Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria
© Avalos Vizcarra et al.; licensee Springer. 2013
Received: 17 June 2013
Accepted: 16 August 2013
Published: 21 August 2013
Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO® 9 with endogenously expressed eGFP, as SYTO® 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions.
Clinically relevant nosocomial infections are frequently caused by adherent bacteria and the subsequent biofilm formation within tissues or on biomaterial surfaces . Surface biofouling commonly starts with the adhesion of individual bacteria that subsequently grow into mature biofilms. To prevent bacterial adhesion and growth already during the pre-biofilm phase, two main surface engineering strategies have been developed so far: non-fouling “stealth” surface coatings that inhibit adhesion of proteins and bacteria [2–4] and bioactive materials, which upon bacterial contact or release of the active molecules interfere with bacterial viability [5–10]. To compare the antimicrobial properties of surface coatings and to study the kinetics of bacterial surface colonization, assays are needed that allow for in situ monitoring of bacterial adhesion and viability. The gold standard for bacterial viability tests has long been quantification of colony forming units (CFU) by plating bacterial suspensions that were incubated with the test surface on nutrient agar . Counting bacterial colonies, which result from plating suspended viable and cultivatable bacteria, however, does not account for the inherent phenotypic heterogeneity and the ability of the bacteria to persist in dormant states [12, 13]. Furthermore, plating assays lack the ability to measure the colonization and viability kinetics directly on the test surface and might not be representative for the surface-attached bacterial population.
Building upon those observations, we present an optimized protocol to probe the viability and growth kinetics of surface-adhering and suspended bacteria using non-toxic concentrations of propidium iodide and Escherichia coli that express the fluorescent protein GFP. Beyond calibrating the assay and monitoring E. coli surface colonization kinetics on bare glass substrates, we demonstrate that this assay is applicable to monitor the inactivation kinetics of E. coli in contact with antimicrobial surface coatings, using dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC) coated glass surfaces as model substrate. To kill bacteria, the quaternary ammonium chloride complexes of surface-bound DMOAC have to directly interact with the bacterial membrane . We previously showed that bacterial fimbriae strongly influence the unspecific adhesion of E. coli to engineered surfaces . Type 1 fimbriae (7 nm diameter, several 100 μm length) protrude from the bacterial membrane thereby preventing the bulk bacterial body from direct interaction with the underlying material surface. To ensure a physical contact of the bacterial membrane with the material surface, we used here the non-fimbriated K-12 derivative AAEC191A E. coli strain. In addition, we highlight the effect of serum protein adsorption on the bactericidal properties of antimicrobial surfaces. We incubated the DMOAC surfaces with fetal bovine serum (FBS) to mimic the physiological situation where serum proteins adsorb to engineered biomaterials upon contact with host body fluids.
Non-fimbriated E. coli AAEC191A bacteria, a derivative of E. coli K-12 MG1655 containing a deletion in the entire fim cluster  was provided by Prof. E. Sokurenko, University of Washington, Seattle, USA. For GFP expression, chemocompetent AAEC191A E. coli were transformed with eGFP pHis plasmid under the control of the tac promoter (AAEC191A pHis-GFP). To obtain E. coli that express eGFP under the control of the constitutive rpsm promoter (AAEC191A rpsm-GFP), E. coli AAEC191A were transformed with the rpsm-GFP plasmid that was extracted from the original fusion library strain MG1655 rpsm-GFP  by Qiaprep Spin Miniprep kit (Qiagen 27106). Transformed bacteria were selected by cultivation on LB agar plates supplemented with either 100 μg/ml ampicillin (pHis-GFP) or 50 μg/ml kanamycin (rpsm-GFP). Bacterial precultures were inoculated from glycerol stocks into LB medium (5 g/l yeast extract, 10 g/l tryptone, 10 g/l NaCl) containing appropriate antibiotics. To induce GFP expression in AAEC191A pHis-GFP E. coli, 0.1 mM Isopropyl-β-D-thiogalactopyranoside (IPTG, Applichem A1008) was added. LB precultures were grown overnight at 37°C under continuous shaking at 180 rpm (Infors Unitron HT). To ensure defined culture conditions for the bacterial growth and viability assays, bacteria from the overnight culture were centrifuged at 1700 g, washed three times and subcultured in 20 ml minimal M9 medium (1x M9 salts (Sigma-Aldrich M6030), 10 mM Mg2SO4 (Sigma-Aldrich 63126), 10 g/l Glucose (Sigma-Aldrich G8270), 0.5 mM CaCl2 (Sigma-Aldrich C5080), 1x MEM vitamins (Gibco 11120), 1x MEM amino acids (Gibco 11130)) supplemented with the appropriate antibiotics and 0.1 mM IPTG for AAEC191A pHis-GFP E. coli. Bacteria were subcultured at 37°C, 180 rpm until exponential growth phase (OD600 = 0.3-0.8). Bacteria were harvested by centrifugation at 1700 g followed by three washing steps. Immediately before the experiment, bacteria were resuspended in M9 medium that contained the strain-specific antibiotics, IPTG as well as 0, 3, 30 μM PI (Sigma-Aldrich, 81845) and 6 μM SYTO® 9 (Invitrogen L13152), respectively.
2.2 Growth curve measurements
Growth curves of suspended bacteria were recorded by turbidity measurements at 600 nm in 96 well plates (Tecan Infinity 200 Pro plate reader). Kinetic measurements were performed every 15 minutes at 37°C and continuous shaking. Bacteria were inoculated to an initial turbidity of 0.01 at 600 nm in M9 medium containing appropriate antibiotics, PI and SYTO® 9.
2.3 Viability assay
For kinetic viability measurements under physiological conditions, bacteria were cultivated in an ibidi® glass bottom flow chamber (ibidi, 80168) within a temperature-controlled microscope incubator to guarantee constant nutrient supply and optimal growth conditions at 37°C. Bioactive surfaces were prepared according to published protocols . Briefly, glass cover slides, that later resemble the bottom slide of the flow chamber, were exposed to air plasma for 15 seconds (Harrick Plasma, PDC-32G) followed by dipping into a 5% (v/v) aqueous DMOAC (Sigma-Aldrich) solution for 1 second, and drying at 105°C overnight. To test the effect of protein pre-incubation on the antimicrobial activity of the DMOAC coatings, slides were incubated in undiluted fetal bovine serum (FBS, Thermo Scientific SH30071.02) for 1 h prior to the assembly of the flow chamber. Bare glass cover slides were attached to the ibidi® chambers as control surfaces. The bacterial suspension (OD600 0.05) in M9 medium containing different concentrations of PI and SYTO® 9, was directly added to the flow chamber and immediately transferred to an epifluorescence microscope (Nikon TE2000-E) for in situ viability monitoring. Adhesion of bacteria to the glass bottom slide was allowed for 5 minutes before the flow chamber was gently washed with 5 ml M9 medium (flow rate 0.01 ml/min) to remove non-adherent bacteria. As control staining at defined time points, the BacLight™ viability kit (Invitrogen, L13152) was used according to the supplier instructions.
The adsorbed dry film thickness of DMOAC and DMOAC + FBS layers on silicone wafers was measured by variable-angle spectroscopic ellipsometry (VASE) using the M2000F variable-angle spectroscopic ellipsometer (J.A. Woollam Co., Inc.). The measurement was performed at 70° relative to the surface normal under ambient conditions. Ellipsometry data were fitted using a cauchy model with parameters for organic layers (n(λ) = Aλ + Bn/λ^2 + Cn/λ^4, with An = 1.45, Bn = 0.01, Cn = 0.0) to obtain dry thickness of adlayers.
2.5 Image segmentation and quantification
To limit the viability analysis to fluorescent E. coli and to eliminate bias in the data analysis based on GFP fluorescence intensity, fluorescence images were thresholded and segmented using the morphological strel algorithm of the image processing toolbox of MATLAB® software (MATLAB, MathWorks; version R2010b) that combines image erosion and dilation operations. The algorithm was included into a semiautomatic image processing workflow that allows for manual adjustment of the thresholding levels of the entire time series as well as individual time frames. Binary masks were generated from the thresholded images and surface-adherent bacteria were counted automatically. The summing of binary masks from consecutive time points allowed for correction of fluorescence signal loss caused by GFP bleaching, washout and degradation of the stained DNA. To prevent false-positive results, binary masks that were not positive for the GFP channel before, were excluded from the PI positive counts to limit the analysis to bacteria that were viable initially. Elimination of x,y drift of time series data was achieved by the register virtual stack slices and transform virtual stack slices plugins of Fiji that were incorporated into a MATLAB® routine using the MIJ java package for bi-directional communication between MATLAB® and ImageJ by D. Sage. The MATLAB® file for the analysis workflow is available in the Additional file 1.
3.1 Impact of SYTO® 9 and propidium iodide concentrations on the growth of suspended E. coli
To confirm that the viable E. coli in the medium supplemented with 3 μM PI were able to replicate, we compared the turbidity increase of a 50% live / 50% isopropanol killed bacterial mixture to cultures containing 100% live and 100% isopropanol treated E. coli (Figure 2a). Within 4 hours, the turbidity increase for the mixed 50% live / 50% dead starting culture did not reach the same level as for the 100% live culture. Those results are consistent with the expected exponential growth rate of viable bacterial batch cultures and thus show that the bacteria replicated normally in 3 μM PI containing medium. To determine if the reduced PI concentration was sufficient to detect dead bacteria in solution, we incubated bacterial batch cultures starting from either 100% live or 50% live / 50% killed bacteria in M9 medium containing 3 and 30 μM PI respectively. The PI fluorescence at 630 nm was subsequently measured by fluorescence spectroscopy over 3 hours (Figure 2b). Supplementing the growth medium with 3 μM PI adequately stained the isopropanol treated bacteria in the 1:1 mixture of live and dead E. coli but did not result in a significant increase of the background fluorescence of the 100% live starting culture. In contrast, supplementing the medium with 30 μM PI significantly increased the PI fluorescence from the 100% live starting culture (Figure 2b), which was consistent with the impaired growth rate under those conditions (Figure 2a), indicating that 30 μM but not 3 μM PI is toxic to E. coli bacteria.
3.2 Viability and growth rate of surface-adhering E. coli is strongly reduced upon long-term incubation in culture medium supplemented with SYTO® 9
To evaluate whether the eGFP/3 μM PI assay is suited for in situ monitoring of bacterial viability and growth on a bioactive model substrate, eGFP-expressing E. coli (AAEC191A pHis-GFP) were incubated on antimicrobial dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC) coated glass surfaces [23, 27] (Figure 3b). Homogeneous DMOAC coating with a dry adlayer thickness of 2.2 nm was confirmed by variable-angle spectroscopic ellipsometry. To allow for a direct contact of the bacterial membrane with the surface-immobilized membrane-active DMOAC molecules, non-fimbriated eGFP expressing E. coli (AAEC191A pHis-GFP) were used . As detected by 3 μM PI staining, all adherent bacteria on the DMOAC surface were killed within 30 minutes of surface incubation and no measurable bacterial growth occurred (Figure 3b, Additional file 1: Figure S2, Additional file 2: Movie S1).
3.3 Pre-incubation of bioactive DMOAC surfaces with serum proteins completely blocked the antimicrobial activity and restored bacterial growth on the surface
To investigate if unspecific protein adsorption would interfere with the bactericidal activity of the DMOAC surfaces, the DMOAC surfaces were pre-incubated with fetal bovine serum (FBS) prior to bacterial incubation. Preconditioning of the antimicrobial surface with serum provides a model system for the rapid protein adsorption on biomaterial surfaces upon contact with host body fluids, notably blood, that can significantly impact the specific and unspecific binding of bacteria to the engineered material [10, 28]. Serum protein adsorption increased the dry adlayer thickness from 2.2 nm for pure DMOAC surfaces to 4.5 nm, as measured by ellipsometry. Using the optimized in situ eGFP/PI assay, we found that serum pre-incubation not only delayed but completely eliminated the bactericidal effect of the DMOAC surfaces on adherent E. coli (Figure 3b, Additional file 3: Movie S2). The bacteria survived and were able to divide on the protein-coated DMOAC surfaces. Division times of the surface-attached E. coli were comparable to those on bare control glass surfaces without bactericidal activity (Figure 3a).
A fluorescence based assay is introduced here that is well suited for the in situ monitoring of the viability and growth kinetics of surface-adhering and suspended bacteria. While we used E. coli as model organism, this assay should be applicable to other bacterial species as well if (i) the commonly used live DNA stains, such as SYTO® 9, are replaced by endogenous eGFP expression and (ii) if the concentrations of propidium iodide (PI) needed to detect dead bacteria is reduced to non-toxic levels (3 μM for E. coli). While most available viability assays are restricted to suspended bacteria (i.e. CFU assay) or optical endpoint determinations (i.e. SYTO® 9/PI LIVE/DEAD BacLight™ viability kit, CTC assays), we show by fluorescence time-lapse microscopy and turbidity measurements, that reducing the PI concentration to 3 μM readily stained dead E. coli on bioactive DMOAC surfaces without disturbing bacterial growth (Figure 2, 3).
Our assay allows viability monitoring of single bacteria and emerging bacterial colonies. We should note though that the assay is not directly transferable to the study of mature biofilms without additional calibrations since the metabolic and genetic profile might change during biofilm mode of growth [29–31], and the synthesis of extracellular polymeric substances (EPS) [32, 33] might influence the bacterial GFP expression as well as the passive diffusion of PI through the biofilm matrix. Furthermore, detection of single bacteria within a dense three-dimensional biofilm matrix by epifluorescence microscopy might be challenging. However, since bacterial surface colonization starts with the adhesion of individual bacteria, the presented assay provides a versatile new tool for high spatial and temporal evaluation of bacterial viability on engineered surface coatings. The assay thus adds to the previously reported eGFP/PI flow cytometry assay that was limited to viability determination of suspended bacteria  and to the eGFP/PI endpoint viability study of groundwater E. coli.
Evaluating bacterial viability on the test surface omits the extraction of the adherent bacteria as required for solution based assays, e.g. CFU counts. Therefore, testing the bacterial viability on the substrate might increase the reliability of the assay since extraction is commonly achieved by ultrasonication or harsh washing procedures, both of which can harm bacteria. Furthermore, all CFU assays require prolonged incubation times for colony growth and are thus not applicable for real-time viability monitoring. As an alternative to extraction, an agar sandwich assay has been suggested to determine the viability of surface-adherent bacteria . This method, however, is prone to errors, since each transferred bacterium will grow into a colony that in turn might overlap with colonies nearby. As an alternative direct optical viability assay of surface-adherent bacteria, the respiratory potential of bacteria can be monitored using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) . The drawback however is that the CTC stain cannot be used for real-time monitoring since CTC disrupts the respiratory chain and is toxic to bacteria. This makes the CTC assay only suitable for end point determinations. We compared the performance of our eGFP/PI assay to the well-established SYTO® 9/PI endpoint dual staining assay and found identical detection efficiencies of dead E. coli (Additional file 1: Figure S2). The SYTO® 9/PI assay itself has been extensively compared to the above-mentioned viability tests [17, 19, 21, 26, 36] and showed comparative results to the solution based CFU assay as well as to other microscopy based endpoint viability protocols including the CTC assay. The added advantage of our assay is the ability to monitor the viability of adherent bacteria in real-time.
For each bacterial strain and species, one needs to optimize the PI concentrations to keep the bacteria viable, as done here for Escherichia coli (E. coli) K-12 MG1655. E. coli K-12 derivatives have been widely used as model strains in surface adhesion and biofilm studies [4, 31, 37–39]. We expressed eGFP from the pHis plasmid under the control of the IPTG inducible tac promoter to replace the growth inhibiting DNA stain SYTO® 9 as live bacterial marker. To exclude negative effects of GFP expression as well as IPTG and antibiotic addition on bacterial viability and adhesion, we compared the adhesion properties and growth kinetics of the K-12 AAEC191A background strain  to a constitutive eGFP expressing strain (rpsm-GFP) and the IPTG inducible pHis-GFP strain (Figure 4a,b). No significant difference in adhesion and growth was observed. Furthermore, no significant difference in the fraction of GFP-expressing bacteria was found for the eGFP expression under the constitutive and inducible promoter used in this study (Figure 4c,d). However, even non-toxic gene products like GFP can have detrimental effects on bacteria when overexpressed , since overexpression of an introduced gene requires a lot of resources and thus might disorganize the bacterial metabolism. Therefore, the inducible IPTG-based expression system, as compared to constitutive expression systems, allows for a control of GFP expression and guarantees a balance between fluorescent and healthy bacteria (Figure 4c,d). Since eGFP is a very stable protein [41, 42] new GFP variants with reduced half-lives, e.g. GFP(LVA), have been suggested to study transient gene expression in bacteria . The enzymatic degradation of unstable GFP(LVA) however, requires a metabolically active viable bacterium (Additional file 1: Figure S3). Thus, the use of GFP(LVA) variants does not improve our assay to limit false-positive detection of dead bacteria.
For live cell imaging, GFP expression in bacteria is used extensively and expression systems are available for many different bacterial species, including clinically relevant strains of Salmonella, Streptococcus, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli O157:H7[41, 44–47]. Plasmid based gene expression is a well-established and long-used method in microbiology. Handling of plasmids is usually easy and versatile and genes, promoters or selection markers can quickly be exchanged and adapted to needs. Compared to plasmids, chromosomal insertions are more complicated and cannot be adapted as easily. Depending on the insertion method used, the gene of interest is inserted in the chromosome at a random location and might disrupt an important chromosomal gene. As for plasmids, selection markers like antibiotic resistances are commonly used for chromosomal insertions, too . Furthermore, chromosomal insertions usually result in the insertion of a single copy of the gene into the chromosome. The pHis plasmid used in this study carries a ColE1-like replicator and occurs at nearly 20 copies per bacterium , each providing the gene of interest. This results in very stable and usually higher expression levels of the gene of interest (here eGFP) than with chromosomal insertions. Plasmids with ColE1-like promoters are stably inherited even without the presence of the corresponding selection agent [50, 51], long-term experiments with GFP expression from a plasmid rather than from a chromosomal insertion are feasible for more than a few hours. For other bacterial species and expression systems, the IPTG level and the concentration of the antibiotic selection marker should be re-evaluated to assure a stable GFP expression without disturbance of bacterial growth.
Finally, we applied our viability assay to highlight the impact of protein adsorption on the antimicrobial activity of engineered DMOAC surfaces. Upon incubation of the DMOAC surfaces with protein-rich fetal bovine serum, bacterial growth on the otherwise bactericidal surface was possible, indicating that the protein layer on top blocked the bioactive quaternary ammonium groups of the DMOAC coating (Figure 3b). The growth rate of the surface-attached E. coli on the control glass and serum-coated DMOAC substrates were identical, illustrating that the design rules for antimicrobial coatings primarily have to be tuned to prevent both, bacterial and protein adsorption since additional bioactive modifications can be lost when the biomaterial gets in contact with protein-rich (host) fluids.
In conclusion, we show that the eGFP/PI assay is suited to study the antimicrobial properties of (bio-) material surface coatings under physiological conditions in real time and with single-bacterium sensitivity. This was so far not possible with the widely used solution based assays (i.e. CFU) or endpoint dual staining protocols (i.e. LIVE/DEAD BacLight™ viability kit, CTC). Possible applications for the assay include studies of bacterial fitness and pathogenicity on biomaterial surfaces using live cell imaging of bacteria as additional readout. While we calibrated and illustrated the advantages of the assay for E. coli, other PI concentrations might have to be employed to optimize the kinetic viability monitoring of other bacterial species. While conventional bacteria viability assays allow for fast endpoint checks without requiring genetic modifications, the eGFP/PI assay presented here constitutes a viability test procedure that requires only one sample and its replicates per time series and is particularly suited for kinetic studies.
The authors thank Prof. Evgeni Sokurenko (University of Washington, Seattle, USA) for providing the E.coli AAEC191A strain and Dr. Peter Kaiser for help on Metamorph journals. We thank Mohammad Divandari and Dr. Edmondo M. Benetti for assistance with ellipsometry measurements and Markus Arnoldini and Prof. Martin Ackermann for providing the MG1655 rpsm-GFP strain. We thank Prof. Marcus Textor for valuable discussions during the design of the assay. Funding from BASF SE, Ludwigshafen, Germany and ETH Zurich (V.V.) is gratefully acknowledged.
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