Microbial Indoles: Key Regulators of Organ Growth and Metabolic Function.

Gut microbes supporting body growth are known but the mechanisms are less well documented. Using the microbial tryptophan metabolite indole, known to regulate prokaryotic cell division and metabolic stress conditions, we mono-colonized germ-free (GF) mice with indole-producing wild-type Escherichia coli (E. coli) or tryptophanase-encoding tnaA knockout mutant indole-non-producing E. coli. Indole mutant E. coli mice showed multiorgan growth retardation and lower levels of glycogen, cholesterol, triglycerides, and glucose, resulting in an energy deficiency despite increased food intake. Detailed analysis revealed a malfunctioning intestine, enlarged cecum, and reduced numbers of enterochromaffin cells, correlating with a metabolic phenotype consisting of impaired gut motility, diminished digestion, and lower energy harvest. Furthermore, indole mutant mice displayed reduction in serum levels of tricarboxylic acid (TCA) cycle intermediates and lipids. In stark contrast, a massive increase in serum melatonin was observed-frequently associated with accelerated oxidative stress and mitochondrial dysfunction. This observational report discloses functional roles of microbe-derived indoles regulating multiple organ functions and extends our previous report of indole-linked regulation of adult neurogenesis. Since indoles decline by age, these results imply a correlation with age-linked organ decline and levels of indoles. Interestingly, increased levels of indole-3-acetic acid, a known indole metabolite, have been shown to correlate with younger biological age, further supporting a link between biological age and levels of microbe-derived indole metabolites. The results presented in this resource paper will be useful for the future design of food intervention studies to reduce accelerated age-linked organ decline.

Life at the borderlands: microbiomes of interfaces critical to One Health.

Microbiomes are foundational components of the environment that provide essential services relating to food security, carbon sequestration, human health, and the overall well-being of ecosystems. Microbiota exert their effects primarily through complex interactions at interfaces with their plant, animal, and human hosts, as well as within the soil environment. This review aims to explore the ecological, evolutionary, and molecular processes governing the establishment and function of microbiome-host relationships, specifically at interfaces critical to One Health-a transdisciplinary framework that recognizes that the health outcomes of people, animals, plants, and the environment are tightly interconnected. Within the context of One Health, the core principles underpinning microbiome assembly will be discussed in detail, including biofilm formation, microbial recruitment strategies, mechanisms of microbial attachment, community succession, and the effect these processes have on host function and health. Finally, this review will catalogue recent advances in microbiology and microbial ecology methods that can be used to profile microbial interfaces, with particular attention to multi-omic, advanced imaging, and modelling approaches. These technologies are essential for delineating the general and specific principles governing microbiome assembly and functions, mapping microbial interconnectivity across varying spatial and temporal scales, and for the establishment of predictive frameworks that will guide the development of targeted microbiome-interventions to deliver One Health outcomes.

The dual GGDEF/EAL domain enzyme PA0285 is a Pseudomonas species housekeeping phosphodiesterase regulating early attachment and biofilm architecture.

Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation.

High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo.

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

RNA is a key component of extracellular DNA networks in Pseudomonas aeruginosa biofilms.

The extracellular matrix of bacterial biofilms consists of diverse components including polysaccharides, proteins and DNA. Extracellular RNA (eRNA) can also be present, contributing to the structural integrity of biofilms. However, technical difficulties related to the low stability of RNA make it difficult to understand the precise roles of eRNA in biofilms. Here, we show that eRNA associates with extracellular DNA (eDNA) to form matrix fibres in Pseudomonas aeruginosa biofilms, and the eRNA is enriched in certain bacterial RNA transcripts. Degradation of eRNA associated with eDNA led to a loss of eDNA fibres and biofilm viscoelasticity. Compared with planktonic and biofilm cells, the biofilm matrix was enriched in specific mRNA transcripts, including lasB (encoding elastase). The mRNA transcripts colocalised with eDNA fibres in the biofilm matrix, as shown by single molecule inexpensive FISH microscopy (smiFISH). The lasB mRNA was also observed in eDNA fibres in a clinical sputum sample positive for P. aeruginosa. Thus, our results indicate that the interaction of specific mRNAs with eDNA facilitates the formation of viscoelastic networks in the matrix of Pseudomonas aeruginosa biofilms.

GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

IMPORTANCE: New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

Escherichia coli displays a conserved membrane proteomic response to a range of alcohols.

BACKGROUND: Alcohol is a good and environment-friendly fuel that can be microbially produced, capable of eliminating many of the limitations of the present-day fossil fuels. However, the inherent toxic nature of alcohols to the microbial cells leads to end-product inhibition that limits large-scale alcohol production by fermentation. Fundamental knowledge about the stress responses of microorganisms to alcohols would greatly facilitate to improve the microbial alcohol tolerance. The current study elucidates and compares the changes in the membrane proteome of Escherichia coli in response to a range of alcohols. RESULTS: Although alcohol toxicity increased exponentially with alcohol chain length (2-6 carbon), similar stress responses were observed in the inner and outer membrane proteome of E. coli in the presence of 2-, 4- and 6-carbon alcohols at the MIC50. This pertains to: (1) increased levels of inner membrane transporters for uptake of energy-producing metabolites, (2) reduced levels of non-essential proteins, associated with anaerobic, carbon starvation and osmotic stress, for energy conservation, (3) increased levels of murein degrading enzymes (MltA, EmtA, MliC and DigH) promoting cell elongation and 4) reduced levels of most outer membrane ß-barrel proteins (LptD, FadL, LamB, TolC and BamA). Major outer membrane ß-barrel protein OmpC, which is known to contribute to ethanol tolerance and membrane integrity, was notably reduced by alcohol stress. While LPS is important for OmpC trimerisation, LPS release by EDTA did not lower OmpC levels. This suggests that LPS release, which is reported under alcohol stress, does not contribute to the reduced levels of OmpC in the presence of alcohol. CONCLUSIONS: Since alcohol primarily targets the integrity of the membrane, maintenance of outer membrane OmpC levels in the presence of alcohol might help in the survival of E. coli to higher alcohol concentrations. The study provides important information about the membrane protein responses of E. coli to a range of alcohols, which can be used to develop targeted strategies for increased microbial alcohol tolerance and hence bioalcohol production.

Increased iron utilization and oxidative stress tolerance in a Vibrio cholerae flrA mutant confers resistance to amoeba predation.

IMPORTANCE: Persistence of V. cholerae in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by heterotrophic protists such as the free-living amoeba A. castellanii. This study explores how a mutant of V. cholerae adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that V. cholerae can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.

Chemically Mediated Interactions with Macroalgae Negatively Affect Coral Health but Induce Limited Changes in Coral Microbiomes.

Allelopathic chemicals facilitated by the direct contact of macroalgae with corals are potentially an important mechanism mediating coral-macroalgal interactions, but only a few studies have explored their impacts on coral health and microbiomes and the coral's ability to recover. We conducted a field experiment on an equatorial urbanized reef to assess the allelopathic effects of four macroalgal species (Bryopsis sp., Endosiphonia horrida, Hypnea pannosa and Lobophora challengeriae) on the health and microbiomes of three coral species (Merulina ampliata, Montipora stellata and Pocillopora acuta). Following 24 h of exposure, crude extracts of all four macroalgal species caused significant coral tissue bleaching and reduction in effective quantum yield. The corals were able to recover within 72 h of the removal of extracts, except those that were exposed to L. challengeriae. While some macroalgal extracts caused an increase in the alpha diversity of coral microbiomes, there were no significant differences in the composition and variability of coral microbiomes between controls and macroalgal extracts at each sampling time point. Nevertheless, DESeq2 differential abundance analyses showed species-specific responses of coral microbiomes. Overall, our findings provide insights on the limited effect of chemically mediated interactions with macroalgae on coral microbiomes and the capacity of corals to recover quickly from the macroalgal chemicals.

Plasma activated water as a pre-treatment strategy in the context of biofilm-infected chronic wounds.

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of plasma activated water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular reactive oxygen and nitrogen species accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

Transcriptomic and metabolomic characterization of antibacterial activity of Melastoma dodecandrum.

Antibacterial resistance poses a significant global threat, necessitating the discovery of new therapeutic agents. Plants are a valuable source of secondary metabolites with demonstrated anticancer and antibacterial properties. In this study, we reveal that Melastoma dodecandrum exhibits both bacteriostatic and bactericidal effects against Pseudomonas aeruginosa and Staphylococcus aureus. Treatment with plant extracts results in membrane damage and a reduction in P.aeruginosa swimming and swarming motility. A comparative analysis of bacterial transcriptomes exposed to M.dodecandrum extracts and four distinct antibiotics indicates that the extracts may trigger similar transcriptomic responses as triclosan, a fatty acid synthesis inhibitor. Activity-guided fractionation suggests that the antibacterial activity is not attributable to hydrolyzable tannins, but to unidentified minor compounds. Additionally, we identified 104 specialized metabolic pathways and demonstrated a high level of transcriptional coordination between these biosynthetic pathways and phytohormones, highlighting potential regulatory mechanisms of antibacterial metabolites in M.dodecandrum.

Machine learning-based detection of adventitious microbes in T-cell therapy cultures using long-read sequencing.

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP <71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated, and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimized experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 colony-forming units (CFU)/mL using metagenomic classification. Machine learning can be coupled with long-read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP <71> organisms to 10 CFU/mL. IMPORTANCE This research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long-read nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

Associational Resistance to Predation by Protists in a Mixed Species Biofilm.

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa ΔrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the ΔrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of ΔrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes.

Pseudomonas aeruginosa is a Gram-negative bacterium that is able to survive and adapt in a multitude of niches as well as thrive within many different hosts. This versatility lies within its large genome of ca. 6 Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widespread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. c-di-GMP is made by diguanylate cyclases and degraded by phosphodiesterases, while the intracellular level of this molecule drives phenotypic responses. Signaling involves the modification of enzymes' or proteins' function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca. 40 genes encoding putative DGCs or PDEs. The combined activity of those enzymes should reflect the overall c-di-GMP concentration, while specific phenotypic outputs could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool of cells grown planktonically, while several mutants display a phenotypic impact on biofilm including initial attachment and maturation. If this observation of minor changes in c-di-GMP level correlating with significant phenotypic impact appears to be true, it further supports the idea of a local vs global c-di-GMP pool. In contrast, there was little to no effect on motility, which differs from previous studies. Our RNA-seq analysis indicated that all PAO1 dgc/pde genes were expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks need to be reconstructed for each strain separately and cannot be extrapolated from one to another.

Bacterial biofilm colonization and succession in tropical marine waters are similar across different types of stone materials used in seawall construction.

Seawalls are important in protecting coastlines from currents, erosion, sea-level rise, and flooding. They are, however, associated with reduced biodiversity, due to their steep orientation, lack of microhabitats, and the materials used in their construction. Hence, there is considerable interest in modifying seawalls to enhance the settlement and diversity of marine organisms, as microbial biofilms play a critical role facilitating algal and invertebrate colonization. We assessed how different stone materials, ranging from aluminosilicates to limestone and concrete, affect biofilm formation. Metagenomic assessment of marine microbial communities indicated no significant impact of material on microbial diversity, irrespective of the diverse surface chemistry and topography. Based on KEGG pathway analysis, surface properties appeared to influence the community composition and function during the initial stages of biofilm development, but this effect disappeared by Day 31. We conclude that marine biofilms converged over time to a generic marine biofilm, rather than the underlying stone substrata type playing a significant role in driving community composition.

The ratio of nicotinic acid to nicotinamide as a microbial biomarker for assessing cell therapy product sterility.

Controlling microbial risks in cell therapy products (CTPs) is important for product safety. Here, we identified the nicotinic acid (NA) to nicotinamide (NAM) ratio as a biomarker that detects a broad spectrum of microbial contaminants in cell cultures. We separately added six different bacterial species into mesenchymal stromal cell and T cell culture and found that NA was uniquely present in these bacteria-contaminated CTPs due to the conversion from NAM by microbial nicotinamidases, which mammals lack. In cells inoculated with 1 × 104 CFUs/mL of different microorganisms, including USP <71> defined organisms, the increase in NA to NAM ratio ranged from 72 to 15,000 times higher than the uncontaminated controls after 24 h. Importantly, only live microorganisms caused increases in this ratio. In cells inoculated with 18 CFUs/mL of Escherichia coli, 20 CFUs/mL of Bacillus subtilis, and 10 CFUs/mL of Candida albicans, significant increase of NA to NAM ratio was detected using LC-MS after 18.5, 12.5, and 24.5 h, respectively. In contrast, compendial sterility test required >24 h to detect the same amount of these three organisms. In conclusion, the NA to NAM ratio is a useful biomarker for detection of early-stage microbial contaminations in CTPs.

Inhibition of the Quorum Sensing System, Elastase Production and Biofilm Formation in Pseudomonas aeruginosa by Psammaplin A and Bisaprasin.

Natural products derived from marine sponges have exhibited bioactivity and, in some cases, serve as potent quorum sensing inhibitory agents that prevent biofilm formation and attenuate virulence factor expression by pathogenic microorganisms. In this study, the inhibitory activity of the psammaplin-type compounds, psammaplin A (1) and bisaprasin (2), isolated from the marine sponge, Aplysinellarhax, are evaluated in quorum sensing inhibitory assays based on the Pseudomonas aeruginosa PAO1 lasB-gfp(ASV) and rhlA-gfp(ASV) biosensor strains. The results indicate that psammaplin A (1) showed moderate inhibition on lasB-gfp expression, but significantly inhibited the QS-gene promoter, rhlA-gfp, with IC50 values at 14.02 µM and 4.99 µM, respectively. In contrast, bisaprasin (2) displayed significant florescence inhibition in both biosensors, PAO1 lasB-gfp and rhlA-gfp, with IC50 values at 3.53 µM and 2.41 µM, respectively. Preliminary analysis suggested the importance of the bromotyrosine and oxime functionalities for QSI activity in these molecules. In addition, psammaplin A and bisaprasin downregulated elastase expression as determined by the standard enzymatic elastase assay, although greater reduction in elastase production was observed with 1 at 50 µM and 100 µM. Furthermore, the study revealed that bisaprasin (2) reduced biofilm formation in P. aeruginosa.