Recombinant NK1 Protein and LEDs: An Innovative Strategy to Counteract Resistant Staphylococcus pseudintermedius and Pseudomonas aeruginosa Strains

TL;DR

This paper proposes a new non-antibiotic strategy using recombinant NK1 protein and LEDs to combat drug-resistant bacteria like Staphylococcus pseudintermedius and Pseudomonas aeruginosa.

Contribution

The novel approach combines recombinant NK1 protein and LEDs to effectively reduce bacterial growth and virulence in resistant strains.

Findings

Combining NK1 and LEDs reduced CFU/ml by up to 85.98% after 24 hours.

The treatment reduced P. aeruginosa motility and biofilm formation by up to 60%.

The strategy showed significant antimicrobial and anti-virulence effects on both planktonic and sessile bacterial phases.

Abstract

The increase in multi-drug-resistant strains represents a global challenge that strongly underlines the importance of the search for new eco-sustainable strategies. The aim of this work was to suggest a non-antibiotic approach to counteract resistant Staphylococcus pseudintermedius and Pseudomonas aeruginosa strains in planktonic and sessile phases. The proposed strategy includes the combination of natural spliced variant of hepatocyte growth factor NK1, a protein produced by recombinant DNA technology in Pichia pastoris expression system and Light-Emitting Diodes (LEDs). The antimicrobial action was determined by Minimum Inhibitory Concentration, Minimum Bactericidal Concentration, and CFU/ml evaluations. The anti-virulence action was performed by measuring P. aeruginosa motility and twitching and anti-S. pseudintermedius and -P. aeruginosa biofilms. Recombinant NK1 and LEDs alone and combined with each other showed relevant antimicrobial and anti-virulence effects. In particular, when NK1 was combined with LEDs, significant CFU/ml reduction (up to 85.98% reduction with respect to the control) after 24 h of contact, P. aeruginosa swimming/swarming/twitching halos reduction (up to 60% reduction with respect to the control) and bacterial anti-biofilm formation action was obtained. The obtained results demonstrated the innovative and interesting non-antibiotic strategy formed by recombinant NK1 protein and LEDs to affect the S. pseudintermedius and P. aeruginosa growth.

The online version contains supplementary material available at 10.1007/s12602-025-10702-3.

Figures

S). When the biofilms were treated with the combination of NK1 and LEDs, major dead cells were observed with respect to the other tested conditions. Although some red blood cells were detected in the *S. pseudintermedius* control group, the amount was not significant and did not affect the comparison of the effects of experimental treatments on biofilm viability.Fig. 6Representative CLSM images of *S. pseudintermedius* and *P. aeruginosa* biofilm formation in the presence of NK1 and LEDs alone and combined with each other. The viable cells are green (SYTO 9) and dead cells are red (propidium i

Inhibition of biofilm formation by using different concentrations of NK1 and LEDs alone and combined with each other on *S. pseudintermedius* 115 and *P. aeruginosa* 700 in terms of detected CFU/ml. *Statistically significant (*p* < 0.05) with respect to the control. Statistically significant (*p* < 0.05)

Anti-biofilm effect of different concentrations of NK1 and LEDs alone and combined with each other on *S. pseudintermedius* 115 (a) and *P. aeruginosa* 700 (b) mature biofilms in terms of detected CFU/ml. *Statistically significant (*p* < 0.05) with respect to the control. Statistically significant (*p* < 0.05)

Full text

Introduction

Nowadays, the increase of multi-drug-resistant (MDR) strains represents a global threat reducing the ability to treat microbial infections. The MDR strains survive, multiply, and spread in the presence of drugs. As a result of Antimicrobial Resistance (AMR), antibiotics and other antimicrobials became ineffective, and infections are difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death [1, 2]. Different studies demonstrate that farm animals are a source of MDR strains such as Salmonella, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and intimate contact with them is responsible for AMR transmission to humans [3, 4]. In industrialized countries, the relationship between companion animals and humans changed, leading to a higher risk of infections and the cross-transmission of AMR traits [5]. Thus, the potential of reverse zoonosis, along with the creation of animal reservoirs that keep the loop of infection and AMR diffusion is, nowadays, gaining a steadily increasing concern [6]. The AMR phenomenon in companion animals involves principally methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-resistant staphylococci, vancomycin-resistant enterococci, carbapenemase-producing enterobacteria, extended spectrum beta-lactamase (ESBL) Gram-negative bacteria, and Pseudomonas aeruginosa [7]. Staphylococcus pseudintermedius, a coagulase-positive staphylococcal species, is the main opportunistic bacterium of canine skin microbiota and can cause several infections, including pyoderma, otitis, abscesses, urinary tract infections (UTIs), and wound infections in dogs and in cats [8]. The β-lactam resistance in MRSP is attributed to penicillin-binding protein 2a (PBP2a), a protein encoded by the methicillin-resistant gene mecA. In addition, another important virulence factor in MRSP is the ability to form biofilm, as it allows the bacterium to adhere to target tissues [9]. In chronic infections, S. pseudintermedius is often associated with P. aeruginosa, especially in infections of hospitalized or vet-visiting companion animals (i.e., dogs, cats and horses), as well as fecal colonization and environmental sites across veterinary clinics/hospitals [8]. de Sousa et al. demonstrated that the increase of MDR P. aeruginosa strains is a global challenge due to several factors, including low outer membrane permeability, the production and expression of the chromosomal AmpCβ-lactamase, and the presence of numerous genes coding for multidrug resistance efflux pumps [10]. The AMR problem is more emphasized when the microorganisms co-exist and co-aggregate each other in the biofilm that increases the tolerance against the conventional treatments. To overcome AMR and tolerance/resistance, alternative and innovative solutions are necessary. Different studies demonstrated the antimicrobial and anti-virulence effects of novel technologies such as Light-Emitting Diodes (LEDs) alone and combined with non-antibiotic compounds against Gram-positive and -negative bacteria [11]. Moreover, LEDs are recognized as eco-sustainable innovative antimicrobials for their manufacturing, transport, and disposal, respecting each product cycle. Antimicrobial action is related to their capability to excite endogenous photosensitive compounds, e.g., porphyrins, present in the bacterial cells, causing the production of reactive oxygen species (ROS) such as hydroxyl radicals, hydrogen peroxide, and singlet oxygen. ROS further react with cellular components, causing cell death [11]. Among the developed antibacterial therapeutic approaches to fight AMR, drugs such as peptides, small molecules targeting bacterial cell membranes, have shown great interest [12]. Perturbation of membrane assembly and/or integrity represents a valid strategy to develop antimicrobials able to overcome intrinsic, spontaneous, and acquired antibiotic resistance in both Gram-positive and -negative bacteria [13–16]. These peptides and small molecules, in addition to their primary action of targeting bacterial membranes, can also act through other secondary mechanisms by interfering with biofilm formation and downregulating virulence genes. Moreover, we focused our studies on the impact of a natural spliced variant of hepatocyte growth factor (HGF), NK1, produced by recombinant DNA technology in Pichia pastoris expression system on some pathophysiological conditions [17–20]. The NK1 protein, consisting of HGF amino-terminal (N) sequence and the first kringle (K1) domain, has the ability to bind, with high affinity, heparan sulfate (HS), and dermatan sulfate (DS) glycosaminoglycans [21]. In all types of cells, the glycosaminoglycan HS is covalently bound to proteoglycans (PG), forming the so-called heparan sulfate proteoglycans (HSPG), which act as intermediaries between the extracellular matrix and intracellular signaling pathways, and regulates a wide range of physio-pathological processes, including cell proliferation, motility, adhesion, apoptosis, wound healing, inflammation, and many others [22, 23]. The biological activity of HSPGs is mainly ascribed to their negatively charged HS side chains that interact with a wide spectrum of protein ligands, thus exerting essential functions in cellular homeostasis as well as in disease [24–27]. The ability to interact with HS side chains of HSPG is widespread among microbial pathogens, including Gram-positive and -negative bacteria, viruses, and parasites [27–30]. These pathogens exploit HSPG on the host cell surface for their attachment, subsequent entry, cell–cell transmission, dissemination, and evasion from host defense mechanisms, thus suggesting that HSPG-pathogen interactions are potential targets for innovative therapeutic approaches for infectious diseases. Indeed, drugs targeting HS side chains of HSPG have been shown to be effective in preventing entry and spread into host cells of different types of bacteria [27–32]. The capability of NK1 to bind HS chains of PG with high affinity [17] enables the recombinant protein to interfere with cellular signaling and related cellular processes regulated by HSPG, thus making it a valuable drug candidate for the treatment of diseases involving HSPG [25, 26, 33]. These findings prompted us to explore the potential antimicrobial activity of recombinant NK1 protein to develop a novel therapeutic approach to counteract AMR. However, the direct impact of NK1, HS binding molecules, on the bacterial structure, virulence factors, and biofilm has never been investigated.

On these bases, the aim of this work was to suggest an eco-sustainable strategy by using NK1 alone and combined with novel technologies (LEDs) to counteract MRSP and MDR P. aeruginosa associated with persistent zoonotic infection that can be transmitted to humans.

Materials and Methods

Synthesis and Purification of Recombinant NK1 Protein

Engineered Pichia pastoris GS115 yeast strain expressing recombinant NK1 available to us [17–20, 25–28, 33] was grown at 30 °C under stirring for 72 h in BMMY medium supplemented with 0.5% methanol (Fisher Scientific, Milan, Italy) every day for the induction of NK1 expression. The recombinant NK1 protein expressed and secreted into the culture medium was purified by affinity chromatography with a heparin column (GE Healthcare). The peak corresponding to NK1 was collected, and the fractions containing the recombinant protein were analyzed by SDS-PAGE. The fractions were further purified by molecular exclusion chromatography G200. Finally, the purified protein was concentrated up to about 1 × 10^−4^ M, dialyzed into 20 mM phosphate buffer (PBS), pH 7.4, and stored at – 20 °C. The purity of recombinant NK1 was evaluated by SDS-PAGE electrophoresis. Protein concentration was assessed by the Bradford method. For the experiments, the used concentration of NK1 ranged from 5 × 10^−5^ to 0.09 10^−5^ M.

Light-Emitting Diodes (LEDs) Device

LED device (TL-06) characterized by 630 nm ± 10 nm FHWM nm-wavelength was used as light source (Alpha strumenti, Italy). The handpiece was constituted by twelve LEDs in a rectangular arrangement (3 × 4 LEDs) at the exit and a surface irradiance of 380 mW/cm^2^. The device emitting light was applied for 17 min [11] and the irradiation was performed by maintaining the light sources stationary in a perpendicular position and at 5 cm from the samples. The exposure time of 17 min is the tested time in the previous work [11] and it showed the best compromise between irradiation time, antimicrobial activity*,* and in vivo application.

Microbial Strains

Staphylococcus pseudintermedius 115 and P. aeruginosa 700 clinical strains were collected in the Microbiology Laboratory of the Department of Veterinary Medicine and Animal Production, University of Naples “Federico II,” Italy. The strains were obtained from dogs presenting with cutaneous disorders at the Veterinary University Teaching Hospital of Naples, where skin samples were collected for bacteriological analysis and antimicrobial susceptibility testing. The strains were characterized for their main virulence factors and antimicrobial resistance profile. The selected bacterial isolates were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Germany) according to the manufacturer’s instructions [9]. For the experiments, the strains were standardized to Optical Density at 600 nm (OD_600_) = 0.125 (~ 10^7^ CFU/ml).

Antimicrobial Action

The antimicrobial action of NK1 and LEDs alone and combined with each other was determined by Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and CFU/ml determination. The LEDs were applied for 17 min. The NK1 MIC and MBC values were carried out according to the CLSI guidelines by the microdilution method [34]. MICs were measured by determining the lowest concentration of NK1 able to inhibit microbial visible growth. MBCs were determined by sub-culturing 10 μl of suspensions from the MICs on Mueller Hinton agar (MHA, OXOID, Milan, Italy). For CFU/ml enumerations, after 17 min (to evaluate the immediately effect after LED irradiation) and 24 h (to evaluate the effect of longer exposure) of contact time with NK1 and LEDs alone and combined with each other, several dilutions were performed, and S. pseudintermedius was spread on Mannitol Salt Agar (MSA, Oxoid, Milan, Italy) and P. aeruginosa was spread on Cetrimide Agar (CET, Oxoid, Milan, Italy). The tested NK1 concentrations ranged from 5 × 10^−5^ M to 0.09 × 10^−5^ M. The plates were incubated in aerobic conditions at 37 °C for 24–48 h.

Antivirulence Action

The NK1 and LEDs, alone and combined with each other, antivirulence effects were determined by P. aeruginosa motility and twitching and S. pseudintermedius and P. aeruginosa anti-biofilms.

Effect on P. aeruginosa Motility and Twitching

The NK1 and LEDs (applied for 17 min) alone and combined with each other were evaluated for their capability to interfere with P. aeruginosa motility by swimming, swarming motility, and twitching [35]. Briefly, for the swimming motility, the standardized cultures were inoculated in the presence of NK1 and/or LEDs at the center of soft agar plates composed of 1% tryptone (Sigma Aldrich, Milan, Italy), 0.5% NaCl (Sigma Aldrich, Milan, Italy), and 0.3% agar (Sigma Aldrich, Milan, Italy). As the control group, the untreated sample was inoculated. For the swarming motility, the standardized cultures were inoculated in the presence of NK1 and/or LEDs at the center of soft agar plates containing 1% peptone (Sigma Aldrich, Milan, Italy), 0.5% NaCl, 0.5% agar, and 0.5% d-glucose (Sigma Aldrich, Milan, Italy). As the control group, the untreated sample was inoculated. All plates were incubated at 37 °C for 24 h and the bacterial halos were recorded. For twitching, the standardized broth cultures were inoculated in the presence of NK1 and/or LEDs to the bottom of the twitching plates consisting of 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, and 1% agar. As the control group, the untreated sample was inoculated. The plates were incubated at 37 °C for 24 h, and then, the agar was removed, and the halo was stained with 0.1% crystal violet and measured.

Effect on S. pseudintermedius and P. aeruginosa Biofilms

The anti-biofilm effect of NK1 and LEDs (applied for 17 min) alone and combined with each other was evaluated in terms of inhibition of biofilm formation and disaggregation of mature biofilm. For biofilm formation, the standardized broth cultures were inoculated in the presence of NK1 and LEDs alone and combined with each other and incubated for 24 h in aerobic conditions at 37 °C. The LEDs were applied for 17 min. For the mature biofilms, 200 μl of standardized broth cultures were inoculated and incubated for 24 h and then treated with NK1 and LEDs (applied for 17 min) alone and combined with each other. After contact times, biomass and CFU/ml enumeration were performed.

For biomass quantification, dry biofilms were stained with 0.1% crystal violet and quantified according to Di Lodovico et al. [35]. For CFU/ml determination, after incubation, each well was washed with PBS and the adhered bacteria were scraped off and resuspended in 200 μl of PBS, transferred to test tubes, vortexed for 2 min, diluted, and spread on MSA for S. pseudintermedius and on CET for P. aeruginosa. The plates were incubated for 24 h at 37 °C. Microscopic observations with live/dead staining prior to spreading confirmed the presence of disaggregated viable cells.

The cell viability in the biofilms was also evaluated by using live/dead staining (Molecular Probes Inc., Invitrogen, San Giuliano Milanese, Italy) and observed by using Confocal Laser Scanning Microscopy (CLSM) [11].

Statistical Analysis

All data were obtained from at least three independent experiments performed in duplicate. Data was shown as the means ± standard deviation. Differences between groups were assessed with one-way analysis of variance (ANOVA). P values ≤ 0.05 were considered statistically significant.

Results

The strains identification performed by MALDI-TOF–MS provided an optimal log score (> 2.0) for S. pseudintermedius 115 and P. aeruginosa 700, and the molecular analysis further confirmed the identification of the genera and species. Furthermore, S. pseudintermedius 115 was identified as a methicillin-resistant strain (MRSP), with its phenotypic resistance to oxacillin confirmed by the detection of the mecA gene. The antimicrobial susceptibility results showed that the strain was resistant to all tested antimicrobials except for vancomycin and linezolid. Additionally, S. pseudintermedius 115 carried the tetM and ermB genes, which were responsible for resistance to tetracycline and erythromycin, respectively. P. aeruginosa 700 was resistant to aztreonam, ceftazidime, ceftriaxone, imipenem, meropenem, piperacillin-tazobactam, and sulfamethoxazole-trimethoprim. ESBL genotypic resistance was identified through the detection of blaPER, while blaVIM and blaGES were simultaneously detected for MBL genotypic resistance. Both strains exhibited concerning antimicrobial resistance profiles.

Table 1 shows NK1 MICs and MBCs against S. pseudintermedius and P. aeruginosa clinical strains. The values were more than the maximum tested NK1 concentration. The results showed no visible growth reduction in the presence of different NK1 concentrations. Table 1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of NK1 (from 5 × 10^−5^ M to 0.09 × 10^−5^ M) against S. pseudintermedius 115 and P. aeruginosa 700StrainsMICMBCS. pseudintermedius 115 > 5 × 10^−5^ M > 5 × 10^−5^ M > 5 × 10^−5^ M > 5 × 10^−5^ MP. aeruginosa 700

The antimicrobial action of NK1 and LEDs alone was evaluated by S. pseudintermedius and P. aeruginosa CFU/ml reduction after 17 min and 24 h of contact time. As shown in Fig. 1a, after 17 min of contact time, a relevant inhibition at each NK1 concentration of S. pseudintermedius growth was recorded. In fact, the CFU/ml in presence of NK1 ranged from 2.08 × 10^6^ ± 8.37 × 10^4^ to 2.96 × 10^6^ ± 1.17 × 10^5^ with respect to the CFU/ml of the control (4.12 × 10^6^ ± 8.67 × 10^5^). A relevant CFU/ml reduction was also obtained with LEDs. No relevant effect was obtained with NK1 against P. aeruginosa after 17 min (Fig. 1b). Instead, a slight reduction was obtained with LEDs (12.5% of CFU/ml reduction). After 24 h of incubation at 37 °C, except with 0.09 × 10^−6^ M of NK1, significant (p < 0.05) S. pseudintermedius CFU/ml reductions were obtained in presence with NK1 showing a remarkable antimicrobial action. Also, in presence with LEDs, a S. pseudintermedius reduction growth was obtained. Regarding P. aeruginosa at 24 h, relevant CFU/ml reductions were recorded with 6 × 10^−6^ M and 3 × 10^−6^ M of NK1 and LEDs (Fig. 1c and d).Fig. 1Staphylococcus pseudintermedius 115 (a, c) and P. aeruginosa 700 (b, d) CFU/ml in presence of different concentrations of NK1 and LEDs after 17 min (a, b) and 24 h (c, d). *Statistically significant (p < 0.05) with respect to the control. **Statistically significant (p < 0.05)

When NK1 was combined with LEDs, an enhanced antimicrobial action was detected, especially after 24 h. In fact, as shown in Fig. 2a, after 17 min of contact time, a significant (p < 0.05) antimicrobial action was detected only against S. pseudintermedius. When NK1 was combined with LEDs, the same trend obtained with the substances separately was detected. The S. pseudintermedius CFU/ml in presence of NK1 ranged from 1.84 × 10^6^ ± 1.52 × 10^5^ to 1.83 × 10^5^ ± 2.60 × 10^6^ with respect to 7.37 × 10^7^ ± 8.91 × 10^6^ CFU/ml of the control. At 24 h, relevant S. pseudintermedius and P. aeruginosa CFU/ml reductions were recorded (Fig. 2c and d). A remarkable potentiated antimicrobial action was obtained when NK1 was combined with LEDs after 24 h. At this time, the microbial growth reduction was registered for all tested strains, with CFU/ml in the presence of NK1 ranging from 1.03 × 10^7^ ± 5.77 × 10^5^ to 1.85 × 10^7^ ± 9.03 × 10^6^ and 1.56 × 10^7^ ± 4.34 × 10^6^ to 2.05 × 10^7^ ± 1.29 × 10^6^ for S. pseudintermedius and P. aeruginosa, respectively. No statistical differences were observed among all tested NK1 concentrations with LEDs showing no significant concentration-dependent differences.Fig. 2Staphylococcus pseudintermedius 115 (a, c) and P. aeruginosa 700 (b, d) CFU/ml in presence of different concentrations of NK1 combined with LEDs after 17 min (a, b) and 24 h (c, d). *Statistically significant (p < 0.05) with respect to the control. **Statistically significant (p < 0.05)

Figure 3 shows the capability of NK1 and LEDs alone and combined with each other to affect P. aeruginosa motility and twitching. NK1 significantly (p < 0.05) reduced the swimming motility by 12.5% with an incremented reduction (p < 0.05) when the protein was combined with LEDs. The combination of NK1 and LEDs also significantly (p < 0.05) affected the swarming motility. Regarding twitching, a remarkable halo reduction was obtained in the presence of all tested treatments with a significant reduction obtained with NK1 + LEDs.Fig. 3Pseudomonas aeruginosa 700 swimming, swarming motility, and twitching in presence of 10^−6^ M of NK1 and LEDs alone and combined with each other. *Statistically significant (p < 0.05) with respect to the control. ** Statistically significant (p < 0.05)

In Table 2, reported are the percentage of biofilm formation biomass produced in the presence of NK1 and LEDs alone and combined with each other. A slight biomass reduction was obtained with all tested strains in the presence of different concentrations of NK1 and LEDs, also when associated with each other, up to 63.92% ± 2.30 of biomass reduction with P. aeruginosa. Regarding S. pseudintermedius, no statistically significant differences were observed with NK1 alone and irradiated with LEDs. With P. aeruginosa, an increase in biomass reduction was obtained when NK1 was irradiated with LEDs, up to 64.22% ± 4.77 of biomass reduction. Table 2. Percentage of biomass reductions of S. pseudintermedius 115 and P. aeruginosa 700 biofilm formation in presence of NK1 and LEDs alone and combined each other. #statistically significant with respect to the controlStrainsPercentage of biomass reductionsNK1 and LEDs aloneNK1 combined with LEDs631.5NK10.70.30.180.09LEDs31.5NK10.70.30.180.09*+ LEDsS. pseudintermedius 11534.25 ± 9.5131.70± 20.5625.98± 17.6414.33± 0.518.92± 1.319.59± 2.049.07± 1.9711.16± 0.6219.38± 4.0114.77± 0.4013.25± 1.175.00± 3.644.23± 0.730.00± 0.00P. aeruginosa70031.20± 0.8525.00± 3.4917.77± 4.3410.12± 6.658.73± 1.458.07± 0.345.12± 0.7763.61#± 1.0264.22#± 4.7763.92#± 2.3060.84#± 7.1622.53± 5.2815.90± 7.338.55± 1.02*10^-5^ M NK1

Figure 4 shows the microbial adherent CFU/ml in the presence of different NK1 concentrations and LEDs alone and combined with each other. A general CFU/ml reduction was obtained when treated with NK1 and LEDs alone and when they were combined. For S. pseudintermedius, the best effect was recorded with 6 × 10^−5^ M of NK1 with 6.10 × 10^9^ ± 1.00 10^8^ with respect to 8.25 × 10^9^ ± 2.12 10^8^ CFU/ml of the control. The LEDs treatment didn’t affect significantly the S. pseudintermedius growth with 7.73 × 10^9^ ± 1.55 × 10^9^ CFU/ml. When treated with NK1 combined with LEDs, a slight CFU/ml reduction was obtained with the best effect with 3 × 10^−5^ M of NK1 plus LEDs for 17 min. A remarkable biofilm inhibition was recorded in the presence of NK1 alone and combined with LEDs against P. aeruginosa. A stable P. aeruginosa effect was detected when NK1 was combined with LEDs, with CFU/ml ranging from 2.57 × 10^7^ ± 5.03 × 10^6^ to 5.86 × 10^8^ ± 9.34 10^6^ with respect to 3.85 × 10^7^ ± 4.57 10^6^ CFU/ml of the control.Fig. 4. Inhibition of biofilm formation by using different concentrations of NK1 and LEDs alone and combined with each other on S. pseudintermedius 115 and P. aeruginosa 700 in terms of detected CFU/ml. *Statistically significant (p < 0.05) with respect to the control. ** Statistically significant (p < 0.05)

Table 3 shows the percentage of reduction of S. pseudintermedius 115 and P. aeruginosa 700 mature biomass production in presence of NK1 and LEDs alone and combined with each other. As shown in Table 3, the reductions were obtained only against S. pseudintermedius in the presence of NK1 with up to 47.22% ± 0.41 of biomass reduction. Table 3. Percentage of mature biomass reductions of S. pseudintermedius 115 and P. aeruginosa 700 mature biomass production in presence of NK1 and LEDs alone and combined each other. #statistically significant with respect to the controlStrainsPercentage of biomass reductionsNK1 and LEDs aloneNK1 combined with LEDsNK1LEDsNK1631.50.70.30.180.0931.50.70.30.180.09*+ LEDsS. pseudintermedius 11547.22#± 0.4147.54#± 0.5146.27#± 3.7446.59#± 0.5045.11#± 0.8742.90#± 1.2326.63± 0.0114.75± 0.7712.46± 8.100.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.00P. aeruginosa7000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.000.00± 0.00*10^-5^ M NK1

As shown in Fig. 5, a relevant S. pseudintermedius anti-biofilm effect was also obtained regarding the microbial CFU/ml of the mature biofilm. In fact, the S. pseudintermedius CFU/ml significantly reduced when treated with NK1 and LEDs alone, but no effect was registered when combined with each other. No effect was detected against P. aeruginosa mature biofilm in the presence of NK1 and LEDs alone and combined with each other.Fig. 5. Anti-biofilm effect of different concentrations of NK1 and LEDs alone and combined with each other on S. pseudintermedius 115 (a) and P. aeruginosa 700 (b) mature biofilms in terms of detected CFU/ml. *Statistically significant (p < 0.05) with respect to the control. ** Statistically significant (p < 0.05)

Figure 6 shows representative CLSM images of S. pseudintermedius and P. aeruginosa biofilm formation in presence of NK1 and LEDs alone and combined with each other. In presence of NK1 and LEDs, a relevant inhibition of bacterial adhesion with all green and viable cells was observed. In these conditions, the biofilms appeared less 3D structured, shorter, and thinner (Fig. 1S). When the biofilms were treated with the combination of NK1 and LEDs, major dead cells were observed with respect to the other tested conditions. Although some red blood cells were detected in the S. pseudintermedius control group, the amount was not significant and did not affect the comparison of the effects of experimental treatments on biofilm viability.Fig. 6. Representative CLSM images of S. pseudintermedius and P. aeruginosa biofilm formation in the presence of NK1 and LEDs alone and combined with each other. The viable cells are green (SYTO 9) and dead cells are red (propidium iodide). Propidium iodide shows a damaged membrane, whereas green-stained bacteria represent viable cells. The images observed at the Zeiss LSM800 microscope (CarlZeiss, Jena, Germany) coupled to an inverted microscope Axio-Observer D1 (CarlZeiss, Jena, Germany) equipped with a Plan Neofluaroil-immersion objective (100 ×/1.45NA) were recorded at an emission wavelength of 500 nm for SYTO 9 and of 635 nm for Propidium iodide, and more fields of view randomly were examined. Original magnification

Discussion

Zoonotic infections associated with MDR strains are very important globally due to the direct impact on human and animal health, representing a global challenge that requires new therapeutical solutions in order to counteract their infections [36]. In particular, S. pseudintermedius infections are commonly associated with canine diseases often related to MRSP strains. The virulence factors of this bacterium are related to adhesion, invasion, toxins, and biofilm formation [37]. The presence of various virulence factors indicates the highly virulent potential of S. pseudintermedius strains, contributing to the zoonotic hazard of this pathogen. In dog chronic wound infections, S. pseudintermedius is often co-aggregated with P. aeruginosa in a poly-microbial biofilm that increases tolerance to traditional antimicrobial treatments [11, 38]. The MDR P. aeruginosa strains are widely spread in hospital-acquired or healthcare-associated infections, underlying the importance of regular disinfection processes and stringent hygiene measures at small animal clinics [39]. The increasing of MDR and tolerant S. pseudintermedius and P. aeruginosa strains strongly underlines the importance of the search for innovative and eco-sustainable solutions to counteract the infections associated with them. The aim of this work was to suggest an eco-sustainable strategy by using NK1 alone and combined with novel technologies (LEDs) to counteract MRSP and MDR P. aeruginosa associated with persistent zoonotic infections that can be transmitted to humans. This innovative approach is based on the use of a recombinant protein (NK1) which, thanks to its high affinity binding with oxidized cysteine residues, has the capability to modulate the activity of structural and functional proteins that are essential for bacterial pathogenicity. The interaction with peroxiredoxin and oxyr, two proteins with oxidized cysteine, can interfere with their antioxidant effect. Noteworthy, recombinant NK1 exerts antimicrobial activity against Gram-positive and -negative bacteria, with the advantage, among others, of avoiding the pitfalls of drugs whose high target specificity can give rise to resistance evolution. The recombinant protein NK1, already tested in cellular and mouse models [33], is not toxic, active at very low concentration, and requires low costs of production in a fermentative way. LEDs’ antimicrobial action is related to the ROS production, in particular hydroxyl radical (OH) and singlet oxygen (^1^O_2_), which can promote toxic effects associated with oxidative stress, culminating in cell death [40]. NK1 and LEDs didn’t show significant antimicrobial action in terms of MIC and MBC, but they reduced the S. pseudintermedius and P. aeruginosa viable cells with a major effect against Gram-positive bacterium with an immediate effect. The irradiation of the protein increased its antimicrobial action at 24 h and against P. aeruginosa. LEDs are able to interact and link P. aeruginosa outer membrane through the interaction with proteins including the pyoverdine system, showing an antibacterial effect. In addition, LEDs irradiation could enhance the NK1 penetration in Gram-negative bacterium with an action on membrane permeabilization. The antimicrobial action of the NK1 and LEDs combination is related to a multi-target action that makes the bacteria unbalanced state and more sensitive to the treatments. In addition, the combination effect of NK1 and LEDs could be associated with inducing stress conditions by the ROS production and inhibition effect of antioxidant proteins. D’Ercole et al. demonstrated the efficacy of LEDs and NaOCl 1% against E. faecalis in 5 min of treatments for oral cavity treatment [41]. P. aeruginosa shows a great capability to adapt and survive over a long term thanks to its flexibility in switching between the planktonic and surface-associated lifestyles by swimming, swarming, and twitching motilities, representing environmental reservoirs, resistance against extreme conditions, and neutralization of the immunological responses of the host [42]. The LEDs treatment induced P. aeruginosa motility increase with respect to the control, favoring the planktonic status that represents a phenotype more susceptible to treatments [35]. NK1 irradiated with LEDs significantly reduced the swimming and swarming motility. The heterogeneity of P. aeruginosa motility is broad, and it is mediated by the flagellum and the multiple flagella, T4P, rhamnolipid biosurfactants, specific bacterial cell density, and nutrient availability [43]. Probably a multi-target action of NK1 and LEDs makes the bacterium less motile, and a motility gene expression could better explain the results.

In addition, P. aeruginosa colonizes and explores abiotic and host surfaces using twitching motility, which is powered by retractile extracellular filaments called type IV pili (T4P) [43]. NK1 combined with LEDs affected the P. aeruginosa virulence, reducing its capability to move and adhere to the surfaces.

Regarding the NK1 and LEDs alone and combined with each other on the bacterial biofilm, the results displayed a remarkable effect especially against S. pseudintermedius and P. aeruginosa biofilm formation. NK1 reduced the microbial biofilm formation due to its capability to interfere with the adhesion.

For the mature biofilm, NK1 affected only the S. pseudintermedius biofilm, disrupting the microbial aggregation. LEDs are widely used against chronic wound biofilms, acting on the biofilm matrix and disrupting the polymicrobial aggregation. Mahmoudi et al. [44] showed a great anti-biofilm effect of the LEDs 630 nm against S. aureus biofilm, significantly reducing the biomass production. Probably, the LEDs anti-biofilm effect is associated with quorum sensing inhibition after treatment. Tan et al. [45] demonstrated the reduced expression of quorum sensing genes in P. aeruginosa biofilm after ALAD-PDT application. When NK1 was combined with LEDs, major dead cells were detected in the CLSM images, demonstrating a potential synergistic antimicrobial action.

The combination of NK1 and LEDs results in more efficacy than the effects obtained with the single molecules in planktonic and sessile phases. In fact, when NK1 was irradiated with LEDs, a potent antimicrobial action (especially at 24 h) and anti-P. aeruginosa motility and anti-biofilm formation were obtained. These remarkable effects were observed with all tested NK1 concentrations and LEDs, showing a significant effect with a low NK1 dose. LEDs (at 630 nm) with protein solutions are widely used in various applications, such as skin care and wound healing, to enhance cell growth, collagen production, and overall tissue regeneration. Red light therapy at 630 nm can stimulate cell proliferation and migration, while protein-based treatments can provide tissue repair [11, 41, 46–48]. In terms of applicability, the proposed solution includes the treatment with NK1 irradiated immediately with 630 nm LEDs and reprocessed with 630 nm LEDs after another 24 h, to increase the effectiveness of the protein. Overall, the NK1 and LEDs combination represents an innovative strategy to counteract resistant S. pseudintermedius and P. aeruginosa strains. When NK1 was irradiated with the LEDs, an increase in NK1 antimicrobial and anti-virulence actions was detected, underlining the use of this innovative combination, reducing the use of antibiotics due to the worrying phenomenon of AMR. Further study will be carried out to better understand the effects of the new tested strategy on S. pseudintermedius and P. aeruginosa gene expression.

Conclusions

The search for innovative and non-antibiotic strategies is an urgent and challenging goal to counteract infection associated with MDR strains. Most of the human microbial diseases are correlated to zoonotic transmission, indicating that the influence of animals in human diseases has increased and is still evolving, affirming the importance of AMR surveillance. The obtained results demonstrate that the recombinant protein NK1 alone and combined with LEDs exhibits: (i) antimicrobial activity against both S. pseudintermedius and P. aeruginosa tested strains; (ii) anti-swimming, -swarming motility, and -twitching; (iii) anti-biofilm formation. The advantages of using recombinant NK1 protein over conventional antimicrobial drugs developed to date include several aspects: the efficacy of HS-binding peptides for the treatment of bacterial infections; the NK1 no-toxic or off-target effects tested in several cellular systems and mouse models of human diseases; the NK1 antimicrobial activity coupled with the established ability of the recombinant protein to bind HSPG on the host cell surface, thus inhibiting bacterial attachment, entry, and spread into target cells; the synthesis of the recombinant protein NK1 carried out by a fermentative way on a large scale at low costs. For these reasons, the combination of recombinant NK1 protein and LEDs represents an innovative strategy to counteract resistant S. pseudintermedius and P. aeruginosa strains, showing significant antimicrobial and anti-virulence actions by using a protein. Novel technologies combined with a recombinant protein are a cheap and eco-sustainable strategy to treat the infections associated with S. pseudintermedius and P. aeruginosa.

Supplementary Information

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