Biofilm-disrupting effects of phage endolysins LysAm24, LysAp22, LysECD7, and LysSi3: breakdown the matrix

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Lendel, Nataliia P. Antonova, Igor V. Grigoriev, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3899892/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Apr, 2024 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 8 You are reading this latest preprint version Abstract The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without the subsequent emergence of endolysin-resistant genotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins’ effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a 2-fold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3. antibiofilm activity biofilm endolysin exopolysaccharides extracellular DNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Bacterial biofilm (BF) is a community of immobilized and phenotypically altered microorganisms embedded in self-produced exogenous polymeric substances (EPSs) (Donlan and Costerton 2002 ; Karygianni et al. 2020 ). In particular, biofilm-related phenotype is formed by various matrix structures and chemical compositions, such as exopolysaccharides, extracellular DNA (eDNA), proteins, lipids and membrane vesicles, low molecular compounds (for example, “quorum sensing” ligands, cyclic di-GMP, accumulated water, and polyvalent ions). These heterogeneous constituents greatly impact biofilm formation, sustainability, availability of nutrients, and tolerance toward antiseptics, antibiotics and bacteriophages. Being one of the most abundant states of bacterial existence both in natural and human-made environments, BFs have great biological significance owing to efficient expansion of the community and increased survival rates under severe conditions (Donlan and Costerton 2002 ; Flemming et al. 2016 ). Specifically, there are challenges in the therapy of diseases associated with Gram-negative opportunistic bacteria, such as Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa , and Escherichia coli , associated with rapid emergence of multidrug-resistance traits and hardly removable biofilms formed within the host organisms and on surfaces of hospital equipment and prosthetic devices. These features together with advanced natural antibiotic resistance led to a notable increase in the probability of negative outcomes, formation of persistent infection and spread of nosocomial infections (Omar et al. 2017 ; Stewart 2015 ). Despite actively developed restrictive measures to control Gram-negative pathogens, there are limited innovative treatment approaches (Giacobbe et al. 2018 ; Prasad et al. 2022 ), while common chemotherapy is often considered as non-effective or excessively deleterious (Eriksson et al. 2022 ). At the same time, the search for reliable antibacterial strategies continues, thus an application of nanoparticles, antibodies, bacteriophages, bacteriophage-derived antimicrobial proteins (also called enzybiotics) and peptides (AMPs) are expected to be highly advanced options as a supplementation to antibiotic therapy or as an alternative treatment strategy (Heselpoth et al. 2021 ; Koo et al. 2017 ). Endolysins are promising peptidoglycan-degrading enzybiotics that are already investigated for clinical relevance due to their pronounced activity and safety (Karau et al., 2023 ). For decades of research on endolysins, no strict evidence of acquired resistance to these proteins has been reported, that is explained particularly by the high fitness costs of most changes in the peptidoglycan, especially across Gram-negative species (Grishin et al. 2020 ). Several studies reveal an ability of endolysins not only to eliminate multidrug resistant bacteria, but also to efficiently disrupt their biofilms (Arroyo-Moreno et al. 2022 ; Gutiérrez et al. 2014 ; Karau et al. 2023 ). However, the key mechanisms of endolysin-biofilm interactions are uncovered, and the investigation is mostly limited to the fact of BFs disruption and prevention of their formation. Whilst interactions between endolysins and EPSs and their effects on the enzymes’ activity are poorly described. Our previous research was focused on recombinant endolysins, muramidases LysAm24, LysAp22, LysSi3 and endopeptidase LysECD7, derived from different bacteriophages infected Gram-negative hosts. Recently, promising safety properties and antibacterial activity of the endolysins against a broad spectrum of Gram-negative representatives were described (Antonova et al. 2019 ), furthermore, highlighted the substantial disruption of 24-h BFs, affected by these enzymes (Vasina et al. 2021 ). However, the catalytic mechanism of endolysins, associated with the hydrolysis of CW (cell-wall) peptidoglycan bonds, is not enough to explain their influence on preformed BFs, degrading complex three-dimensional structures of the matrix, down to individual bacterial cells. In the present study we investigated the composition of biofilms formed by polyresistant strains of A. baumannii and K. pneumoniae , revealing interactions between either of the endolysins and the abundant defensive compounds of preformed BFs. Materials and methods Bacterial strains and culture conditions Acinetobacter baumannii Ts 50 − 16 and Klebsiella pneumoniae F 104 − 14 clinical isolates (collection of the N.F. Gamaleya Federal Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation), were implicated in the present study as biofilm-forming Gram-negative pathogens of the ESKAPE group. A. baumannii Ts 50 − 16 was isolated from patient’s sputum, intensive care unit, and K. pneumoniae F 104 − 14 was obtained from patient’s sputum, outpatient hospital. Both strains were stored at − 80°C and cultivated in pre-autoclaved Tryptic Soy Broth (TSB; SIFIN, Berlin, Germany) at 37°C, at 250 rpm overnight before performing further tests. Recombinant expression and purification of proteins Recombinant endolysins LysAm24, LysAp22, LysECD7, and LysSi3 were obtained as described previously (Vasina et al., 2021 ). In brief, proteins modified with polyhistidine-tag (8 His) were recombinantly expressed in Escherichia coli BL21(DE3) pLysS strain using 1mM β-D-1-thiogalactopyranoside induction at 37°C for 3 h. The cells were harvested by centrifugation (6,000×g for 10 min at 4°C) and incubated with 100 µg/mL lysozyme in lysis buffer (20 mM Tris-HCl, 250 mM NaCl, and 0.1 mM EDTA, pH = 8.0), and disrupted by sonication. Soluble proteins were purified on an NGC Discovery™ 10 FPLC system (Bio-Rad, Hercules, CA, U.S.) with a HisTrap FF column (GE Healthcare, Munich, Germany) pre-charged with Ni 2+ ions. The filtered lysate was mixed with 30 mM imidazole and 1 mM MgCl 2 and loaded on the column pre-equilibrated with binding buffer (20 mM Tris–HCl, 250 mM NaCl, and 30 mM imidazole, pH = 8.0). The fractions were eluted using a linear gradient to 100% elution buffer (20 mM Tris–HCl, 250 mM NaCl, and 500 mM imidazole, pH = 8.0). Resulting protein fractions were dialyzed against 20 mM Tris–HCl buffer (pH = 7.5). The purity of endolysins was determined by 16% SDS-PAGE, and protein concentrations were measured using a spectrophotometer (Implen NanoPhotometer; Implen, München, Germany) at 280 nm and calculated using the predicted extinction coefficients [0.840, 0.831, 1.460, and 1.029 (mg/mL) −1 cm − 1 for LysAm24, LysAp22, LysECD7, and LysSi3, respectively]. The isoelectric points of endolysins LysAm24, LysAp22, LysECD7, and LysSi3 were predicted using the internet-source http://isoelectric.org . Biofilm composition and morphology Macrocolony assays were conducted on solid culture medium with application of three different staining techniques and without any dyes as an intact control. For the formation of BFs, cells from overnight cultures were harvested by centrifugation (6,000×g, 10 min, RT), resuspended in PBS (pH = 7.4), and diluted to achieve a cell density of approximately 3×10 6 CFU/mL. To obtain dual-species biofilms, the appropriate bacterial suspensions were mixed in a 1:1 (v/v) ratio. Thereafter, 7 µL of the suspension was inoculated onto solid TSB agar medium on 90 mm Petri dishes and BFs were grown at 37°C for 48 h without agitation. All images of colonies were acquired using HD automatic colony counter Scan 300 and provided software (Interscience, Saint-Nom-la-Bretèche, France). Alcian blue 8GX 0.3% stain (PanReac AppliChem, Darmstadt, Germany) in a 4% acetic acid aqueous solution (pH = 2.5) was prepared to visualize polysaccharides with abundant carboxyl groups. Five mL of pre-filtered Alcian blue stain were carefully dripped on plates containing macrocolonies and then incubated for 15 min at RT without agitation. Subsequently, the plates were rinsed with 5 mL of deionised distilled water. The results were confirmed by the Congo red stain absorption test (Freeman et al., 1989 ). For this, TSB agar was supplemented with pre-filtered 0.08% Congo red stain (Sigma, U.S.) to prepare agar plates. The 48-h old biofilms were cultured on the medium at 37°C without agitation. The intensity of red staining was considered proportional to the quantity of extracellular polysaccharides and proteins (Ahmad et al., 2020 ). The morphotyping assay was conducted according to (Römling et al. 1998 ) with modifications. In brief, TSB agar was supplemented with a CRCB aqueous mixture, made up of pre-filtered 0.04% Congo red stain and 0.02% Coomassie blue G-250 stain (VWR (Avantor), Radnor, U.S.) to prepare agar-containing Petri dishes. Biofilms were grown on these CRCB agar plates for 24, 48, and 144 hours. Microtiter biofilm formation assay (CV Mtp) Overnight bacterial cultures in TSB were harvested (6,000×g, 10 min, RT), then suspended in PBS (pH = 7.4), diluted in the buffer to achieve a cell density of approximately 3×10 6 CFU/mL. Next, 100 µL of the suspension was added to sterile wells of a 96-well polystyrene cell culture plate, and incubated for 48 h at 37°C and 200 rpm. To obtain dual-species biofilms suspensions of both bacteria were mixed in a 1:1 (v/v) ratio. The wells’ content with planktonic cells was discarded, and the plate was washed three times with 200 µL of PBS (pH = 7.4), then air-dried for approximately 20 min. The dried BFs were stained with a 0.1% aqueous solution of Crystal violet (CV) for 15 min, at RT, followed by triple rinsing with water. The stained content was resolubilized in 200 µL of 33% acetic acid, and OD 590 of the obtained solutions was measured using SPECTROstar NANO spectrophotometer (BMG LABTECH, Ortenberg, Germany). All experiments were performed with quadruplicate technical replicates and repeated at least in two independent assays. The values were normalized by dividing them by the OD 590 of biofilm treated with the blank buffer. The level of biofilm formation was interpreted according to (Stepanović et al. 2007 ). Quantification of eDNA in the biofilm A measurement of eDNA was carried out with DNAse I (PanReac AppliChem, Darmstadt, Germany) treatment of biofilms, grown for 24 and 48 h. BFs were grown as described in the subsection “Microtiter biofilm formation assay” and treated with 100 µL of DNAse I solution (20 µg/mL of DNAse I in 20 mM Tris-HCl buffer, 1 mM CaCl 2 (pH = 7.5)) or the same volume of the buffer at 37°C and 200 rpm, for 2 h. Thereafter, biofilms were rinsed with water, air-dried, and stained with 0.1% Crystal violet, following the OD 590 measurement. The eDNA content was calculated as the difference in BF staining with and w/o of DNAse I treatment (BF biomass reduction). Quantification of proteins content in the biofilm A measurement of proteins’ quantity was conducted for 24- and 48-h old biofilms. Proteinase K (PanReac Applichem, Darmstadt, Germany) in 20 mM Tris-HCl buffer supplemented with 100 mM NaCl, 1 mM CaCl 2 (pH = 7.5), at 50 and 800 µg/mL concentration was added to A. baumannii biofilms; and at 50, 800 and 1600 µg/mL concentration was added to K. pneumoniae biofilms. Preformed biofilms were treated with 100 µL of Proteinase K solutions or blank buffer at 37°C and 200 rpm, for 2 h. Thereafter, treated biofilms were rinsed, air-dried, stained with Crystal violet and analyzed as described in the subsection “Quantification of eDNA in the biofilm”. Antibiofilm activity assessment Mono-species and dual-species biofilms grown for 48 h and prepared for microtiter assay, were treated with either 100 µL of endolysin solutions at concentrations of 100 or 1000 µg/mL, or equal volume of 20 mM Tris-HCl (pH = 7.5) buffer as a negative control, for 2 h at 37°C and 200 rpm. After incubation, biofilms were rinsed twice, air-dried, stained with 0.1% Crystal violet, rinsed again, dissolved and analyzed as was described in the subsection “Microtiter biofilm formation assay”. Microscopy of biofilms Sterile glass coverslips (Hampton Research, Aliso Viejo, CA, U.S.) were plunged into the overnight cultures, diluted in fresh TSB medium on Petri dishes and incubated at 37°C for 48 h without shaking. The slides were then carefully washed three times with sterile distilled water and air-dried. Two slides were treated with 300 µL of 20 mM Tris–HCl (pH = 7.5) control buffer, other pairs of slides were exposed to 300 µL of 100 µg/mL LysAm24, LysAp22, LysECD7, or LysSi3 solutions for 2 h at RT. Afterward, all slides were again washed with water two times. Air-dried slides were stained with a 0.1% aqueous solution of Crystal violet for 15 min at RT. All stained samples were rinsed once with water. Then, half of the slides were immediately rinsed twice and air-dried for microscopy. Another half of the slides were negatively stained to identify acid polysaccharides, which are crucial for antibacterial tolerance in most bacterial capsules and biofilms. A negative stain was performed using the Anthony method (Hughes and Smith 2013 ) with modifications. Following the method, CV-stained slides were submerged into 20% aqueous CuSO 4 for 10 s, rinsed thoroughly with water, and then dried. All slides were imaged using Axiostar Plus Transmitted Light Microscope (Zeiss AG, Jena, Germany) at ×630 magnification. DNA-binding effect of the endolysins To detect interaction between eDNA and the endolysins we obtained genomic DNA of A. baumannii using a modified CTAB method (Minas et al. 2011 ). Briefly, cells from an overnight bacterial culture were harvested, once washed, and solubilized in a CTAB solution (2% w/v cetrimonium bromide (Helicon, Moscow, Russia), 100 mM Tris-HCl, 20 mM EDTA and 1.4 M NaCl, pH = 8.0), ex tempore supplemented with 0.2% β-mercaptoethanol, at 65°C for 40 min. Then, the suspensions were mixed with an equal volume of chloroform-isoamyl alcohol (24:1), and the aqueous phase was collected after centrifugation at 12,000×g for 10 min, RT. The DNA precipitation proceeded in 0.6 volume of isopropanol overnight at – 25°C. The precipitates were harvested at 8,000×g for 15 min and washed three times with 80% ethanol. After ethanol was discarded, the pellets were suspended in 20 mM Tris-HCl (pH = 7.5) buffer with 1 µg/mL RNAse A (PanReac Applichem, Darmstadt, Germany), incubated for 15 min at 37°C, afterwards RNAse A was inactivated at 65°C for 15 min. Quality of the DNA samples was estimated spectrophotometrically, analyzing A260/230 and A260/280, thereafter a concentration of DNA was measured using Qubit DNA HS Assay Kit and Qubit 3.0 fluorometer (Thermo Fisher Scientific Eugene, Oregon, U.S.). Endolysins LysAm24, LysAp22, LysECD7, and LysSi3 solutions in 20 mM Tris-HCl buffer at 10 or 50 ng/µL concentration were mixed with DNA solutions (at a concentration of 20 ng/µL) at a protein:DNA (P:DNA) mass ratio of 2:1 or 10:1 (final volume of the mixtures was 7.5 µL) and incubated for 30 min at RT. Solutions containing either DNA or investigated endolysin in 20 mM Tris-HCl buffer (pH = 7.5) were used as control. The influence of electrostatic interactions was assessed by adding NaCl to final concentrations of 150 or 300 mM to the mixtures (P:DNA w/w ratio of 10:1) during their incubation. Post-incubated solutions were mixed with the 6X DNA Loading Dye (Thermo Fisher Scientific Eugene, Oregon, U.S.) and loaded in 1% agarose gel, containing 0.2 µg/mL ethidium bromide (Helicon, Moscow, Russia). An electrophoretic separation of all samples was performed in the Tris-borate buffer (50 mM Tris-base, 50 mM boric acid, 2 mM EDTA, pH = 8.3) at 80 V for an hour. Imaging was performed using the Gel Doc EZ gel documentation system (Bio-Rad, Hercules, CA, U.S.). To correlate the binding assay with the inhibitory effect of eDNA, an antibacterial activity test was performed. An overnight bacterial culture of A. baumannii was diluted 30-fold in LB broth and grown to the exponential phase (OD 600 = 0.6). Subsequently, the cells were harvested by centrifugation (6,000×g, 10 min) and resuspended in the same volume of PBS (pH = 7.4). Each suspension was diluted in the 20 mM Tris-HCl (pH = 7.5) to a final density of approximately 10 6 cells/mL. Afterward, 100 µL of the bacterial suspensions and 100 µL of the pre-incubated protein-DNA solutions (a final protein concentration was 10 µg/mL, a final DNA concentration was 1 or 5 µg/mL) were mixed in 96-well plates. Buffers with or without DNA were used as the negative controls. The mixtures were incubated at 37°C for 30 min with shaking at 200 rpm and then were diluted 10-fold in PBS (pH = 7.4). Then, 100 µl of each dilution were plated onto LB agar, and the number of bacterial colonies were counted after an overnight incubation at 37°C. All experiments were performed in quadruplicate, and the antibacterial activity was expressed as follows: Antibacterial activity (%) = 100% – (CFUexp/CFUcont) × 100%, where CFUexp is the number of bacterial colonies in the experimental culture plates, and CFUcont is the mean number of bacterial colonies in the control culture plates. Statistical Analysis The data were analyzed and illustrated using GraphPad Prism 9.0 software. According to the results of normality tests (Kolmogorov-Smirnov’s method), data sets were compared using appropriate statistical tests with corrections for multiple comparisons (detailed information on the chosen analysis methods is provided in captions and results). Results Exopolysaccharides content To visualize different EPSs, various staining procedures of bacterial BF-macrocolonies on nutritious media with glucose are often implemented. Cultivation of bacterial macrocolonies on solid medium, containing Congo red (CR) dye is an express test system of biofilm formation properties. CR interacts with most β-folded proteins, glucans with β (1->4) and β (1->3) bonds, and with various polycationic polysaccharides (Puchtler et al. 1962 ). During the 48-h incubation on CR agar, the investigated bacterial colonies accumulated small amounts of the stain and slightly decolorized the medium (Fig. 1 a, the second row). The K. pneumoniae originated macrocolonies with a crystalline red center and sharp, clear edges, while A. baumannii formed homogeneous brown and rough colonies indicating production of moderate quantity of polysaccharides and amyloid proteins. Dual-species colonies resembled those of K. pneumoniae , with a rougher structure and partially colored periphery. A different matrix architecture was observed by staining with polycationic dye Alcian blue, revealing carboxyl-rich polysaccharides (Scott and Dorling 1965 ), located on the air-contacting exterior of formed macrocolonies (Fig. 1 a, the third row). The colonies of K. pneumoniae had a concentric staining, significantly more intense compared to A. baumannii , while the dual-species colonies possessed well resolved dark-blue strands radiating from the center, revealing the conglomerated polycationic and polyanionic polysaccharides’ plots. To investigate the coexistence of bacteria in mixed biofilms the morphotyping assay with culture media containing Coomassie brilliant blue dye (CBB) was done. CBB stains biofilm-associated proteins, and Congo red dye, binding with both proteins and polysaccharides (Nesse et al. 2020 ). The significant heterogeneity of biofilm composition was observed for dual-species colonies (Fig. 1 b and Online Resource Fig. 1 ) combining phenotypic traits of both species and characterized by increased cell motility. Quantification of eDNA The biomass density of either K. pneumoniae or A. baumannii 24-h old biofilms pretreated with DNAse I, was reduced by 20% on average (Fig. 2 ), while the enzymatic treatment of 48-h biofilms led to a decline in the biofilm biomass by 30.9% and 25.9% respectively. Neither the increase in DNAse I concentration (up to tenfold) nor the time of biofilm formation led to a significant reduction in the BFs’ biomass (p > 0.05, two-way ANOVA with Tukey’s correction). Quantification of proteins within biofilms The presence of fibrillar proteins, pili or pilus-like structures within examined biofilms of 24- and 48-h macrocolonies of A. baumannii (brown staining) and K. pneumoniae (red staining) was demonstrated in CRCB-TSB assay (Fig. 1 . b). To quantify the approximate content of proteins within the matrix of biofilms, treatment with Proteinase K (PK) was used. It was shown that biofilms of investigated strains, particularly of A. baumannii , were significantly resistant to proteolysis (Fig. 3 ). No effective concentration of PK was found to disrupt biofilms of A. baumannii , where the addition of 800 µg/mL of PK resulted in a negligible loss of biomass (Fig. 3 a). Thereby, proteins make up relatively small fraction of the A. baumannii biofilm matrix (less than 10–15%), although some EPS proteins might evade a proteolysis, for instance, due to aggregation in fibers, O-glycosylation, or their covering by other compounds of the matrix (Iwashkiw et al. 2012 ). Twenty-four hours old biofilms formed by K. pneumoniae also remained recalcitrant to the activity of PK (Fig. 3 b), that apparently correlated with the results of morphotyping, where 24-h old macrocolonies of K. pneumoniae had no amyloid content (Fig. 1 b, the first row). On the contrary, biofilms of K. pneumoniae , grown for 48 hours, were susceptible to the action of Proteinase K, in a dose-dependent manner in concentrations range 50 to 1600 µg/mL of PK, resulting in the loss up to 45% of biomass at 1600 µg/mL. Notably, simultaneous treatment with DNAse and PK did not lead to a synergistic decline in biomass of the investigated biofilms (Online Resource Fig. 2 ), which indicates tough stability of these BFs, provided by multiple contacts between various EPSs. Activity of the endolysins against 48-h biofilms A pronounced reduction in biomass of 48-h old BFs was observed after 2-h incubation with endolysins as shown by the CV Mtp assay for A. baumannii (Fig. 4 a), K. pneumoniae (Fig. 4 b) or mixed biofilms (Fig. 4 c), which all formed moderate or strong BFs on the polystyrene. Depending on the biofilm-forming bacteria, endolysins acted mostly in a dose-dependent manner. Each protein led to at least a 2-fold loss in the biomass of preformed biofilms, in particular, LysAp22 possessed the strongest disruption of BFs, whilst antibiofilm activity on EPS-rich biofilms by LysSi3 was less evident than that of other endolysins. Exposure to a 1000 µg/mL of LysAp22 solution decreased the biofilms’ biomass up to 33%, 25% and 27% on average; thus K. pneumoniae- related BFs were almost eliminated (Fig. 4 b). Two endolysins, LysECD7 and LysAm24, acted similarly versus mono-species BFs, albeit the impact of LysECD7 on dual-species BFs was comparable to that of LysAp22. The 2-h treatment with LysAm24 or LysECD7 at a concentration of 1000 µg/mL led to a decrease up to approximately 41%, 35% and 27% of A. baumannii , K. pneumoniae or mixed biofilms’ biomass respectively, compared to the controls treated with the buffer. At 100 µg/mL, these proteins exhibited a lower activity. Except for LysSi3, endolysins at a higher concentration removed dual-species biofilms better than low-EPS containing A. baumannii BFs (p < 0.01, Mann-Whitney tests with Holm-Šídák’s correction). LysSi3 acted on mixed biofilms worse than other endolysins, disrupting the biomass by approximately 54%, independently on dose. Exopolysaccharides and biomass staining of biofilms pretreated with endolysins As demonstrated, three-dimensional biofilms were formed after a 48-h incubation of A. baumannii cells in TSB media on glass slides (Fig. 5 ). These aggregates consisted of dense and tightly connected structures (Fig. 5 a), which were disrupted by endolysins to incoherent monolayers (BFs treated with LysAm24, LysAp22, LysSi3), or microcolonies (LysECD7). After destaining with CuSO 4 , light-blue color of the biofilm’s structures indicated the presence of acid, mostly capsular carbohydrates. Otherwise, if there is a lack of these EPSs, the biomass was colored violet. Thus, it allows to distinguish a significant number of cells covered with capsular polysaccharides and also the heterogeneous arrangement of different EPSs, especially on the BF edges (Fig. 5 a’). Biofilms treated with LysAp22 or LysECD7 were poorly stained by CV (Fig. 5 c and d), except for a few areas with dark violet peaks, and did not differ from the cells, additionally rinsed with CuSO 4 , suggesting interactions with carbohydrates, subsequent dissociation, and destabilization of biofilms’ matrix. The BF-disrupting effect of LysSi3 (Fig. 5 e and e’) appeared to be similar to that of LysECD7, however, it was less pronounced, and did not cause evident destabilization of exopolysaccharides, but also efficiently passed through EPSs. A different manner of endolysin-biofilm interactions was observed for LysAm24 (Fig. 5 b and b’). Preparations treated with this endolysin and stained with CV possessed an intense violet color, whereas double-stained biofilms apparently lacked any purple areas that demonstrate the interactions between LysAm24 and EPS, other than acid carbohydrates, providing additional sites for CV staining. Effects of bacterial DNA on endolysins We estimated that in abundance of endolysins (10:1 ratio), the DNA samples incubated with three out of 4 investigated endolysins displayed a notable decline in the electrophoretic mobility of nucleic acids (Fig. 6 a), suggesting the complexes’ formation of endolysin with genomic DNA, which remained in the loading well or formed significant smears. Among the examined proteins, LysAm24 and LysECD7 exhibited the most pronounced DNA-binding activity (Fig. 6 a). However, with the exception of LysAm24, endolysin-eDNA complexes were not detected when the ratio of P:DNA was 2:1. Only LysSi3 showed no interactions with DNA samples in this test. Exogenous nucleic acids led to the inhibition of muramidases’ activity versus free-living A. baumannii cells. At the lower concentration (10:1 ratio), DNA did not impact antibacterial properties of these proteins, whereas it dramatically inhibited them at the ratio of 2:1 (Fig. 6 b). At the same time, endopeptidase LysECD7 remained effective against the bacteria even in the excess of DNA. Discussion Clinically relevant biofilms, formed by A. baumannii and K. pneumoniae species or their combination, are associated with a variety of skin and soft tissue infections, as well as nosocomial infections of the respiratory (Said et al. 2022 ) and urinary tract (Paczosa and Mecsas 2016 ). Because of producing a heterogeneous and thick matrix, these causative agents possess an extreme tolerance towards various antimicrobials (Stewart 2015 ). Herein, we characterized the content of biofilms formed by two bacterial isolates and estimated that 48-h A. baumannii BFs contained a moderate amount of highly deacetylated glycosaminoglycans, acid polysaccharides, eDNA, and a relatively large fraction of the cellular biomass (Online Resource Table 1). For K. pneumoniae , the BF matrix makes up at least half of the dry biofilm’s volume, with a pronounced content of negatively charged polysaccharides, DNA, and non-amyloid proteins. Despite the difference in composition, we have shown that investigated enzymes disrupt heterogeneous biofilms, interacting directly with different BF compounds. All of the endolysins at a concentration of 100 µg/mL led to a decrease in biomass of mono- and dual-species films of Gram-negative bacteria by at least 2–3 times. Moreover, treatment with LysAm24, LysAp22, or LysECD7 at a concentration of 1000 µg/mL was linked to the almost complete removal of the biofilms. Although most endolysins are considered highly specific hydrolases, disrupting the peptidoglycan at specific sites, a side ability to influence compounds of the biofilm matrix could be recognized. Thus, we detected weak interactions between endolysins and exopolysaccharides and eDNA. These interactions, however, were associated with a significant destabilization of the treated biofilm and could be beneficial in terms of bacteriophage ecology. Endolysin-EPS interactions may include effects on adhesion of competitive viruses, availability of phages’ receptors, and formation of cell-wall deficient cells to sustain the host population (Wohlfarth et al. 2023 ). Furthermore, lysins can be engaged in BF-specific signaling pathways due to the products of peptidoglycan hydrolysis (Irazoki et al. 2019 ). Extracellular carbohydrates are a significant component involved in biofilm formation, crucial for stable cell adhesion on various surfaces, and establish multiple contacts with other exogenous molecules (Ostapska et al. 2018 ). They include enterobacterial cellulose (Nesse et al. 2020 ), and poly-β-1,6-N-acetyl-D-glucosamine (PNAG), found in numerous species of bacteria (Choi et al. 2009 ). Capsular polysaccharides, frequently consisting of various negatively and neutrally charged subunits, serve as a physical shield against environmental factors (Paczosa and Mecsas 2016 ; Singh et al. 2019 ), and within BFs modulate surface adhesion (Mann and Wozniak 2012 ; Pompilio et al. 2021 ). Therefore, massive detachment of acid exopolysaccharides by LysAp22 and LysECD7 may impair BF stability, without relevant loss of the antibacterial activity, and subsequently seems to be an intriguing feature for further development of enzybiotics. The exact mechanism of this change is not fully understood. However, it is unlikely to be related to capsule digestion by specific phage depolymerases. In particular, their active sites differ from those of endolysins and structures of the investigated lysins possess no significant similarity with any of this wide group of enzymes. It is worth noting, that exopolysaccharides can trigger changes in the catalytic activity of other enzymes, more related to endolysins. For instance, a polyanionic exopolysaccharide of Xantamonas spp. inhibits egg white lysozyme and lysostaphin, but activates two bacterial muramidases (Stepnaya et al. 2001 ). The results of the eDNA-endolysin binding assay partly correlated with the isoelectric points (pIs) of the lysins: LysAp22 (pI 9.19), LysAm24 (pI 8.89), LysECD7 (pI 8.83) and LysSi3 (pI 8.52), proposing a higher probability of binding with an increase of pI. Hence, unspecified electrostatic forces may determine the interactions between the endolysins and eDNA. However, the differences in pIs between the studied proteins are small; thereby an actual net charge distribution of molecules may be distinct, affecting the binding capacity. Anyway, as shown in Online Resource Fig. 3 , the strength of the endolysin-DNA association was significantly reduced by the addition of NaCl. Thus, LysECD7 partially ceased to bind with DNA in the mixtures, containing 150 and 300 mM NaCl, LysAm24 showed a similar tendency at the lower salt’s concentration, whereas the DNA-binding effect of LysAp22 was evident in the mixture without NaCl. Therefore, the interplay observed between eDNA and the investigated enzymes should be considered nonspecific, and primarily driven by electrostatic binding. Strong electrostatic interactions are suggested to destabilize eDNA in the cases of LysECD7 and possibly LysAm24, serving as a mechanism for the antibiofilm effect of these enzymes within BF areas depleted in free cations, formed by eDNA. LysSi3 is believed to have a reduced antibiofilm activity due to the indirect influence of exogenous nucleic acids, while eDNA affected LysAp22 ambivalently. We suggest that DNA mixed with a bacterial suspension can bind positively charged compounds on the outer membrane (OM) of bacteria and enhance their surface hydrophobicity (Das et al. 2014 ), impairing CW accessibility for either of hydrolases. It is interesting to note, that the investigated endolysins contain positively charged terminal structures putatively involved in permeabilization of membranes, except for LysECD7. The mechanism of passing through OM for LysECD7 is probably based on other, not only electrostatic interactions with a cell’s surface (Antonova et al. 2019 ). There is plenty of findings noted extracellular DNA can be a component of biofilm tolerance exhibited by different species against charged antimicrobial peptides (Batoni et al. 2016 ) and proteins, as well as aminoglycosides (Chiang et al. 2013 ), fluoroquinolones (Tetz et al. 2009 ), and non-phage derived lysozymes. In this regard, eDNA interaction with positively charged structures often found in endolysins’ molecules may also affect their catalytic activity and access to target sites, hampering endolysin-based therapy. On the other hand, enzybiotics may destabilize exposed biofilms through unspecific interactions with extracellular nucleic acids, allowing biofilm control. For example, side antibacterial effects have been reported for egg lysozyme and antistaphylococcal chimeric peptidoglycan hydrolase (Fernández et al. 2017 ; Liu et al. 2023 ). It has been proposed to be based on strong endolysin-DNA binding, that influence the expression of bacterial genes. Another interesting mechanism of biofilm removal by targeting defensive BF compounds has been reviewed for a few AMPs, which possess similarity with semi-conservative structures of the studied proteins (Batoni et al. 2016 ). Therefore, our study reveals contrasting aspects in the antibiofilm activity of the phage endolysins, which deviated from bacterial cell lysis caused by catalytic digestion of the CW peptidoglycan. Specifically, we detected the interaction with the carbohydrate component of the matrix (LysAp22 and LysECD7), as well as the electrostatic binding of polycationic sites of endolysins to eDNA (LysAm24 and LysECD7). These interactions result in the biofilm removal by endolysins, despite a decrease in their initial antibacterial activity. However, this research did not evaluate the influence of abnormal lysin-induced release of various bacterial compounds on the stability of BFs, which could also be compelling. Additional experiments should be conducted to clarify the endolysin-exoproteome interactions (De Gregorio et al. 2015 ), whilst details in endolysin-exopolysaccharides association could be investigated through a mass-spectrometric structural analysis accompanied by a design of possible binding in silico . Treatment of biofilms with the cell-free lysate or products of the peptidoglycan hydrolysis could also provide additional information. Thus, the present and further findings are expected to highlight a complex mechanism underlying the endolysins’ antibiofilm activity, allowing them to successfully evade a biofilm’s defense, provided by its matrix. Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and materials: The data that support the findings of this study are available within the paper and its Supplementary Information. Additionally, the raw data are available from the authors upon reasonable request. Competing interests: The authors have no financial or proprietary interests in any material discussed in this article. Fundings: The study was supported by the Russian Science Foundation (RSF), grant № 23-74-10027, https://rscf.ru/project/23-74-10027/. Authors’ contributions: Conceptualization: Daria V. Vasina; Methodology: Anastasiya M. Lendel; Formal analysis and investigation: Anastasiya M. Lendel, Nataliia P. Antonova, Igor V. Grigoriev, Evgeny V. Usachev; Writing - original draft preparation: Anastasiya M. Lendel; Writing - review and editing: Nataliia P. Antonova, Igor V. Grigoriev, Daria V. Vasina; Funding acquisition: Daria V. Vasina; Resources and project administration: Vladimir A. Gushchin; Supervision: Daria V. Vasina. All authors have read and approved the manuscript. 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Supplementary Files Supplementary26.01.24.docx Graphicalabstract.jpg Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 29 Apr, 2024 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 14 Mar, 2024 Reviews received at journal 14 Mar, 2024 Reviews received at journal 22 Feb, 2024 Reviewers agreed at journal 09 Feb, 2024 Reviewers invited by journal 01 Feb, 2024 Editor assigned by journal 31 Jan, 2024 Submission checks completed at journal 31 Jan, 2024 First submitted to journal 26 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Nataliia","middleName":"P.","lastName":"Antonova","suffix":""},{"id":270454230,"identity":"090f9f18-d904-4059-8dcf-0a4a4838e6b6","order_by":2,"name":"Igor V. Grigoriev","email":"","orcid":"","institution":"N. F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Igor","middleName":"V.","lastName":"Grigoriev","suffix":""},{"id":270454231,"identity":"db62c321-3a35-4b28-9fd7-e55c36cb4964","order_by":3,"name":"Evgeny V. Usachev","email":"","orcid":"","institution":"N. F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Evgeny","middleName":"V.","lastName":"Usachev","suffix":""},{"id":270454232,"identity":"3fc4c4a9-790a-495b-9eb9-41fcc680c6c6","order_by":4,"name":"Vladimir A. Gushchin","email":"","orcid":"","institution":"N. F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Vladimir","middleName":"A.","lastName":"Gushchin","suffix":""},{"id":270454233,"identity":"7fc43d84-34fe-4ab3-a441-01aaf0053847","order_by":5,"name":"Daria V. Vasina","email":"","orcid":"","institution":"N. F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Daria","middleName":"V.","lastName":"Vasina","suffix":""}],"badges":[],"createdAt":"2024-01-26 12:14:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3899892/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3899892/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-024-03999-9","type":"published","date":"2024-04-29T23:28:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50538840,"identity":"27c8b3cf-327f-42ee-8810-791e943fe5a6","added_by":"auto","created_at":"2024-02-02 06:02:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":137470,"visible":true,"origin":"","legend":"\u003cp\u003eMacrocolony staining of \u003cem\u003eK. pneumoniae \u003c/em\u003eand\u003cem\u003e A. baumannii\u003c/em\u003e biofilms components\u003cem\u003e.\u003c/em\u003e a) Extracellular polymers of colonies, grown on solid media for 48 h: TS, TSB agar without staining; CR, TSB agar stained with Congo red, indicating amyloids and spectrum of carbohydrates; AlcB, TSB agar stained with Alcian blue, indicating polysaccharides in colonies’ exterior, but not proteins. b) Formation of EPS-containing structures within colonies, grown on CRCB-TSB agar media for 24 and 48 hours.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/d2362d759130e83d9c623c12.jpg"},{"id":50538847,"identity":"eebdf712-c14d-4ab1-a900-dc38a5ce9746","added_by":"auto","created_at":"2024-02-02 06:02:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107665,"visible":true,"origin":"","legend":"\u003cp\u003eBiomass reduction of 24- and 48-h biofilms of \u003cem\u003eA. baumannii \u003c/em\u003e(a) and \u003cem\u003eK. pneumoniae\u003c/em\u003e (b) after treatment with 20 µg/ml DNAse I (DNAse+, white columns). Each dataset is presented as a normalized mean value ± standard deviation (SD). Only statistically relevant differences from controls and between cohorts of distinct aged biofilms are marked as asterisks: *, p \u0026lt; 0,05; **, p \u0026lt; 0,01 (one-way ANOVA with Tukey’s correction).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/8c168ebfb5299d96199bc16e.jpg"},{"id":50538841,"identity":"170464f0-b316-477b-9b1e-4d7988b61a2c","added_by":"auto","created_at":"2024-02-02 06:02:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56325,"visible":true,"origin":"","legend":"\u003cp\u003eContent of proteins in 24- (grey) and 48-h (black) preformed biofilms of \u003cem\u003eA. baumannii \u003c/em\u003e(a) and K.\u003cem\u003e pneumoniae\u003c/em\u003e (b). Each dataset represents a normalized mean value ± SD. Only statistically relevant differences from controls and between cohorts of 24- and 48-h biofilms are marked as asterisks: *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001 (Mixed-effects analysis with Tukey’s correction)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/a2a58582fe298b6f5c18faf0.jpg"},{"id":50539555,"identity":"20619426-dcb8-4aeb-99ef-7c6f39f9d17e","added_by":"auto","created_at":"2024-02-02 06:18:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176651,"visible":true,"origin":"","legend":"\u003cp\u003eThe activity of endolysins LysAm24, LysAp22, LysECD7, and LysSi3 \u003cem\u003eversus\u003c/em\u003e the 48-h biofilms. a) \u003cem\u003eA. baumannii\u003c/em\u003e biofilms;\u003cem\u003e \u003c/em\u003eb) \u003cem\u003eK. pneumoniae \u003c/em\u003ebiofilms\u003cem\u003e; \u003c/em\u003ec) dual-species (mixed) biofilms. Black columns refer to controls treated with Tris-HCl buffer (pH = 7.5); light grey columns – with 100 µg/mL of endolysins; white columns – biofilms treated with 1000 µg/mL of lysins. Each dataset is demonstrated as normalized mean value ± SD. Only differences between data related to distinct concentrations of enzyme interpreted as follows: ns, p \u0026gt; 0.05; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001, whereas all differences between the buffer- and endolysin-treated groups were supposed highly reliable, p \u0026lt; 0.0001 (one per row Mann-Whitney tests with Holm-Šídák’s correction)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/e1f5c4fd64e649769787740c.jpg"},{"id":50538915,"identity":"14ee19e8-f669-4fd2-a3a8-3a0048531499","added_by":"auto","created_at":"2024-02-02 06:10:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":318114,"visible":true,"origin":"","legend":"\u003cp\u003eMicrophotography of 48-h \u003cem\u003eA.\u0026nbsp;baumannii \u003c/em\u003ebiofilms treated with the endolysins (b-e and b’-e’) or buffer w/o endolysins (a, a’), provided by bright-field microscopy after staining with only 0.1% Crystal violet dye (CV, the upper row), or both CV and 20% CuSO\u003csub\u003e4 \u003c/sub\u003esolutions (Cu, the lower row)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/ac8bd0ffb8e52b4a26c17fe1.jpg"},{"id":50538913,"identity":"f068d192-a213-426f-b19f-d62ced3c2a1b","added_by":"auto","created_at":"2024-02-02 06:10:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":166970,"visible":true,"origin":"","legend":"\u003cp\u003ea) Binding of endolysins to \u003cem\u003eA. baumannii\u003c/em\u003e genomic DNA. The Protein:DNA mass ratios are shown; Cont means only DNA-containing buffer solution; Mr, molecular length in base pairs (bp) of the DNA standards. b) Changes in the antibacterial activity of 10 μg/mL lysins in the presence of DNA at the same ratios, \u003cem\u003eversus\u003c/em\u003e free-living cells of \u003cem\u003eA. baumannii \u003c/em\u003e(only statistically significant differences between data sets are shown, *, p \u0026lt; 0.05; **, p \u0026lt; 0.01 (Kruskal-Wallis test with Dunn’s correction)\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/65748f816b60d011cb3becb3.jpg"},{"id":55695092,"identity":"3ca34360-4cab-4f71-9712-4d49d0f3caf1","added_by":"auto","created_at":"2024-05-02 00:58:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1054743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/4d20b01e-7468-4630-8490-14d5b637c093.pdf"},{"id":50538844,"identity":"05ec5d12-4be2-473f-b4eb-f14f1ad8f93b","added_by":"auto","created_at":"2024-02-02 06:02:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1014580,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary26.01.24.docx","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/e229c8d879b45322189c886c.docx"},{"id":50538916,"identity":"5bf0780e-8da9-4acf-a138-a608dc36883a","added_by":"auto","created_at":"2024-02-02 06:10:04","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":311115,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899892/v1/b64a1b6918cec5569a5efe57.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biofilm-disrupting effects of phage endolysins LysAm24, LysAp22, LysECD7, and LysSi3: breakdown the matrix","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacterial biofilm (BF) is a community of immobilized and phenotypically altered microorganisms embedded in self-produced exogenous polymeric substances (EPSs) (Donlan and Costerton \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Karygianni et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In particular, biofilm-related phenotype is formed by various matrix structures and chemical compositions, such as exopolysaccharides, extracellular DNA (eDNA), proteins, lipids and membrane vesicles, low molecular compounds (for example, \u0026ldquo;quorum sensing\u0026rdquo; ligands, cyclic di-GMP, accumulated water, and polyvalent ions). These heterogeneous constituents greatly impact biofilm formation, sustainability, availability of nutrients, and tolerance toward antiseptics, antibiotics and bacteriophages. Being one of the most abundant states of bacterial existence both in natural and human-made environments, BFs have great biological significance owing to efficient expansion of the community and increased survival rates under severe conditions (Donlan and Costerton \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Flemming et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpecifically, there are challenges in the therapy of diseases associated with Gram-negative opportunistic bacteria, such as \u003cem\u003eAcinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e, associated with rapid emergence of multidrug-resistance traits and hardly removable biofilms formed within the host organisms and on surfaces of hospital equipment and prosthetic devices. These features together with advanced natural antibiotic resistance led to a notable increase in the probability of negative outcomes, formation of persistent infection and spread of nosocomial infections (Omar et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Stewart \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Despite actively developed restrictive measures to control Gram-negative pathogens, there are limited innovative treatment approaches (Giacobbe et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Prasad et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), while common chemotherapy is often considered as non-effective or excessively deleterious (Eriksson et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). At the same time, the search for reliable antibacterial strategies continues, thus an application of nanoparticles, antibodies, bacteriophages, bacteriophage-derived antimicrobial proteins (also called enzybiotics) and peptides (AMPs) are expected to be highly advanced options as a supplementation to antibiotic therapy or as an alternative treatment strategy (Heselpoth et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Koo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEndolysins are promising peptidoglycan-degrading enzybiotics that are already investigated for clinical relevance due to their pronounced activity and safety (Karau et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For decades of research on endolysins, no strict evidence of acquired resistance to these proteins has been reported, that is explained particularly by the high fitness costs of most changes in the peptidoglycan, especially across Gram-negative species (Grishin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several studies reveal an ability of endolysins not only to eliminate multidrug resistant bacteria, but also to efficiently disrupt their biofilms (Arroyo-Moreno et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Guti\u0026eacute;rrez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Karau et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the key mechanisms of endolysin-biofilm interactions are uncovered, and the investigation is mostly limited to the fact of BFs disruption and prevention of their formation. Whilst interactions between endolysins and EPSs and their effects on the enzymes\u0026rsquo; activity are poorly described.\u003c/p\u003e \u003cp\u003eOur previous research was focused on recombinant endolysins, muramidases LysAm24, LysAp22, LysSi3 and endopeptidase LysECD7, derived from different bacteriophages infected Gram-negative hosts. Recently, promising safety properties and antibacterial activity of the endolysins against a broad spectrum of Gram-negative representatives were described (Antonova et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), furthermore, highlighted the substantial disruption of 24-h BFs, affected by these enzymes (Vasina et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the catalytic mechanism of endolysins, associated with the hydrolysis of CW (cell-wall) peptidoglycan bonds, is not enough to explain their influence on preformed BFs, degrading complex three-dimensional structures of the matrix, down to individual bacterial cells. In the present study we investigated the composition of biofilms formed by polyresistant strains of \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e, revealing interactions between either of the endolysins and the abundant defensive compounds of preformed BFs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Ts 50\u0026thinsp;\u0026minus;\u0026thinsp;16 and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e F 104\u0026thinsp;\u0026minus;\u0026thinsp;14 clinical isolates (collection of the N.F. Gamaleya Federal Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation), were implicated in the present study as biofilm-forming Gram-negative pathogens of the ESKAPE group. \u003cem\u003eA. baumannii\u003c/em\u003e Ts 50\u0026thinsp;\u0026minus;\u0026thinsp;16 was isolated from patient\u0026rsquo;s sputum, intensive care unit, and \u003cem\u003eK. pneumoniae\u003c/em\u003e F 104\u0026thinsp;\u0026minus;\u0026thinsp;14 was obtained from patient\u0026rsquo;s sputum, outpatient hospital. Both strains were stored at \u0026minus;\u0026thinsp;80\u0026deg;C and cultivated in pre-autoclaved Tryptic Soy Broth (TSB; SIFIN, Berlin, Germany) at 37\u0026deg;C, at 250 rpm overnight before performing further tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant expression and purification of proteins\u003c/h2\u003e \u003cp\u003eRecombinant endolysins LysAm24, LysAp22, LysECD7, and LysSi3 were obtained as described previously (Vasina et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In brief, proteins modified with polyhistidine-tag (8 His) were recombinantly expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21(DE3) pLysS strain using 1mM β-D-1-thiogalactopyranoside induction at 37\u0026deg;C for 3 h. The cells were harvested by centrifugation (6,000\u0026times;g for 10 min at 4\u0026deg;C) and incubated with 100 \u0026micro;g/mL lysozyme in lysis buffer (20 mM Tris-HCl, 250 mM NaCl, and 0.1 mM EDTA, pH\u0026thinsp;=\u0026thinsp;8.0), and disrupted by sonication. Soluble proteins were purified on an NGC Discovery\u0026trade; 10 FPLC system (Bio-Rad, Hercules, CA, U.S.) with a HisTrap FF column (GE Healthcare, Munich, Germany) pre-charged with Ni\u003csup\u003e2+\u003c/sup\u003e ions. The filtered lysate was mixed with 30 mM imidazole and 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e and loaded on the column pre-equilibrated with binding buffer (20 mM Tris\u0026ndash;HCl, 250 mM NaCl, and 30 mM imidazole, pH\u0026thinsp;=\u0026thinsp;8.0). The fractions were eluted using a linear gradient to 100% elution buffer (20 mM Tris\u0026ndash;HCl, 250 mM NaCl, and 500 mM imidazole, pH\u0026thinsp;=\u0026thinsp;8.0). Resulting protein fractions were dialyzed against 20 mM Tris\u0026ndash;HCl buffer (pH\u0026thinsp;=\u0026thinsp;7.5).\u003c/p\u003e \u003cp\u003eThe purity of endolysins was determined by 16% SDS-PAGE, and protein concentrations were measured using a spectrophotometer (Implen NanoPhotometer; Implen, M\u0026uuml;nchen, Germany) at 280 nm and calculated using the predicted extinction coefficients [0.840, 0.831, 1.460, and 1.029 (mg/mL)\u003csup\u003e\u0026minus;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for LysAm24, LysAp22, LysECD7, and LysSi3, respectively]. The isoelectric points of endolysins LysAm24, LysAp22, LysECD7, and LysSi3 were predicted using the internet-source \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://isoelectric.org\u003c/span\u003e\u003cspan address=\"http://isoelectric.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm composition and morphology\u003c/h2\u003e \u003cp\u003eMacrocolony assays were conducted on solid culture medium with application of three different staining techniques and without any dyes as an intact control. For the formation of BFs, cells from overnight cultures were harvested by centrifugation (6,000\u0026times;g, 10 min, RT), resuspended in PBS (pH\u0026thinsp;=\u0026thinsp;7.4), and diluted to achieve a cell density of approximately 3\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL. To obtain dual-species biofilms, the appropriate bacterial suspensions were mixed in a 1:1 (v/v) ratio. Thereafter, 7 \u0026micro;L of the suspension was inoculated onto solid TSB agar medium on 90 mm Petri dishes and BFs were grown at 37\u0026deg;C for 48 h without agitation. All images of colonies were acquired using HD automatic colony counter Scan 300 and provided software (Interscience, Saint-Nom-la-Bret\u0026egrave;che, France).\u003c/p\u003e \u003cp\u003eAlcian blue 8GX 0.3% stain (PanReac AppliChem, Darmstadt, Germany) in a 4% acetic acid aqueous solution (pH\u0026thinsp;=\u0026thinsp;2.5) was prepared to visualize polysaccharides with abundant carboxyl groups. Five mL of pre-filtered Alcian blue stain were carefully dripped on plates containing macrocolonies and then incubated for 15 min at RT without agitation. Subsequently, the plates were rinsed with 5 mL of deionised distilled water.\u003c/p\u003e \u003cp\u003eThe results were confirmed by the Congo red stain absorption test (Freeman et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). For this, TSB agar was supplemented with pre-filtered 0.08% Congo red stain (Sigma, U.S.) to prepare agar plates. The 48-h old biofilms were cultured on the medium at 37\u0026deg;C without agitation. The intensity of red staining was considered proportional to the quantity of extracellular polysaccharides and proteins (Ahmad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe morphotyping assay was conducted according to (R\u0026ouml;mling et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) with modifications. In brief, TSB agar was supplemented with a CRCB aqueous mixture, made up of pre-filtered 0.04% Congo red stain and 0.02% Coomassie blue G-250 stain (VWR (Avantor), Radnor, U.S.) to prepare agar-containing Petri dishes. Biofilms were grown on these CRCB agar plates for 24, 48, and 144 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMicrotiter biofilm formation assay (CV Mtp)\u003c/h2\u003e \u003cp\u003eOvernight bacterial cultures in TSB were harvested (6,000\u0026times;g, 10 min, RT), then suspended in PBS (pH\u0026thinsp;=\u0026thinsp;7.4), diluted in the buffer to achieve a cell density of approximately 3\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL. Next, 100 \u0026micro;L of the suspension was added to sterile wells of a 96-well polystyrene cell culture plate, and incubated for 48 h at 37\u0026deg;C and 200 rpm. To obtain dual-species biofilms suspensions of both bacteria were mixed in a 1:1 (v/v) ratio. The wells\u0026rsquo; content with planktonic cells was discarded, and the plate was washed three times with 200 \u0026micro;L of PBS (pH\u0026thinsp;=\u0026thinsp;7.4), then air-dried for approximately 20 min. The dried BFs were stained with a 0.1% aqueous solution of Crystal violet (CV) for 15 min, at RT, followed by triple rinsing with water. The stained content was resolubilized in 200 \u0026micro;L of 33% acetic acid, and OD\u003csub\u003e590\u003c/sub\u003e of the obtained solutions was measured using SPECTROstar NANO spectrophotometer (BMG LABTECH, Ortenberg, Germany). All experiments were performed with quadruplicate technical replicates and repeated at least in two independent assays. The values were normalized by dividing them by the OD\u003csub\u003e590\u003c/sub\u003e of biofilm treated with the blank buffer. The level of biofilm formation was interpreted according to (Stepanović et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of eDNA in the biofilm\u003c/h2\u003e \u003cp\u003eA measurement of eDNA was carried out with DNAse I (PanReac AppliChem, Darmstadt, Germany) treatment of biofilms, grown for 24 and 48 h. BFs were grown as described in the subsection \u0026ldquo;Microtiter biofilm formation assay\u0026rdquo; and treated with 100 \u0026micro;L of DNAse I solution (20 \u0026micro;g/mL of DNAse I in 20 mM Tris-HCl buffer, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e (pH\u0026thinsp;=\u0026thinsp;7.5)) or the same volume of the buffer at 37\u0026deg;C and 200 rpm, for 2 h. Thereafter, biofilms were rinsed with water, air-dried, and stained with 0.1% Crystal violet, following the OD\u003csub\u003e590\u003c/sub\u003e measurement. The eDNA content was calculated as the difference in BF staining with and w/o of DNAse I treatment (BF biomass reduction).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of proteins content in the biofilm\u003c/h2\u003e \u003cp\u003eA measurement of proteins\u0026rsquo; quantity was conducted for 24- and 48-h old biofilms. Proteinase K (PanReac Applichem, Darmstadt, Germany) in 20 mM Tris-HCl buffer supplemented with 100 mM NaCl, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e (pH\u0026thinsp;=\u0026thinsp;7.5), at 50 and 800 \u0026micro;g/mL concentration was added to \u003cem\u003eA. baumannii\u003c/em\u003e biofilms; and at 50, 800 and 1600 \u0026micro;g/mL concentration was added to \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilms. Preformed biofilms were treated with 100 \u0026micro;L of Proteinase K solutions or blank buffer at 37\u0026deg;C and 200 rpm, for 2 h. Thereafter, treated biofilms were rinsed, air-dried, stained with Crystal violet and analyzed as described in the subsection \u0026ldquo;Quantification of eDNA in the biofilm\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAntibiofilm activity assessment\u003c/h2\u003e \u003cp\u003eMono-species and dual-species biofilms grown for 48 h and prepared for microtiter assay, were treated with either 100 \u0026micro;L of endolysin solutions at concentrations of 100 or 1000 \u0026micro;g/mL, or equal volume of 20 mM Tris-HCl (pH\u0026thinsp;=\u0026thinsp;7.5) buffer as a negative control, for 2 h at 37\u0026deg;C and 200 rpm. After incubation, biofilms were rinsed twice, air-dried, stained with 0.1% Crystal violet, rinsed again, dissolved and analyzed as was described in the subsection \u0026ldquo;Microtiter biofilm formation assay\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy of biofilms\u003c/h2\u003e \u003cp\u003eSterile glass coverslips (Hampton Research, Aliso Viejo, CA, U.S.) were plunged into the overnight cultures, diluted in fresh TSB medium on Petri dishes and incubated at 37\u0026deg;C for 48 h without shaking. The slides were then carefully washed three times with sterile distilled water and air-dried. Two slides were treated with 300 \u0026micro;L of 20 mM Tris\u0026ndash;HCl (pH\u0026thinsp;=\u0026thinsp;7.5) control buffer, other pairs of slides were exposed to 300 \u0026micro;L of 100 \u0026micro;g/mL LysAm24, LysAp22, LysECD7, or LysSi3 solutions for 2 h at RT. Afterward, all slides were again washed with water two times. Air-dried slides were stained with a 0.1% aqueous solution of Crystal violet for 15 min at RT. All stained samples were rinsed once with water. Then, half of the slides were immediately rinsed twice and air-dried for microscopy. Another half of the slides were negatively stained to identify acid polysaccharides, which are crucial for antibacterial tolerance in most bacterial capsules and biofilms. A negative stain was performed using the Anthony method (Hughes and Smith \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with modifications. Following the method, CV-stained slides were submerged into 20% aqueous CuSO\u003csub\u003e4\u003c/sub\u003e for 10 s, rinsed thoroughly with water, and then dried. All slides were imaged using Axiostar Plus Transmitted Light Microscope (Zeiss AG, Jena, Germany) at \u0026times;630 magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA-binding effect of the endolysins\u003c/h2\u003e \u003cp\u003eTo detect interaction between eDNA and the endolysins we obtained genomic DNA of \u003cem\u003eA. baumannii\u003c/em\u003e using a modified CTAB method (Minas et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Briefly, cells from an overnight bacterial culture were harvested, once washed, and solubilized in a CTAB solution (2% w/v cetrimonium bromide (Helicon, Moscow, Russia), 100 mM Tris-HCl, 20 mM EDTA and 1.4 M NaCl, pH\u0026thinsp;=\u0026thinsp;8.0), \u003cem\u003eex tempore\u003c/em\u003e supplemented with 0.2% β-mercaptoethanol, at 65\u0026deg;C for 40 min. Then, the suspensions were mixed with an equal volume of chloroform-isoamyl alcohol (24:1), and the aqueous phase was collected after centrifugation at 12,000\u0026times;g for 10 min, RT. The DNA precipitation proceeded in 0.6 volume of isopropanol overnight at \u0026ndash; 25\u0026deg;C. The precipitates were harvested at 8,000\u0026times;g for 15 min and washed three times with 80% ethanol. After ethanol was discarded, the pellets were suspended in 20 mM Tris-HCl (pH\u0026thinsp;=\u0026thinsp;7.5) buffer with 1 \u0026micro;g/mL RNAse A (PanReac Applichem, Darmstadt, Germany), incubated for 15 min at 37\u0026deg;C, afterwards RNAse A was inactivated at 65\u0026deg;C for 15 min. Quality of the DNA samples was estimated spectrophotometrically, analyzing A260/230 and A260/280, thereafter a concentration of DNA was measured using Qubit DNA HS Assay Kit and Qubit 3.0 fluorometer (Thermo Fisher Scientific Eugene, Oregon, U.S.).\u003c/p\u003e \u003cp\u003eEndolysins LysAm24, LysAp22, LysECD7, and LysSi3 solutions in 20 mM Tris-HCl buffer at 10 or 50 ng/\u0026micro;L concentration were mixed with DNA solutions (at a concentration of 20 ng/\u0026micro;L) at a protein:DNA (P:DNA) mass ratio of 2:1 or 10:1 (final volume of the mixtures was 7.5 \u0026micro;L) and incubated for 30 min at RT. Solutions containing either DNA or investigated endolysin in 20 mM Tris-HCl buffer (pH\u0026thinsp;=\u0026thinsp;7.5) were used as control. The influence of electrostatic interactions was assessed by adding NaCl to final concentrations of 150 or 300 mM to the mixtures (P:DNA w/w ratio of 10:1) during their incubation. Post-incubated solutions were mixed with the 6X DNA Loading Dye (Thermo Fisher Scientific Eugene, Oregon, U.S.) and loaded in 1% agarose gel, containing 0.2 \u0026micro;g/mL ethidium bromide (Helicon, Moscow, Russia). An electrophoretic separation of all samples was performed in the Tris-borate buffer (50 mM Tris-base, 50 mM boric acid, 2 mM EDTA, pH\u0026thinsp;=\u0026thinsp;8.3) at 80 V for an hour. Imaging was performed using the Gel Doc EZ gel documentation system (Bio-Rad, Hercules, CA, U.S.).\u003c/p\u003e \u003cp\u003eTo correlate the binding assay with the inhibitory effect of eDNA, an antibacterial activity test was performed. An overnight bacterial culture of \u003cem\u003eA. baumannii\u003c/em\u003e was diluted 30-fold in LB broth and grown to the exponential phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6). Subsequently, the cells were harvested by centrifugation (6,000\u0026times;g, 10 min) and resuspended in the same volume of PBS (pH\u0026thinsp;=\u0026thinsp;7.4). Each suspension was diluted in the 20 mM Tris-HCl (pH\u0026thinsp;=\u0026thinsp;7.5) to a final density of approximately 10\u003csup\u003e6\u003c/sup\u003e cells/mL. Afterward, 100 \u0026micro;L of the bacterial suspensions and 100 \u0026micro;L of the pre-incubated protein-DNA solutions (a final protein concentration was 10 \u0026micro;g/mL, a final DNA concentration was 1 or 5 \u0026micro;g/mL) were mixed in 96-well plates. Buffers with or without DNA were used as the negative controls. The mixtures were incubated at 37\u0026deg;C for 30 min with shaking at 200 rpm and then were diluted 10-fold in PBS (pH\u0026thinsp;=\u0026thinsp;7.4). Then, 100 \u0026micro;l of each dilution were plated onto LB agar, and the number of bacterial colonies were counted after an overnight incubation at 37\u0026deg;C. All experiments were performed in quadruplicate, and the antibacterial activity was expressed as follows: Antibacterial activity (%)\u0026thinsp;=\u0026thinsp;100% \u0026ndash; (CFUexp/CFUcont) \u0026times; 100%, where CFUexp is the number of bacterial colonies in the experimental culture plates, and CFUcont is the mean number of bacterial colonies in the control culture plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed and illustrated using GraphPad Prism 9.0 software. According to the results of normality tests (Kolmogorov-Smirnov\u0026rsquo;s method), data sets were compared using appropriate statistical tests with corrections for multiple comparisons (detailed information on the chosen analysis methods is provided in captions and results).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExopolysaccharides content\u003c/h2\u003e \u003cp\u003eTo visualize different EPSs, various staining procedures of bacterial BF-macrocolonies on nutritious media with glucose are often implemented. Cultivation of bacterial macrocolonies on solid medium, containing Congo red (CR) dye is an express test system of biofilm formation properties. CR interacts with most β-folded proteins, glucans with β (1-\u0026gt;4) and β (1-\u0026gt;3) bonds, and with various polycationic polysaccharides (Puchtler et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). During the 48-h incubation on CR agar, the investigated bacterial colonies accumulated small amounts of the stain and slightly decolorized the medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the second row). The \u003cem\u003eK. pneumoniae\u003c/em\u003e originated macrocolonies with a crystalline red center and sharp, clear edges, while \u003cem\u003eA. baumannii\u003c/em\u003e formed homogeneous brown and rough colonies indicating production of moderate quantity of polysaccharides and amyloid proteins. Dual-species colonies resembled those of \u003cem\u003eK. pneumoniae\u003c/em\u003e, with a rougher structure and partially colored periphery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA different matrix architecture was observed by staining with polycationic dye Alcian blue, revealing carboxyl-rich polysaccharides (Scott and Dorling \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1965\u003c/span\u003e), located on the air-contacting exterior of formed macrocolonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the third row). The colonies of \u003cem\u003eK. pneumoniae\u003c/em\u003e had a concentric staining, significantly more intense compared to \u003cem\u003eA. baumannii\u003c/em\u003e, while the dual-species colonies possessed well resolved dark-blue strands radiating from the center, revealing the conglomerated polycationic and polyanionic polysaccharides\u0026rsquo; plots.\u003c/p\u003e \u003cp\u003eTo investigate the coexistence of bacteria in mixed biofilms the morphotyping assay with culture media containing Coomassie brilliant blue dye (CBB) was done. CBB stains biofilm-associated proteins, and Congo red dye, binding with both proteins and polysaccharides (Nesse et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The significant heterogeneity of biofilm composition was observed for dual-species colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and Online Resource Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) combining phenotypic traits of both species and characterized by increased cell motility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of eDNA\u003c/h2\u003e \u003cp\u003eThe biomass density of either \u003cem\u003eK. pneumoniae\u003c/em\u003e or \u003cem\u003eA. baumannii\u003c/em\u003e 24-h old biofilms pretreated with DNAse I, was reduced by 20% on average (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while the enzymatic treatment of 48-h biofilms led to a decline in the biofilm biomass by 30.9% and 25.9% respectively. Neither the increase in DNAse I concentration (up to tenfold) nor the time of biofilm formation led to a significant reduction in the BFs\u0026rsquo; biomass (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, two-way ANOVA with Tukey\u0026rsquo;s correction).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of proteins within biofilms\u003c/h2\u003e \u003cp\u003eThe presence of fibrillar proteins, pili or pilus-like structures within examined biofilms of 24- and 48-h macrocolonies of \u003cem\u003eA. baumannii\u003c/em\u003e (brown staining) and \u003cem\u003eK. pneumoniae\u003c/em\u003e (red staining) was demonstrated in CRCB-TSB assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. b). To quantify the approximate content of proteins within the matrix of biofilms, treatment with Proteinase K (PK) was used. It was shown that biofilms of investigated strains, particularly of \u003cem\u003eA. baumannii\u003c/em\u003e, were significantly resistant to proteolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No effective concentration of PK was found to disrupt biofilms of \u003cem\u003eA. baumannii\u003c/em\u003e, where the addition of 800 \u0026micro;g/mL of PK resulted in a negligible loss of biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Thereby, proteins make up relatively small fraction of the \u003cem\u003eA. baumannii\u003c/em\u003e biofilm matrix (less than 10\u0026ndash;15%), although some EPS proteins might evade a proteolysis, for instance, due to aggregation in fibers, O-glycosylation, or their covering by other compounds of the matrix (Iwashkiw et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwenty-four hours old biofilms formed by \u003cem\u003eK. pneumoniae\u003c/em\u003e also remained recalcitrant to the activity of PK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), that apparently correlated with the results of morphotyping, where 24-h old macrocolonies of \u003cem\u003eK. pneumoniae\u003c/em\u003e had no amyloid content (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, the first row). On the contrary, biofilms of \u003cem\u003eK. pneumoniae\u003c/em\u003e, grown for 48 hours, were susceptible to the action of Proteinase K, in a dose-dependent manner in concentrations range 50 to 1600 \u0026micro;g/mL of PK, resulting in the loss up to 45% of biomass at 1600 \u0026micro;g/mL. Notably, simultaneous treatment with DNAse and PK did not lead to a synergistic decline in biomass of the investigated biofilms (Online Resource Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which indicates tough stability of these BFs, provided by multiple contacts between various EPSs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eActivity of the endolysins against 48-h biofilms\u003c/h2\u003e \u003cp\u003eA pronounced reduction in biomass of 48-h old BFs was observed after 2-h incubation with endolysins as shown by the CV Mtp assay for \u003cem\u003eA. baumannii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), \u003cem\u003eK. pneumoniae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) or mixed biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), which all formed moderate or strong BFs on the polystyrene. Depending on the biofilm-forming bacteria, endolysins acted mostly in a dose-dependent manner. Each protein led to at least a 2-fold loss in the biomass of preformed biofilms, in particular, LysAp22 possessed the strongest disruption of BFs, whilst antibiofilm activity on EPS-rich biofilms by LysSi3 was less evident than that of other endolysins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExposure to a 1000 \u0026micro;g/mL of LysAp22 solution decreased the biofilms\u0026rsquo; biomass up to 33%, 25% and 27% on average; thus \u003cem\u003eK. pneumoniae-\u003c/em\u003erelated BFs were almost eliminated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Two endolysins, LysECD7 and LysAm24, acted similarly \u003cem\u003eversus\u003c/em\u003e mono-species BFs, albeit the impact of LysECD7 on dual-species BFs was comparable to that of LysAp22. The 2-h treatment with LysAm24 or LysECD7 at a concentration of 1000 \u0026micro;g/mL led to a decrease up to approximately 41%, 35% and 27% of \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e or mixed biofilms\u0026rsquo; biomass respectively, compared to the controls treated with the buffer. At 100 \u0026micro;g/mL, these proteins exhibited a lower activity. Except for LysSi3, endolysins at a higher concentration removed dual-species biofilms better than low-EPS containing \u003cem\u003eA. baumannii\u003c/em\u003e BFs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Mann-Whitney tests with Holm-Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s correction). LysSi3 acted on mixed biofilms worse than other endolysins, disrupting the biomass by approximately 54%, independently on dose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eExopolysaccharides and biomass staining of biofilms pretreated with endolysins\u003c/h2\u003e \u003cp\u003eAs demonstrated, three-dimensional biofilms were formed after a 48-h incubation of \u003cem\u003eA. baumannii\u003c/em\u003e cells in TSB media on glass slides (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These aggregates consisted of dense and tightly connected structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), which were disrupted by endolysins to incoherent monolayers (BFs treated with LysAm24, LysAp22, LysSi3), or microcolonies (LysECD7). After destaining with CuSO\u003csub\u003e4\u003c/sub\u003e, light-blue color of the biofilm\u0026rsquo;s structures indicated the presence of acid, mostly capsular carbohydrates. Otherwise, if there is a lack of these EPSs, the biomass was colored violet. Thus, it allows to distinguish a significant number of cells covered with capsular polysaccharides and also the heterogeneous arrangement of different EPSs, especially on the BF edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026rsquo;). Biofilms treated with LysAp22 or LysECD7 were poorly stained by CV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and d), except for a few areas with dark violet peaks, and did not differ from the cells, additionally rinsed with CuSO\u003csub\u003e4\u003c/sub\u003e, suggesting interactions with carbohydrates, subsequent dissociation, and destabilization of biofilms\u0026rsquo; matrix. The BF-disrupting effect of LysSi3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and e\u0026rsquo;) appeared to be similar to that of LysECD7, however, it was less pronounced, and did not cause evident destabilization of exopolysaccharides, but also efficiently passed through EPSs. A different manner of endolysin-biofilm interactions was observed for LysAm24 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and b\u0026rsquo;). Preparations treated with this endolysin and stained with CV possessed an intense violet color, whereas double-stained biofilms apparently lacked any purple areas that demonstrate the interactions between LysAm24 and EPS, other than acid carbohydrates, providing additional sites for CV staining.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffects of bacterial DNA on endolysins\u003c/h2\u003e \u003cp\u003eWe estimated that in abundance of endolysins (10:1 ratio), the DNA samples incubated with three out of 4 investigated endolysins displayed a notable decline in the electrophoretic mobility of nucleic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), suggesting the complexes\u0026rsquo; formation of endolysin with genomic DNA, which remained in the loading well or formed significant smears. Among the examined proteins, LysAm24 and LysECD7 exhibited the most pronounced DNA-binding activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). However, with the exception of LysAm24, endolysin-eDNA complexes were not detected when the ratio of P:DNA was 2:1. Only LysSi3 showed no interactions with DNA samples in this test.\u003c/p\u003e \u003cp\u003eExogenous nucleic acids led to the inhibition of muramidases\u0026rsquo; activity versus free-living \u003cem\u003eA. baumannii\u003c/em\u003e cells. At the lower concentration (10:1 ratio), DNA did not impact antibacterial properties of these proteins, whereas it dramatically inhibited them at the ratio of 2:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). At the same time, endopeptidase LysECD7 remained effective against the bacteria even in the excess of DNA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eClinically relevant biofilms, formed by \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e species or their combination, are associated with a variety of skin and soft tissue infections, as well as nosocomial infections of the respiratory (Said et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and urinary tract (Paczosa and Mecsas \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Because of producing a heterogeneous and thick matrix, these causative agents possess an extreme tolerance towards various antimicrobials (Stewart \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Herein, we characterized the content of biofilms formed by two bacterial isolates and estimated that 48-h \u003cem\u003eA. baumannii\u003c/em\u003e BFs contained a moderate amount of highly deacetylated glycosaminoglycans, acid polysaccharides, eDNA, and a relatively large fraction of the cellular biomass (Online Resource Table\u0026nbsp;1). For \u003cem\u003eK. pneumoniae\u003c/em\u003e, the BF matrix makes up at least half of the dry biofilm\u0026rsquo;s volume, with a pronounced content of negatively charged polysaccharides, DNA, and non-amyloid proteins. Despite the difference in composition, we have shown that investigated enzymes disrupt heterogeneous biofilms, interacting directly with different BF compounds. All of the endolysins at a concentration of 100 \u0026micro;g/mL led to a decrease in biomass of mono- and dual-species films of Gram-negative bacteria by at least 2\u0026ndash;3 times. Moreover, treatment with LysAm24, LysAp22, or LysECD7 at a concentration of 1000 \u0026micro;g/mL was linked to the almost complete removal of the biofilms.\u003c/p\u003e \u003cp\u003eAlthough most endolysins are considered highly specific hydrolases, disrupting the peptidoglycan at specific sites, a side ability to influence compounds of the biofilm matrix could be recognized. Thus, we detected weak interactions between endolysins and exopolysaccharides and eDNA. These interactions, however, were associated with a significant destabilization of the treated biofilm and could be beneficial in terms of bacteriophage ecology. Endolysin-EPS interactions may include effects on adhesion of competitive viruses, availability of phages\u0026rsquo; receptors, and formation of cell-wall deficient cells to sustain the host population (Wohlfarth et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, lysins can be engaged in BF-specific signaling pathways due to the products of peptidoglycan hydrolysis (Irazoki et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Extracellular carbohydrates are a significant component involved in biofilm formation, crucial for stable cell adhesion on various surfaces, and establish multiple contacts with other exogenous molecules (Ostapska et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). They include enterobacterial cellulose (Nesse et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and poly-β-1,6-N-acetyl-D-glucosamine (PNAG), found in numerous species of bacteria (Choi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Capsular polysaccharides, frequently consisting of various negatively and neutrally charged subunits, serve as a physical shield against environmental factors (Paczosa and Mecsas \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and within BFs modulate surface adhesion (Mann and Wozniak \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pompilio et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, massive detachment of acid exopolysaccharides by LysAp22 and LysECD7 may impair BF stability, without relevant loss of the antibacterial activity, and subsequently seems to be an intriguing feature for further development of enzybiotics. The exact mechanism of this change is not fully understood. However, it is unlikely to be related to capsule digestion by specific phage depolymerases. In particular, their active sites differ from those of endolysins and structures of the investigated lysins possess no significant similarity with any of this wide group of enzymes. It is worth noting, that exopolysaccharides can trigger changes in the catalytic activity of other enzymes, more related to endolysins. For instance, a polyanionic exopolysaccharide of \u003cem\u003eXantamonas\u003c/em\u003e spp. inhibits egg white lysozyme and lysostaphin, but activates two bacterial muramidases (Stepnaya et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of the eDNA-endolysin binding assay partly correlated with the isoelectric points (pIs) of the lysins: LysAp22 (pI 9.19), LysAm24 (pI 8.89), LysECD7 (pI 8.83) and LysSi3 (pI 8.52), proposing a higher probability of binding with an increase of pI. Hence, unspecified electrostatic forces may determine the interactions between the endolysins and eDNA. However, the differences in pIs between the studied proteins are small; thereby an actual net charge distribution of molecules may be distinct, affecting the binding capacity. Anyway, as shown in Online Resource Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the strength of the endolysin-DNA association was significantly reduced by the addition of NaCl. Thus, LysECD7 partially ceased to bind with DNA in the mixtures, containing 150 and 300 mM NaCl, LysAm24 showed a similar tendency at the lower salt\u0026rsquo;s concentration, whereas the DNA-binding effect of LysAp22 was evident in the mixture without NaCl. Therefore, the interplay observed between eDNA and the investigated enzymes should be considered nonspecific, and primarily driven by electrostatic binding. Strong electrostatic interactions are suggested to destabilize eDNA in the cases of LysECD7 and possibly LysAm24, serving as a mechanism for the antibiofilm effect of these enzymes within BF areas depleted in free cations, formed by eDNA. LysSi3 is believed to have a reduced antibiofilm activity due to the indirect influence of exogenous nucleic acids, while eDNA affected LysAp22 ambivalently. We suggest that DNA mixed with a bacterial suspension can bind positively charged compounds on the outer membrane (OM) of bacteria and enhance their surface hydrophobicity (Das et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), impairing CW accessibility for either of hydrolases. It is interesting to note, that the investigated endolysins contain positively charged terminal structures putatively involved in permeabilization of membranes, except for LysECD7. The mechanism of passing through OM for LysECD7 is probably based on other, not only electrostatic interactions with a cell\u0026rsquo;s surface (Antonova et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere is plenty of findings noted extracellular DNA can be a component of biofilm tolerance exhibited by different species against charged antimicrobial peptides (Batoni et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and proteins, as well as aminoglycosides (Chiang et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), fluoroquinolones (Tetz et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and non-phage derived lysozymes. In this regard, eDNA interaction with positively charged structures often found in endolysins\u0026rsquo; molecules may also affect their catalytic activity and access to target sites, hampering endolysin-based therapy. On the other hand, enzybiotics may destabilize exposed biofilms through unspecific interactions with extracellular nucleic acids, allowing biofilm control. For example, side antibacterial effects have been reported for egg lysozyme and antistaphylococcal chimeric peptidoglycan hydrolase (Fern\u0026aacute;ndez et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It has been proposed to be based on strong endolysin-DNA binding, that influence the expression of bacterial genes. Another interesting mechanism of biofilm removal by targeting defensive BF compounds has been reviewed for a few AMPs, which possess similarity with semi-conservative structures of the studied proteins (Batoni et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, our study reveals contrasting aspects in the antibiofilm activity of the phage endolysins, which deviated from bacterial cell lysis caused by catalytic digestion of the CW peptidoglycan. Specifically, we detected the interaction with the carbohydrate component of the matrix (LysAp22 and LysECD7), as well as the electrostatic binding of polycationic sites of endolysins to eDNA (LysAm24 and LysECD7). These interactions result in the biofilm removal by endolysins, despite a decrease in their initial antibacterial activity. However, this research did not evaluate the influence of abnormal lysin-induced release of various bacterial compounds on the stability of BFs, which could also be compelling. Additional experiments should be conducted to clarify the endolysin-exoproteome interactions (De Gregorio et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), whilst details in endolysin-exopolysaccharides association could be investigated through a mass-spectrometric structural analysis accompanied by a design of possible binding \u003cem\u003ein silico\u003c/em\u003e. Treatment of biofilms with the cell-free lysate or products of the peptidoglycan hydrolysis could also provide additional information. Thus, the present and further findings are expected to highlight a complex mechanism underlying the endolysins\u0026rsquo; antibiofilm activity, allowing them to successfully evade a biofilm\u0026rsquo;s defense, provided by its matrix.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available within the paper and its Supplementary Information. Additionally, the raw data are available from the authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings:\u0026nbsp;\u003c/strong\u003eThe study was supported by the Russian Science Foundation (RSF), grant № 23-74-10027, https://rscf.ru/project/23-74-10027/.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;\u003c/strong\u003eConceptualization:\u0026nbsp;Daria\u0026nbsp;V.\u0026nbsp;Vasina; Methodology:\u0026nbsp;Anastasiya M. Lendel; Formal analysis and investigation:\u0026nbsp;Anastasiya\u0026nbsp;M.\u0026nbsp;Lendel, Nataliia\u0026nbsp;P.\u0026nbsp;Antonova, Igor\u0026nbsp;V.\u0026nbsp;Grigoriev, Evgeny\u0026nbsp;V.\u0026nbsp;Usachev; Writing - original draft preparation:\u0026nbsp;Anastasiya\u0026nbsp;M.\u0026nbsp;Lendel; Writing - review and editing:\u0026nbsp;Nataliia P. Antonova, Igor V. Grigoriev, Daria V. Vasina; Funding acquisition:\u0026nbsp;Daria\u0026nbsp;V.\u0026nbsp;Vasina; Resources and project administration:\u0026nbsp;Vladimir\u0026nbsp;A.\u0026nbsp;Gushchin; Supervision:\u0026nbsp;Daria\u0026nbsp;V.\u0026nbsp;Vasina. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e: Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad, I., Nygren, E., Khalid, F., Myint, S.L., Uhlin, B.E., 2020. A Cyclic-di-GMP signaling network regulates biofilm formation and surface associated motility of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e 17978. Sci. Rep. 10, 1\u0026ndash;11. https://doi.org/10.1038/s41598-020-58522-5\u003c/li\u003e\n\u003cli\u003eAntonova, N.P., Vasina, D.V., Lendel, A.M., Usachev, E.V., Makarov, V.V., Gintsburg, A.L., Tkachuk, A.P., Gushchin, V.A., 2019. Broad Bactericidal Activity of the Myoviridae Bacteriophage Lysins LysAm24, LysECD7, and LysSi3 against Gram-Negative ESKAPE Pathogens. 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Microbiol. 8, 387\u0026ndash;399. https://doi.org/10.1038/s41564-022-01317-3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"antibiofilm activity, biofilm, endolysin, exopolysaccharides, extracellular DNA","lastPublishedDoi":"10.21203/rs.3.rs-3899892/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3899892/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without the subsequent emergence of endolysin-resistant genotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins’ effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a 2-fold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3.","manuscriptTitle":"Biofilm-disrupting effects of phage endolysins LysAm24, LysAp22, LysECD7, and LysSi3: breakdown the matrix","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-02 06:01:59","doi":"10.21203/rs.3.rs-3899892/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-14T14:12:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-14T13:26:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-22T14:55:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a6e2a618-b5e0-45d3-ac2b-59e2580ffc9c","date":"2024-02-09T15:46:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-01T10:08:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-31T18:59:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-31T13:24:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2024-01-26T12:07:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0aaed329-05d1-4b0a-aaba-edd879e32479","owner":[],"postedDate":"February 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T23:28:13+00:00","versionOfRecord":{"articleIdentity":"rs-3899892","link":"https://doi.org/10.1007/s11274-024-03999-9","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2024-04-29 23:28:13","publishedOnDateReadable":"April 29th, 2024"},"versionCreatedAt":"2024-02-02 06:01:59","video":"","vorDoi":"10.1007/s11274-024-03999-9","vorDoiUrl":"https://doi.org/10.1007/s11274-024-03999-9","workflowStages":[]},"version":"v1","identity":"rs-3899892","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3899892","identity":"rs-3899892","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}