Wild-type Biofilm Research Articles

Structural characterization and disinfection assessment of isogenic E.coli biofilms using impedance spectroscopy with microelectrode arrays

Structural characterization and disinfection assessment of isogenic E.coli biofilms using impedance spectroscopy with microelectrode arrays

Thiol Starvation Induces Redox-Mediated Dysregulation of Escherichia coli Biofilm Components.

A hallmark of bacterial biofilms is the production of an extracellular matrix (ECM) that encases and protects the community from environmental stressors. Biofilm formation is an integral portion of the uropathogenic Escherichia coli (UPEC) life cycle. Approximately 2% of UPEC isolates are cysteine auxotrophs. Here, we investigated how cysteine homeostasis impacted UPEC UTI89 strain biofilm formation and, specifically, the production of the ECM components curli and cellulose. Cysteine auxotrophs produced less cellulose and slightly more curli compared to wild-type (WT) strains, and cysteine auxotrophs formed smooth, nonrugose colonies. Cellulose production was restored in cysteine auxotrophs when YfiR was inactivated. YfiR is a redox-sensitive regulator of the diguanylate cyclase, YfiN. The production of curli, a temperature-regulated appendage, was independent of temperature in UTI89 cysteine auxotrophs. In a screen of UPEC isolates, we found that ∼60% of UPEC cysteine auxotrophs produced curli at 37°C, but only ∼2% of cysteine prototrophic UPEC isolates produced curli at 37°C. Interestingly, sublethal concentrations of amdinocillin and trimethoprim-sulfamethoxazole inhibited curli production, whereas strains auxotrophic for cysteine continued to produce curli even in the presence of amdinocillin and trimethoprim-sulfamethoxazole. The dysregulation of ECM components and resistance to amdinocillin in cysteine auxotrophs may be linked to hyperoxidation, since the addition of exogenous cysteine or glutathione restored WT biofilm phenotypes to mutants unable to produce cysteine and glutathione.IMPORTANCE Uropathogenic Escherichia coli (UPEC) bacteria are the predominant causative agent of urinary tract infections (UTIs). UTIs account for billions of dollars of financial burden annually to the health care industry in the United States. Biofilms are an important aspect of the UPEC pathogenesis cascade and for the establishment of chronic infections. Approximately 2% of UPEC isolates from UTIs are cysteine auxotrophs, yet there is relatively little known about the biofilm formation of UPEC cysteine auxotrophs. Here we show that cysteine auxotrophs have dysregulated biofilm components due to a change in the redox state of the periplasm. Additionally, we show the relationship between cysteine auxotrophs, biofilms, and antibiotics frequently used to treat UTIs.

Mitigation of a nitrate reducing Pseudomonas aeruginosa biofilm and anaerobic biocorrosion using ciprofloxacin enhanced by D-tyrosine

Pseudomonas aeruginosa (PA) is a ubiquitous microbe. It can form recalcitrant biofilms in clinical and industrial settings. PA biofilms cause infections in patients. They also cause biocorrosion of medical implants. In this work, D-tyrosine (D-tyr) was investigated as an antimicrobial enhancer for ciprofloxacin (CIP) against a wild-type PA biofilm (strain PAO1) on C1018 carbon steel in a strictly anaerobic condition. Seven-day biofilm prevention test results demonstrated that 2 ppm (w/w) D-tyr enhanced 30 ppm CIP by achieving extra 2-log sessile cell reduction compared with the 30 ppm CIP alone treatment. The cocktail of 30 ppm CIP + 2 ppm D-tyr achieved similar efficacy as the 80 ppm CIP alone treatment in the biofilm prevention test. Results also indicated that the enhanced antimicrobial treatment reduced weight loss and pitting corrosion. In the 3-hour biofilm removal test, the cocktail of 80 ppm CIP + 5 ppm D-tyr achieved extra 1.5-log reduction in sessile cell count compared with the 80 ppm CIP alone treatment. The cocktail of 80 ppm CIP + 5 ppm D-tyr achieved better efficacy than the 150 ppm CIP alone treatment in the biofilm removal test.

PA3225 Is a Transcriptional Repressor of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa.

ABSTRACTThe tssABC1 locus is part of the Hcp secretion island I (HSI-I) type VI secretion system (T6SS) in Pseudomonas aeruginosa. Previous work implicated the tssC1 gene in P. aeruginosa biofilm-specific antibiotic resistance, and tssC1 is preferentially expressed in biofilms compared to planktonic cells. Using a DNA-dependent protein pulldown approach, we discovered that PA3225, an uncharacterized LysR-type transcriptional regulator, specifically bound to the tssABC1 upstream regulatory region. The deletion of PA3225 led to a 2-fold decrease in tssA1 expression levels in planktonic cells compared to the wild type, and tssA1 expression was slightly reduced in ΔPA3225 biofilms compared to wild-type biofilms. Intriguingly, further investigations revealed that the ΔPA3225 mutant was less susceptible to multiple, structurally unrelated antibiotics with various mechanisms of action when grown planktonically. The ΔPA3225 mutant was additionally more resistant to ciprofloxacin when grown in a biofilm. The decreased antibiotic susceptibility of the ΔPA3225 strain was linked to the transcriptional upregulation of the MexAB-OprM efflux pump. By using transcriptome sequencing (RNA-seq), other PA3225-regulated genes were identified, and the products of these genes, such as the putative ABC transporter PA3228, may also contribute to antibiotic resistance.

A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human respiratory track, where it utilizes cell-cell communication systems to coordinate population-level behaviors. We reasoned that secreted peptides that are highly expressed during infection are pivotal for virulence. Thus, we used in silico pattern searches to define a pneumococcal secretome and analyzed the transcriptome of the clinically important PMEN1 lineage to identify which peptide-encoding genes are highly expressed in vivo. In this study, we characterized virulence peptide 1 (vp1), a highly expressed Gly-Gly peptide-encoding gene in chinchilla middle ear effusions. The vp1 gene is widely distributed across pneumococcus as well as encoded in related species. Studies in the chinchilla model of middle ear infection demonstrated that VP1 is a virulence determinant. The vp1 gene is positively regulated by a transcription factor from the Rgg family and its cognate SHP (short hydrophobic peptide). In vitro data indicated that VP1 promotes increased thickness and biomass for biofilms grown on chinchilla middle ear epithelial cells. Furthermore, the wild-type biofilm is restored with the exogenous addition of synthetic VP1. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.

Macrophage Migration Is Impaired within Candida albicans Biofilms.

Candida albicans is an opportunistic fungal pathogen that infects immunocompromised patients. Infection control requires phagocytosis by innate immune cells, including macrophages. Migration towards, and subsequent recognition of, C. albicans fungal cell wall components by macrophages is critical for phagocytosis. Using live-cell imaging of phagocytosis, the macrophage cell line J774.1 showed enhanced movement in response to C. albicans cell wall mutants, particularly during the first 30 min, irrespective of the infection ratio. However, phagocyte migration was reduced up to 2-fold within a C. albicans biofilm compared to planktonic fungal cells. Biofilms formed from C. albicans glycosylation mutant cells also inhibited macrophage migration to a similar extent as wildtype Candida biofilms, suggesting that the physical structure of the biofilm, rather than polysaccharide matrix composition, may hamper phagocyte migration. These data illustrate differential macrophage migratory capacities, dependent upon the form of C. albicans encountered. Impaired migration of macrophages within a C. albicans biofilm may contribute to the recalcitrant nature of clinical infections in which biofilm formation occurs.

Divide and conquer: the Pseudomonas aeruginosa two-component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits.

The opportunistic pathogen Pseudomonas aeruginosa forms antimicrobial resistant biofilms through sequential steps requiring several two-component regulatory systems. The sensor-regulator hybrid SagS plays a central role in biofilm development by enabling the switch from the planktonic to the biofilm mode of growth, and by facilitating the transition of biofilm cells to a highly tolerant state. However, the mechanism by which SagS accomplishes both functions is unknown. SagS harbours a periplasmic sensory HmsP, and phosphorelay HisKA and Rec domains. SagS domain was used as constructs and site-directed mutagenesis to elucidate how SagS performs its dual functions. It was demonstrated that HisKA-Rec and the phospho-signalling between SagS and BfiS contribute to the switch to the biofilm mode of growth, but not to the tolerant state. Instead, expression of SagS domain constructs harbouring HmsP rendered ΔsagS biofilm cells as recalcitrant to antimicrobial agents as wild-type biofilms, likely by restoring BrlR production and cellular c-di-GMP levels to wild-type levels. Restoration of biofilm tolerance by HmsP was independent of biofilm biomass accumulation, RsmA, RsmYZ, HptB and BfiSR-downstream targets. Our findings thus suggest that SagS likely makes use of a "divide-and-conquer" mechanism to regulate its dual switch function, by activating two distinct regulatory networks via its individual domains.

Staphylococcal β-Toxin Modulates Human Aortic Endothelial Cell and Platelet Function through Sphingomyelinase and Biofilm Ligase Activities.

ABSTRACTStaphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE). IE is an endovascular infection of native and prosthetic valves and the lining of the heart; it is characterized by the formation of cauliflower-like “vegetations” composed of fibrin, platelets, other host factors, bacteria, and bacterial products. β-Toxin is an S. aureus virulence factor that contributes to the microorganism’s ability to cause IE. This cytolysin has two enzymatic activities: sphingomyelinase (SMase) and biofilm ligase. Although both activities have functions in a rabbit model of IE, the mechanism(s) by which β-toxin directly affects human cells and is involved in the infectious process has not been elucidated. Here, we compared the in vitro effects of purified recombinant wild-type β-toxin, SMase-deficient β-toxin (H289N), and biofilm ligase-deficient β-toxin (H162A and/or D163A) on human aortic endothelial cells (HAECs) and platelets. β-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8) from these cells by both SMase and biofilm ligase activities. β-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1). HAECs treated with β-toxin displayed granular membrane morphology not seen in treatment with the SMase-deficient mutant. The altered morphology resulted in two possibly separable activities, cell rounding and redistribution of cell membranes into granules, which were not the result of endosome production from the Golgi apparatus or lysosomes. β-Toxin directly aggregated rabbit platelets via SMase activity.

Real-Time Monitoring of nfxB Mutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin.

Biofilm infections caused by Pseudomonas aeruginosa are frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation in nfxB, encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence of nfxB mutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with an mCherry tag for constitutive red fluorescence and chromosomal transcriptional fusion between the P mexCD promoter and gfp leading to green fluorescence upon mutation of nfxB We observed a rapid development of nfxB mutants by live confocal laser scanning microscopy (CLSM) imaging of the flow cell biofilm (reaching 80 to 90% of the whole population) when treated with 1/10 minimal biofilm inhibitory concentration of CIP for 24 h and 96 h. Based on the observed developmental stages, we propose that nfxB mutants emerged de novo in the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. Identical nfxB mutations were found in fluorescent colonies from the same flow cell biofilm, especially in 24-h biofilms, suggesting selection and clonal expansion of the mutants during biofilm growth. Our findings point at the significant role of high-enough antibiotic dosages or appropriate combination therapy to avoid the emergence of resistant mutants in biofilms.

Synthetic analogs of rhamnolipids modulate structured biofilms formed by rhamnolipid-nonproducing mutant of Pseudomonas aeruginosa

Rhamnolipids secreted by Pseudomonas aeruginosa are required for the bacteria to form biofilm efficiently and form biofilm with internal structures including pores and channels. In this work, we explore the effect of a class of synthetic analogs of rhamnolipids at controlling (promoting and inhibiting) the biofilm formation activities of a non-rhamnolipid-producing strain – rhlA – of P. aeruginosa. This class of rhamnolipid analogs is known to modulate the swarming motilities of wild-type PAO1 and rhlA mutant, but its effect on biofilm formation of rhlA mutant is unknown. We show that small structural details of these molecules are important for the bioactivities, but do not affect the general physical properties of the molecules. The bioactive synthetic analogs of rhamnolipids promote biofilm formation by rhlA mutant at low concentrations, but inhibit the biofilm formation at high concentrations. To explore the internal structures formed by the biofilms, we first demonstrate that wild-type biofilms are formed with substantial topography (hills and valleys) when the sample is under shaking conditions. Using this observation as a comparison, we found that synthetic analogs of rhamnolipids promoted structured (porous) biofilm of rhlA mutant, at intermediate concentrations between the low ones that promoted biofilm formation and the high ones that inhibited biofilm formation. This study suggests a potential chemical signaling approach to control multiple bacterial activities.

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

Effects of chromosomal deletion of the operon encoding the multiple resistance and pH-related antiporter in Vibrio cholerae.

To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.

Structural and recovery mechanisms of 3D dental pulp cell microtissues challenged with Streptococcusmutans in extracellular matrix environment.

Cariopathogen Streptococcus mutans exists in infected dental pulp of deciduous teeth and is frequently linked with heart diseases. Organotypic (3D) dental pulp stem cell (DPSC) cultures/microtissues, developed to mimic the physiological conditions in vivo, were utilized to assess the bacterial impact on their (i) 3D structural configuration and (ii) recovery mechanisms. The cultures, developed in extracellular matrix (ECM) bio-scaffold (Matrigel™), interacted with WT and GFP-tagged bacterial biofilms by permitting their infiltration through the ECM. Challenged cell constructs were visualized by F-actin/nuclei staining. Their pluripotency (Sox2) and differentiation (osteocalcin) markers were assessed by immunocytochemistry. Secreted mineral was detected by alizarin red, and 3D structural arrangements were analysed by epi-fluorescence and confocal scanning microscopy. Bacterial biofilm/ECM-embedded DPSC interactions appeared in distinct areas of the microtissues. Bacterial attachment to the cell surface occurred without evidence of invasion. Surface architecture of the challenged versus unchallenged microtissues was apparently unaltered. However, significant increases in thickness (138.42 vs 106.51 µm) and bacterial penetration were detected in challenged structures causing canal-like microstructures with various diameters (12.94 -42.88 µm) and average diameter of 20.66 to 33.42 µm per microtissue. Challenged constructs expressed pluripotency and differentiation markers and secreted the mineral. Presented model shows strong potential for assessing pulp-pathogen interactions in vivo. S. mutans infiltrated and penetrated the microtissues but did not invade the cells or compromise major cell repair mechanisms. These findings would suggest reexamining the role of S. mutans as an endodontic pathogen and investigating DPSC resistance to its pathogenicity.

Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus

Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms.

Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies

Biofilms are recalcitrant and raise safety problems in the food industry. In this study, the role of CabA, an extracellular matrix protein, in the resistance of the biofilms of Vibrio vulnificus, a foodborne pathogen, to decontamination strategies was investigated. Biofilms of the cabA mutant revealed reduced resistance to detachment by vibration and disinfection by sodium hypochlorite compared to the biofilms of the parental wild type in vitro. The reduced resistance of the cabA mutant biofilms was complemented by introducing a recombinant cabA, indicating that the reduced resistance of the cabA mutant biofilms is caused by the inactivation of cabA. The expression of cabA was induced in cells bound to oyster, the primary vehicle of the pathogen. The cabA mutant biofilms on oyster are defective in biomass and resistance to detachment and disinfection. The bacterial cells in the wild-type biofilms are clustered by filaments which are not apparent in the cabA mutant biofilms. The combined results indicated that CabA contributes to the structural integrity of V. vulnificus biofilms possibly by forming filaments in the matrix and thus rendering the biofilms robust, suggesting that CabA could be a target to control V. vulnificus biofilms on oyster.

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

ABSTRACTIntrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

Type IV pili promote early biofilm formation by Clostridium difficile.

Increasing morbidity and mortality from Clostridium difficile infection (CDI) present an enormous challenge to healthcare systems. Clostridium difficile express type IV pili (T4P), but their function remains unclear. Many chronic and recurrent bacterial infections result from biofilms, surface-associated bacterial communities embedded in an extracellular matrix. CDI may be biofilm mediated; T4P are important for biofilm formation in a number of organisms. We evaluate the role of T4P in C. difficile biofilm formation using RNA sequencing, mutagenesis and complementation of the gene encoding the major pilin pilA1, and microscopy. RNA sequencing demonstrates that, in comparison to other growth phenotypes, C. difficile growing in a biofilm has a distinct RNA expression profile, with significant differences in T4P gene expression. Microscopy of T4P-expressing and T4P-deficient strains suggests that T4P play an important role in early biofilm formation. A non-piliated pilA1 mutant forms an initial biofilm of significantly reduced mass and thickness in comparison to the wild type. Complementation of the pilA1 mutant strain leads to formation of a biofilm which resembles the wild-type biofilm. These findings suggest that T4P play an important role in early biofilm formation. Novel strategies for confronting biofilm infections are emerging; our data suggest that similar strategies should be investigated in CDI.

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based self-resistance, controlled by a simple circuit, in a Gram-negative pathogen.

Dissecting the regulation of bile-induced biofilm formation in Staphylococcus aureus.

Aspiration of bile into the cystic fibrosis (CF) lung has emerged as a prognostic factor for reduced microbial lung biodiversity and the establishment of often fatal, chronic pathogen infections. Staphylococcus aureus is one of the earliest pathogens detected in the lungs of children with CF, and once established as a chronic infection, strategies for its eradication become limited. Several lung pathogens are stimulated to produce biofilms in vitro in the presence of bile. In this study, we further investigated the effects of bile on S. aureus biofilm formation. Most clinical S. aureus strains and the laboratory strain RN4220 were stimulated to form biofilms with sub-inhibitory concentrations of bovine bile. Additionally, we observed bile-induced sensitivity to aminoglycosides, which we exploited in a bursa aurealis transposon screen to isolate mutants reduced in aminoglycoside sensitivity and augmented in bile-induced biofilm formation. We identified five mutants that exhibited hypersensitivity to bile with respect to bile-induced biofilm formation, three of which carried transposon insertions within gene clusters involved in wall teichoic acid (WTA) biosynthesis or transport. Strain TM4 carried an insertion between the divergently oriented tagH and tagG genes, which encode the putative WTA membrane translocation apparatus. Ectopic expression of tagG in TM4 restored a wild-type bile-induced biofilm response, suggesting that reduced translocation of WTA in TM4 induced sensitivity to bile and enhanced the bile-induced biofilm formation response. We propose that WTA may be important for protecting S. aureus against exposure to bile and that bile-induced biofilm formation may be an evolved response to protect cells from bile-induced cell lysis.

Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production.

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.