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

The findings come from a team led by Hsin-Yu Wang, Chun Ming How, Yong-Shan Li, Yuqing Mao, Thanh H. Nguyen and Chia-Cheng Wei, who set out to answer a question that has become increasingly urgent in food safety research: do non-antibiotic chemicals, particularly the fungicides sprayed widely on fruit and vegetable crops, push bacteria toward resistance or heightened virulence? Prior work has hinted at the danger. Azoxystrobin and carbendazim have been shown to enrich bacterial resistomes in nematode guts, tebuconazole can promote the spread of multidrug-resistant plasmids in soil bacteria, and chlorothalonil facilitates metabolic adaptation in soil microbial communities. But whether such exposure produces phenotypic resistance and increased pathogenicity in a major foodborne pathogen remained largely untested.

Bacillus cereus was an obvious candidate for scrutiny. The Gram-positive, spore-forming organism is found in 36 to 45 percent of dairy products, vegetables, beans and cereals, and it is capable of causing food poisoning, eye infections, anthrax-like progressive pneumonia, fulminant sepsis and central nervous system infections. Multidrug-resistant strains of the species have already emerged in hospital wastewater, and its versatility—including the ability to build biofilms of varied architecture—makes any shift in its behavior a serious public health concern.

The researchers first screened eight widely used fungicides against B. cereus: chlorothalonil (CHT), propineb (PRO), tebuconazole (TEB), azoxystrobin, propiconazole, mancozeb, carbendazim and triadimefon. Three of them—CHT at 8 micromolar, TEB at 500 micromolar and PRO at 175 micromolar—completely inhibited bacterial growth within 24 hours and were selected for long-term adaptation experiments. The design was demanding: every day for 30 days, the bacteria endured a three-hour fungicide challenge followed by recovery and regrowth in fresh medium. Survival trajectories differed by compound. Under chlorothalonil, survival dipped to about 70 percent on day one but rebounded within 24 hours. Tebuconazole initially halved the population before recovery stabilized around day eight. Propineb proved the harshest pressure, dropping survival below five percent on day six before the bacteria clawed back to stable levels by day twelve. The bacterium, in short, adapted to all three chemical regimes.

Whole-genome sequencing of the adapted lineages delivered a surprising verdict: no meaningful genetic mutations. Phylogenetic comparison against reference strains and variant-calling analyses found the treated bacteria essentially identical to their ancestors. Instead of classical, mutation-driven resistance, the adaptation appears to be physiological—a reversible, non-heritable tolerance state akin to the persister-cell and stress-response phenomena documented in bacteria subjected to repeated antibiotic cycles. Similar patterns have been reported when Listeria monocytogenes and uropathogenic Escherichia coli were exposed to disinfectants such as benzalkonium chloride and triclosan, with minimum inhibitory concentrations rising without stable genetic change.

The phenotypic consequences, however, were substantial. Biofilm formation—often a shield against both immune attack and antimicrobial agents—was initially suppressed during early exposure but rose significantly in tebuconazole-adapted bacteria from day ten onward and climbed markedly in propineb-exposed cells by day ten. Statistical testing confirmed significant effects of the fungicide treatment, the duration of exposure, and their interaction on biofilm output. Antibiotic challenge assays revealed a parallel erosion of susceptibility. Bacteria adapted to chlorothalonil grew significantly better than controls in gentamicin at 4, 6 and 8 micrograms per milliliter; tebuconazole- and propineb-adapted lineages also outgrew controls at key gentamicin doses, and chlorothalonil- and tebuconazole-adapted cells showed improved growth at 8 micrograms per milliliter of tetracycline. Because no growth occurred at concentrations of 16 micrograms per milliliter or above, the strains do not meet formal clinical criteria for resistance—but the shift toward tolerance was clear and reproducible.

The most dramatic result emerged in living hosts. Using the nematode Caenorhabditis elegans, a genetically tractable infection model whose intestinal epithelium provides a biologically meaningful readout of colonization and killing, the team measured how fungicide-adapted bacteria fared against unadapted controls. All three adapted lineages killed worms significantly faster than the parent strain, with log-rank tests showing p values below 0.001. Tebuconazole-adapted bacteria were especially aggressive: worm survival collapsed within two days, and by day three most of the animals were dead. Follow-up colonization assays showed that tebuconazole-adapted B. cereus also established significantly higher intestinal loads in the worms, indicating that the fungicide had promoted persistence within the host gut, not merely faster killing.

Transcriptional profiling of the tebuconazole-adapted lineage offers a mechanistic window into these changes. Quantitative real-time PCR revealed significantly elevated expression of genes encoding the non-hemolytic enterotoxin (nheC) and the hemolysin BL complex (hblA, hblC and hblD)—toxins that disrupt intestinal epithelial cells—alongside upregulation of purC and purL, which support purine biosynthesis and extracellular DNA release during early biofilm formation, and calY, a bifunctional matrix protein that promotes adhesion to host tissues. The efflux-pump gene smr was also induced, a plausible explanation for the reduced antibiotic susceptibility, and one that echoes efflux upregulation seen in stressed Mycobacterium tuberculosis. Importantly, the elevated virulence and resistance gene expression persisted even when the adapted bacteria were subsequently exposed to gentamicin, suggesting that the stress-adapted state complicates antibiotic treatment rather than simply surviving it.

The authors are careful to frame the work as hazard identification rather than a direct portrait of what happens on farms or in food. The experiments used a single reference strain, BCRC15850, and the exposure concentrations—particularly 500 micromolar tebuconazole and 175 micromolar propineb—exceed the residue levels typically reported on treated foods, although the chlorothalonil dose is of the same order of magnitude as residues found in some food commodities. Local bioavailable concentrations in soil and produce depend on moisture, adsorption, formulation and degradation, so the laboratory model of recurrent acute stress cannot be directly translated into field-level risk estimates. Nor should the transcriptional findings be generalized beyond the tebuconazole lineage without confirming that chlorothalonil- and propineb-adapted bacteria share the same regulatory program. Nonetheless, the study reveals an understudied scenario in which persistent sublethal chemical stress can harden a major foodborne pathogen—improving its resilience, deepening its virulence and weakening the grip of frontline antibiotics—without any mutational fingerprint. Whether such phenotypes persist after fungicide withdrawal, and whether they arise in the genetically diverse field isolates that actually contaminate the food supply, are the questions the team now hopes will drive the next round of research.

Subject of Research: Effects of long-term fungicide exposure on adaptation, antibiotic tolerance and virulence of the foodborne pathogen Bacillus cereus

Article Title: Long-term fungicide exposure promotes bacterial adaptation and increases virulence of foodborne pathogen Bacillus cereus in Caenorhabditis elegans

Article References: Wang, H.-Y., How, C. M., Li, Y.-S., Mao, Y., Nguyen, T. H., & Wei, C.-C. (2026). Long-term fungicide exposure promotes bacterial adaptation and increases virulence of foodborne pathogen Bacillus cereus in Caenorhabditis elegans. Current Research in Food Science, 13, Article 101572. https://doi.org/10.1016/j.crfs.2026.101572

Image Credits: AI Generated

DOI: 10.1016/j.crfs.2026.101572

Keywords: Bacillus cereus, fungicide exposure, antimicrobial tolerance, biofilm formation, Caenorhabditis elegans, tebuconazole, chlorothalonil, propineb, virulence genes, food safety, foodborne pathogen, efflux pumps

The history of dual therapy with ceftriaxone and azithromycin illustrates how quickly the situation can change. The combination was introduced as a strategic response to concerns about ceftriaxone monotherapy failure, and it carried the added benefit of activity against Chlamydia trachomatis and Mycoplasma genitalium, which frequently co-occur with gonorrhea. In its early years the strategy worked in many settings, because most ceftriaxone-resistant gonococcal strains were still susceptible to azithromycin, and the dual approach helped slow the international spread of ceftriaxone resistance. Over the past five years, however, azithromycin resistance has risen disturbingly across the globe, undermining the drug’s value within dual therapy and threatening the efficacy of current treatment protocols. Contributing factors include extensive and often inappropriate antibiotic use, suboptimal diagnostic practices, limited surveillance systems, and delayed updates to clinical guidelines.

The future therapeutic landscape appears increasingly precarious. Two novel antimicrobials, zoliflodacin and gepotidacin, have recently been approved for uncomplicated gonorrhea, yet few additional agents are in clinical development. At the same time, resistance to ceftriaxone itself continues to climb in many regions, and strains with concurrent resistance to both ceftriaxone and azithromycin have now been reported in several WHO regions. High treatment costs further restrict access in resource-limited settings, where surveillance and control measures are often weakest, compounding the risk that resistance emerges and spreads undetected. Without new therapeutic strategies, enhanced global surveillance and robust research investment, gonorrhea could become progressively harder to treat, with rising morbidity, complications and treatment failures.

Azithromycin is an oral macrolide long used against chlamydial and mycoplasmal infections, and it once demonstrated good efficacy against gonococci as well. Its widespread availability, particularly where antibiotics can be obtained without prescription or regulation, has contributed significantly to the development of resistance. Over the past decade, azithromycin resistance has steadily increased in both high- and low-income countries, with marked geographic variability shaped by local treatment practices, surveillance infrastructure and antibiotic consumption patterns. Although azithromycin is no longer routinely recommended for gonorrhea in many settings, rising resistance limits its remaining utility and underscores why continued antimicrobial resistance surveillance matters.

The numbers tell a sobering story. A retrospective genomic surveillance study by Daniel Golparian and colleagues, published in The Lancet Microbe, examined N. gonorrhoeae isolates collected across 21 European Economic Area countries in 2020 through the European Gonococcal Antimicrobial Surveillance Programme. It found that azithromycin resistance, defined by the EUCAST epidemiological cut-off of MIC greater than 1 mg/L, had risen to 9.4 percent of isolates, up from 8.0 percent in 2018. In the United States, resistance climbed from 0.01 percent in 1992 to 0.4 percent by 2012, then surged fourfold from 0.6 percent in 2013 to 2.5 percent in 2014, reaching 4.6 percent in 2018 and 5.9 percent by 2019 among isolates with elevated MICs. Globally, the WHO Gonococcal Antimicrobial Surveillance Programme reported that 46 of 53 countries, or 87 percent, detected azithromycin-resistant isolates in 2022, and 36 countries reported resistance in at least 5 percent of isolates.

Regional heterogeneity is striking. In the WHO Western Pacific region, seven of eight reporting countries detected resistant isolates in 2022, with Brunei, Cambodia, China, Japan and Singapore each reporting resistance in at least 5 percent of isolates, while Australia and New Zealand recorded lower levels. China’s Gonococcal Resistance Surveillance Program documented azithromycin resistance reaching 16.9 percent in 2022, a substantial increase over the preceding five years, whereas all isolates reported from the Philippines remained susceptible. Surveillance itself faces structural challenges: while high-income countries maintain robust systems, reporting is inconsistent in low- and middle-income countries due to constraints in laboratory capacity, funding and infrastructure. Only 77 countries and territories reported gonococcal resistance data to WHO GASP during 2019 to 2022, and the absence of data from parts of Africa and Southeast Asia means the true global extent of resistance, and of dual-therapy treatment failures, may be significantly underreported.

Understanding the molecular machinery behind resistance clarifies why surveillance is difficult. Azithromycin inhibits bacterial protein synthesis by binding the peptidyltransferase region of domain V of the 23S rRNA in the 50S ribosomal subunit, blocking peptide exit. Resistance arises through several mechanisms: mutations in the 23S rRNA itself, particularly A2059G and C2611T, alter the drug-binding site; the A2059G transition in all four rRNA alleles produces extraordinarily high MICs of 256 to 4096 mg/L, while C2611T yields moderate resistance of 2 to 32 mg/L. Acquired erm genes encoding rRNA methylases can modify the target, though they have become rare in recent isolates. Mutations in rplD and rplV, encoding ribosomal proteins L4 and L22, moderately raise MICs, and efflux pump overexpression adds another layer of resistance.

Efflux is now central to the story. The MtrCDE pump, the best-characterized system, expels macrolides when its regulator MtrR is disabled by mutations such as G45D, or when promoter mutations, including a single-nucleotide deletion in a 13-base-pair inverted repeat, increase pump transcription. Mosaic mtr alleles, acquired through recombination with sequences from Neisseria meningitidis or Neisseria lactamica, substantially raise azithromycin MICs by altering both the pump and its regulation. The MacAB and mef-encoded efflux systems may also contribute, though they remain less studied in gonococci. Collectively, these mechanisms reveal a complex and geographically variable genetic architecture of resistance.

That variability is precisely what undermines molecular diagnostics. Historically, 23S rRNA mutations were the predominant, best-characterized determinants, and systematic reviews confirm that assays targeting A2059G accurately detect high-level resistance while C2611T assays identify moderate resistance. But the European genomic surveillance revealed a decisive shift: only 10.5 percent of azithromycin-resistant isolates in 2020 carried 23S rRNA mutations, down from 24.1 percent in 2018, while roughly 89 percent instead carried mtrD/mtrR promoter mosaic 2 or semi-mosaic mtrD variants. The authors attribute the resistance surge to the expansion of specific NG-STAR clonal complexes, notably CC63, CC168 and CC213 harboring the mosaic promoter, plus the emergence of novel CC1031 carrying a semi-mosaic mtrD variant. An assay built for the old targets would miss the dominant circulating resistance genotypes.

The implications for public health are clear. The WHO advocates molecular assays that detect resistance markers to guide therapy and support stewardship, but their validity, accuracy and specificity falter when marker distributions differ across regions; in areas dominated by mtrD mosaic variants, a 23S rRNA-focused assay would yield a very low positive predictive value. Tailoring molecular targets to local genetic epidemiology is therefore essential. In low- and middle-income countries where routine whole-genome sequencing is impractical, a pragmatic sentinel-site strategy, with representative laboratories performing susceptibility testing and periodically referring isolates to regional reference centers for genomic characterization, offers a feasible alternative. A standardized minimum dataset reported to WHO GASP, covering susceptibility results, specimen source, geography, collection year and resistance determinants, would enable continuous refinement of regionally calibrated assays. The shift from dual therapy toward ceftriaxone monotherapy may also reshape selection pressure on azithromycin resistance, though the magnitude and direction of that effect remain uncertain, since macrolide use for other infections, fitness effects and lineage dynamics all play a role. Sustained phenotypic and genomic surveillance, novel antimicrobials and combination therapies, and collaboration among researchers, clinicians and public health stakeholders will determine whether gonorrhea remains treatable in the decade ahead.

Subject of Research: Heterogeneity of azithromycin resistance markers in Neisseria gonorrhoeae and the challenges of implementing molecular assays for routine antimicrobial resistance surveillance

Article Title: Heterogeneity of azithromycin resistance markers in Neisseria gonorrhoeae and challenges in implementing molecular assays for routine surveillance

Article References: Heterogeneity of azithromycin resistance markers in Neisseria gonorrhoeae and challenges in implementing molecular assays for routine surveillance. (n.d.). https://doi.org/10.1016/j.nmni.2026.101838

Image Credits: AI Generated

DOI: 10.1016/j.nmni.2026.101838

Keywords: Neisseria gonorrhoeae, azithromycin resistance, antimicrobial resistance, molecular surveillance, 23S rRNA mutations, mtr mosaic variants, efflux pumps, WHO GASP, ceftriaxone, gonorrhea treatment, genomic epidemiology, molecular diagnostics