Regulation and Therapeutic Intervention of Bacterial Biofilms

Recently, quorum sensing (QS) inhibitors (QSIs) have been combined with antibiotics to enhance antibiofilm efficacy in vitro and in vivo. However, targeting QS signals alone is not enough to prevent bacterial infections. Drug resistance and recurrence of biofilms makes it difficult to eradicate. Herein, photodynamic therapy (PDT) is selected to unite QSIs and antibiotics. A synergistically antibiofilm system, which combines QSIs, antibiotics, and PDT based on hollow carbon nitride spheres (HCNSs) is envisaged. First, HCNS provides the multidrug delivering ability, enabling QSIs and antibiotics to be released in sequence. Subsequently, multistage releases sensitize bacteria effectively, potentiating the chemotherapeutic effects of the antibiotics. Finally, the integration of QSIs and PDT not only minimizes the possibility of drug resistance, but also overcomes the problem of limited mass and extension of PDT. Even after 48 h of incubation, the bacterial biofilm is obviously inhibited. And its biofilm disperse efficiency exceeds 48% (compared with QSI‐potentiated chemotherapy group) and 40% (compared with PDT group). Besides, the inhibition of the QS system influences phenotypes related to virulence factor production and surface hydrophobicity, which weaken biofilm invasion and formation. Eventually, this system is applied to disperse bacterial biofilm in vivo. Overall, PDT and QS modulation are devoted to eradicate drug resistance and recurrence of the biofilm.

Quorum sensing (QS) is a critical bacterial communication system regulating behaviors like biofilm formation, virulence, and antibiotic resistance. This review highlights QS’s role in polymicrobial infections, where bacterial species interactions enhance antibiotic resistance. We examine QS mechanisms, such as acyl-homoserine lactones (AHLs) in Gram-negative bacteria and autoinducing peptides (AIPs) in Gram-positive bacteria, and their impact on biofilm-associated antibiotic resistance. The challenges uniquely associated with polymicrobial infections, such as those found in cystic fibrosis lung infections, chronic wound infections, and medical device infections, are also summarized. Furthermore, we explore various laboratory models, including flow cells and dual-species culture models, used to study QS interactions in polymicrobial environments. The review also discusses promising quorum sensing inhibitors (QSIs), such as furanones and AHL analogs, which have demonstrated efficacy in reducing biofilm formation and virulence in laboratory and clinical studies. By addressing the interplay between QS and antibiotic resistance, this paper aims to advance therapeutic strategies that disrupt bacterial communication and improve antibiotic efficacy, ultimately mitigating the global challenge of antibiotic resistance in polymicrobial infections.

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C(12)-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an alpha-beta-alpha fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-A resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.

Quorum sensing (QS) is a coordination of a group of organisms to exhibit a specific action. It is an acquired social behavior presented to perform either symbiotic or pathogenic activity; however, most of the cases in the absence of QS, the decision to execute certain actions has not been performed. Therefore, QS is also termed as “collective decisions”; it is induced and executed by signaling molecules when the signaling molecule crosses a certain threshold. QS phenomenon is shown by many bacteria and fungi and yeast; recently, viruses also have shown to communicate via QS. In modern research era, study of QS is of most interest for the majority of human healthcare as well as animal health reasons. In general, a key approach perceived is inhibition of QS in case of infections or biofilm formation. Inhibition of QS can reduce the initiation of disease and its severity. Hence, inhibition of QS is of significant interest in human and animal healthcare for developing diagnostic and therapeutic tool. Inhibition of QS is an emerging tool to perform the antimicrobial activity by using targeting agents at either three different levels: production, spread, or acceptance of the signal. The current review primarily emphasizes diverse mechanisms of QS, its inhibition, recent challenges and advances in the field, and its clinical implications in human and animal healthcare.

The implications of quorum sensing inhibition in bacterial antibiotic resistance- with a special focus on aquaculture

The rise of multidrug resistance (MDR) bacteria in nosocomial and health-care institutions is widespread and is currently recognized as a major medical challenge. Mechanisms of bacterial resistance, namely, quorum sensing (QS), biofilm formation, and efflux pumps, have been identified as critical biological processes in MDR bacteria. Following previous reports on the activity of phenothiazines against mechanisms of bacterial resistance, in this work we focus on the synthesis of xanthene derivatives aiming to discover phenothiazine bioisosteres with improved activity. Four compounds were obtained from the conjugation of xanthydrol with sulfonamides and aniline and were fully characterized. Their antibacterial activity was assessed considering their minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative strains, efflux pump inhibition, influence on biofilm formation and quorum-sensing (QS) inhibition. It was observed that the MIC of all the tested compounds was above 64 µg/mL The four 9-xanthenyl derivatives obtained, particularly the xanthene sulfonamide derivatives 3b and 3c, showed promising results on QS inhibition with a reduction of pigment production of 48 and 41 mm, and on biofilm formation with a reduction of 78 and 79%, respectively.

Acute hepatopancreatic necrosis disease (AHPND) is a disease that has caused significant losses to shrimp farming since 2009. The primary mechanism of this disease involves the binary toxins PirAvp and PirBvp, which are produced by specific strains of Vibrio parahaemolyticus, and which lead to significant damage to the hepatopancreatic cells of shrimps. Recent studies on the pathology of AHPND have also highlighted the role of the Vibrio quorum sensing (QS) system, which affects growth, virulence, and biofilm regulation in Vibrio species. For example, deletion of the qseC gene reduces the virulence of the AHPND-causative V. parahaemolyticus. Most importantly, the QS regulators LuxOvp and AphBvp have been implicated as they control the growth-phase-dependent expression of the pirAvp/pirBvp genes. Additionally, given the growing problem of antibiotic resistance, this article reviews several alternative control strategies targeting the QS system, including QS inhibition using natural products, biofloc technology, and the development of small-molecule inhibitors against AphBvp. Finally, we also discussed the potential of using probiotics to enhance shrimp disease resistance through QS inhibition, highlighting the feasibility of targeting the QS system for AHPND control.

Antibiotic resistance is a major problem and a major global health concern. In total, there are 16 million deaths yearly from infectious diseases, and at least 65% of infectious diseases are caused by microbial communities that proliferate through the formation of biofilms. Antibiotic overuse has resulted in the evolution of multidrug-resistant (MDR) microbial strains. As a result, there is now much more interest in non-antibiotic therapies for bacterial infections. Among these revolutionary, non-traditional medications is quorum sensing inhibitors (QSIs). Bacterial cell-to-cell communication is known as quorum sensing (QS), and it is mediated by tiny diffusible signaling molecules known as autoinducers (AIs). QS is dependent on the density of the bacterial population. QS is used by Gram-negative and Gram-positive bacteria to control a wide range of processes; in both scenarios, QS entails the synthesis, identification, and reaction to signaling chemicals, also known as auto-inducers. Since the usual processes regulated by QS are the expression of virulence factors and the creation of biofilms, QS is being investigated as an alternative solution to antibiotic resistance. Consequently, the use of QS-inhibiting agents, such as QSIs and quorum quenching (QQ) enzymes, to interfere with QS seems like a good strategy to prevent bacterial infections. This review sheds light on QS inhibition strategy and mechanisms and discusses how using this approach can aid in winning the battle against resistant bacteria.

Bacteria communicate through a system known as quorum sensing (QS), which allows them to coordinate their behavior in response to changes in cell density. This process involves the production, secretion, and detection of small extracellular signaling molecules, usually called autoinducers (AI). QS regulates a wide range of genes and functions, including biofilm formation and dispersal, swarming motility, production of virulence factors, antibiotic resistance, and bioluminescence, among others. QS inhibition has become a promising antivirulence strategy against antibiotic-resistant bacteria as it exerts a lower selective pressure compared to traditional antibiotics. In the bacterium Chromobacterium violaceum ATCC 12472, the QS proteins CviI/CviR, which are homologous to the LuxI/LuxR-type QS proteins of Aliivibrio fisheri, control many genes, including those responsible for producing the purple pigment violacein. C. violaceum has been widely used as a biosensor strain in QS inhibition studies, particularly the inhibition of violacein production. Many compounds have the potential to inhibit QS by binding to QS proteins, either LuxI or LuxR homologues, disrupting QS circuits. This protocol describes the method for quantifying violacein produced by C. violaceum ATCC 12472 and its inhibition by bioactive compounds at concentrations that do not affect bacterial growth. The assay described here demonstrates a significant reduction in violacein production by the tested compounds. This study highlights the potential for using this protocol to screen promising candidates in QS inhibition research.

Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.

Bacterial biofilms are responsible for many persistent infections by many clinically relevant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilms are much more resistant to conventional antibiotics than their planktonic counterparts. Quorum sensing, an intercellular communication system, controls pathogenesis and biofilm formation in most bacterial species. Quorum sensing provides an important pharmacological target since its inhibition does not provide a selective pressure for resistance. In this study, we investigated the quorum sensing and biofilm inhibitory activities of 126 plant extracts from 71 species collected from neotropical rainforests in Costa Rica. Quorum sensing and biofilm interference were assessed using a modified disc diffusion bioassay with Chromobacterium violaceum ATCC 12,472 and a spectrophotometric bioassay with Pseudomonas aeruginosa PA14, respectively. Species with significant anti-quorum sensing and/or anti-biofilm activities belonged to the Meliaceae, Melastomataceae, Lepidobotryaceae, Sapindaceae, and Simaroubaceae families. IC50 values ranged from 45 to 266 µg/mL. Extracts of these active species could lead to future development of botanical treatments for biofilm-associated infections.

Agricultural crops around the world are attacked by approximately 3,000–10,000 species of pest insect. There is increasing interest in resolving this problem using environmentally friendly approaches. Wolbachia (Hertig), an insect endosymbiont, can modulate host reproduction and offspring sex through cytoplasmic incompatibility (CI). The incompatible insect technique (IIT) based on CI-Wolbachia is a promising biological control method. Previous studies have reported an association between CI and Wolbachia density, which may involve a quorum sensing (QS) mechanism. In this study, we investigated the effect of manipulating QS in Wolbachia using several chemicals including 3O-C12-HSL; C2HSL; spermidine (QS inducers), 4-phenylbutanoyl; and 4-NPO (QS inhibitors) on American serpentine leafminer (Liriomyza trifolii [Burgess]), an agricultural pest. The results showed that inducing QS with 3O-C12-HSL decreased the proportion of hatched eggs and increased Wolbachia density, whereas QS inhibition with 4-phenylbutanoyl had the opposite effects. Thus, manipulating QS in Wolbachia can alter cell density and the proportion of hatched eggs in the host L. trifolii, thereby reducing the number of insect progeny. These findings provide evidence supporting the potential efficacy of the IIT based on CI-Wolbachia for the environmentally friendly control of insect pest populations.

Biofilm formation is mainly controlled by quorum sensing (QS). To prevent biofilm formation on stainless steel, the QS inhibitory (QSI) effect of 10 essential oil (EO) components on dual-species bacterial biofilm (Erwinia carotovora and Pseudomonas fluorescens) were screened. Geraniol showed the best QSI effect on dual-species biofilm with an inhibition rate of 61.27%. Meanwhile, geraniol at the sub-MIC significantly restrained the swimming, swarming, twitching motilities, exopolysaccharide production, and biofilm biomass of the two bacterial biofilms. The reduction of QS regulatory factors was possibly attributed to the inhibition of AI-2 signal molecule and thereby controlled the formation of biofilm. The viable cells in the dual-species biofilm formed on stainless steel were also decreased by using geraniol at sub-MICs. Such an inhibition also showed a good dose-dependence. Results of this study indicated that geraniol can be utilized as a QSI to achieve biofilm control on foods. Practical applications EOs can be utilized as a group of natural food preservers. However, high value and significant flavoring impact restrict their applications in the food industry. Facing these issues, taken Eos as QSI would be helpful to achieve microbial control at a low concentration level. Geraniol is an important component in many essential oils. In this study, the QSI effect of geraniol on the AI-2 signal molecule production, and biofilm formation in Erwinia carotovora and Pseudomonas fluorescens were elucidated. These findings would support that geraniol can be further developed as a promising strategy to achieve postharvest preservation of vegetables.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell-cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2, and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

Streptococcus suis (S. suis) is an important zoonotic pathogen. It mainly uses quorum sensing (QS) to adapt to complex and changeable environments. QS is a universal cell-to-cell communication system that has been widely studied for its physiological functions, including the regulation of bacterial adhesion, virulence, and biofilm formation. Quorum sensing inhibitors (QSIs) are highly effective at interfering with the QS system and bacteria have trouble developing resistance to them. We review the current research status of the S. suis LuxS/AI-2 QS system and QSIs. Studies showed that by inhibiting the formation of AI-2, targeting the LuxS protein, inhibiting the expression of luxs gene can control the LuxS/AI-2 QS system of S. suis. Other potential QSIs targets are summarized, which may be preventing and treating S. suis infections, including AI-2 production, transmission, LuxS protein, blockage of AI-2 binding to receptors, AI-2-mediated QS. Since antibiotics are becoming increasingly ineffective due to the emergence of resistant bacteria, including S. suis, it is thus critical to find new antibacterial drugs with different mechanisms of action. QSIs provide hope for the development of such drugs.