1. Introduction
Clay has been employed as a natural remedy since prehistory. Aside from its healing and soothing properties, clay has been investigated for its antibacterial activities, exemplified by the successful application of French green clay in the treatment of Buruli ulcer, a necrotizing cutaneous infection caused by
Mycobacterium ulcerans [
1] and the antibacterial efficacy of clay leachates against
Escherichia coli and methicillin-resistant
Staphylococcus aureus (MRSA) [
2]. These studies have stimulated interest in the identification of clays from other localities that possess antibacterial activity and could be utilized as new antibacterial agents.
In the food industry, studies have found a high degree of cross-contamination of various pathogens including
Salmonella typhimurium, a Gram-negative enterobacterial pathogen and
Staphylococcus aureus, a Gram-positive species also associated with skin infections due to inadequate cleaning and disinfection of cutting boards [
3,
4]. The most common disinfecting agents used to control bacterial proliferation in the food industry are the peroxygens and chlorine-based compounds. In Malaysia,
Salmonella and
S. aureus have been isolated from multiple food environments, but their survival against hydrated clays has not been investigated. In view of emerging science describing clay as an efficient killing agent against several bacterial pathogens, this study investigates the antibacterial activity of clay from Selangor, Malaysia against food-borne bacterial pathogens in an effort to demonstrate the potential of naturally produced clay as alternative sanitizing agents for the food industry.
Only a small proportion of clay minerals have been proven to be antibacterial [
5,
6,
7]. In Malaysia, clay-rich soils are derived from erosion of metamorphic and sedimentary rocks, and marine alluvium deposits [
8]. Locally sourced antibacterial clay has not yet been reported from this region. The search for antibacterial clays should be focused on locations where clay deposits possess characteristics similar to those identified in previous studies. Soil types with a high clay content, acidic pH [
9,
10] and high levels of Al
3+ and reduced Fe
2+ [
6] were located around Selangor, Malaysia based on data compiled by Paramananthan [
8].
The antibacterial activity of clays is somewhat variable, since no natural clay minerals are precisely the same due to differences in mineralogical and geochemical composition. However, the antibacterial activity of clay leachates is widely reported to be due to metal ion toxicity released from the clay mineral interlayer. Although clays have fundamental structural and chemical characteristics in common, each clay mineral has its own unique properties that determine how it will associate with other species. Cunningham et al. [
9] and Williams [
10] reported the importance of pH as a crucial parameter in preliminary assessment of antibacterial activity, as an acidic environment of hydrated clay minerals contributes to antibacterial efficacy by increasing the availability and toxicity of metal ions. Investigation of the antibacterial activity of natural clays against methicillin-resistant
Staphylococcus aureus (MRSA) revealed that illite and montmorillonite perform better than kaolinite, with the high cation-exchange capacity (CEC) of clay allowing free exchange of metal ions from the surface of the particles into the surrounding medium [
11]. In fact, clay minerals with high CEC have been targeted in the creation of synthetic antibacterial materials [
12,
13]. Known antibacterial metal ions, such Ag, Cu, Fe and Al, were commonly selected as active ingredients due to their strong inhibitory and bactericidal effects [
14,
15,
16,
17]. Previously, clay minerals with single metal ion incorporation were actively sought [
18,
19]. However, more recent studies suggest that antibacterial activity is due to multiple metal species working in concert, particularly in the presence of elevated iron concentrations [
6,
9].
This study aims to discover clays with antibacterial activity in the tropical soils in Malaysia and to examine their antibacterial activity against representative food-borne pathogens. Although different in geological setting, knowing the properties of antibacterial clays from published work helped with the screening and selection of clays with potential antibacterial activity. The outcome of this study will serve to guide future studies aimed at screening clays from soil samples for antibacterial activity and evaluating their potential as sanitizing agents in the food industry.
3. Discussion
The aim of this study was to investigate the potential antibacterial properties of clays obtained from tropical soils. Most of the known antibacterial clays, such as the French Green and Oregon Mineral Technologies (OMT) clays, are of hydrothermal origin and contain mixed illite-smectite (expandable clay) minerals [
5,
6,
23,
24]. In searching for clays with antibacterial activity in the tropical region, six types of soil with high clay content were selected for this study. However, our preliminary results showed that high clay content in soil samples did not necessarily influence their antibacterial properties. Munchong and Batu Anam soils (>70% clay) were expected to have better potential for antibacterial activity, as clays have an important role in buffering water pH to conditions where metal ions are soluble [
10]. It was also anticipated that a more acidic leachate would prove to be superior in antibacterial activity, as metal ions become more bioavailable and potentially more toxic in a low-pH solution [
25]. Our preliminary disc diffusion testing showed that the acidic condition of the six clay solutions was not effective enough to inhibit the bacterial growth by itself. The antibacterial activity of Batu Anam and Bernam clay soils (pH > 5) against
Salmonella typhimurium was not significantly different from Carey and Serkat (pH < 3) or Melaka (pH 5). Only the Munchong clay leachate with a pH 3.60 produced a significantly greater zone of inhibition (11.00 ± 0.71) when tested against the Gram-negative
Salmonella typhimurium. In contrast to the Gram-negative bacterial screening, the Carey leachate showed the greatest activity (7.63 ± 0.48) against the Gram-positive
S. aureus. The remaining clays with differing levels of acidity were less effective at inhibiting bacterial growth. In general, the leachates showed larger zones of inhibition with
Salmonella typhimurium relative to
S. aureus, indicating that Gram-negative bacteria are more susceptible. The variation found in these initial experiments may indicate that the soil preparation method and assay by disc diffusion have some limitations as means of determining the antibacterial efficacy of different types of clay samples.
Sieving served as a simple approach to collect and rapidly prepare the six soil samples. The leachates were prepared by the sieving method (untreated clay), in which larger grains of sand and silt, having particle sizes of 50–500 μm and 2–50 μm respectively, are likely to be retained. It is possible that aggregation of the clay particles and inconsistent distribution of the clay fraction could negatively affect their antibacterial activities. Particle-size fractionation is often undertaken to ensure optimal yield of the fine-sized clay mineral fraction (<2 µm) through either sedimentation or centrifugation methods. Antibacterial clays sourced from hydrothermal mineral deposits, which are mineral rich, were simply air-dried, ground and sieved before testing to prevent oxidation that could influence antibacterial activity [
24]. Other studies on antibacterial clays employed the centrifugation method [
26,
27]. Particle size analysis revealed that Carey has a silty clay loam texture with less than 35% clay content, whereas the clay fraction was dominant (>70%) in Munchong soil. Due to the high volume of organic matter in our soil samples [
8], the sedimentation approach was undertaken to recover the clay fraction, thus yielding the treated clay. The sedimentation method involves pre-treatment with hydrogen peroxide for organic matter removal and Calgon solution as a dispersing agent to produce the treated samples. However, such chemical treatment can have an effect on the dissolution of mineral constituents [
28]; hence, samples were washed thoroughly with distilled water to minimise this effect.
The treated Carey clays (suspension and leachate) proved the most effective at killing
Salmonella typhimurium. However, the viability of
S. aureus was also reduced by the Carey clay suspension (>3 log) and leachate (<2 log), respectively. The untreated Carey and all the Munchong clays showed little or no antibacterial activity against either bacterial species. The difference in susceptibility between Gram-negative and Gram-positive bacteria has been observed with clay mineral leachates previously [
9,
10,
26]. Otto et al. [
29] reported bactericidal activity on the Gram-negative
Escherichia coli, whereas a methicillin-resistant
S. aureus strain was killed at a slower rate. This is likely to be due to differences in the cell-envelope architectures of these bacteria, with the outer membrane of Gram-negatives, absent from Gram-positives, representing a permeability barrier to toxic components. Caflisch et al. [
30] reported that OMT clay suspensions are more effective in killing multiple strains of representative Gram-negative (
E. coli) and Gram-positive (
S. aureus) bacteria than clay leachates at the same concentration. While the previous study employed clay at 200 mg/mL in both suspensions and leachates, our study used a higher concentration (500 mg/mL), although the outcome is similar. These findings support a direct antibacterial contribution for clay minerals, rather than metal ion toxicity in the solution chemistry being the sole factor, as previously concluded with clay leachates [
31].
We found that both untreated Carey and Munchong samples possess low CEC values, whereas the CEC values of both treated clays were higher. This fits with a higher distribution of clay in the treated sample, as the larger particles have been removed during the purification procedure for treating the clay. The increased yield of clays in the treated samples creates a larger surface area due to the finer particle size, providing enhanced clay–bacterial contacts in antibacterial assays. Although this explanation fits with the antibacterial properties of the Carey treated clays, it does not account for the reduced efficacy of the Munchong treated clays. Hence, the higher portion of clay in samples is not the sole contributor to their antibacterial properties.
The activity could be influenced by the pH of the sample, as acidic conditions should favour metal ion solubility [
9]. As noted previously, pH alone is not the only factor contributing to antibacterial activity. While both
Salmonella typhimurium and
S. aureus are sensitive to acidic conditions [
32,
33],
Salmonella typhimurium displays better capacity to survive low-pH stress (pH 3.3) [
34]. However, in an acidic environment, abundant protons saturate metal binding sites in the solution, maximising the concentration of soluble metal ions. Metal ions become increasingly more bioavailable and potentially more toxic in a low-pH solution [
25]. It seems likely that the abundance of metal ions in treated Carey clay samples, especially in an acidic environment, contributes to bacterial killing due to metal ion toxicity.
Carey is an acid sulfate soil, characterised by the presence of pyrite and high Al and Fe content [
22]. XRD analysis confirmed the presence of pyrite, as well as magnetite in the Carey sample. The pyrite and magnetite minerals are sulfur and iron composite minerals, which could potentially contribute as sources of sulfur and iron in the clay leachate and suspension. In our study however, sulfur content was not measured. Future evaluation could be performed to analyse the potential toxicity caused by metal ions and the influence of pH on antibacterial activity.
In our effort to further determine the role of metal toxicity, we discovered that the soluble metal content was higher in treated Carey clay samples compared to the untreated ones. This finding fits with the greater antibacterial activity of treated Carey, which is also more acidic than its untreated counterpart, as judged by the bacterial log reduction value. These results are similar to previous studies which have shown that metal ion toxicity is responsible for bactericidal activity against
E. coli and
S. aureus (MRSA), with toxicity directly associated with the released metal ions (Fe
2+, Cu
2+ and Zn
2+) in an acidic environment [
2,
31]. Aqueous leachates derived from known antibacterial clays tend to have high levels of Mg, Al and Ca, with some containing exceptionally high concentrations of Fe [
10]. Previous work has concluded that Ca was not significant in bacterial cell death [
5,
35], hence we did not include analysis of Ca levels in our study. For OMT clay minerals, the dissolution of reduced Fe
2+ and Al
3+ was identified as the active antibacterial component, acting in concert to damage bacterial membranes [
6]. Speciation modelling by Otto and Haydel [
31] suggests that increasing the soluble metal ions Cu
2+ and Fe
2+ are the key to increasing the antibacterial activity of the leachates.
The available metals (Al, As and Cu) were higher in the treated Munchong than the untreated leachate by 280-, 80- and 4.5-fold, respectively. The higher soluble metal content could be attributed to the higher clay percentage in the treated sample, as evidenced by their larger CEC values compared to the untreated sample. However, metal analysis revealed much lower Fe, Mg, Mn, Ni, and Zn leachate content compared to both of the Carey leachate samples. This could mean that the metal content in untreated and treated Munchong is below the threshold value that could cause death of bacteria. Analysis of soluble metals in the suspension form of the antibacterial treated Carey sample revealed a significantly higher Ag and Mg content, although much lower amounts of Al, Cu and Fe in comparison to its leachate form. Both leachates and suspension killed
Salmonella typhimurium, while the suspension displayed slightly greater antibacterial activity than the leachate when tested against
S. aureus. Although the differences observed here were relatively small, previous studies have indicated that the continuous supply of metals in clay suspensions results in greater antibacterial efficacy compared to leachates [
6].
Relative to the other three non-antibacterial leachates, the antibacterial treated Carey leachate contains significantly higher levels of available Ag, Al, Cu, Fe, Mg, Mn, Ni and Zn. Treated Carey clay has a greater CEC in comparison to the sieved sample due to increased finer clay fractions in the sample. The elevated quantities of these metals suggest that a greater amount of elements is concentrated in the finer clay fractions, as previously suggested by Williams [
10], and is only released into solution upon hydration. The high total Al and Fe content in the treated Munchong clay did not seem to improve its antibacterial activity. Al is abundant as it provides the structural framework of clay minerals, but may not necessarily be present in a bioactive form. Similarly, Fe may be present in pyrite and hematite within the clay minerals but may not be readily available in a reduced state with its associated higher toxicity. On the other hand, the available metals in leachates, when present above the threshold amount, can cause death of bacteria due to metal toxicity. Williams et al. [
5] reported that the leachate of antibacterial OMT clay contained significantly higher Al, Fe, Cu and Pb content, with the values of 23.9, 51.6, 0.23 and 0.00002 mg/L, respectively. These are among the metals associated with
E. coli death. Although the metal threshold value was not determined, we can conclude, based on earlier studies, that Fe content is likely to make significant contribution to the antibacterial effectiveness of the sample against the test bacteria.
In future work, it would be helpful to determine the amount of the active antibacterial form of Fe (Fe2+) and how this correlates with bacterial toxicity. Further analysis of clay stability and the time course of antibacterial activity would help clarify the contribution that pH makes to metal release and the antibacterial efficacy of the Carey clay. Investigation of the mechanism of action (membrane permeability, cell surface damage and oxidative stress production) would also help to validate the clay materials as a marketable and consumable mineral-based disinfectant/sanitizing agent. In the context of Halal food assurance, the use of clays would be beneficial, not only for Islamic cleansing procedures but for effective removal of bacterial hazards along the food supply chain.