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. 2024 May 25:2024:8917136.
doi: 10.1155/2024/8917136. eCollection 2024.

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy

Omar Zmerli  1   2 , Sara Bellali  1 , Gabriel Haddad  1   2 , Rim Iwaza  1   2 , Akiko Hisada  3 , Erino Matsumoto  3 , Yusuke Ominami  4 , Didier Raoult  5 , Jacques Bou Khalil  1   2
Affiliations

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy

Omar Zmerli et al. Int J Microbiol. .

Abstract

Background: Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin.

Methods: Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes.

Results: We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method.

Conclusions: We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

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Conflict of interest statement

The authors would like to declare that DR was a consultant in microbiology for Hitachi High-Tech Corporation from March 2018 until March 2021. YO is employed by Hitachi High-Tech Corporation. AH and EM are employed by Hitachi, Ltd. Personal fees for GH, SB, and JBK are paid through a collaborative contract from the Hitachi High-Tech Corporation. OZ and RI declare no relevant competing interests. A patent application for this methodology is pending (PCT/FR2021/052468).

Figures

Figure 1
Figure 1
Workflow of the SEM-AST assay detailing the steps for sample preparation from culture to SEM observation.
Figure 2
Figure 2
Colistin SEM-AST assay applied to Colistin-sensitive and Colistin-resistant strains of Acinetobacter baumannii. (a) Evolution of the percentage of viable bacteria for Colistin-resistant strains exposed to Colistin for 120 minutes. (b) SEM images of Colistin-resistant strains exposed to Colistin for 120 minutes. (c) Evolution of the percentage of viable bacteria for Colistin-sensitive strains exposed to Colistin for 120 minutes. (ns: not significant / ∗: p ≤ 0.05 / ∗∗: p ≤ 0.01) (d) SEM images of Colistin-sensitive strains exposed to Colistin for 120 minutes demonstrating morphologic changes (dysmorphism (red arrow), inflation (blue arrow), and fusion (yellow arrow)).
Figure 3
Figure 3
Colistin SEM-AST assay applied to Colistin-sensitive and Colistin-resistant strains of Enterobacter cloacae. (a) Evolution of the percentage of viable bacteria for Colistin-resistant strains exposed to Colistin for 120 minutes. (b) SEM images of Colistin-resistant strains exposed to Colistin for 120 minutes. (c) Evolution of the percentage of viable bacteria for Colistin-sensitive strains exposed to Colistin for 120 minutes. (ns: not significant / ∗∗∗: p ≤ 0.001/ ∗∗∗∗: p ≤ 0.0001) (d) SEM images of Colistin-sensitive strains exposed to Colistin for 120 minutes demonstrating morphologic changes (dysmorphism (red arrow), inflation (blue arrow), and lysis (orange arrow)).
Figure 4
Figure 4
Colistin SEM-AST assay applied to Colistin-sensitive and Colistin-resistant strains of E. coli. (a) Evolution of the percentage of viable bacteria for Colistin-resistant strains exposed to Colistin for 120 minutes. (b) SEM images of Colistin-resistant strains exposed to Colistin for 120 minutes. (c) Evolution of the percentage of viable bacteria for Colistin-sensitive strains exposed to Colistin for 120 minutes. (ns: not significant / ∗∗∗∗: p ≤ 0.0001) (d) SEM images of Colistin-sensitive strains exposed to Colistin for 120 minutes demonstrating morphologic changes (inflation (blue arrow) and lysis (orange arrow)).
Figure 5
Figure 5
Colistin SEM-AST assay applied to Colistin-sensitive and Colistin-resistant strains of K. pneumoniae. (a) Evolution of the percentage of viable bacteria for Colistin-resistant strains exposed to Colistin for 120 minutes. (b) SEM images of Colistin-resistant strains exposed to Colistin for 120 minutes. (c) Evolution of the percentage of viable bacteria for Colistin-sensitive strains exposed to Colistin for 120 minutes. (ns: not significant / ∗∗∗∗: p ≤ 0.0001) (d) SEM images of Colistin-sensitive strains exposed to Colistin for 120 minutes demonstrating morphologic changes (inflation (blue arrow), fusion (yellow arrow), and lysis (orange arrow)).
Figure 6
Figure 6
Colistin SEM-AST assay applied to Colistin-sensitive and Colistin-resistant strains of P. aeruginosa. (a) Evolution of the percentage of viable bacteria for Colistin-resistant strains exposed to Colistin for 120 minutes. (b) SEM images of Colistin-resistant strains exposed to Colistin for 120 minutes. (c) Evolution of the percentage of viable bacteria for Colistin-sensitive strains exposed to Colistin for 120 minutes. (ns: not significant / ∗∗: p ≤ 0.01 / ∗∗∗∗: p ≤ 0.0001) (d) SEM images of Colistin-sensitive strains exposed to Colistin for 120 minutes demonstrating morphologic changes (inflation (blue arrow), lysis (yellow arrow), and a granular pattern (green star)).
Figure 7
Figure 7
Evolution of percentage of viable bacteria following Colistin exposure. Observation of Colistin SEM-AST assay applied to Colistin-sensitive strains of five clinically relevant species over all experimental time points.
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