A significant challenge in bacterial detection is the identification of viable bacteria over debris, specifically post decontamination. Of increasing concern are antibiotic resistant strains that require accurate and rapid post decontamination analysis. Current strategies are fraught with disadvantages and most of them are not selective for viable bacteria. However, bacteria are critically dependent upon iron sequestration, synthesizing and releasing siderophores (SDPs) to tightly bind iron, with the subsequent uptake of iron bound SDPs. This is a highly conserved process that occurs only in intact bacteria. Herein we report a facile method to use bacterial SDPs to selectively and rapidly identify only viable bacteria in complex matrices, and discriminate them from their dead counterparts. Desferrioxamine B (Desf B) tethered to a glass slide is used to specifically capture viable bacteria from a mixture of viable and dead Escherichia coli, as demonstrated by fluorescence microscopy. We re- port both direct conjugation of Desf B on thin-film-coated glass slides as well as biotin-streptavidin conjugation strategies, both of which are successful in the said goal. We have analyzed the density of images obtained upon fluorescence staining using edge detection with a Canny edge detector. This novel application of a software analysis tool originally developed for satellite imaging to biological staining allows for accurate quantitation of observed data.
Recent Escherichia coli (E. coli) outbreaks in Europe have resulted in over 4300 cases of infection leading to 50 deaths [
Using siderophores (SDPs)—molecules employed by the bacteria to sequester vital iron within a host—show promise as a way to selectively interrogate for viable bacteria [
Recent studies have utilized immobilized SDPs to identify pathogens and the receptors that bind the iron transporting species [10,11], highlighting the effective use of surface bound SDP’s. Furthermore, use of SDPsupported particles has been reported for aqueous iron detection at low concentrations [
The method reported herein differentiates from that of Kim et al. [
1) Covalent SDP attachment to the glass surface, which leads to greater structural integrity, and allows significantly simpler technology; 2) An innovative method to quantify fluorescence imaging, namely by using a software package initially developed for interpreting satellite images, and 3) Use of surface functionalization chemistry that facilitates direct tethering of a variety of SDPs while minimizing non-specific binding.
We elected to assess this method with Desf B, a bacterial SDP characteristic of Streptomyces species, although it is recognized by various bacteria including E. coli [
We herein report a robust, facile and rapid method for the detection of viable bacteria using a tethered SDP capture (
Glass microscope slides (“Fisher’s Finest”) were purchased from Thermo-Fisher (NJ, USA). Mesylated salt of Desf B and Diglycolic anhydride was obtained from Sigma Aldrich (MO, USA). Ethanol was obtained from Pharmco/Aaper (CT, USA). All other solvents and buffers were obtained from Sigma-Aldrich or Thermo-Fisher. 3-Aminopropylmethyldiethoxysilane (APMDES) was purchased from Gelest (PA, USA), while polyethylene glycol (PEG) reagents were obtained from Quanta Biodesign® (OH, USA); these materials were used as received without further purification. Live/Dead BacLight-TM bacterial viability kit was purchased from Invitrogen. Luria-Bertani Agar media and all other cell culture requirements were obtained from Invitrogen Photosciences, Inc. (CA, USA). Fluorescence microscopy was used to characterize the live/dead staining (Olympus IX81 microscope with FITC band-pass, (DIFCO, NY,
USA). The UV photomask was designed at the Los Alamos National Laboratory and fabricated by Photosciences, Inc. (CA, USA). Fluorescence microscopy was used to characterize the live/dead staining (Olympus IX81 microscope with FITC band-pass, TRITC bandpass, and FITC long-pass filter sets, and a DP71 camera) (Olympus America Inc. (PA, USA)).
Due to the commercial abundance of and facile functionalization of Desf B, we chose to evaluate it as a model SDP for the selective capture of viable E. coli, on a robust, chemically modified surface. Two methods were tested (Figures 2 and 3). First, the SDP was covalently tethered to the glass surface (direct method) and used to capture viable bacteria (
In both the methods, patterned, PEG-modified thin films were prepared as described previously [
The attachment was carried out using standard peptide coupling methods (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N-diisopropylethylamine (DIEA), and N-methylpyrrolidinone (NMP)) at room temperature, overnight. After rinsing with NMP, acetone, and ethanol; drying under a stream of argon; and contact angle measurement; the Fmocgroups were removed (20% piperidine/NMP, 2 × 15 min). The slides were rinsed thoroughly with NMP, and the resulting amines were capped with either a carboxylic acid using diglycolic anhydride (for direct binding) or biotin using N-hydroxysuccinimidylbiotin (for indirect biotin/streptavidin mediated capture). Both reactions utilized DIEA in NMP. The slides were rinsed thoroughly (NMP, acetone, ethanol) and dried under a stream of argon. Irradiation of the slides with UV/O3 through a chrome photomask (500 μm feature size) removed the thin film in certain areas while leaving the carboxylic acidor biotin-terminated film in the masked areas. The cleaned areas were “backfilled,” i.e., reacted with another portion of APMDES and the methoxy-terminated PEG chain, to create patterned surfaces. This procedure allowed for the incorporation of an intrinsic negative control (in the regions that only contained methoxy groups) while controlling the number of reactive groups present on the surfaces (by the percentage of Fmocamine-protected PEG reagent included in the PEG deposition solution). The subsequent reactants could only conjugate to the portions of the film that contain the carboxylic acid terminus/biotin functionality (circular areas in Figures 2 and 3), but not in the areas with only methoxy-terminated SAM (remaining area of the glass slide in Figures 2 and 3). In previous studies using a waveguide-based method, we determined that methoxy-ter-
minated SAMs as functional surfaces minimized nonspecific interactions in complex biological samples [
Both attachment strategies utilized the terminal amine of Desf B, which is not critical to iron binding. For direct tethering (
For indirect tethering (biotin-streptavidin chemistry), the glass slides with biotinterminated regions were incubated in streptavidin solution (2 mM) for 10 min, and were thoroughly washed. The biotinylated Desf B, or E. coli incubated with biotinylated Desf B was then added on to the functionalized glass slides to bind the SDP on the patterned glass slide.
For the evaluation of specific binding, E. coli was grown overnight in Luria Bertani media at 37˚C. The bacterial cells were harvested, re-suspended in phosphate buffered saline (PBS), and split into two portions. To one portion of the bacterial suspension, isopropanol (80%) was added to kill the bacterial cells. After incubation for 2 hrs, the cell suspension was washed thoroughly with PBS. This method is expected to kill 100% of bacterial cells. The dead cell lysates were not clarified and contain SDP receptor residues and other cell components.
To confirm this expectation, assessment of viability was confirmed by incubation (48 hr) in Luria Bertani agar media at 37˚C for both live and dead bacterial suspensions. Live and dead bacterial suspensions were mixed in equal proportion, and labeled using a Live/ Dead Bac-LightTM bacterial viability kit that differentially stains living and dead bacterial cells. Bac-LightTM kit consists of two nucleic acid stains, SYTO 9 and propidium iodide. SYTO 9 (green) penetrates most of the membranes freely whereas propidium iodide (red) only penetrates the damaged membranes and also reduces SYTO 9 fluorescence when both the dyes are present. Using this method, viable cells are dyed green, and dead cells, red. Live/dead E. coli labeled mixture (at different dilutions (CFU/ml)) was added to functionalized glass slides, and incubated for 45 min in a hydration chamber at room temperature. The glass slides were then washed with PBS or PBS with 0.01% Tween-20 to remove the unbound bacteria (1 ml wash volume, 2 washes), and imaged using fluorescence microscopy (Figures 2-4). This allows for the removal of non-specifically associated components, while preserving any covalent interactions of relevance to the assay. To test the sensitivity of the method, Live/dead E. coli labeled mixture was serially diluted in PBS and was added on to the glass slides. The bacterial suspensions were plated on the agar plate to obtain colony-forming units per ml (CFU/ml), which indicates the limit of detection of the assay.
The fluorescence images collected upon SDP capture of E. coli were subjected to edge detection using a Canny edge detector for identification of edge pixels around each site. A Delaunay triangulation of the edge points was computed to obtain proximity graph. Triangle edges of length greater than three pixel units were rejected to obtain polygons that tightly bound the sites (
Direct conjugation of Desf B was highly effective in the capture of viable bacteria (
We used fluorescence microscopy for the feasibility demonstration shown here, but the approach can conceptually be integrated into other readout formats. The approach is directly compatible with reagent-free methods like interferometry and impedance-based detection. With the use of fluorescently labeled reporter ligands (antibody or aptamers), the binding of viable bacteria to surface SDPs can be read in any fluorescence or absorbance reader with appropriately functionalized plates.
With the indirect tethering strategy (
and incubated. Second, biotinyl-Desf B was pre-incubated with labeled E. coli, and the mixture was added to the streptavidin-coated thin films. Both methods were able to selectively capture viable E. coli. However, the pre-incubation method resulted in less non-specific binding (
Our quantification strategy (
In summary, we have developed a universal, simple yet robust platform that can distinguish between live and dead bacteria using the SDP-mediated bacterial iron uptake mechanism. The novel aspects of this approach are 1) use of bacterial SDPs for determination of viability; 2) use of self-assembled monolayer chemistry for surface functionalization that intrinsically resists non-specific interactions in complex biological samples; 3) flexibility of functionalization chemistry to multiple tethering strategies; 4) incorporation of an intrinsic control with patterned surfaces for accurate data interpretation; and 5) unique adaptation of a software application for the rapid and accurate translation of the observed images to quantitative data, with statistical relevance. Fluorescence microscopy is used as the readout in the current study, but the approach is not limited to any single transduction strategy. Although the method is universal in that all bacterial pathogens secrete SDPs, the widespread application of this approach relies on the availability of SDPs, either from commercial sources or using synthetic strategies. It is likely that a wider variety of SDPs will become available as SDPs become an essential part of biological detection; by sharing our strategy, we hope to help drive this innovation.
A simple method for viable bacterial capture can have far reaching application for pathogen detection, especially for identifying medical and food borne pathogens, and in assessing effectiveness of decontamination methods. Without the “live or dead” ambiguity, use of antibody recognition should be thoroughly revitalized, as after SDP capture, recognition can take place with the knowledge of observing a known live pathogen. Efforts are currently underway to apply this technology to further pathogens, develop multiplex readout assays, and provide a useable platform for application of a rapid assay to assess bacterial viability.
We thank the Los Alamos LDRD Exploratory Research (HM and JS) program for funding. The authors thank Drs. Basil I. Swanson and Jennifer S. Martinez, Los Alamos National Laboratory, for technical suggestions and discussions.