Biofilm Environment Research Articles

P-1371. Identifying Occurrences of Plasmid-Borne Antimicrobial Resistance Gene Transfer between Diverse Bacterial Species within Environmental Biofilms Using Proximity-Ligation Sequencing

Abstract Background Antimicrobial resistance is a critical global concern. Pathogens may become resistant through acquisition of mobile genetic elements harboring antimicrobial resistance genes (ARGs). Environmental biofilms provide an excellent venue for ARG transfer via mobile elements. Mobile ARG dissemination pathways in the environment require a better understanding. Here we use proximity-ligation (Hi-C) and metagenomic sequencing to pair plasmids and ARGs to their bacterial hosts and present evidence of plasmid sharing between bacterial species. Methods Eight environmental biofilm swabs collected from water sources in Sindh Province, Pakistan were equally aliquoted for preparation by 1) DNA cross-linking for proximity ligation and Hi-C metagenomic sequencing, and 2) traditional shotgun metagenomic sequencing. Metagenomic Hi-C was used to analyze the proximity-ligation and shotgun sequencing data to construct metagenomic-assembled genomes, determine if ARGs were chromosomally- or plasmid-located, and associate mobile genetic elements and ARGs with their host cells. Results Actinomycetia, Gammaproteobacteria, and Alphaproteobacteria were the most frequently detected taxa in most samples, with Bacteroidia also being at higher numbers than other taxa in most samples. Multiple classes of ARGs in the sample were identified as being located on plasmids, including aminoglycoside ARGs (e.g. aph(3'), aph(6)), beta-lactam RGs (blaCTX-M-15, blaEC, blaTEM-1, blaOKP-A), macrolide RGs (ere(A), erm(X), mph(A), mef(C), mph(G)), quinolone RG qnrS1, sulfonamide RG sul1, and several tetracycline and metal ARGs. TetC, blaA2, and aac(3)I, were only found on bacterial chromosomes. Analysis of unique assembled ARG-bearing plasmids and taxonomic clusters revealed plasmid sharing between bacterial species, some of which were not closely related taxa. Conclusion Mobile ARGs are common in environmental biofilms. These ARGs have the potential to spread within biofilms between species, as evidenced by shared plasmid between different bacterial species. The environment should be considered an important source for mobile ARG dissemination between bacterial species. Disclosures All Authors: No reported disclosures

Acetylation of alginate enables the production of inks that mimic the chemical properties of P. aeruginosa biofilm.

The reason why certain bacteria, e.g., Pseudomonas aeruginosa (PA), produce acetylated alginate (Alg) in their biofilms remains one of the most intriguing facts in microbiology. Being the main structural component of the secreted biofilm, like the one formed in the lungs of cystic fibrosis (CF) patients, Alg plays a crucial role in protecting the bacteria from environmental stress and potential threats. Nonetheless, to investigate the PA biofilm environment and its lack of susceptibility to antibiotic treatment, the currently developed in vitro biofilm models use native seaweed Alg, which is a non-acetylated Alg. The role of the acetyl side group on the backbone of bacterial Alg has never been elucidated, and the transposition of experimental results obtained from such systems to clinical conditions (e.g., to treat CF-infection) may be hazardous. We systematically investigated the influence of acetylation on the physico-chemical and mechanical properties of Alg in solution and Ca2+-crosslinked hydrogels. Furthermore, we assessed how the acetylation influenced the interaction of Alg with tobramycin, a common aminoglycoside antibiotic for PA. Our study revealed that the degree of acetylation directly impacts the viscosity and Young's Modulus of Alg in a pH-dependent manner. Acetylation increased the mesh size in biofilm-like Alg hydrogels, directly influencing antibiotic penetration. Our results provide essential insights to create more clinically relevant in vitro infection models to test the efficacy of new drugs or to better understand the 3D microenvironment of PA biofilms.

Structural, Morphological, and Antibacterial Attributes of Graphene Oxide Prepared by Hummers' and Brodie's Methods.

Graphite oxidation to graphene oxide (GO) is carried out using methods developed by Brodie (GO-B) and Hummers (GO-H). However, a comparison of the antibacterial properties based on the physicochemical properties has not been performed. Therefore, this paper outlines a comparative analysis of GO-H and GO-B on antibacterial efficacy against Gram-positive and Gram-negative bacterial cultures and biofilms in an aqueous environment and discusses which of the properties of these GO nanomaterials have the most significant impact on the antibacterial activity of these materials. Synthesis of GO with Brodie's and modified Hummers' methods was followed by an evaluation of their structural, morphological, and physicochemical properties by Raman, FTIR, UV-vis spectroscopy, and X-ray diffraction (XRD). The GO-B surface appeared more oxidized than that of GO-H, which could be crucial for interactions with bacteria. According to our results, GO-B demonstrated notably superior anti-biofilm efficacy. Despite its higher production cost, GO-B exhibits more excellent capabilities in combating bacterial biofilms than GO-H.

Effect of neutral electrolyzed water on biofilm formed by meat-related Listeria monocytogenes: Intraspecies variability and influence of the growth surface material.

Listeria monocytogenes raises major challenges for the food industry. Due to its capacity to form biofilms, this pathogen can persist in processing environments and contaminate the final products. Neutral electrolyzed water (NEW) may offer a promising and eco-friendly method for controlling L. monocytogenes biofilms, though current in vitro studies on its antibiofilm activity are limited and often focused on reference strains. In this study, we assessed the effect of NEW on biofilms formed by meat-related and reference L. monocytogenes strains on polystyrene and stainless steel. Forty wild-type strains isolated from meat products and processing environments were firstly screened for their biofilm-forming abilities and classified as weak (30%; 12/40), moderate (55%; 22/40), and strong (15%; 6/40) biofilm producers. Twenty-two wild-type and two reference strains were selected for the eradication assays, performed by treating the biofilms with NEW for 9minutes of total contact time. In silico functional enrichment analysis and the visualization of biofilms by scanning electron microscopy (SEM) were also performed. The NEW treatment resulted in a greater average reduction of viable cells in biofilms formed on polystyrene (4.3±1.0 log10 CFU/cm2) compared to stainless steel (2.9±2.0 log10 CFU/cm2), and a remarkable intraspecies variability was observed. SEM images revealed higher structural damage on biofilms formed on polystyrene. Functional enrichment analysis suggested that clustered regularly interspaced short palindromic repeats (CRISPR)-associated elements could be involved in resistance to the treatments. NEW could be a promising additional tool to mitigate L. monocytogenes biofilms in meat processing environments, although its effect varied with surface material and strain-specific characteristics.

Characterization of Serratia marcescens: Prodigiosin Synthesis under Various Environmental Conditions and Biofilm Formation

Characterization of Serratia marcescens: Prodigiosin Synthesis under Various Environmental Conditions and Biofilm Formation

Biofilms in Food Processing: A Comprehensive Review of Understanding Formation, Challenges, and Future Directions

Biofilms are complex communities of microorganisms attached to the surfaces and can cause cross-contamination of foods. Biofilms are irreversibly linked with a surface inside an extracellular polymeric substance matrix (EPS) which is challenging for the food industry. There exists an urgent need for disinfectants or new technologies to restrict reversible and irreversible attachment which are the main cause of surface tension of microorganisms that leads to surface adhesion. Many articles have reported the negative effects of biofilm production in the food industry and the role of microorganisms such as Bacillus cereus, and Listeria monocytogenes in foodborne diseases. This review paper discusses the formation and characteristics of Biofilm, the structure and composition of biofilms, factors influencing biofilm development, the presence of biofilms in food processing environments, the type of microorganisms present in the biofilms, and strategies and controlling measures for preventing biofilm formation. The article also discussed some positive impacts of biofilms in various applications.

ZnO‐CuS/F127 Hydrogels with Multienzyme Properties for Implant‐Related Infection Therapy by Inhibiting Bacterial Arginine Biosynthesis and Promoting Tissue Repair

AbstractImplant‐related infections are characterized by the formation of bacterial biofilms. Current treatments have various drawbacks. Nanozymes with enzyme‐like activity can produce highly toxic substances to kill bacteria and remove biofilms without inducing drug resistance. However, it is difficult for current monometallic nanozymes to function well in complex biofilm environments. Therefore, the development of multimetallic nanozymes with efficient multienzyme activities is crucial. In the present study, bimetallic nanozyme, ZnO‐CuS nanoflowers with peroxidase (POD), glutathione oxidase (GSH‐Px), and catalase (CAT) activity are successfully synthesized via calcination and loaded into F127 hydrogels for the treatment of implant‐related infections. The ability of ZnO‐CuS nanoflowers to bind bacteria is key for efficient antimicrobial activity. In addition, ZnO‐CuS nanoflowers with H2O2 disrupt the metabolism of MRSA, including arginine synthesis, nucleotide excision repair, energy metabolism, and protein synthesis. ZnO‐CuS/F127 hydrogel in combination with H2O2 has been demonstrated to be effective in clearing biofilm infection and facilitating the switch of M1 macrophages to M2‐repairative phenotype macrophages for the treatment of implant infections in mice. Furthermore, ZnO‐CuS/F127 hydrogels have favorable biosafety, and their toxicity is negligible. ZnO‐CuS/F127 hydrogel has provided a promising biomedical strategy for the healing of implant‐related infections, highlighting the potential of bimetallic nanozymes for clinical applications.

TcaR is an important transcriptional regulator involved in environmental stress response and virulence in foodborne Staphylococcus aureus

Staphylococcus aureus is a pathogenic microorganism that widely exists in milk and dairy products, posing a great challenge to food safety and human health. Although TcaR is known to be the only transcriptional regulator in the teicoplanin-associated operon, its function in stress response and virulence has not been investigated. In this study, the role of TcaR in environmental stress tolerance, biofilm formation, antibiotic sensitivity and virulence was investigated in foodborne S. aureus. Deletion of tcaR induced the biofilm formation and environmental stress (heat, desiccation and H2O2) tolerance of S. aureus. The tcaR mutants showed reduced susceptibility to vancomycin and teicoplanin. Additionally, mutation of tcaR resulted in decreased hemolytic activity, suggesting weakened virulence and pathogenicity of S. aureus. This paper is the first time to report the functions of TcaR in virulence and environmental tolerance, providing a new potential target gene for revealing mechanism of stress resistance in foodborne S. aureus and contributing to prevent food poisoning caused by this pathogenic microorganism.

Biofilms in modern CaCO3-supersaturated freshwater environments reveal viral proxies

Biofilms are mucilaginous-organic layers produced by microbial activity including viruses. Growing biofilms form microbial mats which enhance sediment stability by binding particles with extracellular polymeric substances and promoting growth through nutrient cycling and organic matter accumulation. They preferentially develop at the sediment-water interface of both marine and non-marine environments, and upon the growing surfaces of modern tufa and travertine. In this context, however, little is known about the factors, environmental or anthropogenic, which affect viral communities in freshwater spring settings. To explore this issue, geochemical and metagenomic data were subjected to multidimensional analyses (Principal Component Analysis, Classical Multidimensional Scaling, Partial Least Squares analysis and cluster analysis based on beta-diversity), and these show that viral composition is specific and dependent on environment. Indeed, waters precipitating tufa and travertine do vary in their geochemistry with their viruses showing distinct variability between sites. These differences between virus groups allow the formulation of a viral proxy, based on the Caudoviricetes/Megaviricetes ratio established on the most abundant groups of viruses. This ratio may be potentially used in analysing ancient DNA preserved in carbonate formations as an additional source of information on the microbiological community during sedimentation.

Effect of novel non-coding small RNA111 of Pseudomonas aeruginosa on bacterial virulence

Pseudomonas aeruginosa is an important gram-negative pathogen that often causes skin, respiratory, and bloodstream infections. The virulence of P. aeruginosa is regulated by sRNAs through a complex network, which is still incompletely understood. This study employed transcriptome sequencing (RNA-Seq) to screen sRNA 111, and real-time quantitative PCR was conducted for validation. In addition, an overexpression strain of sRNA111 was constructed and the effects of overexpression of sRNA111 on virulence factors such as biofilm formation, pyocyanin, swarming motility, inflammatory factors, and biofilm-associated factors were analyzed. The results revealed that sRNA111 might be closely related to the formation of P. aeruginosa biofilm. Compared to the vector, sRNA111 was found to enhance P. aeruginosa biofilm formation while also suppressing swarming motility, invasive ability, and adhesion capability. Meanwhile, sRNA111 up-regulated the expression of biofilm-related genes, such as Anr, pel family, polB, recA, etc., more significantly in the biofilm environment compared with the wild strain. In addition, sRNA111 effectively promoted the expression of inflammatory factors in the biofilm environment compared with the planktonic environment. These results implied that sRNA111 regulates the formation of P. aeruginosa biofilm and other virulence factors and further modulates the process of infection immunity.

The application of ozone within the food industry, mode of action, current and future applications, and regulatory compliance.

The food industry faces numerous challenges today, with the prevention and reduction of microbial contamination being a critical focus. While traditional chemical-based methods are effective and widely used, rising energy costs, the development of microbial tolerances, and growing awareness of the ecological impact of chemical biocides have renewed interest in novel biocides. Ozone, in both its gaseous and aqueous forms, is recognized as a potent disinfectant against bacteria, viruses, and fungi due to its high oxidation potential. Our review highlights several studies on the applications of ozone within the food industry, including its use for surface and aerosol disinfection and its capacity to reduce viable Listeria monocytogenes, a pertinent foodborne pathogen harbouring environmental and biocide stress tolerances and biofilm former. We also explore the use of ozone in food treatment and preservation, specifically on blueberries, apples, carrots, cabbage, and cherry tomatoes. While ozone is an effective disinfectant, it is important to consider material incompatibility, and the risks associated with prolonged human exposure to high concentrations. Nevertheless, for certain applications, ozone proves to be an efficacious and valuable alternative or complementary method for microbial control. Compliance with the biocide products regulation will require ozone device manufacturers to produce proven efficacy and safety data in line with British standards based on European standards (BS EN), and researchers to propose adaptations to account for ozone's unique properties.

PH‐sensitive size/surface charge‐adaptive quaternary ammonium salt copolymer micelles for antibiofilm activity against Staphylococcus aureus

AbstractThe low penetration of antibacterial materials within biofilms results in a 1000‐fold decrease in bactericidal efficiency, complicating the complete removal of biofilms and potentially leading to persistent and recurrent bacterial infections that significantly impact human health. In this study, we present a multifunctional pH‐responsive antibacterial material based on random copolymers poly(dimethylaminoethyl methacrylate decyl ammonium bromide‐co‐polyethylene glycol methacrylate) copolymer (PQACs10‐co‐PEGMA10). The PQACs10‐co‐PEGMA10 copolymers could self‐assemble into micelles in selective solvent water. With the stealth function of polyethylene glycol (PEG), the PQACs10‐co‐PEGMA10 micelles can rapidly penetrate biofilms in a physiological environment and exhibit excellent antibacterial activity by exposing the quaternary ammonium salt (QACs) in an acidic microenvironment to eliminate biofilm. Furthermore, PQACs10‐co‐PEGMA10 micelles were applied as coatings using a dropwise method. The PEG chain formed hydration layer and the QACs chain for sterilization can hinders bacterial adhesion and proliferation, thereby preventing biofilm formation. The results show that the minimal inhibit concentration values and minimum biofilm eradication concentrations of the PQACs10‐co‐PEGMA10 micelles against Gram‐positive Staphylococcus aureus were 64 and 128 μg/mL, respectively. The antifouling and antibacterial rates of micelle coating against S. aureus were more than 99.99%. Taken together, the pH‐responsive PQACs10‐co‐PEGMA10 micelles demonstrate a good ability to clear and prevent biofilms, holding promise for complete biofilm removal and a reduction in biofilm‐related infections.

Phage communities in household-related biofilms correlate with bacterial hosts

The average American spends 93% of their time in built environments, almost 70% of that is in their place of residence. Human health and well-being are intrinsically tied to the quality of our personal environments and the microbiomes that populate them. Conversely, the built environment microbiome is seeded, formed, and re-shaped by occupant behavior, cleaning, personal hygiene and food choices, as well as geographic location and variability in infrastructure. Here, we focus on the presence of viruses in household biofilms, specifically in showerheads and on toothbrushes. Bacteriophage, viruses that infect bacteria with high host specificity, have been shown to drive microbial community structure and function through host infection and horizontal gene transfer in environmental systems. Due to the dynamic environment, with extreme temperature changes, periods of wetting/drying and exposure to hygiene/cleaning products, in addition to low biomass and transient nature of indoor microbiomes, we hypothesize that phage host infection in these unique built environments are different from environmental biofilm interactions. We approach the hypothesis using metagenomics, querying 34 toothbrush and 92 showerhead metagenomes. Representative of biofilms in the built environment, these interfaces demonstrate distinct levels of occupant interaction. We identified 22 complete, 232 high quality, and 362 medium quality viral OTUs. Viral community richness correlated with bacterial richness but not Shannon or Simpson indices. Of quality viral OTUs with sufficient coverage (614), 532 were connected with 32 bacterial families, of which only Sphingomonadaceae, Burkholderiaceae, and Caulobacteraceae are found in both toothbrushes and showerheads. Low average nucleotide identity to reference sequences and a high proportion of open reading frames annotated as hypothetical or unknown indicate that these environments harbor many novel and uncharacterized phage. The results of this study reveal the paucity of information available on bacteriophage in indoor environments and indicate a need for more virus-focused methods for DNA extraction and specific sequencing aimed at understanding viral impact on the microbiome in the built environment.

Staphylococcus aureus in Dairy Industry: Enterotoxin Production, Biofilm Formation, and Use of Lactic Acid Bacteria for Its Biocontrol.

Staphylococcus aureus is a well-known pathogen capable of producing enterotoxins during bacterial growth in contaminated food, and the ingestion of such preformed toxins is one of the major causes of food poisoning around the world. Nowadays 33 staphylococcal enterotoxins (SEs) and SE-like toxins have been described, but nearly 95% of confirmed foodborne outbreaks are attributed to classical enterotoxins SEA, SEB, SEC, SED, and SEE. The natural habitat of S. aureus includes the skin and mucous membranes of both humans and animals, allowing the contamination of milk, its derivatives, and the processing facilities. S. aureus is well known for the ability to form biofilms in food processing environments, which contributes to its persistence and cross-contamination in food. The biocontrol of S. aureus in foods by lactic acid bacteria (LAB) and their bacteriocins has been studied for many years. Recently, LAB and their metabolites have also been explored for controlling S. aureus biofilms. LAB are used in fermented foods since in ancient times and nowadays characterized strains (or their purified bacteriocin) can be intentionally added to prolong food shelf-life and to control the growth of potentially pathogenic bacteria. Regarding the use of these microorganism and their metabolites (such as organic acids and bacteriocins) to prevent biofilm development or for biofilm removal, it is possible to conclude that a complex network behind the antagonistic activity remains poorly understood at the molecular level. The use of approaches that allow the characterization of these interactions is necessary to enhance our understanding of the mechanisms that govern the inhibitory activity of LAB against S. aureus biofilms in food processing environments.

Redox Concomitant Formation Method for Fabrication of Cu(I)-MOF/Polymer Composites with Antifouling Properties.

Marine biofouling, which is one of the technical challenges hindering the growth of the marine economy, has been controlled using cuprous oxide (Cu2O) nanoparticles due to the exceptional antifouling properties of Cu(I) ions. However, Cu2O nanoparticles have encountered bottlenecks due to explosive releases of Cu+ ions, high toxicity at elevated doses, and long-term instability. Here, we present a novel method called Redox Concomitant Formation (RCF) for fabricating a hierarchical Cu(I) metal-organic framework polypyrrole (Cu(I)-MOF/PPy) composite. This method enables in situ phase transition via successive redox reactions that change the chemical valence state and coordination mode of Cu(II)-MOF, resulting in a new structure of Cu(I)-MOF while creating a PPy layer surrounded by the hierarchical structure. Owing to the steady release of Cu+ ions from the Cu(I) sites and photothermal properties of PPy, Cu(I)-MOF/PPy exhibits superior and broad-spectrum resistance to marine bacteria, algae, and surface-adhered biofilms in complex biological environments, as well as long-term stability, resulting in 100 % eradication efficiency under solar-driven heating. Mechanistic insights into successive structural redox reactions and formation using the RCF method are provided in detail, enabling the fabrication of novel MOFs with the desired composition and structure for a wide range of potential applications.

Redox Concomitant Formation Method for Fabrication of Cu(I)−MOF/Polymer Composites with Antifouling Properties

AbstractMarine biofouling, which is one of the technical challenges hindering the growth of the marine economy, has been controlled using cuprous oxide (Cu2O) nanoparticles due to the exceptional antifouling properties of Cu(I) ions. However, Cu2O nanoparticles have encountered bottlenecks due to explosive releases of Cu+ ions, high toxicity at elevated doses, and long‐term instability. Here, we present a novel method called Redox Concomitant Formation (RCF) for fabricating a hierarchical Cu(I) metal–organic framework polypyrrole (Cu(I)−MOF/PPy) composite. This method enables in situ phase transition via successive redox reactions that change the chemical valence state and coordination mode of Cu(II)−MOF, resulting in a new structure of Cu(I)−MOF while creating a PPy layer surrounded by the hierarchical structure. Owing to the steady release of Cu+ ions from the Cu(I) sites and photothermal properties of PPy, Cu(I)−MOF/PPy exhibits superior and broad‐spectrum resistance to marine bacteria, algae, and surface‐adhered biofilms in complex biological environments, as well as long‐term stability, resulting in 100 % eradication efficiency under solar‐driven heating. Mechanistic insights into successive structural redox reactions and formation using the RCF method are provided in detail, enabling the fabrication of novel MOFs with the desired composition and structure for a wide range of potential applications.

2D Janus carrier-enabled trojan horse: Gallium delivery for the sequential therapy of biofilm associated infection

Biofilm-associated infections (BAIs) continue to pose a major challenge in the medical field. Nanomedicine, in particular, promises significant advances in combating BAIs through the introduction of a variety of nanomaterials and nano-antimicrobial strategies. However, studies to date have primarily focused on the removal of the bacterial biofilm and neglect the subsequent post-biofilm therapeutic measures for BAIs, rendering pure anti-biofilm strategies insufficient for the holistic recovery of affected patients. Herein, we construct an emerging dual-functional composite nanosheet (SiHx@Ga) that responds to pHs fluctuation in the biofilm microenvironment to enable a sequential therapy of BAIs. In the acidic environment of biofilm, SiHx@Ga employs the self-sensitized photothermal Trojan horse strategy to effectively impair the reactive oxygen species (ROS) defense system while triggering oxidative stress and lipid peroxidation of bacteria, engendering potent antibacterial and anti-biofilm effects. Surprisingly, in the post-treatment phase, SiHx@Ga adsorbs free pathogenic nucleic acids released after biofilm destruction, generates hydrogen with ROS-scavenging and promotes macrophage polarization to the M2 type, effectively mitigating damaging inflammatory burst and promoting tissue healing. This well-orchestrated strategy provides a sequential therapy of BAIs by utilizing microenvironmental variations, offering a conceptual paradigm shift in the field of nanomedicine anti-infectives.

Suspended particulate matter-biofilm aggregates benefit microcystin removal in turbulent water but trigger toxicity toward Daphnia magna

Suspended particulate matter (SPM) and biofilm are critical in removing contaminants in aquatic environments, but the environmental behavior and ecological toxicity of SPM-biofilm aggregates modulated by turbulence intensities are largely unknown. This study determined the removal pathways of microcystin-LR (MC-LR) by SPM and its biofilm under different turbulence intensities (2.25 × 10–3, 1.01 × 10–2, and 1.80 × 10–2 m2/s3). Then, we evaluated the toxicity of SPM-biofilm aggregates to Daphnia magna. The results revealed that SPM contributed to the adsorption of MC-LR, and the removal of MC-LR can be accelerated with biofilm formation on SPM, with 95.66 % to 97.45 % reduction in MC-LR concentration under the studied turbulence intensities. Higher turbulence intensity triggered more frequent contact of SPM and MC-LR, formed compact but smaller clusters of SPM-biofilm aggregates, and enhanced the abundance of mlrA and mlrB; thus benefiting the adsorption, biosorption, and biodegradation of MC-LR. Furthermore, the SPM-biofilm aggregates formed in turbulent water triggered oxidative stress to Daphnia magna, while a weak lethal toxic effect was identified under moderate turbulence intensity. The results indicate that the toxicity of SPM-biofilm aggregates fail to display a linear relationship with turbulence intensity. These findings offer new perspectives on understanding the environmental behavior and ecological outcomes of SPM and its biofilms in turbulent aquatic environments.

Can photocatalysis inhibit interspecies bacterial cooperation to quench the formation of robust complex bacterial biofilms in water environments?

Bacterial biofilms pose significant a public health risk as an environmental reservoir for opportunistic aquatic bacterial pathogens. Understanding the interspecies roles of complex bacterial biofilms under different stimuli and regulatory mechanisms of stress responses is the key to controlling their dissemination. Herein, two-species mixture (TSM) biofilms (Staphylococcus aureus and Pseudomonas aeruginosa) were constructed in a flowthrough reactor. Compared with the single-species biofilms, the TSM biofilm had higher growth activity to reach maturity faster, forming a staggered community structure. Moreover, the TSM biofilm exhibited greatly improved resistance to different antibiotics (16−128 times higher), especially to those that act on protein synthesis and cell membrane integrity, when compared to single planktonic microorganisms. In the presence of stimuli, photocatalysis effectively inactivated the TSM biofilm within 10 h, a 4-fold shorter inactivation time compared to UVC irradiation. In addition, photocatalysis effectively depleted the extracellular polymers of the TSM biofilm and inhibited secretion of their interspecies quorum sensing signaling molecule autoinducer-2 (AI-2). However, the expression of AI-2 induced related virulence factors, and biofilm growth-related genes were initially up-regulated 3 − 10 fold for the TSM biofilm within the first 2 − 4 h of photocatalysis, followed by significant down-regulation. Furthermore, the addition of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione effectively delayed the photocatalytic inactivation efficiency of the TSM biofilm compared to the control. These results suggest that photocatalysis can effectively inactivate biofilms by inhibiting interspecies cooperation by quenching AI-2 in the TSM biofilm. This work sheds light on controlling biofilms in public health engineering systems.

Distribution of extracellular adhesins in environmental biofilms and flocs: Reimagining the microbial structure

Extracellular cellular adhesins facilitate microbial aggregation; however, most of the information about extracellular adhesins is based on pure culture studies. In this study, we characterized the hydrophobic characteristics and distribution of the extracellular adhesins in environmental biofilms and flocs. The hydrophobic characteristics of the extracellular adhesins were studied by sonicating the microbial aggregates to disperse the cells and by fractionating them using the microbial adhesion to the hydrocarbon method. Furthermore, we probed environmental biofilms and flocs using immunohistochemistry coupled with confocal laser scanning microscopy for reimaging the microbial aggregates based on extracellular adhesins. Small flocs have a relatively dispersed distribution of extracellular adhesins (flagella, fimbriae, pili, and amyloid adhesins). The stratified distribution of extracellular adhesins was observed in environmental biofilms. It was observed that the pili and amyloid adhesins were predominantly present in the core of biofilms, whereas flagella and fimbriae were present in the outer layer of the microbial aggregates. The dispersion of microbial aggregates is one of the limiting factors that challenge the sustainable application of wastewater treatment processes. Greater attention to the components of extracellular protein (such as the adhesins) is required to understand the aggregation of dispersible environmental microbial aggregates.