Isotachophoretic Fluorescence in Situ Hybridization of Intact Bacterial

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Isotachophoretic Fluorescence in situ Hybridization of intact bacterial cells Sui Ching Phung, Joan Marc Cabot, Mirek Macka, Shane M. Powell, Rosanne M Guijt, and Michael C. Breadmore Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 22, 2017

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Analytical Chemistry

Isotachophoretic Fluorescence in situ Hybridization of Intact Bacterial Cells. Sui C Phung1 Joan M Cabot1,2 Mirek Macka1 Shane M Powell3 Rosanne M Guijt1,4,5 Michael Breadmore1*. 1

Australia Centre for Research on Separation Science, School of Physical Sciences- Chemistry, University of Tasmania, Tasmania, Australia

2

ARC Centre of Excellence for Electromaterials Science, School of Physical Sciences- Chemistry, University of Tasmania, Tasmania, Australia 3

Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Tasmania, Australia

4

Pharmacy, School of Medicine, University of Tasmania, Tasmania, Australia

5

Microfluidic Chipshop, GmbH, Stockholmer Strasse, Germany

*

Corresponding author: Email: [email protected] Fax: +61-3-6226-2686

KEYWORDS : Fluorescence in situ Hybridization, Capillary Isotachophoresis, bacterial cells ABSTRACT: A counter-pressure assisted capillary isotachophoresis method in combination with a sieving matrix and ionic spacer was used to perform in-line fluorescence in situ hybridization (FISH) of bacterial cells. A high concentration of sieving matrix (1.8% w/v HEC) was introduced at one end of the capillary and the bacterial cells were suspended in the spacer electrolyte for injection. Using a 2 min injection with 18 psi counter-pressure, 50% of the cells injected into the capillary were hybridized with the fluorescently labeled oligonucleotide and the excess unhybridized probe was separated from the hybridized cell-probe complexes in a two-stage ITP method. With an LOD (6.0 x 104 cells/mL) comparable with the CE analysis of a sample processed using an offline FISH protocol, the total analysis time was reduced from 2.5 hr to 30 mins. With the selection of the appropriate hybridized probe, this approach can be used for specific detection of bacterial cells in aqueous samples.

1. Introduction Rapid and robust alternative methods for enumeration of pathogenic microorganisms have evolved significantly over the last decade. These include the use of immunological and molecular techniques as an alternative to the classical standard reference method – the plate count method1. Plate count is the method is used for the detection of bacteria by regulatory agencies and all new methods have to be validated against this1-4. It is low cost and provides exquisite sensitivity, being able to quantify 4 colony forming units per mL (CFU/mL) and 1 CFU/25 g of solid material5. However, it is time consuming with 24-48 h of incubation time, the process is laborious, success dependent on the suitability of the growth medium and the process is difficult to automate1,5. Alternate methods, such as immunological techniques, lack the sensitivity at low cell concentrations and hence enrichment is required prior analysis1,6 7. Using PCR, a single bacteria in a sample could theoretically be detected, but inhibitors may be present in the sample matrix or poor purity of the target nucleic acids may cause false negatives1,6,8. Despite the fact that care has to be taken by having the right controls in place, nucleic acid based methods have allowed detection of bacterial cells based on DNA/RNA sequences without enrichment.

Fluorescence in situ hybridization (FISH) is a molecular technique that is widely used for microbial identification, quantification and, in combination with other techniques, for characterization of phylogenetically defined microbial populations. The assay relies on the selectivity of binding of a labeled nucleotide probe to a complementary sequence9. Hybridization will only occur when the target is present in the cell, providing specificity by matching the probe with a target-specific sequence. FISH is sensitive by targeting the highly abundant ribosomal RNA (rRNA)1,2,10-12 with a reported detection limit of 102 cells/mL for drinking water4,13,14. A detection limit between 103-104 cells/mL is more routinely obtained because when using a microscope for detection, at least one cell is required per field of view to provide unambiguous results2. For the detection of bacterial cells, FISH provides the capability of detecting so called ‘viable and non-culturable’ (VBNC) pathogens2, addressing a known shortfall of the plate count method. The target rRNA used in FISH is typically either the 16S rRNA of the small ribosomal subunit or 23S rRNA of the large ribosomal subunit12. Delong et al. reported FISH for phylogenetic identification of single microbial cells11, and

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Amann et al. combined FISH with flow cytometry to analyse a mixed microbial population10. Although FISH is rapid in comparison to the plate count method, the fixing and staining take about two hours before detection. The Santiago group demonstrated rapid hybridization of nucleic acids in solution using isotachophoresis (ITP) with a sequence specific hybridization probe, reporting a 960fold and 14,000-fold increase in the rate of hybridization15. By focusing the target and probe nucleotides together in a very small volume at the ITP interface, their initial work used a molecular beacon15,16, increases the fluorescence upon hybridization. This was followed by using a permanent fluorophore for hybridization by using a sieving matrix and spacer ion to separate the unhybridized probe from the hybridized nucleotides17. The Bercovici group used a low electrophoretic mobility of peptide nucleic acid probe (PNA) that does not focus under ITP unless the probe is bound to the target sequences. This eliminates the use of spacer ions and sieving matrix to separate the unbound probe18. The same group also demonstrated the use of ITP with a counter-pressure to focus antimicrobial peptides at a fixed position within a microfluidic channel for continuous bacterial detection without cell lysis19. The peptides target the bacteria by nonspecific binding to the negatively charged outer membrane, and thus while it provides the ability to selectivity detect bacteria, it cannot distinguish between different bacteria. Lantz et al. demonstrated selective bacteria detection by using a FISH probe for CE analysis of Salmonella typhimurium in a mixed population20. In this work, the cells were fixed and FISH-stained off-line before injection for CE analysis. This paper examines ITP accelerated hybridization for in-line FISH staining of intact bacterial cells. A counter-pressure is used to extend the hybridization process, where the FISH probe and bacterial cells are focused in a small volume, followed by the separation of the unhybridized probe from the cells using a sieving matrix and appropriate spacer ions. The in-line FISH staining and subsequent analysis took 30 min, a significant improvement in analysis speed compared with the 2.5 hr required for the traditional off-line FISH. The staining efficiency using the in-line method was 50% of the off-line method, making this new approach – which is compatible with automation – an attractive alternative to routine methods for bacterial cells enumeration.

2.

Methods and Materials 2.1 Chemicals Tris (hydroxymethyl)aminomethane) (Tris), ≥ 99.95%, hydroxyl ethyl cellulose (HEC), (Mw 250 000), polypyrolidine (PVP), (Mw 1300 kDA), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) 99.5%, 2-(NMorpholino)ethanesulfonic acid hydrate, magnesium chloride anhydrous (MgCl2), 4-Morpholineethanesulfonic acid (MES) ≥99.5%, dimethyl sulfoxide anhydrous (DMSO) ≥ 99.9%, 1 M Tris HCl (pH 8.0), and coumarin 344 were purchased from Sigma Aldrich (St. Louis, USA). Luria Bertani media (LB, 1 L LB – 10 g tryptone, 5 g yeast extract, 10 g NaCl), and LB agar plates were prepared in house. Tryptone, yeast extract, and Phosphate Buffered Saline (PBS) were purchased from Oxoid (Hampshire, England). Sodium

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chloride was purchased from Univar (Sevenhill, Australia). Agar was purchased from Gelita (Beaudesert, Australia).

2.2 Electrolytes For all ITP experiments, the leading electrolyte (LE) used was 50 mM TRIS HCl (pH 8.0) with 0.05% w/v HEC, 5 mM MgCl2 0.1% v/v DMSO. The terminating electrolyte (TE) was 50 mM TRIS HEPES (pH 8.0) with 0.1% v/v DMSO. The spacer electrolyte (SE) used was 50 mM MES adjusted with TRIS to pH 7.8 with 0.1% v/v DMSO. The sieving matrix used was 50 mM TRIS HCl (pH 8.0) + 1.8% HEC w/v. All solutions were prepared using 18.2 MΩ cm-1 Milli-Q water (Millipore, Bedford, MA, USA).

2.3 Bacterial growth The model bacteria, Escherichia coli (E. coli) strain TOP10 was obtained from Invitrogen (Mulgrave, Australia) and Pseudomonas aeruginosa (P. aeruginosa) strain was obtained in house from the microbiology culture collection (School of Land and Food, University of Tasmania). The bacterial cells were grown in solid LB media at 37°C and, when necessary, in liquid LB broth at 37°C with shaking. To obtain E. coli and P. aeruginosa cells for ITP experiments, a colony from a stock plate of each of the E. coli and P. aeruginosa cultures were inoculated in LB broth and incubated overnight at 37 °C with shaking. To harvest the cells, 10 mL of the overnight cell culture was transferred to a sterile 15 mL centrifuge tube and centrifuged for 5 min at 3600 rpm at room temperature (model: Universal 16 A, Hettich, Zentrifugen, Tuttlingen, Germany). The supernatant was carefully discarded and the cell pellet was resuspended in sterile PBS. This suspension was stored at 4°C until required and remained viable for up to one week. Cell numbers of E. coli and P. aeruginosa colony forming units (CFU equivalent to cells) was determined by the plate count enumeration method. 2.4 Sample preparation Bacterial cells: 100-500 µL of bacterial cells from the PBS stock was centrifuged for 5 min at 3600 rpm at room temperature (Model: Eppendorf 5424, Hamburg, Germany), the supernatant was discarded and 100 µL of 50 mM SE was added and mixed by gentle vortex for 20 s. This washing step was repeated once. Final volume for analysis of the cells was 500 µL in SE solution with 0.1% v/v DMSO. Most of the bacterial cells in the SE solution were still intact and alive and were able to grow if plated onto the LB agar as long as they had not been injected for in-line ITP analysis. During the ITP analysis, bacterial cells that were 6 days old in PBS solution can still focus in the in-line ITP interface although most of the cells at 6 days old might have been dead due to absence of nutrients but remain intact. Therefore, the cells concentration is reported as cells/mL not CFU as we did not examined whether the injected cells were live or dead.

2.5 DNA probes and hybridization Three DNA probes targeting the 16S rRNA of bacterial cells were used which are Enterobacteriaceae specific: 5’ TGC TCT CGC GAG GTC GCT TCT CTT 3’21, P. aeruginosa (PseaerA) sequence probe: 5’ GGT AAC CGT CCC CCT TGC 3’22 and Enterobacteriaceae-2 specific: 5’CCC CCT CTT TGG TCT TGC 3’23. The 3’ end for both of the Enterobacteriaceae probes were labelled with 6 – carboxyfluorescein


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(FAM) and the 3’ end of PseaerA probe was labeled with a cyanine dye (Cy5). All probes were custom synthesized with HPLC-purification by Intergrated DNA Technology (Coralville, IA). After receipt, the probes were resuspended in Nuclease Free water at a final concentration of 100 µM and stored at -80 °C. A secondary stock was prepared from the main stock and suspended in the LE solution at a final concentration of 26 µM. For analysis the secondary stock of the probe was diluted with the LE solutions to a final concentration of 2.6 µM.

2.6 Capillary electrophoresis All CE experiments were carried out on a Beckman Coulter P/ACE MDQ Capillary Electrophoresis System equipped with 488-nm laser module. An external bench top laser with output at 635 nm (Thorlabs, New Jerseys, USA) was fibre coupled to the second laser port on Beckman Coulter. Experiments were conducted using a bare fused silica capillary (Polymicro Technology, AZ, USA) of 50 µm id with total length of 40 cm (effective length to detector, 30 cm). The capillary was maintained at a temperature of 25°C. 2.7 Capillary conditioning and electrophoresis A new capillary was pre-conditioned at 20 psi in the following order: 1M NaOH (30 min), milli-Q water (20 min), 1M HCl (20 min), Milli-Q water (10 min) followed by 1 % w/v PVP at 45 psi for 45 min. Finally, the capillary was conditioned with 50 mM TRIS HCl (pH 8.0), 5 mM MgCl2, 0.05% w/v HEC at 16 kV for 10 min. Each analysis began by flushing with 1.8% w/v HEC for 8 min at 75 psi, 2.6 µM probe in LE solution for 9 min at 10 psi, electrokinetic (EKI) injection of sample in SE solution at -16 kV (400 V/cm) with counter pressure of 18 psi for 2 min, followed by EKI injection of TE solution at – 16kV, (400 V/cm).

3.

Results and Discussion 3.1 In-line ITP FISH staining of bacteria In previous work, we employed ITP with a generic intercalation dye (SYTO 9) to quantitate cells down to 135 cells/mL (without the use of counter-pressure)24, which was reduced to 78 cells/mL when a counter-pressure was introduced25.While effective in the detection of bacterial cells, SYTO 9 is a universal dye, hence it was not possible to introduce specificity in the staining and quantification. Specificity can be introduced through the use of sequence-specific hybridization probes as demonstrated by Lantz et al.18, but to do so, the cells needed to be fixed and stained off-line first. Here, we added DMSO to the electrolyte to improve the staining efficiency as it is known for its ability to enhance transport across biological barrier26,27 However, only 0.1% v/v DMSO was used for cell permeabilization in this study as when higher concentration of DMSO were used, the mobility of the cells and probe were affected and the capillary coating did not last more than 20 injections. The Santiago group demonstrated in-capillary/chip hybridization of nucleotides using ITP with 103-104 improvements in hybridization kinetics15, the premise of this work is to use ITP to enhance the hybridization kinetics for the staining of intact bacterial cells with a FISH probe.A schematic explaining the proposed method is shown in figure 1. The aim of the protocol is to (1) focus both bacterial cells and FISH probe at the ITP boundary for rapid hybridization, (2) separate the unhybrid-

ized FISH probe and bacterial cells into two distinct ITP bands for quantitation of the stained cells. After hybridization, this requires the establishment of a second ITP boundary, separating the unhybridized probe and cells. It is known from the literature that in ITP a second boundary can be created through the use of a spacer ion with appropriate mobility. Because the probe and cells need to be in the same band in stage (1) and separated in stage (2), the ITP system from stage (1) moves into a sieving matrix where the mobility of the cells is reduced owing to their size. This means that the mobility of the bacterial cells and free probe in free solution has to be between the leader and spacer to allow for rapid hybridization (Figure 1 iii) in stage (1). When entering the sieving matrix for stage (2) the mobility of the bacterial cells is lowered to a value between the spacer and the terminator, leaving the unhybridized probe between the leader and spacer (figure 1v). To meet all these conditions, chloride was selected as leader, HEPES as terminator, MES as spacer and the sieving matrix was 1.8% HEC. To provide sufficient time for hybridization to occur prior to reaching the sieving matrix, a counter-pressure was used to counter the movement of the ITP band23 and also to slowly fill the capillary with the sieving matrix from the outlet during the injection/hybridization stage (figure 1ii). The counter-pressure and applied voltage balancing the movement of the ITP boundary were optimized so that at least 10% of the total capillary length was left without the sieving matrix, to provide sufficient time and space for the hybridization to occur in free solution. This was established by conducting an experiment using the neutral fluorescent dye coumarin 334 to mark the position of the free solution and the position of the hybridization probe was monitored to optimize the counter-pressure assisted ITP method. The optimized procedure was as follows: First, the capillary was filled with 1.8% HEC in 50 mM TRIS HCl (pH 8.0). A plug of LE containing 2.6 µM of the FISH probe was injected into the capillary for 9 min at 10 psi. The inlet was changed to the sample vial containing the bacterial cells suspended in SE. A voltage of -16 kV and a counterpressure of 18 psi were applied for 2 min to inject the cells into the capillary and begin the ITP hybridization process. After 2 min, the counter-pressure was removed and the inlet vial was changed to TE and outlet vial was changed to LE (without probe). Again, a voltage of -16 kV was used to continue with the hybridization until the ITP boundary enters the sieving matrix. Here, the cells are slowed down enabling the spacer ions overtake. The unhybridized FISH probe is focused between the LE and spacer ions while the cells are focused between the spacer and TE (figure 1v). The counter-pressure ITP protocol was applied to samples containing either E. coli or P. aeruginosa using their respective probe in the LE. Figure 2A shows the isotachopherograms obtained for different cell concentrations of E. coli, while figure 2B shows isotachopherograms for P. aeruginosa. These results clearly demonstrate in-line staining of the cells with the FISH probe with peaks for the bacterial cells well separated from the excess unhybridized probe. The inserts in each show the linearity plots for both E. coli and P. aeruginosa cells with good linearity obtained for both. From the calibration curves, the LOD of E. coli was calculated to be 6.8 x 107 cells/mL and 2.7 x 105 cells/mL for P. aeruginosa. The two-order magnitude difference between the LOD of E. coli and P. aeruginosa could be due to the laser used (20 mW 635 nm vs. 2 mW 488



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nm), photobleaching of the fluorophore and/or to differences in the amount of ribosome in the cells.

3.2 Specificity of in-line staining The probes used above were selected from the literature to evaluate the specificity of the probes and the ITP process. An Enterobacteriaceae probe was selected for E. coli based on the sequence at 1251 – 1274 of the E. coli 16S rRNA which is a highly conserved region that allows in situ hybridization of Enterobacteriaceae without a false positive reaction19. The oligonucleotide was attached with a 6-FAM fluorophore allowing detection with excitation at 488 nm. The PseaerA probe was selected for P. aeruginosa because it is reported to specifically bind to P. aeruginosa22, and was labelled with Cy5 allowing excitation with at 635 nm. To demonstrate selectivity of the probes, experiments were performed using E. coli with the PseaerA probe, and with P. aeruginosa with the Enterobacteriaceae probe. The results, shown in Figure 3, show that the Enterobacteriaceae probe provided a detectable response with P. aeruginosa but E. coli cells did not provide any response with the PseaerA probe. A nucleotide check of the Enterobacteriaceae probe with 16S rRNA of P. aeruginosa using BLAST identified a 7 bp match which explains the response of P. aeruginosa with the probe previously reported to be specific for Enterobacteriaceae. A similar check was performed with the PseaerA probe and E. coli 16S rRNA sequence with no nucleotide matches, indicating no hybridization would occur between the cells and the probe. Due to a detectable signal response between the Enterobacteriaceae probe with P. aeruginosa, an alternative probe was selected from the literature23 with no bp match when checked against P. aeruginosa genomes with BLAST. This probe is labeled as Enterobacteriaceae-2. Experiments were repeated using the same probe and cell concentration of E. coli and P. aeruginosa. From figure 3C, when E. coli was injected, a second ITP band was observed at 7.2 min but when the Enterobacteriaceae-2 probe was used in combination with P. aeruginosa, no peak was observed at 7.2 min, demonstrating inline selective staining of bacterial cells. These results show that specificity is possible using in-line ITP for FISH of bacterial cells. However, hybridization stringency is an issue as there may be a response if there are any bp matches between the probe and the 16S rRNA of the bacterial cells (figure 3A). Nonetheless, this issue is also a problem with ex-situ FISH staining of nucleic acids as well, and may be overcome with careful probe designs. Experiments were then conducted using the PseaerA probe with a mixed community of both E. coli and P. aeruginosa. The ratio of E. coli to P. aeruginosa cells was varied with a fixed concentration of P. aeruginosa while increasing the amount of E. coli. This was performed with 2.6 µM PseaerA probe. When the concentration of the E. coli increased in the mixture, the signal of P. aeruginosa decreased (figure 4A). There were two possible reasons: uptake of the hybridization probe into the E. coli cells without binding and/or electrokinetic discrimination. To examine whether the E. coli cells were taking up the probe, the effect of the concentration of the probe was examined first by increasing the concentration of the P. aeruginosa selective PseaerA probe while keeping the concentration of P. aeruginosa (2.7 x 106 cells/mL) constant. The results are shown in

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Figure 4B, where it can be seen that as the probe concentration increases, the signal of the second ITP peak (hybridized cells) increases. The signal increased by 4.7 and 5.3-fold when the probe concentration was increased from 2.6 to 26 and 52 µM, respectively. While the highest response was with 52 µM, higher concentrations were not examined due to the cost and concentration of the probe. Given the marginal improvement with 52 µM, 26 µM of probe was used for further evaluation. The mixed culture experiments were repeated with this higher amount of probe. The data in Figure 4A with the higher concentration of probe shows a higher detector response, but the signal still decreased as the amount of E. coli in the sample was increased. Normalizing the response to the initial signal without any E. coli (figure 4C), it can be seen that with the higher concentration of probe, the relative decrease is less; approximately 90% of the signal was retained when the E. coli was less than 50 times excess of P. aeruginosa. In higher excesses, the signal decreased more rapidly suggesting that there is an influence of the non-target cells on the staining of target cells. This currently limits applicability to samples with a relatively even microbial community, but may be addressed through further optimization of the ITP conditions and the probe used. The initial slight decrease in response at this higher concentration of probe suggests that there is also some electrokinetic bias. The use of hydrodynamic injection would be a potential solution to this issue, but as demonstrated previously, would significantly lower detection limits compared with electrokinetic injection.

3.3 In-line versus off-line cells staining To benchmark the performance of the in-line staining process, samples stained off-line using a conventional FISH protocol were compared with the in-line ITP approach. A 1 mL sample of of 1.0 x 105 cells/mL of P. aeruginosa was fixed with ethanol:PBS (60:40) based on the procedure described by Lantz et al.20. The fixed cells were then stained using the FISH protocol from Glöckner et al.29 in solution in an Eppendorf tube instead of on a membrane. Once the cells were stained, excess probe was removed using washing buffer, and the off-line stained cells were suspended into SE. A sample containing 100 µL of the cell suspension was injected using the counterpressure ITP method, without probe in the LE. The in-line method used the same number of cells suspended in spacer electrolyte that contained 0.1 v/v of DMSO for permeabilization of the probe into the cells during ITP. Probe was added to the LE at a concentration of 26 µM. Representative isotachopherograms are shown in figure 5A with the peak for the in-line stained cells being 50% (n=3) of the off-line value. The confirmation for this proportion of staining was obtained by collecting the off-line and in-line stained cells on black filters for cell counting with a fluorescent microscope. From figure 5B(i), 17 field views were counted and based on the area of the filter and the depth field of the microscope, the average of the total cell count of the off-line FISH cells without ITP was 2.24 x 107 cells/mL, from a total of 2.50 x 107, which indicated that the off-line FISH method hybridized ~90% of the bacterial cells in the solution. The ITP band was collected in a sample vial and filtered onto a separate black filter which yielded 5.32 x 106 cells/mL, again confirming that the in-line approach stains approximately 50% of the cells (inset table in figure 5B(ii)). While this is worse than the off-line process, it is much simpler and quicker, and only compromises the sensitivity by a factor of 2.


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To understand the improvement in hybridization efficiency, the time that the cells are exposed to the probe in the first ITP zone separation without the sieving matrix was examined by changing the volume of the leader in the capillary prior to injection of the cells. Figure 6 shows isotachopherograms when the injection time of the leader (corresponding to panel ii in figure 1) is varied from 1-9 min. As can be seen, the longer the injection, the higher the response of the cell peakwith the maximum response reach at 7 min and longer. This corresponds to filling the capillary 60% to the window. Assuming that the cells spend 60% of their migration time in the first ITP zone, this means they spend 255 s in the ITP boundary with the probe. To compare with non-ITP staining, probe and cells were suspended in the same electrolyte and incubated at room temperature for 30 min (1800 s) to 3 days (259,200 s), without prior fixing of the cells. Prior to analysis by ITP, excess probe was removed via centrifugation and the cells resuspended to eliminate any influence of the ITP process. However, no peak was observed under any conditions, indicating significant enhancement of penetration of the probe into the cells by using ITP, by at least a factor of 1000. The experiment did not proceed longer than 3 days due to reduced detection response of the free fluorophore. When compared to off-line FISH staining, Lantz et al.18 reported a LOD of 1.9 x 104 cells/mL for their CE based method analyzing a sample after fixing the cells and incubating for 30 min. The LOD reported here of 6.0 x 104 cells/mL is three times higher due to the reduced staining efficiency, and other differences including different light sources, PMTs and fluorophores. As a last experiment, the LOD was confirmed by analyzing a sample prepared using the off-line FISH protocol with a concentration of 3.0 x 104 cells/mL and comparing this with in-line FISH protocol staining a sample containing 6.0 x 104 cells/mL. The respective isotachopherograms are shown in figure 5C, with the peaks at about three times the noise for both analyses confirming the loss of a factor 2 in sensitivity for the in-line FISH protocol. While the loss in sensitivity is not ideal, there are significant gains in time, and elimination of the fixation step means that this approach may have applicability for continuous monitoring systems. However, the limit of detection is not sufficient for some applications, such as monitoring drinking water supplies, which require significantly lower detection limits. These can be achieved both by conventional plate count methods and real-time PCR, however at the expense of time, cost and automation. Inter- and intra-day repeatability was acceptable, at 4.35% and 4.05% RSD for E. coli, respectively, and the range was linear over 2 orders of magnitude. The limited linear range may be problematic for samples with high concentrations of cells, as the single ITP boundary into which the cells and probe are focused may reach steady-state leading to their separation in discrete plateau according to mobility.

Conclusions A dual stage ITP protocol for the in-line hybridization and subsequent quantitation of bacteria was developed. Using ITP to enhance hybridization kinetics, samples containing bacterial cells can be analyzed in 30 min including sample preparation. In the first ITP stage, the bacterial cells and free probe are brought in close proximity at the ITP boundary. In the second stage, the bacterial cells are separated from the free probe through the use of a sieving matrix to reduce the effective

mobility of the cells. The protocol allows for the detection of bacterial cells down to 6.0 x 104 cells/mL while the selectivity is achieved through the use of sequence-specific fluorescently labelled oligonucleotide probes. The presence of other bacterial strains within the sample only has a minimal impact on the obtained signal when less than a 50 times excess of other strains is present. When other strains are present at higher levels, the signal intensity is compromised due to uptake, not binding, of the probe into the other cells reducing the amount of probe available for hybridizing with the target RNA. At high concentrations, an electrokinetic bias is suspected but to eliminate this through the use of hydrodynamic injection could significantly compromise the sensitivity. Further improvements in the type of fluorophore or the use of large volume hydrodynamic injection could overcome this limitation. Considering the sensitivity and selectivity, and the compatibility of the in-line FISH approach with automation, makes this protocol an attractive alternative for the quantitation of bacterial cells in aqueous samples.

4. Acknowledgements MCB and MM acknowledge the ARC for Future fellowships (FT130100101, FT120100559, respectively). RMG acknowledges Alexander von Humboldt Fellowship. JMC acknowledge the ARC Centre of Excellence Scheme (Project Number CE 140100012). The authors have declared no conflict of interest

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(12) Amann, R.; Fuchs, B. M. Nat Rev Micro 2008, 6 (5), 339–348. (13) Hammes, F.; Berney, M.; Wang, Y.; Vital, M.; Köster, O.; Egli, T. Water Res 2008, 42 (1-2), 269–277. (14) Hammes, F.; Broger, T.; Weilenmann, H.-U.; Vital, M.; Helbing, J.; Bosshart, U.; Huber, P.; Peter Odermatt, R.; Sonnleitner, B. Cytometry 2012, 81A (6), 508–516. (15) Bercovici, M.; Han, C. M.; Liao, J. C.; Santiago, J. G. Proc Natl Acad of Sci USA 2012, 109 (28), 11127–11132. (16) Bahga, S. S.; Han, C. M.; Santiago, J. G. Analyst 2012, 138 (1), 87–90. (17) Eid, C.; Garcia-Schwarz, G.; Santiago, J. G. Analyst 2013, 138 (11), 3117–3120. (18) Ostromohov, N.; Schwartz, O.; Bercovici, M. Anal Chem 2015, 87 (18), 9459–9466. (19) Schwartz, O.; Bercovici, M. Anal Chem 2014, 86 (20), 10106–10113. (20) Lantz, A. W.; Brehm-Stecher, B. F.; Armstrong, D. W. Electrophoresis 2008, 29 (12), 2477–2484. (21) Ootsubo, M.; Shimizu, T.; Tanaka, R.; Sawabe, T.; Tajima, K.; Yoshimizu, M.; Ezura, Y.; Ezaki, T.; Oyaizu, H. J Appl Microbiol 2002, 93 (1), 60–68. (22) Hogardt, M.; Trebesius, K.; Geiger, A. M.; Hornef, M.; Rosenecker, J.; Heesemann, J. J. Clin. Microbiol. 2000, 38, 818–825.

Figure 1: Schematic of Counter-pressure assisted ITP for Inline fluorescence in situ hybridization. (i) The capillary is filled with 1.8% w/v HEC dissolved in 50 mM TRIS HCl (pH8.0). (ii) The solution at the inlet is switched to LE containing the probe (2.6 µM) and pumped into the capillary at 10 psi for 9 min. (iii) The solution at the inlet is changed to the bacterial cells in spacer electrolyte, applying a voltage of – 16kV electrokinetically inject the cells and spacer into the capillary. A counter-pressure (18 psi) was applied from the outlet to inject the 1.8% HEC into the capillary while the probe and cells focus at the ITP boundary and hybridize.(iv) After 2 min, the counter pressure is removed and the solution at the inlet is changed to TE while the outlet solution is replaced with LE. (v) When the voltage is switched back on, the ITP system migrates towards the sieving matrix where the cells are slowed down, allowing the spacer to form a band between the unhybridized probe band and cells.

(23) Kempf, V. A.; Trebesius, K.; Autenrieth, I. B. J. Clin. Microbiol. 2000, 38 (2), 830–838. (24) Phung, S. C.; Nai, Y. H.; Powell, S. M.; Macka, M.; Breadmore, M. C. Electrophoresis 2013, 34 (11), 1657–1662. (25) Phung, S. C.; Nai, Y. H.; Macka, M.; Powell, S. M.; Guijt, R. M.; Breadmore, M. C. Anal Bioanal Chem 2015, 407 (23), 6995–7002. (26)

Kligman, A. M. JAMA 1965, 193, 796–804.

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Figure 2: (A) Isotachopherogram of different concentrations of E. coli analyzed with Enterobacteriaceae probe. Inset is the calibration curve of different E. coli concentration vs average peak area (n=3). (B) Isotachophoregoram of P. aeruginosa cells analyzed with PseaerA probe. Inset is the calibration curve of different P. aeruginosa concentration vs average peak area (n=3). Protocol as illustrated in Figure 1 using the following solutions LE 50 mM TRIS HCl (pH 8.0) with 0.05% w/v HEC, 5 mM MgCl2 0.1% v/v DMSO; TE 50 mM TRIS HEPES (pH 8.0) with 0.1% v/v DMSO; SE 50 mM MES adjusted with TRIS to pH 7.8 with 0.1% v/v DMSO.

Figure 3: (A) Isotachopherogram of E. coli and P. aeruginosa analyzed using 2.6 µM Enterobacteriaceae probe. (B) Isotachopherogram of E. coli and P. aeruginosa cells analysed using 2.6 µM of PseaerA probe. (C) Isotachopherogram of E. coli and P. aeruginosa analyzed using 2.6 µM of Enterobacteriaceae-2 probe.



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Figure 4: (A) Graph of 2.6 µM and 26µM PseaerA concentrations vs different E. coli mixture mix with constant P. aeruginosa cell concentration. (B) Isotachopherogram of different PseaerA probe concentrations using constant P. aeruginosa cell concentration (2.7 x 106 cells/mL). (C) Graph response of 2.6 µM and 26µM PseaerA concentrations vs different concentrations of E. coli mixture with constant concentration of P. aeruginosa cells.

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Figure 5: (A) Isotachopherogram comparison between in-line FISH ITP of P. aeruginosa cells and off-line FISH ITP of P. aeruginosa cells at constant P. aeruginosa concentration. (B) (i) Image of cell count of cells analyzed using counter pressure ITP method for both off-line and in-line with same cell concentrations.(ii) Inset of the total cell counts for each methods. (C): Isotachopherogram of 3.0 x 104 cells/mL stained off-line FISH method and 6.0 x 104 cells/mL stained in-line FISH method using counter pressure ITP.

Figure 6: Isotachopherogram of different flushing time of LE solution with 26µM Enterobacteriaceae- 2 probe with 2.8 x 107 cells/mL of E. coli.


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Isotachophoretic Fluorescence in Situ Hybridization of Intact Bacterial

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