Sensitive Pathogenic Bacteria Research Articles

A dual-channel electrochemical biosensor enables concurrent detection of pathogens and antibiotic resistance

Diarrheagenic E. coli infections, commonly treated with β-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the β-lactam resistance marker, β-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a β -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For β-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, β-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and β-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and β-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.

Exo-III Enzyme-Assisted Triple Cycle Signal Amplifications for Sensitive and Accurate Identification of Pathogenic Bacteria.

Early determination of infectious pathogens is vitally important to select appropriate antibiotics, and to manage nosocomial infection. Herein, we propose a target recognition triggered triple signal amplification-based approach for sensitive pathogenic bacteria detection. In the proposed approach, a double-strand DNA probe (capture probe) that is composed of an aptamer sequence and a primer sequence is designed for specific identification of target bacteria and initiation of following triple signal amplification. After recognition of target bacteria, primer sequence is released from capture probe to bind with the designed H1 probe, forming a blunt terminal in the H1 probe. Exonuclease-III (Exo-III enzyme) specifically recognizes the blunt terminal in H1 probe and degrades the sequence from 3' terminal, resulting a single-strand DNA to induce the following signal amplification. Eventually, the approach exhibits a low detection limit of 36cfu/mL with a broad dynamic range. The high selectivity endows the method a promising prospective for clinical sample analysis.

Sensitive and Portable Detection of Bacteria Using Exonuclease-III (Exo-III) Assisted Signal Amplification and Personal Glucose Meters.

Despite major advances in combating pathogenic bacteria caused infection, infectious diseases remain a major cause of death today. A variety of bacteria detection approaches have been established by exploiting the aptamer to specifically identify target. However, these methods require specific equipment or laboratory based instruments, which is expensive and not easily available to the public. To deal with this issue, we develop here a portable and sensitive pathogenic bacteria detection approach through integrating capture probe based recognition and Exo-III assisted signal amplification. In this method, a designed capture probe identifies the surface protein of target bacteria, leading to the release of blocker sequence. The released blocker sequence mediates Exo-III enzyme based signal amplification, generating a large amount of signal strand DNA (ssDNA) sequences. The obtained ssDNA works as a linker to conjugate streptavidin magnetic beads (SMBs) with DNA invertase. After magnet based enrichment and washing away of free DNA invertase, the SMBs-DNA invertase complex is used to catalyze the hydrolysis of sucrose into glucose for PGM readout. Based on this, the approach exhibits a 6 order of magnitudes in detecting Staphylococcus Aureus (S. aureus) with the limit of detection as low as 78cfu/mL. In addition, the capability to specifically detect target bacteria and high repeatability (relative standard deviation, 4.44%) endows the method wider applicable scenes. In all, the established shows a promising prospect in the detection of bacteria pathogen and the early-diagnosis of bacterial infections.

The Mechanism of Bacterial Resistance and Potential Bacteriostatic Strategies.

Bacterial drug resistance is rapidly developing as one of the greatest threats to human health. Bacteria will adopt corresponding strategies to crack the inhibitory effect of antibiotics according to the antibacterial mechanism of antibiotics, involving the mutation of drug target, secreting hydrolase, and discharging antibiotics out of cells through an efflux pump, etc. In recent years, bacteria are found to constantly evolve new resistance mechanisms to antibiotics, including target protective protein, changes in cell morphology, and so on, endowing them with multiple defense systems against antibiotics, leading to the emergence of multi-drug resistant (MDR) bacteria and the unavailability of drugs in clinics. Correspondingly, researchers attempt to uncover the mystery of bacterial resistance to develop more convenient and effective antibacterial strategies. Although traditional antibiotics still play a significant role in the treatment of diseases caused by sensitive pathogenic bacteria, they gradually lose efficacy in the MDR bacteria. Therefore, highly effective antibacterial compounds, such as phage therapy and CRISPER-Cas precision therapy, are gaining an increasing amount of attention, and are considered to be the treatments with the moist potential with regard to resistance against MDR in the future. In this review, nine identified drug resistance mechanisms are summarized, which enhance the retention rate of bacteria under the action of antibiotics and promote the distribution of drug-resistant bacteria (DRB) in the population. Afterwards, three kinds of potential antibacterial methods are introduced, in which new antibacterial compounds exhibit broad application prospects with different action mechanisms, the phage therapy has been successfully applied to infectious diseases caused by super bacteria, and the CRISPER-Cas precision therapy as a new technology can edit drug-resistant genes in pathogenic bacteria at the gene level, with high accuracy and flexibility. These antibacterial methods will provide more options for clinical treatment, and will greatly alleviate the current drug-resistant crisis.

Highly sensitive and label-free digital detection of whole cell E. coli with Interferometric Reflectance Imaging

Bacterial infectious diseases are a major threat to human health. Timely and sensitive pathogenic bacteria detection is crucial in bacterial contaminations identification and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards developing new biosensors. Biosensors offering label-free, whole bacteria detection are highly desirable over those relying on label-based or pathogenic molecular components detection. The major advantage is eliminating the additional time and cost required for labeling or extracting the desired bacterial components. Here, we demonstrate rapid, sensitive and label-free Escherichia coli (E. coli) detection utilizing interferometric reflectance imaging enhancement allowing visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate an extrapolated limit of detection of 2.2 CFU/ml from experimental data in buffer solution with no sample preparation. To the best of our knowledge, this level of sensitivity for whole E. coli detection is unprecedented in label-free biosensing. The specificity of our biosensor is validated by comparing the response to target bacteria E. coli and non-target bacteria S. aureus, K. pneumonia and P. aeruginosa. The biosensor’s performance in tap water proves that its detection capability is unaffected by the sample complexity. Furthermore, our sensor platform provides high optical magnification imaging and thus validation of recorded detection events as the target bacteria based on morphological characterization. Therefore, our sensitive and label-free detection method offers new perspectives for direct bacterial detection in real matrices and clinical samples.

Point-of-Care Testing of Pathogenic Bacteria at the Single-Colony Level via Gas Pressure Readout Using Aptamer-Coated Magnetic CuFe2O4 and Vancomycin-Capped Platinum Nanoparticles.

Pressure measurements are performed everyday with simple devices, and in the field of analytical chemistry the pressure-based signaling strategy offers two important advantages, signal amplification and particular applicability in point-of-care settings. Herein, by using vancomycin (Van)-functionalized platinum nanoparticles (PtNPs@Van) and aptamer-coated magnetic CuFe2O4 nanoprobes dual-recognition units integrated with a catalyzed breakdown of H2O2 for O2 generation, we demonstrated that gas pressure can be used as a readout means for highly sensitive pathogenic bacteria identification and quantification. Using Staphylococcus aureus ( S. aureus) as a test case, integration of the molecular dual-recognition component with the catalyzed gas-generation reaction leads to a significant pressure change (Δ P), and the correlation between the concentration of S. aureus and the Δ P signal was found to be linear from 5.0 to 1.0 × 104 cfu/mL with a detection limit of 1.0 cfu/mL. Other nontarget bacteria show negative results, verifying the high specificity of the present strategy. When employed to assay S. aureus in saliva and milk samples, the approach shows recoveries from 93.3% to 107.1% with relative standard derivation (RSD) less than 8.8%. By the integration of catalyzed gas-generation reaction with the designed molecular recognition event, obviously the pressure-based signaling strategy could facilitate pathogenic bacteria identification and quantification not only in the laboratory but also in point-of-care settings, which could have great potential in the application of food safety and infectious disease diagnosis.

Determination of Antimicrobial Effects of Probiotic Lactic Acid Bacteria and Garlic Extract Against Some Foodborn Pathogenic Bacteria

In this study, it was investigated that the inhibition effect of some lactic acid bacteria (Lactobacillus acidophilus NCC68, Lactobacillus casei Shirota, Lactobacillus rhamnosus (Ezal, commercial starter cultures)) which possessed with probiotic characteristics, against Bacillus cereus, Salmonella Enteritidis, Escherichia coli, Escherichia coli 0157:H7 ATCC 35150 and Staphylococcus aureus ATCC 25923. Besides, the inhibitory effect of probiotic cultures which used with meat and meat product additives that garlic extract over the antagonistic effects of sensitive pathogens were investigated in vitro. Consequently, the whole of lactic acid bacteria and garlic extract which were used in this study, showed inhibition effects against all selected pathogenic bacteria. Staphylococcus aureus ATCC 25923 was determined as the most sensitive pathogenic bacteria while Bacillus cereus was the most resistant bacteria against lactic acid bacteria and garlic extract. There was a distinctive increase in inhibition effects were observed by used of a combination with lactic acid bacteria and garlic extract.

Graphene-interfaced electrical biosensor for label-free and sensitive detection of foodborne pathogenic E. coli O157:H7

E. coli O157:H7 is an enterohemorrhagic bacteria responsible for serious foodborne outbreaks that causes diarrhoea, fever and vomiting in humans. Recent foodborne E. coli outbreaks has left a serious concern to public health. Therefore, there is an increasing demand for a simple, rapid and sensitive method for pathogen detection in contaminated foods. In this study, we developed a label-free electrical biosensor interfaced with graphene for sensitive detection of pathogenic bacteria. This biosensor was fabricated by interfacing graphene with interdigitated microelectrodes of capacitors that were biofunctionalized with E. coli O157:H7 specific antibodies for sensitive pathogenic bacteria detection. Here, graphene nanostructures on the sensor surface provided superior chemical properties such as high carrier mobility and biocompatibility with antibodies and bacteria. The sensors transduced the signal based on changes in dielectric properties (capacitance) through (i) polarization of captured cell-surface charges, (ii) cells' internal bioactivity, (iii) cell-wall's electronegativity or dipole moment and their relaxation and (iv) charge carrier mobility of graphene that modulated the electrical properties once the pathogenic E. coli O157:H7 captured on the sensor surface. Sensitive capacitance changes thus observed with graphene based capacitors were specific to E. coli O157:H7 strain with a sensitivity as low as 10–100 cells/ml. The proposed graphene based electrical biosensor provided advantages of speed, sensitivity, specificity and in-situ bacterial detection with no chemical mediators, represents a versatile approach for detection of a wide variety of other pathogens.

Antibacterial activity of supernatant obtained from Weissella koreensis and Lactobacillus sakei on the growth of pathogenic bacteria

This study was carried out to obtain basic data for the industrial use of Weissella koreensis and Lactobacillus sakei. The antibacterial activity of supernatants obtained from W. koreensis and L. sakei were tested against pathogenic bacteria such as Escherichia coli KCCM 11234, Salmonella enteritidis KCCM 3313, Salmonella enteritidis KCCM 12021, Salmonella typhimurium KCCM 40253, and Salmonella typhimurium KCCM 15. The supernatant of L. sakei showed antibacterial activity against E. coli KCCM 11234, S. enteritidis KCCM 12021, and S. typhimurium KCCM 15, while the supernatant of W. koreensis showed antibacterial activity against E. coli KCCM 11234 and S. enteritidis KCCM 12021. The effect of pH changes and heat treatment on antibacterial activity of the supernatants was examined using the sensitive pathogenic bacteria (E. coli KCCM 11234, S. enteritidis KCCM 12021 and S. typhimurium KCCM 15). Antibacterial activity against sensitive pathogenic bacteria was maintained under heat treatment at all temperatures, but there was no antibacterial activity associated with pH modification. Furthermore, it was confirmed that the antibacterial activity of the supernatants obtained from W. koreensis and L. sakei was a result of organic acids including, lactic, acetic, phosphoric, succinic, pyroglutamic, citric, malic, and formic acids. Therefore, the present study showed that the organic acids produced by L. sakei and W. koreensis exhibited a strong antibacterial activity against pathogenic bacteria. Moreover, in the food industry, these organic acids have the potential to inhibit the growth of pathogenic bacteria and improve the quality of stored food.

Multifunctional graphene magnetic nanosheet decorated with chitosan for highly sensitive detection of pathogenic bacteria.

Multifunctional graphene magnetic nanosheet decorated with chitosan (GMCS) is demonstrated as a promising biosensor for fluorescence spectroscopy and it can be also applied for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for sensitive pathogenic bacteria detection. Graphene-magnetic@chitosan (GMCS) can be applied for multiple applications such as fluorescence biosensor, fluorescence background suppressor, acting as co-matrix, enriching nanoprobe, and separation by external magnets for pathogenic bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) present in aqueous suspension or blood colloids. GMCS has been prepared and characterized using various techniques including TEM, UV, FTIR, XRD, Raman and fluorescence spectroscopy. Due to the non-covalent interactions among the pathogenic bacteria with the GMCS, fluorescence enhancement takes place as a function of bacterial cell number with a low number of cell detection (4.5 × 102 to 5.0 × 102 cfu mL-1) and wide linear dynamic range which indicates that the GMCS approach is an excellent quantitative methodology and highly sensitive approach for detection of bacteria cells. Graphene works intrinsically as a fluorescence quencher for blood fluorophores and cell autofluorescence as graphene typically has a long life time, which allows detection of the bacteria signals by direct fluorescence measurements. Because of the high fluorescence enhancement efficiency, and large surface area (to volume ratio) of graphene, GMCS exhibits extraordinarily high sensitivity for complex (blood) sample analysis. GMCS also can be applied as an efficient separation and preconcentration nanoprobe for surface assisted laser desorption/ionization (SALDI) and enhances the ionization of bacterial biomolecules during MALDI-MS analysis. The bacterial cell suspension was concentrated from 1 mL to 10 μL via an external magnetic field, thus it can enhance bacteria detection for MALDI-MS analysis. GMCS is an outstanding approach which is a rapid, sensitive, culture free technique and low cost biosensor for pathogenic bacteria detection.

Promoting resistance by the emission of antibiotics from hospitals and households into effluent

There is growing concern about bacterial resistance to antimicrobials. The majority of antibiotics used are only partially metabolized after administration, and are released via patient excreta into the municipal sewage system. Data on the use of antibiotics and their emission into hospital effluent are not available. Antibiotic consumption in Germany was calculated on the basis of five hospitals of varying size and medical service spectrum and on prescriptions issued by medical practitioners. The predicted environmental concentration (PEC) was calculated for hospital effluent and for municipal sewage. The PECs were compared both with published minimum inhibitory concentrations (MIC50) for sensitive pathogenic bacteria and with the predicted no-effect concentrations (PNECs). The amount of antibiotics emitted into hospital effluent may reach and exceed the MIC50 of susceptible pathogenic bacteria. The PEC/PNEC ratio is highest for hospital effluent (in some cases 10-20 times the MIC50) and frequently > 1 for municipal sewage. PECs are high enough for some compounds to have a PEC/PNEC ratio > 1 even in surface water. The volume of antibiotics used in hospitals and private households and released into effluent and municipal sewage indicates a selection pressure on bacteria. Steps should be taken to reduce the risk by proper handling of antibiotics and their residues both in hospitals and by private users.