Silver Nanoparticles Conjugated with BK510Lys Endolysin for Gram-Negative Bacteria Inhibition

Abstract

Featured Application

Featured Application: Biopharmaceuticals for veterinary use.

Abstract

In the last decade, the problems caused by the excessive use of antibiotics and the emergence of multi-resistant patterns in Gram-negative bacteria caused the generation of several alternatives and/or replacements, including endolysins and the use of silver nanomaterials (AgNPs). It has been shown that the AgNPs help overcome the bacterial lipopolysaccharide barrier and allow the endolysins to reach the cell wall; therefore, in the present investigation, conjugates of BK510 commercial endolysins (BK510Lys) with metallic nanoparticles were made, which eliminated the obstacle that represents the outer membrane of Gram-negative bacteria. AgNPs were produced by green synthesis using Lepidium virginicum extract as a reducing agent, and diameters of 16.06 ± 4.23 nm were obtained. The endolysins BK510Lys and AgNPs were used to generate conjugates that were characterized and evaluated against gram-negative and gram-positive bacteria of veterinary interest. The results indicated that using the conjugates at a concentration of 0.01 mg/mL, a 2:1 ratio, a temperature of 40 °C, and at pH 5, the inhibitory effect was greater than that of AgNPs (0.5 µg/mL) in more than 65% of the Gram-negative bacteria analyzed. Furthermore, it was determined that for both BK510Lys and the AgNP-BK510Lys conjugate, the only factor that had a significant effect on the lytic activity was their concentration. Finally, we concluded that based on the promising results obtained, future follow-up can be conducted for the formulation of highly specific alternative drugs for super-resistant Gram-negative bacteria.

1. Introduction

Antimicrobial resistance (AMR) is a growing and life-threatening problem in both human and veterinary medicine, especially in surgical patients [1,2,3]. AMR further complicates nosocomial infections by increasing morbidity, mortality, and costs. This occurs when a pathogen develops resistance to one or more agents to which the pathogen was previously sensitive. Recent studies show that the main pathogens present around the world are Gram-negative bacteria [1,2,3,4]; some nosocomial predominant microorganisms are Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. [4]. Being the last three on the critical priority list of bacteria for which new antibiotics are urgently needed, published by the World Health Organization in 2017 [5].

This problem prompted the development of new strategies for the therapy of infectious diseases, including strategies for denaturalizing the lipopolysaccharides (LPS) present in these bacteria. A very promising system for this purpose is the one formed by nanoparticles and endolysins [6,7].

Silver nanoparticles (AgNPs) have been widely used in biomedical studies in recent years due to their wide variety of applications in this field. Some recent studies have shown the strong antimicrobial activity of green synthesized AgNPs against Gram-negative, Gram-positive, and/or antibiotic-resistant bacteria [8,9,10].

To enhance the antimicrobial activity and reduce the toxicity of AgNPs, some studies have focused on making conjugates with biological molecules, such as peptides [11], bacteriophages [12,13], and endolysins [6,14].

Endolysins belong to the enzymatic group of peptidoglycan hydrolases, which break down the peptidoglycan structure. They are produced in bacterial cells infected by bacteriophages at the end of their lytic cycle and accumulate in the cytoplasm of the infected cell while the bacteriophage particles mature [15].

Studies realized by Ciepluch et al. [16] recorded the interactions of PEGyled dendronized AgNPs with P. aeruginosa LPS in the presence of lysozymes and KP27 endolysin. They found that PEGyled dendronized AgNPs with endolysins overcame the LPS barrier and enhanced the antibacterial effect more efficiently than lysozymes.

BK510Lys is an endolysin from the Staphylococcus aureus bacteriophage BK510, isolated from cows diagnosed with mastitis [17], which has shown high effectiveness against Gram-positive bacteria [18]. However, its efficiency against Gram-negative bacteria is very low. Therefore, the present research aims to conjugate BK510Lys with AgNPs (Synthesized by a green route with Lepidium virginicum extract as a reducing agent) to increase its efficiency in inhibiting Gram-negative bacteria of veterinary interest.

2. Materials and Methods

2.1. Preparation of Lepidium Virginicum Extract

The extract was prepared with fresh leaves and stems of the Lepidium virginicum plant. 100 g of leaves and stems were selected, washed with distilled water, and placed in a conventional oven (Memmert UN110) at 70 °C for 8 h. The dehydrated leaves and stems were pulverized in an electric grain mill (Insely 1000A), and Screening was carried out with two stainless steel sieves, number 10 mesh and number 19 mesh (Gilson company inc., complies with ASTM E11, Lewis Center, OH, USA). Subsequently, 2 g of L. virginicum powder was placed in a 100 mL beaker, and 60 mL of ethanol was added. The prepared mixture was placed on a heating rack (IKA, C-MAG HS 7, Staufen, Germany) and brought to a temperature of 50 °C for 20 h with constant stirring at 80 rpm. Finally, the resulting ethanolic solution was filtered and stored in a Falcon tube at 4 °C for later use.

2.2. AgNPs Green Synthesis Methodology and Characterization

Silver nanoparticles were synthesized using a green methodology using the extract of the L. virginicum plant. 90 mL of silver nitrate (AgNO₃, Sigma Aldrich CAS 7761-88-8, Saint Louis, MI, USA) solution was prepared at an initial concentration of 5 mM and brought to a temperature of 85 °C. Subsequently, 10 mL of L. virginicum extract was added as a reducing agent, and the chemical reaction was maintained at 85 °C for 60 min. The final solution was allowed to cool to room temperature and placed in Falcon tubes for further characterization.

2.3. Characterization of the AgNPs Obtained by Green Synthesis

The presence of AgNPs was verified using a UV-Vis spectrophotometer in a scanning range of 300 to 600 nm (Thermo Fisher Scientific NanoDrop 2000c, Waltham, MA, USA). The morphology and diameter of the AgNPs were characterized using a Transmission Electron Microscope (TEM) at 80 kV (JEOL-JEM1010). The samples were placed on a copper mesh grid coated with Ted Pella brand carbon substrate. Finally, to determine the surface charge of the nanoparticles, the measurement of the Z potential was carried out using the Z-potential analyzer (Z-sizer Nano ZS, Malvern Panalytical, Malvern, UK).

2.4. AgNPs and BK510Lys Endolysin Conjugation

BK510Lys is an endolysin produced and commercialized by SEDEBIO (Fagox-EL, pharmaceutical grade). Nanoparticles-endolysins conjugates (AgNP-BK510Lys) were created using AgNPs sized between 10 and 20 nm, with concentrations of 0.5 µg/mL and 0.22 mg/mL for AgNPs and BK510Lys, respectively. The mixture was made and stirred at 120 rpm for 15 min at 35 °C. Three different ratios (AgNPs:BK510Lys) were tested: 2:1, 1:1, and 1:2. The verification of the conjugation of AgNPs was carried out using the TEM.

2.5. Antimicrobial Efficiency Test

Reactivated cultures of S. aureus, Listeria sp., E. coli, Klebsiella, P. vulgaris, P. mirabillis, P. aeruginosa, and Salmonella sp. (all strains provided by the SEDEBIO Sample Bank) were used. A colony of each culture was sown in flasks (Schott Duran, 100 mL volume) with 50 mL of nutrient broth (BD Bioxon, Eysins, Switzerland) and incubated (incubator FELISA model FE-132AD) at 35 °C for 24 h.

After the incubation, 100 µL of each culture was spread on plates with Mueller Hinton agar (BD Bioxon). Using the “spot-on-lawn technique” [17], the different conjugate solutions were added (Table 1).

For each culture, the minimum inhibitory amount (MIA) of an antibiotic was added, which also served as a positive control. The MIA for each antibiotic was selected based on previous tests indicating the inhibition corresponding to each bacterium (Table 2). For each culture, and to compare the effects independently, a BK510Lys solution (E) and an AgNPs solution (A) were added at a concentration of 0.22 mg/mL and 0.5 µg/mL, respectively. Finally, nutritive broth was used as a negative control.

All the plates were incubated at 35 °C for 24 h. Once time had passed, the inhibitory halos were measured using a vernier.

2.6. Optimal Parameters Determination

Using an experimental design by Box Behnken (3³), the three factors and the three levels were established for BK510Lys and AgNP-BK510Lys, as shown in Table 3. The response variable was the lytic activity, defined as:

(1)$LA={\displaystyle \frac{{∆OD}_{600}}{\left(t_f-t_0\right)\left(E\right)}}\left[=\right]{\displaystyle \frac{{OD}_{600}}{\left(\mathrm{s}\right)\left(\mathrm{mg}\right)}}$

where:

LA = lytic activity $\left[=\right]{\displaystyle \frac{{OD}_{600}}{\left(\mathrm{s}\right)\left(\mathrm{mg}\right)}}$

ΔOD₆₀₀ = Optical Density Difference at 600 nm

t_f = Final time $\left[=\right]$ seconds

t₀ = Initial time $\left[=\right]$ seconds

E = Enzyme (BK510Lys or AgNP-BK510Lys) concentration $\left[=\right]$ mg.

The BK510Lys systems were prepared with 100 µL of the endolysin (Fagox-EL, pharmaceutical grade) solution (to obtain a mass of 0.14, 0.70, and 1.25 mg) and 900 µL aliquots of S. aureus cell suspension, at an OD₆₀₀ between 0.400 and 0.500 in 1.5 mL microtubes (Axygen, Union City, CA, USA). The systems were incubated for 15 min and 5 h at 20, 30, and 40 °C, according to the experimental design; the OD₆₀₀ was measured at each time. The pH value was adjusted at 5, 7, and 9 according to the experimental design (using TRIS-HCl and TRIS-base, Sigma Aldrich). Each experiment was evaluated in duplicate.

The AgNP-BK510Lys (2:1 ratio) systems were prepared with 100 µL of the conjugates solution (to obtain a mass of 0.01, 0.07, and 0.13 mg) and 900 µL aliquots of S. aureus cell suspension, at an OD₆₀₀ between 0.400 and 0.500 in 1.5 mL microtubes. The systems were incubated for 15 min and 5 h at 20, 30, and 40 °C; according to the experimental design, the OD₆₀₀ was measured at each time. The pH values were adjusted at 5, 7, and 9, according to the experimental design, using TRIS-HCl and TRIS-base. Each experiment was evaluated in duplicate.

The positive control was prepared using 5 µL of ciprofloxacin (Sigma Aldrich, 17850-5G-F) at 5 mg/mL and 955 µL aliquots of S. aureus cell suspension. The negative control was prepared using 5 µL of Phosphate Buffer (Meyer, reactive grade) and 955 µL aliquots of S. aureus cell suspension.

3. Results

3.1. AgNP-BK510Lys Characterization and Antimicrobial Efficiency

Nanoparticles with spherical morphology with diameters distributed in the range of 10 to 25 nm (average diameters of 16.06 ± 4.23 nm) were obtained (Figure 1a,c). A resonant peak was observed in the AgNPs solution at 425 nm (Figure 1b), also reported by Maliszewska et al. [19]. Figure 1a,c shows the AgNPs micrographs. It is observed that the size of the nanoparticles is homogeneously dispersed, and some organic remains of L. virginicum extract are present. The measured surface charge of the silver nanoparticles was negative, with a value of −38.7 mV (Figure 1d).

Figure 2 shows the AgNP-BK510Lys micrograph at a 1:1 ratio; it is clearly observed that the AgNPs remain completely within the endolysins (Figure 2a). TEM image (Figure 2b) reveals the presence of dispersed BK510Lys clusters with sizes ranging from 200 to 300 nm.

Table 4 and Figure 3 show the inhibition diameters obtained for each strain tested. All strains showed inhibition with AgNPs, and only S. aureus with BK510Lys endolysins. Similar results for AgNPs were previously reported [20,21].

3.2. BK510Lys and AgNP-BK510Lys Optimal Parameters

The experiments were set up in three blocks, one for each temperature tested. Table 5 shows the lytic activities found for each experiment. The highest LA obtained, 2.69 × 10⁻³ ΔOD/(s)(mg), was at pH 5, 30 °C and 0.14 mg/mL of BK510Lys, while the lowest value, 0.46 × 10⁻³ ΔOD/s*mg, was at pH 5, 40 °C and 0.07 mg/mL.

While for AgNP-BK510Lys, the highest LA obtained, 83.3 × 10⁻³ ΔOD/(s)(mg), was at pH 9, 30 °C and 0.01 mg/mL of conjugate, while the lowest value, 0.09 × 10⁻³ ΔOD/(s) (mg), was at pH 9, 40 °C and 0.07 mg/mL.

Using the equations obtained from the regression analysis of BK510Lys and AgNP-BK510Lys data (equations 2 and 3, respectively), response surfaces were obtained for different combinations of factors for the same response variable. The graphs are shown in Figure 4 (BK510Lys) and Figure 5 (AgNP-BK510Lys).

LA = 5.66173 − 0.094(T) − 0.092(pH) − 6.699(BK510Lys) + 0.002(T)² + 0.001(T)(pH) − 0.004(T)(BK510Lys) − 0.025(pH)²+ 0.423(pH)(BK510Lys) + 1.890(BK510Lys)²(2)

LA = −22.1275 + 6.09583(T) − 12.6125(pH) − 157.338(AgNP-BK510Lys) − 0.0192917(T)² − 0.36375(T)(pH) − 20.8333(T)(AgNP-BK510Lys) + 1.83021(pH)² − 46.6667(pH)(AgNP-BK510Lys) + 4790.51(AgNP-BK510Lys)²(3)

The values to maximize the LA using the data obtained were:

• BK510Lys: pH 5.00, temperature of 20.0 °C, and BK510Lys concentration of 0.14 mg/mL, for a lytic activity of 2.74 × 10⁻³ ΔDO/(s)(mg).

• AgNP-BK510Lys: pH 5.00, temperature of 39.9 °C, and BK510Lys concentration of 0.01 mg/mL, for a lytic activity of 88.80 × 10⁻³ ΔDO/(s)(mg).

To determine if there was any significant effect of the factors studied, or some combination of these, an analysis of variance (ANOVA) was performed for the lytic activity. The ANOVA table (Table 6 and Table 7 for BK510Lys and AgNP-BK510Lys, respectively) partitions the variability in LA into separate pieces for each of the effects.

Figure 6 shows the Standardized Pareto Chart for BK510Lys (a) and AgNP-BK510Lys (b). As in the ANOVA, it is observed that there is one significant effect: BK510Lys concentration and AgNP-BK510 concentration, respectively.

4. Discussion

4.1. AgNP-BK510Lys Characterization and Antimicrobial Efficiency

AgNPs were synthesized using silver nitrate (AgNO₃) and the reducing agent L. virginicum extract; the use of any other agent to enhance the stability of AgNPs was not necessary. The successful synthesis of AgNPs was visually confirmed by the color change of the solution from green to brown, indicative of nanoparticle formation.

According to Ismail et al. [22], UV–Vis absorption spectroscopy is very suitable for detecting the wavelength and absorption corresponding to the surface plasmon resonance band. Additionally, previous studies have established relationships between particle size, bandwidth, and solution coloration with the maximum wavelength of absorption bands displayed in electronic spectra [23].

Zeta potential measurements play a crucial role in providing valuable data about nanoparticle agglomeration, distribution, and stability in solution [24]. The AgNPs sample exhibited a Zeta potential value close to −38.7 mV, indicating a high level of stability for the nanoparticles in the solution. The nanoparticles demonstrated high stability over a period of approximately 40 days, as observed from consistent size and Zeta potential values.

TEM has a high accuracy and reliability for determining the shape, distribution, size, and composition of AgNPs. TEM images showed that the sample is composed of well-dispersed spherical AgNPs, an average diameter of 16.06 ± 4.23 nm for the AgNPs, and a small size, indicative of an efficient synthesis process (Figure 1).

TEM images for AgNPs-BK510Lys (Figure 2) suggest that the AgNPs are dispersed inside the BK510Lys clusters and prove the formation of AgNPs-BK510Lys conjugates.

Average diameters of uncoated AgNPs were obtained between 10 and 25 nm (Figure 1c). With the conjugation process, the variation of the diameters ranged from 50 nm to 90 nm on average, which indicated the surface coating with endolysins (Figure 2). It is observed in Figure 2a,b that the nanoparticles coated with endolysins maintain a hemispherical morphology and form clusters of coated nanoparticles.

It is known that electrostatic interactions, hydrophobic interactions, and hydrogen bonding are commonly implicated in the protein-nanoparticle binding process [25]. Nayak et al. [26] studied the adsorption behavior of lactoferrin onto silver nanoparticles using the isothermal titration calorimetry technique (ITC). The findings elucidate that there were no substantial impacts on the protein’s conformation and stability caused by van der Waals interactions and hydrogen bonding.

In a separate study, Coelho et al. [14] studied the AgNPs-PlyB221 endolysin conjugates using the ITC analysis. They found that PlyB221’s adsorption onto the AgNPs interface occurred spontaneously, and the primary interactions governing this process were van der Waals and hydrogen bond interactions.

In the case of AgNPs-BK510Lys, the mechanism is still unknown and will need to be studied in greater depth in the future.

Table 4 shows the inhibition diameters obtained for each strain tested. All strains showed inhibition with AgNPs, and only S. aureus with BK510Lys endolysins. This was to be expected as there are several references on the antimicrobial properties of AgNPs [20,21], and since the endolysin comes from a S. aureus bacteriophage, it should present inhibition.

It is observed that in the conjugates, there is a synergy that allows for greater efficiency, compared to the AgNPs, in the strains of S. aureus, E. coli, Salmonella sp., and Klebsiella sp. (Figure 3a), being 24, 24, 100 and 40% higher, respectively. In the strains of Listeria sp. and P. vulgaris, the efficiency was maintained, whereas, in the strains of P. mirabillis and P. aeruginosa (Figure 3b), it decreased by 17 and 29%, respectively.

This synergy has also been demonstrated with more complex systems such as peptides-AgNPs [11], endolysin-dendron-PEG or endolysin-dendron [6,15,27], bacteriophage-AgNPs [12,13,28], and endolysin-AgNPs [14].

Coelho et al. [14] studied the lytic activity of AgNPs, PlyB221 endolysin, and AgNPs-PlyB221 conjugate in Bacillus cereus cultures. The AgNPs conjugated with PlyB221 (8.125 × 10⁻⁶ M) were more effective in reducing turbidity than the chloramphenicol (positive control), AgNPs, and PlyB221 endolysin, promoting an 80% reduction in turbidity. Any Gram-negative bacteria were tested in their study.

Ciepluch et al. [16] studied spectrometrically by measuring the optical density changes of the bacterial culture at 600 nm, the effect of PEGylated and unmodified dendronized AgNPs combined with KP27 endolysin on wild-type P. aeruginosa PAO1 and its LPS knock-out mutant (1wbpL). The synergic effect was seen in the presence of PEGylated and unPEGylated dend-AgNPs-KP27 at concentrations of 10, 20, and 50 µg/mL. The greatest inhibitory effect occurred at the lower concentration (10 µg/mL), as in the present study.

A suggested mechanism that modulates enzymatic activity is the interaction of metal ions with specific amino acid residues in different domains of endolysins [23,29]. According to Zhao et al. [30], the lytic activity of endolysin can be affected by divalent metal ions, while other metal ions may exert an inhibitory effect.

Given the results obtained in the antibiograms, it is possible to obtain a stable solution that allows the inhibitory capacity to increase, compared to each one separately. Therefore, the ratio chosen to continue studying the conjugate was 2:1.

4.2. BK510Lys and AgNP-BK510Lys Optimal Parameters

To enhance the lytic activity of BK510Lys and AgNP-BK510Lys, an experimental design for response surface methodology Box–Behnken was carried out. Data showed that to obtain the maximum experimental lytic activity, the optimal conditions of endolysin and conjugate were pH 5.00, temperature of 20.0 °C and endolysin concentration of 0.14 mg/mL and, pH 5.00, temperature of 40.0 °C and conjugate concentration of 0.01 mg/mL, respectively.

Filatova et al. [31,32] reported that, under similar conditions to those studied in the present work, the endolysin LysK from bacteriophage K of S. aureus presented a similar lytic activity (Table 8).

There is not much information about the kinetic parameters of conjugate nanoparticles-endolysins (Table 9). Nevertheless, all the results hold significant implications for the development of biocontrol agents aimed at discerning and selectively eliminating super-resistant bacteria. More studies are necessary to enhance the applications in veterinary, specifically for Gram-negative bacterial diseases.

To determine if there is any significant effect of pH, temperature, endolysin or conjugate concentration, or some combination of these, an analysis of variance (ANOVA) for the lytic activity was performed. The ANOVA table (Table 6 and Table 7, for BK510Lys and AgNP-BK510Lys, respectively) partitions the variability in LA into separate pieces for each of the effects. Then, it tests the statistical significance of each effect by comparing the mean square against an estimate of the experimental error. In both cases, when one of the effects has P-values less than 0.05, indicating that they are significantly different from zero at the 95.0% confidence level: BK510Lys and AgNP-BK510Lys concentration, respectively.

To determine the magnitude and importance of the effects, a Standardized Pareto Chart was made for BK510Lys (Figure 6a) and AgNP-BK510Lys (Figure 6b). This shows that the main effects were:

• BK510Lys: Concentration (C).

• AgNP-BK510Lys: Concentration (C).

The ANOVA analysis, the Standardized Pareto Chart, and the response surface show the great importance and effect of endolysin concentration in the LA of BK510Lys and the concentration of conjugate in the LA of this. Therefore, these factors must be considered to continue deeper studies of the mechanism of action and applications of endolysin and AgNP-BK510Lys conjugates.

5. Conclusions

Endolysin BK510Lys was successfully conjugated with stable AgNPs obtained by green synthesis using L. virginicum extract as a reducing agent, with average diameters of 16.06 ± 4.23 nm and a surface charge of −38.7 mV. Using the conjugates at a concentration of 0.01 mg/mL and a 2:1 ratio at a temperature of 40 °C and pH 5, the inhibitory effect was greater than AgNPs (0.5 µg/mL) in more than 65% of Gram-negative bacteria analyzed. Furthermore, it was determined that for both BK510Lys and the AgNP-BK510Lys conjugate, the only factor that had a significant effect on the lytic activity was their concentration. This study provides significant and promising data to advance towards the creation of highly specific drugs for super-resistant Gram-negative bacteria.

Author Contributions

D.R.S.; methodology, investigation, writing original draft preparation, funding acquisition, J.C.M.E.; writing, review and editing, A.G.V.M.; methodology, investigation, A.A.L.A.; formal analysis visualization, funding acquisition R.A.S.C.; validation and resources, A.J.G.C.; project administration, validation, supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No data was used for the research described in the article.

Acknowledgments

The authors thank all colleagues who assisted in this study. The authors also thank Fabio de Jesús Martínez Maciel for support in the digital design of the graphic abstract.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1. AgNPs characterization: (a) micrograph obtained in TEM microscopy of green synthesized AgNPs; (b) Normalized average absorbance spectrum: resonant peak observed in the AgNPs solution scanned from 300 to 600 nm; (c) Average diameter distribution. (d) Zeta potential measurement.

Figure 2. Representative TEM micrographs corresponding to AgNPs coated with endolysins: (a) Micrograph with 100 nm reference bar: Representative red circles as a reference for the size of the conjugated nanoparticles; (b) Micrograph with 200 nm reference bar: Red arrows indicating the presence of conjugated nanoparticles.

Figure 3. Samples of the cultures spread in Petri dishes with Luria Bertani medium, incubated at 37 °C for 24 h, tested at different ratios of AgNP-BK510Lys: (a) S. aureus; (b) Listeria sp.; (c) E. coli; (d) Salmonella sp.; (e) Klebsiella sp.; (f) P. vulgaris; (g) P. mirabillis; (h) P. aeruginosa.

Figure 3. Samples of the cultures spread in Petri dishes with Luria Bertani medium, incubated at 37 °C for 24 h, tested at different ratios of AgNP-BK510Lys: (a) S. aureus; (b) Listeria sp.; (c) E. coli; (d) Salmonella sp.; (e) Klebsiella sp.; (f) P. vulgaris; (g) P. mirabillis; (h) P. aeruginosa.

Figure 4. Response surface for different factors interactions for BK510Lys: (a) Temperature and pH; (b) pH and BK510Lys concentration; (c) Temperature and BK510Lys concentration.

Figure 5. Response surface for different factors interactions for AgNP-BK510Lys: (a) Temperature and pH; (b) pH and AgNP-BK510Lys concentration; (c) Temperature and AgNP-BK510Lys concentration.

Figure 6. Standardized Pareto chart for Lytic Activity using: (a) BK510Lys; (b) AgNP-BK510Lys. Grey (+) and blue (−).

Table 1

Experimental design for antimicrobial efficiency test of AgNP-BK510Lys. Gentamicin (Sigma Aldrich, G1397); Ciprofloxacin (Sigma Aldrich, 17850-5G-F); Amoxicillin + Clavulanic acid (Amoxiclav vet, Pisa Laboratory).

Solution		Amount
AgNP-BK510Lys	2:1	1.0 µg/mL:0.22 µg/mL
	1:1	0.5 µg/mL:0.22 µg/mL
	1:2	0.5 µg/mL:0.44 µg/mL
Antibiotic	C+	Gentamicin, 10 µLCiprofloxacin, 5 µLAmoxicillin + Clavulanic acid, 10 µL
Nutrient broth	C−	10 µL

Table 2

Antibiotics are used for each bacteria strain. Gentamicin (Sigma Aldrich, G1397); Ciprofloxacin (Sigma Aldrich, 17850-5G-F); Amoxicillin + Clavulanic acid (Amoxiclav vet, Pisa Laboratory).

Bacteria	Antibiotic	Concentration
Staphylococcus aureus	Gentamicin	10 mg/mL
Listeria sp.	Gentamicin	10 mg/mL
Escherichia coli	Gentamicin	10 mg/mL
Salmonella typhimurium	Ciprofloxacin	5 mg/mL
Klebsiella sp.	Gentamicin	10 mg/mL
Proteus vulgaris	Ciprofloxacin	5 mg/mL
Proteus mirabillis	Amoxicillin + Clavulanic acid	10 mg/mL
Pseudomonas aeruginosa	Ciprofloxacin	5 mg/mL

Table 3

Experimental design Box Behnken for BK510Lys and AgNP-BK510Lys.

System	BK510Lys			AgNP-BK510Lys
	Temperature (°C)	pH	Bk510Lys (mg/mL)	Temperature (°C)	pH	AgNP-BK510Lys(mg/mL)
1	20	5	0.70	20	5	0.07
2	40	5	0.70	40	5	0.07
3	20	9	0.70	20	9	0.07
4	40	9	0.70	40	9	0.07
5	20	7	0.14	20	7	0.01
6	40	7	0.14	40	7	0.01
7	20	7	1.25	20	7	0.13
8	40	7	1.25	40	7	0.13
9	30	5	0.14	30	5	0.01
10	30	9	0.14	30	9	0.01
11	30	5	1.25	30	5	0.13
12	30	9	1.25	30	9	0.13
13	30	7	0.70	30	7	0.07
14	30	7	0.70	30	7	0.07
15	30	7	0.70	30	7	0.07

Table 4

Inhibition zones (mm) under different conditions and for the different strains tested.

Strain	C− (Nutritive Broth)	C+ (ATB)	AgNPs	BK510Lys	A+E (2:1)	A+E (1:1)	A+E (1:2)
S. aureus	0	24	10	12	12	10	8
Listeria sp.	0	24	10	0	10	8	0
E. coli	0	26	12	0	14	12	10
S. typhimurium	0	48	8	0	16	16	8
Klebsiella sp.	0	20	10	0	14	12	10
P. vulgaris	0	56	10	0	10	10	6
P. mirabillis	0	46	12	0	10	8	8
P. aeruginosa	0	44	14	0	10	8	0

Table 5

Lytic activity of BK510Lys and AgNP-BK510Lys (2:1 ratio).

System	BK510Lys LA (ΔDO/(s)(mg)) ×10−3	AgNP-BK510Lys LA (ΔDO/(s)(mg)) ×10−3
1	0.83	23.8
2	0.46	45.9
3	0.96	7.9
4	0.68	0.9
5	2.12	26.4
6	2.28	81.2
7	0.59	2.9
8	0.67	7.7
9	2.69	51.0
10	0.72	83.3
11	0.69	5.5
12	0.53	15.4
13	0.69	19.4
14	0.69	11.0
15	0.70	12.3

Table 6

Analysis of Variance for lytic activity of BK510Lys.

Source	Sum of Squares	Df	Mean Square	F-Ratio	p-Value
A: Temperature	0.0210125	1	0.0210125	0.10	0.7600
B: pH	0.427812	1	0.427812	2.12	0.2051
C: BK510Lys	3.645	1	3.645	18.07	0.0081
AA	0.0719391	1	0.0719391	0.36	0.5764
AB	0.002025	1	0.002025	0.01	0.9241
AC	0.0016	1	0.0016	0.01	0.9325
BB	0.0372314	1	0.0372314	0.18	0.6854
BC	0.8836	1	0.8836	4.38	0.0906
CC	1.25103	1	1.25103	6.20	0.0551
Total error	1.00869	5	0.201738
Total (corr.)	7.36037	14

Table 7

Analysis of Variance for lytic activity of AgNP-BK510Lys.

Source	Sum of Squares	Df	Mean Square	F-Ratio	p-Value
A: Temperature	697.511	1	697.511	2.11	0.2058
B: pH	43.7113	1	43.7113	0.13	0.7308
C: AgNP-BK510Lys	5533.52	1	5533.52	16.76	0.0094
AA	13.7416	1	13.7416	0.04	0.8464
AB	211.703	1	211.703	0.64	0.4596
AC	625.0	1	625.0	1.89	0.2273
BB	197.888	1	197.888	0.60	0.4738
BC	125.44	1	125.44	0.38	0.5646
CC	1098.16	1	1098.16	3.33	0.1278
Total error	1650.74	5	330.148
Total (corr.)	10,169.9	14

Table 8

Results comparison of similar studies with S. aureus bacteriophages endolysins.

Parameter	BK510LYS	LYSK [30]	LYSH5 [31]	Lys-phiSA012 [28]
pH	5.00	5	7.00	7.00
Temperature (°C)	20.0	30	37.0	37.0
Lytic Activity (ΔDO/(s)(mg))	2.74 × 10−3	1.5 × 10−3	0.19	0.11
Endolysin concentration (mg/mL)	0.14	0.5	0.3	0.109

Table 9

Results comparison of similar studies with conjugates nanoparticles-endolysins.

Parameter	AgNP-BK510Lys	AgNPs-PlyB221 [13]	PEG-dend-AgNPs-KP27 [6]
pH	5.00	DNS *	7.2–7.4
Temperature (°C)	39.9	30.0	37.0
Lytic Activity (ΔDO/(s)(mg))	0.088	DNS *	82 ***
Conjugate concentration (mg/mL)	0.01	8.125 × 10−6 **	0.01

* Data not show (DNS); ** Molar concentration; *** % of control (OD₆₀₀).

References

1. Lemiech-Mirowska, E.; Kiersnowska, Z.M.; Michałkiewicz, M.; Depta, A.; Marczak, M. Nosocomial infections as one of the most important problems of healthcare system. Ann. Agric. Environ. Med.; 2021; 28, pp. 361-366. [DOI: https://dx.doi.org/10.26444/aaem/122629] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34558254]

2. Rohini, C.; Kumar, A.V.; Sravanthi, M.; Kumar, E. Incidence of nosocomial infections associated with surgical procedures. J. Livest. Sci.; 2023; 14, pp. 300-303. [DOI: https://dx.doi.org/10.33259/JLivestSci.2023.300-303]

3. Ahmed, S.K.; Hussein, S.; Qurbani, K.; Ibrahim, R.H.; Fareeq, A.; Mahmood, K.A.; Mohamed, M.G. Antimicrobial resistance: Impacts, challenges, and future prospects. J. Med. Surg. Public Health; 2024; 2, 100081. [DOI: https://dx.doi.org/10.1016/j.glmedi.2024.100081]

4. Zendri, F.; Isgren, C.M.; Devaney, J.; Schmidt, V.; Rankin, R.; Timofte, D. Resistome-based surveillance identifies ESKAPE pathogens as the predominant gram-negative organisms circulating in veterinary hospitals. Front. Microbiol.; 2023; 14, 1252216. [DOI: https://dx.doi.org/10.3389/fmicb.2023.1252216] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37744932]

5. World Health Organization: WHO. WHO Publishes List of Bacteria for Which New Antibiotics Are Urgently Needed. 27 February 2017; Available online: https://www.who.int/es/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed (accessed on 25 June 2024).

6. Ciepluch, K.; Maciejewska, B.; Gałczyńska, K.; Kuc-Ciepluch, D.; Bryszewska, M.; Appelhans, D.; Drulis-Kawa, Z.; Arabski, M. The influence of cationic dendrimers on antibacterial activity of phage endolysin against P. aeruginosa cells. Bioorg. Chem.; 2019; 91, pp. 103-121. [DOI: https://dx.doi.org/10.1016/j.bioorg.2019.103121] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31310881]

7. Shemyakin, I.G.; Firstova, V.V.; Fursova, N.K.; Abaev, I.V.; Filippovich, S.Y.; Ignatov, S.G.; Dyatlov, I.A. Next-generation antibiotics, bacteriophage endolysins, and nanomaterials for combating pathogens. Biochemistry; 2020; 85, pp. 1374-1388. [DOI: https://dx.doi.org/10.1134/S0006297920110085] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33280580]

8. Wasilewska, A.; Klekotka, U.; Zambrzycka, M.; Zambrowski, G.; Święcicka, I.; Kalska-Szostko, B. Physico-chemical properties and antimicrobial activity of silver nanoparticles fabricated by green synthesis. Food Chem.; 2023; 400, 133960. [DOI: https://dx.doi.org/10.1016/j.foodchem.2022.133960] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36063680]

9. Akter, S.; Lee, S.; Siddiqi, M.Z.; Balusamy, S.R.; Ashrafudoulla, M.; Rupa, E.J.; Huq, M.A. Ecofriendly Synthesis of Silver Nanoparticles by Terrabacter humi sp. nov. and Their Antibacterial Application against Antibiotic-Resistant Pathogens. Int. J. Mol. Sci.; 2020; 21, 9746. [DOI: https://dx.doi.org/10.3390/ijms21249746] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33371231]

10. Martínez Espinosa, J.C.; Carrera Cerritos, R.; Ramírez Morales, M.A.; Sánchez Guerrero, K.P.; Silva Contreras, R.A.; Macías, J.H. Characterization of Silver Nanoparticles Obtained by a Green Route and Their Evaluation in the Bacterium of Pseudomonas aeruginosa. Crystals; 2020; 10, 395. [DOI: https://dx.doi.org/10.3390/cryst10050395]

11. Li, W.; Li, Y.; Sun, P.; Zhang, N.; Zhao, Y.; Qin, S.; Zhao, Y. Antimicrobial peptide-modified silver nanoparticles for enhancing the antibacterial efficacy. RSC Adv.; 2020; 10, pp. 38746-38754. [DOI: https://dx.doi.org/10.1039/D0RA05640E]

12. Manoharadas, S.; Altaf, M.; Alrefaei, A.F.; Devasia, R.M.; Hadj, A.Y.M.B.; Abuhasil, M.S.A. Concerted dispersion of Staphylococcus aureus biofilm by bacteriophage and ‘green synthesized’ silver nanoparticles. RSC Adv.; 2021; 11, pp. 1420-1429. [DOI: https://dx.doi.org/10.1039/D0RA09725J] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35424119]

13. Szymczak, M.; Pankowski, J.A.; Kwiatek, A.; Grygorcewicz, B.; Karczewska-Golec, J.; Sadowska, K.; Golec, P. An effective antibiofilm strategy based on bacteriophages armed with silver nanoparticles. Sci. Rep.; 2024; 14, 9088. [DOI: https://dx.doi.org/10.1038/s41598-024-59866-y] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38643290]

14. Coelho, F.; Zapata, A.; Machado, T.R.; Canduri, F.; Zucolotto, V. Exploring the agricultural potential of AgNPs/PlyB221 endolysin bioconjugates as enhanced biocontrol agents. Biocatal. Agric. Biotechnol.; 2024; 56, 103040. [DOI: https://dx.doi.org/10.1016/j.bcab.2024.103040]

15. Young, R. Phage lysis: Three steps, three choices, one outcome. J. Microbiol.; 2014; 52, pp. 243-258. [DOI: https://dx.doi.org/10.1007/s12275-014-4087-z] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24585055]

16. Ciepluch, K.; Skrzyniarz, K.; Barrios-Gumiel, A.; Quintana, S.; Sánchez-Nieves, J.; Maciejewska, B.; Drulis-Kawa, Z.; Arabski, M. Dendronized silver nanoparticles as bacterial membrane permeabilizers and their interactions with P. aeruginosa lipopolysaccharides, lysozymes, and phage-derived endolysins. Front. Microb.; 2019; 10, 2771. [DOI: https://dx.doi.org/10.3389/fmicb.2019.02771] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31866964]

17. Varela, O.D.F.; Barboza, C.J.E.; González, M.J.; León, G.M.F.; Valencia, P.M.; Lechuga, A.A.A.; Sánchez, F.C.G.; Ledezma, G.F.; Gutierrez, C.A.J. Antibiotic susceptibility of Staphylococcus aureus isolated from subclinical bovine mastitis cases and in vitro efficacy of bacteriophage. Vet. Res. Commun.; 2018; 42, pp. 243-250. [DOI: https://dx.doi.org/10.1007/s11259-018-9730-4] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30043292]

18. Ramírez-Sáenz, D.; Rubio-Cortes, M.I.; Valdivia-Mancillas, A.G.; García-Pereda, Y.M.; Custodio-Villagómez, C.U.; Lechuga-Arana, A.A.; Gutiérrez-Chávez, A.J. Mammary gland physiological response to BK510 bacteriophage and endolysins based treatment against bovine mastitis. J. Bioeng. Biomed. Res.; 2022; 6, pp. 32-39.

19. Maliszewska, I.; Szewczyk, K.; Waszak, K. Biological synthesis of silver nanoparticles. J. Phys. Conf. Ser.; 2009; 146, 012025. [DOI: https://dx.doi.org/10.1088/1742-6596/146/1/012025]

20. Morones, J.R.; Elechiguerra, J.L.; Camacho, A.; Holt, K.; Kouri, J.B.; Ramírez, J.T.; Yacaman, M.J. The bactericidal effect of silver nanoparticles. Nanotechnology; 2005; 16, pp. 2346-2353. [DOI: https://dx.doi.org/10.1088/0957-4484/16/10/059]

21. Nair, L.S.; Laurencin, C.T. Silver Nanoparticles: Synthesis and Therapeutic Applications. J. Biomed. Nanotechnol.; 2007; 3, pp. 301-316. [DOI: https://dx.doi.org/10.1166/jbn.2007.041]

22. Ismail, M.; Gul, S.; Khan, M.; Khan, M.; Asiri, A.; Khan, S. Medicago polymorpha-mediated antibacterial silver nanoparticles in the reduction of methyl orange. Green Process. Synth.; 2019; 8, pp. 118-127. [DOI: https://dx.doi.org/10.1515/gps-2018-0030]

23. Solomon, S.; Bahadory, M.; Jeyarajasingam, A.V.; Rutkowsky, S.A.; Boritz, C.; Mulfinger, L.M. Synthesis and study of silver nanoparticles. J. Chem. Edu.; 2007; 84, 322. [DOI: https://dx.doi.org/10.1021/ed084p322]

24. Edis, Z.; Haj Bloukh, S.; Ibrahim, M.R.; Abu Sara, H. “Smart” antimicrobial nanocomplexes with potential to decrease surgical site infections (SSI). Pharmaceutics; 2020; 12, 361. [DOI: https://dx.doi.org/10.3390/pharmaceutics12040361] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32326601]

25. Cavalcanti, I.D.L.; Xavier, F.H.; Magalhães, N.S.S.; De Britto Lira Nogueira, M.C. Isothermal titration calorimetry (ITC) as a promising tool in pharmaceutical nanotechnology. Int. J. Pharm.; 2023; 641, 123063. [DOI: https://dx.doi.org/10.1016/j.ijpharm.2023.123063]

26. Nayak, P.S.; Borah, S.M.; Gogoi, H.; Asthana, S.; Bhatnagar, R.; Jha, A.N.; Jha, S. Lactoferrin adsorption onto silver nanoparticle interface: Implications of corona on protein conformation, nanoparticle cytotoxicity and the formulation adjuvanticity. Chem. Eng. J.; 2019; 361, pp. 470-484. [DOI: https://dx.doi.org/10.1016/j.cej.2018.12.084]

27. Ciepluch, K.; Skrzyniarz, K.; Zdańska, J.; Barrios-Gumiel, A.; Sánchez-Nieves, J.; De La Mata, F.J.; Maciejewska, B.; Drulis-Kawa, Z.; Bryszewska, M.; Arabski, M. PEGylation of dendronized silver nanoparticles increases the binding affinity of antimicrobial proteins. J. Mol. Liq.; 2020; 319, 114339. [DOI: https://dx.doi.org/10.1016/j.molliq.2020.114339]

28. Elsayed, M.M.; Elkenany, R.M.; Zakari, A.I.; Badawy, B.M. Isolation and characterization of bacteriophages for combating multidrug-resistant Listeria monocytogenes from dairy cattle farms in conjugation with silver nanoparticles. BMC Microbiol.; 2023; 23, 146. [DOI: https://dx.doi.org/10.1186/s12866-023-02893-y] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37217869]

29. Fujiki, J.; Nakamura, T.; Furusawa, T.; Ohno, H.; Takahashi, H.; Kitana, J.; Usui, M.; Higuchi, H.; Tanji, Y.; Tsutsumi, Y. et al. Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage. Pharmaceuticals; 2018; 11, 25. [DOI: https://dx.doi.org/10.3390/ph11010025] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29495305]

30. Zhao, X.; Li, L.; Zhang, Q.; Li, M.; Hu, M.; Luo, Y.; Xu, X.; Chen, Y.; Liu, Y. Characterization of the Clostridium perfringens phage endolysin cpp-lys and its application on lettuce. Int. J. Food Microb.; 2023; 405, 110343. [DOI: https://dx.doi.org/10.1016/j.ijfoodmicro.2023.110343]

31. Filatova, L.Y.; Becker, S.C.; Donovan, D.M.; Gladilin, A.K.; Klyachko, N.L. LysK, the enzyme lysing Staphylococcus aureus cells: Specific kinetic features and approaches towards stabilization. Biochimie; 2010; 92, pp. 507-513. [DOI: https://dx.doi.org/10.1016/j.biochi.2010.01.026]

32. Obeso, J.M.; Martínez, B.; Rodríguez, A.; García, P. Lytic activity of the recombinant staphylococcal bacteriophage ΦH5 endolysin active against Staphylococcus aureus in milk. Int. J. Food Microb.; 2008; 128, pp. 212-218. [DOI: https://dx.doi.org/10.1016/j.ijfoodmicro.2008.08.010] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18809219]