Influence of nanoscale-modified apatite-type calcium phosphates on the biofilm formation by pathogenic microorganisms

Nanoparticles (25–50 nm) of chemically modified calciumphosphates Ca10−x−yM II xNay(PO4)6−z(CO3)z(OH)2 (M – Cu2+, Zn2+) were synthesized via a wet precipitation method at room temperature. The Fourier-transform infrared spectroscopy data confirmed the partial substitution of PO4 → CO3 (B-type) in apatite-type structure. The influence of prepared phosphates on biofilm formation by pathogenic microorganisms was investigated. It was found that the samples Na,CO3-hydroxyapatite (HAP) and Na+, Zn,CO3-HAP (5–20mM)had thehighest inhibitory effect on biofilm formation by Staphylococcus aureus strains. The sample Na+, CO3-HAP had the slight influence on the formation of the biofilm by Pseudomonas aeruginosa, while for the samples Na+, Cu2+, CO3-HAP and Na+, Zn2+, CO3HAP such an effect was not detected. According to transmission electron microscopy data, a correlation between the activity of synthesized apatite-relatedmodified calcium phosphates in the processes of biofilm formation and their ability to adhere to the surface of bacterial cells was established. The prepared samples can be used for the design of effective materials with antibacterial activity for medicine.


Introduction
At present, apatite-type calcium phosphate (Ca 10 (PO 4 ) 6 (OH) 2 ), hydroxyapatite [HAP]) and its chemically modified analogues owing to its biocompatibility, bioactivity and similarity with bone tissue are widely used in medicine, in particular, orthopedics and dentistry for bone restoration and coating of orthopedic and dental implants, for oral care, for remineralization of carious lesions of enamel and protection of teeth from caries [1][2][3][4]. The surface of these materials due to their physicochemical properties modulates enhanced adhesion of osteoblasts, increases the strength of adhesion of implants to bone tissue, but, unfortunately, this feature can also promote bacterial adhesion. Bacterial infections associated with implants and medical devices are a serious clinical problem. Such infections are difficult to treat with antibacterial agents because the bacteria that cause the infection can form biofilms on the surface of the implant [5][6][7][8].
Bacterial biofilms are complex structures of microbial cells, which are embedded in an extracellular matrix and attached to biotic or abiotic surfaces. The biofilm matrix consists of exopolysaccharides, proteins, teichoic acids, lipids and DNAs [9,10]. Biofilms can protect planktonic bacteria from the immune system of the host by disrupting the activation of complement system and phagocytes, and increasing pathogenic bacteria resistance to antibiotics around 1,000-fold [11][12][13][14].
Three-quarters of all biofilm-related infections on medical devices are formed by Staphylococcus aureus, Staphylococcus epidermidis or Pseudomonas aeruginosa [15]. It should be noted that different factors influence the formation of biofilms such as the type and surface on characteristics of biomaterial as well as the type of bacteria and their concentration, temperature, pH and nutrients. Hence, creation of materials with special characteristics, which reduce bacterial adhesion and inhibit the formation of bacterial biofilms, is very important. Investigation of the influence of chemical and morphological modifications of apatite-related calcium phosphate on the biofilm formation by the most common strains is important for the design of effective biomaterials. This will further avoid the development of pathological processes while using materials based on apatite.
A lot of literature data are devoted to the study of the antimicrobial properties of HAPs doped with different cations [16][17][18][19][20][21][22][23][24][25][26]. It was shown that Ag-HAP exhibits strong antibacterial activity [17,18]. The silver ions are slowly released from the interior of the HAP, which discloses its bactericidal activity throughout the material surrounding it [19]. HAPs doped with Cu 2+ have inhibitory effect on the growth of Escherichia coli [20]. The antibacterial behavior of Zn 2+ -doped HAP has also been studied [21,22], while its nanorods are reported to have improved performance against oral cavity bacteria [23]. A significant decrease in the number of viable Staphylococcus aureus bacteria was found after in contact with Zn-doped HAP [24]. The incorporation of Mg 2+ , Ni 2+ and SiO 4 ions into the HAP structure also showed in vitro antibacterial activity against Escherichia coli and Pseudomonas aeruginosa [25,26]. Predoi et al. showed that Ce-HAP suspensions had a biocide effect against Escherichia coli and Candida albicans microbial strains [27]. Thus, one of the approaches to enhance the antimicrobial properties of apatite-based materials is modification of initial matrix by cations with antibacterial properties, in particular silver (Ag + ), zinc (Zn 2+ ) or copper (Cu 2+ ). Another promising approach is the fabrication of HAP composite with antibacterial properties such as nano HAP-ZnO composites [28] and HAP-Ag [29].
The main goal of the present work was to investigate the influence of chemically modified apatite-related calcium phosphate on the biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa strains. These cultures are the most important pathogens causing bone and joint infections. The apatite-related calcium phosphates which contained different dopants (Na + , CO 3 2 or Na + , Zn 2+ , CO 3 2− or Na + , Cu 2+ , CO 3 2− ) were synthesized and their antibiofilm effect were evaluated. Such investigation is important for the creation of effective materials with antimicrobial activity for medical purposes.

Preparation of chemically modified calcium phosphates
Chemically modified apatite-related calcium phosphates (Na + , CO 3 2− -HAP, Na + , Zn 2+ , CO 3 2− -HAP and Na + , Cu 2+ ,  = 14). The concentration of all used solutions was 0.1 M. The obtained precipitate was stirred for 15 min, and then solid was filtered and washed with distilled water to eliminate any residual salts. The powders were dried at 100°C for 80 h. The prepared Na + , CO 3 2− -HAP was also annealed at a temperature of 700°C for 1 h. Drying oven SingleDISPLAY and muffle furnace SNOL-7.2/1100 (TermoPro-601 temperature controller) were used for drying and annealing of samples.

Characterization of prepared calcium phosphates
The phase composition of prepared samples was determined by powder X-ray diffraction (XRD) method. Diffractograms were recorded using Shimadzu XRD-6000 diffractometer with Cu-Kα radiation (λ = 1.54178 Å, 2θ = 5-90°, step size = 0.01°). The presence of different anion types in the synthesized phase was detected by Fourier-transform infrared spectroscopy (FTIR). PerkinElmer Spectrum BX spectrometer was used in the frequency range of 400-4,000 cm −1 at 1 cm −1 resolution for samples pressed in KBr pellets.
Surface characteristics (shape, size and elemental composition) of samples were investigated by scanning electron microscopy (SEM; FEI Quanta 400 ESEM instrument) with EDX analizator (Genesis 4000 instrument).
The amount of calcium, sodium, zinc, copper and phosphorus was additionally defined by atomic absorption spectroscopy (Thermo Electron M-Series instrument) after dissolution of the particles in hydrochloric acid, while the amount of carbon was measured using CHN elemental analysis (Elementar Analysensysteme).

Antibacterial activity
The effect of synthesized modified calcium phosphates on the biofilm formation by pathogenic microorganisms was investigated according to the study by Rode et al. [30] with some modifications. All prepared samples were sterilized before use by autoclaving for 30 min at 112°C (0.75 atm). The reference cultures Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 9027 were used for the investigation of antibiofilm effect of the synthesized samples. These cultures are medically relevant bacterial strains and the most important pathogens causing bone and joint infections. S. aureus is a gram-positive facultative anaerobic cocci and P. aeruginosa is a gram-negative facultative anaerobic rod. S. aureus and P. aeruginosa are highly virulent multidrug-resistant strains that establish dangerous infections and in many instances are very difficult to be treated with the existing medicines [31][32][33]. Almost 70% biofilmrelated infections on medical devices are formed by S. aureus or P. aeruginosa [15,34].
Suspensions of 24 h bacterial culture such as S. aureus and P. aeruginosa strains were grown on tryptone soy broth at 37°C. Opportunistic strains were grown at 37°C for 18 h on liquid culture mediumtryptone soy broth (HiMedia).
Different amounts (5, 10 and 20 mM) of the synthesized samples were added to 96-well plates with sterile nutrient medium. Then the inoculum (10 7 CFU/mL) was added. All systems were incubated at 37°C for 24 h.
The optical density was measured as absorbance at λ = 630 nm using Reader Multiskan FC (Thermo Fisher Scientific, USA). The culture fluid was poured off, and the wells were washed three times with sterile phosphate-buffered saline (PBS) buffer. The plates were air dried for 30 min and then stained with 0.1% solution of crystalline violet (Sigma, USA). The dye was poured off after 20 min of exposure at room temperature and washed five times with PBS buffer. The plates were air dried for 30 min at room temperature and 0.1 mL of 95% ethanol was added to the wells. After 30 min, the optical density was measured as absorbance at 492 nm.
The morphologic characterization of the synthesized samples (10 mM suspension) with opportunistic pathogens 24 h culture was analyzed using a transmission electron microscope JEM-1400 (JEOL, Japan). Investigated suspension of samples with some microorganisms was applied on the surface of a carbon mesh and dried at room temperature.
The statistical analysis of data was performed using the program ≪STATISTICA 7.0≫ (StatSoft, Inc. USA). The post-hoc-test using the criterion of LSD was used for assessing the reliability of quantitative indicators of differences in different strains. The difference was considered significant at P ≤ 0.05.
Ethical approval: The conducted research is not related to either human or animal use.

Results and discussion
According to the XRD data, synthesized samples are poorly crystalline apatite-related calcium phosphates (Figure 1a). Their powder diffractograms contained two wide reflexes in the ranges 2θ = 23-26°and 31-35°, which are characteristic for apatite-related calcium phosphates [35,36]. Samples did not contain the crystalline impurities. Elemental analysis results showed that the prepared calcium phosphates contained Na + -0.2-0.3 wt%, C -1.16-1.33 wt%, Cu 2+ -1.0 wt% or Zn 2+ -1.9 wt% ( Table 1). FTIR spectra of all synthesized phosphates are similar with respect to intensity and position of vibration bands, and a general view of these spectra is typical for apatite-type calcium phosphates (Figure 1b). Intensive characteristic modes of symmetric and asymmetric stretching vibrations type structure [35,36]. SEM data demonstrated that all synthesized samples contained spherical particles with sizes of 25-50 nm, which did not depend on their chemical composition (Figure 2a, c and d).
Taking into account that biofilm formation depends not only on chemical composition of materials but also their surface characteristics, the influence of particle size of apatite-related phase on its biological properties has also been investigated. On this way, sodium-containing phosphate Ca 10−x Na x (PO 4 ) 6−z (CO 3 ) z (OH) 2 was annealed at 700°C. As shown in ref. [36], such heating resulted in the particle aggregation and crystallite growth (300-500 nm) without changes in phase composition.
Infections associated with the formation of bacterial biofilms on implants are mainly caused by staphylococci. The biofilm of these bacteria on the surface of the implant protects microorganisms from the immune system of the host and also increases their resistance against antibiotics [6,15,37]. Bacterial biofilm formation is a complex multi-stage biological process that involves the adhesion of planktonic bacteria to the surface, proliferation, subsequent accumulation of cell biomass in the form of a multilayer structure containing a polymeric extracellular matrix, maturation and propagation of biofilm fragments [6,7,9,10].
Biofilm formation can be assessed by various methods. One of the standard methods of research on   the presence of biofilm is crystal violet analysis due to the quantitative determination of the dye bound to bacterial cells on polystyrene. Therefore, the study of the influence of the synthesized samples: Na + , CO 3 2− -HAP (particle size of 25-50 nm), Na + ,CO 3 2− -HAP (particle size of 300-500 nm), Na + , Zn 2+ , CO 3 2− -HAP and Na + , Cu 2+ , CO 3 2− -HAP on biofilm formation by the S. aureus strain was performed by this method for 24 h. It was found that all synthesized samples inhibited the biofilm formation by the S. aureus strain (Figure 3). The samples Na + , CO 3 2− -HAP (particles of size 25-50 nm) and Na + , Zn 2+ , CO 3 2− -HAP had the highest inhibitory effect on the biofilm formation. Thus, increase in amount of sample Na + , CO 3 2− -HAP from 5 to 10 mM and 20 mM led to decrease in the coefficient of formation of the biofilm by the test strain on 55.3, 76.3 and 84.2%, respectively ( Figure 3). In particular, for the Na + , Zn 2+ , CO 3 2− -HAP, the decrease in the biofilm coefficient is slightly higher (63.2, 81.6 and 79.7% for amount of sample 5, 10 and 20 mM, respectively). This result indicates that modification of apatite-type structure by 2 wt% of Zn 2+ ions leads to an increase in inhibitory effect of phosphate on biofilm formation by the S. aureus strain.
It should be noted that antibiofilm-forming activity of Zn-containing apatite to the resistant strains of Staphylococcus aureus was previously demonstrated in refs [38,39]. Inhibition of biofilm formation by Zn-doped apatites can be caused by general Zn 2+ toxicity to bacteria above physiological concentrations but also other biofilm-specific mechanisms of action could be involved. For example, it has been proposed that sublethal Zn 2+ or Ag + concentrations could affect biofilm formation by interfering with quorum sensing [40] or modulate amyloid fibril formation [41]. Other samples Na + , Cu 2+ , CO 3 2− -HAP and Na + , CO 3 2− -HAP (particles of size 300-500 nm) inhibited biofilm formation to a lesser extent (Figure 3). It should be noted that increase in the particle size of Na + , CO 3 2− -HAP from 25-50 nm to 300-500 nm at concentrations of 5 and 20 mM led to a significant decrease in phosphate activity (Figure 3). However, the effective use of Na + , Cu 2+ , CO 3 2− -HAP resulted in a slight inhibitory effect on the biofilm formation of S. aureus. It is known from the literature data that gram-positive microorganisms, in particular, the S. aureus strains have acquired resistance to the toxic effects of copper [16,42]. Analysis of influence of the synthesized samples on the biofilm formation by the P. aeruginosa strain showed the lower activity and only for samples that did not contain Cu 2+ and Zn 2+ ions ( Figure 4). Thus, in the presence of the sample Na + , CO 3 2− -HAP (with particle sizes in the range of 25-50 nm) the biofilm coefficient decreased only on 28, 49.3 and 41.3% at the amounts of phosphate equal to 5, 10 and 20 mM, respectively.
Inhibition of biofilm formation on 50% compared to the control was found for both samples Na + , CO 3 2− -HAP with particle size of 25-50 and 300-500 nm at their amount of 10 mM. Thus, the obtained results indicate that the activity of the synthesized chemically modified calcium phosphates to the inhibition of biofilm formation by test cultures S. aureus and P. aeruginosa depends on the size of their nanoparticles and nature of dopants.
The similar results of significant antimicrobial action of modified apatite-type calcium phosphates against grampositive microorganisms (S. aureus) in comparison with gram-negative (P. aeruginosa) were previously reported in ref. [43]. Therefore, the decrease in biofilm formation of opportunistic pathogens under the action of synthesized chemically modified calcium phosphates may be associated with a decrease in the number of viable bacteria due to mechanical destruction of bacterial cells on the surface of nanoscale structures. In addition, similar antibacterial effect was also recorded in other nanostructured oxide materials [44,45].
The interaction of synthesized modified calcium phosphates with opportunistic pathogens was investigated by the transmission electron microscopy (TEM) method ( Figures 5 and 6).
It was found that the influence of the synthesized samples on biofilm formation by microorganisms correlates with their ability to attach to the surface of bacterial cells. The high adhesion of Na + , CO 3 2− -HAP and Na + , Zn 2+ , CO 3 2− -HAP samples to S. aureus inhibited the biofilm formation ( Figure 5). A slight adhesion of gram-negative strain of P. aeruginosa to phosphates may explain the low inhibitory activity of sample Na + , CO 3 2− -HAP and its absence for samples Na + , Cu 2+ , CO 3 2− -HAP and Na + , Zn 2+ , CO 3 2− -HAP in the biofilm formation ( Figure 6).

Conclusions
The nanoscale (25-50 nm) particles of chemically modified calcium phosphates for the potential orthopedic application were synthesized by the precipitation method Scale is 500 nm. and their influence on biofilm formation by pathogenic microorganisms was investigated. The prepared apatiterelated calcium phosphates Ca 10−x−y M II x Na y (PO 4 ) 6−z (CO 3 ) z (OH) 2 (M II -Cu 2+ , Zn 2+ ) contained 0.2-0.3 wt% -Na + , 1.16-1.33 wt% -C and 1.0 wt% -Cu 2+ or 1.9 wt% -Zn 2+ . According to FTIR data, the partial substitution of phosphate by carbonate groups occurred in apatite-type structure. Differences in the ability of synthesized modified calcium phosphates to inhibit biofilm formation by grampositive (S. aureus) and gram-negative (P. aeruginosa) bacteria were found. Our results indicated that all investigated samples had an inhibitory effect on the biofilm formation of S. aureus, the highest effect was found for samples Na + , CO 3 2− -HAP and Na + , Zn 2+ , CO 3 2− -HAP (at the investigated amounts of 5-20 mM). The sample of Na + , CO 3 2− -HAP with particle sizes in the range of 25-50 nm (at the concentration range of 5-20 mM) and 300-500 nm (at the concentration of 10 mM) had the significantly less influence on the formation of the biofilm by P. aeruginosa. For samples Na + , Cu 2+ , CO 3 2− -HAP and Na + , Zn 2+ , CO 3 2− -HAP, inhibitory effect on biofilm formation by test strain of P. aeruginosa was not detected. TEM data showed that the activity of calcium phosphates in the processes of biofilm formation by opportunistic pathogens depended on their ability to adhere to the surface of bacterial cells. The obtained results demonstrated the prospects of using synthesized apatite-related chemically modified calcium phosphates in the development of materials with antimicrobial activity for biomedical purposes.

Conflict of interest:
The authors declare no conflict of interest.
Data availability statement: All data generated or analyzed during this study are included in this published article.