Probiotic Bacterium Defies Tetracycline Through Efflux Pump, Study Finds

Probiotic bacteria are prized for their health benefits, but when a candidate strain carries antibiotic resistance, regulators and food scientists take notice. A new study from researchers at Gachon University, Dankook University, and Konkuk University in South Korea has dissected exactly how the probiotic candidate Lentilactobacillus buchneri KU200793 withstands tetracycline, one of the most widely used classes of antibiotics in medicine and animal husbandry. The work, published in Food Science and Biotechnology, combines genome annotation, biochemical assays, and functional translation experiments to build a layered picture of a resistance phenotype that turns out to be both multifactorial and, in a crucial sense, reversible.

The stakes of such an investigation are considerable. Tetracycline antibiotics work by binding to the 30S ribosomal subunit of bacteria, blocking the attachment of aminoacyl transfer RNA and thereby halting protein synthesis. Decades of clinical and agricultural use have driven the spread of resistance across bacterial communities, and lactic acid bacteria used in food fermentations are no exception. Because probiotic strains are consumed live and may reside temporarily in the gut, any resistance genes they carry could, in principle, be transferred to commensal or pathogenic bacteria. The European Food Safety Authority therefore sets microbiological cut-off values for antibiotic susceptibility in microorganisms intended for food or feed applications, and strains exceeding those thresholds face heightened scrutiny before they can be considered safe.

That is precisely the situation with L. buchneri KU200793, a strain previously isolated from Korean fermented foods and characterized for its probiotic properties and neuroprotective effects. When the research team, led by corresponding author Young-Seo Park, measured the minimum inhibitory concentration of tetracycline against the strain using standardized methods for non-enterococcal lactic acid bacteria, they arrived at a value of 128 milligrams per liter. That figure exceeds the EFSA cut-off, meaning the strain would formally be classified as resistant and would need a mechanistic explanation before any safety assessment could proceed. The question the researchers posed was deceptively simple: is this resistance intrinsic and benign, or does it involve genes that could spread?

To answer it, the team first turned to the genome. Annotation of the KU200793 sequence revealed a suite of genes plausibly linked to antibiotic tolerance, including transporters belonging to the major facilitator superfamily and to the ATP-binding cassette family, a PmrA-type protein, and a transcriptional regulator of the TetR family. These categories are familiar players in resistance biology. Major facilitator superfamily pumps and ABC transporters can expel antibiotics from the cell before the drugs reach their ribosomal targets, while TetR-family regulators commonly control the expression of efflux systems in response to tetracycline itself. Notably, the analysis did not identify the classic acquired tetracycline resistance determinants, such as ribosomal protection genes of the tet(M) type or enzymatic inactivation genes, which are the elements most often mobilized horizontally between bacterial species.

The genomic picture was reinforced by comparisons at the drug’s actual target. Tetracycline resistance in many bacteria arises through mutations in the 30S ribosomal protein S10 or in the 16S ribosomal RNA that alter the antibiotic binding site. When the researchers compared these sequences in KU200793 with those of the tetracycline-susceptible reference strain L. buchneri ATCC 4005, they found no mutations at all. The ribosome of the resistant strain, in other words, appears structurally unremarkable, which pointed the investigation away from target modification and toward processes acting elsewhere in the cell.

One alternative hypothesis involved chemical inactivation of the drug itself. Some bacteria degrade or modify antibiotics extracellularly, rendering them harmless before uptake. The team used high-performance liquid chromatography to examine whether components released by tetracycline-exposed cultures of KU200793 affected the stability of the drug in the surrounding medium. The results suggested that extracellular components from exposed cultures may influence tetracycline stability, but the evidence did not support direct enzymatic inactivation as the mechanism. Whatever was happening outside the cell, it was not the straightforward destruction of the antibiotic molecule that characterizes classic inactivation-based resistance.

The decisive experiment came from pharmacological dissection of efflux. Efflux pumps are powered by the proton motive force or by ATP hydrolysis, and certain compounds can collapse that energy supply and disable the pumps. Chlorpromazine, a compound long used as an inhibitor of efflux-mediated resistance in studies dating back to work on fluoroquinolone-resistant Staphylococcus aureus, was applied to KU200793 alongside tetracycline. The effect was dramatic: the minimum inhibitory concentration of tetracycline dropped sixteen-fold, from 128 milligrams per liter to just 8 milligrams per liter. A sixteen-fold reduction upon efflux inhibition is strong functional evidence that active export is the dominant determinant of the resistance phenotype, converting a strain that exceeds regulatory thresholds into one that would fall within them if its pumps were silenced.

Yet the story did not end with efflux. The researchers also probed cell envelope permeability by measuring alkaline phosphatase activity, an enzyme whose accessibility to substrates depends on the outer layers of the Gram-positive cell wall. Reduced alkaline phosphatase activity in the resistant strain provided supportive evidence that decreased permeability contributes to the phenotype, limiting how much tetracycline enters the cell in the first place. Resistance built on restricted permeability is considered less worrisome from a horizontal gene transfer standpoint than resistance encoded by discrete, mobile resistance genes, because it reflects the strain’s own architectural and physiological traits rather than an acquired genetic element.

The final layer of evidence came from an elegant cell-free translation assay. Using a system in which green fluorescent protein is synthesized outside a living cell, the team tested whether cellular factors from KU200793 could restore translation in the presence of tetracycline. Partial restoration of cell-free GFP synthesis indicated that inducible translation-associated factors contribute to the strain’s ability to keep making proteins under antibiotic pressure. Together with the efflux and permeability findings, this positions the resistance of KU200793 as a multifactorial phenotype in which no single mechanism is solely responsible, but in which chlorpromazine-sensitive efflux clearly plays the leading role.

For the probiotics industry, the implications are twofold. On one hand, the absence of canonical acquired resistance genes, ribosomal target mutations, and direct drug inactivation is reassuring: the strain’s tolerance appears rooted in its intrinsic physiology rather than in a transferable genetic package. On the other hand, the study demonstrates the depth of characterization that modern safety assessment demands, and it offers a template for how genome annotation, inhibitor-based functional tests, and biochemical assays can be combined to distinguish benign intrinsic tolerance from genuine risk. As candidate probiotic strains continue to move from fermented foods into regulated applications, mechanistic studies of this kind will increasingly determine which organisms earn a place in the food supply, and the case of L. buchneri KU200793 shows that a sixteen-fold drop in resistance with a single inhibitor can speak louder than any genome annotation alone.

Subject of Research: Tetracycline resistance mechanisms in the probiotic bacterium Lentilactobacillus buchneri KU200793

Article Title: Molecular characterization of tetracycline resistance mechanisms in the probiotic strain Lentilactobacillus buchneri KU200793

Article References: Kang, Y., Jeong, H., Kang, D.-K., Paik, H. D., & Park, Y.-S. (2026). Molecular characterization of tetracycline resistance mechanisms in the probiotic strain Lentilactobacillus buchneri KU200793. Food Science and Biotechnology. https://doi.org/10.1007/s10068-026-02296-5

Image Credits: AI Generated

DOI: 10.1007/s10068-026-02296-5

Keywords: probiotics, Lentilactobacillus buchneri, tetracycline resistance, antibiotic resistance, efflux pumps, lactic acid bacteria, food safety, EFSA, minimum inhibitory concentration, chlorpromazine, genome annotation, cell envelope permeability