Physical and chemical factors influencing antimicrobial activity of the drugs
SEM images reveal that
S. aureus adhered to the surface of the active drug samples. Surface nature (surface roughness and charge) of the drug particles can influence the adhesion of the bacterial species to the surface of the drug particles [
34]. Ion oxides are characterized by rough surfaces despite the smooth cleavage surfaces in mica and surface of clay minerals [
17,
34,
35]. Therefore, the contact between mineral particles and the bacteria could be expected to be strong in the drugs that were rich in iron oxides (ABL1, ABL2 and AbBh) compared to AbCh that contained a relatively low amount of iron oxide (Fig.
3).
Electrostatic attraction between the microbes and the drug particles is another factor that would contribute to microbe lethality. Bacterial cell wall has a net negative charge both in Gram-positive and -negative bacteria due to the presence of teichoic acids (rich in phosphate), phospholipids and lipo-polysaccharides in their structure [
33,
36]. Iron oxides on the other hand have a net positive charge over a wide range of pH [
2‐
7] conditions. Negatively charged bacteria could readily adhere to the drug particles (ABL1, ABL2 and AbBh) that are rich in positively charged iron oxide sites [
37,
38]. Drug particles of AbCh could be having a relatively low negative surface charge compared to the other drugs due to the presence of high fractions of clay minerals [
39,
40]. The electrostatic repulsions between the microorganism and AbCh drug particles could be a factor contributing to the inactivity of AbCh drug.
Although electrostatic attractions could be expected in both Gram-positive and -negative bacterial species, the antimicrobial effect was observed in the current study only in Gram-positive bacteria. This difference in observation could be explained by the dissimilarities of Gram-positive and Gram-negative bacterial cell walls. The cell wall of Gram-positive bacteria has a thick layer of peptidoglycan while Gram-negative bacteria have a thick lipid bilayer on the outside, which is selectively permeable, in addition to the thin peptidoglycan layer. The peptidoglycan layers of Gram-positive bacteria are more permeable than the lipid bilayer of the Gram-negative bacteria. Due to this reason, Gram-positive bacteria are much more susceptible to antibiotics than Gram-negative bacteria [
41‐
44]. Similarly, the antimicrobial cations (see below) of the drugs used in the current study are less likely to permeate through the lipid bilayer of Gram-negative bacteria than through the peptidoglycan layer of Gram-positive bacteria. Thus, the drugs were more effective against the Gram-positive organisms than the Gram-negative organisms. Further, drug permeability through the fungal membranes could be slower than the bacterial membranes due to the presence of the chitin cell wall in fungi, and this could be a reason for the drugs to be ineffective against
C. albicans.
Although the physical properties of the drugs appear to influence antimicrobial activity, all the drugs were inactive when the tests were done using the solid growth media (well diffusion assay and agar dilution assay) probably due to the limited diffusion of material at a 10 mg/ml concentration. Chavadi [
45] has observed antimicrobial activity of a mica drug at a much higher concentration (20 mg/ml) against
S. aureus and
E. coli by the well diffusion assay. Since the results of a diffusion assay could depend on the concentration and water-solubility of a particular antibiotic in addition to physical properties of the antibiotic, Chavadi’s results [
45] observed in the well diffusion assay could be attributed to both physical properties and chemical constituents of the drugs. According to the results obtained in the current study where the broth dilution method (Miles and Misra) displayed antimicrobial activity (at 10 mg/ml), it could be that the drug particles were easily dispersed in water (broth) enabling better contact with the microbes when compared to the solid medium in the well diffusion assay.
When considering the antimicrobial results in relation to chemical constituents of the drugs, iron-rich drugs exhibited relatively high antimicrobial activity. Iron is present mostly as iron oxides in the drugs. However, the conventional iron oxide is not considered as an antimicrobial agent unless it is in the form of nanoparticles [
36,
46]. In addition, iron oxides are known to be antibacterial when the metal ions are released to the bacterial environment in aqueous medium [
1,
47]. The current results support the above contentions as the drugs showed their activity only when allowed to disperse in the aqueous BHI growth medium. This could be due to the greater interaction of drug particles with the microbes in liquid media. Further, antimicrobial cations released to the growth medium could easily penetrate through the bacterial cell wall in aqueous medium.
The CEC of a mineral indicates the amount of its exchangeable cations. If an antimicrobial mineral (specifically clay) carries effective antimicrobial cations, the mineral could act as a suitable antimicrobial agent; Fe
2+, Zn
2+, Mn
2+, Cu
2+ and Pb
2+ cations have shown antimicrobial properties in previous studies [
33,
48]. AbCh drug possess high CEC presumably due to the presence of significantly high clay fractions. However, its inactivity as well as growth enhancement in microorganisms indicate that the presence of relatively high amounts of exchangeable cations (Na
+, K
+, and Ca
2+) in AbCh sustains and promotes the growth of rather than causing lethality to the microorganisms; Na
+ ions are essential to the microorganisms for optimum growth [
49] while Ca
2+ and K
+ ions can enhance the growth of microorganisms [
50]. Hence, the antimicrobial activity of the investigated drugs of the current study is determined by their physical and chemical properties.
The doses of the drugs that are lethal to brine shrimp nauplii appear to exceed the 10 mg/ml level. Therefore, the water soluble preparations of the drugs at a concentration of 10 mg/ml could be used as promising antimicrobial agents devoid of cytotoxicity.