Nadeera Dilshan Wickramasinghea,
A. H. Janaka Sampatha,
Chandrika M. Nanayakkarab,
K. M. Nalin de Silvaa and
Rohini M. de Silva*a
aCentre for Advanced Materials and Devices (CAMD), Department of Chemistry, University of Colombo, Colombo 00300, Sri Lanka. E-mail: rohini@chem.cmb.ac.lk; Fax: +94714406263
bDepartment of Plant Sciences, University of Colombo, Colombo 00300, Sri Lanka
First published on 22nd January 2024
The emergence of resistance in detrimental pathogenic bacteria towards well-recognized antibiotics has greatly impacted global medicine, consequently exploring potent antibacterial compounds is becoming a potential area of research. Although photocatalytic metal oxides have been extensively explored in this regard, their applicability is diminished due to the requirement of photon energy. Therefore, in our study, we explored the light-independent antibacterial effect of two unexplored titanium species, known as metatitanic acid (MTA) and potassium titanate, against Staphylococcus aureus, Escherichia coli, and Pseudomonas spp. using the disk diffusion method in Luria–Bertani agar medium, where the well-known antibiotic, gentamicin, was used as the positive control. These two titanium compounds were readily synthesized through a novel process which was originally developed for the extraction of TiO2 from ilmenite. The synthesized MTA was characterized using FT-IR, Raman spectroscopy, XRD, TGA, UV-visible spectroscopy, and SEM. According to our findings, both MTA and potassium titanate exhibited superior light-independent antibacterial properties, where for some concentrations, the effect was even greater than gentamicin. However, nano-TiO2 totally failed as an antibacterial compound against the tested three strains under dark conditions.
For this purpose, inorganic nanoparticles such as titanium dioxide,5–8 zinc oxide,9–13 copper oxide,14–16 and silver17–19 have been widely studied as potential antibacterial compounds and most of them are currently used in clinics.20,21 Of these materials, titanium species have obtained higher merits due to their excellent photocatalytic activity, leading to the formation of reactive oxygen species (ROS) such as hydroxyl, oxo, and peroxo in the presence of UV radiation exposure.22,23 These high-energy species can attack the bacterium, negatively contributing to its vital processes and inhibiting growth.24 Since it is essential to have an irradiation source like UV, there is a greater difficulty in applying this to large-scale clinical uses. Therefore, it would be beneficial and practical if materials exhibited light-independent antibacterial activity.
Titanic acid species are acidic substances having titanium, oxygen, and hydrogen. Although there is a wide variety of titanic acids, including poly acids with layered structures, metatitanic acid (MTA), which has scarcely been studied, is an isolated form with the chemical formula H2TiO3.25–28 The antibacterial effect of MTA and other titanate species has not been heavily investigated.29 Synthesis of MTA using conventional methods is associated with significant drawbacks, including higher time consumption, use of non-greener harsh conditions, and utilization of expensively prepared titanium dioxide.27,28 We isolated MTA as an intermediate in our ongoing research to develop a cost-effective and greener process for TiO2 from Pulmoddai ilmenite in Sri Lanka. Here, in this study, we offered to prepare MTA as a valuable intermediate in the synthesis process of TiO2 from the Pulmoddai ilmenite in Sri Lanka (awaiting patent). We explored the capacity of MTA and its potassium salt in the quest for light-independent antibacterial materials.
This study investigated the light-independent antibacterial effect of MTA and potassium titanate against Gram-negative and Gram-positive bacterial species, namely, Staphylococcus aureus, Escherichia coli, and Pseudomonas spp. In addition to the light-independent activity, the light-dependent activity of the two compounds was also evaluated and compared with respect to three bacterial strains. Additionally, these titanium intermediates were compared with the antibacterial activity of nano-TiO2 (nano-titania) under light and dark conditions.
Fig. 3 X-Ray diffraction pattern of (a) ilmenite raw material (b) TiO2 (bulk) and (c) synthesized MTA. |
The corresponding narrow and sharp diffraction peaks in the obtained XRD pattern can prove the crystalline nature of synthesized MTA. However, most importantly, hydrous titania (TiO2·H2O) in the anatase phase is erroneously described in literature as ‘metatitanic acid’ (H2TiO3) due to some structural similarities between the two compounds, thereby showing that ‘metatitanic acid’ exists as an amorphous compound owing to the presence of broader peaks at the same 2θ angles of the XRD spectrum of anatase phase crystalline TiO2.34,35 Conversely, it is clear that the obtained XRD pattern for synthesized MTA is entirely different compared to that of anatase TiO2, where the characteristic peak of anatase TiO2 at 2θ = 47.82° is completely absent in the XRD pattern of synthesized MTA, although the reflection around 2θ = 25° (25.11° for anatase TiO2 and 24.54° for MTA) appears to be common for both the compounds. However, the presence of intensive sharp peaks such as 2θ = 17.03°, 22.05°, and 26.71°, which were not observed for TiO2, firmly verifies that H2TiO3 possesses a different crystalline structure compared to that of TiO2·H2O. In previously reported work, investigation of proton exchange with lithium titanate (Li2TiO3) to generate protonated form, H2TiO3, (MTA) possesses major reflections in the XRD pattern at 2θ = 18.8° and 36.5°.27 Therefore, sharp XRD peaks at 2θ = 17.77° and 37.04° observed with our synthesized MTA can be sensibly attributed to the crystalline structure of MTA, which is different from anatase TiO2·H2O.
The mean inhibition zone diameters for different concentrations of MTA, potassium titanate, and nano-titania against those mentioned above three bacterial strains (Pseudomonas spp., E. coli, and S. aureus) were calculated and plotted as shown in Fig. 10 and 11.
Fig. 10 Mean inhibition zone diameter shown by different concentrations of MTA against Pseudomonas spp., E. coli and S. aureus, under dark conditions. |
Fig. 11 Mean inhibition zone diameters shown by different concentrations of potassium titanate against Pseudomonas spp., E. coli and S. aureus, under dark conditions. |
According to the one-way ANOVA, the mean inhibition zone diameter significantly differed among the different concentrations of the MTA (p < 0.05) against Pseudomonas spp. at a 5% significant level in both dark and light conditions.
Under dark conditions, as shown in Fig. 7(a) and 10, standard antibiotic gentamicin against Pseudomonas spp. recorded the highest mean inhibition zone diameter of 17.5 ± 0.4 mm. The second (17.1 ± 0.7 mm), third (16.5 ± 0.4 mm), and fourth (16.3 ± 0.6 mm) highest mean inhibition zone diameters against Pseudomonas spp. were given by the MTA concentrations 400 mg L−1, 500 mg L−1 and 450 mg L−1, respectively, under dark conditions. According to the multiple mean comparison test, there were no significant differences with the mean inhibition zone diameter of gentamicin at 400 mg L−1. These results indicated significantly high antibacterial activity of MTA at above three concentrations against the Pseudomonas spp. However, significantly lower mean inhibition zone diameters were given for the rest of the six concentrations, indicating low antibacterial activity at reduced concentrations. The improved antibacterial activity of the compound at higher concentrations could be accredited to the enhanced interaction between MTA and the bacteria, where the optimum MTA concentration was found to be 400 mg L−1. However, the weakening of the antibacterial activity with higher MTA concentrations of 450 mg L−1 and 500 mg L−1 can be attributed to unexpected effects such as saturation or precipitation of the compound under these exceedingly high concentrations, as MTA is prone to get precipitated, causing lower interactions with the bacteria.
However, as shown in Fig. 12(a), the presence of light has further improved the antibacterial activity of MTA against Pseudomonas spp. The highest mean inhibition diameter (20.2 ± 0.4 mm) resulted from the MTA concentration of 400 mg L−1, exhibiting its superiority even compared to gentamicin (17.9 ± 0.7) under light exposure.
Compared to Pseudomonas spp., MTA showed elevated antibacterial activity against E. coli. In terms of both light-dependent and light-independent activity, the mean inhibition zone diameter demonstrated for E. coli significantly differed among the different concentrations (p < 0.05) of MTA at a 5% significant level.
Under dark conditions, the highest mean inhibition zone diameter of 24.9 ± 0.5 mm was observed for E. coli in 500 mg L−1 of MTA (Fig. 7(b) and 10). The second highest mean inhibition zone diameter of 19.9 ± 1.0 mm was shown by the concentration of 400 mg L−1 of MTA. These two concentrations resulted in significantly higher mean inhibition zone diameters for E. coli, where the effect of MTA was greater than that of gentamicin, even in the absence of light. There was no significant difference between MTA and gentamicin for mean inhibition zone diameters recorded at 450 mg L−1 and 350 mg L−1 of MTA. Lower concentrations of MTA (100–300 mg L−1) had shown significantly lower mean inhibition zone diameters than gentamicin. The effectiveness of MTA in inhibiting the growth of E. coli under dark conditions was clearly evident by the experiment's results. Fig. 10 reveals that, compared to the Pseudomonas spp., E. coli was more susceptible to the light-independent antibacterial activity of MTA at the concentrations of 500 mg L−1 and 400 mg L−1.
However, the irradiation of light further increased the antibacterial action of MTA, compared to dark conditions, resulting in higher inhibition zone diameters against E. coli (Fig. 12(b)). Likewise, the mean inhibition zone at the MTA concentration of 500 mg L−1 could be expanded from the diameter of 24.9 ± 0.5 mm up to 25.5 ± 0.2 mm in the presence of light. Similarly, the mean inhibition zone diameter could be widened from 19.9 ± 1.0 mm up to 21.4 ± 0.9 mm with light assistance for the 400 mg L−1 MTA concentration. These results further conclude that the photon energy can enhance the antibacterial capability of MTA.
Concerning S. aureus, the inhibitory effect was significantly different among the different concentrations of MTA (p < 0.05) at a 5% significant level. According to Fig. 7(c) and 10, the highest activities in the dark condition, as reflected by mean inhibition zone diameters (20.4 ± 1.7 mm and 19.7 ± 0.6 mm), were recorded at concentrations of 300 mg L−1 and 350 mg L−1 of MTA, respectively, which is also higher than the activity of gentamicin (17.3 ± 0.3 mm). The remaining concentrations showed growth inhibition (500 mg L−1, 250 mg L−1, and 200 mg L−1), which was not as high as gentamicin. Among the tested nine concentrations, five showed intense antibacterial activity against S. aureus, confirming the effectiveness of the MTA as an antibacterial compound. As observed with the two above bacterial species, the antibacterial action of MTA under the influence of light was higher for S. aureus as well (Fig. 12(c)). Mean inhibition zone diameters were 22.6 ± 0.2 mm and 21.9 ± 0.7 mm for 300 mg L−1 and 350 mg L−1 respectively. In outline, MTA against three bacterial strains tested under dark conditions revealed that the Gram-positive bacteria were more affected by the MTA, and it is a more powerful antibacterial compound than the standard gentamicin, even under dark conditions. Optimized MTA concentrations can be listed as 500 mg L−1, 400 mg L−1, and 300 mg L−1 for E. coli, Pseudomonas spp., and S. aureus respectively. However, optimum MTA concentration was found to vary depending upon the bacterial species, and this variation could be reasonably ascribed to the membrane permeability, resistance mechanisms, genetic makeup, and different metabolic pathways of the bacteria, resulting in disparities in the susceptibility to external compounds.2–4
Fig. 8(a) and 11 reveal results that we obtained with potassium titanate against Pseudomonas spp. Accordingly, potassium titanate could be recognized as a more potent antibacterial agent in dark and light conditions. The mean inhibition zone diameters significantly differed among the different concentrations of potassium titanate (p < 0.05) at a 5% significant level. The highest mean inhibition zone diameter of 18.3 ± 1.2 mm was observed in the 500 mg L−1 concentration of potassium titanate, and it was significantly higher than that of the 400 mg L−1 concentration of gentamicin. The 400 mg L−1 gave the second-highest mean inhibition zone (16.4 ± 0.8 mm), and it was not significantly higher than the third-highest mean inhibition zone (15.7 ± 0.3 mm) given by gentamicin. Potassium titanate concentrations of 250 mg L−1 and 450 mg L−1 also recorded mean inhibition zone diameters, which were not significantly different from those of gentamicin. Four concentrations of potassium titanate (300 mg L−1, 200 mg L−1, 150 mg L−1, and 100 mg L−1) showed significantly lower mean inhibition zone diameters against Pseudomonas spp. enhanced antibacterial activity of potassium titanate against Pseudomonas spp. was observed for all nine concentrations under the influence of light (Fig. 13(a)). The maximum light-dependent action against Pseudomonas spp. was also revealed for the potassium titanate concentration of 500 mg L−1, resulting in a mean inhibition diameter of 20.1 ± 0.7 mm. Overall results concluded the suitability of potassium titanate for the control of growth of Pseudomonas spp. due to the markedly higher activities recorded.
Overall, both light-dependent and light-independent antibacterial activity of potassium titanate recorded against E. coli was at lower levels than that of Pseudomonas spp. The mean inhibition zone diameter measured against E. coli for different compound concentrations was significantly different at a 5% significant level (p < 0.05).
Following Fig. 8(b) and 11, the 350 mg L−1 of potassium titanate recorded the highest mean inhibition zone diameter of 14.7 ± 1.3 mm, under dark conditions and was not significantly higher than the inhibition zones recorded by 400 mg L−1 of gentamicin. Concentrations of 400 mg L−1, 500 mg L−1 and 450 mg L−1 of potassium titanate recorded inhibition zone diameters of 14.1 ± 0.8 mm, 13.8 ± 0.2 mm and 12.7 ± 0.9 mm, respectively. However, among the tested nine concentrations of potassium titanate, five concentrations gave a non-significant reaction to that of gentamicin. These results also indicated the effective antibacterial activity of potassium titanate against E. coli, even without photon energy, exerting a similar effect as the standard antibiotic gentamicin. However, under the influence of light, a greater mean inhibition zone diameter (16.2 ± 0.9 mm) was recorded for potassium titanate in the concentration of 350 mg L−1, compared to gentamicin (14 ± 0.4 mm) against E. coli.
Moreover, comparatively higher antibacterial activity of potassium titanate against S. aureus was observed than other two species with significantly different (p < 0.05) mean inhibition zones at a 5% significant level. Fig. 8(c) and 11 disclose that the highest inhibition zone diameter of 18.6 ± 0.5 mm was recorded by the potassium titanate concentration of 500 mg L−1 followed by 450 mg L−1 (17.1 ± 0.3 mm), under dark conditions. These two values were not notably higher than the third-highest mean inhibition zone diameter of 16.1 ± 1.8 mm recorded by gentamicin. Six concentrations of potassium titanate recorded significantly lower mean inhibition zone diameters against S. aureus than gentamicin, showing reduced antibacterial activity at lower concentrations.
The light-dependent activity of potassium titanate was only slightly elevated against S. aureus, where the highest inhibition zone diameter (19.2 ± 0.8 mm) was recorded for the concentration of 500 mg L−1 (Fig. 13(c)).
When the light-independent antibacterial activity of potassium titanate was compared against three bacteria, potassium titanate is a more potent antibacterial compound against Pseudomonas spp. and S. aureus than E. coli. Potassium titanate concentrations can be optimized as 500 mg L−1 for both Pseudomonas spp. and S. aureus, whereas 350 mg L−1 for E. coli, under dark conditions. Further, several concentrations of potassium titanate exhibited high antibacterial activity compared to gentamicin at 400 mg L−1.
The third compound, nano-titania did not show antibacterial activity at all nine concentrations against E. coli, S. aureus, and Pseudomonas spp. (Fig. 9(a–c)) under the dark conditions used in the experiment. However, as revealed in Fig. 14(a–c), nano-titania was successful against the three bacterial strains only in the presence of light, where the mean inhibition zone diameter, recorded for the bacterial species in the presence of different concentrations of nano-titania, was significantly different at a 5% significant level (p < 0.05). The highest inhibition zone diameters of 14.9 ± 0.4 mm, 14.3 ± 0.9 mm, and 12.7 ± 0.7 mm were obtained for Pseudomonas spp., S. aureus, and E. coli, respectively, at the nano-titania concentration of 400 mg L−1.
Fig. 15 depicts the comparison of the susceptibility of the three bacterial strains, i.e., Pseudomonas spp., E. coli, and S. aureus, towards the optimized concentrations of MTA, potassium titanate, and nano-titania, under both dark and light conditions. Both Pseudomonas spp. and S. aureus possess a greater vulnerability towards MTA and potassium titanate than nano-titania in the presence and absence of light. However, E. coli was more affected by MTA than potassium titanate in dark and light conditions. In accordance with the measured inhibition zone diameters, it is clear that the light-independent antibacterial activity of both MTA and potassium titanate was even greater than the light-dependent antibacterial activity of nano-titania. Since the light-independent activity becomes significant in clinical applications, it was evident that MTA and potassium titanate gain merits over nano-titania as effective antibacterial agents. The light-dependent antibacterial activity of nano-titania and the enhanced activity of MTA and potassium titanate under light exposure can be reasonably assigned to the photocatalytic damage to the bacteria caused by Reactive Oxygen Species (ROS), which can be produced by these three compounds in the presence of photon energy.36–44 Most significantly, the light-independent antibacterial activity of MTA and potassium titanate should be explained without regard to the photocatalytic effect due to the absence of photon energy. Although MTA and potassium titanate have not been explored in antimicrobial applications under dark conditions by previous studies, the mechanism of light-independent action of the two species against bacteria can be assessed under similar compounds reported in the literature.45–50 The electron-capturing ability of the Schottky junction created between Fe and titanium oxide has been attributed to the cause of oxidation damage to S. aureus under dark conditions.45 A similar study reports the light-independent antibacterial action of ZnO nanoparticles against S. aureus and Klebsiella pneumoniae, as its attachment to bacterial cell wall. The impact of ZnO on the bacteria is related to the local dissolution of adsorbed ZnO and thus elevated Zn2+ concentration in the bacterial cytoplasm.46 Light-independent antimicrobial activity of ZnO against E. coli has also been related to ROS generated from the surface of the compound47 as well as defects on ZnO surface.50 The significance of possessing surface defects on antimicrobial compounds has been further verified through the contact-based antimicrobial action of MgO against E. coli, S. epidermidis and Pseudomonas spp.49
In our study, both MTA and potassium titanate were successful towards both Gram-positive and Gram-negative bacterial strains, implying several possible pathways for their mechanism of action in the absence of light. Therefore, the light-independent mechanism can be predicted as follows (Fig. 16).
Fig. 16 Proposed mechanisms for the light-independent antibacterial action of MTA and potassium titanate. |
The light-independent mechanism of action can be mainly supposed to be through the interaction of the antipathogenic material with the bacterial cell wall, eventually exerting harmful effects on the microbe due to the membrane's disintegration and the cytoplasmic material's leakage. Adsorption of bacteria on the antimicrobial material is expected to improve the extent of interactions with the cell wall and, therefore, alters the cell membrane integrity even without photon energy.27,46,48 Moreover, being titanium-based catalytic materials, the bactericidal potential of MTA and potassium titanate can be related to their super-hydrophilic properties as well as to their defect sites, which is predicted to possess a greater oxidation tendency due to the presence of oxygen vacancies and Ti4+ in higher concentrations. The presence of such defects causes the generation of ROS upon contact with O2, and ROS generated can profoundly have a detrimental impact on pathogens.47–50 Furthermore, it can be suggested that the direct contact of MTA and potassium titanate with bacterial cell walls leads to phagocytosis of the titanium species by microbes, resulting in alterations of the cytoplasm composition and thereby, hindering vital biochemical processes of the cell.48,51
MTA exhibited light-independent antibacterial properties against all three bacterial species at 100–500 mg L−1 concentrations. For E. coli, 400 mg L−1 and 500 mg L−1 concentrations showed superior antibacterial properties compared to the positive control, gentamicin. For S. aureus, the light-independent effect of 300 mg L−1 and 350 mg L−1 MTA concentrations was also more significant than gentamicin. For Pseudomonas spp. under dark conditions, optimum MTA concentrations of 400–500 mg L−1 were more effective; however, the effect was comparatively lower than that of gentamicin.
Greater antibacterial activity of potassium titanate was found against the three bacterial species. Significantly, the antibacterial activity of potassium titanate (500 mg L−1) under dark conditions was superior against Pseudomonas spp. over the positive control. The concentration of potassium titanate could be optimized as 350 mg L−1 and 500 mg L−1 for E. coli and S. aureus, respectively; however, the light-independent antibacterial effect of the compound was not more significant than that of gentamicin. A well-known antibacterial agent, nano-titania, completely failed to inhibit the growth of any of the bacteria tested under dark conditions. The antibacterial activity of all three titanium compounds, i.e., MTA, potassium titanate, and nano-titania, was enhanced under the influence of light against all three bacterial species. This study identified the light-independent antibacterial effect of MTA and potassium titanate as superior to the light-dependent effect of nano-titania. Another important point is that both MTA and potassium titanate are effective against Gram-positive and Gram-negative bacterial strains, i.e., showing a broad spectrum of activity in both dark and light conditions, an uncommon phenomenon concerning antibacterial compounds. Hence, these two compounds will have a definite advantage over common antibiotics in clinical applications.
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