Microdialysis Sampling of Quorum Sensing Homoserine Lactones

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Microdialysis Sampling of Quorum Sensing Homoserine Lactones during Biofilm Formation Alda Diaz Perez, Kaleb Kougl, Thaddeus W. Vasicek, Rohana Liyanage, Jackson Lay, and Julie A. Stenken Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05168 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 13, 2019

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Analytical Chemistry

Microdialysis Sampling of Quorum Sensing Homoserine Lactones during Biofilm Formation Alda Diaz Perez, Kaleb Kougl, Thaddeus W. Vasicek, Rohana Liyanage, Jackson Lay, and Julie A. Stenken* Department of Chemistry and Biochemistry University of Arkansas * Corresponding Author: Phone: 479-575-7018; email [email protected] Abstract Bacteria communicate chemically through a system called quorum sensing. In this work, microdialysis sampling procedures were optimized to collect quorum sensing molecules produced during in situ biofilm formation directly on the polymeric semipermeable membrane of the microdialysis probe.

V. harveyi, a gram-negative bacterium, was used as the model

organism and releases variable chain length acyl-homoserine lactones (AHLs) and acyl-oxohomoserine lactones (AOHLs) as signaling molecules during quorum sensing. Eliciting biofilm formation required coating fetal bovine serum onto the polyethersulfone microdialysis membrane. Dialysates were collected in different experiments either during or after biofilm formation directly on a microdialysis probe. Continuous sampling of C4-AHL, C6-AHL, C8-AHL, C6-OXO-AHL, and C12-OXO-AHL was achieved over a period of up to 4 days. The AHLs and AOHLs in dialysates were concentrated with solid phase extraction and quantified using LC-MS. Dialysate concentrations obtained for the AOHLs and AHLs ranged between 1 and 100 ppb (ng/mL) and varied between sampling days.

This work demonstrates the initial use of

microdialysis sampling to collect quorum sensing signaling chemicals during biofilm formation by a gram-negative bacterial species.

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Introduction Bacteria communicate through a chemical mechanism termed quorum sensing (QS).1, 2 Different species of bacteria use unique signaling molecules that are emitted as beacons to determine their species density within their microenvironment. Under certain conditions, particularly when a reduction of nutrients occurs, these QS signals are amplified resulting in diverse phenotypic changes, matrix production, and a collection of bacteria called a biofilm.3 Biofilm formation is a dynamic process that involves 1) transport and initial attachment of the microbes, 2) surface attachment, 3) microcolony formation, 4) biofilm maturation and 5) detachment and dispersion of the cells.4

Biofilm formation has been suggested to be a

protection mechanism for the bacteria allowing them to function as a single unit. . Quorum sensing systems include low molecular weight molecules allowing intercellular communication resulting in biofilm formation, virulence, and antibiotic resistance.1, 2, 5 For gramnegative bacteria studied in this work, the most prevalent QS signals are N-acyl-L-homoserine lactones (AHLs) and N-oxo-acyl-homoserine lactones (AOHLs) whose structures are shown in Figure S1. AHLs are known to mediate cross-talk between different species of microbial communities.6 It is important to note that not just one type of AHL or AOHL is released during quorum sensing, but different types. Gram-positive bacteria as well as some gram-negative bacteria have signaling chemicals that are typically peptides.7 The discovered and reported quorum sensing signaling chemicals known for different types of bacteria continue to grow.8, 9 Quantitative measurement of quorum sensing signals is challenging since typical concentrations are in the low nanomolar range (~ 1 ppb/1 ng/mL).10, 11 Common methods for measurement of quorum sensing chemicals include different engineered biosensors, chromatographic separations coupled with mass spectrometric detectors, or spectroscopic identification.12-15 Quorum sensing detection methods have been reviewed.16 Due to the low nM (ng/mL) detection limits needed, the variety of AHLs/AOHLs emitted, as well as the chemical similarity of the various AHLs and AOHLs, LC-MS/MS techniques are commonly used for these


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quantitative measurements due to the requirements for separation, quantitation and confirmation of the different analytes.11 Microdialysis is a sampling technique that has been widely used for collection of various solutes from localized and chemically-complex environments, particularly from the extracellular fluid space (ECF) of tissues.

17, 18

The primary advantages of this sampling technique include

the small probe size (200 to 500 microns in width) and its ability to directly sample from numerous types of complex matrices. Microdialysis probes are thus well suited for interrogating the chemical signaling occurring during biofilm development. The concentrations of analyte in the perfusion fluid relative to the sample medium is defined by the dialysis probe’s extraction efficiency,

, where Coutlet, Cinlet, and Csample are the analyte concentrations in the

outlet, inlet and sample, respectively. Since it is expected that analyte concentrations in the dialysis probe are less than those in the surrounding medium, the EE is used to relate collected concentrations to external concentrations. In this work, we describe the optimization procedures necessary to create an initial use of microdialysis sampling as an in situ tool to study quorum sensing processes occurring during localized biofilm formation. Biofilm formation used in this study involved static means using the tube-adherence method to obtain the biofilm.19

Different procedures were necessary to

optimize the model biofilm system. A particular focus was needed to promote biofilm formation directly onto the microdialysis membrane. The data presented here demonstrates that microdialysis sampling can be used as a model system to collect AHLs and AOHLs during various stages of biofilm formation.

These procedures are a necessary first step toward

integrating microdialysis sampling as a means to follow biofilm formation, quorum sensing communication, and ultimately immune system response in both in vitro as well as in vivo settings.



Materials and Methods Materials and reagents Vibrio harveyi (ATCC BAA-1116) was purchased from American Type Culture Collection (Manassas, VA). Marine broth (BD 279110) and 22 g needles (Hamilton 90134) were obtained from VWR (Radnor, PA); HPLC grade water, methanol, formic acid, and acetonitrile were obtained from Fisher Scientific (Hanover Park, IL). Fetal bovine serum (FBS) was obtained from Sigma-Aldrich (St. Louis, MO), and Bond Elut-PPL with a 50 mg bed capacity was obtained from Agilent Technologies (Santa Clara, CA). N-[(3S)-tetrahydro-2-oxo-3-furanyl]-butanamide (C4-AHL), N-[(3S)-tetrahydro-2-oxo-3-furanyl]-hexanamide (C6-AHL), N-[(3S)-tetrahydro-2-oxo3-furanyl]-octanamide (C8-AHL), 3-oxo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-hexanamide (C6OXO-AHL), and 3-oxo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-dodecanamide (C12-OXO-AHL) were obtained from Cayman Chemical (Ann Arbor, MI).

Microdialysis All microdialysis sampling was performed using CMA 20 probes, 10-mm membrane length, with a polyethersulfone membrane and 100 kDa molecular weight cutoff (Harvard Apparatus, Holliston, MA). The microdialysis probes were perfused using a BAS Bee microdialysis pump (Bioanalytical Systems Inc, West Lafayette, IN) and 3 mL sterile syringes (VWR) connected with 22 g needles (Hamilton 90134). Probes were placed into neat fetal bovine serum (FBS) solutions overnight prior to placement into bacterial cultures.

Bacteria strain and growth conditions Vibrio harveyi (V. harveyi) with strain designation BB120 was used. The growth medium for V. harveyi contained 37 g/L of marine broth in HPLC grade water and was autoclaved at 121 °C, prior to use.

The bacteria CFU/mL was measured indirectly using an optical density


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measurement with a spectrophotometer at 600 nm. The bacteria were incubated to a final concentration of approximately 1 × 107 CFU/mL, which occurs during their stationary growth phase. The microdialysis probe was incubated in Vibrio harveyi for 4 days at 30°C in a plate shaker at 300 RPM.20 A 1% (v/v) solution of safranin in HPLC grade water was made to stain for the gram-negative bacteria. Biofilms were stained by placing the probe in the safranin solution for 10 mins at room temperature followed by two successive washes with HPLC-grade water.

Microdialysis Optimization Procedures. Standard extraction efficiency determination: The extraction efficiency of C4-AHL, C6-OXO-AHL, C6-AHL, C8-AHL, and C12-OXOAHL was determined using spiked samples (2 µg/mL, 2000 ppb) into the marine broth with a perfusion fluid flow rate through the dialysis probe between 1.5 and 4.5 µL/min.

These

experiments were performed using a clean microdialysis probe without FBS coating.

Biofilm effect on extraction efficiency. An important aspect of the work was to determine the potential and level of extraction efficiency reduction for the targeted AHL and AOHL compounds during biofilm formation on the dialysis probes. To determine if biofilm growth on the semipermeable membrane blocked the diffusion of QS chemicals from the sampling medium to the probe, the probe membranes were treated with the FBS as noted above. Then, a treatment probe was incubated with V. harveyi for 4 days at 30 °C to promote biofilm formation. After the incubation, an untreated control (CC) probe pulled from a new package and treatment probe with biofilm attached (CT) were used to collect a wide range of molecules. For this experiment, fluorescein isothiocyanate-dextran (4, 10 and 20 kDa) was prepared (100 µM concentrations) in marine broth, the probe was then immersed in these solutions and perfused with phosphate buffered saline (PBS) solution (137



mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) or the marine broth. The rationale for using the FITC-dextrans rather than the AHLs/AOHLs is discussed below.

Sample preparation and HPLC-MS/MS quantification AHLs and AOHLs were extracted with Bond Elut-PPL solid-phase extraction (SPE) device with a 50 mg bed capacity (Agilent Technologies, Santa Clara, CA) connected with a vacuum manifold. To extract AHLs from the marine broth medium or Ringer’s solution (Ringer's solution was prepared using 7.2g sodium chloride, 0.17 g calcium chloride and 0.37g of potassium chloride in 1 L of HPLC water at pH 7.3-7.4), the cartridge was washed using 2 mL of 0.1 % v/v of formic acid in methanol and conditioned with 2 mL of 0.1% v/v of formic acid in HPLC grade water. Then, 1.0 mL of the broth medium collected as the dialysate was loaded into the SPE cartridge. The cartridge was washed with 2 mL of 0.1 % v/v of formic acid in HPLC grade water. Finally, to extract AHLs and AOHLs from the SPE cartridge, the cartridge was loaded with 2 mL of 0.1% of formic acid in methanol. To dry the sample, a CentriVap Vacuum was used at room temperature for 3 hours. Samples were reconstituted in 100 µL HPLC grade water and stored at -80 °C until analysis. LC-MS/MS analysis was performed using a Shimadzu UPLC-20A/LC-30A in line with the Shimadzu 8060 triple quadrupole mass spectrometer with a heated electrospray source (positive-ion mode). Separations were performed using a C18 column (2.1 x 50 mm, 1.9 µm particle size, Shimadzu, UHPLC check out kit) with a linear gradient comprised of 0.1% formic acid (FA) in HPLC grade water/ 0.1% FA in acetonitrile ramped at a rate of 7.5% acetonitrile/min over 10 min. The flow rate was 0.3 mL/min. Sample volumes of 1 µL were injected. The three most intense multiple reaction monitoring (MRM) fragments for each AHL and AOHL standard was first fully optimized at their corresponding retention time windows using Shimadzu Labsolution software,Version 5.89 (Table S2). From the three MRM channels, the MRM event corresponding to the most intense fragment ion was used as the “quant” ion and the other two


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fragment ions were used as reference ions for identification purposes. Reference ion ratios were calculated compared to the “quant” ion and unknown samples were required to meet within 30% of the reference ion ratio of the standard as this is the minimum required by the software program. All AHL and AOHL standards and samples were analyzed three times.

Stability of AHLs and AOHLs Since the microdialysis sampling process was occurring continuously within a single culture, a stability experiment was performed by incubating samples with standards for varying time points, ranging from 1 day to 5 days in single day intervals. For this experiment, five separate groups (n=3) containing 1-mL of marine broth medium spiked to 5 µg/mL with C4-AHL, C6-AHL, C6-OXO-AHL, C8-AHL, and C12-OXO-AHL were used. The solution was maintained at 30C.

Each group was then removed and immediately processed using the Bond Elut PPL

SPE cartridges with the eluates stored at -80°C until chemical analysis. Chemical analysis of all samples occurred within two days of the last collected stability sample.

Quorum sensing chemical collection during biofilm formation The quorum sensing chemicals were measured from dialysates under different biofilm and sampling conditions described below. To do this, two groups were prepared (Figure S2). For the first group, the probe was incubated with neat FBS overnight, followed by incubation with 1 x 107 CFU/mL of V. harveyi for 4 days in marine broth for 24 hours, then the microdialysis collection process was initiated using Ringer’s solution at 5 µL/min with collection times at 5 and 20 hrs after the initiation of the perfusion infusion (Figure S2A).

All experiments were

maintained at 30°C and the 5 and 20 hr collection times were maintained for four days (e.g., 15 hrs and then 9 hr sampling periods).

For the second group (Figure S2B), the probe was

incubated in neat FBS overnight, and then the probe was incubated with 1 x 107 CFU/mL of V. harveyi in a plate shaker for 4 days at 30°C and then sampling commenced on day 5 through



day 8. The marine broth for all sampling procedures remains throughout the sampling process and is not changed. All collected dialysates were stored at -80C prior to work up. Results and Discussion Chromatographic Separation. Chromatographic separation was achieved between the standard AHLs and AOHLs which showed a consistent retention time and a predictable increase in the retention as the acyl chain length increased (Figure S3). LC-MS/MS quantitation for quorum sensing compounds is necessary to quantify different QS chemicals due to the LOD requirements in the ppb range.21-23 In this study, LC-MS/MS with MRM was applied to quantify the AHLs and AOHLs in the dialyates. The instrument LOD was found to be in the low ppb range for C4-AHL, C6-OXOAHL, C6-AHL, C8-AHL and C12-OXO-AHL (~ 34 to 70 pM), which is comparable to results in published literature (Table S1).6, 21, 22

Sample preparation. Bacteria and the formed biofilms can be sensitive to chemical changes within their environment. We were initially concerned that a loss of nutrients dialyzed from the probe may cause a lack of biofilm formation. This would necessitate using marine broth as the perfusion medium which would require sample clean up before LC/MS-MS analysis. While this did not turn out to be the case, all samples were prepared with solid phase extraction before quantitation. We had additional concerns that concentrations of the quorum sensing signals might be too low to quantify from the collected dialysates. We chose to use a higher perfusion fluid flow rate through the dialysis probe to obtain more analyte mass and thus use the sample preparation process as a means to concentrate the collected dialysates. For low molecular weight solutes, mass recovery in microdialysis sampling increases with increasing flow rates.2426


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For marine broth processing, the Bond Elut-PPL material was used as it has been previously shown to extract the AHLs with higher recovery than other stationary phase materials without significant loss of the hydrophilic C4-AHL.22 Bond-Elut PPL is a styrene-divinylbenzene (SDVB) polymer that has been modified with a proprietary nonpolar surface and is reported to retain polar analytes. Retention of polar analytes is needed as the estimated octanol-water partition coefficient (log P) values for the acyl homoserine lactones are such that they are mostly hydrophilic (C4 AHL [-1.1]; C6 AHL [-0.04], C6-oxo [-0.04] and C12-oxo [2.02], no data was available for the C8-AHL).27-31 The absolute mass recovery (n=6) using the Bond Elut-PPL SPE for spiked samples from the marine broth was calculated and found for C4-AHL (72 ± 3%), C6-

Figure 1: Absolute recovery values for the AHL/AOHLs using Bond-elut-PPL solid phase extraction (SPE). Spikes (5 µg/mL) of acyl-oxo-homoserine lactone (AHLs) and acyl-homoserine lactone (OXOAHLs) in marine broth were used. The data represent mean  SD, n=6 different spike measurements.

OXO-AHL (100 ± 6%), C6-AHL (100 ± 6%), C8-AHL (100 ± 17%) and C12-OXO-AHL (95 ±14%) shown in Figure 1. The loss of the C4-AHL during the sample preparation process is not surprising as it is the most hydrophilic of the AHLs and likely lost during sample processing. The mass recovery values found by this work using the marine broth are similar to those



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previously reported for agricultural based bacterial quorum sensing.22 The volume of samples was reduced by 10-fold resulting in an additional 10-fold increase in concentration recovery, which has been applied for all reported sample concentrations.

Stability of AHLs Since microdialysis sampling was performed continuously over many days, it was important to determine the stability of the AHL and AOHL compounds within the marine broth. A decrease was observed for AHL and AOHL concentrations from spiked marine broth samples (Figure 2), leading to approximate hydrolysis decay of 8 x 10-8 10-5

for C6-AHL, 5.8 x 10-6

for C6-OXO-AHL, 6.6 x 10-6

for C8-AHL and 8.8 x 10-6

for C4-AHL, 1.5 x

for C12-OXO-AHL. What this data suggests is that

during the microdialysis sampling period the collected lactones are likely generated in situ as they degrade by 80% over a two-day period in the marine broth, likely due to the hydrolysis of the lactone ring.32 The LC-MS method was not set up to monitor or identify degraded lactones. Additionally, other species of gram- negative bacteria actually use their quorum sensing

Figure 2: AHL/AOHL stability over 5 days. Samples were spiked to 4 µg/mL and incubated in marine broth at 37 °C, for up to 5 days. At each day, each sample was prepared and stored in -80 °C. Error bars denote mean  SD, n=3 for each day. (18 total samples including the stock).


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chemicals as a food source.33 Due to these uncertainties, the concentration values reported for the AHLs and AOHLs are those found directly in the dialysates and are not corrected for these alterations in concentrations caused by the hydrolytic instability or biological processes that may remove these chemicals from their environment.

Initial Biofilm Formation. Multiple techniques for creating in vitro biofilm have been described for different bacterial species using different types of in vitro devices (e.g., Calgary device, drip flow.).22 Despite the critical needs in biofilm research, there is no absolute consensus with respect to the preferred ways to perform in vitro biofilm studies. The most important goals seem to be ensuring in vitro models meet the needs of the research question posed. 34 While the PES membranes used in the microdialysis probes are considered proprietary, it is known these hollow-fiber membranes are derived from kidney dialyzer units. As such, the membrane materials typically prevent protein and biological fouling. In this work, we found that V. harveyi did not attach to untreated microdialysis membranes and favored attaching to the interface between the plastic tubing and the membrane. It has been reported that different physicochemical properties of membranes affect bacterial adhesion and biofilm formation.35 To promote biofilm formation and growth onto the membrane, we had to coat the membrane with fetal bovine serum (FBS) overnight prior to immersion into V. harveyi culture. Biofilm was only observed on membranes incubated with FBS overnight. It is known that different proteins including fibronectin, fibrinogen, vitronectin, and elastin have a role in the initial bacterial attachment to various surfaces.36 Protein adsorption onto the polyethersulfone semi-permeable membrane was necessary in order to observe the formation of biofilm growth into the microdialysis probe (Figure 3).



Figure 3: (A) Microdialysis probe incubated in fetal bovine serum for 24 hours and then in V. harveyi for 4 days at 37 °C. Picture showed a probe coated with Vibrio harveyi around the polymeric semipermeable membrane and (B) control probe incubated with V. harveyi. Probes are stained with safranin.

Fouling effects: Microdialysis extraction efficiency is affected by complex samples that can foul the membrane resulting in reduced extraction efficiency.37 Biofilm formation around the microdialysis probe would be expected to cause restricted diffusion of the QS compounds through the membrane pores. First, it was evaluated if the growth of the biofilm around the semipermeable membrane blocked the diffusion of QS chemicals from the broth into the probe. A probe that has a biofilm was placed into a marine broth spiked with AHLs and AOHLs. With this approach, it appeared the presence of quorum sensing signals in the external solution caused phenotypic changes and disruption (release) of pockets of the biofilm. Moreover, the bacteria embedded within the biofilm may also react to the external quorum sensing chemicals


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by releasing additional AHL or AOHLs and as such may not represent a valid measure of the extraction efficiency and/or how it changes with the potential for increased mass transport resistance with the biofilm. Due to these observations, we believed such an approach was not a valid way to attempt to address diffusivity issues through the surrounding biofilm on the dialysis probe. For this reason, we chose to use a series of FITC-labeled dextrans to assess potential diffusivity differences between a biofilm-adhered membrane vs. a clean membrane on the CMA/20 microdialysis probes. We also used this approach to assess the viscosity effect on the

Figure 4: Extraction efficiency comparison for different microdialysis conditions and perfusion fluids. Extraction efficiency was calculated using a 100 µM spike medium for FITC-4, FITC-10 and FITC-20 was calculated using a flow rate at the flow rates listed. For this experiment, PBS or marine broth was used as a perfusion fluid. Both PBS and CC are probes without biofilm and perfused with PBS or marine broth (CC). CT is a probe with biofilm and perfused with marine broth. Error bars denote mean  SD, n=3 probes.

extraction efficiency from the marine broth. Figure 4 shows a decrease of EE% at 1.5 µL/min from 35% to 20% for FITC-4 (4000 Da) between using PBS vs. the marine broth as a perfusion fluid. No difference in the extraction efficiency was observed between a probe with a biofilm



(CT) vs. a clean probe with no biofilm (CC) using the marine broth. Intuitively, the attachment of biofilm to the membrane would be expected to add more mass transport resistance during the microdialysis sampling process. However, there have been different biofilm mass transport analyses using low molecular weight antibiotics that demonstrate effective diffusivity through different biofilms is dependent upon the bacterial-producing biofilm strain as well as the specific antibiotic.38 The data on solute diffusion through biofilms as to whether transport is significantly hindered or not is mixed.39 The use of the marine broth as the perfusion fluid resulted in a nearly 50% decrease in extraction efficiency for FITC dextrans. To assess the approximate extraction efficiency of the AHL and AOHLs, marine broth was spiked with these solutes and standard in vitro microdialysis sampling procedures were performed with PBS as the perfusion fluid. At the 5 µL/min flow rate used in these studies, we estimate a nearly 10% EE for the AHLs and AOHLs quantified in this work (Figure 5).

Figure 5: Extraction efficiency through control microdialysis probes. The data represent the mean  SD, n=3 probes.


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Biofilm Formation After optimization of the LC-MS/MS quantitation method, sample preparation and microdialysis process, we performed different timed experiments to assess QS production during biofilm formation and maturation. Two separate experiments were performed. In the first set of experiments, the probe was incubated in fetal bovine serum overnight, then transferred to the marine broth containing V. harveyi for 24 hrs prior to initiating and then QS was collected continuously for 4 days. To obtain enough sample for the sample preparation process, collection occurred over four days at 5 µL/min for 4 days. Figure 6 shows that a decrease of AHLs at day 1 from 5 hrs to 20 hrs occurred; however, it is also important to note a steady concentration of AHLs were observed from day 2 through day 4. The high concentrations observed at 5 hours on day 1 are likely an accumulation of AHLs in 24 hours period which the probe was incubated, but not perfused. On the other hand, C6-OXO-AHL decreased in concentration from day 1 until day 2, and then the concentration of C6-OXO-AHL was below of the instrument detection limit (Table S1). The steady concentration of AHLs in the medium could be explained by the presence of nutrients; however, a decrease of nutrients could change the concentration of AHLs in the microenvironment. For example, in the next experiment, the probe was incubated overnight in FBS and then incubated in marine broth with 1.0 x 106 CFU/mL of V. harveyi for 4 days at 30°C, and then collected continuously for 4 days, (Figure 6B). In this experiment, Figures 7a and 7b shows an increase of AHLs and C-12-oxo AHL from days 5 to days 8. This increase of concentration of AHLs can be explained due to the decrease of nutrient in the medium and the increase of biofilm formation. By comparing Figures 6 and 7, it is possible to observe the presence of AHLs and AOHLs during biofilm formation and how these concentrations change during the development of the biofilm onto the surface of the microdialysis probe. The production of AHLs can be amplified or repressed possibly due to the presence of nutrients in the microenvironment.3



Figure 6: AHL/AOHL recovered from microdialysis probes immersed in Vibrio harveyi and collected over a four day period. A flow rate of 5 μL/min was used and samples were collected at 5 and 20 hours. Each sample was collected from day 1 (24 hours after inoculation) through day 4 (observable biofilm). (A) Mass recovery for C4AHL, C6-AHL, and C8-AHL and (B) for C6-OXO-AHL and C12-OXO-AHL. The data are represented as three separate microdialysis probes in three separate cultures. Error bars represent the SD among these three samples.

The predominant AHL/AOHLs that were quantified in the dialysate samples were the C4, C6-oxo and C12-oxo-AHLs.

There is known tremendous diversity in the various quorum

sensing chemicals in the AHL class produced by the marine Vibrio species.40 Vibrio harveyi has been identified as producing the C4-AHL.41 The variety of these AHLs continue to be reported


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noting recent report of different gram-negative bacterial species producing AHLs that had not been previously described.21 The role that various AHLs play as either signals or cues remains

Figure 7: AHL/AOHL recovered from microdialysis probes immersed in Vibrio harveyi and collected over a four day period after the probe had visible biofilm. A flow rate of 5 μL/min was used and samples were collected at 5 and 20 hours. Each sample was collected from day 1 (24 hours after inoculation) through day 4 (observable 17 biofilm). (A) Mass recovery for C4-AHL, C6AHL, and C8-AHL and (B) for C6-OXO-AHL and C12-OXO-AHL. The data are represented as ACS in Paragon Plus Environment three separate microdialysis probes three separate cultures. Error bars represent the SD among these three samples.


an active research area that will continue to demand new and improved analytical chemistry approaches.42

Conclusion An initial in vitro model system using the tube-adherence method for quorum sensing detection during biofilm development has been created using microdialysis sampling with 100 kDa MWCO polyethersulfone membranes. Our choice for these membranes was based on a longer-term interest in collecting larger molecular weight solutes in this system particularly signaling proteins from macrophages. Differences in bacterial adhesion to different surface chemistries will likely require slightly different methods for obtaining attached biofilm . Given the variety of bacterial species, it is likely that obtaining biofilms derived from different species onto the microdialysis membrane will require different coating reagents to promote bacterial adhesion. Many other types of biofilm formation processes have been extensively reviewed in the literature.19

The use of microdialysis in these processes has not been described, but if

experiments are designed such that the ~ 500 micron (i.d.) × 4 or 10 mm membranes could be embedded into the biofilm process, there is nothing that precludes the use of the microdialysis sampling technique for other biofilm formation processes. With the V. harveyi created biofilm, the presence of biofilm attached to the microdialysis probe did not seem to affect the ability to extract the different AHLs and AOHLs. Low ppb (ng/mL) concentrations of the quorum sensing chemicals were monitored over a period of many days. This process is the first step of many necessary to be able to consider the use of this model system for studies in vivo. Another limitation include the need to determine how flux or removal of quorum sensing signals effects biofilm development long term. Given the historical use of microdialysis sampling in vivo, much additional research remains to determine how effective this new model system will be with respect to further understanding different


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fundamental aspects of biofilms. Getting in vitro models that can ultimately be used for in vivo measurements and assessment is of ongoing interest within the biofilm community.43

Acknowledgements: We acknowledge NSF (1641166) and a University of Arkansas Honors College grant (KK) for funding.

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