Fluorescein-labeled beta-lactamase mutant for high-throughput screening of bacterial beta-lactamases against beta-lactam antibiotics

The increasing emergence of new bacterial beta-lactamases that can efficiently hydrolyze beta-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for beta-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled beta-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen beta-lactamases, including the Bacillus cereus beta-lactamase I (PenPC), B. cereus beta-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various beta-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible Omega-loop, which is close to the enzyme's active site, with a cysteine residue on a class A beta-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of beta-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial beta-lactamases and beta-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial beta-lactamases and beta-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of beta-lactamase-resistant antibiotics from beta-lactamase-sensitive ones in the screening of bacterial beta-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent beta-lactam antibiotics for treatment of bacterial infections.     

Combination of immunosensor detection with viability testing and confirmation using the polymerase chain reaction and culture

Rapid and accurate differential determination of viable versus nonviable microbes is critical for formulation of an appropriate response after pathogen detection. Sensors for rapid bacterial identification can be used for applications ranging from environmental monitoring and homeland defense to food process monitoring, but few provide viability information. This study combines the rapid screening capability of the array biosensor using an immunoassay format with methods for determination of viability. Additionally, cells captured by the immobilized antibodies can be cultured following fluorescence imaging to further confirm viability and for cell population expansion for further characterization, e.g., strain identification or antibiotic susceptibility testing. Finally, we demonstrate analysis of captured bacteria using the polymerase chain reaction (PCR). PCR results for waveguide-captured cells were 3 orders of magnitude more sensitive than the fluorescence immunoassay and can also provide additional genetic information on the captured microbes. These approaches can be used to rapidly detect and distinguish viable versus nonviable and pathogenic versus nonpathogenic captured organisms, provide culture materials for further analysis on a shorter time scale, and assess the efficacy of decontamination or sterilization procedures.     

Detection and quantification of bacterial autofluorescence at the single-cell level by a laboratory-built high-sensitivity flow cytometer

Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.     

In Vivo Dynamic Monitoring of Bacterial Infection by NIR‐II Fluorescence Imaging

Time window of antibiotic administration is a critical but long‐neglected point in the treatment of bacterial infection, as unnecessary prolonged antibiotics are increasingly causing catastrophic drug‐resistance. Here, a second near‐infrared (NIR‐II) fluorescence imaging strategy based on lead sulfide quantum dots (PbS QDs) is presented to dynamically monitor bacterial infection in vivo in a real‐time manner. The prepared PbS QDs not only provide a low detection limit (10⁴ CFU mL^?1) of four typical bacteria strains in vitro but also show a particularly high labeling efficiency with Escherichia coli (E. coli). The NIR‐II in vivo imaging results reveal that the number of invading bacteria first decreases after post‐injection, then increases from 1 d to 1 week and drop again over time in infected mouse models. Meanwhile, there is a simultaneous variation of dendritic cells, neutrophils, macrophages, and CD8+ T lymphocytes against bacterial infection at the same time points. Notably, the infected mouse self‐heals eventually without antibiotic treatment, as a robust immune system can successfully prevent further health deterioration. The NIR‐II imaging approach enables real‐time monitoring of bacterial infection in vivo, thus facilitating spatiotemporal deciphering of time window for antibiotic treatment.     

Antibiotic-Free Antibacterial Strategies Enabled by Nanomaterials: Progress and Perspectives

Bacterial infection is one of the top ten leading causes of death globally and the worst killer in low-income countries. The overuse of antibiotics leads to ever-increasing antibiotic resistance, posing a severe threat to human health. Recent advances in nanotechnology provide new opportunities to address the challenges in bacterial infection by killing germs without using antibiotics. Antibiotic-free antibacterial strategies enabled by advanced nanomaterials are presented. Nanomaterials are classified on the basis of their mode of action: nanomaterials with intrinsic or light-mediated bactericidal properties and others that serve as vehicles for the delivery of natural antibacterial compounds. Specific attention is given to antibacterial mechanisms and the structure–performance relationship. Practical antibacterial applications employing these antibiotic-free strategies are also introduced. Current challenges in this field and future perspectives are presented to stimulate new technologies and their translation to fight against bacterial infection.     

Targeted photothermal lysis of the pathogenic bacteria, Pseudomonas aeruginosa, with gold nanorods

Increases in the prevalence of antibiotic resistant bacteria require new approaches for the treatment of infectious bacterial pathogens. It is now clear that a nanotechnology-driven approach using nanoparticles to selectively target and destroy pathogenic bacteria can be successfully implemented. We have explored this approach by using gold nanorods that have been covalently linked to primary antibodies to selectively destroy the pathogenic Gram-negative bacterium, Pseudomonas aeruginosa. We find that, following nanorod attachment to the bacterial cell surface, exposure to near-infrared radiation results in a significant reduction in bacterial cell viability.     

纳米材料在电化学传感器检测抗生素中的应用进展

抗生素自发现至今,由于其可以阻碍细菌的生长,被广泛应用于预防和治疗细菌的感染疾病上。但是抗生素在畜牧业、农业等方面的滥用滥排导致抗生素污染,极大地威胁水源地的安全,导致细菌耐药性增强、给环境及人类健康带来重大的危害。因此,抗生素的检测近年来得到了广泛的关注,而大多抗生素都具有电化学活性。基于此,纳米修饰电极可以使抗生素在电解质中的电化学氧化或还原反应增强,从而促进其灵敏度的提高,使电化学传感器可以检测到各类抗生素。本文详细介绍了用于检测抗生素的各种纳米材料修饰电极的电化学传感器及其性能。最后,讨论了纳米材料电化学传感器在抗生素检测中面临的挑战和发展前景。     

Recent advances in hydrogel-based anti-infective coatings

Bacterial infections associated with biomedical devices and implants have posed a great challenge to global healthcare systems.These infections are mainly caused by bacterial biofilm formed on the surface of biomaterials,protecting the encapsulated bacteria from conventional antibiotic treatment and attack of the immune system.As the bacterial biofilm is difficult to eradicate,bactericidal and antifouling coatings have emerged as promising strategies to prevent biofilm formation and subsequent infections.Hydrogels with three-dimensional crosslinked hydrophilic networks,tunable mechanical property and large drug-loading capacity are desirable coating materials,which can kill bacteria and/or prevent bacterial adhesion on the surface,inhibiting biofilm formation.Herein,we review recent developments of hydrogels as anti-infective coatings.Particularly,we highlight two chemical approaches(graft-from and graft-to),which have been used to immobilize hydrogels on surfaces,and present advances in the development of bactericidal(contact-killing and antimicrobial-releasing),antifouling(hydrophilic polymer network)and bifunctional hydrogel coatings with both bactericidal and antifouling activities.In addition,the challenges of hydrogel coatings for clinical applications are discussed,and future research directions of anti-infective hydrogel coatings are proposed.     

Biodegradable Antibacterial Polymeric Nanosystems: A New Hope to Cope with Multidrug‐Resistant Bacteria

The human society is faced with daunting threats from bacterial infections. Over decades, a variety of antibacterial polymeric nanosystems have exhibited great promise for the eradication of multidrug‐resistant bacteria and persistent biofilms by enhancing bacterial recognition and binding capabilities. In this Review, the “state‐of‐the‐art” biodegradable antibacterial polymeric nanosystems, which could respond to bacteria environments (e.g., acidity or bacterial enzymes) for controlled antibiotic release or multimodal antibacterial treatment, are summarized. The current antibacterial polymeric nanosystems can be categorized into antibiotic‐containing and intrinsic antibacterial nanosystems. The antibiotic‐containing polymeric nanosystems include antibiotic‐encapsulated nanocarriers (e.g., polymeric micelles, vesicles, nanogels) and antibiotic‐conjugated polymer nanosystems for the delivery of antibiotic drugs. On the other hand, the intrinsic antibacterial polymer nanosystems containing bactericidal moieties such as quaternary ammonium groups, phosphonium groups, polycations, antimicrobial peptides (AMPs), and their synthetic mimics, are also described. The biodegradability of the nanosystems can be rendered by the incorporation of labile chemical linkages, such as carbonate, ester, amide, and phosphoester bonds. The design and synthesis of the degradable polymeric building blocks and their fabrications into nanosystems are also explicated, together with their plausible action mechanisms and potential biomedical applications. The perspectives of the current research in this field are also described.     

Antimicrobial effects of nanofiber poly(caprolactone) tissue scaffolds releasing rifampicin

This study quantified the antibiotic release kinetics and subsequent bactericidal efficacy of rifampicin (RIF) against Gram-positive and Gram-negative bacteria under in vitro static conditions. Antibiotic-loaded scaffolds were fabricated by electrospinning poly(caprolactone) (PCL) with 10% or 20% (w/w) RIF. Scaffold fiber diameter and RIF loading were characterized, and RIF release kinetics were measured. RIF-releasing and RIF-free scaffolds were inoculated with Pseudomonas aeruginosa and Staphylococcus epidermidis, and the suspended concentration live and dead bacteria were determined by fluorescent microscopy. Adherent bacteria and biofilm formation were examined using scanning electron microscopy. Mean fiber diameters were 557 ± 399 nm for RIF-free, 402 ± 225 nm for 10% RIF, and 665 ± 402 nm for 20% RIF scaffolds. RIF release kinetics exhibited a short-burst release during the first hour, followed by a 7 h, zero-order release during which both RIF scaffolds released ~50% of their initial RIF mass loading. P. aeruginosa and S. epidermidis suspended cell populations proliferated in accordance with logarithmic growth models when exposed to control scaffolds; however both RIF-containing scaffolds completely inhibited bacterial growth in suspension and, subsequently, prevented biofilm formation within the scaffolds through the first 6 h.     

Enhanced antibacterial effect of azlocillin in conjugation with silver nanoparticles against Pseudomonas aeruginosa

In recent years, the problems associated with bacterial resistance to antibiotics caused nanodrugs to be considered as a new way for infectious diseases treatment. The main purpose of this study was to develop a new agent against Pseudomonas aeruginosa, a very difficult bacterium to treat, based on azlocillin antibiotic and silver nanoparticles (AgNPs). Azlocillin was conjugated with AgNPs by chemical methods and its antimicrobial activity was studied against P. aeruginosa using well diffusion agar method. Then, minimum inhibitory concentration and minimum bactericidal concentration of the new conjugate was specified with macro‐dilution method. The animal study showed the considerable enhanced antibacterial effect of azlocillin in conjugation with AgNPs against P. aeruginosa in comparison with azlocillin alone, AgNPs alone and azlocillin in combination with AgNPs.Inspec keywords: antibacterial activity, silver, nanoparticles, organic compounds, microorganisms, drugs, nanomedicine, biomedical materials, diseases, diffusion, nanofabricationOther keywords: Ag, macrodilution method, minimum bactericidal concentration, minimum inhibitory concentration, well diffusion agar method, P. aeruginosa, antimicrobial activity, chemical methods, azlocillin antibiotic nanoparticles, infectious diseases treatment, nanodrugs, bacterial resistance, Pseudomonas aeruginosa, silver nanoparticles, antibacterial effect     

Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction

The clinically important vancomycin antibiotic inhibits the growth of pathogens such as Staphylococcus aureus by blocking cell wall synthesis through specific recognition of nascent peptidoglycan terminating in D-Ala-D-Ala. Here, we demonstrate the ability of single-molecule atomic force microscopy with antibiotic-modified tips to measure the specific binding forces of vancomycin and to map individual ligands on living bacteria. The single-molecule approach presented here provides new opportunities for understanding the binding mechanisms of antibiotics and for exploring the architecture of bacterial cell walls.     

Monitoring the mode of action of antibiotics using Raman spectroscopy: investigating subinhibitory effects of amikacin on Pseudomonas aeruginosa

During the last 20 years the rate at which new antimicrobial agents are produced has decreased dramatically, with concomitant increase in the number of pathogens that are becoming multidrug resistant. Together these have created a patient healthcare risk and this is of great concern. A crucial aspect for the discovery of new antibiotics is the development of new techniques that allow rapid and accurate characterization of the mode of action of the pharmacophore. In this work UV resonance Raman (UVRR) spectroscopy has been developed to monitor the concentration effect of antibiotics on bacterial cells. UVRR was conducted at 244 nm and spectra were collected in typically 60 s. Supervised multivariate analysis and 2D correlation spectroscopy were used to evaluate whether the UVRR spectra contained valuable information that could be used to study the mode of action of antibiotics. The clustering pattern in the discriminant factors space correlated directly to the concentration of amikacin, and partial least squares (PLS) regression analysis of the UVRR spectra was able to predict the concentration of amikacin to which bacterial cells had been exposed. 2D correlation spectroscopy contour maps indicated that spectral changes due to the presence of amikacin in the growth media occur according to the known mode of action of the studied antibiotic. Therefore, we conclude that UVRR spectroscopy, when coupled with chemometrics and 2D correlation spectroscopy, constitutes a powerful approach for the development and screening of new antibiotics.     

Effects of ozone nano-bubble water on periodontopathic bacteria and oral cells - in vitro studies

The aims of the present study were to evaluate the bactericidal activity of a new antiseptic agent, ozone nano-bubble water (NBW3), against periodontopathogenic bacteria and to assess the cytotoxicity of NBW3 against human oral cells. The bactericidal activities of NBW3 against representative periodontopathogenic bacteria, Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) were evaluated using in vitro time-kill assays. The cytotoxicity of NBW3 was evaluated using three-dimensional human buccal and gingival tissue models. The numbers of colony forming units (CFUs)/mL of P. gingivalis and A. actinomycetemcomitans exposed to NBW3 dropped to below the lower limit of detection (<10 CFUs mL⁻¹) after only 0.5 min of exposure. There were only minor decreases in the viability of oral tissue cells after 24 h of exposure to NBW3. These results suggest that NBW3 possesses potent bactericidal activity against representative periodontopathogenic bacteria and is not cytotoxic to cells of human oral tissues. The use of NBW3 as an adjunct to periodontal therapy would be promising.     

Single Upconversion Nanoparticle–Bacterium Cotrapping for Single‐Bacterium Labeling and Analysis

Detecting and analyzing pathogenic bacteria in an effective and reliable manner is crucial for the diagnosis of acute bacterial infection and initial antibiotic therapy. However, the precise labeling and analysis of bacteria at the single‐bacterium level are a technical challenge but very important to reveal important details about the heterogeneity of cells and responds to environment. This study demonstrates an optical strategy for single‐bacterium labeling and analysis by the cotrapping of single upconversion nanoparticles (UCNPs) and bacteria together. A single UCNP with an average size of ≈120 nm is first optically trapped. Both ends of a single bacterium are then trapped and labeled with single UCNPs emitting green light. The labeled bacterium can be flexibly moved to designated locations for further analysis. Signals from bacteria of different sizes are detected in real time for single‐bacterium analysis. This cotrapping method provides a new approach for single‐pathogenic‐bacterium labeling, detection, and real‐time analysis at the single‐particle and single‐bacterium level.     

Antimicrobial activity of UV-irradiated nylon film for packaging applications

Antimicrobial packaging could enhance food storage life and safety. An antimicrobial moiety that is permanently bound to the polymer surface and does not leach has particular appeal. The use of 193 nm UV irradiation to convert amide groups on the surface of nylon to amines having antimicrobial activity has been reported previously. We prepared materials accordingly and explored their mode of action and activity against pathogens. Three food related bacterial strains, Staphylococcus aureus, Pseudomonas fluorescens and Enterococcus faecalis were exposed to antimicrobial film in 0.2 M sodium phosphate buffer (pH 7.0). Samples were held shaken at 100 r.p.m. in a 25°C incubator. The antimicrobial film was effective in reduction of microbial concentration in the bulk fluid for all food-related bacteria tested. The effectiveness was dependent on the bacterial strain. Adsorption of bacterial cells diminished the effectiveness of amine groups. Experimental results indicate that the decrease in concentration of bacterial cells in bulk fluid is more likely to be the bactericidal action than adsorption of live cells. © 1998 John Wiley & Sons, Ltd.     

Hydrothermal synthesis of fluorescent silicon nanoparticles using maleic acid as surface-stabilizing ligands

Water-soluble silicon nanoparticles (SiNPs) have been synthesized with photoluminescence quantum yield more than 32% via a hydrothermal treatment of 3-aminopropyltriethoxysilane as silicon sources and maleic acid (MA) as surface-stabilizing ligands. Prepared SiNPs showed the presence of carboxylic acid groups through the incorporation of MA. The presence of 48.8 and 51.2% of Si–Si and Si–O binding was observed in the resulting carboxylic acid-functionalized SiNPs (COOH-SiNPs). As revealed by the fluorescence lifetime images, COOH-SiNPs possesses several fluorophores mainly composed of above Si–Si binding inside of single particle, which explains the excitation-dependent fluorescence emission behavior of COOH-SiNPs. Also, the presence of oxides mainly composed of Si–O binding and MA on the surface of COOH-SiNPs provides long-term stability for both fluorescence and dispersion. The potential use of COOH-SiNPs as fluorescence bioimaging agents for cellular media has been demonstrated. COOH-SiNPs showed excellent cell viability more than 91% for both MDAMB and MDCK cells even in 1,000 ppm concentration, and multicolor fluorescence imaging (blue, green, and red) of MDAMB cells was successfully accomplished with different excitation wavelengths.     

Enhanced detection of live bacteria using a dendrimer thin film in an optical biosensor

Here we describe the detection of live Pseudomonas aeruginosa using a sensing film containing a fourth-generation hydroxy-terminated polyamidoamine (PAMAM) dendrimer (i.e., G4-OH) and SYTOX Green fluorescent nucleic acid stain. The films are configured on simple, disposable plastic coupons or optical fibers and are interrogated using a miniature fiber-optic spectrometer. SYTOX Green is generally considered a dead cell stain because it is not able to cross the membranes of live cells. In the presence of PAMAM-OH (G4-OH) in water, the bacterial cell becomes permeable to the SYTOX dye and the fluorescence is significantly enhanced. The fluorescence increases with the bacteria concentration, and the intensity at 5.4 x 10(7) cells mL(-1) is 350% higher than the liquid controls without PAMAM-OH. We also demonstrate that dendrimers stabilize the sensing film. After drying and desiccation, the SYTOX Green/PAMAM-OH films are still able to quantitatively detect P. aeruginosa in water. Incorporation of glucose into the SYTOX Green/ PAMAM-OH film may improve the homogeneity of the film and enhances the fluorescence signal an additional 11-25%.     

Quantitative imaging of gene expression in individual bacterial cells by chemiluminescence

Recent gene expression studies at the single bacterial cell level have primarily used green fluorescent protein (GFP) as the reporter. However, fluorescence monitoring has intrinsic limitations, such as GFP maturation time, high background, and photobleaching. To overcome those problems, we introduce the alternative approach of chemiluminescence (CL) detection with firefly luciferase as the probe. Firefly luciferase is roughly 100 times more efficient and is faster in generating CL than bacterial luciferase but requires the introduction of luciferin, a species that is not native to bacteria. The difficulty of luciferin diffusion into the cells was solved by making use of cell membrane leakage during bacteria dehydration. In this scheme, the overall sensitivity of the system approaches the single protein molecule level. Quantitative studies of gene expression in BL21 and XLU102 bacteria can thus be performed.     

Ultrasensitive reporter protein detection in genetically engineered bacteria

We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response. Although we use arsenic inducible bioreporters here, the method is extensible to gfp/egfp bioreporters that are responsive to other substances.