Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9, 26

Effects of Antibacterial Mineral Leachates on the Cellular Ultrastructure, Morphology, and Membrane Integrity of Escherichia Coli and Methicillin-Resistant Staphylococcus Aureus

Affiliations

Effects of Antibacterial Mineral Leachates on the Cellular Ultrastructure, Morphology, and Membrane Integrity of Escherichia Coli and Methicillin-Resistant Staphylococcus Aureus

Caitlin C Otto et al. Ann Clin Microbiol Antimicrob.

Abstract

Background: We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates.

Methods: We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Baclight LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity.

Results: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates.

Conclusions: These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.

Figures

Figure 1
Figure 1
E. coli (a) and MRSA (b) survival in CB07 and BY07 mineral leachates for 24 h. Values represent the mean CFU and SD of at least three independent experiments. The dotted line represents the limit of CFU detection.
Figure 2
Figure 2
SEM images of E. coli (a, c, e, g, i) and MRSA (b, d, f, h, j) after 24 h incubation in broth, dH2O, pH 3.6 buffer, CB07-L, or BY07-L. Scale bar = 500 nm.
Figure 3
Figure 3
Frequency of characteristics observed in three independent replicates of SEM images of E. coli (a) and MRSA (b). For each independent replicate, at least 200 cells were visualized and scored for the observed characteristic.
Figure 4
Figure 4
TEM images of E. coli (a, c, e, g, i) and MRSA (b, d, f, h, j) after 24 h incubation in broth, dH2O, pH 3.6 buffer, CB07-L, or BY07-L. Scale bar = 200 nm. Electron-dense region, asterisk (a); mesosome-like structure, striped arrow (f); granules, black arrowheads (g, i); cytoplasmic condensation, white arrowheads (g, i); cytoplasmic disruption, black arrows (h, j); cytoplasmic membrane disruptions, white arrows (h, j).
Figure 5
Figure 5
(a) Frequency of characteristics observed in three independent replicates of TEM images of E. coli (a) and MRSA (b). For each independent replicate, at least 60 cells were visualized and scored for the observed characteristic.
Figure 6
Figure 6
E. coli (a) viability determined by CFU plate counts following 1 h exposure to CB07-L and BY07-L. MRSA (a) viability determined by CFU plate counts following 15 h exposure to mineral leachates. The dotted line represents the limit of CFU detection.
Figure 7
Figure 7
Flow cytometric analysis of E. coli (a, c, e, g, i) and MRSA (b, d, f, h, j) after incubation in broth, dH2O, pH 3.6 buffer, CB07-L, or BY07-L. Two red fluorescent populations were observed with a higher fluorescence intensity (black arrow) and lower fluorescence intensity (white arrow). Flow cytometric analyses and percentages were derived from a single representative experiment. Average values from at least three independent replicate experiments are reported in parentheses. Percentages denote the number of cells that remained impermeable to PI, and therefore fluoresced green. Green fluorescence was detected on channel FL1 with a 525 nm ± 10 nm bandpass filter for SYTO9; red fluorescence was detected on channel FL3 with a ≥ 620 bandpass filter for PI.
Figure 8
Figure 8
Forward-scatter and side-scatter dot plots of E. coli (a, c, e, g, i) and MRSA (b, d, f, h, j) determined by flow cytometry after incubation in broth, dH2O, pH 3.6 buffer, CB07-L, or BY07-L. Flow cytometric dot plot analyses and percentages were derived from a single representative experiment. Average values from at least three independent replicate experiments are reported in parentheses. Percentages denote the number of cells falling inside the gate.

Similar articles

See all similar articles

Cited by 14 PubMed Central articles

See all "Cited by" articles

References

    1. Chastre J. Evolving problems with resistant pathogens. Clin Microbiol Infect. 2008;14(Suppl 3):3–14. doi: 10.1111/j.1469-0691.2008.01958.x. - DOI - PubMed
    1. Carretero M. Clay minerals and their beneficial effects upon human health. A review. Appl Clay Sci. 2002;21(3-4):155–163. doi: 10.1016/S0169-1317(01)00085-0. - DOI
    1. Ziegler JL. Geophagy: a vestige of palaeonutrition. Trop Med Int Health. 1997;2(7):609–611. doi: 10.1046/j.1365-3156.1997.d01-359.x. - DOI - PubMed
    1. Vermeer DE, Ferrell RE Jr. Nigerian geophagical clay: a traditional antidiarrheal pharmaceutical. Science. 1985;227(4687):634–636. doi: 10.1126/science.3969552. - DOI - PubMed
    1. Cunningham TM, Koehl JL, Summers JS, Haydel SE. pH-dependent metal ion toxicity influences the antibacterial activity of two natural mineral mixtures. PLoS ONE. 2010;5(3):e9456. doi: 10.1371/journal.pone.0009456. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback