuous monitoring of pathogenic bacteria is increasingly becoming a global public health prime target. Electrochemical biosensors are among the strategies that can be utilized for accomplishing that goal with promising potential. In recent years, identifying target biological analytes by interacting with bioreceptors modified electrodes is among the most commonly used detection techniques in electrochemical biosensing strategies. The commonly employed bioreceptors are nucleic acid molecules (DNA or RNA), proteins, antibodies, enzymes, organisms, tissues, and biomimetic components such as molecularly imprinted polymers. Despite the advancement in electrochemical biosensing, developing a reliable and effective biosensor for detecting pathogenic bacteria is still in the infancy stage with so much room for growth. A major milestone in addressing some of the issues and improving the detection pathway is the investigation of specific bacterial detection techniques. The present study covers the fundamental concepts of electrochemical biosensors, human PB illnesses, and the latest electrochemical biosensors based on bioreceptor elements that are designed to detect specific pathogenic bacteria. This study aims to assist researchers with the most up-to-date research work in the field of bio-electrochemical pathogenic bacteria detection and monitoring.

Rapid detection and occurrence of foodborne pathogens in minimally processed vegetables: a review

SummaryThe increased consumption of minimally processed vegetables (MPV) in various countries is related to the continued interest of consumers in seeking practical and healthy food items. Due to multiple processing steps, MPV can be contaminated by several foodborne pathogens that pose significant health risks to consumers. The use of rapid techniques to detect pathogens in ready‐to‐eat (RTE) foods such as MPV is therefore essential to provide high quality and safe products. This review aims to provide a comprehensive description of molecular‐based techniques for rapid detection of pathogenic bacteria in MPV, and their occurrence reported in studies published in the last 10 years. The main pathogens detected using rapid methods were Salmonella spp., Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Shigella spp., and Campylobacter jejuni. Molecular‐based techniques included real‐time polymerase chain reaction (PCR), multiplex PCR, matrix‐assisted laser desorption ionisation time of flight mass spectrometry (MALDI‐TOF MS), and denaturing gradient gel electrophoresis (DGGE). The data indicate high incidences of pathogenic bacteria in MPV, stressing the need for their rapid detection in these products to prevent associated health risks. Further studies should be carried out to increase the sensitivity of molecular‐based techniques and prevent false positives due to undesirable non‐specific PCR amplifications.

Magnetic Fe3O4@MOF@aptamer-mediated entropy-driven fluorescence biosensor for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria

Rapid and early identification of pathogenic bacteria in food and clinical samples is critical for infectious disease control and precision medicine. In this study, we present a novel Magnetic Fe3O4@metal–organic framework (MOF)@Aptamer-based Entropy-driven fluorescence biosensor combined with mesophilic Clostridium butyricumArgonaute (CbAgo) for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria, termed the MAEA biosensor. In this study, magnetic zirconium-based MOF nanoparticles were modified with a phosphorylated aptamer as an efficient signal converter, which recognized the pathogens and released the complementary DNA to activate the entropy-driven circuit reaction. The highly efficient catalytic DNA circuit and isothermal enzyme-free format produce large amounts of guide DNA. CbAgo exerts precise and highly efficient endonuclease activity under the guidance of guide DNA, which makes the MAEA biosensor useful for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria with high sensitivity (<102 CFU/mL). This study demonstrates a highly selective and reliable magnetic Fe3O4@MOF@aptamer-based biosensor using CbAgo for simultaneously detecting multiple pathogenic bacteria, providing a potential tool for other infectious disease pathogens with convenient operation and high sensitivity.

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Precise, Sensitive Detection of Viable Foodborne Pathogenic Bacteria with a 6-Order Dynamic Range via Digital Rolling Circle Amplification.

The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one. The one-target-one-amplicon characteristic of dRCA enables high sensitivity and a broad quantitative detection range, conferring a detection limit of 10 CFU/mL and a dynamic range of 6 orders. dRCA can detect rare viable bacteria, even at a proportion as low as 0.1%, which is 50 times more sensitive than the live/dead staining method. The high sensitivity for detecting viable bacteria accommodates dRCA for assessing sterilization efficiency. Based on the assay, we found that, for pasteurization, slightly elevating the temperature to 68 °C can reduce the heating time to 10 min, which may minimize nutrient degradation caused by high-temperature exposure. The assay can serve as a precise tool for estimating the contamination by viable pathogenic bacteria and assessing sterilization, which facilitates food safety control.

An NMR immunosensor based on streptavidin-biotin signal amplification for rapid detection of Salmonella in milk

Sensitive and accurate detection of harmful pathogenic bacteria in food is of great significance to public health. In this paper, a nuclear magnetic resonance (NMR) sensor based on biological signal amplification was constructed. The carboxymethyl dextran (CMD) was used as both the probe skeleton and the chelating agent to realize the fast chelating adsorption of the signal source gadolinium ion. Finally, the magnetic complex and biotinylated antibody formed an immune probe through the biological bridge to achieve rapid capture of Salmonella. In addition, non-target bacteria such as Proteus vulgaris and Listeria monocytogenes were used to test the specificity of the constructed sensor. The sensor can detect Salmonella within 1.5 h under optimal conditions, the detection limit in milk is 7.4 × 103 CFU mL−1, and the RSD (Relative standard deviation) of reproducibility experiment is 2.35%, which has good reproducibility.

UV-ozone treated glass fiber based lateral flow DNA extraction platform integrated with LAMP for rapid detection of pathogen bacteria in whole blood

The need for a fast and reliable platform for the detection of pathogens in point-of-care (POC) applications is increasing day by day. Molecular detection of pathogens is made possible by a good DNA isolation method, which allows for efficient and pure extraction from the cell. To address this need, our work presents a new lateral flow platform based on UV ozone-treated glass fiber pads for high-throughput DNA isolation from very low volumes of bacterial samples. The platform we developed mainly consists of UV ozone-treated glass fiber, which serves as the essential component binding DNA, and a cellulose-based absorbent pad acting as a reservoir for washing buffers and undesired substances from bacterial cells. UV ozone treatment increased the DNA recovery efficiency of glass fiber by 2.5 times compared to untreated glass fiber. qPCR results confirmed the isolation of pure DNA from both gram-negative and gram-positive bacteria samples performed on the developed platform. For five different bacteria concentrations (10–109 cfu/mL), a good linearity between Cq values and bacteria concentrations was obtained. A minimum concentration of 10 cfu/mL could be detected for both bacterial samples of 10 µL. The developed DNA extraction platform was also integrated with a portable heater for the LAMP-based detection application. We successfully demonstrated that the proposed extraction platform, combined with the colorimetric LAMP-based system, can detect Staphylococcus aureus bacteria spiked in whole blood samples in under one hour, including both extraction and detection processes. Our simplified paper-based platform will be a good alternative for the DNA extraction and molecular detection of pathogens without the need for complex laboratory equipment in POC applications.

Antimicrobial Peptide-Based Sensor for the Electrochemical Detection of Methicillin-Resistant Staphylococcus Aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive bacterium that colonizes on the skin and nasal track of humans, which can lead to serious illnesses such as skin and soft tissue infections, pneumonia, and septicemia.MRSA is resistant to an entire class of antibiotics called beta-lactams, including penicillin, amoxicillin, and oxacillin, making it more difficult to treat. Culture based methods and molecular based methods like PCR are currently being used for the detection of MRSA, but these methods possess the drawbacks of being slow and expensive, respectively. Thus, we focused on developing a reagentless, cost-effective, near real-time detection method for intact MRSA cells.Antimicrobial peptides (AMPs) possess the ability to interact with bacterial cells by inhibiting their growth or by lysing them.However, this initial interaction between the cationic AMPs and the negatively charged bacterial cells can be used for the detection of pathogens like MRSA. Here, we used a MRSA-specific antimicrobial peptide, DP7, to fabricate an electrochemical peptide-based (E-PB) sensor. The DP7 probe was further modified with a methylene blue redox label and an alkanethiol chain which allows for the formation of a thiol-gold self-assembled monolayer. The sensor surface was passivated with both 6-mercapto-1-hexanol and a thiolated tetra-thymine sequence to prevent surface fouling. Electrochemical detection of MRSA was accomplished using alternating current voltammetry. Control experiments were performed using two gram-positive bacteria, Bacillus subtilis and Staphylococcus epidermidis, and two gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. The DP7 sensor responded to MRSA in a concentration-dependent manner and was proven to be selective for MRSA over the other four microbes. Specifically, the % signal suppression obtained for MRSA was much larger when compared to those obtained for the other microbes. The same behavior was observed in pure buffer and 5% synthetic wound fluid. In addition, the detection time was relatively short, with signal saturation in less than 60 min. Overall, the DP7 sensor has demonstrated to possess attributes similar to other E-PB sensors. It has the potential to be developed as a rapid, reliable, and cost-effective diagnostic tool for MRSA, which will be useful in preventing the spread of the pathogen in the community. Furthermore, this sensing platform is versatile and can be redesigned for detection of other pathogenic bacteria.

Swift and precise detection of unlabeled pathogens using a nanogap electrode impedimetric sensor facilitated by electrokinetics

For the protection of human health and environment, there is a growing demand for high-performance, user-friendly biosensors for the prompt detection of pathogenic bacteria in samples containing various substances. We present a nanogap electrode-based purely electrical impedimetric sensor that utilizes the dielectrophoresis (DEP) mechanism. Our nanogap sensor can directly and sensitively detect pathogens present at concentrations as low as 1–10 cells/assay in buffers and drinking milk without the need for separation, purification, or specific ligand binding. This is achieved by minimizing the electrical double-layer effect and electrode polarization in nanogap impedance sensors, reducing signal loss. In addition, even at low DEP voltages, nanogap sensors can quickly establish strong DEP forces between the nanogap electrodes to control the spatial concentration of pathogens around the electrodes. This activates and stabilizes inter-electrode signal transmission along the nanogap-aligned pathogens, increasing sensitivity and reducing errors during repeated measurements. The DEP-enabled nanogap impedance sensor developed in this study is valuable for a variety of pathogen detection and monitoring systems including point-of-care testing (POCT) as it can detect pathogens in diverse samples containing multiple substances quickly and with high sensitivity, is compatible with complex solutions such as food and beverages, and provides highly reproducible results without the need for separate binding and separation processes.

ITO-Coated Vertical Nanowires-Based Biosensors for the Label-Free Detection of Escherichia coli

A major concern in public health is the direct in-situ detection of pathogenic bacteria in food, beverages, and the air [1]. This study utilizes ITO-coated vertical nanowires (ITO-VNWs) as sensing substrates for the detection of Escherichia coli (E. coli). The innovation in this research lies in the direct detection of various concentrations of E. coli using boronic acid (BA)-modified ITO-VNWs connected to a benchtop E4980A precision LCR meter. Impedance spectra were expressed in terms of real and imaginary parts, corresponding to resistance (R) and reactance (X). Serial dilutions of E. coli were prepared in saline for bacterial enumeration and tested using the standard plate count method. Subsequently, 100 μl of each bacterial dilutions (107, 106, 105, 104, 103, 102, and 101 CFU/ml) were applied to BA-modified ITO-VNWs. In summary, BA-modified ITO-VNWs demonstrated accurate and distinct detection of varying concentrations of E. coli. This accomplishment highlights the promising potential of our BA-modified ITO-VNW biosensor for precise bacterial detection across diverse applications. Further exploration and optimization of this platform could lead to advancements in the rapid and specific identification of pathogenic bacteria, making it an invaluable tool in the realm of public health and environmental safety. REFERENCES [1] A. Ahmed, J.V. Rushworth, N.A. Hirst, P.A. Millner, Biosensors for whole-cell bacterial detection, Clin Microbiol Rev 27(3) (2014) 631-46. Figure 1

Rapid detection of Salmonella typhimurium in food samples using electrochemical sensor

Rapid and accurate detection of pathogenic bacteria is crucial for ensuring food safety and public health. In this study, we aimed to develop a novel molecularly imprinted polymer sensor based on screen-printed electrodes for the specific detection of Salmonella typhimurium. The sensor was constructed by electropolymerizing dopamine in the presence of Salmonella typhimurium on the electrode surface, followed by the removal of the bacteria to create specific binding cavities. The sensor demonstrated excellent specificity for Salmonella typhimurium, with a detection limit approximately of 101 CFU/ml and a detection time of only 4 min, with the sensor accurately differentiating Salmonella typhimurium from other common foodborne pathogens such as Escherichia coli and Listeria monocytogenes. The performance of the sensor was validated using real food samples, including pork and milk, showing its well suited for rapid and on-site detection of pathogenic bacteria. The development of this molecularly imprinted polymer with electrochemical sensor represents a significant advancement in the field of electrochemical biosensors, offering a promising tool for food safety monitoring and public health protection.

Investigation and detection of multiple antibiotic-resistant pathogenic bacteria in municipal wastewater of Dhaka city

BackgroundWater pollution in densely populated urban areas, mainly from municipal wastewater, poses a significant threat. Pathogenic bacteria, such as Vibrio spp. and fecal coliform, endanger public health and the environment. Additionally, antibiotic-resistant bacteria in wastewater complicate treatment and heighten public health concerns.MethodsThe study sampled municipal wastewater from ten Dhaka neighborhoods, selecting treatment plants, sewage outlets, and various collection points using meticulous techniques for representative samples. Bacteriological and biochemical analyses were conducted using standardized methods. Antimicrobial susceptibility testing (AST) was performed with the disk diffusion method against 13 widely used antibiotics.ResultsAll sampled areas exhibited positive results for Vibrio spp., fecal coliform, E. coli, and Salmonella spp. Varying bacterial concentrations were observed, with the highest concentration of TVC, total vibrio spp., and total fecal coliform, total E. coli count, and total Salmonella spp. were found in Uttara (1.9 × 104 CFU/ml), Bangshal (1.8 × 102 CFU/ml), and Lalbag (2.1 × 103 CFU/ml), Mirpur (3.70 × 102 CFU/ml), and Lalbag (6 × 102 CFU/ml) respectively. AST results revealed significant resistance among all bacterial species to various antibiotics. Specifically, Vibrio spp. showed 100% resistance to cefuroxime, fecal coliform exhibited 90% resistance to cephradine, E. coli demonstrated 60% resistance to cephradine, and Salmonella spp. displayed 90% resistance to ampicillin.ConclusionThe study highlights the existence of multiple antibiotic-resistant bacteria in Dhaka's wastewater. Addressing antibiotic resistance is essential to manage the risks of multiple antibiotic-resistant infections and maintain antibiotic effectiveness. These implications are critical for various stakeholders, including public health officials, policymakers, environmentalists, and urban planners.

Hook-Like DNAzyme-Activated Autocatalytic Biosensor for the Universal Detection of Pathogenic Bacteria.

DNAzyme-based assays have found extensive utility in pathogenic bacteria detection but often suffer from limited sensitivity and specificity. The integration of a signal amplification strategy could address this challenge, while the existing combination methods require extensive modification to accommodate various DNAzymes, limiting the wide-spectrum bacteria detection. We introduced a novel hook-like DNAzyme-activated autocatalytic nucleic acid circuit for universal pathogenic bacteria detection. The hook-like connector DNA was employed to seamlessly integrate the recognition element DNAzyme with the isothermal enzyme-free autocatalytic hybridization chain reaction and catalytic hairpin assembly for robust exponential signal amplification. This innovative autocatalytic circuit substantially amplifies the output signals from the DNAzyme recognition module, effectively overcoming DNAzyme's inherent sensitivity constraints in pathogen identification. The biosensor exhibits a strong linear response within a range of 1.5 × 103 to 3.7 × 107 CFU/mL, achieving a detection limit of 1.3 × 103 CFU/mL. Noted that the sensor's adaptability as a universal detection platform is established by simply modifying the hook-like connector module, enabling the detection of various pathogenic bacteria of considerable public health importance reported by the World Health Organization, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella typhimurium. Additionally, the specificity of DNAzyme in bacterial detection is markedly improved due to the signal amplification process of the autocatalytic circuit. This hook-like DNAzyme-activated autocatalytic platform presents a versatile, sensitive, and specific approach for pathogenic bacteria detection, promising to significantly expand the applications of DNAzyme in bacteria detection.