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Scanning Electron Microscope: A New Potential Tool to Replace Gram Staining for Microbe Identification in Blood Cultures

Haddad, Gabriel, Bellali, Sara, Takakura, Tatsuki, Fontanini, Anthony, Ominami, Yusuke, Bou Khalil, Jacques, Raoult, Didier·2021·Microorganisms
DOI 10.3390/microorganisms9061170PMC PMC8227564
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Abstract

Blood culture is currently the most commonly used method for diagnosing sepsis and bloodstream infections. However, the long turn-around-time to achieve microbe identification remains a major concern for clinical microbiology laboratories. Gram staining for preliminary identification remains the gold standard. We developed a new rapid strategy using a tabletop scanning electron microscope (SEM) and compared its performance with Gram staining for the detection of micro-organisms and preliminary identification directly from blood cultures. We first optimised the sample preparation for twelve samples simultaneously, saving time on imaging. In this work, SEM proved its ability to identify bacteria and yeasts in morphotypes up to the genus level in some cases. We blindly tested 1075 blood cultures and compared our results to the Gram staining preliminary identification, with MALDI-TOF/MS as a reference. This method presents major advantages such as a fast microbe identification, within an hour of the blood culture being detected positive, low preparation costs, and data traceability. This SEM identification strategy can be developed into an automated assay from the sample preparation, micrograph acquisition, and identification process. This strategy could revolutionise urgent microbiological diagnosis of infectious diseases.

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Crossref · 10.3390/microorganisms9061170

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Figure 1 Optimisation of the sample preparation for imaging micro-organisms of 12 positive blood cultures. Reducing the sample preparation time was crucial since we aimed for early and fast identification of the microbes. This protocol was optimised and adapted after many modifications. Protocol 4 showed the best yield for image acquisition and analysis.
Figure 1 Optimisation of the sample preparation for imaging micro-organisms of 12 positive blood cultures. Reducing the sample preparation time was crucial since we aimed for early and fast identification of the microbes. This protocol was optimised and adapted after many modifications. Protocol 4 showed the best yield for image acquisition and analysis.CC BY · PubMed Central
Figure 2 Timelines comparing routinely used methods for SEM identification. (upper) Gram staining starting from the positive blood culture, an essential step in redirecting the choice of the agar medium for the subculture. MALDI TOF MS identification is then applied to the colonies grown after 4–24 h of incubation. (lower) SEM identification starting from the positive blood culture through the identification of the microbe within one hour following the simple SEM strategy we developed.
Figure 2 Timelines comparing routinely used methods for SEM identification. (upper) Gram staining starting from the positive blood culture, an essential step in redirecting the choice of the agar medium for the subculture. MALDI TOF MS identification is then applied to the colonies grown after 4–24 h of incubation. (lower) SEM identification starting from the positive blood culture through the identification of the microbe within one hour following the simple SEM strategy we developed.CC BY · PubMed Central
Figure 3 Merging steps 1, 2, and 3 allowed us to build a robust analysis for microbe identification.
Figure 3 Merging steps 1, 2, and 3 allowed us to build a robust analysis for microbe identification.CC BY · PubMed Central
Figure 4 Microbe classification and identification criteria acquired using Gram staining versus SEM with MALDI-TOF/MS as a reference. Identification information (shape, cell wall, cell size, subgroup-specific feature, genus-specific feature) acquired for each of the species is indicated with a tick mark, missing information is indicated with a red cross. The total Scheme 100. artificial blood cultures inoculated with various Acinetobacter sp. and Enterobacteria. We were able to blindly identify 82% of the Acinetobacter according to the presence of these structures.
Figure 4 Microbe classification and identification criteria acquired using Gram staining versus SEM with MALDI-TOF/MS as a reference. Identification information (shape, cell wall, cell size, subgroup-specific feature, genus-specific feature) acquired for each of the species is indicated with a tick mark, missing information is indicated with a red cross. The total Scheme 100. artificial blood cultures inoculated with various Acinetobacter sp. and Enterobacteria. We were able to blindly identify 82% of the Acinetobacter according to the presence of these structures.CC BY · PubMed Central
Figure 5 SEM identification results compared to those of MALDI-TOF/MS as a reference method in (A) mono-microbial blood cultures and (B) poly-microbial blood cultures. Validated: same identification results; Discordant: different identification results; Incomplete: Routine methods showed more microbes than did SEM.
Figure 5 SEM identification results compared to those of MALDI-TOF/MS as a reference method in (A) mono-microbial blood cultures and (B) poly-microbial blood cultures. Validated: same identification results; Discordant: different identification results; Incomplete: Routine methods showed more microbes than did SEM.CC BY · PubMed Central

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Metadata

Authors
Haddad, G., Bellali, S., Takakura, T., Fontanini, A., Ominami, Y., Bou Khalil, J., & Raoult, D.
Venue
Microorganisms
Publisher
MDPI AG
Published
2021-05-28
Volume
9
Issue
6
Pages
1170
ISSN
2076-2607
DOI
10.3390/microorganisms9061170
PMC
PMC8227564
Cited by
20
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
Fondation Méditerranée Infection (ANR-10-IAHU-03)

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