Mechanisms Of Enhanced Antibacterial Activity By Reduced Chitosan-intercalated Nontronite

The proliferation of antibiotic resistant human pathogens resulted from the misuse and overuse of antibiotics in healthcare,compels us to seek alternative antibacterial compounds.Previous studies have documented the antibacterial activity of natural clay assemblages.These clay assemblages can kill bacteria at non-neutral pH?<4.5 or>10?via soluble metal ions(e.g.,Fe²⁺and Al³⁺),which were obtained via clay interlayer cation exchange and mineral dissolution.Our group recently identified the bactericidal role of structural Fe?II?in reduced iron-containing clays?RIC?.However,the repulsion between negatively-charged bacteria and clay surface makes this process inefficient,especially considering the short life-span of·OH?in nanoseconds?.The objective of this study is to improve the bactericidal efficiency of clays via enhanced proportions of effective ROS,e.g.,the fraction of ROS that effectively react with bacterial cells,by reversing their surface charge from negative to positive.To achieve this objective,positively-charged chitosan,was intercalated into nontronite NAu-2.Chitosan-intercalated NAu-2?C-NAu-2?was chemically reduced to obtain reduced C-NAu-2?rC-NAu-2?.Relative to reduced nontronite?rNAu-2?,the antibacterial activity of rC-NAu-2 is higher and more persistent over a wider pH range of 6-8,even at a lower concentration.rNAu-2 was only bactericidal at pH 6 but not over pH 6-7,while rC-NAu-2 was antibacterial regardless of the pH values.Even at pH 6,rNAu-2 with a low concentration of Fe?II??3.13 mM?was not adequate to eliminate E.coli DH5?,but it was sufficient to completely kill E.coli DH5?by rC-NAu-2.Moreover,rC-NAu-2 successfully extended the antibacterial capacity from 24 h for rNAu-2 to 24 h.The close spatial association between positively-charged rC-NAu-2and negatively-charged bacteria increases the chances of bacterial cell membrane attack by extracellular ROS,influx of soluble Fe²⁺,and yield of intracellular ROS.In contrast to rNAu-2 treated E.coli DH5?cells,where membrane damage and intracellular ROS/Fe accumulation are restricted to the polar regions,the close bacteria-clay association in rC-NAu-2 results in a nonselective membrane damage and more uniform intracellular ROS/Fe distribution across whole bacterial cells.These results advance the antibacterial model by highlighting the importance of bacteria-clay interactions to the antibacterial activity of Fe-bearing clays.The idea of effective ROS is of great importance for future synthesizing novel mineral-based antibacterial drugs.