The impact of the hly gene deletion on biofilm formation and antibiotic sensitivity in Listeria monocytogenes.

Listeria monocytogenes is the causative agent of human listeriosis which has high hospitalization and mortality rates for individuals with weakened immune systems. The survival and dissemination of L. monocytogenes in adverse environments can be reinforced by the formation of biofilms. Therefore, this study aimed to understand the mechanisms underlying listerial biofilm development. Given that both nutrient availability and quorum sensing (QS) have been known as the factors influencing biofilm development, we hypothesized that the signal from a sentinel metabolite S‐adenosylmethionine (SAM) and Agr‐based QS could be synchronous in L. monocytogenes to modulate nutrient availability, the synthesis of extracellular polymeric substances (EPSs), and biofilm formation. We performed biofilm assays and quantitative real‐time PCR to investigate how biofilm volumes and the expression of genes for the synthesis of EPS were affected by SAM supplementation, agr deletion, or both. We found that exogenously applied SAM induced biofilm formation and that the expression of genes encoding the EPS synthesis machineries was regulated by SAM and/or Agr QS. Moreover, the gene transcription of components acting in the methyl cycle for SAM synthesis and Agr QS was affected by the signals from the other system. In summary, we reveal an interconnection at the transcriptional level between metabolism and QS in L. monocytogenes and highlight the critical role of metabolite‐oriented QS in biofilm development.

Listeria monocytogenes is a major foodborne pathogen in the United States that is capable of forming sanitizer-tolerant biofilms on diverse food contact surfaces and under varying temperature conditions. A plethora of research in the last decade has explored the potential of phytochemicals as antibiofilm agents. However, the low solubility of phytochemicals is a significant challenge that needs to be addressed to develop plant-based disinfectants that can be applied in the industry for controlling L. monocytogenes biofilms and improving food safety. This study investigated the efficacy of eugenol nanoemulsion (EGNE) in inhibiting biofilm formation in two strains of L. monocytogenes (Scott A and AT19115) on stainless steel surfaces at two temperatures (25 or 10°C). In addition, the effect of EGNE on pathogen motility, extracellular polymeric substances (EPS) production, eDNA production, and quorum sensing activity during biofilm formation was studied using standard bioassays. Moreover, the efficacy of EGNE in killing mature L. monocytogenes biofilm was also investigated against both the strains and temperature combinations. All experiments had a completely randomized design with duplicate samples and were repeated at least three times. EGNE had a particle size of ~75 nm, a polydispersity index of 0.25, and a high negative surface charge. EGNE 700 mg/L inhibited L. monocytogenes biofilm formation significantly by ~1.89 log in 72 h at 25°C and ~1.25 log on day 16 at 10°C, when compared to control (p < 0.05). EGNE at 2,750 mg/L concentration completely inactivated (~7 log CFU/coupon reduction as compared to control) L. monocytogenes biofilm cells developed at 25 or 10°C as early as 1 min of treatment time (p < 0.05). In addition, EGNE was able to significantly reduce the motility, EPS, eDNA production, and quorum sensing activity which plays a major role in biofilm formation. Both L. monocytogenes Scott A and AT19115 strains exhibited similar sensitivity to EGNE treatments. The results suggest that EGNE could potentially be used as a natural sanitizer to effectively control L. monocytogenes biofilms in food processing environments.

We develop a multiphasic hydrodynamic theory for biofilms taking into account interactions among various bacterial phenotypes, extracellular polymeric substance (EPS), quorum sensing (QS) molecules, solvent, and antibiotics. In the model, bacteria are classified into down-regulated QS, up-regulated QS, and non-QS cells based on their QS ability. The model is first benchmarked against an experiment yielding an excellent fit to experimental measurements on the concentration of QS molecules and the cell density during biofilm development. It is then applied to study development of heterogeneous structures in biofilms due to interactions of QS regulation, hydrodynamics, and antimicrobial treatment. Our 3D numerical simulations have confirmed that (i). QS is beneficial for biofilm development in a long run by building a robust EPS population to protect the biofilm; (ii). biofilms located upstream can induce QS downstream when the colonies are close enough spatially; (iii). QS induction may not be fully operational and can even be compromised in strong laminar flows; (v). the hydrodynamic stress alters the biofilm morphology. Through further numerical investigations, our model suggests that (i). QS-regulated EPS production contributes to the structural formation of heterogeneous biofilms; (ii) QS down-regulated cells tend to grow at the surface of the biofilm while QS up-regulated ones tend to grow in the bulk; (iii) when nutrient supply is sufficient, QS induction might be more effective upstream than downstream; (iv) QS may be of little benefit in a short timescale in term of fighting against invading strain/species; (v) the material properties of biomass (bacteria and EPS) have strong impact on the dilution of QS molecules under strong shear flow. In addition, with this modeling framework, hydrodynamic details and rheological quantities associated with biofilm formation under QS regulation can be resolved.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Part 1 Biofilms in the food and beverage industries: Biofilms in the food and beverage industries: an introduction Molecular mechanisms involved in biofilm formation by food-associated bacteria Methods for imaging and quantifying the structure of biofilms in food processing and other environments Monitoring of biofilms in the food and beverage industries A centralized database for use in studying bacterial biofilms and quorum sensing in food processing and other environments: MicroBQs. Part 2 Microorganisms and their metabolites in biofilms: Biofilm formation by food spoilage microorganisms in food processing environments Biofilm formation by Listeria monocytogenes and transfer to foods Biofilm formation by Salmonella in food processing environments Biofilm formation by Gram-positive bacteria including Staphylococcus aureus, Mycobacterium avium and Enterococcus spp in food processing environments Biofilm formation by spore-forming bacteria in food processing environments. Part 3 Biofilm prevention, inactivation and removal and beneficial biofilms: Food contact surfaces, surface soiling and biofilm formation Cleaning and sanitation in food processing environments for the prevention of biofilm formation and biofilm removal Novel methods for biofilm control and removal from food processing equipment. Part 4 Biofilms in particular food industry sectors: Biofilms in red meat processing Biofilms in dairy processing Biofilms and brewing Biofilms in poultry processing Beneficial biofilms: wastewater and other industrial applications Biofilms in fish processing Biofilms in fresh fruit and vegetables. Part 5 Appendix: Sampling and quantification of biofilms in food processing and other environments.

Application and mechanism of quorum sensing in biological wastewater treatment systems: Current state and future prospects

Biofilms are the communities of microorganisms, especially bacteria attached to a biotic or abiotic surface. These biofilms live in a self-sustained matrix and produce different substances called extracellular polymeric substances (EPS) which are responsible for the pathogenicity of a number of bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Klebsiella pneumoniae, Escherichia coli, etc. These EPS substance makes it difficult to eradicate the biofilm present on the surface. Biofilm formation is a five-step process. Biofilms can be monospecies or multispecies. In biofilms, cells communicate via Quorum Sensing (QS). QS is the regulation of gene expression in bacteria with respect to changes in cell population density. In QS, bacteria produce various signaling molecules called Auto-inducers (AI). AI concentration increases as the bacterial population increases. Bacteria respond to these AIs results in an alteration of gene expression, which results in the release of various virulence factors. QS involves a two-component signaling process which is different for both Gram-positive and Gram-negative bacteria. QS and EPS make the bacteria resistant to various antibiotics, which make the eradication difficult and hence requires more effective treatment. This article discusses the biofilm structure, phenomenon of biofilm formation, signaling, and pathogenicity to highlight the understanding of processes involved in biofilm formation.

Biofilm formation in marine Streptomyces is a dynamic yet poorly understood process, that limits their functional exploitation. This study investigated the biofilm development and extracellular polymeric substances (EPS) synthesis by Streptomyces nigra strain KDS4, a promising marine Actinobacterium, aiming to optimize EPS yield and characterize its bio-functional properties. Biofilm and EPS formation began with spore germination and hyphal growth, maturing by 60h with dense hyphal intertwining, sporulation, and EPS secretion, followed by dispersal at 84h. The bacterium showed the highest biofilm height (~ 2.35μm) over the polypropylene substrate. Upregulated expression of the cslA gene, associated with biofilm matrix production, was confirmed during biofilm development. Structural analysis of EPS revealed α- and β-glycosidic linkages, hydroxyl, and alkyne groups, along with an amorphous morphology and diverse elemental composition. EPS exhibited thermal transition up to 300°C and antioxidant and emulsifying properties. Notably, EPS demonstrated hydrogel-forming capability, with 5% (wt/wt) EPS-based hydrogel exhibiting rapid gelation (73s), high porosity and pore size (31.66μm), excellent swelling (53.54%), and strong viscoelasticity (G' > G''). At 20% (wt/wt) EPS, the hydrogel achieved a compressive strength of 36.83kPa, demonstrating its mechanical robustness. These findings highlight S. nigra strain KDS4 as a promising source of multifunctional EPS for sustainable environmental and biomedical applications. While the study provides detailed in vitro insights, evaluation of EPS functionality and biocompatibility remain to be explored. Future work should focus on scale-up production, structural-functional correlations, and validation of EPS-based hydrogels in environmental remediation and biomedical models.

Members of the genus Burkholderia are known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterized Burkholderia thailandensis biofilm development under flow conditions and sought to determine whether QS contributes to this process. B. thailandensis biofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by "dome" structures filled with biofilm matrix material. We showed that this process was dependent on QS. B. thailandensis has three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the three B. thailandensis QS systems, we show that QS-1 is required for proper biofilm development, since a btaR1 mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. The btaR1 mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions. The saprophyte Burkholderia thailandensis is a close relative of the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms, B. thailandensis is an ideal model organism for investigating questions in Burkholderia physiology. In this study, we characterized B. thailandensis biofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows that B. thailandensis produces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience of B. thailandensis biofilms against changes in the nutritional environment.

Biofilms are assemblages of bacteria embedded within a matrix of extracellular polymeric substances (EPS) attached to a substratum. The process of biofilm formation is a complex phenomenon regulated by the intracellular and intercellular signaling systems. Various secondary messenger molecules such as cyclic dimeric guanosine 3′,5′-monophosphate (c-di-GMP), cyclic adenosine 3′,5′-monophosphate (cAMP), and cyclic dimeric adenosine 3′,5′-monophosphate (c-di-AMP) are involved in complex signaling networks to regulate biofilm development in several bacteria. Moreover, the cell to cell communication system known as Quorum Sensing (QS) also regulates biofilm formation via diverse mechanisms in various bacterial species. Bacteria often switch to the biofilm lifestyle in the presence of toxic pollutants to improve their survivability. Bacteria within a biofilm possess several advantages with regard to the degradation of harmful pollutants, such as increased protection within the biofilm to resist the toxic pollutants, synthesis of extracellular polymeric substances (EPS) that helps in the sequestration of pollutants, elevated catabolic gene expression within the biofilm microenvironment, higher cell density possessing a large pool of genetic resources, adhesion ability to a wide range of substrata, and metabolic heterogeneity. Therefore, a comprehensive account of the various factors regulating biofilm development would provide valuable insights to modulate biofilm formation for improved bioremediation practices. This review summarizes the complex regulatory networks that influence biofilm development in bacteria, with a major focus on the applications of bacterial biofilms for environmental restoration.

Biofilm development plays a pivotal role in the bioleaching process. The attachment of the acidophilic chemolithotrophic Acidithiobacillus ferrooxidans to mineral surfaces is mediated by extracellular polymeric substances (EPS) involved in biofilm development. Previous work suggests that EPS composition of A. ferrooxidans is adapted to the energy source and, accordingly, the bacterium must be able to sense the surface to which attachment occurs with the consequent triggering of the expression of different EPS-genes. Quorum sensing (QS) is recognized as one of the main regulators of biofilm formation. A. ferrooxidans possesses a functional QS type AI-1 system and the analysis of culture supernatants revealed us that this bacterium is able to synthesize nine different homoserine lactones (AHLs) whose acyl-chain lengths oscillate between 8 and 16 carbons and include an alcohol or a ketone function at the C3 position. The transcription levels of the afeI gene encoding for the AHL synthase are higher in cells grown in sulfur and thiosulfate media than in iron-grown cells, suggesting that biofilm formation in A. ferrooxidans would be regulated by the QS type AI-1 system. In the present study, the effect of several synthetic AHLs and analogues on the attachment of A. ferrooxidans to pyrite was analyzed. Preliminary results suggest that some of these molecules are changing the bacterial attachment to pyrite.

Involvement of biofilm formation process during phosphate (P) solubilization by rhizobacterial strains is not clearly understood. Scanning electron microscopic observations revealed prominent biofilm development on tricalcium phosphate as well as on four different rock phosphate granules by two P solubilizing rhizobacteria viz. Burkholderia tropica P4 and B. unamae P9. Variation in the biofilm developments were also observed depending on the total P content of insoluble P used. Biofilm quantification suggested a strong correlation between the amounts of available P and degrees of biofilm formation. Lower concentrations of soluble P directed both the organisms towards compact biofilm development with maximum substratum coverage. Variation in the production of extracellular polymeric substances (EPS) in the similar pattern also suggested its close relationship with biofilm formation by the isolates. Presence of BraI/R quorum sensing (QS) system in both the organisms were detected by PCR amplification and sequencing of two QS associated genes viz. braR and rsaL, which are probably responsible for biofilm formation during P solubilization process. Overall observations help to hypothesize for the first time that, biofilm on insoluble P granules creates a close environment for better functioning of organic acids secreted by Burkholderia strains for maximum P solubilization during P deficient conditions.

The advances from at least two major research areas, biofilms and bacterial quorum sensing, have led us to begin to appreciate the concept that bacteria can organize into groups, form well-organized communities, and communicate with each other for coordinated activities or social life that was once believed to be restricted to multicellular organisms. Bacteria with altered physiological activities (biofilm phenotypes) are known to result largely from bacterial social behaviors controlled by quorum sensing or other mechanisms when they are living in biofilms. Understanding bacterial social behaviors and their molecular mechanisms in the development of biofilms will greatly facilitate the development of novel strategies in the prevention and treatment of biofilm infections. In 1998, researchers first described the role of las quorum sensing in biofilm formation of Pseudomonas aeruginosa. The biofilms formed by the mutant were also dispersed by the addition of the detergent sodium dodecyl sulfate. This finding suggests that quorum sensing plays an important role in the development of bacterial biofilms. More importantly, this study suggests an inextricable connection between two bacterial social behaviors, quorum sensing and biofilm formation. In P. aeruginosa organism, quorum sensing is highly complex and consists of two interlinked N-acyl-homoserine lactone (AHL) dependent regulatory circuits, which are modulated by numerous regulators acting at both the transcriptional and posttranscriptional levels. The chapter discusses how might quorum sensing signal molecules function in biofilms. Quorum sensing is emerging as an integral component of bacterial global gene regulatory networks responsible for bacterial adaptation in biofilms.

Quorum sensing (QS) is a cell-to-cell communication system, which is used by many bacteria to regulate diverse gene expression in response to changes in population density. Bacteria recognize the differences in cell density by sensing the concentration of signal molecules such as N-acyl-homoserine lactones (AHL) and autoinducer-2 (AI-2). In particular, QS plays a key role in biofilm formation, which is a specific bacterial group behavior. Biofilms are dense aggregates of packed microbial communities that grow on surfaces, and are embedded in a self-produced matrix of extracellular polymeric substances (EPS). QS regulates biofilm dispersal as well as the production of EPS. In some bacteria, biofilm formations are regulated by c-di-GMP-mediated signaling as well as QS, thus the two signaling systems are mutually connected. Biofilms are one of the major virulence factors in pathogenic bacteria. In addition, they cause numerous problems in industrial fields, such as the biofouling of pipes, tanks and membrane bioreactors (MBR). Therefore, the interference of QS, referred to as quorum quenching (QQ) has received a great deal of attention. To inhibit biofilm formation, several strategies to disrupt bacterial QS have been reported, and many enzymes which can degrade or modify the signal molecule AHL have been studied. QQ enzymes, such as AHL-lactonase, AHL-acylase, and oxidoreductases may offer great potential for the effective control of biofilm formation and membrane biofouling in the future. This review describes the process of bacterial QS, biofilm formation, and the close relationship between them. Finally, QQ enzymes and their applications for the reduction of biofouling are also discussed.

Simple SummaryQuorum sensing in ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacteria denotes a global threat to public health. The acquisition of antimicrobial resistance genes, virulence production, and biofilm formation by ESKAPE pathogens has reduced the treatment options for serious infections. QS has been well recognized as being involved in the pathogenesis and antibiotic resistance. More understanding of QS mechanistic would also aid in the prediction of underlying or even unknown mechanisms of antimicrobial resistance and bacterial pathogenesis. In this review, we describe the known antibiotic resistance and pathogenesis caused by QS as well as the strategies to control QS in these pathogens.A clique of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) bugs is the utmost causative agent responsible for multidrug resistance in hospital settings. These microorganisms employ a type of cell–cell communication termed ‘quorum sensing (QS) system’ to mediate population density and synchronously control the genes that modulate drug resistance and pathogenic behaviors. In this article, we focused on the present understanding of the prevailing QS system in ESKAPE pathogens. Basically, the QS component consisted of an autoinducer synthase, a ligand (e.g., acyl homoserine lactones/peptide hormones), and a transcriptional regulator. QS mediated expression of the bacterial capsule, iron acquisition, adherence factors, synthesis of lipopolysaccharide, poly-N-acetylglucosamine (PNAG) biosynthesis, motility, as well as biofilm development allow bacteria to promote an antimicrobial-resistant population that can escape the action of traditional drugs and endorse a divergent virulence production. The increasing prevalence of these harmful threats to infection control, as well as the urgent need for effective antimicrobial strategies to combat them, serve to highlight the important anti-QS strategies developed to address the difficulty of treating microorganisms.