System For Detection Of Bacteria Research Articles

Construction of on-site DNA pre-treatment device and rapid electrochemical biosensor set for Escherichia coli detection in milk

Restaurants and cafeterias are constantly exposed to bacterial contamination. In such environments, it is highly needed the rapid bacteria detection system with simple pre-treatment device. To solve this problem, this study presented the pre-treatment device and Rapid electrochemical (REC) biosensor set for detecting Escherichia coli (E. coli) gene in milk sample. The pre-treatment device is consisted of tetraethyl orthosilicate (TEOS)-coated bead that operated with sonication method, and it can effectively remove the E. coli residues and extract E. coli DNA well. By analyzing the sequences of various foodborne pathogens, we selected the highly variable region sequences of E. coli DNA to use as the bioprobe for REC biosensor. The selected thiol-modified bioprobe was immobilized onto the fabricated biosensor electrode. For detecting the extracted E. coli gene fragment rapidly, the alternating current electrothermal flow (ACEF) technique was introduced to REC-biosensor. ACEF technique minimize the target-bioprobe binding time to 10 mins. Using the extracted E. coli DNA fragments with the pre-treatment device, the proposed REC-biosensor shows the limit of detection (LOD) in milk samples was 9.235 × 10-5 ng/μL, and the target detection accuracy was 92.5 %, with a maximal error rate up to 6.730 %. Moreover, the biosensor demonstrated high selectivity in electrochemical performance evaluation (Matrix effect = 7.425 %). The developed pre-treatment device provides simple and quick preprocessing time (within 20 min) of E. coli, and REC biosensor provides the rapid detection of E. coli (within 10 min). In conclusion, the proposed pre-treatment device and biosensor set can be easily applied to other types of bacteria, virus or germs in the near future.

A cost-effective and field-deployable sensing system for chip-integrated detection of bacteria with the naked eye

To tailor the broadly applied polymerase chain reaction (PCR) technology to point-of-care settings, an intuitive, time-saving, and cost-effective method to determine the DNA amplification results has significant practical potential. Herein we present an economical and field-deployable sensing system that enables naked-eye readout of PCR results. The proposed system is powered by an amplification enhancer to greatly enhance the positive-to-background ratio and a palm-sized readout device to assist on-site applications. High specificity, as well as single-copy sensitivity, was achieved in detecting Salmonella enterica and other bacterial pathogens. Using the proposed system, we identified the pathogenic bacterial strains in artificially contaminated food samples easily, quickly, and reliably. Also crucial for point-of-care testing, this naked-eye readout system applies to microfluidic platforms with the sample volumes scaled down to 1 μL. The time savings, portability, and cost-effectiveness of the established sensing system make it transformative for on-chip bacteria detection in point-of-care and field settings, especially in resource-limited areas.

Point-of-care bacteria detection system—integrating a bacteria test strip and a color analysis application

Point-of-care bacteria detection system—integrating a bacteria test strip and a color analysis application

Integration of Microscopic Image Capturing System for Automatic Detection of Mycobacterium Tuberculosis Bacteria

The Ministry of Health of the Republic of Indonesia is running a program to eliminate Tuberculosis (TB) by 2030. At the Primary Health Care level, AFB (acid-fast bacteria) examination confirms the TB diagnosis. In this process, the patient's sputum is prepared in the form of preparation and observed by the laboratory analyst through the lens of a microscope. The reporting process to establish this diagnosis requires calculating the number of TB bacteria in 100 fields of view per preparation. This manual microscopic observation process is tedious, and the reading results are subjective. This study offers an integrated design for automatic microscopic imaging with a computer-integrated TB bacteria detection system. The process of taking pictures is automatically obtained with the help of a driving motor added to the microscope. With the addition of this motor, the process of taking microscopic images for 100 fields of view takes ±450 seconds. The proposed system integration process can reduce laboratory analysts' work fatigue in conducting microscopic observations manually. The TB bacteria detection system utilizes the working principle of image processing techniques by combining color-deconvolution, segmentation, and contour-detection methods. The comparative value of the TB object detection system with experts resulted in a sensitivity value of 77% and a specificity value of 68%. However, the low detection rate is because the image obtained is still blurry. Thus, further investigation is needed to determine the driving motor's movement rate and the right timing for taking microscopic images so that the resulting image is not blurry. The final result that is the focus of this paper is the successful integration of the system carried out between the motor drive system on the preparation stand and the TB bacteria detection system to become a unified system.

TBC Bacteria Detection in Microscopic Image With Watershed Countur Method

Tuberculosis (TB) is an infectious disease that can be detected using a sputum sample. TB cases in Indonesia have spread throughout the region; the highest cases are in West Java. This problem makes the government do some handling and prevention of TB disease. The Bandung City Health Office (DKKB) conducted a cross-test to diagnose TB using a sputum sample. So in this study, a TB bacteria detection system, namely Mycobacterium Tuberculosis (MTB), will be made in sputum samples and their number to diagnose TB. Detection and calculation of the number of MTB are done by processing the image on the sputum sample using the watershed contour detection method. In this study, sputum sample data were obtained from DKKB. The acquisition of microscopic images at each point of the field of view is carried out using an SLR camera connected directly to the microscope to replace the function of the ocular lens. In this study, the microscopic sputum sample images were classified into positive and negative using the watershed and colorspace methods and were tested on a total of 90 microscopic images. From the system testing results, the system accuracy level is 100%, and the system precision is 100% for the detection of TB diagnosis. The system processing time averaged 5.811 seconds for 90 images used.

An automatic centrifugal system for rapid detection of bacteria based on immunomagnetic separation and recombinase aided amplification.

This study reported an automatic centrifugal system for rapid quantification of foodborne pathogenic bacteria based on immunomagnetic separation (IMS) for target bacteria enrichment and recombinase aided amplification (RAA) for nucleic acid detection. First, target bacteria were captured by immune magnetic nanoparticles (MNPs) to form magnetic bacteria, which were purified and enriched by magnetic separation. Then, nucleic acid extraction buffer was used to extract genomic DNA of magnetic bacteria and dissolve lyophilized RAA reagent. Finally, isothermal amplification and fluorescent detection were conducted for bacteria quantification. Bacteria magnetic separation, nucleic acid extraction and fluorescent RAA detection were elaborately achieved in a centrifugal disc with unique functional chambers and multistage siphon channels. A supporting device was developed to automatically and successively perform the programmed centrifugal protocol, including temperature control for isothermal amplification and fluorescence detection for real-time RAA analysis. Under optimal conditions, this centrifugal system enabled Salmonella detection as low as 10 CFU mL-1 in spiked chicken samples in 1 h with average recovery of 105.6% and average standard deviation of 8.4%. It has been demonstrated as an alternative for rapid detection of Salmonella.

Early detection and classification of live bacteria using time-lapse coherent imaging and deep learning

Early identification of pathogenic bacteria in food, water, and bodily fluids is very important and yet challenging, owing to sample complexities and large sample volumes that need to be rapidly screened. Existing screening methods based on plate counting or molecular analysis present various tradeoffs with regard to the detection time, accuracy/sensitivity, cost, and sample preparation complexity. Here, we present a computational live bacteria detection system that periodically captures coherent microscopy images of bacterial growth inside a 60-mm-diameter agar plate and analyses these time-lapsed holograms using deep neural networks for the rapid detection of bacterial growth and the classification of the corresponding species. The performance of our system was demonstrated by the rapid detection of Escherichia coli and total coliform bacteria (i.e., Klebsiella aerogenes and Klebsiella pneumoniae subsp. pneumoniae) in water samples, shortening the detection time by >12 h compared to the Environmental Protection Agency (EPA)-approved methods. Using the preincubation of samples in growth media, our system achieved a limit of detection (LOD) of ~1 colony forming unit (CFU)/L in ≤9 h of total test time. This platform is highly cost-effective (~$0.6/test) and has high-throughput with a scanning speed of 24 cm2/min over the entire plate surface, making it highly suitable for integration with the existing methods currently used for bacteria detection on agar plates. Powered by deep learning, this automated and cost-effective live bacteria detection platform can be transformative for a wide range of applications in microbiology by significantly reducing the detection time and automating the identification of colonies without labelling or the need for an expert.

Recyclable metal nanoparticle-immobilized polymer dot on montmorillonite for alkaline phosphatase-based colorimetric sensor with photothermal ablation of Bacteria

Development of simultaneous bacteria detection and eradication with simple, rapid, and reusable material is important in addressing bacterial contamination issues. In this study, we utilized the expression of alkaline phosphatase (ALP) from bacteria to design fluorescence ON/OFF system for bacteria detection, also using metal oxide nanoparticle for obtaining antibacterial activity and recyclability. The fluorescent-based biosensor with antibacterial activity was prepared by intercalating ALP-sensitive polymer dot (PD) containing β-cyclodextrin (β-CD) onto montmorillonite (MMT) as loading matrix via ionic exchange reaction, followed by immobilization of magnetic iron oxide (Fe3O4) and NIR-responsive cesium tungsten oxide (CsWO3). The PD-βCD-MMT/Fe3O4–CsWO3 nanocomposite exhibited strong fluorescence intensity, which was quenched in the presence of bacterial ALP (0–1000 U/L) due to hydrolysis of p-nitrophenyl phosphate (NPP) into p-nitrophenol (NP) in the hydrophobic site of β-CD. Furthermore, the nanocomposite could detect both gram-negative Escherichia coli and gram-positive Staphylococcus aureus in the range of 101–107 CFU/mL (LOD 5.09 and 4.62 CFU/mL, respectively), and showed high antibacterial activity against bacteria by generating photothermal heat under 5 min NIR irradiation, causing damage to bacterial cells. This material also demonstrated recyclability via magnetic field exposure due to the presence of Fe3O4. In addition, the fluorescence can be recovered following pH shock and re-conjugation of β-CD molecules. After 4 cycles, nanocomposite still showed stable photothermal effects and fluorescence-based bacteria detection. Thus, this reusable material offers promising approach for simultaneous bacteria detection and killing, which is simple, rapid, and effective.

A simple mix-and-read bacteria detection system based on a DNAzyme and a molecular beacon.

Here, we describe a simple mix-and-read method for the detection of specific bacterial strains that uses a DNAzyme and a molecular beacon to generate a signal. We have greatly improved upon the previously described DNAzyme-based bacteria detection method by eliminating a tedious preparation step while maintaining detection sensitivity.

Detection and discrimination of Shigella sonnei and Shigella flexneri based on vacuolar responses in Saccharomyces cerevisiae

This study provided a system for bacteria detection based on a lysosome-like-vacuole response in the yeast Saccharomyces cerevisiae. Vacuoles are factors known to activate the immune system in the presence of foreign substances. Here, Shigella sonnei and Shigella flexneri were exposed to yeast to analyze the alteration of vacuolar enzymes. The ability to detect the bacteria was evaluated by confocal microscopy after exposing and staining vacuoles with LysoTracker. Results showed that the treatment of yeast with these bacteria increased the number of red vacuole-like organelles surrounding yeast nuclei. Thus, vacuole alteration can be used as a biomarker for bacteria detection. Next, the expression of vacuolar enzymes under the influence of bacteria was examined using two-dimensional gel electrophoresis (2-DE) method for screening specific biomarkers for each Shigella strain. Finally, the recombinant yeasts that contained biomarkers fused to different fluorescent proteins confirmed the ability of yeast to detect these two Shigella strains at concentrations ranging from 10 to 100 CFU/mL.

An Affordable Microsphere-Based Device for Visual Assessment of Water Quality.

This work developed a prototype of an affordable, long-term water quality detection device that provides a visual readout upon detecting bacterial contamination. This device prototype consists of: (1) enzyme-releasing microspheres that lyse bacteria present in a sample, (2) microspheres that release probes that bind the DNA of the lysed bacteria, and (3) a detector region consisting of gold nanoparticles. The probes bind bacterial DNA, forming complexes. These complexes induce aggregation of the gold nanoparticles located in the detector region. The nanoparticle aggregation process causes a red to blue color change, providing a visual indicator of contamination being detected. Our group fabricated and characterized microspheres made of poly (ε-caprolactone) that released lysozyme (an enzyme that degrades bacterial cell walls) and hairpin DNA probes that bind to regions of the Escherichia coli genome over a 28-day time course. The released lysozyme retained its ability to lyse bacteria. We then showed that combining these components with gold nanoparticles followed by exposure to an E. coli-contaminated water sample (concentrations tested—106 and 108 cells/mL) resulted in a dramatic red to blue color change. Overall, this device represents a novel low-cost system for long term detection of bacteria in a water supply and other applications.

Drug resistance and protoporphyrin ferrochelatase of Ralstonia mannitolilytica

To investigate the drug resistance, β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding genes of Ralstonia mannitolilytica, and to explore its structure and pathogenic function. The strain was isolated by plate streaking method and identified by automatic bacteria detection system and 16S RNA gene PCR. Microdilution method was applied for drug susceptibility test. β-lactamases, extended spectrum β-lactamases (ESBL) and carbapenemases were detected using nitrocefin-disk, Kirby-Bauer disk, and Hodge test, respectively. Five β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene of the isolate were amplified by PCR for sequencing. Bioinformatic softwares were used to analyze the structure and function of the product of protoporphyrin ferrochelatase-encoding gene. A strain belonging to Ralstonia mannitolilytica was isolated. This isolate was sensitive to cefepime, ciprofloxacin, ofloxacin and tigecycline, but resistant to five penicillins, four cephalosporins and two carbapenems antibiotics. The isolate produced β-lactamases but did not produce ESBL and carbapenemases. The isolate had five distinct β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene. The product of protoporphyrin ferrochelatase-encoding gene contained two functional domains of protoporphyrin ferrochelatase belonging to type Ⅱ chelatase superfamily that presented the most closely genetic relationship with the protoporphyrin ferrochelatase of Neisseria meningidis. The isolate of Ralstonia mannitolilytica has a higher resistance to β-lactam antibiotics and its β-lactamase-encoding genes are different with the common bacterial β-lactamase-encoding genes. Protoporphyrin ferrochelatase may act as an important virulence factor of Ralstonia mannitolilytica.

Quantamatrix Multiplexed Assay Platform system for direct detection of bacteria and antibiotic resistance determinants in positive blood culture bottles

ObjectivesRapid and accurate identification of the causative pathogens of bloodstream infections (BSIs) is crucial for initiating appropriate antimicrobial therapy, which decreases the related morbidity and mortality rates. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay system, the Quantamatrix Multiplexed Assay Platform (QMAP) system, obtained directly from blood culture bottles, to simultaneously detect the presence of bacteria and identify the genes for antibiotic resistance. MethodsThe QMAP system was used to evaluate 619 blood culture bottles from patients with BSIs and to compare the results of conventional culture methods. ResultsUsing conventional bacterial cultures as the reference standard, the sensitivity, specificity, positive predictive value, and negative predictive value of the QMAP system for detection of bacterial pathogens in positive blood culture (PBC) samples were 99.8% (n=592, 95% CI 0.9852-1.000, p <0.001), 100% (95% CI 0.983-1.000, p <0.001), 100% (95% CI 0.9922-1.000, p <0.001), and 99.5% (95% CI 0.9695-1.000, p <0.001), respectively. In addition, sensitivity and specificity of the QMAP system for identification of the genes for antibiotic resistance were 99.4% (n=158, 95% CI 0.9617-0.9999, p <0.009) and 99.6% (95% CI 0.9763-0.9999, p <0.0001), respectively. ConclusionsObtaining results using the QMAP system takes about 3 hr, while culture methods can take 48–72 hr. Therefore, analysis using the QMAP system is rapid and reliable for characterizing causative pathogens in BSIs.

Modular development of an inline monitoring system for waterborne pathogens in raw and drinking water

The state-of-the-art monitoring of drinking water hygiene is based on the cultivation and enumeration of indicator bacteria. Despite its proven reliability, this approach has the disadvantages of being (a) relatively slow and (b) limited to a small number of indicator bacteria. Ideally, alternative methods would be less time-consuming while providing information about a larger set of hygienically relevant microorganisms including viruses. In this paper, we present insights into the design of a modular concentration and detection system for bacteria, bacteriophages and viruses. Following further validation, this or similar techniques have the potential to extend and speed up the monitoring of raw and drinking water hygiene in the future. The system consists of different modules for the concentration of microorganisms, an amplification and detection unit that includes a module for the differentiation between live and dead microorganisms, and an automated system for decision support and self-diagnosis. The ongoing testing under controlled laboratory conditions and real-life conditions in the water supply industry yields further system improvements. Moreover, the increased sensitivity and broader range of microbiological parameters emphasize the need for a reconsideration of the currently used criteria for the assessment of (drinking) water hygiene.

Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis

The propidium monoazide (PMA) coupled with PCR (viability PCR) is used in foodborne pathogen detection in order to detect only viable bacteria. Originally presented to fully remove the signal of dead bacteria, the limits of the viability PCR rapidly came out in the literature. In this study, the use of PMA in a viability-qPCR (v-qPCR) was assessed on viable and dead cells of Salmonella enterica subsp. enterica serovar Enteritidis. The PMA treatment protocol was modified (dark incubation duration, concentration of PMA) to evaluate if a complete negative signal of dead Salmonella was possible. However, none of these modifications was found to improve the removal of the remaining qPCR signal observed in the presence of dead bacteria. The present research also underlines that PMA may unexpectedly decrease the qPCR signal observed on living S. Enteritidis at low concentration. Finally, the use of S. Enteritidis cells killed by processes altering or not the cell-wall/membrane gives us a clue to answering the question about the non-total extinction of the signal of dead cells sample in the v-qPCR assay. Indeed, the data strongly indicate that the remaining qPCR signal observed in non-culturable cells does not only depend on the cell-wall/membrane integrity of the bacteria. According to these results, the authors suggest that for a rapid and reliable foodborne bacteria detection system, an enrichment followed by a qPCR analysis should be preferred to a v-qPCR.

Novel System to Detect Bacteria in Real Time in Aquatic Environments

Bacteria tests are conducted for quality control in many different industries. However, the cultivation method takes a long period of time to obtain results and there are more than a few bacteria that are difficult to cultivate. We have focused on the autofluorescence substance in the bacteria to detect them, and developed a sensor to measure the bacteria in real-time, without any pretreatments or addition of any reagents. This system uses a 405nm laser focused on the sample flowing through the flow-cell in order to detect the fluorescent light from the bacteria as well as scattered light. Fluorescent light and scattered light are separated by a dichroic mirror, and the number of viable particles (bacteria) and that of non-viable particles are obtained. We tested this system using fluorescent polystyrene latex particles and several bacterial strains, and confirmed that it had good detection capability. We believe that this system will become a next-generation bacteria detection system and help the introduction of PAT (process analytical technology) to all areas where real time and on-site detection is needed.

BiotecVisions 2012, November

BiotecVisions 2012, November

Design of Bioluminescence Sensor System for Rapid Detection of Bacteria in Samples without Cultivation

Based on the principle of ATP bioluminescent reaction, we design a portable bioluminescence sensor system to detect bacteria in samples rapidly, which included an optical sensing cell, a flow injection unit and a center processing unit. A fast ATP extraction technique was developed by using trichloro acetic acid as extraction reagent Experimental results showed that the intracellular ATP could be released from bacterial cells sufficiently within 5 min by TCA In the presence of Lucifer ace the reaction between ATP and lucifer5in would generate light and the luminescence intensity RLU was proportional to the amount of bacteria existed in the sample. The dynamic range of the sensor system is 10-107 CFU /ml. The linear regression coefficient was 0.976, and the detection limit was 10CFU /ml. The whole testing time needed was about 10 min. Compared with the plate count method, bacteria concentration in samples could be detected rapidly by using the portable sensor system without cultivation.

Mice that exclusively express TLR4 on endothelial cells can efficiently clear a lethal systemic Gram-negative bacterial infection

Recognition of LPS by TLR4 on immune sentinel cells such as macrophages is thought to be key to the recruitment of neutrophils to sites of infection with Gram-negative bacteria. To explore whether endothelial TLR4 plays a role in this process, we engineered and imaged mice that expressed TLR4 exclusively on endothelium (known herein as EndotheliumTLR4 mice). Local administration of LPS into tissue induced comparable neutrophil recruitment in EndotheliumTLR4 and wild-type mice. Following systemic LPS or intraperitoneal E. coli administration, most neutrophils were sequestered in the lungs of wild-type mice and did not accumulate at primary sites of infection. In contrast, EndotheliumTLR4 mice showed reduced pulmonary capillary neutrophil sequestration over the first 24 hours; as a result, they mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wild-type mice in the first 48 hours. In fact, the only defect we detected in EndotheliumTLR4 mice was a failure to accumulate neutrophils in the lungs following intratracheal administration of LPS; this response required TLR4 on bone marrow-derived immune cells. Therefore, endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow-derived immune cells are critical for pathogen detection at barrier sites. Nonendothelial TLR4 contributes to failure to accumulate neutrophils at primary infection sites in a disseminated systemic infection.

Culture‐based bacterial detection systems for platelets: the effect of time prior to sampling and duration of incubation required for detection with aerobic culture

Bacterial contamination of platelet (PLT) products occurs at low concentrations requiring a period of incubation for growth to minimize sampling error. Culture-based detection methods also need sufficient incubation time; together these periods may limit the useful life of PLTs. This study characterizes the impact of sampling and detection times with two commercially available bacteria detection products. Apheresis PLTs inoculated with nine bacterial species at low concentrations were sampled immediately and 24 hours after inoculation. Test results were analyzed after incubation at 16, 20, and 24 hours after sampling with two bacterial detection systems. When sampled immediately after inoculation, two commercially available bacterial detection systems (BacT/ALERT, bioMérieux; and eBDS, Pall Corp.) failed to detect some PLTs inoculated with Staphylococcus epidermidis, Serratia liquefaciens, or Pseudomonas aeruginosa and S. epidermidis, S. liquefaciens, Bacillus cereus, or P. aeruginosa, respectively. The BacT/ALERT was better at 20 hours (p<0.02), but not at 16 or 24 hours for Time 0 sampling. When sampling occurred 24 hours after inoculation, there were no difference between the two systems. Results suggest that for either bacteria detection system, holding PLTs for 24 hours before sampling improves the detection sensitivity for PLTs contaminated with low concentrations of bacteria, and longer incubation periods improve detection.