Targeting Quorum Sensing LsrR Protein in E. coli: A Computational Approach to Screen the Plant Bioactive Compounds as Inhibitors of Biofilm Formation in Urinary Tract Infections.

The rapid emergence of bacterial resistance to antibiotics has greatly pressed the need for novel therapeutics. One of the strategies bacteria use to enhance their resistance toward antibiotics is to embed themselves into polymeric matrices known as a biofi lm. Bacteria in a biofi lm state are highly resistant towards antibiotics and immune response, thereby more diffi cult to eradicate. Hence, antibiofi lm agents are considered an alternative strategy to unravel problems regarding bacterial antibiotic resistance. Our lab has focused on bacterial communication process called quorum sensing (QS), which is crucial for bacteria to conduct group behaviors such as biofi lm. Cysteine residue of LasR, key QS protein regulator, has been suggested to play an important role in QS-mediated biofi lm formation. In addition, cysteine-containing biomolecules have been implicated in bacterial pathogenesis. Herein, we explored the ability of thiol-reactive molecules for their ability to interfere with biofi lm formation in pathogenic Gram-negative Pseudomonas aeruginosa (P. aeruginosa). We hypothesized that the thiol-reactive molecules could potentially react with sulfhydryl group of cysteine residue of crucial QS enzyme or of cysteine-containing biomolecules that may lead to the reduce in the ability of the pathogen to form biofi lm. Carbonylacrylic compounds were previously reported to rapidly undergo thiol-Michael reaction with cysteine-containing proteins under physiological conditions. Total of three new carbonylacrylic derivatives were synthesized and tested for their anti-biofi lm activity. All synthesized compounds could inhibit biofi lm formation in a concentration-dependent manner without toxicity to bacteria. Derivative 1a exhibited the most potent antibiofi lm activity with IC50 of 85 μM.

Escherichia coli assemble functional amyloid fibers termed curli that contribute to bacterial adhesion, biofilm formation, and host pathogenesis. We developed a cell-based high-throughput screen to identify inhibitors of curli-mediated adhesion in the laboratory strain MC4100 and curli-associated biofilm formation in the uropathogenic E. coli clinical isolate UTI89. Inhibitors of biofilm formation can operate through many mechanisms, and such inhibitors could hold therapeutic value in preventing and treating urinary tract infections. The curli-specific screen allows the identification of compounds that inhibit either curli expression, curli biogenesis, or adhesion by normally produced curli. In screening the NIH Clinical Collection of 446 compounds, we identified rifapentine as a potent inhibitor in both of these screens. Rifapentine is an antibiotic used to treat tuberculosis that targets RNA polymerase, but prevents curli-dependent adhesion and biofilm formation in E. coli at concentrations below those that affect viability. Rifapentine inhibits curli production and prevents biofilm formation on plastic, on agar, and at the air-liquid interface by inhibiting curli gene transcription. Comparisons with a cephalosporin antibiotic further revealed that curli production is not affected by standard antibiotic treatment and cell killing pressure. Thus, we reveal a new role independent of killing activity for rifapentine as an inhibitor of curli and curli-mediated biofilm formation.

Bacterial urinary tract infections (UTIs) are the most common nosocomial infections, accounting for about 40% of all hospital-acquired infections. The bacterial spectrum of nosocomial UTIs is broad and the treatment of UTIs is becoming difficult owing to the emergence of drug resistance. Therefore, it is reasonable to investigate novel and alternative therapeutic strategies to treat UTIs. Since UTIs are caused by uropathogens with quorum sensing (QS)-dependent biofilm forming abilities, interruption of QS systems may be a novel approach to combat drug resistance. In the present study, a methanol extract (and hexane extract derived from it) of the medicinal plant Hyptis suaveolens (L.) were shown to have anti-QS activity against the biosensor strain Chromobacterium violaceum (ATCC 12472). Furthermore, the hexane extract of H. suaveolens (HEHS) inhibited biofilm formation by uropathogens such as Escherichia coli, Proteus vulgaris, Proteus mirabilis, Klebsiella pneumoniae and Serratia marcescens. HEHS promotes the loosening of biofilm architecture and strongly inhibits in vitro biofilm formation by uropathogens, which was more apparent from microscopic images. In addition to this, HEHS reduces the production of QS-dependent virulence factors like protease and hemolysin, along with motility. The partial purification and GC-MS analysis of the active fraction revealed the presence of several therapeutically important compounds which may synergistically act on the uropathogens and possibly reduce the QS-dependent phenotypes. These findings suggest HEHS as potential phytotherapeutic agent which can be employed to formulate protective strategies against biofilm linked infections caused by uropathogens.

Quorum sensing (QS) is a bacterial cell-to-cell communication mechanism that plays an essential role in bacterial pathogenesis. QS governs bacterial behavior and controls biofilm formation, which in turn contributes to antibiotic resistance. Therefore, identifying and synthesizing novel compounds to overcome QS and inhibit biofilm formation are essential. Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications, including antibacterial, antifungal, anticoagulant, antioxidant, anticancer, and anti-inflammatory properties. Additionally, coumarins are capable of QS rewiring and biofilm formation inhibition, leading to higher susceptibility to antimicrobial agents and less antibiotic resistance. Therefore, in this review, we aim to provide an overview of QS and biofilm formation. This review also discusses the role of natural and synthesized coumarins in controlling QS, inhibiting biofilm formation, and inducing synergy in antibiotic–coumarin combinations. Hence, this review emphasizes the potential of coumarin compounds to act as antibacterial agents and demonstrates their ability to alleviate antibiotic resistance.

The formation of biofilm by bacteria confers resistance to biocides and presents problems in medical and veterinary clinical settings. Here we report the effect of carvacrol, one of the major antimicrobial components of oregano oil, on the formation of biofilms and its activity on existing biofilms. Assays were carried out in polystyrene microplates to observe (a) the effect of 0–0.8 mM carvacrol on the formation of biofilms by selected bacterial pathogens over 24 h and (b) the effect of 0–8 mM carvacrol on the stability of pre-formed biofilms. Carvacrol was able to inhibit the formation of biofilms of Chromobacterium violaceum ATCC 12472, Salmonella enterica subsp. Typhimurium DT104, and Staphylococcus aureus 0074, while it showed no effect on formation of Pseudomonas aeruginosa (field isolate) biofilms. This inhibitory effect of carvacrol was observed at sub-lethal concentrations (<0.5 mM) where no effect was seen on total bacterial numbers, indicating that carvacrol's bactericidal effect was not causing the observed inhibition of biofilm formation. In contrast, carvacrol had (up to 8 mM) very little or no activity against existing biofilms of the bacteria described, showing that formation of the biofilm also confers protection against this compound. Since quorum sensing is an essential part of biofilm formation, the effect of carvacrol on quorum sensing of C. violaceum was also studied. Sub-MIC concentrations of carvacrol reduced expression of cviI (a gene coding for the N-acyl-L-homoserine lactone synthase), production of violacein (pigmentation) and chitinase activity (both regulated by quorum sensing) at concentrations coinciding with carvacrol's inhibiting effect on biofilm formation. These results indicate that carvacrol's activity in inhibition of biofilm formation may be related to the disruption of quorum sensing.

Cutibacterium acnes is an opportunistic pathogen in acne vulgaris. C. acnes produces autoinducer-2 (AI-2）, a signaling molecule used for communication known as quorum sensing (QS）. In C. acnes, QS reportedly upregulates biofilm formation leading to resistance against bactericidal agents. In this study, we analyzed how heparinoid affected QS and biofilm formation of the opportunistic pathogen C. acnes. We also verified whether heparinoid would suppress biofilm formation and enhance the efficacy of the bactericidal agent 4-isopropyl-3-methylphenol (IPMP) against C. acnes biofilms. We ran an AI-2 bioassay using Vibrio harveyi ATCC BBA-1121. Heparinoid exhibited inhibitory activity against AI-2 at concentrations of 0.003-0.005%, suggesting an AI-2 analog-derived or C. acnes culture supernatant-derived inhibition of the AI-2 activity. To evaluate how heparinoid suppresses biofilm formation in C. acnes, we completed a biofilm assay in 96-well plates. We also evaluated the bactericidal activity of IPMP against the C. acnes biofilm prepared with or without heparinoid. Heparinoid inhibited C. acnes biofilm formation and IPMP bactericidal efficacy increased upon heparinoid-mediated suppression of biofilm formation. In this study, we clarified that heparinoid inhibits the AI-2-mediated QS of C. acnes, thereby suppressing biofilm formation and increasing IPMP bactericidal efficacy, potentially suppressing acne vulgaris.

Quorum sensing (QS) is a population-density-dependent communication process of microorganisms to coordinate their activities by producing and detecting low-molecular-weight signal molecules. In pathogenic bacteria, the property controlled by QS is often related to infectivity, e.g., biofilm formation. Molecular encapsulation of the QS signals is an innovative method to prevent the signals binding to the receptors and to attenuate QS. Cyclodextrins (CDs) may form an inclusion complex with the signals, thus reducing the communication (quorum quenching, QQ). A systematic study was performed with α-, β-cyclodextrin, and their random methylated, quaternary amino and polymer derivatives to evaluate and compare their effects on the biofilm formation of Pseudomonas aeruginosa. To examine the concentration-, temperature- and time-dependency of the QQ effect, the CDs were applied at a 0.1–12.5 mM concentration range, and biofilm formation was studied after 6, 24, 48 and 72 h at 22 and 30 °C. According to the results, the QS mechanism was significantly inhibited; the size of the cavity, the structure of the substituents, as well as the monomeric or polymeric character together with the concentration of the CDs have been identified as key influencing factors of biofilm formation. Statistically determined effective concentration values demonstrated outstanding efficiency (higher than 80% inhibition) of α-CD and its random methylated and polymer derivatives both on the short and long term. In summary, the potential value of CDs as inhibitors of QS should be considered since the inhibition of biofilm formation could significantly impact human health and the environment.

Quorum sensing (QS) is a cell-to-cell communication mechanism through which microorganisms can sense their population density and adjust their physiology by producing and detecting small signaling molecules called autoinducers (AIs). QS influences various aspects of microbial physiology, including virulence and pathogenesis by bacterial pathogens, biofilm formation, sporulation, antimicrobial resistance, etc. Lactic acid bacteria (LAB) have been used for centuries in food fermentation to improve sensory and nutritional profiles and preserve against spoilage and pathogenic microflora. This study investigated the potential of foodborne LAB of various genera, including Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, to interfere with the QS system of bacterial pathogens. For this, cell-free supernatants (CFSs) of 89 LAB foodborne isolates were collected by centrifugation following a 20 h culture (at 30 °C) in quarter-strength Brain Heart Infusion (BHI) broth. The pH of all CFSs was adjusted to 6.5 and sterilized by filtration. The anti-QS activity of the sterilized and neutralized CFSs was initially screened using the biosensor strains Chromobacterium violaceum 026 and Agrobacterium tumefaciens NTL4 (pZLR4) through an agar well diffusion assay that can detect the inhibition of the QS system that is based on acylated homoserine lactones (AHLs), which are used as AIs by Gram-negative bacteria. Additionally, all the CFSs were also screened for interference with the autoinducer 2 (AI-2) QS system that is mostly used for interspecies communication by both Gram-positive and Gram-negative bacteria. This was assessed using a luminescence bioassay with the Vibrio harveyi BAA-1117 biosensor strain. The results indicate that none of the LAB CFSs could inhibit AHL-based QS. However, 61.8% (55/89) of the CFSs induced luminescence in V. harveyi BAA-1117, while the remaining 38.2% (34/89) of the samples were capable of inhibiting AI-2-based QS. In the next steps, the most representative of these latter AI-2 interfering LAB isolates will be investigated for possible inhibition of biofilm formation by some important foodborne bacterial pathogens.

Virulence factors and biofilms constitute attractive targets for the prevention of infections caused by multidrug-resistant bacteria. Among alkyl gallates, propyl gallate (PG) and octyl gallate (OG) are used as food preservatives. Here we found that alkyl gallates differentially affect virulence, biofilm formation, and quorum sensing (QS) in Pseudomonas aeruginosa. Ethyl gallate (EG), PG, and butyl gallate (BG) inhibited biofilm formation and virulence factors including elastase, pyocyanin, and rhamnolipid, in P. aeruginosa without affecting cell viability by antagonizing the QS receptors LasR and RhlR. PG exhibited the most potent activity. Interestingly, hexyl gallate (HG) inhibited the production of rhamnolipid and pyocyanin but did not affect elastase production or biofilm formation. Notably, OG inhibited the production of rhamnolipid and pyocyanin but stimulated elastase production and biofilm formation. Analysis of QS signaling molecule production and QS gene expression suggested that HG inhibited RhlR, while OG activated LasR but inhibited PqsR. This mechanism was confirmed using QS mutants. Additionally, PG prevented the virulence of P. aeruginosa in Caenorhabditis elegans and a mouse model. This is the first report of the differential effects of alkyl gallates on QS systems and PG has great potential as an inhibitor of the virulence and biofilm formation of P. aeruginosa.

The unabated abuse of antibiotics has created a selection pressure that has resulted in the development of antimicrobial resistance (AMR) among pathogenic bacteria. AMR has become a global health concern in recent times and is responsible for a high number of mortalities occurring across the globe. Owing to the slow development of antibiotics, new chemotherapeutic antimicrobials with a novel mode of action is required urgently. Therefore, in the current investigation, we green synthesized a nanocomposite comprising zinc oxide nanoparticles functionalized with extracellular polysaccharide xanthan gum (ZnO@XG). Synthesized nanomaterial was characterized by structurally and morphologically using UV-visible spectroscopy, XRD, FTIR, BET, SEM and TEM. Subinhibitory concentrations of ZnO@XG were used to determine quorum sensing inhibitory activity against Gram-negative pathogens, Chromobacterium violaceum, and Serratia marcescens. ZnO@XG reduced quorum sensing (QS) regulated virulence factors such as violacein (61%), chitinase (70%) in C. violaceum and prodigiosin (71%) and protease (72%) in S. marcescens at 128 µg/mL concentration. Significant (p ≤ 0.05) inhibition of biofilm formation as well as preformed mature biofilms was also recorded along with the impaired production of EPS, swarming motility and cell surface hydrophobicity in both the test pathogens. The findings of this study clearly highlight the potency of ZnO@XG against the QS controlled virulence factors of drug-resistant pathogens that may be developed as effective inhibitors of QS and biofilms to mitigate the threat of multidrug resistance (MDR). ZnO@XG may be used alone or in combination with antimicrobial drugs against MDR bacterial pathogens. Further, it can be utilized in the food industry to counter the menace of contamination and spoilage caused by the formation of biofilms.

Background: Quorum sensing (QS) is one of the main regulatory systems which have various effects on populations of microorganisms. This process has been found in a diverse array of microorganisms (various bacterial taxa, microalgae and fungi). QS is required for different activities of microorganisms such as, virulence factor secretion, motility, competence, biofilm and sporulation. There are different molecules as signals in disparate microorganisms. Biofilm formation is one of the significant functions of QS. Biofilms are groups of microorganisms that are tied to a surface (biotic or abiotic). One of the remarkable effects of biofilm formation seems to be the persistence against hostile environmental condistions. Biofilm formation have been widely reported as a pathogenesis strategy in microorganisms. Here we describe QS and biofilm formation in some important microorganisms and describe some of the suggested strategies for eradication of microbial biofilms. Conclusion: Inhibition of biofilms formation can have detectable effects on the treatment of infectous diseases. In this line, multiple approaches have been suggested to inhibit the biofilm formation by microorganisms.

As a complex microbial aggregate, biofilm is a group behavior of bacterial ability to adapt to the environment. Bacteria produce biofilm substrates that enhance their tolerance to stress and cause microbial infections. Biofilm infection is usually closely related to virulence, pathogenicity, and even life-threatening to immunocompromised patients. Therefore, studying bacterial biofilm generation and regulatory mechanisms has become one of the most important fields. It is well known that biofilm formation involves group behavior and relies on complex regulation of quorum sensing (QS). A series of small molecule compounds such as indole, AI-2 (autoinducer-2), AHL (N-acyl-homoserine lactone), AIP (auto-inducing peptide), and DSF (diffusible signal factor) are widely available intraspecific or interspecific signaling molecules, with regulatory functions on a wide range of physiological activities of bacteria, including biofilm formation. Given that various bacteria employ QS mechanisms to regulate biofilm formation, inhibition of QS becomes a promising potential strategy for the treatment of bacterial infections. Here, we describe how bacterial intraspecific and interspecific signaling molecules regulate the mechanism of biofilm formation and dispersion. This may contribute to anti-biofilm active molecules and provide ideas or directions for studies on controlling bacterial infections by inhibiting biofilm formation through QS. KEY POINTS: • The formation and hazard of biofilm have been discussed. • The effects of quorum sensing on biofilm formation have been highlighted. • The inhibition of biofilm through quorum sensing has been discussed and highlighted.

Biofilm inhibition based on controlling the transmembrane transport and extracellular accumulation of quorum sensing signals

Acinetobacter baumannii causes several nosocomial infections and poses major threat when it is multidrug resistant. Even pan drug-resistant strains have been reported in some countries. The intensive care unit (ICU) mortality rate ranged from 45.6% to 60.9% and it is as high as 84.3% when ventilator-associated pneumonia was caused by XDR (extensively drug resistant) A. baumannii. Acinetobacter baumannii constituted 9.4% of all Gram-negative organisms throughout the hospital and 22.6% in the ICUs according to a study carried out in an Indian hospital. One of the major factors contributing to drug resistance in A. baumannii infections is biofilm development. Quorum sensing (QS) facilitates biofilm formation and therefore the search for 'quorum quenchers' has increased recently. Such compounds are expected to inhibit biofilm formation and hence reduce/prevent development of drug resistance in the bacteria. Some of these compounds also target synthesis of some virulence factors (VF). Several candidate drugs have been identified and are at various stages of drug development. Since quorum quenching, inhibition of biofilm formation and inhibition of VF synthesis do not pose any threat to the DNA replication and cell division of the bacteria, chances of resistance development to such compounds is presumably rare. Thus, these compounds ideally qualify as adjunct therapeutics and could be administered along with an antibiotic to reduce chances of resistance development and also to increase the effectiveness of antimicrobial therapy. This review describes the state-of-art in QS process in Gram-negative bacteria in general and in A. baumannii in particular. This article elaborates the nature of QS mediators, their characteristics, and the methods for their detection and quantification. Various potential sites in the QS pathway have been highlighted as drug targets and the candidate quorum quenchers which inhibit the mediator's synthesis or function are enlisted.

N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1