Antibiotic protection assay revisited: Metronidazole is unable to completely eliminate Porphyromonas gingivalis

Intracellular bacterial survival is widely studied in host-microbiome interactions. The antibiotic protection assay is often used to quantify intracellular bacteria. This method uses gentamicin to kill extracellular bacteria, where the bacteria that remain inside host cells survive the treatment. However, gentamicin is ineffective against anaerobic bacteria, such as Porphyromonas gingivalis. To remedy this, metronidazole is often incorporated. However, the effectiveness of this adaptation seems not to be validated properly. The aim of this study was to show the ineffectiveness of metronidazole to eliminate extracellular P. gingivalis in vitro. Microscopy showed uptake of P. gingivalis by murine J774A.1 macrophages and primary human macrophages. However, quantification of intracellular bacteria was unreliable as the control without macrophages contained significant numbers of viable bacteria. Upon testing metronidazole under assay conditions, P. gingivalis survived within the tested timeframes. Next, it was attempted to find a suitable alternative antibiotic compound to use in the antibiotic protection assay. The MIC and MBC were therefore determined for various alternative antibiotics and antimicrobial peptides. None of the included antibiotics effectively killed P. gingivalis. Antimicrobial peptides cycloLL-37 and D-LL-31 were effective against P. gingivalis, but were toxic to macrophages under the conditions used as determined using a Lactate dehydrogenase-based cytotoxicity assay. To conclude, metronidazole and gentamicin seem unsuitable for the antibiotic protection assay for the strict anaerobic oral bacterium P. gingivalis. To be able to determine viable intracellular P. gingivalis, alternative bactericidal agents should be found which, under assay conditions, eliminate extracellular P. gingivalis without entering or affecting mammalian cells.