METHODS article
Front. Microbiol.
Sec. Antimicrobials, Resistance and Chemotherapy
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1582371
This article is part of the Research TopicEmerging Antimicrobials: Sources, Mechanisms of Action, Spectrum of Activity, Combination Antimicrobial Therapy, and Resistance MechanismsView all 40 articles
A streamlined method to determine the antibiotic resistance of plaque-forming predatory bacteria
Provisionally accepted
- 1University of Kiel, Kiel, Germany
- 2Max Planck Institute for Evolutionary Biology, Plön, Schleswig-Holstein, Germany
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Introduction Antimicrobial resistance (AMR) is a critical global health issue caused by antibiotic overuse, leading to the rise of multi-resistant pathogens such as in bacteria of the ESKAPE group. Alternative or combination therapies, including bacteriophages and plaque-forming predatory bacteria, are being explored in response. Bdellovibrio bacteriovorus, a Gram-negative bacterial predator belonging to the Bdellovibrio and like organisms (BALOs), can kill other Gram-negative bacteria after the periplasmic invasion, including multidrug-resistant pathogens. However, a combined treatment of antibiotics and plaque-forming predatory bacteria requires the predatory bacteria to be resistant to the antibiotic. The predator’s unique growth requirements limit standardized AMR testing methods. Material and methods We propose a streamlined three-step protocol to measure AMR in plaque-forming predatory bacteria. It requires the (i) cultivation of a dense Bdellovibrio culture with a suitable prey strain, followed by (ii) a double-layered agar plaque assay using a prey strain resistant to the antibiotic of interest, and (iii) the application of E-test strips for minimum inhibitory concentration (MIC) determination. We apply the method to the commonly used strain B. bacteriovorus HD100. We use P. aeruginosa H03 as prey for MIC determination for five antibiotics. Results Our results show consistent MICs for B. bacteriovorus HD100 across independent experiments. Reliable MIC determination for meropenem was limited by P. aeruginosa H03 susceptibility to this antibiotic. Further, we observed a positive association between MIC values and predator inoculum concentration for ceftazidime, ciprofloxacin, and gentamicin. Prolonged incubation time increased MIC values, notably for ciprofloxacin. While resistant to piperacillin, predator plaques were absent on plates with piperacillin-tazobactam combinations. Conclusion The streamlined approach described here to determine MICs in plaque-forming predatory bacteria proves effective and robust, when using a suitable (i.e. resistant) prey. It provides a starting point for the joint study of antibiotics and plaque-forming predatory bacteria.
Keywords: Bdellovibrio, antibiotic, plaque assay, E-tests, antibiotic resistance
Received: 24 Feb 2025; Accepted: 14 Jul 2025.
Copyright: © 2025 Wülbern, Carstensen, Buchholz, Schulenburg and Johnke. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Julia Johnke, University of Kiel, Kiel, Germany
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