Method and Measuring Device for Determination of the Growth Rate of Biofilm

Abstract

A method for determination of the growth rate of biofilm (7) using an electrical impedance analyses is disclosed. The method comprises the steps of: bringing a culture medium fluid (3) in contact to an electrode structure (4a, 4b), having biofilm (7) grown within the fluid culture medium (3) with the biofilm (7) arranged in distance to the electrodes structure (4a, 4b), so that the fluid culture medium (3) is placed between the growing biofilm (7) and the electrode structure (4a, 4b); measuring the impedance of the electrodes structure (4a, 4b) over a monitoring time, and determining the growth rate of the biofilm (7) as a function of the reduction rate of the impedance values measured on the electrode structure (4a, 4b).

Claims

1. A method for determination of the growth rate of biofilm using an electrical impedance analysis, comprising: bringing a fluid culture medium in contact with an electrode structure, growing biofilm within the fluid culture medium with the biofilm arranged at a distance relative to the electrode structure so that the fluid culture medium is between the biofilm which is being grown and the electrode structure, measuring impedance values of the electrode structure over a monitoring time, and determining a growth rate of the biofilm as a function of a reduction rate of the impedance values measured on the electrode structure.

2. The method according to claim 1, wherein the determining step is performed by scaling the measured impedance values and determining the growth rate of the biofilm as a function of the reduction rate of the scaled impedance value.

3. The method according to claim 2, wherein the step of scaling the measured impedance values is performed with respect to related reference impedance values measured on a same or similar electrode structure without biofilm.

4. The method according to claim 3, further comprising the steps of determining scaled cell index values as a function of the impedance values measured at different frequencies and of related reference impedance values without biofilm, and determining the growth rate of the biofilm in proportion to a reduction rate of said scaled cell index values.

5. The method according to claim 1 wherein the step of determining the growth rate of the biofilm is performed as a function of a reduction rate of a reactance portion of the measured impedance values, wherein the impedance values comprise the reactance portion and a resistance portion.

6. The method according to claim 1 wherein the step of determining the growth rate of the biofilm is performed after expiration of a specific growth time, the specific growth time being expired at the earliest when a continuous reduction of the impedance values measured on the electrode structure occurs.

7. The method according to claim 1 wherein the step of determining the growth rate of biofilm is performed for species having an ability of forming pellicles.

8. The method according to claim 1 wherein the biofilm comprises Pseudomonas aeruginosa.

9. The method according to claim 1 further comprising the step of characterizing substances added to the fluid culture medium for their ability to modulate growth of the biofilm.

10. A measuring device for determining a growth rate of biofilm, comprising: at least one electrode structure, an impedance measuring unit electrically connected or connectable to the electrode structure for measuring impedance of the electrode structure, and at least one receptacle for storing a fluid culture medium, wherein the at least one receptacle is arranged for being coupled to the electrode structure such that the fluid culture medium is able to contact the electrode structure, an evaluation unit arranged for determining the growth rate of biofilm located or locatable at a distance from the electrode structure such that the fluid culture medium is between the electrode structure and the biofilm being grown, and wherein the evaluation unit evaluates growth of the biofilm as a function of a reduction rate of the impedance values measured on the electrode structure.

11. The measuring device according to claim 10, wherein the at least one of receptacle comprises a chamber having a closed bottom, and the electrode structure is arranged at the bottom of the chamber in an internal space of the chamber.

12. The measuring device according to claim 10, wherein the at least one receptacle comprises an upper chamber, a lower chamber, and a microporous membrane between the upper chamber and the lower chamber, and wherein the electrode structure is arranged adjacent to the microporous membrane in an interconnection area of the upper chamber and the lower chamber, and wherein the upper chamber is configured to store the fluid culture medium with the biofilm growing in the upper chamber.

13. The measuring device according to claim 10 wherein the electrode structure comprises a pair of comb-type finger electrodes, wherein fingers of the pair of comb-type finger electrodes are alternately arranged adjacent and at a distance from each other such that adjacent fingers extend in opposite directions from each other.

14. The measuring device according to claim 10 wherein the at least one receptacle includes a plurality of receptacles, wherein each receptacle of the plurality of receptacles comprises a pair of electrodes having a first electrode and a second electrode, and wherein at least two of the first electrodes are electrically interconnected to each other, and wherein the second electrodes are individually controllable by the impedance measuring unit.

15. The measuring device according to claim 10 wherein the evaluation unit is arranged for determining the growth rate of the biofilm by scaling the measured impedance values and determining the growth rate of the biofilm as a function of the reduction rate of the scaled impedance value.

16. A method for characterizing substances for their ability to modulate growth of a biofilm, comprising: growing the biofilm in a measuring device which comprises at least one electrode structure, an impedance measuring unit electrically connected or connectable to the electrode structure for measuring impedance of the electrode structure, and at least one receptacle for storing a fluid culture medium, wherein the at least one receptacle is arranged for being coupled to the electrode structure such that the fluid culture medium is able to contact the electrode structure; and evaluating the biofilm with an evaluation unit arranged for determining the growth rate of the biofilm located or locatable at a distance from the electrode structure such that the fluid culture medium is between the electrode structure and the biofilm being grown, and wherein the evaluation unit evaluates growth of the biofilm as a function of a reduction rate of the impedance values measured on the electrode structure.

17. The method of claim 7 wherein said species comprises a pel-genotype.

Description

[0044] The invention is described by use of exemplary embodiments with the enclosed drawings. It shows:

[0045] FIG. 1—Block diagram of a first embodiment of a measuring device for determining the biofilm growth rate;

[0046] FIG. 2—Block diagram of a second embodiment of a measuring device for determining the biofilm growth rate comprising a microporous membrane;

[0047] FIG. 3—Diagram of crystal violet (CV) by monitoring absorption at 550 nm staining measurements of biofilms formed by PA14 (wild type WT, ΔpelA, and ΔpqsA) in a fluid culture medium LB at various timepoints (FIG. 3A), compared to the cell index measurements (FIG. 3B) of these strains under equal conditions;

[0048] FIG. 4—Diagram of the cell index over the time for determining the PA14 biofilm growth rate in a fluid culture medium LB with ΔpelA, wild type WT and ΔpqsA;

[0049] FIG. 5—Diagram of PA 14 biofilms determined after 43 hours with crystal violet by monitoring absorption at 620 nm;

[0050] FIG. 6—Diagram of the RK cell index per hour for PA14 biofilms determined by impedance spectroscopy.

[0051] FIG. 7a)—Diagram of the cell index over the time for a control experiment after transplantation of biofilm, the addition of PBS and the LB medium as negative control.

[0052] FIG. 7b)—Diagram of the cell index over the time for an experiment where paraffin was also added in different amounts (10-100 μL) to wells (B) to mimic pellicle biofilm, given paraffin has no dielectric constant its presence showed no effect on the capacitance.

[0053] FIG. 8a-c)—Diagram showing the impact of ciprofloxacin on biofilm formation of PA14.

[0054] FIG. 9a-c)—Diagram showing the impact of tobramycin on biofilm formation of PA14.

[0055] FIG. 10a-c)—Diagram showing the impact of meropenem on biofilm formation of PA14.

[0056] FIG. 1 shows a block diagram of a measuring device 1 for determining the growth rate of biofilm. The measuring device 1 comprises a plurality of receptacles 2a, 2b provided for storing a fluid culture medium 3. Each receptacle 2a, 2b comprising an electrode structure 4a, 4b. In the exemplary embodiment, each electrode structure 4a, 4b is placed at the bottom in the interior space of a chamber 5 formed by a related receptacle 2a, 2b. The receptacles 2a, 2b are placed such, that the fluid culture medium 3 is able to contact the related electrode structure 4a, 4b, wherein one electrode acts as measurement electrode 6a and the other as counter electrode 6b. The electrode structures 4a, 4b each are formed by a pair of comb-type finger electrodes. Each comb-type finger electrode comprising a plurality of fingers extending in the same direction and being placed adjacent to each other like a comb. The pair of comb-type finger electrodes are interdigitating arranged such that the fingers of the pair of comb-type finger electrodes being alternately placed adjacent and in distance from each other such that the adjacent fingers extending in opposite directions from each other.

[0057] When injecting biomass into the fluid culture medium 3, a biofilm 7 is growing within the respective chamber 5 and the biofilm 7 is floating on top of the fluid culture medium 3.

[0058] Other than in the prior art, by use of fluid culture medium 3 and having the biofilm 7 floating on top of the fluid culture medium 3, the biofilm 7 will not adhere to the electrode structure 4a, 4b.

[0059] For all exemplarily described embodiments of the invention and other variants, the impedance of the electrode structures 4a, 4b are each measured by use of an impedance measuring unit 8 in electrical connection to the electrode structures 4a, 4b. In order to measure the impedance, a measuring signal, e.g. a AC sinus wave, is applied to a pair of electrodes of the selected electrode structure 4a, 4b, i.e. the measurement electrode 6a and the counter electrode 6b, and the signal amplitude and phase is then measured. For example when keeping the current constant, the voltage amplitude and phase between the measurement signal and the measured signal will vary as a function of the capacitance. When applying a measuring signal with constant voltage, the current amplitude and the phase angel will vary accordingly. Thus, the impedance measuring unit 8 is arranged to measure the impedance value of a selected electrode structure 4a, 4b. The impedance measuring unit 8 may have a multiplexer for selecting one of the plurality of electrode structures 4a, 4b after the other in order to perform fast impedance measurement for a plurality of receptacles 2a, 2b and their related electrode structures 4a, 4b. The impedance is the sum of the ohmic resistance and the complex reactance:

Z=Z.sub.R+Z.sub.C=R+1/jωC

where ω is the frequency of the measurement signal, C the capacity and R the ohmic resistance.

[0060] The capacity C is related to the reactance with a kind of exponential function. The capacity C is linear proportional to In (Z).

[0061] The growth rate of biofilm 7 floating on the fluid culture medium 3 is a function of the reduction rate of the reactance and the increase rate of the capacitance C.

[0062] Therefore, the measuring device 1 comprises an evaluation unit 9 arranged for determining the growth rate of biofilm 7 being located in distance to the electrode structure 4a, 4b with the fluid culture medium 3 placed between the electrode structure 4a, 4b and the biofilm 7 as a function of the reduction rate of the impedance values measured on the related electrode structure 4a, 4b by use of the impedance measuring unit 8.

[0063] FIG. 2 shows a schematic block diagram of a second embodiment of a measuring device 1. In this second embodiment, the receptacles 2a, 2b each comprising an upper chamber 5a and a lower chamber 5b. The electrode structure 4a, 4b each is fixedly mounted at the bottom of the related lower chamber 5b. The upper chambers 5a are provided for storing the fluid culture medium 3a, 3b and for being placed in the related lower chamber 5b after inserting the fluid culture medium 3 into the related upper chamber 5a and optionally also into the lower chamber 5b. The upper chambers 5a are closed with a microporous membrane 10 at their bottom. When injecting biomass into the fluid culture medium 3, a biofilm 7 is growing within the upper chamber 5a. The biofilm 7 is floating on top of the fluid culture medium 3 and cells are hindered to reach the electrode structure 4a, 4b at the bottom of the respective lower chamber 5b and to adhere to said electrode structure 4a, 4b.

[0064] Thus, when inserting the upper chamber 5a in the corresponding lower chamber 5b, the fluid culture medium 3 will be placed on top on the electrode structure 4a and 4b and the biofilm 7 will grow in distance to the electrode structure 4a, 4b. The microporous membrane 10 has the effect of acting against cells adhering to the electrode structure 4a, 4b, so that interfering effects to the measurement result are reduced. The pore size of the microporous membrane 10 is, for this purpose, preferably smaller than the mean cell diameter.

[0065] FIG. 3 shows a diagram of the cell index for biofilms of Pseudomonas aeruginosa measured by use of the impedance spectroscopy device xCELLigence® Real-time cell analyzer according to the prior art (FIG. 3B), in comparison to a standard method for biofilm quantification that is based on staining of the biofilm with crystal violet (FIG. 3A). The diagram shows that the characteristic change in cell index occurs at time periods (ca. 30-45 h) that are featured by significant growth of biofilm.

[0066] The experiment was repeated for a number of n with n=12 for the well type WT and pelA and pqsA mutants. For the medium control n was equal to 8. ΔpelA is a mutant of the wild type WT without the pelA gene, showing only planktonic growth of biofilm. ΔpqsA is a deletion mutant showing growth of biofilm with an increased capacity as compared to the wild type WT. ΔpelA grown within the fluid culture medium provides an almost horizontal line without significant reduction. Thus, there is no biofilm growing within this sample. With the ΔpelA probe it is also verified that the method according to the present invention is not sensitive for planktonic cells.

[0067] In the example according to diagram of FIG. 3, the cell index is calculated based upon the scaled impedance.

[0068] FIG. 4 shows a diagram of the cell index over the time for a plurality of probes measured with the xCELLigence® Real-time cell analyzer for biofilm of Pseudomonas aeruginosa. The biomass Pseudomonas aeruginosa had been measured as an exemplary biomass. Eventually also other strains can be analyzed. The 96 E-Plate had been used for a measuring time of 43 hours. The fluid culture medium LB had been introduced into the receptacles together with wild type (WT) PA14, ΔpelA deletion mutant or ΔpqsA, respectively.

[0069] When placing a fluid culture medium LB between the electrode structure 4a, 4b and the biofilm 7, the value of the cell index shows a fast decrease within a starting phase, a following increase and, after expiration of the specific growth time, e.g. of ca. 20 hours, an almost linear decrease. A significant linear reduction of the negative cell index occurs after about 35 hours and more preferably in the time frame of the 37 hours to 42 hours. Within this time frame of 37 hours to 42 hours, the directional cell index RK=(cell index[t2]−cell index[t1])/(t2−t1) is decreasing. Thus, for such a short time frame, the value of the cell index change provides the directional cell index RK, which corresponds to the linear relation between the capacitance and the reactance having the general form of a straight line. Optionally, and in particular in case of evaluating longer time frames, the growth rate of biofilm can be calculated as function of the natural logarithm of the cell index, e.g. by the formula RK=In(cell index[t2]−cell index[t1])/(t2−t1).

[0070] The directional cell index RK has, in this example, a value of −7.25E-03/h±7.56E-04 for the biofilm growth rate of WT.

[0071] For the biofilm growth rate of ΔpqsA, the directional cell, index RK has a value,of −1.07E-02/h±1.01E-03.

[0072] The measurements had been taken for a number of 12 samples.

[0073] The biofilm growth rate can be determined according the impedance measurement with fluid culture medium placed between the electrode structure and the biofilm. The biofilm growth rate is proportional to the directional coefficient of the capacitance, e.g. the gradient of (Z) or (cell index) when considering the value of Z or the related cell index. For longer time frames linearizing by use of the natural, logarithm might be necessary, so that biofilm growth rate is proportional to the gradient of In (Z) or In (cell index).

[0074] Thus, the biofilm growth rate is directional proportional to

(cell index(t2)−(cell index (t1)).

and for longer, time frames between t1 and t2 by use of the natural logarithm

In(cell index(t2)−cell index (t1)).

[0075] The variable t1 is the first time of the measurement window after a specific growth time e.g. 37 hours, while t2 is the end time of measuring time, e.g. 42 hours.

[0076] The cell index or impedance curve shows a continuous reduction within this time measurement time frame t1 to t2.

[0077] FIG. 5 is a diagram of various types of PA14 biofilms measured by use of the 96-Well-Plate after 43 hours by use of the fluid culture medium LB with crystal violet analysis by monitoring absorption at 620 nm.

[0078] It is obvious that compared to the wild type WT alone, the addition of arginine (L-Arg) to the wild type WT inhibits the growth of biofilm.

[0079] FIG. 6 shows a diagram of the RK cell indices obtained for various types of PA14 biofilms grown with the fluid culture medium LB by use of the impedance analysis with a 96-xCELLigence® Well-Plate within the evaluation time of 37 to 42 hours.

[0080] For ΔpqsA the highest negative RK cell index occurs, which is related to the biofilm growth rate. The use of 0.4% or 0.8% Arginine (L-Arg) reduces the biofilm growth rate by about ½.

[0081] Adding 0.4% or 0.8% Arginine (L-Arg) to the wild type WT results in reduction of the biofilm growth rate compared to the wild type WT without the presence of L-Arg. Again the directional cell index RK of about −0.004/h is directly proportional to the biofilm growth rate.

[0082] The factor r2 provides information on the average straightness of the line in the section considered for determining the biofilm growth rate.

[0083] As can be seen when comparing the diagrams in FIG. 5 and FIG. 6, the standard deviation can be significantly decreased and the distinctiveness can be increased by impedance analysis of the biofilm growth rate when measuring the reduction of the reactance with fluid culture medium between the biofilm and the electrode structure. The impedance analysis allows measuring of a plurality of probes in parallel within short time.

[0084] The measuring device can also be provided in front of an integrated circuit or integrated chip array.

[0085] In particular, the growth time of biofilm can be shortened by reducing the volume of the fluid culture medium 3 (LB). Due to the reduced volume cells reach earlier a threshold of cell density where they are going to produce biofilm.

[0086] FIGS. 3 and 4 shows that the growth of biofilm does not occur continuously but in different phases. The method for measuring the biofilm growth when the capacitance increases and the reactance decreases can not only be used for determining Pseudomonas aeruginosa PA14 biofilm growth. Other classes of biomaterials exhibit the same effect, in particular all ESKAPE organisms. ESKAPE pathogens are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species.

[0087] When monitoring the different phases of biofilm growth with their characteristic changes of the cell index curve over the measurement time, it is possible to define suitable times for taking probes of biomass from the receptacles in order to analyze the probe as required. Thus, the cell index curve over the time measured with the possible presence of biofilm floating on the fluid culture medium and not significantly adhering to the electrode structure provides an indicator for the appropriate timing of samples to be taken from the biofilm.

[0088] In order to probe the specific effect of the pellicle biofilm on the cell index, two experiments had been performed.

[0089] FIG. 7a) shows the result of the Cell index over the time measured with the Xcelligence® system, where Biofilm was transplanted to an adjacent receptacle with a metal loop to measure the effect of its presence on the impedance spectroscopy (n=4) compared to the addition of 10, 20, 40, and 80 μl PBS buffer (n=1)—the average value is shown—and no addition (LB medium, n=8).

[0090] FIG. 7b) shows the result of the Cell index over the time measured with the Xcelligence® system, where paraffin was also added in different amounts (10-100 μL) to wells (n=4) in order to mimic pellicle biofilm. The results were compared to the sole LB medium (n=8). Given paraffin has no dielectric constant its presence showed no effect on the capacitance.

[0091] First, a pellicle biofilm obtained by growing PA14 in LB medium for 72 h was transplanted, with a metal loop into wells filled with LB medium. In control experiments, PBS buffer was added to LB medium as shown in FIG. 7a).

[0092] Secondly, as shown in FIG. 7b), paraffin (10-100 μl) was added as an artificial substrate for comparison. Paraffin is immiscible with water and therefore floats on top of the hydrophilic medium due to its lower density. Because its chemical composition consists of alkanes, paraffin doesn't have a dielectric constant, in contrast to the pellicle biofilm of P. aeruginosa. For both experiments, the Xcelligence® system was calibrated once and paused before addition. It can be observed, that the slope of decrease after transplantation of biofilm was steeper than that obtained for an addition of PBS alone and the LB medium. This effect cannot be explained by the addition of an immiscible layer on top of the LB medium, as the addition of paraffin led to unchanged slopes of cell index.

[0093] The impedance method has also been used to study the effect of antibiotics on biofilm formation of P. aeruginosa PA14. The results of the impact of ciprofloxacin (A), tobramycin (B), and meropenem (C) on biofilm formation of PA14 are shown in FIGS. 8 to 10. As examples, the antibiotics ciprofloxacin (FIGS. 8a)-c)), tobramycin (FIGS. 9a)-c)) and meropenem (FIGS. 10a)-c)) have been tested. All three antibiotics changed the impedance curves in a concentration-dependent manner, indicating effects on biofilm formation. Interestingly, impedance spectroscopy could monitor an effect on biofilm formation at antibiotic concentrations that did not impact planktonic growth at the same period of time. For example, the addition of 0.13 μg/mL ciprofloxacin clearly changed the impedance curve reflecting altered biofilm formation, although planktonic growth was not impaired at this concentration. At a concentration of 0.25 μg/mL, steady biofilm formation was not observed anymore, but planktonic growth was still possible.

[0094] The effect on impedance was quantified by three parameters at varying antibiotic concentrations:

[0095] The first parameter is the time of onset of a linear decrease of the cell index, (FIGS. 8a), 9a) and 10a)).

[0096] The second parameter is the duration of a linear decrease of the cell index. This duration was determined by a linear trendline that was fitted to the data with an r.sup.2 value of 0.97 or higher. In the graphs of FIGS. 8a), 9a) and 10a), the black vertical bars represent the time period over which the biofilm formation rate was measured to be steady, based on a straight line assigned with an r.sup.2 of at least 0.97 (n=3 or 4).

[0097] The third parameter was the cell index slope during that time period (FIGS. 8b), 9b) and 10b)). These graphs show the concentration dependency of the biofilm formation expressed in the slope/ h of the cell index (n=3 or 4).

[0098] For ciprofloxacin and tobramycin, a shallower decline could be observed at increasing concentrations, indicating an impaired biofilm formation. For meropenem on the other hand, the increasing cell index slope suggests that it induced biofilm formation at concentrations between 0.063 and 0.25 μg/ml; this effect vanished at 0.5 μg/ml. Especially at the highest tested concentrations, all antibiotics led to later onsets of linear declines of impedance; at the same time, the period of linear decline was longer compared to untreated samples, indicating a delayed and attenuated biofilm formation.

[0099] FIGS. 8c) to 10c) show the result of the control experiment by measuring the OD600 value over the time with UV spectroscopy. OD600 indicates the absorbance, or optical density, of a sample measured at a wavelength of 600 nm and indicates the concentration of cells in a liquid. These graphs of FIGS. 8c), 9c) and 10c) represent growth curves of PA14 at various concentrations over time (n=4). At t=0 the OD600 was installed at 0.1.

[0100] The method allows determining the effect of other classes of modulators, for example [0101] standard of care drugs used for the treatment of P. aeruginosa, including aminoglycosides (e.g. Amikacin, Tobramycin) Fluorochinolones (e.g. Ciprofloxacin, Levofloxacin, Ofloxacin) beta-lactam antibiotics (e.g. Meropenem, Doropenem, Imipenem, Piperacillin, Cefepime); [0102] Compounds that have shown activity against P. aeruginosa in vitro and/or in vivo models (e.g. POL7080, Eravacyclin, Omadacycline, Plazomicine, Ceftazidime-Avibactam, Ceftolozane/Tazobactam); [0103] tool compounds with known positive or negative effects on of biofilm growth (e.g. arginine and casamino acid); [0104] agonists and antagonists of quorum sensing (e.g. from the classes of AHL- or PQS-analogs); [0105] Virulence factors (e.g. compounds interfering with secretion systems like anti-PcrV antibodies).

[0106] The results of the method for determining the growth of biofilm can be used to present online phenotypical effects of the biofilm growth. Thus, molecular changes can be made visible that are related to [0107] Membrane composition [0108] Metabolites [0109] Proteome [0110] Transcriptome [0111] Fluxome.