Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy
Abstract Rapid determination of drug efficacy against bacterial pathogens is needed to detect potentially resistant bacteria and allow for more rational use of antimicrobials. As an indicator of the antimicrobial effect for rapid detection, we found changes in image brightness in antimicrobial-affec...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-38198-3 |
| _version_ | 1797778909640523776 |
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| author | Akiko Hisada Erino Matsumoto Ryo Hirano Mami Konomi Jacques Yaacoub Bou Khalil Didier Raoult Yusuke Ominami |
| author_facet | Akiko Hisada Erino Matsumoto Ryo Hirano Mami Konomi Jacques Yaacoub Bou Khalil Didier Raoult Yusuke Ominami |
| author_sort | Akiko Hisada |
| collection | DOAJ |
| description | Abstract Rapid determination of drug efficacy against bacterial pathogens is needed to detect potentially resistant bacteria and allow for more rational use of antimicrobials. As an indicator of the antimicrobial effect for rapid detection, we found changes in image brightness in antimicrobial-affected bacteria by scanning electron microscopy (SEM). The cell envelopes of unaffected bacteria were stained with phosphotungstic acid (PTA), whereas the entire cells of affected bacteria were stained. Since tungsten density increases backscattered electron intensity, brighter bacterial images indicate lethal damage. We propose a simplified method for determining antimicrobial efficacy by detecting damage that occurs immediately after drug administration using tabletop SEM. This method enabled the visualization of microscopic deformations while distinguishing bacterial-cell-envelope damage on gram-negative bacteria due to image-brightness change. Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa were exposed to imipenem and colistin, which affect the cell envelope through different mechanisms. Classification of single-cell images based on brightness was quantified for approximately 500 bacteria per sample, and the bright images predominated within 5 to 60 min of antimicrobial treatment, depending on the species. Using intracellular PTA staining and characteristic deformations as indicators, it was possible to determine the efficacy of antimicrobials in causing bacterial-cell-envelope damage. |
| first_indexed | 2024-03-12T23:24:19Z |
| format | Article |
| id | doaj.art-764002567e0e46c5a1ed1d3ce41d34a6 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-12T23:24:19Z |
| publishDate | 2023-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-764002567e0e46c5a1ed1d3ce41d34a62023-07-16T11:15:57ZengNature PortfolioScientific Reports2045-23222023-07-0113111010.1038/s41598-023-38198-3Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopyAkiko Hisada0Erino Matsumoto1Ryo Hirano2Mami Konomi3Jacques Yaacoub Bou Khalil4Didier Raoult5Yusuke Ominami6Healthcare Innovation Center, Research and Development Group, Hitachi, Ltd.Healthcare Innovation Center, Research and Development Group, Hitachi, Ltd.Core Technology and Solutions Group, Hitachi High-Tech CorporationCore Technology and Solutions Group, Hitachi High-Tech CorporationInstitut Hospitalo-Universitaire Méditerranée InfectionConsulting Infection MarseilleCore Technology and Solutions Group, Hitachi High-Tech CorporationAbstract Rapid determination of drug efficacy against bacterial pathogens is needed to detect potentially resistant bacteria and allow for more rational use of antimicrobials. As an indicator of the antimicrobial effect for rapid detection, we found changes in image brightness in antimicrobial-affected bacteria by scanning electron microscopy (SEM). The cell envelopes of unaffected bacteria were stained with phosphotungstic acid (PTA), whereas the entire cells of affected bacteria were stained. Since tungsten density increases backscattered electron intensity, brighter bacterial images indicate lethal damage. We propose a simplified method for determining antimicrobial efficacy by detecting damage that occurs immediately after drug administration using tabletop SEM. This method enabled the visualization of microscopic deformations while distinguishing bacterial-cell-envelope damage on gram-negative bacteria due to image-brightness change. Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa were exposed to imipenem and colistin, which affect the cell envelope through different mechanisms. Classification of single-cell images based on brightness was quantified for approximately 500 bacteria per sample, and the bright images predominated within 5 to 60 min of antimicrobial treatment, depending on the species. Using intracellular PTA staining and characteristic deformations as indicators, it was possible to determine the efficacy of antimicrobials in causing bacterial-cell-envelope damage.https://doi.org/10.1038/s41598-023-38198-3 |
| spellingShingle | Akiko Hisada Erino Matsumoto Ryo Hirano Mami Konomi Jacques Yaacoub Bou Khalil Didier Raoult Yusuke Ominami Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy Scientific Reports |
| title | Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy |
| title_full | Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy |
| title_fullStr | Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy |
| title_full_unstemmed | Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy |
| title_short | Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy |
| title_sort | detection of antimicrobial impact on gram negative bacterial cell envelope based on single cell imaging by scanning electron microscopy |
| url | https://doi.org/10.1038/s41598-023-38198-3 |
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