Promote Biofilm Formation Research Articles

Biofilm-based biocatalysis for β-cyclodextrin production by the surface-display of β-cyclodextrin glycosyltransferase in Bacillus subtilis

β-cyclodextrin (β-CD) is an important cyclic oligosaccharide, which is widely applicated in foods, environmental protection, and cosmetics, primarily prepared from enzymatic synthesis in traditional industry. However, several challenges persist, including cumbersome processes and difficulties in achieving continuous fermentation and catalysis. This research introduced a biofilm-based immobilized fermentation, integrating with enzyme catalysis system of surface display in Bacillus subtilis. The bslA gene was selected to construct the surface display system due to its ability to promote biofilm formation and serve as an anchorin. Compared to free cell catalysis, the biofilm-based immobilized catalysis expanded the temperature range to 40–70 and the pH range to 5–7.5. During the continuous catalysis process, by the 13th batch, the relative activity remained around 52%, and the conversion rate exceeded 36%, similar to the single-batch free cell catalysis. These findings provide valuable insights and effective strategies for the industrial production of β-CD and other biochemicals through continuous catalysis.

The Quorum Sensing Mechanism of Aggregatibacter actinomycetemcomitans in Pathogenesis of Periodontitis: Systematic Literature Review

Background: Periodontitis is one of the most prevalent oral diseases worldwide and a significant public health concern. Periodontitis characterized by inflammation of the tissues supporting the teeth. It is primarily caused by bacteria in the subgingival biofilm, with A. actinomycetemcomitans and P. gingivalis as a key pathogens. These bacteria contribute to oral dysbiosis, leading to periodontal tissue damage. A. actinomycetemcomitans is particularly virulent, utilizing quorum sensing (QS) to regulate biofilm formation and enhance its pathogenicity, playing a significant role in the development and progression of periodontitis. Objective: This study aims to explain the quorum sensing mechanism in the pathogenesis of periodontitis caused by A. actinomycetemcomitans. Methods: This study employed a systematic literature review design by searching for articles based on predetermined inclusion and exclusion criteria. The search strategy used keywords and boolean operators across three databases (PubMed, Ebsco, ScienceDirect). Results: The variation between wild-type (Aa-WT) and LuxS mutant (Aa-LuxS) strains of A. actinomycetemcomitans impacts its pathogenicity. Quorum sensing (QS) interactions between A. actinomycetemcomitans and other periodontopathogens (P. gingivalis, T. denticola, P. intermedia) amplify virulence, promoting biofilm formation and disease progression. AI-2 regulates key processes, underscoring A. actinomycetemcomitans significant role in periodontitis. Conclusion: The quorum sensing mechanism in A. actinomycetemcomitans, through AI-2 production, is essential for microbial communication and biofilm formation, influencing the bacterium's virulence and role in periodontitis.

The chemoreceptor controlling the Wsp-like transduction pathway in Halomonas titanicae KHS3 binds and responds to purine derivatives.

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of this pathway results in colony morphology alterations and an increased ability to form biofilm. Such characteristics resemble the behavior of the Wsp pathway of Pseudomonas. In this work, we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. The purine derivatives guanine and hypoxanthine were identified as ligands of the recombinantly produced Htc10 ligand-binding domain, with dissociation constants in the micromolar range, and its structure was solved by X-ray protein crystallography. The sensor domain of Htc10 adopts a double Cache folding, with ligands bound to the membrane-distal pocket. A high-resolution structure of the occupied guanine-binding pocketallowed the identification of residues involved in ligand recognition. Such residues were validated by site-directed mutagenesis and isothermal titration calorimetry analyses of the protein variants. Moreover, heterologous expression of Htc10 in a Pseudomonas putida mutant lacking the native Wsp chemoreceptor promoted biofilm formation, a phenotype that was further enhanced by Htc10-specific ligands. To our knowledge, this is the first description of binding specificity of a chemoreceptor that controls the activity of an associated diguanylate cyclase, opening the way for dynamic studies of the signaling behavior of this kind of sensory complex.

Polymicrobial interactions between Staphylococcus aureus and Pseudomonas aeruginosa promote biofilm formation and persistence in chronic wound infections.

Chronic, non-healing wounds are a leading cause of prolonged patient morbidity and mortality due to biofilm-associated, polymicrobial infections. Staphylococcus aureus and Pseudomonas aeruginosa are the most frequently co-isolated pathogens from chronic wound infections. Competitive interactions between these pathogens contribute to enhanced virulence, persistence, and antimicrobial tolerance. P. aeruginosa utilizes the extracellular proteases LasB, LasA, and AprA to degrade S. aureus surface structures, disrupt cellular physiology, and induce cell lysis, gaining a competitive advantage during co-infection. S. aureus evades P. aeruginosa by employing aggregation mechanisms to form biofilms. The cell wall protein SasG is implicated in S. aureus biofilm formation by facilitating intercellular aggregation upon cleavage by an extracellular protease. We have previously shown that proteolysis by a host protease can induce aggregation. In this study, we report that P. aeruginosa proteases LasA, LasB, and AprA cleave SasG to induce S. aureus aggregation. We demonstrate that SasG contributes to S. aureus biofilm formation in response to interactions with P. aeruginosa proteases by quantifying aggregation, SasG degradation, and proteolytic kinetics. Additionally, we assess the role of SasG in influencing S. aureus biofilm architecture during co-infection in vivo, chronic wound co-infections. This work provides further knowledge of some of the principal interactions that contribute to S. aureus persistence within chronic wounds co-infected with P. aeruginosa, and their impact on healing and infection outcomes.

Nebivolol, an FDA-Approved Drug, Has General Antibacterial and Antibiofilm Effects and Increases Pseudomonas aeruginosa Tolerance to Ciprofloxacin.

The repurposing of approved drugs for new activities is gaining widespread attention, including drugs that have antibacterial properties. Nevertheless, besides the benefits of repurposing drugs, the discovery of new antibiotic activity in commonly used medicines raises concerns about inducing antibiotic tolerance and resistance due to the stress produced by the drugs. We found that nebivolol, which is used to treat hypertension, also has antibacterial activity. The antibacterial activity of nebivolol was tested by disc diffusion and kinetic O.D. Antibiofilm activity was determined by crystal violet staining. Nebivolol has antibiotic and antibiofilm activity against several bacteria. However, its effect on Pseudomonas aeruginosa's growth is limited, and it promotes biofilm formation. In addition, P. aeruginosa exposure to nebivolol induces resistance to ciprofloxacin but increases sensitivity to tobramycin. Nebivolol has antibiotic activity against several bacteria tested but is less effective and possibly detrimental in P. aeruginosa infections. The use of nebivolol, together with other antibiotics, should be further tested and carefully considered.

Functional analysis of cyclic diguanylate-modulating proteins in Vibrio fischeri.

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid (Euprymna scolopes) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri. The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V. fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions.

Unveiling the impact of antibiotic stress on biofilm formation and expression of toxin-antitoxin system genes in Clostridium difficile clinical isolates.

The study investigates how antibiotics affect biofilm formation and toxin gene expression in Clostridium difficile, which is essential for its survival and persistence. The study confirmed 25 strains of C. difficile and assessed biofilm formation. The MIC of metronidazole and vancomycin was determined through agar dilution, and the impact of sub-MIC levels on biofilm formation and eradication was investigated. Additionally, Real-time PCR was used to analyze the expression levels of target genes related to antibiotic treatment. We found that certain genes, such as the ImmA/IrrE system, were associated with increased biofilm formation in isolates. Sub-MIC antibiotic levels influenced gene expression related to biofilm activities, particularly emphasizing the importance of toxin-antitoxin systems. The results suggest that antibiotics at sub-MIC levels may play a signaling role in promoting biofilm formation and gene expression in C. difficile. Our study suggests that toxin and antitoxin genes may impact C. difficile biofilm formation, while antibiotics could signal biofilm strengthening and gene expression increase.

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

ABSTRACT The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in S. aureus.

The Epiphyte Bacillus sp. G2112 Produces a Large Diversity of Nobilamide Peptides That Promote Biofilm Formation in Pseudomonads and Mycobacterium aurum.

Bacillus sp. G2112, an isolate from cucumber plants that inhibited plant pathogens, produces not only surfactins, iturins, and fengycins common to many Bacillus spp., but also a large variety of N-acyl-(depsi)peptides related to A-3302-B and nobilamides. Four known and fourteen previously unreported nobilamide peptides were characterized using high-resolution mass spectrometry, tandem mass spectrometry, and NMR. The stereochemistry of the amino acids of nobilamide peptides was determined using Marfey's method. The diversity of nobilamide peptides from Bacillus sp. G2112 resulted from the incorporation of different acyl groups and amino acids in the sequence. The peptides occur in linear or cyclic form. In addition, a truncated N-acetylpentapeptide was produced. Agar diffusion assays with selected nobilamide peptides against plant pathogens and human pathogens revealed that A-3302-B and its N-acyl homologs, A-3302-A and nobilamide J, exhibited powerful antibiotic activity (at 5 µg/hole) against Lysinibacillus sphaericus that can cause severe sepsis and bacteremia in patients. Moreover, nobilamide peptides from Bacillus sp. G2112 strongly promoted biofilm formation in the Gram-positive Mycobacterium aurum and Gram-negative pseudomonads. Structurally diverse nobilamides from Bacillus sp. G2112, whether linear or cyclic, penta and heptapeptides, induced biofilm formation, suggesting that the common N-acetyl-D-Phe-D-Leu-L-Phe-D-allo-Thr-L-Val amino acid sequence motif is important for the biofilm-inducing activity.

Biofilm formation of Lactiplantibacillus plantarum food isolates under flow and resistance to disinfectant agents

Bacterial biofilms formed in food processing environments can be resilient against cleaning and disinfection causing recontamination and spoilage of foods. We investigated the biofilm formation of six Lactiplantibacillus plantarum food spoilage isolates (FBR1-FBR6) using WCFS1 as a reference strain, and examined the impact of benzalkonium chloride (BKC) and peracetic acid (PAA) on planktonic and biofilm cells formed under static and dynamic flow conditions. We used a custom-designed setup composed of a 48-well plate with 0.8 ml culture volumes. We quantified biofilm formation under static and dynamic flow conditions with a flow rate of 3.2 ml/h using plate counting, Crystal Violet (CV) staining, and fluorescence staining techniques. Our findings revealed significant differences in biofilm formation and disinfectant resistance among studied strains and cell types. We observed that flow promoted biofilm formation in some strains and increased the number of culturable cells within biofilms in all strains. Furthermore, biofilm cells demonstrated higher resistance to disinfectants in comparison to planktonic cells for certain strains. Interestingly, cells from dispersed under flow biofilms show higher resistance to disinfectants than cells from static biofilms. The results indicate the importance of flow conditions in influencing L. plantarum food isolates biofilm formation and disinfection resistance, which may have implications for product contamination and spoilage risks.

Porous hollow microspheres based on industrial solid waste enhance biomethane recovery from corn straw

The increasing production of industrial solid waste requires better disposal solutions. Porous hollow microspheres (PHM) are small inorganic materials with high surface area and adsorption capacity, but their potential for use in anaerobic digestion (AD) has not been explored. With PHM as additive, the effects of different industrial solid wastes (waste glass, steel slag, and fly ash) with different loadings (2 %–8 %), respectively, on the AD of corn straw were investigated in this study. The results showed that PHM could supplement trace elements and promote biofilm formation, which effectively shortened the lag period (25.00–60.87 %) and increased the methane yield (4.75 %–16.28 %). The 2 % PHM loading based on steel slag gave the highest methane yield (300.16 NmL/g VSadd). Microbial and PICRUSt2 analyses indicated that PHM enriched hydrolytic and acidogenic bacteria, increased the abundance of methanogenesis-related enzyme genes. This study provides a theoretical basis for the comprehensive utilization of coupled industrial and agricultural wastes.

Interspecies synergistic interactions mediated by cofactor exchange enhance stress tolerance by inducing biofilm formation.

Metabolic interactions (also known as cross-feeding) are thought to be ubiquitous in microbial communities. Cross-feeding is the basis for many positive interactions (e.g., mutualism) and is a primary driver of microbial community assembly. In this study, a combination of multi-omics analysis and metabolic modeling simulation was used to reveal the metabolic interactions of a synthetic consortium under hyperosmotic stress. Interspecies cofactor exchange was found to promote biofilm formation under hyperosmotic stress. This provides a new perspective for understanding the role of metabolic interactions in microbial communities to enhance environmental adaptation, which is significant for improving the efficiency of production activities and environmental bioremediation.

Effect of antibiotics and antibiotic by-products on the chlorine resistance of biofilms in drinking water distribution systems

This study investigated the effects of tetracycline (TC) and sulfadiazine (SD), along with their degradation products, on biofilm chlorine resistance. The results showed that low concentrations of antibiotics and their degradation products promoted biofilm formation on tube surfaces and increased resistance to chlorine. There was a positive correlation between antibiotic levels, degradation products, and biofilm chlorine resistance within a specific concentration range. SD had a more significant impact on biofilm chlorine resistance compared to TC. The presence of both antibiotics and their degradation products in water led to an increase in biofilm chlorine resistance, especially when combined with 0.3 mg/L sodium hypochlorite. The study shows a positive association between antibiotic and degradation product concentrations and the biofilms' improved chlorine resistance, a phenomenon that could have major consequences for water treatment efforts. Additionally, higher concentrations of antibiotics and their degradation products were associated with reduced biodiversity within the biofilm. Overall, the findings suggest that the presence of antibiotics and their degradation products can influence biofilm formation, alter bacterial species composition, and enhance biofilm resistance to chlorine in drinking water distribution systems. This fresh perspective on the relationship between pharmaceutical residues and microbial communities in water systems could transform our approach to assuring water safety and combatting antimicrobial resistance.

Molecular and phenotypic characterization of biofilm formation and antimicrobial resistance patterns of uropathogenic staphylococcus haemolyticus isolates in Casablanca, Morocco

This study aimed to establish the correlation between antibiotic resistance and biofilm formation by Staphylococcus haemolyticus and to examine the impact of sub-inhibitory concentrations of antibiotics (sub-MICs) on biofilm formation.Antibiotic susceptibility testing was conducted using the disk diffusion method, and biofilm formation was determined using Congo red agar and microtiter plate methods. Antibiotic resistance and biofilm-associated genes were detected using polymerase chain reaction.The majority of the twenty-one S. haemolyticus isolates were multidrug-resistant, methicillin-resistant (MRSH) and biofilm producers, including 43 % of moderate biofilm producers. A significant correlation was observed between MRSH and MSSH isolates in terms of biofilm production. Vancomycin, gentamicin, and ciprofloxacin at their sub-MICs tended to promote biofilm formation. The eno gene was present in 76.2 % of strains, followed by aap, and atlE.This study revealed a strong correlation between the biofilm-forming ability and antibiotic resistance in S. haemolyticus, which underlines a crucial public health issue.

Enhancement of nitrogen removal in constructed wetlands through boron mud and elemental sulfur addition: Regulation of sulfur and oxygen cycling

Constructed wetlands (CWs) utilizing elemental sulfur (S0) as an electron donor are effective for nitrate (NO3–-N) removal from low C/N wastewater. However, the limited bioavailability of chemical S0 (chem-S0) due to low water solubility and inadequate mass transfer impedes complete denitrification. In this study, boron mud waste and S0 (S0&BM) were processed and combined to convert chem-S0 to biological S0 (bio-S0) in CWs. The employment of S0&BM improved NO3–-N and total nitrogen (TN) removal rates by 15.36 % and 10.55 % compared to S0 alone. The 15N isotope tracing experiment unveiled the pathways of N2O production and demonstrated that S0&BM facilitated full denitrification, resulting in a higher percentage of nitrogen being degraded in a non-polluting form (54.70 %). Releasing trace amounts of boron and magnesium in S0&BM mitigated the excessive acidification caused by the sulfur autotrophic process and promoted biofilm formation, thereby pronouncing vertical oxygen fluctuations. Meanwhile, oxygen vacancies generated in the S0&BM promoted the closed-loop sulfur cycle, starting from the oxidation of chem-S0 to sulfate (SO42-), the important process of SO42--reducing bacteria using partial organic compounds as electron donors to reduce SO42- to sulfide (S2-) was realized, and finally the S2- was oxidized to bio-S0 with better properties. S0&BM enriched functional microbial communities in the biofilm related to nitrogen and sulfur transformation. Functional gene analysis showed an increase in the narG, nirS, norB, and nosZ genes supporting denitrification. The dsrA and sqr genes responsible for the sulfur cycle exhibited the highest abundance. This realization allowed for the utilization of waste materials for treatment, enhancing the energy efficiency of carbon reduction and pollution reduction in CWs.

BolA-like protein (IbaG) promotes biofilm formation and pathogenicity of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium widespread in temperate and tropical coastal waters; it is considered to be the most frequent cause of Vibrio-associated gastroenteritis in many countries. BolA-like proteins, which reportedly affect various growth and metabolic processes including flagellar synthesis in bacteria, are widely conserved from prokaryotes to eukaryotes. However, the effects exerted by BolA-like proteins on V. parahaemolyticus remain unclear, and thus require further investigation. In this study, our purpose was to investigate the role played by BolA-like protein (IbaG) in the pathogenicity of V. parahaemolyticus. We used homologous recombination to obtain the deletion strain ΔibaG and investigated the biological role of BolA family protein IbaG in V. parahaemolyticus. Our results showed that IbaG is a bacterial transcription factor that negatively modulates swimming capacity. Furthermore, overexpressing IbaG enhanced the capabilities of V. parahaemolyticus for swarming and biofilm formation. In addition, inactivation of ibaG in V. parahaemolyticus SH112 impaired its capacity for colonizing the heart, liver, spleen, and kidneys, and reduced visceral tissue damage, thereby leading to diminished virulence, compared with the wild-type strain. Finally, RNA-sequencing revealed 53 upregulated and 71 downregulated genes in the deletion strain ΔibaG. KEGG enrichment analysis showed that the two-component system, quorum sensing, bacterial secretion system, and numerous amino acid metabolism pathways had been altered due to the inactivation of ibaG. The results of this study indicated that IbaG exerts a considerable effect on gene regulation, motility, biofilm formation, and pathogenicity of V. parahaemolyticus. To the best of our knowledge, this is the first systematic study on the role played by IbaG in V. parahaemolyticus infections. Thus, our findings may lead to a better understanding of the metabolic processes involved in bacterial infections and provide a basis for the prevention and control of such infections.

Biochar, microbes, and biochar-microbe synergistic treatment of chlorinated hydrocarbons in groundwater: a review.

Dehalogenating bacteria are still deficient when targeted to deal with chlorinated hydrocarbons (CHCs) contamination: e.g., slow metabolic rates, limited substrate range, formation of toxic intermediates. To enhance its dechlorination capacity, biochar and its composites with appropriate surface activity and biocompatibility are selected for coupled dechlorination. Because of its special surface physical and chemical properties, it promotes biofilm formation by dehalogenating bacteria on its surface and improves the living environment for dehalogenating bacteria. Next, biochar and its composites provide active sites for the removal of CHCs through adsorption, activation and catalysis. These sites can be specific metal centers, functional groups or structural defects. Under microbial mediation, these sites can undergo activation and catalytic cycles, thereby increasing dechlorination efficiency. However, there is a lack of systematic understanding of the mechanisms of dechlorination in biogenic and abiogenic systems based on biochar. Therefore, this article comprehensively summarizes the recent research progress of biochar and its composites as a "Taiwan balm" for the degradation of CHCs in terms of adsorption, catalysis, improvement of microbial community structure and promotion of degradation and metabolism of CHCs. The removal efficiency, influencing factors and reaction mechanism of the degraded CHCs were also discussed. The following conclusions were drawn, in the pure biochar system, the CHCs are fixed to its surface by adsorption through chemical bonds on its surface; the biochar composite material relies on persistent free radicals and electron shuttle mechanisms to react with CHCs, disrupting their molecular structure and reducing them; biochar-coupled microorganisms reduce CHCs primarily by forming an "electron shuttle bridge" between biological and non-biological organisms. Finally, the experimental directions to be carried out in the future are suggested to explore the optimal solution to improve the treatment efficiency of CHCs in water.

Nitrogen conversion performance of a polypropylene carrier designed to promote biofilm formation through foaming

The performance of plastic carriers in a moving bed biofilm reactor used for water treatment is strongly determined by the carrier material and its shape. Only a few reports have described the effect of foaming on biofilm formation and nitrogen conversion by polypropylene (PP) carriers. Here, we investigated a PP foam carrier for its biofilm forming ability and the resulting biofilm’s ability to remove nitrogen compounds. The results revealed that foaming increased the amounts of denitrifying bacterial biofilms attached to PP by approximately 44 times, indicating that unevenness by foaming promoted biofilm formation. Biofilms were also formed using activated sludges from nitrification and denitrification of a wastewater treatment facility. Amplicon sequencing analysis of the biofilms revealed that the families Xanthomonadaceae and Comamonadaceae, including nitrifying bacteria, were enriched in nitrifying sludge-derived biofilms, whereas Dechloromonas sp. and family Pseudomonadaceae, including denitrifying bacteria, were enriched in denitrifying sludge-derived biofilms. Furthermore, nitrifying sludge-derived biofilms completely removed ammonia, and its removal ability was superior to that of the original sludge. Denitrification by denitrifying sludge-derived biofilms was promoted by the addition of fumaric acid. Both nitrifying and denitrifying sludge-derived biofilms could remove nitrogen compounds from 1.4 mM ammonia-containing synthetic wastewater. Finally, the effect of biomass addition on the carrier was investigated. The addition of composted seaweed waste to PP foam carriers enhanced the formation of denitrifying sludge-derived biofilms by approximately 2.2 times, and the denitrification reaction by biofilms was promoted. These results revealed that waste biomass can be used to further enhance PP foam carrier performance.

Candida parapsilosis: Heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host's immune response. In C. parapsilosis, three Saps have been identified: Sapp1, Sapp2 and Sapp3. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in a cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 fluorescence arbitrary units (FAU), with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

Exogenous putrescine plays a switch-like influence on the pH stress adaptability of biofilm-based activated sludge.

Microbial community adaptability to pH stress plays a crucial role in biofilm formation. This study aims to investigate the regulatory mechanisms of exogenous putrescine on pH stress, as well as enhance understanding and application for the technical measures and molecular mechanisms of biofilm regulation. Findings demonstrated that exogenous putrescine acted as a switch-like distributor affecting microorganism pH stress, thus promoting biofilm formation under acid conditions while inhibiting it under alkaline conditions. As pH decreases, the protonation degree of putrescine increases, making putrescine more readily adsorbed. Protonated exogenous putrescine could increase cell membrane permeability, facilitating its entry into the cell. Subsequently, putrescine consumed intracellular H+ by enhancing the glutamate-based acid resistance strategy and the γ-aminobutyric acid metabolic pathway to reduce acid stress on cells. Furthermore, putrescine stimulated ATPase expression, allowing for better utilization of energy in H+ transmembrane transport and enhancing oxidative phosphorylation activity. However, putrescine protonation was limited under alkaline conditions, and the intracellular H+ consumption further exacerbated alkali stress and inhibits cellular metabolic activity. Exogenous putrescine promoted the proportion of fungi and acidophilic bacteria under acidic stress and alkaliphilic bacteria under alkali stress while having a limited impact on fungi in alkaline biofilms. Increasing Bdellovibrio under alkali conditions with putrescine further aggravated the biofilm decomposition. This research shed light on the unclear relationship between exogenous putrescine, environmental pH, and pH stress adaptability of biofilm. By judiciously employing putrescine, biofilm formation could be controlled to meet the needs of engineering applications with different characteristics.IMPORTANCEThe objective of this study is to unravel the regulatory mechanism by which exogenous putrescine influences biofilm pH stress adaptability and understand the role of environmental pH in this intricate process. Our findings revealed that exogenous putrescine functioned as a switch-like distributor affecting the pH stress adaptability of biofilm-based activated sludge, which promoted energy utilization for growth and reproduction processes under acidic conditions while limiting biofilm development to conserve energy under alkaline conditions. This study not only clarified the previously ambiguous relationship between exogenous putrescine, environmental pH, and biofilm pH stress adaptability but also offered fresh insights into enhancing biofilm stability within extreme environments. Through the modulation of energy utilization, exerting control over biofilm growth and achieving more effective engineering goals could be possible.