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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

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10.1038/s41598-024-67716-0
Article
Quantitative study of early-stage transient bacterial adhesion to bioactive glass and glass ceramics: atomic force microscopic observations
Gour Shivani 12
Mukherjee Abhijit 1
Balani Kantesh kbalani@iitk.ac.in

2
Dhami Navdeep K. Navdeep.dhami@curtin.edu.au

13
1 https://ror.org/02n415q13 grid.1032.0 0000 0004 0375 4078 School of Civil and Mechanical Engineering, Curtin University, Bentley, WA 6102 Australia
2 grid.417965.8 0000 0000 8702 0100 Department of Material Science and Engineering, Indian Institute of Technology, Kanpur, UP 208016 India
3 https://ror.org/02n415q13 grid.1032.0 0000 0004 0375 4078 School of Molecular and Life Sciences, Curtin University, Bentley, WA 6102 Australia
2 9 2024
2 9 2024
2024
14 2033628 2 2024
15 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Antimicrobial potential of bioactive glass (BAG) makes it promising for implant applications, specifically overcoming the toxicity concerns associated with traditional antibacterial nanoparticles. The 58S composition of BAG (with high Ca and absence of Na) has been known to exhibit excellent bioactivity and antibacterial behaviour, but the mechanisms behind have not been investigated in detail. In this pioneering study, we are using Atomic Force Microscopy (AFM) to gain insights into 58S BAG’s adhesive interactions with planktonic cells of both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria; along with the impact of crystallinity on antibacterial properties. We have recorded greater bacterial inhibition by amorphous BAG compared to semi-crystalline glass–ceramics and stronger effect against gram-negative bacteria via conventional long-term antibacterial tests. AFM force distance curves has illustrated substantial bonding between bacteria and BAG within the initial one second (observed at a gap of 250 ms) of contact, with multiple binding events. Further, stronger adhesion of BAG with E.coli (~ 6 nN) compared to S. aureus (~ 3 nN) has been found which can be attributed to more adhesive nano-domains (size effect) distributed uniformly on E.coli surface. This study has revealed direct evidence of impact of contact time and 58S BAG’s crystalline phase on bacterial adhesion and antimicrobial behaviour. Current study has successfully demonstrated the mode and mechanisms of initial bacterial adhesion with 58S BAG. The outcome can pave the way towards improving the designing of implant surfaces for a range of biomedical applications.

Keywords

Atomic force microscopy (AFM)
Bioactive glass
Force–distance measurement
Bacterial adhesion
Bio-mineral AFM probe
Subject terms

Microbiology
Materials science
http://dx.doi.org/10.13039/501100001409 Department of Science and Technology, Ministry of Science and Technology, India DST/SJF/ETA-02-2016-17 DST/SJF/ETA-02-2016-17 Gour Shivani Balani Kantesh http://dx.doi.org/10.13039/501100001797 Curtin University of Technology CIPRS Gour Shivani issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Increasing antimicrobial resistance to antibiotics and rising number of hospital and community acquired infections pose a huge health threat worldwide resulting in poor wound healing and implant failures. Therefore, use of antimicrobial materials for medical implants and coatings is a prudent strategy to tackle implant-related infections and promote healthy wound healing1,2. In the recent years, several studies on antibacterial and anti-biofilm activity of different compositions of bioactive glasses, a class of synthetic biomaterials, have been reported in literature3. Osteostimulation, osseointegration, and angeogenic potential of bioactive glasses have been well established since their advent in 1969, thereby making these ideal for bone and tissue replacement4.

Silica-based bioactive glasses form a strong bond with both calcified and soft tissues by dissolving in bodily fluid and leading to precipitation of hydroxycarbonate apatite (HCA) on the surface5. These bioactive glasses are used clinically for treating bone defects in jaw and orthopaedics caused by traumatic injuries, genetic conditions or disease. Their ability to bond with bones and promote bone regeneration has led to applications in coatings for metal implants, bone grafting, spondylitis treatment, oral and maxillofacial prosthesis and toothpastes for treating tooth hypersensitivity6.

Dissolution of bioactive glass in bodily fluids leads to an increase in local pH from 7 to 10 and higher osmotic pressure due to leaching out of alkali ions which is believed to be the mechanisms driving their antimicrobial action7–9. Bioglass 45S5 (45% SiO2–24.5% Na2O–24.5% CaO–6% P2O5) slurry is used in dental procedures to sterilise the root canal site by raising the local pH up to 12 and it also has the added incentive of imparting bioactive particles to the implant site10. In a 2009 study, S. Hu et al. reported the formation of needle- like 45S5 Bioglass® debris, when 1 ml of E. coli suspension was subjected to 100 mg of 45S5 Bioglass® powder for 1 h, leading to bacterial cell walls damage and ultimately causing cell lysis. The study used melt derived 45S5 Bioglass® powders. This has since been accepted as yet another antibacterial mechanism at play. According to the study, adhesion of bacteria to bioactive glass actually increases its bactericidal activity as close proximity to bacterial cells is important for the above mechanisms to be effective11. Since bacteria experience a continuous shear force due to bodily fluids in-vivo, measuring transient adhesion forces within one second of contact between bacteria and implant material gives a more realistic idea about the likelihood of bacterial arrest on implant surface. Shear force is shown to enhance adhesion residence time of bacteria but the number of adhering bacteria decreases with increase in shear12,13. It is also important to note that the rise in pH in-vivo is more localised than and not as high as in-vitro tests reported in literature due to buffer effect within the body8,14. Therefore, it is important to study nanoscale interactions between bioactive glass and bacteria for small time scales to get a better understanding of the antibacterial mechanism.

The antibacterial effect of bioactive glass is affected by chemical composition, rate of dissolution in test environment and a decrease in particle size usually results in more antibacterial effect in case of bioactive glasses with high SiO2 content15. Clinical trials conducted using bioactive glass S53P4 (53% SiO2–23% Na2O–20% CaO–4% P2O5) for bone grafts and treatment of chronic osteomyelitis has shown a lot of promise16. Based on retrospective single centre cohort studies conducted on 2717,18 and 319 people and retrospective multicentre cohort studies conducted on 1120 people S53P4 bioactive glass granules have exhibited a 90% success rate in treatment of osteomyelitis. Bacteria of genus Staphylococcus are responsible for majority of orthopaedic infections like osteomyelitis, septic arthritis and prosthetic joint infection21,22. Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis and Escherichia coli are the pathogens most commonly responsible for osteomyelitis23. Staphylococcus aureus is most frequently the causative organism for septic arthritis and S. aureus and S. epidermidis account for 65% of prosthetic joint infections24,25. Antibiotics become less effective against these infections due to the formation of protective biofilms. Several studies have been conducted which compare the antibacterial effect of different bioactive glasses against various strains of bacteria26–31. A 2010 study conducted by Mortazavi et al. compared antibacterial effect of three compositions of bioactive glass 58S (57.72 wt% SiO2–35.09 wt% CaO–7.1 wt% P2O5), 63S (62.17 wt% SiO2–28.47 wt% CaO–9.25 wt% P2O5) and 72S (72.88 wt% SiO2–17.49 wt% CaO–9.56 wt% P2O5) against gram positive and gram negative aerobic bacteria. 58S showed the highest antibacterial efficacy with the lowest minimum bactericidal concentration due to higher concentration of calcium resulting in faster dissolution higher pH. Based on bacterial broth dilution method, 58S bioactive glass showed bactericidal activity at concentrations of 50 mg/ml and 100 mg/ml against E. coli, S. aureus and P. aeruginosa whereas 63S showed bactericidal activity at 100 mg/ml and 72S showed no bactericidal effect26. An in vivo study conducted on a rabbit model by Wheeler et al.21 compared the resorption and bone ingrowth using sol–gel 58S, 45S5 and sol–gel 77S bioactive glass grafts (critical sized cancellous defect filled with particulates of bioactive glass). The 12 week study showed highest bone to graft ratio for 58S graft and stiffness of closest to that of natural bone, better than 45S5 bioactive glass which is the first and most widely used composition. Despite its antibacterial efficacy and excellent bioactivity 58S bioactive glass has received relatively less attention in research studies. In this study, we explore the antibacterial effect of 58S bioactive glass in great detail with special focus on initial adhesion.

Initial adhesion between bacteria and biomaterial surface is influenced by a variety of factors like environment conditions, material surface characteristics, bacterial characteristics and presence of serum or tissue proteins21. Gram positive and gram-negative bacteria interact differently with biomaterial surface due to different surface adhesins and AFM studies have been used to better understand these interactions32–34. Gram-negative bacteria express fimbrial, non-fimbrial and discrete polysaccharide adhesins whereas gram-positive bacteria express “Microbial surface components recognizing adhesive matrix molecules” (MSCRAMMs) and pilus fibres which form by covalently bonded pilin units. Fibronectin-binding proteins (FnBPs) are expressed by both35. An AFM single cell force spectroscopy study reported binding strength in the range of ~ 0.05 to ~ 2 nN for gram-positive adhesin-ligand interaction36. AFM nanomechanical studies have revealed the importance of outer membrane of gram-negative E. coli in determining its stiffness and strength37 which in turn affects its resistance to antimicrobial treatments34. Several Single cell force spectroscopy (SCFS) AFM studies have been conducted to measure the adhesion force between bacteria and biomaterial. For example, a study reported higher adhesion between stainless steel 316 and gram-negative bacteria (8.53 ± 1.40 nN and 7.88 ± 0.94 nN) when compared to gram-positive bacteria (1.44 ± 0.21 nN) for 0–60 s of contact time38. However, there are no studies reporting AFM adhesion measurements between bioactive glasses and bacteria. We address this gap by using AFM force spectroscopy to measure adhesion force between bioactive glass 58S and gram-positive S. aureus and gram-negative E. coli for a contact time of 0–1 s.

Contact time also plays a crucial role in bacterial adhesion. The forces responsible for bond strengthening between a surface and cell increase exponentially till they plateau according to the following function39:Ft=F0+F∞-F0exp-tτk

where t = time, F(t) = adhesion force at time t, F0 = adhesion force before bond maturation, F∞ = adhesion force after bond strengthening, τk = characteristic time constant.

With increase in contact time, bacterial adhesion transitions from reversible to irreversible adhesion which is characterised by an increase in number of minor peaks observed in force–distance curves. Initial adhesion can be divided into two stages: (1) primary or docking stage which involves physiochemical interactions between surface and planktonic bacteria. In this stage, the adhesion is reversible. (2) Secondary or locking stage which involves molecular and cellular interactions40. This initial attachment or initial part of adhesion and the transition from one adhesion stage to another with increase in contact time is what we call transient adhesion in this study. Studying transient response of bacteria is important to understand the dynamics of bacterial adhesion in early stages and beginning of transition from reversible to irreversible adhesion.

Physiochemical interactions associated with bacterial adhesion can be classified as short- range and long-range interactions. Long-range forces are non-specific and act at distances > 50 nm, they are responsible for transporting bacteria to the surface. Short-range interactions, which include chemical bond like hydrogen bonding, ionic, dipole and hydrophobic interactions, act at distances < 5 nm and lead to stronger attachment of bacteria to the surface which makes phase two of adhesion possible. Atomic force microscopy (AFM) is a very powerful tool to study biological samples41–47. AFM measures the attractive and repulsive forces between a sharp tip and sample surface by measuring the deflection of cantilever using optical laser, making it a highly sensitive technique which can be used to measure forces from pN to 100 nN range. The interaction between single cells or molecules and material of interest can be achieved by attaching cells, single molecules or bio-mineral to tip-less cantilevers or beads and measuring detachment forces48.

Poisson analysis overcomes the concerns, mainly the macroscopic scale, associated with surface thermodynamic analysis using measured contact angles. Another important advantage of Poisson analysis over other statistical methods is that it does not require large number of measurements which prevents damage of bacterial surface49. When using Poisson analysis, we can get enough data from 7 to 8 spots with a minimum of 10 force distance curves measured at each spot. However, for more robust results, increasing the number of measurements to around 20 per spot is recommended50.

In this study, we will focus on analysis the interaction between bacteria and modified AFM tip in primary or docking stage in order to:Quantify the initial adhesion force, between gram positive and gram negative planktonic bacterial cells with 58S BAG and study the effect of contact time (up to 1 s) on adhesion force in the primary or docking stage.

Improve fundamental understanding of bacterial adhesion to BAG 58S by observing the effect of nano-mechanical properties of bacterial surface and decoupling the long- and short-range interactions.

Study the effect of crystalline phase of BAG on initial bacterial adhesion and long term antibacterial behaviour.

To achieve the goals, we first prepared bio-mineral AFM probes by attaching bio-mineral powder on tip-less cantilevers and immobilised planktonic bacteria on glass slides to make AFM samples51. We used E. coli and S. aureus as the model gram negative and gram-positive bacteria to study the interaction with bioactive glass. The bio-mineral probes were used to carry out AFM force–distance measurements in a fluid cell. In this first of its kind study, we measured the transient adhesion force between bacteria and biomaterial samples and observed the effect of contact time. We used Poisson analysis to de-couple the short-range and long-range forces acting between BG58S-C800 and bacteria to shed some light on the nature of adhesive forces. The bioactive glass powders were prepared in house using sol–gel synthesis and the calcination temperatures were varied to study the effect of degree of crystallinity of 58S bioactive glass: at 800 °C (BG58S-C800: amorphous), 900 °C (BG58S-C900: semi-crystalline) and 1000 °C (BG58S-C1000: semi-crystalline). Long term antibacterial tests were also carried out to get a realistic idea of how the initial adhesion affects the antibacterial action of bio-minerals. We hypothesize that the outcome of this study will help in understanding the antibacterial effect of BG58S. The initial transient interactions between planktonic bacteria and bioactive glass combined with the long-term antibacterial studies can in turn help to shed light on likelihood of bacterial infection and biofilm formation for different crystalline phases. This fundamental knowledge can be used as a starting step for designing antibacterial implant surfaces and may encourage further investigation of antibacterial behaviour of 58S bioactive glass under different conditions and against different bacteria.

Materials and methods

Synthesis of bioactive glass 58S nanopowders

58S bioactive glass (60 mol%SiO2, 36 mol%CaO, 4 mol% P2O5) was prepared using sol–gel method and a modified version of Stöber process. We adopted the method used by J. Fan et al. for 58S bioactive glass synthesis52. Briefly, 9.85 ml tetraethyl orthosilicate (TEOS Sigma-Aldrich Si(OC2H5)4 ≥ 99.0%(GC)) was dissolved in a solution of 60 ml ethanol and 120 ml DI water and the pH of solution was adjusted to 2 using citric acid. 7.693 g of calcium nitrate tetrahydrate (Sigma-Aldrich, ≥ 99.0% Ca(NO3)2·4H2O) was mixed with the solution using magnetic stirrer. In a separate container 1.078 g diammonium hydrogen phosphate ((NH4)2HPO4; Aldrich) and 2% PEG-20000 solution was dissolved in 1.5 L water and the pH was adjusted to 11 using ammonium hydroxide (28% NH3 in H2O; Sigma-Aldrich). The solution containing TEOS and calcium nitrate was slowly dripped into the solution containing diammonium hydrogen phosphate and stirred vigorously using magnetic stirrer while maintaining a pH of 11. The white gel precipitate was washed with DI water and aged in a sealed container for 1 day and then dried in a drying oven at 90 °C for 1 day. The dried gels was then calcined in a muffle furnace at 800 °C (BG58S-C800), 900 °C (BG58S-C900) and 1000 °C (BG58S-C1000) to obtain bioactive glass and glass–ceramics with varying degree of crystallinity. Bioactive glass pellets were made using hydraulic press at 5 Mpa for 60 s. Green pellets of 9 mm diameter and 3.5 mm thickness were produced and subsequently sintered before use.

Phase, composition and microstructural characterization of 58S BAG

Phase information of nano-powders was collected by X-ray diffraction using PANalytical X-pert diffractometer (Panalytical, USA). CuKα radiation (λ = 0.154 nm) and a step size of 0.02° were used for a scan range of 10–50°. Whereas, microstructural images of powders were taken using field-emission scanning electron microscope (Nova nanoSEM 450, FEI; Thermo Fisher Scientific, Waltham, MA) and the elemental analysis of the powders was performed using Energy Dispersive X-ray Microanalysis (EDX).

Bacterial strains and cultivation

Strains E. coli ATC25404 and S. aureus ATC25923 were purchased from In Vitro Technologies Pty Ltd. Australia. Both 5-ml cultures were cultivated in Luria–Bertani medium (Difco) at 37 °C and in a rotary shaker at 150 rpm and harvested in mid-exponential growth phase. Bacterial growth was monitored by measuring optical density at 600 nm and by measuring CFU/ml using serial dilution method.

Antibacterial tests

Two antibacterial tests were performed to examine the antibacterial activity of bioactive glass and glass ceramics over 24–96 h: Kirby–Bauer disk diffusion test to visually examine the bacterial growth near bioactive glass pellets and colony forming unit (CFU) enumeration test to measure the growth of bacteria in presence of bioactive glass over a period of 96 h26,53. The change in local pH ensuing from glass and glass ceramic resorption when exposed to the growth medium was also measured. The pH measurements were carried out for BG58S-C800, BG58S-C900 and BG58S-C1000 over the period of 96 h.

For Kirby–Bauer disk diffusion test, 100 µL of E. coli and S. aureus cultures with 5 × 105 CFU/ml bacteria was spread on sterilised nutrient agar plates. The plates were incubated at room temperature for 10 min. Then, sterilised pellets of BG58S-C800, BG58S-C900 and BG58S-C1000 were placed on the plate and incubated for 1 day at 37 °C. The zone of inhibition (ZoI) formed around the pellets was measured using compound microscope images.

For CFU enumeration test, 100 mg/ml of BG58S-C800, BG58S-C900 and BG58S-C1000 powders was dissolved in LB medium using magnetic stirrer26. 200 µL of fresh E. coli and S. aureus culture with OD600 of 0.1–0.5 was added to the solution and incubated for 96 h. Bacterial growth was measured using serial dilution method and counting the CFUs which were then converted to CFU/ml using dilution factor. The experiments were performed in triplicates. Bacterial solution without bio-mineral was used as control. The pH of biomineral powder and LB medium mixture was measured using pH meter at regular intervals till 96 h.

Planktonic bacteria immobilization and AFM imaging

Live gram positive and gram negative planktonic bacteria were immobilized on glass slides for AFM imaging and force–distance measurement. The glass slides were prepared by first cleaning with soap and ethanol. The slides were then immersed in Piranha solution (30% H2O2, 20% H2O and 50% H2SO4 (conc.) (v/v)) for 2 h and rinsed with sterile DI water. Cells were harvested by centrifugation at 5000 g for 10 min at 5 °C, washed three times in PBS solution and then resuspended in PBS at an optical density of 0.1 at 600 nm. Clean glass slides were prepped with a drop of 1% poly-l-lysine and 20 µL drop of bacterial suspension was added on the glass slides and incubated for 1 h at room temperature. The slides were gently rinsed with sterile DI water to remove loosely attached bacteria. The samples were stored in an ice-box and imaged within 2 h of preparation to ensure that the cells are alive.

Cells were imaged using Peak force QNM of Dimension FastScan AFM (Bruker, USA) in AFM-liquid cell containing PBS (pH 7.2). The AFM images were taken while simultaneously viewing the cells under optical microscope. 3D peak force imaging, modulus mapping, height mapping, deformation mapping and adhesion force mapping of live cell surface was performed using ScanAsyst Air silicon nitride probe with a spring constant of 0.4 N/m using tapping mode. The probes were calibrated in liquid before measurement. A scan rate of 0.5 Hz and 512 line pixel was set for a scan size of 2 µm for detailed observation.

Bioactive glass AFM probe preparation

Bioactive glass probes were prepared by attaching bioactive glass powder on tip-less AFM cantilever using Bruker Dimension FastScan AFM. Epoxy glue (Araldite, Australia) was attached to Bruker MLCT-O10 silicon nitride tip-less cantilever using contact mode of AFM. The same cantilever was then approached, while viewing simultaneously under optical microscope, to a glass slide containing bioactive glass powder agglomerate (< 1 µm) using AFM contact mode and held in contact with the agglomerate particle for 10 s to ensure attachment. The probe was allowed to cure for 24 h before use. The probes were cleaned with ethanol and UV to remove any loosely attached particles or biological matter and observed under the microscope to ensure that they are fit for use.

AFM force–distance measurements

The adhesion measurements were performed using Dimension Icon AFM (Bruker, USA) in AFM-liquid cell containing PBS (pH 7.2). The bio-mineral probes, with a spring constant of 0.02 N/m, were approached and retracted using piezo sensor several times and measurements were taken at different spots on a cell. For averaging adhesion values of each bacteria-biomineral system, we repeated the experiment with 3 identical probes and on 3 different cells for each probe.

In our method, we reversed the position of bacteria and bio-interface used in traditional Single Cell Force Spectroscopy (SCFS). We used bio-mineral probes instead of live cell probes as they are more durable and therefore reduce the number of probes required to ensure that F–D curves are measured for live cells54.

All force–distance curves were recorded using an approach and retraction speed of 2 μm/s and maximum loading force of 1nN. The adhesion force between BG58S-C800, BG58S-C900 and BG58S-C1000 probes and planktonic cells of E. coli and S. aureus was measured for 5 contact times (0 s, 250 ms, 500 ms, 750 ms and 1 s). The probes were cleaned with ethanol and UV to remove any biological matter before and after use and monitored using before every use using optical microscope. They were also tested on clean glass slide to ensure that no protein residue is left attached to them before any measurement. The probes were calibrated in liquid before measurement.

Poisson analysis

Poisson analysis was conducted by recording 10–20 force–distance curves at 8 distinct points on bacteria. The number of peaks for each force distance curve was calculated. A histogram of force was generated. The fit of force data to Poisson distribution was judged by coefficient of correlation (R2) value. Subsequently, the mean force (λF) and variance (σ2F) was computed for each spot. Linear regression of mean force (λF) and variance (σ2F) was used to decouple the short-range (fSR) and long-range (FLR) forces corresponding to adhesion based on the following equation:σF2=fSR×λF-fSR×FLR

Statistical analysis

All experiments were carried out in triplicates. All retraction curves were analysed using NanoScope analysis 1.9 and MATLAB R2020b. Poisson analysis was carried out using MATLAB R2020b. All values sets used for calculating mean and standard deviation are considered significant at p < 0.05.

Results and discussion

Microstructure and phase analysis

The XRD pattern in Fig. 1a shows a broad hump and absence of any crystalline peaks confirming that the powders calcined at 800 °C (58S-C800) are completely amorphous. XRD patterns of BG58S-C900 (Fig. 1a) and BG58S-C1000 (Fig. 1a) powders calcined at 900 °C and 1000 °C respectively show a broad hump with emergence of peaks corresponding to pseudowollastonite (Ca3Si3O9), wollastonite (CaSiO3), quartz (SiO2) and calcite phase (CaCO3) indicating the beginning of crystallisation. In this work, we have explored the effects of this phase change on initial adhesion and antibacterial behaviour of bioactive glass.Figure 1 (a) X-ray diffraction patterns of BG58S-C800, BG58S-C900 and BG58S-C1000 calcined at 800 °C, 900 °C and 1000 °C respectively, (b) scanning electron microscopy image of BG58S-C800 powder, (c) energy dispersive X-ray microanalysis (EDX) of BG58S-C800 powders.

The powder size (Fig. 1b) is obtained in the size range of 50–80 nm. The presence of calcium, silicon and phosphorous is confirmed via elemental quantification (Fig. 1c). The Si:Ca:(P2) molar ratio of the powder was calculated using EDX elemental analysis. The ratio 60.68 mol% Si 34.86 mol% Ca and 4.46 mol% (P2) is observed to be consistent with bioactive glass 58S composition, thereby confirming successful synthesis of BG58S bioglass55.

Antibacterial tests

The results of disc diffusion test show bactericidal activity of BG58S-C800, BG58S-C900 and BG58S-C1000 (Fig. 2). There is a distinct zone of inhibition around the BG58S-C800 and BG58S-C900 samples, whereas the BG58S-C1000 disc shows no zone of inhibition. The dissolution is higher for more amorphous samples (for both E. coli and S. aureus) and, consequently, the zone of inhibition is also larger.Figure 2 Kirby Bauer disk diffusion test: zone of inhibition (ZOI) around samples indicate bactericidal effect of (a) BG58S-C800 against E.coli , (b) BG58S-C900 against E.coli (c) BG58S-C1000 against E. coli (d) BG58S-C800 against S. aureus, (e) BG58S-C900 against S. aureus (f) BG58S-C1000 S. aureus.

In Fig. 3a, a significant decrease in E. coli viability is observed in the presence of BG58S-C800 and BG58S-C900 when compared to the control sample. S. aureus’ growth is also greatly affected due to the presence of BG58S-C800 and BG58S-C900 (Fig. 3b) but the effect is less pronounced when compared to E. coli in Fig. 3a. This shows that BG58S-C800 and BG58S-C900 inhibit bacterial growth and they are more potent against gram-negative bacteria. Semi-crystalline BG58S-C1000, however, does not restrain the growth of either bacteria.Figure 3 CFU enumeration test: (a) growth rate of E. coli in presence of different bio-ceramics, (b) growth rate of S. aureus in presence of different bio-ceramics, (c) schematic representation of dissolution and antibacterial activity bioactive glass and glass ceramics and effect of by crystalline phase (d) pH change in growth media.

A higher antibacterial activity and bacterial resistance is observed for more amorphous phases calcined at higher temperatures. This can be understood by observing the change in pH with time in Fig. 3d. Increase in pH is due to dissolution of bioactive glass in fluid which leads to leaching out of ions, demonstrated schematically in Fig. 3c. Therefore, completely amorphous bioactive glass is more antibacterial than semi-crystalline phases calcined at higher temperatures. Also, the antibacterial effect is stronger against gram negative E. coli, this can be due to a thinner peptidoglycan layer as compared to gram positive S. aureus56.

AFM images and biomechanical mapping

AFM images of live planktonic cells captured the structural details and biomechanical properties of the cell surface. E. coli cell (Fig. 4A1,A2) has concentric oblong ring like patterns and S. aureus cell (Fig. 4B1,B2) has an uneven morphology with nano-ridge like patterns. These features correspond to accumulation of macromolecules on the bacterial surface which are characteristic of a bacterial strain. High resolution images of such macromolecules (proteins, lipids) have been reported in literature57. These macromolecules include surface proteins which are responsible for initial adhesion of bacteria.Figure 4 AFM peak force error image, modulus map, adhesion map and height map of bacterial cells on glass slides using ScanAsyst air silicon nitride tip (A1) peak force images of E. coli planktonic cell, (A2) height map of E. coli planktonic cell, (A3) adhesion map of E. coli planktonic cell, (A4) LogDMTModulus (logarithmic of elastic modulus based on DMT model) map of E. coli planktonic cell, (B1) peak force image of E. coli planktonic cell, (B2) height map of S. aureus planktonic cell, (B3) adhesion map of S. aureus planktonic cell, (B4) LogDMTModulus map of S. aureus planktonic cell.

E. coli adhesion map (Fig. 4A3) visualises and delineates regions/nano-domains of heightened adhesive activity and reduced adhesive interaction on the cell’s surface. Adhesion values up to 750 pN are measured in adhesive parts of the surface and repulsion up to 970 pN observed in other areas. Note that these adhesion maps are measured using silicon nitride tip in AFM tapping mode and these values are with respect to the AFM tips’ interaction with glass surface. This information helps us understand the findings of adhesion measurements (F–D curves) better. Similar observations are made in case of S. aureus cell surface (Fig. 4B3), the distribution of adhesive and non-adhesive domains is not as symmetric as in case of E. coli. The highly adhesive domains show adhesion values as high as 3.8 nN and other areas have shown repulsive forces as high as 640 pN. The LogDMTModulus (logarithmic of elastic modulus based on DMT model58 values are also mapped (Fig. 4A4,B4)) giving us a deeper insight on the effect of macromolecule placement on cell stiffness. E. coli cells are relatively larger in size providing more surface area for adhesive bonds to form. Further, the uneven distribution of adhesive domains on S. aureus cell envelop further limits the area available for adhesion59.

Adhesion forces between bioactive glass and planktonic bacteria

Representative force–distance curves (retraction) and adhesion force histograms are summarized in Fig. 5. The major and minor peaks of a retraction curve may correspond to any of the following events: breaking of receptor-ligand bond, protein unfolding, mechanical protein stretching or de-adhesion of single cell-biointerface. A closer look at the retraction curve shape and force values gives us a lot of information about molecular events at play60.Figure 5 Representative retraction curves and adhesion force (Fadh) values for interaction of BG58S-C800, BG58S-C900 with planktonic bacteria at different contact times (0–1 s) (a) representative curves (retraction) of BG58S-C800 probe with e. coli, (b) force histograms showing distribution of adhesion force values of BG58S-C800 probe with e. coli, (c) representative curves (retraction) of BG58S-C800 probe with s aureus, (d) force histograms showing distribution of adhesion force values of BG58S-C800 probe with s aureus, (e) representative curves (retraction) of BG58S-C900 probe with e. coli, (f) force histograms showing distribution of adhesion force values of BG58S-C900 probe with e. coli, (g) representative curves (retraction) of BG58S-C900 probe with s. aureus, (h) force histograms showing distribution of adhesion force values of BG58S-C900 probe with s. aureus.

In Fig. 5, the adhesion force values increase for all bacteria-bio mineral system with every 250 ms increment in contact time. A decrease in adhesion force observed with increase in crystallinity. The representative curves (Fig. 5a,c,e,g) show an increase in number of minor peaks and Fig. 6d shows an increase in number of total peaks which indicate an increase in number of bonds formed between bacteria and probe. Each peak corresponds to either of the following: a protein unbinding event, ligand-receptor bond breakage or protein unzipping depending on the shape of the retraction curve61. In Fig. 5a, multiple binding events are observed between E. coli and BG58S-C800 and the number of binding events increase with time. The area under the curve is also much larger (Fig. 6c) and increases with contact time. In Fig. 5e, maximum number of ligand-receptor bonds are observed indicating a very strong bonding in a small contact time, indicating a higher likelihood of transition of E. coli BG58S-C900 adhesion from reversible to irreversible. The area under the curve is higher in E. coli (Fig. 6d) indicating a higher work of adhesion required for detachment as compared to S. aureus (Fig. 5c,g). In Fig. 5c, protein unbinding are unzipping events are largely responsible for bond strengthening between S. aureus planktonic cell and BG58S-C800. More number of binding events are observed (Fig. 5c-inset) which indicates that more ligand receptor bonds are forming between biomineral tip and bacteria in short period of time. In Fig. 5g, protein unzipping and unbinding are primarily responsible for adhesion strength and the number of unbinding events is the minimum of all cases indicating a relatively weak adhesion.Figure 6 Mean adhesion force (Fadh) and standard deviation values of F–D curves and average work of adhesion and average number of peaks for F–D curves using biomineral probes (a) BG58S-C800 probe with E. coli and S. aureus planktonic cell sample (b) BG58S-C900 probe with E. coli and S. aureus planktonic cell sample, (c) mean work of adhesion and standard deviation for F–D curves: BG58S-C800 probe and planktonic bacteria (d) average number of peaks and standard deviation for each F–D curves: BG58S-C800 probe and planktonic bacteria.

Fadh values are the adhesion force values corresponding to the biggest de-adhesion peak. The average Fadh and standard deviation is plotted in Fig. 6. Figure 6a shows an increase in average adhesion force between both bacteria and BG58S-C800 by ~ 0.25 nN with each 250 ms increment of contact time. The spread of adhesion force values is higher for E. coli than in case of S. aureus, with adhesion force values as high as 6 nN as compared to 3 nN for S. aureus within 1 s of contact. This can be understood by revisiting the adhesion maps of both cells in Fig. 4 and observing the contrast in adhesion values over both cell surfaces. E. coli has more adhesion sites distributed throughout the cell. In general, there is a decrease in adhesion between bacteria and bio-mineral with increase in crystallinity.

A low initial adhesion indicates a lower chance of bacterial colonisation. However, a high initial adhesion does not mean a high chance of bacterial infection in case of bioactive glass, given their antibacterial mechanism (Fig. 3). The bioactive glass and glass ceramics form a much stronger bond with E. coli as indicated both by the higher de-adhesion force values and larger area under the curve. Bond strengthening between bacteria and bio-mineral is comprised of short-range and long-range force components. Poisson analysis is used to decouple these forces and calculate the change in their contributions with contact time.

Poisson analysis

The values calculated from adhesion force analysis of E. coli and S. aureus with BG58S-C800 are listed in Table 1. A negative force value indicates attraction and a positive force value indicates repulsion. The high value of R2 (~ 0.9 and higher) in most cases indicate a good fit of our values to the model in general. However, low values R2 (less than 0.5) are observed in some cases (S. aureus at 0 s and 250 ms) can be due to very low contact times at which the measurements were carried out and improve as we increase the contact times to values over 500 ms.Table 1 Short-range (fSR) and long-range (FLR) force values calculated using Poisson analysis for interaction between bacteria and BG58S-C800.

Time (ms)	Escheria coli	Staphylococcus aureus	
fSR (nN)	FLR(nN)	R2	fSR(nN)	FLR(nN)	R2	
0	− 0.04 ± 0.00	− 0.03 ± 0.00	0.99384	− 0.03 ± 0.01	0.12 ± 0.00	0.42845	
250 ms	− 1.35 ± 0.13	− 0.20 ± 0.09	0.9711	− 0.15 ± 0.05	− 0.21 ± 0.03	0.49286	
500 ms	− 2.11 ± 0.10	− 0.24 ± 0.11	0.99036	− 0.17 ± 0.01	0.19 ± 0.01	0.95698	
750 ms	− 3.32 ± 0.08	− 0.70 ± 0.09	0.99633	− 0.39 ± 0.08	− 0.50 ± 0.08	0.88097	
1 s	− 5.05 ± 0.25	− 1.07 ± 0.31	0.98143	− 0.26 ± 0.05	0.24 ± 0.05	0.86619	

Short-range interactions between bacteria and surface are responsible for permanent adhesion of bacteria to a surface62. We observe that the bond-strengthening between E. coli and bioactive glass is mainly driven by short-range forces (fSR = − 5.05 ± 0.25 nN at 1 s), the long-range contributions are much lower (FLR =  1.07 ± 0.31 nN at 1 s). For S. aureus, the short-range force values increase with time but not as sharply as E. coli. An effect of this was observed in adhesion measurements (Fig. 5b) where we saw the adhesion force histograms show much higher force values for E. coli adhesion. This also explains the larger work of adhesion i.e. area under the retraction curve observed for E. coli (3.17 × 10–15 J at 1 s) compared to that for S. aureus (3.78 × 10–16 J at 1 s). We can conclude, based on short-range force contributions and number of peaks, that E. coli is likely to adhere much sooner than S. aureus and therefore is more likely get arrested and form a strong irreversible bond with BG58S-C800. This is due to the stronger short-range interactions made possible by the many adhesive nano-domains spread over E. coli surface.

It is important to note that force values calculated using Poisson analysis comprise of forces associated with major and minor deadhesion peaks and not just the Fadh reported in Fig. 5 i.e. it takes into account all the adhesive events50. Therefore, the force values reported in Table 1 and Fig. 7, which were calculated using Poisson analysis, may be much higher than the average values reported in Fig. 6, which only considers the adhesion force value of the largest de-adhesion peak.Figure 7 Schematic representation of (a) AFM force–distance measurement using bio-mineral probe, (b) bacterial interaction with amorphous bioactive glass 58S based on poisson analysis.

Figure 7 schematically summarises the key aspects of this AFM study. Figure 7a illustrates the preparation of bio-mineral probe and force spectroscopy measurements. Figure 7b conveys the key findings of this study. As observed in Fig. 4, the adhesive nano-domains cover a greater area on E. coli surface than on S. aureus surface. This implies a greater number of binding sites (Fig. 6d) and more binding proteins on the E. coli surface as compared to that of S. aureus surface. The results is more number of adhesive interactions and binding events, which are indicted by major and minor peaks in representative graphs reported in Fig. 5 and total number of peaks and energy reported in Fig. 6, for E. coli planktonic cells as compared to S. aureus planktonic cells. The outcome of these interactions is the different adhesive force values reported in Table 1.

Conclusion

In this study, the use of AFM for nanomechanical analysis has provided insights on transient adhesion between 58S bioactive glass and S. aureus gram positive and E.coli gram negative bacteria surfaces.

The conclusions of the study can be summarised as follows:Long term antibacterial studies confirm that completely amorphous bioactive glass is more antibacterial than the semi-crystalline glass ceramics, which may be attributed to increased pH encouraging greater dissolution of amorphous phase and leaching out of ions. The antibacterial effect is observed to be stronger against gram negative E. coli, ascribed to its thinner cell envelope of peptidoglycan layer than that of gram-positive S. aureus.

Analysis of AFM force–distance curves for interaction between BG58S bioactive glass and bacteria shows a similar average adhesion values (0.3 nN at 0 s to 1.4 nN at 1 s) for both E. coli and S. aureus. However, average work of adhesion values are much higher E. coli (3.17 × 10–15 J at 1 s) compared to that for S. aureus (3.78 × 10–16 J at 1 s) indicating a much stronger adhesion.

Greater number of minor adhesion peaks for E. coli (2.61 per F–D measurement at 1 s) compared to that for S. aureus (1.41 per F–D measurement at 1 s) suggest a much stronger bond and beginning of transition from reversible to irreversible adhesion.

Semi-crystalline BG58S-C900 shows strong transient adhesion (0.18 nN at 0 s to 3.56 nN at 1 s) with E. coli and lower antibacterial effect in long term studies. Therefore, it is not a recommended antibacterial bio-mineral.

Poisson analysis confirms bond strengthening due to increase in attractive short-range forces, the effect being more significant for E. coli (0.04 nN at 0 s to 5.05 nN at 1 s) than S. aureus (0.03 nN at 0 s to 0.26 nN at 1 s). Stronger transient adhesion of E. coli to bioactive glass 58S is explained by presence of more adhesive nano-domain on the cell surface and in general larger cell size compared to S. aureus.

Further research can be carried out comparing different compositions of bioactive glasses and their response to other commonly prevalent bacteria. Single molecular force spectroscopy studies can also be carried out to identify the dominant proteins responsible for adhesion and designing strategies for reducing bacterial adhesion.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-67716-0.

Acknowledgements

The authors acknowledge Curtin University for supporting with funds for HDR scholarship and research at Biologically activated materials laboratory in the School of Civil and Mechanical Engineering. Dr. Thomas Becker is acknowledged for AFM training and support. Kantesh Balani acknowledges Swarnajayanti fellowship, DST, Govt. of India (project no. DST/SJF/ETA-02-2016-17). Dr Navdeep K Dhami acknowledges Australian Research Council Discovery grant (DP220101990) for the funding. Mr. Indrajeet Singh is acknowledged for assisting with creating the schematic.

Author contributions

SG designed and performed the experiments, analyzed the data and wrote the main manuscript. ND, AM and KB supervised the experiments, analysed the results and edited the manuscript. All the authors reviewed the manuscript.

Data availability

Authors declare that all data supporting the findings of the study are available within the paper and its Supplementary Information files.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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