Antimicrobial materials have been used in various environments. However, the activity of cells at a distance from the antimicrobial materials has not been elucidated. In the present study, the cell growth of Escherichia coli NBRC 3972 was observed at different distances (0 - 300 μm) from the antimicrobial surface under various nutrient concentrations, from full strength nutrient broth (NB) to 1/40 NB. Under higher nutrient concentrations, NB and 1/2 NB, no antimicrobial effect on cell growth was observed at any distance from the surface. Under lower nutrient concentrations, 1/10 NB and 1/40 NB, the growth of cells directly contacting the antimicrobial surface (at 0 μm from the surface) was blocked immediately after inoculation on the surface. However, at distances of 100 - 300 μm from the surface, the cells grew normally for a while, and then stopped the growth; earlier growth discontinuation was observed for cells closer to the surface. It was suggested that the antimicrobial agent (silver ions) is released from the antimicrobial surface into the medium and that the diffusion of the silver ions may influence the lag in the antimicrobial effects observed at distances away from the antimicrobial surface. The present study reveals the possibility that antimicrobial activity in the environments where the antimicrobial material is used depends on the distance from the surface and the surrounding nutrient concentrations.
Antimicrobial materials have been used in various contexts worldwide [1-6]. However, concerns are being raised regarding the overuse of antimicrobial materials and the consequent emergence of bacterial resistance [7-10]. The range of bacterial responses to antimicrobial materials is extensive. Thus, precise evaluation of the antimicrobial activity of various antimicrobial materials is important for their further application and to prevent problems caused by the improper utilization of these materials.
The activity of antimicrobial materials is conventionally estimated by using an indirect method, in accordance with ISO 22196 guidelines (JIS Z2801; 2000) [
In environments, bacteria often form biofilms on the surface of substrates and show resistances to antimicrobial agents [7,8]. Previously, we have clarified that the nutrient ion (, , ,) concentrations in biofilms were much higher than those in the surrounding environment [
In the present study, the growth of Escherichia coli NBRC 3972 on antimicrobial and non-antimicrobial materials was investigated at various distances from the surface and at various nutrient concentrations, by modifying the method used in our previous study [
In the present study, the strain E. coli NBRC 3972 (isolated from human feces [
The microculture method used in the previous study [
When bacterial cells grow in a medium on non-antimicrobial or antimicrobial material, they increase in cell number over time and form microscale colonies. We observed the microscale colonies at a vertical distance of 0, 100, 200, and 300 µm from the surface through the microscope in 2 fields of view (width of 1 field, 160 × 220 µm) and photographed the colonies intermittently at 30 - min intervals. The distances from the solid surface were adjusted using the scale attached to the adjustment handle of the microscope. In the previous study, the colony area was estimated for growth rate constants. On the other hand, in the present study, we determined the number of cells per micro-colony for more precise measurements.
In the present study, the growth of E. coli NBRC 3972 at different distances (0, 100, 200, and 300 µm) from antimicrobial and non-antimicrobial surfaces was directly observed under a microscope using the modified microculture method (
The growth rate constants for E. coli in full-strength NB
agar medium on the non-antimicrobial surface (GPPS BL) were similar for all the different distances from the surface (
On the antimicrobial material (GPPS 1.0%), the growth rate constants for E. coli were similar to those observed on the non-antimicrobial material at every distance for NB and 1/2 NB (
In the previous study, we found that antimicrobial activity on the surface was evident when cells were cultured under low nutrient concentrations, and we deduced that antimicrobial materials retard the nutrient uptake or nutrient utilization efficiency of cells or block the initiation of the cell cycle [
The number of cells per colony after the cessation of cell growth at different distances from the antimicrobial and non-antimicrobial surfaces was investigated. E. coli colonies incubated with NB on the antimicrobial material contained a similar number of cells as those on the nonantimicrobial material at all distances (
E. coli colonies start to grow from a single cell, with the number of cells per colony increasing until the cessation of growth. Therefore, the number of cells per colony after the cessation of growth depends on the growth rate and the growth period of the cells. The growth rate constants were similar between the antimicrobial and non-antimicrobial material, though no cell growth was observed at the antimicrobial surface (0 µm), as shown in
On the non-antimicrobial and antimicrobial surfaces (0 µm) under NB conditions and on the non-antimicrobial surface (0 µm) under 1/2 NB conditions, the number of cells per colony was greater than that at the other distances under the same nutrient concentrations. Although the growth rate constants under these conditions were similar to those at other distances, the growth periods were longer than those at the other distances (more than 1440 min). On these surfaces, the colonies spread two-dimensionally along the surface, whereas the colonies at other distances enlarged three-dimensionally. In two-dimensionally spreading colonies, more cells may be able to contact the surrounding nutrients than those in three-dimensionally enlarging colonies, leading to a longer period of nutrient uptake and resulting in colonies with a greater number of cells.
The antimicrobial material used in the present study
contained an inorganic antimicrobial agent (Novaron AG1100) composed of silver-based ion exchangers in hexagonal phosphate zirconium and an organic material, polystyrene. Novaron is presumed to exert its antimicrobial effect through silver ions released from the material and/or activated oxygen generated from water in the presence of Novaron and light, according to the manufacturer’s description (US Food and Drug Administration approved). In a preliminary experiment to avoid the light effect, we performed a microculture experiment under constant dark conditions and compared the final number of cells per colony to those of the present study. The antimicrobial effect was the same as that in the present study (data not shown). Thus, the silver ions seem to be the main factor generating the antimicrobial effect in our experiments. The release rate of silver ions from the material, although quite small, has been reported to be enhanced in the presence of amino acids and proteins [
In the environments in which antimicrobial surfaces are used, bacteria often form biofilms on the surface. The biofilms formed on the antimicrobial surface may inhibit antimicrobial activity; this phenomenon may be due to the decrease in the susceptibility of microbes to materials loaded with antibiotics, and this decrease is caused by the production of extracellular polymeric substances [7, 8]. On the basis of the results of the present study, it may be concluded that the three-dimensional structure [
The present study showed that the cell growth of E. coli NBRC 3972 directly contacting the antimicrobial surface (at 0 µm from the surface) was blocked immediately after inoculation on the surface. However, the cell growth at distances of 100 - 300 µm from the antimicrobial surface was started normally for a while, and then stopped; earlier growth termination was observed for cells closer to the surface. This delay of the growth termination was more obvious under lower nutrient concentrations (1/10 and 1/40 NB). To our best knowledge, the present study is the first to report that the antimicrobial effect on bacterial cells depends on the distance from the antimicrobial surface. In the environments in which antimicrobial surfaces are used, the antimicrobial activity may be weakened by the formation of biofilms because of the three-dimensional structure and the high nutrient concentrations in biofilms.
This study was supported by a fund from the Society of Industrial Technology for Antimicrobial Articles, Japan. We express our appreciation to this Society for providing the antimicrobial and non-antimicrobial materials for this study.