Biofilm Tolerance Emerges as Key Driver of Antibiotic Treatment Failure

Bacteria living in biofilms, the structured communities encased in a self-produced matrix that form on wounds, medical devices and airways, often survive antibiotic treatment without carrying any classical resistance gene. A new review published in Frontiers in Microbiology argues that this survival frequently begins as antimicrobial tolerance, a reversible physiological state, rather than genetically encoded resistance, and that the two phenomena form a conditional continuum in which short-term survival can, under the right selective circumstances, pave the way for stable resistance. The authors, a team from Vidyasagar University and collaborating institutions in India, synthesize evidence from evolutionary microbiology, molecular regulation and single-cell biology to map how biofilm populations adapt under antimicrobial pressure, and to identify biological dependencies that new therapies might exploit before resistance becomes fixed.

The review draws a careful line between related but distinct concepts. Antimicrobial resistance is a heritable reduction in susceptibility caused by mutations or acquired resistance determinants, and it raises the minimum inhibitory concentration of a drug. Antimicrobial tolerance, by contrast, is a transient physiological state that lets bacteria survive brief drug exposure without necessarily changing the MIC, and it reverses when favorable growth conditions return. Persistence describes the survival of a population or subpopulation during treatment, while persister cells are specialized phenotypic variants that withstand lethal exposure and resume growth once the stress is removed. Persisters are generally genetically susceptible; their resistance is phenotypic, not heritable. Adaptive resistance, a temporary reduction in susceptibility induced by environmental signals, completes the picture.

Importantly, the authors stress that tolerance is not an obligatory stepping stone to resistance. Tolerant and persistent populations can raise the probability that resistance evolves, because they keep viable cells alive during antimicrobial exposure, creating opportunities for selection of resistant variants, genetic diversification or horizontal gene transfer. But resistance can also arise directly through selection of pre-existing variants already present in a genetically diverse population, without new mutations arising during treatment. Stress-associated mutagenesis, involving DNA damage responses, oxidative stress and repair deficiencies, has been demonstrated in certain experimental settings, yet its contribution depends strongly on bacterial species, environmental conditions and the intensity and duration of exposure. The review therefore frames resistance evolution as the combined outcome of genetic variation, ecological selection, survival of adaptive subpopulations and gene exchange, rather than a simple consequence of elevated mutation rates.

Biofilm heterogeneity provides the ecological stage for these processes. The three-dimensional architecture of a biofilm generates gradients of oxygen, nutrients, pH and antimicrobial concentration. Cells near the surface tend to be metabolically active, while cells in deeper layers grow slowly or enter dormant-like states. This matters because many antibiotics require active cellular processes: beta-lactams disrupt ongoing cell-wall synthesis, aminoglycosides are taken up through energy-dependent transport, and fluoroquinolones target DNA replication. Slow-growing or metabolically reduced cells can survive exposure to these drugs without any genetic resistance. The review emphasizes that metabolic heterogeneity is best understood as a physiological determinant of antimicrobial response rather than a resistance mechanism in itself, and that regulatory systems such as the stringent response, oxidative-stress pathways, quorum sensing and cyclic di-GMP signalling govern the transitions between growth and survival states.

The extracellular polymeric substance matrix adds another layer of protection, though the authors caution against viewing it as an absolute barrier. Many antimicrobials do penetrate biofilms, yet tolerance persists because of physiological heterogeneity and persister formation. In Pseudomonas aeruginosa, the matrix consists mainly of alginate, with the polysaccharides Pel and Psl also contributing. Pel, positively charged at physiological pH, shields the bacterium against the aminoglycosides gentamicin and tobramycin, and its resistance properties are enhanced through interactions with extracellular DNA, which can chelate cationic antibiotics. The DNABII family of histone-like proteins stabilizes eDNA structures at the vertices of Holliday junction DNA, reinforcing the matrix. In Escherichia coli, the matrix contains phosphoethanolamine-modified cellulose and amyloid protein fibres. Matrix-associated enzymes also contribute: P. aeruginosa secretes beta-lactamases into the biofilm, and aminoglycoside-modifying enzymes, including phosphotransferases, acetyltransferases and nucleotidyltransferases, are upregulated in biofilms, reducing drug binding to bacterial ribosomes.

Regulatory networks coordinate much of this adaptation. Nutrient limitation activates the stringent response, in which the alarmone guanosine 5′-(tri)diphosphate 3′-diphosphate, produced via SpoT and relA, downregulates growth-associated genes and upregulates survival functions. An E. coli mutant unable to produce this alarmone was found to be more susceptible to penicillin, and the stringent response also confers tolerance in Mycobacterium tuberculosis, Bacillus subtilis and Streptomyces coelicolor. Two-component systems add further protection: under low magnesium, the Salmonella PhoPQ system activates genes that defend against cationic antimicrobial peptides such as polymyxin B and colistin, while PmrAB, working with PhoPQ, adds palmitate and 4-amino-arabinose to lipid A, opposing drug entry. Similar systems operate in P. aeruginosa, Klebsiella pneumoniae and Yersinia pestis. Oxidative stress management is equally central: P. aeruginosa biofilms treated with ciprofloxacin showed elevated reactive oxygen species, and katA catalase mutants produced more ROS and were more sensitive to the drug, while the hydroxyl-radical scavenger thiourea increased biofilm survivability. In Burkholderia cepacia complex biofilms, deleting the katB catalase reduced survival under tobramycin and ciprofloxacin treatment by roughly 40-fold and 30-fold, respectively, compared with wild type.

Quorum sensing and cyclic di-GMP signalling tie individual physiology to community behaviour. Biofilms formed by a P. aeruginosa strain lacking the quorum-sensing regulators LasR and RhlR were considerably more sensitive to tobramycin than wild-type biofilms, and a quorum-sensing-deficient agrD mutant of Staphylococcus aureus showed biofilm-specific reductions in rifampin resistance. In Enterococcus faecalis, the quorum-regulated gelE protease and the fsr system were required for biofilm resistance to linezolid, daptomycin and gentamicin. Inhibiting the quorum-sensing receptor TraP with hamamelitannin increased the efficacy of several antibiotics, including cephalosporins, vancomycin and daptomycin, against pre-established S. aureus biofilms. Meanwhile, c-di-GMP, the second messenger governing the switch between motile and sessile lifestyles, reaches estimated levels of 75 to 110 picomoles per milligram in P. aeruginosa biofilms, compared with less than 30 picomoles per milligram in planktonic cells. Elevated c-di-GMP contributes to antimicrobial resistance through the transcription factor BrlR, whose expression is higher in clinical cystic fibrosis isolates than in the laboratory strain PAO1, and brlR mutant biofilms were roughly fourfold less resistant to tobramycin than wild-type biofilms.

Small regulatory RNAs and efflux pumps add further adaptive capacity. Trans-encoded sRNAs such as SroC in Salmonella, SprX in S. aureus, SdsR and MicF in E. coli, and DsrA, which upregulates the MdtEF efflux pump, each modulate resistance to specific antibiotics through repression or activation of target genes. Efflux systems such as MexAB-OprM in P. aeruginosa, AcrAB-TolC in Gram-negative bacteria and NorA in S. aureus can be constitutively overexpressed, producing stable resistance, but they can also be transiently induced by antimicrobial exposure or oxidative stress, yielding adaptive tolerance without a fixed resistance phenotype. The review stresses that whether increased efflux reflects temporary physiology or evolutionary stabilization determines its significance, positioning efflux systems as an interface between adaptation and resistance evolution rather than simple resistance mechanisms.

Biofilms also create ecological conditions favorable to horizontal gene transfer, though the authors urge caution in generalizing. In a multidrug-resistant S. aureus strain, plasmid transfer frequency in biofilm cells was reported to increase roughly ten-thousand-fold compared with planktonic cells, and Enterococcus faecalis biofilm cells showed about a twofold increase in plasmid copy number. Integrase expression, a marker of integron activity that can mobilize resistance genes, was reported to rise approximately 100-fold in biofilm cells relative to planktonic populations. Transfer efficiency nonetheless depends on donor-recipient compatibility, the mobile genetic element involved, environmental conditions and the specific mechanism, whether transformation, conjugation or transduction. The review concludes that biofilms should be regarded as ecological environments in which gene transfer can occur when conditions align, not as inherently efficient hotspots for resistance dissemination.

On the therapeutic front, the review proposes targeting the vulnerabilities that biofilm adaptation itself creates, while noting that nearly all such strategies remain experimental or preclinical. Matrix sensitization with DNase I, dispersin B, proteases or alginate lyases can loosen biofilm structure and improve antimicrobial access, but dispersal alone may release viable cells, supporting a combined disrupt-and-kill approach. Quorum-quenching, c-di-GMP modulation, persister-directed strategies such as metabolic resensitization and toxin-antitoxin targeting, and efflux pump inhibitors each face limitations from pathway redundancy, species specificity and toxicity. Bacteriophage therapy, synthetic antimicrobial peptides of the PS1 series, which inhibited drug-resistant P. aeruginosa and S. aureus biofilms in preclinical models, and nanoparticle delivery systems including chitosan, zinc oxide and silver formulations show promise in animal or laboratory studies but lack human biofilm evidence. CRISPR interference targeting the luxS quorum-sensing gene reduced E. coli biofilm formation in proof-of-concept work. The authors argue that combination therapies must be validated for pharmacodynamic interaction rather than assumed synergistic, and that translation requires clinically representative models, since static assays and monospecies systems poorly capture the polymicrobial, host-associated complexity of real infections, where metabolic cross-feeding and shared matrix structures reshape antimicrobial responses.

Subject of Research: Molecular mechanisms linking biofilm antimicrobial tolerance to resistance evolution and emerging anti-biofilm therapeutic strategies

Article Title: From biofilm tolerance to antimicrobial resistance: molecular adaptation strategies and emerging therapeutic vulnerabilities in resistant pathogens

Article References: Guchhait, K. C., Dey, S., Pradhan, S. R., Panda, A. K., & Ghosh, C. (2026). From biofilm tolerance to antimicrobial resistance: molecular adaptation strategies and emerging therapeutic vulnerabilities in resistant pathogens. Frontiers in Microbiology, 17, Article 1988464. https://doi.org/10.3389/fmicb.2026.1988464

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1988464

Keywords: biofilms, antimicrobial resistance, antimicrobial tolerance, persister cells, quorum sensing, c-di-GMP signalling, efflux pumps, horizontal gene transfer, extracellular polymeric substance, stringent response, anti-biofilm therapy, bacteriophage therapy