The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin—CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.


Introduction
Pseudomonas aeruginosa is one of the critical pathogens that the World Health Organization (WHO) includes in the list of priority pathogens with an urgent need for search and development of new antibiotics. Infections caused by this Gram-negative species are difficult to treat because of the multidrug resistance nature and are especially problematic in immunocompromised individuals or patients suffering from cystic fibrosis [1][2][3][4]. The bacterial outer membrane (OM) is the main protective shield of P. aeruginosa against

Synthesis and Characterization of Cationic CBS Dendrimers Homo-and Heterofunctionalized with Ammonium Groups and Polyethylene Glycol
Previously, we observed that cationic CBS dendrimers of low generation also containing a sulfur atom at the periphery showed no bactericidal activity, whereas analogous dendrimers of higher generation were rather active. This difference was attributed to the lack of hydrophobicity in the low-generation dendrimer [20]. Herein, we increased the hydrophobic framework of a cationic CBS dendrimer of low generation intending to improve its antibacterial properties but without increasing the number of cationic groups.
First, the precursor dendrimer with four vinyl groups was obtained from tetravinylsilane by hydrosilylation and alkenylation, following the same procedure to grow CBS dendrimers (see experimental methodology), but in this case, instead of increasing the generation, only the branches were enlarged. The surface of this dendrimer was modified with the corresponding thiol derivative by thiol-ene reaction, under ultraviolet light conditions [20]. Then, dimethylammonium groups were neutralized with sodium hydrocarbonate. Finally, the quaternization of dimethylamine groups was carried out in presence of an excess of iodomethane ( Figure 1). All reactions have been monitored by nuclear magnetic resonance (NMR) spectroscopy and the final product (4, Figure 2) was characterized by 1 H-NMR, 13 C-NMR, elemental analysis and mass spectrometry.

Synthesis and Characterization of Cationic CBS Dendrimers Homo-and Heterofunctionalized with Ammonium Groups and Polyethylene Glycol
Previously, we observed that cationic CBS dendrimers of low generation also containing a sulfur atom at the periphery showed no bactericidal activity, whereas analogous dendrimers of higher generation were rather active. This difference was attributed to the lack of hydrophobicity in the low-generation dendrimer [20]. Herein, we increased the hydrophobic framework of a cationic CBS dendrimer of low generation intending to improve its antibacterial properties but without increasing the number of cationic groups.
First, the precursor dendrimer with four vinyl groups was obtained from tetravinylsilane by hydrosilylation and alkenylation, following the same procedure to grow CBS dendrimers (see experimental methodology), but in this case, instead of increasing the generation, only the branches were enlarged. The surface of this dendrimer was modified with the corresponding thiol derivative by thiol-ene reaction, under ultraviolet light conditions [20]. Then, dimethylammonium groups were neutralized with sodium hydrocarbonate. Finally, the quaternization of dimethylamine groups was carried out in presence of an excess of iodomethane ( Figure 1). All reactions have been monitored by nuclear magnetic resonance (NMR) spectroscopy and the final product (4, Figure 2) was characterized by 1 H-NMR, 13 C-NMR, elemental analysis and mass spectrometry.  In addition, to introduce a biocompatible group, i.e., PEG, in the surface of the dendrimers [24,25] and to determine the influence of the PEG's length in the bactericidal properties of these cationic CBS dendrimers, two heterofunctionalized CBS dendrimers with cationic trimethylammonium groups and a PEG moiety (PEG-800 (7) and PEG-2K (10)) were prepared ( Figure 2).
These new dendrimers were obtained by also employing thiol-ene. In a first step, the thiol-PEG reagent was added and, after checking by NMR the modification of the dendrimer surface with this ligand, the complete transformation of the surface was accomplished by the addition of 2-(dimethylamino)ethanethiol hydrochloride over the previous solution ( Figure 1). Compounds 7 (PEG-800) and 10 (PEG-2K) were characterized by NMR spectroscopy and elemental analysis.
Finally, for all the cationic systems, we replaced the iodide anion (I − ) with a chloride anion (Cl -) using an excess of the Amberlite IRA-402, Cl − form. Thanks to this anion exchange, we increased the solubility of cationic dendrimers.

Antibacterial Activity of Dendrimers against P. aeruginosa
The antibacterial activity of homofunctionalized (4) and heterofunctionalized (7, 10) cationic CBS dendrimers was studied in P. aeruginosa planktonic cells and biofilm. Our data showed that treatment with compound 4 (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 32 mg/L) was more effective than that with 7 and 10 dendrimers (MIC and MBC of 256 for both compounds) on planktonic cells (Table 1). Thus, PEG heterofunctionalization of dendritic systems decreased the antibacterial activity. A similar effect was observed for the biofilm-forming culture as well as its degradation but at a higher concentration of dendrimers. In addition, to introduce a biocompatible group, i.e., PEG, in the surface of the dendrimers [24,25] and to determine the influence of the PEG's length in the bactericidal properties of these cationic CBS dendrimers, two heterofunctionalized CBS dendrimers with cationic trimethylammonium groups and a PEG moiety (PEG-800 (7) and PEG-2K (10)) were prepared ( Figure 2).
These new dendrimers were obtained by also employing thiol-ene. In a first step, the thiol-PEG reagent was added and, after checking by NMR the modification of the dendrimer surface with this ligand, the complete transformation of the surface was accomplished by the addition of 2-(dimethylamino)ethanethiol hydrochloride over the previous solution ( Figure 1). Compounds 7 (PEG-800) and 10 (PEG-2K) were characterized by NMR spectroscopy and elemental analysis.
Finally, for all the cationic systems, we replaced the iodide anion (I − ) with a chloride anion (Cl -) using an excess of the Amberlite IRA-402, Cl − form. Thanks to this anion exchange, we increased the solubility of cationic dendrimers.

Antibacterial Activity of Dendrimers against P. aeruginosa
The antibacterial activity of homofunctionalized (4) and heterofunctionalized (7, 10) cationic CBS dendrimers was studied in P. aeruginosa planktonic cells and biofilm. Our data showed that treatment with compound 4 (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 32 mg/L) was more effective than that with 7 and 10 dendrimers (MIC and MBC of 256 for both compounds) on planktonic cells (Table 1). Thus, PEG heterofunctionalization of dendritic systems decreased the antibacterial activity. A similar effect was observed for the biofilm-forming culture as well as its degradation but at a higher concentration of dendrimers. The cell morphology changes after incubation with tested dendrimers at concentrations at and below their MBCs ( Figure 3) were examined using scanning electron microscopy (SEM). Cells showed an absence of smooth walls on their surface, and we even found flattened cells (Figure 3, white arrows). The altered morphology of elongated bacterial planktonic cells was observed after treatment with all tested dendrimers. The cell density in the presence of compound 4 was the lowest compared to the control and pegylated 7 and 10 samples.  The cell morphology changes after incubation with tested dendrimers at concentrations at and below their MBCs ( Figure 3) were examined using scanning electron microscopy (SEM). Cells showed an absence of smooth walls on their surface, and we even found flattened cells (Figure 3, white arrows). The altered morphology of elongated bacterial planktonic cells was observed after treatment with all tested dendrimers. The cell density in the presence of compound 4 was the lowest compared to the control and pegylated 7 and 10 samples.

Diffusion Properties and Antibiofilm Activity of Dendrimers on P. aeruginosa PAO1 Biofilm
For laser interferometry and fluorescence study, the most active CBS dendrimers, one functionalized only with ammonium groups (4) and one with PEG800 and ammonium groups (7), were selected. Compound 4, as a fully cationic dendrimer, can interact with the negatively charged biofilm thereby becoming trapped in the matrix, in contrast to the PEGylated dendrimer version 7. Thus, no diffusion of compound 4 through the biofilm layer was observed by laser interferometry, whereas dendrimer 7 was able to efficiently pass through the mature PAO1 biofilm (9.83 × 10 −9 mol after 40 min) ( Figure 4A). The above results corresponded with the biofilm-forming bacteria eradication measured by fluorescence microscopy ( Figure 4B). The live/dead cell staining showed that the cationic CBS dendrimer (4) has much higher anti-biofilm properties compared to dendrimer

Diffusion Properties and Antibiofilm Activity of Dendrimers on P. aeruginosa PAO1 Biofilm
For laser interferometry and fluorescence study, the most active CBS dendrimers, one functionalized only with ammonium groups (4) and one with PEG800 and ammonium groups (7), were selected. Compound 4, as a fully cationic dendrimer, can interact with the negatively charged biofilm thereby becoming trapped in the matrix, in contrast to the PEGylated dendrimer version 7. Thus, no diffusion of compound 4 through the biofilm layer was observed by laser interferometry, whereas dendrimer 7 was able to efficiently pass through the mature PAO1 biofilm (9.83 × 10 −9 mol after 40 min) ( Figure 4A). The above results corresponded with the biofilm-forming bacteria eradication measured by fluorescence microscopy ( Figure 4B). The live/dead cell staining showed that the cationic CBS dendrimer (4) has much higher anti-biofilm properties compared to dendrimer functionalized with PEG800 (7). Nevertheless, both types of dendrimers were able to kill bacteria compared to the non-treated control. functionalized with PEG800 (7). Nevertheless, both types of dendrimers were able to kill bacteria compared to the non-treated control.

Antibacterial Activity of Dendrimer Combined with Endolysin against P. aeruginosa PAO1
Endolysins are a type of enzyme that can be a biocide against both Gram-positive and Gram-negative bacteria [27]. However, with some single exceptions of endolysins reviewed by Gerstmans et al. that permeate the OM barrier per se, the exogenous application of native phage endolysins on Gram-negatives is rather ineffective [28]. Nevertheless, our previous studies on cationic dendrimers with different skeleton nature showed their positive impact on the improvement of endolysin antibacterial activity [22,26,29]. In this work, KP27 endolysin with the activity of L-alanyl-D-glutamate endopeptidase was mixed with the CBS dendrimer functionalized with ammonium groups (NMe3 + ) alone (4) or with PEG (PEG800 (7), PEG2K (10)) to verify a possible synergic effect ( Figure 5). The antibacterial properties were evaluated on PAO1 wild-type and its ΔwbpL knock-out mutant lacking lipopolysaccharide O-chain.

Antibacterial Activity of Dendrimer Combined with Endolysin against P. aeruginosa PAO1
Endolysins are a type of enzyme that can be a biocide against both Gram-positive and Gram-negative bacteria [27]. However, with some single exceptions of endolysins reviewed by Gerstmans et al. that permeate the OM barrier per se, the exogenous application of native phage endolysins on Gram-negatives is rather ineffective [28]. Nevertheless, our previous studies on cationic dendrimers with different skeleton nature showed their positive impact on the improvement of endolysin antibacterial activity [22,26,29]. In this work, KP27 endolysin with the activity of L-alanyl-D-glutamate endopeptidase was mixed with the CBS dendrimer functionalized with ammonium groups (NMe 3 + ) alone (4) or with PEG (PEG800 (7), PEG2K (10)) to verify a possible synergic effect ( It turned out that PEGylation of CBS dendrimers alone and in combination with phage-derived endolysin reduced their antibacterial properties against P. aeruginosa It turned out that PEGylation of CBS dendrimers alone and in combination with phage-derived endolysin reduced their antibacterial properties against P. aeruginosa PAO1 wild-type and its ∆wbpL mutant. The CBS dendrimer functionalized with ammonium groups (-NMe 3 + ) (4) at the concentration of 56 mg/L showed a statistically significant decreased optical density of PAO1 cells (p = 0.031 compared to untreated control; One-Way ANOVA test). The antibacterial effect was higher for the combination with endolysin compared to dendrimer alone (p = 0.042). A similar effect of compound 4 was observed for the ∆wbpL mutant (p = 0.016) but the antagonistic effect of endolysin was not observed. It indicates the role of antigen O from LPS structure on the antibacterial effect of dendrimer in combination with endolysin.

Discussion
The first aim of this study was to evaluate the antibacterial activity of CBS dendrimers functionalized with ammonium groups (-NMe 3 + ) alone or with PEG (PEG800, PEG2K). The dendrimers were tested against planktonic (alone or in combination with endolysin) and biofilm-forming P. aeruginosa cells.
Results in planktonic bacteria showed that PEGylation of the dendrimers (7 and 10; MBC at 256 mg/L) decreases the antibacterial activities giving the MBC of dendrimers eight times higher than that of homofunctionalized CBS dendrimers (4; MBC at 32 mg/L). The tendency to diminish the bactericide effect when dendrimers are PEGylated might be associated with the decrease in the number of cationic ammonium groups and the shielding of the positive charges by the PEG chains. These facts can be correlated with the decrease of the electrostatic interactions between dendrimers and the negatively-charged bacterial surface. It might promote nanopores formation [30]. The effect of PEGylation on the reducing antibacterial properties of dendrimers was previously observed in the case of the G3 and G5 PAMAM dendrimers. It was observed for a higher number of PEG ligands and, consequently, a lower number of ammonium groups [31]. On the other hand, metallic nanoparticles functionalized with cationic CBS dendrons (different generations) and PEG chains, showed the same trend, probably because the PEG and the dendrons had similar size and the PEG chains could not shield the positive charge of ammonium groups [32].
In addition, the presence of long PEG chains also reduced the antibiofilm properties of CBS dendrimers. Regarding the biofilm-damaging (minimum biofilm damaging concentration, MBDC) and -eradicating concentrations (minimum biofilm eradication concentration, MBEC) against an established P. aeruginosa biofilm, dendrimer 4 was the best biocide with an MBDC of 128 mg/L and MBEC of 512 mg/L. The lower antibacterial activity of PE-Gylated CBS dendrimers could be related to the effectiveness of their diffusion through a mature P. aeruginosa biofilm. The results showed that dendrimer 4 interacted with sessile cells in contrast to PEGylated compound 7, in which PEGylation reduced the positive charge and the biocidal activity.
The molecular weights of these three dendrimers are notably different. For this reason, we compared the activity taking into account molar concentration (see Tables S1 and S2, Supporting Information). UnPEGylated dendrimer 4 was the most active with this consideration also. However, among both PEGylated dendrimers, dendrimer 10, with the longest PEG chain, proved to be slightly more active per molecule than dendrimer 7, with the shortest PEG chain. This indicates that the long PEG chain exerts a positive action regarding antibacterial activity when compared with the short one.
On the other hand, images obtained using SEM allowed us to observe the formation of long filaments, especially after treatment with unPEGylated dendrimer 4 (16 mg/L) and PEGylated dendrimer 10 (256 mg/L). This result suggests that treatment with these dendrimers affected the cell septation during the cell division process. The filamentation commonly originates as a consequence of DNA damage or stress conditions [33]. In particular, the inhibition of PBP-3 activity is related to filamentous bacterial morphology [34]. The few cells we found at the MBC concentration were damaged. The absence of viability of these cells was corroborated by the drop plate method, suggesting that the cells could have DNA damage [33].
The next aim of this study was to evaluate the biocide effect of endolysin in combination with tested dendrimers against P. aeruginosa as the representative of Gram-negative bacteria. While the endolysins of Gram-positive specific phages show high bactericidal activity, the lytic activity of endolysins against Gram-negatives is limited by the presence of a protective OM. Although some endolysins that are able to pass the OM barrier and act exogenously against Gram-negative bacteria have already been described, most of them must be assisted by membrane destabilizers. There are two general approaches to support the lytic activity of endolysins.
The first approach relies on the fusion of endolysin with cationic peptides (mainly by genetic engineering) and is based on observation of endolysins, which have an intrinsic antibacterial activity against Gram-negatives. This natural activity depends on the presence of short but highly positively charged domains, which have been proved to be strategic for the antibacterial function of Thermus phage Ts2631 endolysin [35], Acinetobacter baumannii phage lysin PlyF307 [36] and LysC lysin encoded by Clostridium intestinale [37].
The second approach relies on a combination of endolysin with some OM-permeabilizing agents such as organic acids, liposomes, antibiotics or dendrimers [38][39][40]. The enhanced effect of endolysin mixed with unPEGylated dendrimer against P. aeruginosa cells might indicate the synergistic mechanism, in which positively charged dendrimers enhanced the OM's permeability to peptidoglycan-degrading enzyme. This effect confronted the results observed for endolysin combination with amine PPI dendrimers, where no improvement of the antibacterial activity was seen against the PAO1 strain [26]. However, when endolysin was combined with AgNP modified with PEG and cationic CBS dendrons, the bactericidal activity increased notably. Therefore, cationic CBS systems targeting lipopolysaccharides might be useful for OM barrier disruption, thus endolysin to penetrate to the periplasmic space and reach the peptidoglycan [22]. It is also worth noting that in the case of the PEG800 dendrimer, the antimicrobial effect is weaker for the ∆wbpL mutant compared to that for the wild-type strain. This suggests that dendrimers must first interact with the O-chain (the carbohydrate moiety) of the LPS and then act on the membrane. Our previous study on dendronized silver nanoparticles indisputably shows that PEGylated dendronized AgNPs can complex with LPS, while the LPS-non-PEGylated NP complex formation effect is not favored due to the tendency of non-PEGylated dendronized AgNPs to aggregate [22].
This study confirmed an idea for an antibacterial system consisting of cationic dendrimers and lytic proteins such as endolysin for the design of future pharmaceutical strategies.

Bacterial Strains
The antibacterial activity of biocides was tested using P. aeruginosa wild-type strain from the Colección Española de Cultivos Tipo (CECT) 108 of the Department of Biotechnology and Biomedicine, Universidad de Alcalá (Spain). Additionally, the effect of combined cationic CBS dendrimers with endolysin was tested on P. aeruginosa ATCC15692 (PAO1) wild-type strain obtained from the Department of Biochemistry and Genetics, Jan Kochanowski University (Kielce, Poland) and its knock-out ∆wbpL mutant deficient in the lipopolysaccharide (LPS) biosynthesis (lack of A-band and B-band in the O-antigen) provided by Andrew M. Kropinski from the Laboratory of Foodborne Zoonoses, Guelph, ON, Canada.

Analysis of Antibacterial Properties of Dendrimers against Planktonic Cells
The antibacterial activity on planktonic P. aeruginosa PAO1 cells was studied following the ISO 20776-1:2006 protocol. Thirteen concentrations of each dendrimer, in the range of 0.125 to 512 mg/L, were tested in triplicate in 96-well microtiter plates. Control wells were included in all experiments: P. aeruginosa culture in tryptic soy broth (TSB) medium without biocide (positive control), TSB medium containing the biocide (negative control) and wells containing the TSB medium only (negative control). The final volume of tested samples was 200 µL containing 5 × 10 5 CFU/mL of bacterial culture (CFU, colony-forming units). Samples were incubated for 20 h at 37 • C, and the absorbance at 630 nm was measured by the microplate reader ELX808iu (BioTek Instruments, Winooski, VT, USA) to determine the minimal inhibitory concentration (MIC). Then, 5 µL of each sample was deposited on plate count agar (PCA) for 24 h at 37 • C to detect the minimal bactericidal concentration (MBC). Relationship between antibacterial concentration in mg/L and µM used in this work is presented in Table S1. The data of antibacterial activity of tested cationic CBS dendrimers in µM are presented in Table S2.

Biofilm Formation and Quantification Assay
The protocol previously described by Peppoloni et al. was used to prepare a P. aeruginosa CECT 108 biofilm [41]. Bacteria were grown in PCA at 37 • C for 24 h, and then, several colonies were inoculated in TSB at 37 • C and 150 rpm for another 20 h. The inoculum solution was prepared by resuspending P. aeruginosa CECT 108 cells in TSB supplemented with 0.4% (wt./wt.) of glucose until an optical density of 0.08-0.1 (10 8 CFU/mL) at 625 nm. Subsequently, 100 µL of bacterial culture and 100 µL of TSB were added into sterile 96-wells plates to allow the biofilm formation. After 24 h of incubation at 37 • C, biofilm production was checked using a crystal violet assay [42]. Biofilm quantification was carried out by reading the absorbance of a 96-well plate at 630 nm in a microtiter plate reader (Epoch TM , BioTek Instruments, Winooski, VT, USA).

Inhibition of P. aeruginosa Biofilm Formation
The analysis of cationic CBS dendrimers' (4, 7, 10) activity on biofilm formation was carried out as follows. Briefly, 50 µL of P. aeruginosa CECT 108 (10 8 CFU/mL in 2× supplemented with TSB medium) was treated with 50 µL of different doses of each dendrimer (range of initial concentrations from 16 to 1024 mg/L) in a 96-well microtiter plate, for 24 h at 37 • C. Each treatment was evaluated in triplicate. Additionally, all experiments included a positive control (dendrimer-free control) and negative controls (dendrimer without inoculum and culture medium). The minimal biofilm formation inhibitory concentration (MBIC) was determined using a resazurin colorimetric assay, and MBBC, defined as the minimum bactericidal concentration in biofilm, was detected using the drop plate method (Section 4.4.4.).

Antibiofilm Activity Testing
For determining the antibiofilm properties of dendrimers, the supernatant was discarded from the mature biofilm, and cells not adhering to the surface were removed by washing twice with 250 µL of sterile PBS. Then, 100 µL of each dendrimer sample at the concentrations range 16-1024 mg/L was added in triplicate and supplemented with a TSB medium. The plate was incubated at 37 • C for 24 h. In all experiments, positive and negative controls were included. The resazurin colorimetric protocol was followed to determine the minimum biofilm damaging concentration (MBDC) and considered as the lowest concentration that could affect P. aeruginosa CECT 108 metabolic activity, while the minimum biofilm eradication concentration (MBEC) was obtained using the drop plate method (Section 4.4.4).

Resazurin Assay and Drop Plate Method
Biofilm cultures after treatment with dendrimers at the concentration range 16-1024 mg/L at 37 • C for 24 h were washed twice with sterile PBS. Then, 100 µL of sterile PBS and 20 µL of resazurin solution were added to each well, and samples were incubated in the dark for 24 h at 37 • C. The reduction of resazurin, in metabolically active cells, was measured by the absorbance at 570 and 600 nm using a microplate reader EpochTM (BioTek Instruments, Winooski, VT, USA). Results obtained using the resazurin assay were confirmed by inoculating 5 µL of each dendrimer-treated culture and controls on plate count agar [33,34].

Scanning Electron Microscopy
Scanning electron microscopy (SEM) was performed to evaluate morphological changes in P. aeruginosa cell wall after treatment with dendrimers in TSB at 37 • C for 24 h. The samples for SEM were prepared following the methodology previously described [43,44]. Critical-point drying was carried out with a Polaron CPD7501 critical-point drying system and sputter coated with 200 Å gold-palladium using a Polaron E5400. Scanning electron microscopy was performed at 5-15 kV in a Zeiss DSM 950 SEM.

Laser Interferometry
The diffusion of cationic CBS dendrimers (4, 7) through PAO1 biofilm was determined experimentally using the laser interferometry method [45][46][47]. A biofilm of PAO1 was formed in TSB (tryptic soy broth) medium for 72 h in stationary conditions at 37 • C on a PET membrane with pores of diameter 1 µm as an element of BD Falcon™ Cell Culture Inserts. The laser interferometric system consists of a two-beam Mach-Zehnder interferometer with a He-Ne laser type HN 40P (Carl Zeiss" Jena, Germany), two cuvettes made with optical glass of high uniformity, a TV-CCD camera and a computer with software for the acquisition and processing of interference images (interferograms). The P. aeruginosa POA1 biofilm formed on the PET membrane was placed between the cuvettes. The lower cuvette contained a dendrimer-water solution at (9.06 × 10 −4 mol/L). The bottom cuvette was filled with pure water. The transport properties of dendrimers through the P. aeruginosa PAO1 biofilm were measured using the above laser interferometry system based on the obtained interferograms (Figure 6). on a PET membrane with pores of diameter 1 μm as an element of BD Falcon™ Cell Culture Inserts. The laser interferometric system consists of a two-beam Mach-Zehnder interferometer with a He-Ne laser type HN 40P (Carl Zeiss,, Jena, Germany), two cuvettes made with optical glass of high uniformity, a TV-CCD camera and a computer with software for the acquisition and processing of interference images (interferograms). The P. aeruginosa POA1 biofilm formed on the PET membrane was placed between the cuvettes. The lower cuvette contained a dendrimer-water solution at (9.06 × 10 −4 mol/L). The bottom cuvette was filled with pure water. The transport properties of dendrimers through the P. aeruginosa PAO1 biofilm were measured using the above laser interferometry system based on the obtained interferograms ( Figure 6). Figure 6. Experimental setup of the interferometric investigations of the dendrimers' diffusion through a P. aeruginosa PAO1 biofilm. The scheme presents a Mach-Zehnder interferometer with a He-Ne laser and a system for the acquisition and processing of interference images. The laser light was spatially filtered and transformed by the beam expander into a parallel beam ca. 80 mm wide and then split into two beams. The first beam passed through the investigated membrane system parallel to the membrane surface, while the second passed directly through the compensation plate to the light detection system. As a consequence of the superimposition of these beams, respective interference images were generated. The images depend on the refraction coefficient of the solute, which in turn depends on the substance concentration. When the solute is uniform, the interference fringes are straight, and they bend when a concentration gradient appears.
A computer image-processing system, complete with dedicated software, enables mathematical analysis of interferograms shown on the system screen. The interferograms, which appear due to the interference of laser beams, were determined by the refraction coefficient of the solute, which in turn depends on substance concentration. When the solute is uniform, the interference fringes are straight, and they bend when a concentration gradient appears. The basis for the determination of the spatiotemporal substance concentration distribution (e.g., concentration profile), C(x,t), is the proportionality coefficient between changes in substance concentration and the corresponding changes of the solution's refractive index, ∆n(x,t). The correlation between concentration and refractive index of water solutions is refractometrically determined. The concentration profile, C(x,t), is determined by the deviation d(x,t) of the fringes from a straight course. Since the concentration C(x,t) and the refraction coefficient are assumed to be linear [3]: Figure 6. Experimental setup of the interferometric investigations of the dendrimers' diffusion through a P. aeruginosa PAO1 biofilm. The scheme presents a Mach-Zehnder interferometer with a He-Ne laser and a system for the acquisition and processing of interference images. The laser light was spatially filtered and transformed by the beam expander into a parallel beam ca. 80 mm wide and then split into two beams. The first beam passed through the investigated membrane system parallel to the membrane surface, while the second passed directly through the compensation plate to the light detection system. As a consequence of the superimposition of these beams, respective interference images were generated. The images depend on the refraction coefficient of the solute, which in turn depends on the substance concentration. When the solute is uniform, the interference fringes are straight, and they bend when a concentration gradient appears.
A computer image-processing system, complete with dedicated software, enables mathematical analysis of interferograms shown on the system screen. The interferograms, which appear due to the interference of laser beams, were determined by the refraction coefficient of the solute, which in turn depends on substance concentration. When the solute is uniform, the interference fringes are straight, and they bend when a concentration gradient appears. The basis for the determination of the spatiotemporal substance concentration distribution (e.g., concentration profile), C(x,t), is the proportionality coefficient between changes in substance concentration and the corresponding changes of the solution's refractive index, ∆n(x,t). The correlation between concentration and refractive index of water solutions is refractometrically determined. The concentration profile, C(x,t), is determined by the deviation d(x,t) of the fringes from a straight course. Since the concentration C(x,t) and the refraction coefficient are assumed to be linear [3]: where C(x,t) denotes the concentration of dendrimer at a point situated at the distance x from the biofilm-water interface; C 0 is the initial substance concentration; a is the proportionality constant between the concentration and the refraction index (a = 1.975 mol/L for the compound 4 and a = 0.478 for the compound 7 dendrimers in PBS solution); λ is the wavelength of the laser light, 632.8 nm; h is the distance between the fringes in the field where they are straight lines and f is the thickness of the solution layer in the measurement cuvette. By recording the interferograms over a given time interval (2 min), one can reconstruct the concentration profiles at different times. The interferograms were recorded, and the concentration profiles for each interferogram were reconstructed. Based on concentration profiles, one can determine the transport parameters of substance quantity, such as substance quantity after time t (N(t)): where S is the surface of biofilm (S = 7 × 10 −5 m 2 ), and δ is defined as the distance between the biofilm-PBS interface and the point at which the deviation of the interference fringe from its straight-line run amounts to 10% of the fringe thickness.

Fluorescence Microscopy
The P. aeruginosa PAO1 mature biofilm was incubated with cationic CBS dendrimers (4, 7) at 9.06 × 10 −4 mol/L (as in laser interferometry study) for 24 h at 37 • C and stained at room temperature in the dark for 20 min with a mixture of two dyes according to the manufacturer's protocol (FilmTracer™, Invitrogen, Waltham, USA). The mixtures included two compounds: SYTO ® 9, a green-fluorescent nucleic acid stain, and a redfluorescent nucleic acid stain, propidium iodide. The PAO1 biofilm was observed with a confocal microscope, Nikon A1R, no longer than 30 min after staining. Interpretation of the microscopic observations was based on the stained cells. Dyes differ both in their spectral characteristics and in their ability to infiltrate healthy bacterial cells. Bacterial cells with green fluorescence are interpreted as alive, whereas bacteria with damaged membranes show red fluorescence.

Antibacterial Activity of Endolysin Combined with Dendrimers
The purified, recombinant endolysin (with the activity of L-alanyl-D-glutamate endopeptidase) derived from the Klebsiella KP27 lytic phage was prepared according to methods described previously [22]. Pooled endolysin was dialyzed against phosphatebuffered saline (PBS), pH = 7.4 (1 mL of protein against 10 L of buffer). The concentration of recombinant endolysin was determined fluorimetrically (Qubit ® Protein Assay Kit, Molecular Probes, Thermo Fischer Scientific, Waltham, USA). The antibacterial activity of dendrimers was tested using P. aeruginosa PAO1 wild-type and its ∆wbpL mutant deficient in the biosynthesis of A-band and B-band O-antigens. The antigen-O-lacking mutant was chosen to check the possible effect of this part of LPS on the antibacterial properties of the tested dendrimers. In addition, the dendrimer was combined with phage KP27 endolysin, as a peptidoglycan-degrading enzyme. The antibacterial activity was measured on an exponentially growing P. aeruginosa culture (OD 600 ) by a spectrometric method using Microplate Reader TECAN Infinite 200 PRO (Tecan Group Ltd., Männedorf, Switzerland). Each dendrimer, alone and in combination with endolysin (5 mmol/L), was added to the bacterial culture, and the bactericidal effect was determined.

Conclusions
In conclusion, unPEGylated cationic CBS dendrimers are more efficient as a biocide against P. aeruginosa than the PEGylated versions. The synergic antibacterial effect was associated with the destabilization of the OM layer by unPEGylated dendrimer and enhanced further by phage-derived endolysin. Finding a good balance in tested dendritic systems and their combination with PG-degrading enzymes, such as endolysin, could be the way toward the future development of biocides against P. aeruginosa.