Molecular Mechanism For Response Of Escherichia Coli O157:H7 To Goethite

Iron oxide,an important component of soil,impacts on the fate of bacteria in soil profoundly because of its prevailing and colloidal properties.Iron oxide is known to be detrimental to the activity of bacteria through physical contact and oxidative stress in its interaction with them.Foodborne bacterial pathogens can be introduced into soil via manure application and wastewater irrigation,and their persistence in soil has caused numerous outbreaks of foodborne illness.Given that iron oxide is one of the most active component parts in soil,and it is antibacterial,elucidating the mechanism for the response of bacterial pathogens to iron oxide is essential for knowledge of the fate of these pathogens in soil and for control of foodborne illness.However,until now little is known about the molecular mechanism for phenotypic response of foodborne bacterial pathogens to iron oxide,especially at single-cell level.Therefore,Escherichia coli O157:H7(a representative foodborne pathogen)and goethite(a typical iron oxide)were selected for study;biochemical methods,such as fluorescence indicator 2?,7?-dichlorodihydrofluorescein diacetate(DCFH-DA)for detecting intracellular reactive oxygen species(ROS)level,Live/Dead Bac Light viability assay and bicinchoninic acid(BCA)for determining total protein concentration,and modern instruments including Raman spectrometer,fluorescent inverted microscope,transmission electron microscope and microplate reader were utilized to explore the effect of goethite on bacterial growth,bacterial membrane integrity and intracellular ROS level and to reveal phenotypic response of bacteria to goethite at single-cell level.The main results obtained are as follows:(1)The inhibition of E.coli O157:H7 growth by goethite and the effects of goethite concentration and size were identified.By measuring optical density at 600 nm(OD₆₀₀)and the total protein concentration,it was found that as the concentration of nano-sized goethite increased,bacterial growth was gradually inhibited.Compared to 200 mg/L of nano-sized goethite,the same concentration of micro-sized goethite had stronger inhibition effect on bacterial growth.(2)The impact of goethite size on membrane integrity and intracellular ROS level of E.coli O157:H7 was elucidated.It was shown in epifluorescence images that after exposure to two kinds of nano-sized goethite the degrees of bacterial membrane integrity were 30.6±3.5%and 37.2±4.7%,respectively,whereas this figure for micro-sized goethite was lower,18.8±1.1%.The results of intracellular ROS level showed that intracellular ROS level with nano-sized goethite was significantly higher than that with micro-sized counterpart.These findings suggested that the antibacterial mechanism of goethite is size-dependent;the production of ROS is the main antibacterial mechanism of nano-sized goethite,while micro-sized goethite displays antibacterial activity mainly through membrane damage.(3)The molecular mechanism for response of E.coli O157:H7 to nano-and micro-sized goethite at single-cell level was revealed.There were similarities in biomolecular response to nano-and micro-sized goethite:the accumulation of ribonucleoside-related substances,the reduction in protein concentration and the loss of outer membrane substances(lipopolysaccharide&outer membrane porins,LPS&OMPs).But due to the size-dependent antibacterial mechanism of goethite,there were also differences in biomolecular response.Since nano-sized goethite-induced intracellular ROS level was higher,RNA damage may have been severer,ribonucleoside-related substances accumulating more.At the same time,bacterial response to membrane damage encouraged energy metabolism,glycogen being broken down to produce adenosine 5?-triphosphate(ATP),with a decrease in glycogen concentration and an increase in ATP concentration.By contrast,after exposure to micro-sized goethite,because of lower intracellular ROS level,ribonucleoside-related substances accumulated less.More serious membrane damage may have resulted in more decrease in the level of LPS&OMPs.Consequently,greater loss of cellular components was likely to hamper the process by which glycogen was broken down,with high concentration of glycogen and low concentration of ATP.