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PubMed2026

Microblasting Wound Dressings Mechanically Disrupt Polymicrobial Biofilms to Enhance Healing in Treatment-Resistant Wounds.

Treatment-resistant wounds driven by polymicrobial biofilms are a major clinical challenge, affecting millions globally and leading to chronic inflammation, persistent pain, and poor healing outcomes. These wounds are characterized by mature biofilms reinforced by dense extracellular polymeric substances, which confer strong tolerance to conventional treatments. Despite emerging technologies, such as nanoparticles, bacteriophages, and engineered enzymes, effective clearance of established biofilms remains challenging. Here, we develop a microblasting wound dressing (µBLAST) that delivers spatially confined mechano-chemical disruption at the tissue-biofilm interface to remove viscoelastic biofilm matrices and promote tissue regeneration. The µBLAST is assembled by embedding MnO2-doped diatom biosilica beneath an H2O2-releasing cellulose mesh, enabling localized catalytic microbubble generation within biofilm matrices. Confined expansion and rupture of oxygen bubbles produce localized mechanical stress sufficient to dislodge mature, antibiotic-resistant polymicrobial biofilms, while sustained H2O2 release prolongs particle activity. In a murine wound model infected with mature P. aeruginosa and methicillin-resistant S. aureus biofilms, µBLAST treatment significantly reduces biofilm burden, accelerates re-epithelialization, promotes hair regrowth, and mitigates inflammation. Moreover, µBLAST enhances antibiotic efficacy, suppressing biofilm regrowth even at ten-fold reduced drug doses. These findings highlight confined mechano-chemical biofilm disruption as a therapeutic strategy for treating mature, antibiotic-resistant biofilm infections and promoting tissue regeneration.

Biofilms↗

PubMed2025

Proteomic comparison of epidemic Australian Bordetella pertussis biofilm cells.

Bordetella pertussis causes whooping cough, a severe respiratory infectious disease. Studies have compared the currently dominant single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele, ptxP3) and previously dominant SNP cluster II (ptxP1) strains as planktonic cells. Since biofilm formation is linked with B. pertussis pathogenesis in vivo, this study compared the biofilm formation capabilities of representative strains of cluster I and cluster II. Confocal laser scanning microscopy found that the cluster I strain had a denser biofilm structure compared to the cluster II strain. Differences in protein abundance of the biofilm cells were then compared using tandem mass tagging and high-resolution multiple reaction monitoring. In total, 1,453 proteins were identified, of which 40 proteins had significant differential abundance between the two strains in biofilm conditions. Of particular interest was a large increase in the abundance of energy metabolism proteins (cytochrome proteins PetABC and BP3650) in the cluster I strain. When the abundance of these proteins was compared between six additional strains from each cluster, it was found that the protein abundance varied between all strains. These findings suggest that there are large levels of individual proteomic diversity between B. pertussis strains in biofilm conditions despite the highly conserved genome of the species. Overall, this study revealed visual differences in biofilm structure between B. pertussis strains and highlighted strain-specific variation in protein abundance that dominates potential cluster-specific changes that may be linked with the dominance of cluster I strains.IMPORTANCEBordetella pertussis causes whooping cough. The currently circulating cluster I strains have taken over previously dominant cluster II strains. It is important to understand the reasons behind this evolution to develop new strategies against the pathogen. Recent studies have shown that B. pertussis can form biofilms during infection. This study compared the biofilm formation capabilities of a cluster I and a cluster II strain and identified visual differences in the biofilms. The protein abundance between these strains grown in biofilms was compared, and proteins identified with varied abundance were measured with additional strains from each cluster. It was found that despite the highly conserved genetics of the species, there was varied protein abundance between the additional strains. This study highlights that strain-specific variation in protein abundance during biofilm conditions may dominate the cluster-specific changes that may be linked to the dominance of cluster I strains.

Bordetella pertussis↗

PubMed2026

Metagenomic insights into mechanisms of coral larval settlement induction and inhibition by marine biofilms.

BACKGROUND: Biofilms are essential to larval settlement in many marine invertebrates, yet the mechanisms driving settlement induction or inhibition in corals remain poorly resolved. This challenge lies in the vast taxonomic and functional diversity of marine biofilms, making it difficult to identify cues associated with settlement. To address this, we analysed the metagenomes of biofilms used to induce settlement (attachment and metamorphosis) of four broadcast-spawning non-acroporid coral species: Dipsastrea favus, Platygyra sinensis, Echinophyllia aspera and Porites lobata. Biofilms were developed for one or two months, under light or dark treatments, with light biofilms inducing significantly higher settlement than dark biofilms. RESULTS: Gene composition varied strongly among treatments, with light biofilms enriched in genes encoding carotenoid biosynthesis and nitrate reduction, while dark biofilms encoded more genes for denitrification and nitric oxide production. Modelling revealed the abundance of genes encoding GABA biosynthesis and the type III secretion system (SS) were positively associated with settlement, while genes encoding the type II secretion system, flagellar and lipopolysaccharides were negatively associated. Genes predicted to promote settlement were concentrated in metagenome assembled genomes (MAGs) assigned to Flavobacteriaceae, Rhodobacteraceae and Pirellulaceae, consistent with previous research identifying these lineages as potential inducers. While we detected homologues of some biosynthesis genes for the settlement-inducing compounds cycloprodigiosin and tetrabromopyrrole in the MAGs, pathways were incomplete suggesting additional compounds promote settlement on these biofilms. CONCLUSIONS: These findings link biofilm metagenomics to coral larval settlement for the first time, suggesting carotenoids may attract larvae to biofilm surfaces, while GABA may promote searching and attachment. Additional compounds, for example cycloprodigiosin, tetrabromopyrrole or effector proteins, may be required to complete metamorphosis, however the specific compounds responsible likely vary across biofilm communities and suggest multiple mechanisms can lead to settlement. Simultaneously, elevated levels of nitric oxide, type II SS exudates or an abundance of flagellar potentially inhibit the settlement process. This study advances our understanding of the complex microbial processes underpinning coral larval settlement.

Biofilm↗

PubMed2026

Amino acid reprogramming and biofilm-specific tricarboxylate transporters in PET-degrading Piscinibacter sakaiensis.

Plastic-degrading bacteria predominantly colonize polymer surfaces as biofilms, yet it remains unclear whether the biofilm phenotype contributes to metabolism beyond retaining extracellular enzymes. Here, we combine population-level RNA-sequencing across three conditions-biofilm cells on polyethylene terephthalate (PET), planktonic cells incubated with PET, and planktonic cells on maltose-with single-cell Raman spectroscopy to characterize the PET response of Piscinibacter sakaiensis (formerly Ideonella sakaiensis). This integrated approach reveals two metabolically distinct response layers. A carbon-source-driven response shared by all PET-exposed cells is dominated by a broad amino acid reprogramming, led by upregulation of branched-chain amino acid transport genes, enhanced serine biosynthesis, and reduced chemotaxis. A biofilm-specific layer selectively induces tripartite tricarboxylate transporter genes from three distinct genomic loci. This transcriptional feature is accompanied by a single-cell phenotype consistent with a protein-rich and saturated membrane. These results suggest that biofilm formation is not limited to enzyme retention but is associated with selective activation of transport systems, consistent with a putative role in capturing PET-derived intermediates at the polymer interface. This two-layer model separates general metabolic adaptation to PET from biofilm-specific functions and provides a framework for understanding how surface-associated bacterial physiology contributes to plastic degradation.IMPORTANCEPolyethylene terephthalate (PET) degradation in natural and engineered environments is largely mediated by surface-attached microbial communities, yet the physiological role of biofilm state during plastic degradation remains poorly understood. Using the model PET degrader Piscinibacter sakaiensis, we show that biofilm-associated cells are not simply retained near the polymer surface but exhibit a distinct metabolic program characterized by selective induction of tripartite tricarboxylate transporters. In contrast, extensive amino acid reprogramming occurs in both biofilm and planktonic PET-exposed cells, indicating that it is driven by carbon source rather than surface attachment. These findings reveal that PET degradation involves two separable physiological layers: a general metabolic response to PET-derived carbon shared across cell phenotypes, and a biofilm-specific transport response potentially linked to substrate capture at the plastic interface. This work advances our understanding of how microbial physiology is organized during plastic biodegradation and identifies transport processes as previously unrecognized components of PET-degrading biofilms.

PET biodegradation↗

PubMed2026

Biofilm-derived curli and Z-DNA shape anti-DNA antibody responses during Salmonella infections.

Antibodies to Z-DNA, a non-canonical DNA conformation with a left-handed zigzag backbone, are abundant in the serum of patients with systemic lupus erythematosus (SLE), with levels increasing with disease activity and flares. As SLE is associated with bacterial infections, and as extracellular DNA (eDNA) within biofilms of several bacterial species has been shown to adopt the Z-DNA conformation, bacterial Z-DNA may represent a source of immunogenic Z-DNA in SLE and other related autoimmune conditions. In these studies, we investigated whether eDNA in Salmonella biofilms also contained Z-DNA and whether such Z-DNA could elicit an antibody response. Using antibody-based staining approaches, we observed abundant eDNA in Salmonella enterica serovar Typhimurium (STm) biofilms in both the Z- and canonical B-DNA configurations, consistent with the highly Z-prone nature of the GC-rich Salmonella genome. To assess the functional contribution of these DNA conformations to biofilm integrity, biofilms were treated with DNase I, which lacks enzymatic activity against Z-DNA, or with benzonase, a nonspecific nuclease that degrades both B- and Z-DNA. DNase I treatment applied after biofilm maturation was less effective at thinning biofilms than treatment during early biofilm formation, a pattern also observed with benzonase treatment. Purified curli:DNA complexes contained Z-DNA and, when administered intraperitoneally to mice, elicited robust anti-Z-DNA antibody responses. Similarly, infection with invasive STm induced the production of anti-Z-DNA antibodies in vivo. Moreover, STm infection in mice fed a diet that promotes biofilm development was associated with increased Z-DNA levels in the cecal lumen and elevated anti-DNA antibody responses. Collectively, these findings suggest that Z-DNA, likely formed by extruded Salmonella genomic DNA, and embedded within curli:DNA complexes of STm biofilms, triggers a host immune response and drives anti-Z-DNA antibody production. This work provides mechanistic insight into how bacterial infections and diet-dependent modulation of biofilm formation may contribute to anti-Z-DNA antibody responses in autoimmune diseases like SLE.

Animals↗

PubMed2026

Interface-dependent V. parahaemolyticus biofilm under varying temperatures, media, and oxygen conditions: implications for seafood safety.

Vibrio parahaemolyticus biofilms play a critical role in pathogen persistence in marine and seafood-processing environments, where oxygen availability, temperature, and surface interfaces vary widely. This study investigated biofilm development by three strains on partially submerged stainless-steel coupons under gas-liquid-wall (GLW) and fully submerged (SM) interfaces. Viable cell counts (log₁₀CFU/cm2) along with normalized protein concentration per viable cell (nProt) and normalized polysaccharide concentration per viable cell (nPol) were measured, under aerobic and anaerobic conditions across a temperature range of 15-30 °C, using tryptic soy broth with 3% NaCl (TSB) and seawater-based medium (SW). GLW biofilms consistently exhibited higher cell counts (6.4-7.3 log₁₀CFU/cm2) compared to SM biofilms (5.9-6.3 log₁₀CFU/cm2), suggesting that enhanced oxygen diffusion promotes bacterial proliferation. Conversely, SM biofilms exhibited significantly higher nProt and nPol levels (p < 0.001), indicating increased production of the extracellular polymeric substance (EPS) matrix under low-oxygen, high-nutrient conditions. Microscopy and three-dimensional surface plot analyses revealed relatively uniform biofilm layers at the GLW interface, whereas SM biofilms formed heterogeneous, tower-like structures. EPS production was further influenced by medium composition, oxygen, and temperature. SM biofilms grown in SW exhibited significantly higher nProt and nPol than those in TSB under aerobic conditions (p < 0.001), indicating enhanced matrix stabilization. Under anaerobic conditions at 15 °C, nProt and nPol were higher, whereas under aerobic conditions, peak nProt and nPol occurred at elevated temperatures. These findings highlight a trade-off between bacterial growth and matrix production and provide insight into biofilm adaptation and persistence in seafood-processing environments. These insights may help develop improved biofilm control and seafood safety management.

Biofilms↗

PubMed2026

Disruption of efflux activity reduces biofilm formation through multiple pathways.

Free-swimming bacteria must undergo large-scale changes in gene expression to form structured, aggregated biofilm communities. These regulatory changes are susceptible to environmental stimuli such as exposure to antimicrobials, which can affect adhesion, biofilm matrix production, pathogenicity and multidrug susceptibility. Previously, we found that genetic or chemical inactivation of efflux activity in Escherichia coli and Salmonella Typhimurium disrupts biofilm formation with a wide range of pathways sensitive to efflux inhibition, including reduced expression of csgD, a major regulator of biofilm matrix production. How the regulatory networks controlling efflux activity and biofilm formation overlap and how perturbing efflux impacts biofilm formation is still unclear. To address this, we used a combination of directed evolution experiments and large-scale functional genomics screens (TraDIS-Xpress) to identify the genes and pathways affecting efflux activity and biofilm formation in Salmonella enterica serovar Typhimurium and E. coli. This work describes the landscape of pathways linking efflux activity and biofilm formation. Whilst no singular gene or pathway was found to control the link between the two phenotypes, we propose changes in membrane potential following efflux inactivation are sensed through multiple response regulators that each in turn contribute to repression of biofilm development. These include the two-component signal transduction system EnvZ-OmpR and AraC/XylS family transcriptional regulators, RamA and MarA, which have extensive overlapping regulons and demonstrate high degrees of functional redundancy. This work deepens our understanding of the regulatory networks governing efflux activity and biofilm formation in Enterobacteriaceae and highlights the level of overlapping regulation and functional redundancy between them.

Salmonella typhimurium↗

PubMed2025

Analysis of gene expression within individual cells reveals spatiotemporal patterns underlying Vibrio cholerae biofilm development.

Bacteria commonly exist in multicellular, surface-attached communities called biofilms. Biofilms are central to ecology, medicine, and industry. The Vibrio cholerae pathogen forms biofilms from single founder cells that, via cell division, mature into three-dimensional structures with distinct, yet reproducible, regional architectures. To define mechanisms underlying biofilm developmental transitions, we establish a single-molecule fluorescence in situ hybridization (smFISH) approach that enables accurate quantitation of spatiotemporal gene-expression patterns in biofilms at cell-scale resolution. smFISH analyses of V. cholerae biofilm regulatory and structural genes demonstrate that, as biofilms mature, overall matrix gene expression decreases, and simultaneously, a pattern emerges in which matrix gene expression becomes largely confined to peripheral biofilm cells. Both quorum sensing and c-di-GMP-signaling are required to generate the proper temporal pattern of matrix gene expression. Quorum sensing signaling is uniform across the biofilm, and thus, c-di-GMP-signaling alone sets the regional matrix gene expression pattern. The smFISH strategy provides insight into mechanisms conferring particular fates to individual biofilm cells.

Biofilms↗

PubMed2026

Evaluation of sequential phage-antibiotic therapy reveals enhanced biofilm control with meropenem and colistin in clinical MDR hypervirulent Klebsiella pneumoniae strain.

AIMS: The convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae (MDR-HvKp) has narrowed treatment options. Despite growing interest in phage-antibiotic synergy, this study evaluates the underexplored combinatorial effects of phage and antibiotics, including drug-specific interactions and sequence dependency, against the biofilm-forming MDR-HvKp clinical strain. METHODS AND RESULTS: A T5-like Klebsiella bacteriophage, Round, within the genus Webervirus, was therapeutically and genomically characterized. A biofilm-forming clinical strain, Kleb_134, was used to evaluate in vitro phage-antibiotic interactions with meropenem, colistin, and tigecycline in planktonic and biofilm models.In planktonic assays, phage combinations with meropenem and colistin resulted in a multi-log CFU reduction compared to monotherapies, whereas reduced efficacy was observed with tigecycline. In biofilm assays, pre-phage treatment followed by antibiotic exposure demonstrated the strongest biofilm reduction. Drug-specific and sequence-dependent effects were evident. Meropenem-phage combinations reduced biofilm biomass by 2.85-fold (high phage titre) and 3.8-fold (low phage titre), while colistin-phage combinations achieved reductions of 8.4-fold (high phage titre) and 2.8-fold (low phage titre). CONCLUSIONS: Sequential phage-antibiotic treatment was effective against MDR-HvKp biofilms, with pre-phage exposure enhancing antibiotic access through biofilm disruption. The bacteriostatic nature of tigecycline reduced efficacy by affecting phage replication. These findings highlight the importance of treatment sequence and antibiotic selection, and extend existing knowledge in optimizing therapeutic outcomes in MDR-HvKp infections.

Biofilms↗

PubMed2025

Biofilms in clinical infection: pathophysiology, diagnosis, and the evolving therapeutic landscape.

Biofilms are structured communities of microorganisms encased in a self-produced polymeric matrix that typically adhere to surfaces. Recent research, however, has revealed that non-attached aggregates share many common traits with the surface-dependent biofilms. This mode of bacterial growth provides enhanced protection against antibiotics and resistance to host immune defenses. Biofilms require higher antibiotic concentrations than those needed to inhibit planktonic bacteria, necessitating prolonged high-dose and combination therapies to achieve effective eradication. This increased resistance is attributed to multiple factors, including the protective extracellular matrix, reduced metabolic activity of bacteria within the biofilm, and also the ability of bacterial genomes to rapidly adjust in response to environmental changes. Diagnostic modalities such as sonication, tissue culture, and polymerase chain reaction-based assays currently dominate clinical diagnostics of biofilm infections due to their practicality, cost-effectiveness, and proven reliability. Recent research has led to innovative treatment strategies that target biofilm structure, enhance drug delivery, and modulate host-pathogen interactions. This review summarizes our current knowledge of biofilm formation, explores the current techniques for detecting microbial biofilms, and discusses future perspectives for advancing diagnostic and therapeutic strategies.

Biofilms↗

PubMed2026

Characterization of carABpyrB operon and role of pyrE in Francisella novicida biofilm.

Pyrimidine biosynthesis is essential for bacterial growth, but its role in regulating biofilm formation in Francisella (F.) novicida remains poorly defined. In this study, we experimentally defined the carABpyrB operon in F. novicida and investigated how disruption of the de novo pyrimidine biosynthesis pathway affects growth and biofilm formation under nutrient-restricted conditions. Reverse transcriptase PCR confirmed co-transcription of carA, carB, and pyrB, and promoter prediction identified two putative σ70-dependent promoter regions upstream of carA. Transposon mutants disrupted in carA, carB, and pyrB exhibited pronounced growth defects in Chamberlain's Defined Medium that were restored by uracil supplementation, confirming pyrimidine auxotrophy and functional disruption of de novo pyrimidine biosynthesis. We then extended this analysis to additional genes in the pyrimidine biosynthetic pathway and assessed biofilm formation in modified Mueller-Hinton broth, a nutrient-restricted condition. In this medium, carA, carB, pyrB, and pyrE mutants exhibited growth deficiencies; however, the pyrE mutant uniquely produced significantly more biofilm than the wild type. This phenotype remained evident even without growth normalization, with the pyrE mutant producing 3.3-fold more biofilm than wild type, despite impaired growth, and increased to 11.8-fold when normalized to growth. Quantitative PCR demonstrated that uracil supplementation represses carA, carB, and pyrB transcription, consistent with feedback regulation of the pathway. Together, these findings indicate that pyrimidine limitation is not simply a growth-limiting condition but can alter biofilm regulation, with pyrE disruption revealing a strong association between de novo pyrimidine biosynthesis and biofilm formation.

Biofilms↗

PubMed2024

A review focusing on mechanisms and ecological risks of enrichment and propagation of antibiotic resistance genes and mobile genetic elements by microplastic biofilms.

Microplastics (MPs) are emerging ubiquitous pollutants in aquatic environment and have received extensive global attention. In addition to the traditional studies related to the toxicity of MPs and their carrier effects, their unique surface-induced biofilm formation also increases the ecotoxicity potential of MPs from multiple perspectives. In this review, the ecological risks of MPs biofilms were summarized and assessed in detail from several aspects, including the formation and factors affecting the development of MPs biofilms, the selective enrichment and propagation mechanisms of current pollution status of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in MPs biofilms, the dominant bacterial communities in MPs biofilms, as well as the potential risks of ARGs and MGEs transferring from MPs biofilms to aquatic organisms. On this basis, this paper also put forward the inadequacy and prospects of the current research and revealed that the MGEs-mediated ARG propagation on MPs under actual environmental conditions and the ecological risk of the transmission of ARGs and MGEs to aquatic organisms and human beings are hot spots for future research. Relevant research from the perspective of MPs biofilm should be carried out as soon as possible to provide support for the ecological pollution prevention and control of MPs.

Biofilms↗

PubMed2026

Microplastics as vectors for microbial pollutants: Biofilm-associated transfer of pathogens and antibiotic resistance genes in zebrafish intestine.

As composite carriers of microorganisms and pollutants, biofilm-attached microplastics (MPs) serve as potential vectors for the environmental migration and biotransmission of antibiotic resistance genes (ARGs) and pathogens. In this study, traditional polypropylene (PP) and biodegradable polylactic acid (PLA) MPs were used to investigate the interference effects of biofilms-attached MPs on gut microbiota and ARGs transmission, through a combination of laboratory biofilm cultivation, zebrafish (Danio rerio) exposure simulations, metagenomic sequencing, and metabolomic profiling. Results showed that MP biofilms likely induced gut dysbiosis and were associated with altered diversity and abundance of pathogens and ARGs. At the phylum level, Nitrospira was transferred from PP biofilms to the gut. At the genus level, 23 genera were transferred from MP biofilms, with PLA (23 genera) showing higher transfer capacity than PP (4 genera). Notably, two human pathogens, one opportunistic pathogen, and two ARGs (adeF and oqxB) were specifically transferred from PLA biofilms, highlighting the unique dissemination risk of biodegradable MPs. Mechanistically, MPs may activate mobile genetic elements (e.g., Tn916 transposon) through metabolic remodeling and quorum sensing, thereby promoting horizontal gene transfer and ARGs dissemination within the gut. Our findings highlight the potential role of MPs as carriers of microorganisms and ARGs, underscoring the biotransmission risks of antibiotic resistance caused by composite pollution.

Animals↗

PubMed2026

Identification of genetic determinants that promote biofilm growth under heterotrophic conditions in Cupriavidus necator using transposon enrichment.

Cupriavidus necator is a metabolically versatile β-proteobacterium of growing interest for auto- and heterotrophic bioprocesses, yet the genetic determinants governing its biofilm formation remain largely uncharacterized, particularly under process-relevant heterotrophic conditions. Here, we applied a forward-genetics transposon-enrichment approach to identify loci which promote surface-associated growth. A high-density mini-Tn5 mutant library (26,185 insertion clones, exceeding the >17,000 required for genome-wide coverage) was cultivated as a biofilm in a microfluidic flow-cell system on fructose for 168 h, and the surface-associated community was characterized by deep sequencing. Twelve genes showed significantly elevated insertion frequencies, several with documented links to biofilm formation in other bacteria, including the ferrous-iron uptake system (feoA/feoB), galU, and a GSDEF/EAL dual-domain protein. The gene B2043 (E6A55_RS29530), encoding this c-di-GMP-metabolizing protein, was selected for validation by markerless deletion. Under static conditions, the ΔB2043 mutant showed a 1.69 ± 0.06-fold increase in biofilm-associated biomass (p = 5.16 × 10-15). Under flow-through conditions, the mutant attached faster, entered exponential growth ∼10 h earlier, reached its biovolume plateau ∼16 h earlier than the wild-type, and formed distinct tower-like structures. These results identify B2043 as a negative regulator of biofilm formation acting predominantly during attachment, provide the first experimental evidence for c-di-GMP-dependent biofilm regulation in C. necator H16, and establish a functional-genomics framework - together with eleven further candidate loci - for engineering productive biofilms in this organism.

Biofilm formation↗

PubMed2021

Affordable oral health care: dental biofilm disruption using chloroplast made enzymes with chewing gum delivery.

Current approaches for oral health care rely on procedures that are unaffordable to impoverished populations, whereas aerosolized droplets in the dental clinic and poor oral hygiene may contribute to spread of several infectious diseases including COVID-19, requiring new solutions for dental biofilm/plaque treatment at home. Plant cells have been used to produce monoclonal antibodies or antimicrobial peptides for topical applications to decrease colonization of pathogenic microbes on dental surface. Therefore, we investigated an affordable method for dental biofilm disruption by expressing lipase, dextranase or mutanase in plant cells via the chloroplast genome. Antibiotic resistance gene used to engineer foreign genes into the chloroplast genome were subsequently removed using direct repeats flanking the aadA gene and enzymes were successfully expressed in marker-free lettuce transplastomic lines. Equivalent enzyme units of plant-derived lipase performed better than purified commercial enzymes against biofilms, specifically targeting fungal hyphae formation. Combination of lipase with dextranase and mutanase suppressed biofilm development by degrading the biofilm matrix, with concomitant reduction of bacterial and fungal accumulation. In chewing gum tablets formulated with freeze-dried plant cells, expressed protein was stable up to 3 years at ambient temperature and was efficiently released in a time-dependent manner using a mechanical chewing simulator device. Development of edible plant cells expressing enzymes eliminates the need for purification and cold-chain transportation, providing a potential translatable therapeutic approach. Biofilm disruption through plant enzymes and chewing gum-based delivery offers an effective and affordable dental biofilm control at home particularly for populations with minimal oral care access.

Biofilms↗

PubMed2025

A novel regulation on the developmental checkpoint protein Sda that controls sporulation and biofilm formation in Bacillus subtilis.

UNLABELLED: Biofilm formation by Bacillus subtilis is triggered by an unusually simple environmental sensing mechanism. Certain serine codons, the four TCN codons (N for A, T, C, or G), in the gene for the biofilm repressor SinR caused lowered SinR translation and subsequent biofilm induction during transition from exponential to stationary growth. Global ribosome profiling showed that ribosomes pause when translating the four UCN (U for T on the mRNA) serine codons on mRNA, but not the two AGC/AGU serine codons. We proposed a serine codon hierarchy (AGC/AGT vs TCN) in that genes enriched in the TCN serine codons may experience reduced translation efficiency when serine is limited. In this study, we designed an algorithm to score all protein-coding genes in B. subtilis NCIB3610 based on the serine codon hierarchy. We generated a short list of 50 genes that could be subject to regulation by this novel mechanism. We further investigated one such gene from the list, sda, which encodes a developmental checkpoint protein regulating both sporulation and biofilm formation. We showed that synonymously switching the TCN serine codons to AGC in sda led to delayed biofilm formation and sporulation. This engineered strain also outgrew strains with other synonymously substituted sda alleles (TCN) in competition assays for biofilm formation and sporulation. Finally, we showed that the AGC serine codon substitutions in sda elevated the Sda protein levels. This serine codon hierarchy-based novel signaling mechanism could be exploited by bacteria in adapting to stationary phase and regulating important biological processes. IMPORTANCE: Genome-wide ribosome profiling in Bacillus subtilis shows that under serine limitation, ribosomes pause on the four TCN (N for A, C, G, and T), but not AGC/AGT serine codons, during translation at a global scale. This serine codon hierarchy (AGC/T vs TCN) differentially influences the translation efficiency of genes enriched in certain serine codons. In this study, we designed an algorithm to score all 4,000+ genes in the B. subtilis genome and generated a list of 50 genes that could be subject to this novel serine codon hierarchy-mediated regulation. We further investigated one such gene, sda, encoding a developmental checkpoint protein. We show that sda and cell developments controlled by Sda are also regulated by this novel mechanism.

Bacillus subtilis↗

PubMed2026

Building biofilms for saline hydrogenotrophic denitrification from contrasting origins: Convergent acclimation, divergent performance.

Hydrogenotrophic denitrification is promising for deep nitrogen removal from saline, low-C/N wastewaters, but rapid establishment of stable biofilms at high salinity remains challenging. Here, two saline-adapted inocula from two representative, functionally contrasting habitats-a functionally-diversified inoculum from mangrove sediment and a functionally-focused inoculum from seabed sediment-were acclimated in parallel H2-based membrane biofilm reactors at constant 3.5% salinity. The Diverse-derived biofilm required 80 d to reach steady state and achieved only partial denitrification with 61.1% nitrate removal and considerable nitrite accumulation. In contrast, the Focus-derived biofilm rapidly established complete denitrification within ∼40 d, which was maintained for >50 d, with effluent NOx- below 1 mg-N·L-1 and 98.7% nitrate removal. Microbiome analyses showed that identical operation promoted convergence in community structure and enriched similar community-level functional potentials. However, genome-resolved analysis revealed distinct source-dependent functional organization among dominant microbial populations. Complete denitrifiers co-encoding denitrifying, hydrogenotrophic, and autotrophic functions were preferentially enriched in the Focus-derived biofilm, whereas these functions remained partitioned among different dominant populations in the Diverse-derived biofilm, coinciding with less complete denitrification. These findings indicate that saline hydrogenotrophic denitrification performance depends not only on which functions are enriched at the community level, but also on how key functions become organized among microbial populations, providing a previously overlooked criterion for inoculum selection in saline biological nitrogen control.

Complete denitrification↗

PubMed2026

Outbreaks of fluconazole-resistant Candida parapsilosis are driven by low-biofilm-producing isolates that emerge under host selection.

Candida parapsilosis is a major human fungal pathogen, with recent global outbreaks driven by fluconazole-resistant (FLCR-Cp) isolates that are difficult to eradicate and associated with poor clinical outcomes. However, the microbial traits enabling persistence of these outbreak lineages remain poorly defined. Here, we show that FLCR-Cp isolates responsible for prolonged, multi-country outbreaks consistently exhibit a striking low-biofilm-producing (LBP) phenotype. Contrary to the prevailing view that robust biofilm formation promotes persistence, LBP strains displayed enhanced stress tolerance, increased cell wall masking, and reduced immune recognition. These traits conferred resistance to neutrophil and macrophage killing and enhanced survival in immune cell-rich organs during systemic infection. Genome-wide transcriptomic profiling revealed extensive metabolic and regulatory rewiring in LBP strains. Whole-genome sequencing (WGS) of a global isolate collection further demonstrated that the LBP phenotype has emerged independently multiple times, supporting convergent evolution under host selection. Functional genomic analyses suggest that biofilm attenuation arises through multigenic changes, and disruption of key biofilm-associated transcriptional regulators enhanced fitness during immune interactions. Together, our findings overturn the assumption that robust biofilm formation drives outbreak persistence and instead identify biofilm attenuation as an adaptive tradeoff that promotes immune evasion and long-term survival. These results redefine our understanding of C. parapsilosis adaptation during healthcare-associated outbreaks and shift attention toward host-driven evolutionary processes than environmental persistence alone.

Biofilms↗