ORCID Profile
0000-0003-2068-8589
Current Organisations
University of Southampton
,
Boston Children's Hospital
,
National Biofilms Innovation Centre (NBIC)
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Publisher: American Society for Microbiology
Date: 11-2006
DOI: 10.1128/JB.00779-06
Abstract: Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa , we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, Δ nirS ) did not disperse, whereas a NO reductase mutant (Δ norCB ) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.
Publisher: American Society for Microbiology
Date: 08-2006
DOI: 10.1128/AEM.00449-06
Abstract: Pseudalteromonas tunicata and Roseobacter gallaeciensis are biofilm-forming marine bacteria that are often found in association with the surface of the green alga Ulva australis . They are thought to benefit the plant host by producing inhibitory compounds that are active against common fouling organisms. We investigated factors that influence the ability of P. tunicata and R. gallaeciensis to attach to and colonize the plant surface and also the competitive interactions that occur between these organisms and other isolates from U. australis during biofilm formation on the plant surface. A surprisingly high number of P. tunicata cells, at least 10 8 cells ml −1 , were required for colonization and establishment of a population of cells that persists on axenic surfaces of U. australis . Factors that enhanced colonization of P. tunicata included inoculation in the dark and pregrowth of inocula in medium containing cellobiose as the sole carbon source (cellulose is a major surface polymer of U. australis ). It was also found that P. tunicata requires the presence of a mixed microbial community to colonize effectively. In contrast, R. gallaeciensis effectively colonized the plant surface under all conditions tested. Studies of competitive interactions on the plant surface revealed that P. tunicata was numerically dominant compared with all other bacterial isolates tested (except R. gallaeciensis ), and this dominance was linked to production of the antibacterial protein AlpP. Generally, P. tunicata was able to coexist with competing strains, and each strain existed as microcolonies in spatially segregated regions of the plant. R. gallaeciensis was numerically dominant compared with all strains tested and was able to invade and disperse preestablished biofilms. This study highlighted the fact that microbial colonization of U. australis surfaces is a dynamic process and demonstrated the differences in colonization strategies exhibited by the epiphytic bacteria P. tunicata and R. gallaeciensis .
Publisher: Springer Science and Business Media LLC
Date: 10-02-2017
DOI: 10.1038/SREP42166
Abstract: The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825 EAL ) and PA1727 (MucR EAL ). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825 EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.
Publisher: Elsevier BV
Date: 09-2008
Publisher: SAGE Publications
Date: 08-06-2015
Abstract: Acidogenic bacteria within dental plaque biofilms are the causative agents of caries. Consequently, maintenance of a healthy oral environment with efficient biofilm removal strategies is important to limit caries, as well as halt progression to gingivitis and periodontitis. Recently, a novel cleaning device has been described using an ultrasonically activated stream (UAS) to generate a cavitation cloud of bubbles in a freely flowing water stream that has demonstrated the capacity to be effective at biofilm removal. In this study, UAS was evaluated for its ability to remove biofilms of the cariogenic pathogen Streptococcus mutans UA159, as well as Actinomyces naeslundii ATCC 12104 and Streptococcus oralis ATCC 9811, grown on machine-etched glass slides to generate a reproducible complex surface and artificial teeth from a typodont training model. Biofilm removal was assessed both visually and microscopically using high-speed videography, confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM). Analysis by CSLM demonstrated a statistically significant 99.9% removal of S. mutans biofilms exposed to the UAS for 10 s, relative to both untreated control biofilms and biofilms exposed to the water stream alone without ultrasonic activation ( P 0.05). The water stream alone showed no statistically significant difference in removal compared with the untreated control ( P = 0.24). High-speed videography demonstrated a rapid rate (151 mm 2 in 1 s) of biofilm removal. The UAS was also highly effective at S. mutans, A. naeslundii, and S. oralis biofilm removal from machine-etched glass and S. mutans from typodont surfaces with complex topography. Consequently, UAS technology represents a potentially effective method for biofilm removal and improved oral hygiene.
Publisher: Public Library of Science (PLoS)
Date: 13-05-2009
Publisher: Wiley
Date: 13-07-2022
Abstract: Coordination states of metal‐organic materials are known to dictate their physicochemical properties and applications in various fields. However, understanding and controlling coordination sites in metal‐organic systems is challenging. Herein, we report the synthesis of site‐selective coordinated metal‐phenolic networks (MPNs) using flavonoids as coordination modulators. The site‐selective coordination was systematically investigated experimentally and computationally using ligands with one, two, and multiple different coordination sites. Tuning the multimodal Fe coordination with catechol, carbonyl, and hydroxyl groups within the MPNs enabled the facile engineering of erse physicochemical properties including size, selective permeability (20–2000 kDa), and pH‐dependent degradability. This study expands our understanding of metal‐phenolic chemistry and provides new routes for the rational design of structurally tailorable coordination‐based materials.
Publisher: American Society for Microbiology
Date: 04-2016
DOI: 10.1128/AAC.02432-15
Abstract: Streptococcus pneumoniae is one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because, although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing to the antibiotic tolerance and persistence of S. pneumoniae in chronic or recurrent OM. We investigated the treatment of pneumococcal biofilms with nitric oxide (NO), an endogenous signaling molecule and therapeutic agent that has been demonstrated to trigger biofilm dispersal in other bacterial species. We hypothesized that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and that higher concentrations exert direct antibacterial effects. Unlike in many other bacterial species, low concentrations of NO did not result in S. pneumoniae biofilm dispersal. Instead, treatment of both in vitro biofilms and ex vivo adenoid tissue s les (a reservoir for S. pneumoniae biofilms) with low concentrations of NO enhanced pneumococcal killing when combined with amoxicillin-clavulanic acid, an antibiotic commonly used to treat chronic OM. Quantitative proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) identified 13 proteins that were differentially expressed following low-concentration NO treatment, 85% of which function in metabolism or translation. Treatment with low-concentration NO, therefore, appears to modulate pneumococcal metabolism and may represent a novel therapeutic approach to reduce antibiotic tolerance in pneumococcal biofilms.
Publisher: Elsevier BV
Date: 02-2012
DOI: 10.1016/J.JTBI.2011.10.007
Abstract: Biofilms are currently recognised as the predominant bacterial life-style and it has been suggested that biofilm development is influenced by a number of different processes such as adhesion, detachment, mass transport, quorum sensing, cell death and active dispersal. One of the least understood processes and its effects on biofilm development is cell death. However, experimental studies suggest that bacterial death is an important process during biofilm development and many studies show a relationship between cell death and dispersal in microbial biofilms. We present a model of the process of cell death during biofilm development, with a particular focus on the spatial localisation of cell death or cell damage. Three rules governing cell death or cell damage were evaluated which compared the effects of starvation, damage accumulation, and viability during biofilm development and were also used to design laboratory based experiments to test the model. Results from model simulations show that actively growing biofilms develop steep nutrient gradients within the interior of the biofilm that affect neighbouring microcolonies resulting in cell death and detachment. Two of the rules indicated that high substrate concentrations lead to accelerated cell death, in contrast to the third rule, based on the accumulation of damage, which predicted earlier cell death for biofilms grown with low substrate concentrations. Comparison of the modelling results with experimental results suggests that cell death is favoured under low nutrient conditions and that the accumulation of damage may be the main cause of cell death during biofilm development.
Publisher: Wiley
Date: 24-03-2023
Abstract: Flexible metal‐organic materials are of growing interest owing to their ability to undergo reversible structural transformations under external stimuli. Here, we report flexible metal‐phenolic networks (MPNs) featuring stimuli‐responsive behavior to erse solute guests. The competitive coordination of metal ions to phenolic ligands of multiple coordination sites and solute guests (e.g., glucose) primarily determines the responsive behavior of the MPNs, as revealed experimentally and computationally. Glucose molecules can be embedded into the dynamic MPNs upon mixing, leading to the reconfiguration of the metal‐organic networks and thus changes in their physicochemical properties for targeting applications. This study expands the library of stimuli‐responsive flexible metal‐organic materials and the understanding of intermolecular interactions between metal‐organic materials and solute guests, which is essential for the rational design of responsive materials for various applications.
Publisher: Oxford University Press (OUP)
Date: 17-09-2019
DOI: 10.1093/JAC/DKZ378
Abstract: The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.
Publisher: SPIE
Date: 03-10-2022
DOI: 10.1117/12.2635324
Publisher: American Society for Microbiology
Date: 2015
DOI: 10.1128/AAC.03676-14
Abstract: Periprosthetic infection (PI) causes significant morbidity and mortality after fixation and joint arthroplasty and has been extensively linked to the formation of bacterial biofilms. Poly(methyl methacrylate) (PMMA), as a cement or as beads, is commonly used for antibiotic release to the site of infection but displays variable elution kinetics and also represents a potential nidus for infection, therefore requiring surgical removal once antibiotics have eluted. Absorbable cements have shown improved elution of a wider range of antibiotics and, crucially, complete biodegradation, but limited data exist as to their antimicrobial and antibiofilm efficacy. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin, or vancomycin-tobramycin dual treatment (in a 1:0.24 [wt/wt] ratio) were assessed for their abilities to eradicate planktonic methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis relative to that of PMMA beads. The ability of the calcium sulfate beads to prevent biofilm formation over multiple days and to eradicate preformed biofilms was studied using a combination of viable cell counts, confocal microscopy, and scanning electron microscopy of the bead surface. Biofilm bacteria displayed a greater tolerance to the antibiotics than their planktonic counterparts. Antibiotic-loaded beads were able to kill planktonic cultures of 10 6 CFU/ml, prevent bacterial colonization, and significantly reduce biofilm formation over multiple days. However, established biofilms were harder to eradicate. These data further demonstrate the difficulty in clearing established biofilms therefore, early preventive measures are key to reducing the risk of PI. Synthetic calcium sulfate loaded with antibiotics has the potential to reduce or eliminate biofilm formation on adjacent periprosthetic tissue and prosthesis material and, thus, to reduce the rates of periprosthetic infection.
Publisher: American Society for Microbiology
Date: 06-2006
DOI: 10.1128/AEM.03022-05
Abstract: Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis , were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae , Shewanella japonica , Dokdonia donghaensis , and Acinetobacter lwoffii , were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by % in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata , the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.
Publisher: Frontiers Media SA
Date: 04-08-2022
DOI: 10.3389/FCIMB.2022.956808
Abstract: Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs + and NOMBs + . Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was ided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%) and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB + and NOMB + was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for ex le, in chronic wounds.
Publisher: American Society for Microbiology
Date: 21-04-2020
DOI: 10.1128/AAC.02073-19
Abstract: Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a non iding, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against non iding Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and ision proteins, confirming its potential as a treatment for S. aureus biofilm infections.
Publisher: American Society for Microbiology
Date: 04-2005
DOI: 10.1128/AEM.71.4.1729-1736.2005
Abstract: Pseudoalteromonas tunicata is a biofilm-forming marine bacterium that is often found in association with the surface of eukaryotic organisms. It produces a range of extracellular inhibitory compounds, including an antibacterial protein (AlpP) thought to be beneficial for P. tunicata during competition for space and nutrients on surfaces. As part of our studies on the interactions between P. tunicata and the epiphytic bacterial community on the marine plant Ulva lactuca , we investigated the hypothesis that P. tunicata is a superior competitor compared with other bacteria isolated from the plant. A number of U. lactuca bacterial isolates were (i) identified by 16S rRNA gene sequencing, (ii) characterized for the production of or sensitivity to extracellular antibacterial proteins, and (iii) labeled with a fluorescent color tag (either the red fluorescent protein DsRed or green fluorescent protein). We then grew single- and mixed-species bacterial biofilms containing P. tunicata in glass flow cell reactors. In pure culture, all the marine isolates formed biofilms containing microcolony structures within 72 h. However, in mixed-species biofilms, P. tunicata removed the competing strain unless its competitor was relatively insensitive to AlpP ( Pseudoalteromonas gracilis ) or produced strong inhibitory activity against P. tunicata ( Roseobacter gallaeciensis ). Moreover, biofilm studies conducted with an AlpP − mutant of P. tunicata indicated that the mutant was less competitive when it was introduced into preestablished biofilms, suggesting that AlpP has a role during competitive biofilm formation. When single-species biofilms were allowed to form microcolonies before the introduction of a competitor, these microcolonies coexisted with P. tunicata for extended periods of time before they were removed. Two marine bacteria ( R. gallaeciensis and P. tunicata ) were superior competitors in this study. Our data suggest that this dominance can be attributed to the ability of these organisms to rapidly form microcolonies and their ability to produce extracellular antibacterial compounds.
Publisher: Public Library of Science (PLoS)
Date: 16-06-2017
Publisher: Springer Science and Business Media LLC
Date: 08-12-2021
DOI: 10.1038/S41396-021-01157-9
Abstract: Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.
Publisher: Microbiology Society
Date: 11-2005
Publisher: American Society for Microbiology
Date: 15-03-2009
DOI: 10.1128/JB.01437-08
Abstract: Contact-dependent growth inhibition (CDI) is a mechanism identified in Escherichia coli by which bacteria expressing two-partner secretion proteins encoded by cdiA and cdiB bind to BamA in the outer membranes of target cells and inhibit their growth. A third gene in the cluster, cdiI , encodes a small protein that is necessary and sufficient to confer immunity to CDI, thereby preventing cells expressing the cdiBA genes from inhibiting their own growth. In this study, the cdiI gene was placed under araBAD promoter control to modulate levels of the immunity protein and thereby induce CDI by removal of arabinose. This CDI autoinhibition system was used for metabolic analyses of a single population of E. coli cells undergoing CDI. Contact-inhibited cells showed altered cell morphology, including the presence of filaments. Notably, CDI was reversible, as evidenced by resumption of cell growth and normal cellular morphology following induction of the CdiI immunity protein. Recovery of cells from CDI also required an energy source. Cells undergoing CDI showed a significant, reversible downregulation of metabolic parameters, including aerobic respiration, proton motive force (Δp), and steady-state ATP levels. It is unclear whether the decrease in respiration and/or Δp is directly involved in growth inhibition, but a role for ATP in the CDI mechanism was ruled out using an atp mutant. Consistent with the observed decrease in Δp, the phage shock response was induced in cells undergoing CDI but not in recovering cells, based on analysis of levels of pspA mRNA.
Publisher: Microbiology Society
Date: 10-2004
Abstract: Pseudomonas aeruginosa is capable of synthesizing polyhydroxyalkanoic acids (PHAs) and rhamnolipids, both of which are composed of 3-hydroxydecanoic acids connected by ester bonds, as well as synthesizing the biofilm matrix polymer alginate. In order to study the influence of PHA biosynthesis on rhamnolipid and alginate biosynthesis, as well as stress tolerance and biofilm formation, isogenic knock-out mutants deficient in PHA biosynthesis were generated for P. aeruginosa PAO1 and the alginate-overproducing P. aeruginosa FRD1. A gentamicin-resistance cassette was inserted replacing the 3' region of phaC1, the whole of phaZ and the 5' region of phaC2. Gas chromatography/mass spectrometry analysis showed that PHA accumulation was completely abolished in both strains. Interestingly, this gene replacement did not abolish rhamnolipid production. Thus, as previously suggested, the PHA synthase is not directly involved in rhamnolipid biosynthesis. In the PHA-negative mutant of mucoid FRD1 alginate biosynthesis was not affected, whereas in the PHA-negative PAO1 mutant an almost threefold increase in biosynthesis was observed compared to the wild-type. Consistently, PHA accumulation in FRD1 contributed only 4.7 % of cell dry weight, which is fourfold less than in PAO1. These data suggest that PHA biosynthesis and alginate biosynthesis are in competition with respect to a common precursor. The surface attachment and biofilm development of the PHA-negative mutants were also compared to those of wild-type strains in glass flow-cell reactors. PHA-negative mutants of P. aeruginosa PAO1 and FRD1 showed reduced attachment to glass. However, the PAO1 PHA-negative mutant, in contrast to the wild-type, formed a stable biofilm with large, distinct and differentiated microcolonies characteristic of alginate-overproducing strains of P. aeruginosa. The stress tolerance of PHA-negative mutants with respect to elevated temperature was strongly impaired. These data indicated a functional role for PHA in stress response and tolerance.
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1016/J.NIOX.2017.02.006
Abstract: Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3'-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with β-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following β-lactam cleavage mediated by transpeptidases enicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (β-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks β-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of β-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the β-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections.
Publisher: Wiley
Date: 11-11-2014
Publisher: American Society for Microbiology
Date: 12-2004
DOI: 10.1128/JB.186.23.8066-8073.2004
Abstract: A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa , phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa , including a putative postsegregational killing operon.
Publisher: American Society for Microbiology
Date: 12-2001
DOI: 10.1128/AEM.67.12.5614-5620.2001
Abstract: Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686–690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts ( r 2 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2- n -octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid ( -min) loss of fluorescence of greater than 90% when used at concentrations of 150 μg of available chlorine ml −1 and 500 μg ml −1 , respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells ( r 2 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with % loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 10-2019
Publisher: Wiley
Date: 07-08-2019
Publisher: Microbiology Society
Date: 10-2006
Abstract: This study demonstrates that attachment of the marine bacterium Pseudoalteromonas tunicata to the cellulose-containing surface of the green alga Ulva australis is mediated by a mannose-sensitive haemagglutinin (MSHA-like) pilus. We have identified an MSHA pilus biogenesis gene locus in P. tunicata , termed mshI1I2JKLMNEGFBACDOPQ , which shows significant homology, with respect to its genetic characteristics and organization, to the MSHA pilus biogenesis gene locus of Vibrio cholerae . Electron microscopy studies revealed that P. tunicata wild-type cells express flexible pili peritrichously arranged on the cell surface. A P. tunicata mutant (SM5) with a transposon insertion in the mshJ region displayed a non-piliated phenotype. Using SM5, it has been demonstrated that the MSHA pilus promotes attachment of P. tunicata wild-type cells in polystyrene microtitre plates, as well as to microcrystalline cellulose and to the living surface of U. australis . P. tunicata also demonstrated increased pilus production in response to cellulose and its monomer constituent cellobiose. The MSHA pilus thus functions as a determinant of attachment in P. tunicata , and it is proposed that an understanding of surface sensing mechanisms displayed by P. tunicata will provide insight into specific ecological interactions that occur between this bacterium and higher marine organisms.
Publisher: American Society for Microbiology
Date: 22-12-2021
DOI: 10.1128/SPECTRUM.01026-21
Abstract: To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to effect movement.
Publisher: American Society for Microbiology
Date: 13-04-2023
DOI: 10.1128/SPECTRUM.03099-22
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen of high medical importance and is one of the main pathogens responsible for the mortality of patients with cystic fibrosis. In addition to inherited antibiotic resistance, P. aeruginosa can form biofilms, defined as communities of microorganisms embedded in a self-produced matrix of extracellular polymeric substances adhering to each other and/or to a surface.
Publisher: Wiley
Date: 24-03-2023
Abstract: Flexible metal‐organic materials are of growing interest owing to their ability to undergo reversible structural transformations under external stimuli. Here, we report flexible metal‐phenolic networks (MPNs) featuring stimuli‐responsive behavior to erse solute guests. The competitive coordination of metal ions to phenolic ligands of multiple coordination sites and solute guests (e.g., glucose) primarily determines the responsive behavior of the MPNs, as revealed experimentally and computationally. Glucose molecules can be embedded into the dynamic MPNs upon mixing, leading to the reconfiguration of the metal‐organic networks and thus changes in their physicochemical properties for targeting applications. This study expands the library of stimuli‐responsive flexible metal‐organic materials and the understanding of intermolecular interactions between metal‐organic materials and solute guests, which is essential for the rational design of responsive materials for various applications.
Publisher: Elsevier BV
Date: 06-2015
Publisher: American Society for Microbiology
Date: 15-12-2007
DOI: 10.1128/AEM.01543-07
Abstract: Bacteria that produce inhibitory compounds on the surface of marine algae are thought to contribute to the defense of the host plant against colonization of fouling organisms. However, the number of bacterial cells necessary to defend against fouling on the plant surface is not known. Pseudoalteromonas tunicata and Phaeobacter sp. strain 2.10 (formerly Roseobacter gallaeciensis ) are marine bacteria often found in association with the alga Ulva australis and produce a range of extracellular inhibitory compounds against common fouling organisms. P. tunicata and Phaeobacter sp. strain 2.10 biofilms with cell densities ranging from 10 2 to 10 8 cells cm −2 were established on polystyrene petri dishes. Attachment and settlement assays were performed with marine fungi (uncharacterized isolates from U. australis ), marine bacteria ( Pseudoalteromonas gracilis , Alteromonas sp., and Cellulophaga fucicola ), invertebrate larvae ( Bugula neritina ), and algal spores ( Polysiphonia sp.) and gametes ( U. australis ). Remarkably low cell densities (10 2 to 10 3 cells cm −2 ) of P. tunicata were effective in preventing settlement of algal spores and marine fungi in petri dishes. P. tunicata also prevented settlement of invertebrate larvae at densities of 10 4 to 10 5 cells cm −2 . Similarly, low cell densities (10 3 to 10 4 cells cm −2 ) of Phaeobacter sp. strain 2.10 had antilarval and antibacterial activity. Previously, it has been shown that abundance of P. tunicata on marine eukaryotic hosts is low ( × 10 3 cells cm −2 ) (T. L. Skovhus et al., Appl. Environ. Microbiol. 70:2373-2382, 2004). Despite such low numbers of P. tunicata on U. australis in situ, our data suggest that P. tunicata and Phaeobacter sp. strain 2.10 are present in sufficient quantities on the plant to inhibit fouling organisms. This strongly supports the hypothesis that P. tunicata and Phaeobacter sp. strain 2.10 can play a role in defense against fouling on U. australis at cell densities that commonly occur in situ.
Publisher: Elsevier BV
Date: 11-2020
Publisher: Microbiology Society
Date: 12-2008
DOI: 10.1099/JMM.0.2008/005009-0
Abstract: Transmissible Pseudomonas aeruginosa clones potentially pose a serious threat to cystic fibrosis (CF) patients. The AES-1 clone has been found to infect up to 40 % of patients in five CF centres in eastern Australia. Studies were carried out on clonal and non-clonal (NC) isolates from chronically infected CF patients, and the reference strain PAO1, to gain insight into the properties of AES-1. The transcriptomes of AES-1 and NC isolates, and of PAO1, grown planktonically and as a 72 h biofilm were compared using PAO1 microarrays. Microarray data were validated using real-time PCR. Overall, most differentially expressed genes were downregulated. AES-1 differentially expressed bacteriophage genes, novel motility genes, and virulence and quorum-sensing-related genes, compared with both PAO1 and NC. AES-1 but not NC biofilms significantly downregulated aerobic respiration genes compared with planktonic growth, suggesting enhanced anaerobic/microaerophilic growth by AES-1. Biofilm measurement showed that AES-1 formed significantly larger and thicker biofilms than NC or PAO1 isolates. This may be related to expression of the gene PA0729, encoding a biofilm-enhancing bacteriophage, identified by PCR in all AES-1 but few NC isolates ( n =42). Links with the Liverpool epidemic strain included the presence of PA0729 and the absence of the bacteriophage gene cluster PA0632–PA0639. No common markers were found with the Manchester strain. No particular differentially expressed gene in AES-1 could definitively be ascribed a role in its infectivity, thus increasing the likelihood that AES-1 infectivity is multi-factorial and possibly involves novel genes. This study extends our understanding of the transcriptomic and genetic differences between clonal and NC strains of P. aeruginosa from CF lung.
Publisher: Springer Science and Business Media LLC
Date: 25-03-2017
Publisher: Oxford University Press (OUP)
Date: 03-2003
Publisher: Oxford University Press (OUP)
Date: 03-2022
DOI: 10.1111/LAM.13618
Abstract: The human oral cavity is host to a erse microbiota. Much of what is known about the behaviour of oral microbes derives from studies of in idual or several cultivated species, situations which do not totally reflect the function of organisms within more complex microbiota or multispecies biofilms. The number of validated models that allow examination of the role that biofilms play during oral cavity colonization is also limited. The CDC biofilm reactor is a standard method that has been deployed to study interactions between members of human microbiotas allowing studies to be completed during an extended period under conditions where nutrient availability, and washout of waste products are controlled. The objective of this work was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum to study the development, reproducibility and stability of the oral microbiota. By employing deep sequencing of the variable regions of the 16S rRNA gene, we found that the CDC biofilm reactor could be used to efficiently cultivate microbiota containing all six major phyla previously identified as the core saliva microbiota. After an acclimatisation period, communities in each reactor stabilised. Replicate reactors were predominately populated by a shared core microbiota variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units. We conclude that the CDC biofilm reactor can be used to cultivate communities that replicate key features of the human oral cavity and is a useful tool to facilitate studies of the dynamics of these communities.
Publisher: Elsevier BV
Date: 09-2017
Publisher: SAGE Publications
Date: 11-09-2019
Abstract: Chronic rhinosinusitis (CRS) is a common condition which affects the quality of life of millions of patients worldwide and has a significant impact on health-care resources. While Staphylococcus aureus bacterial biofilms play an important role in this disease, antimicrobial therapy is rarely effective and may promote antibiotic resistance. Thus, development of novel biofilm-targeting and antibiotic-sparing therapies is highly desirable and urgently required. This in vitro study evaluated the antimicrobial activity of a novel synthetic honey-equivalent product which was designed to have the same reactive oxygen release profile as the engineered honey SurgihoneyRO™. Treatment efficacy was investigated by assessment of planktonic growth, biofilm viability, thickness, and biomass using 12 CRS-related S. aureus mucosal bacterial strains. Both SurgihoneyRO™ and the synthetic honey-equivalent product inhibited growth of planktonic methicillin-resistant and methicillin-sensitive S. aureus strains, with the synthetic honey-equivalent product exhibiting a lower minimum inhibitory concentration. Treatment of established S. aureus biofilms reduced biofilm viability with 24-hour treatment resulting in a 2-log reduction in viability of biofilms formed by methicillin-resistant strains and a 1-log reduction in biofilms formed by methicillin-sensitive strains. This preliminary study shows that the synthetic honey-equivalent product provides marked antimicrobial activity against S. aureus biofilms, with the potential for development in the clinical setting as an adjunctive biofilm-targeted therapy in CRS. The ultimate aim of such a product would be to reduce the need for antibiotics, steroids, and invasive surgical procedures in CRS patients as well as improving clinical outcomes following endoscopic sinus surgery.
Publisher: BMJ
Date: 04-2011
Publisher: Frontiers Media SA
Date: 23-07-2018
Publisher: Oxford University Press (OUP)
Date: 11-2012
Publisher: IOP Publishing
Date: 02-12-2016
Publisher: Oxford University Press (OUP)
Date: 15-04-2016
DOI: 10.1093/GBE/EVW072
Publisher: Springer Science and Business Media LLC
Date: 26-05-2022
DOI: 10.1038/S41522-022-00306-Y
Abstract: The increasing awareness of the significance of microbial biofilms across different sectors is continuously revealing new areas of opportunity in the development of innovative technologies in translational research, which can address their detrimental effects, as well as exploit their benefits. Due to the extent of sectors affected by microbial biofilms, capturing their real financial impact has been difficult. This perspective highlights this impact globally, based on figures identified in a recent in-depth market analysis commissioned by the UK’s National Biofilms Innovation Centre (NBIC). The outputs from this analysis and the workshops organised by NBIC on its research strategic themes have revealed the breath of opportunities for translational research in microbial biofilms. However, there are still many outstanding scientific and technological challenges which must be addressed in order to catalyse these opportunities. This perspective discusses some of these challenges.
Publisher: Cold Spring Harbor Laboratory
Date: 08-02-2022
DOI: 10.1101/2022.02.08.479540
Abstract: Current methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescence in-situ hybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the in idual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR.
Publisher: Wiley
Date: 12-12-2005
DOI: 10.1111/J.1365-2958.2005.05008.X
Abstract: Pseudomonas aeruginosa produces extracellular DNA which functions as a cell-to-cell interconnecting matrix component in biofilms. Comparison of extracellular DNA and chromosomal DNA by the use of polymerase chain reaction and Southern analysis suggested that the extracellular DNA is similar to whole-genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular beta-galactosidase released from lacZ-containing P. aeruginosa strains was assessed. Experiments with the wild type and lasIrhlI, pqsA, pqsL and fliMpilA mutants indicated that the extracellular DNA is generated via a mechanism which is dependent on acyl homoserine lactone and Pseudomonas quinolone signalling, as well as on flagella and type IV pili. Microscopic investigation of flow chamber-grown wild-type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom-shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk-forming bacteria and the cap-forming bacteria. Biofilms formed by lasIrhlI, pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild-type biofilm.
Publisher: American Society for Microbiology
Date: 08-2005
DOI: 10.1128/AEM.71.8.4407-4413.2005
Abstract: The psl gene cluster, comprising 15 cotranscribed genes from Pseudomonas aeruginosa , was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the psl gene cluster and the function of the first gene in this cluster, the pslA gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free pslA deletion mutant of P. aeruginosa PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the pslA gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid P. aeruginosa . The promoter region of the psl gene cluster, which encodes PslA-PslO, was identified by rapid lification of cDNA 5′ ends. Promoter assays using transcriptional fusions to lacZ and gfp indicated a constitutive expression of the psl cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the psl cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the psl operon enables efficient attachment to surfaces and that regulated localized psl operon expression is required for biofilm differentiation.
Publisher: Elsevier BV
Date: 12-2003
DOI: 10.1016/J.MIB.2003.10.014
Abstract: The development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms. Recent advances in the field provide new insight into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity.
Publisher: Wiley
Date: 26-03-2008
Publisher: Public Library of Science (PLoS)
Date: 23-07-2008
Publisher: American Society for Microbiology
Date: 08-2006
DOI: 10.1128/AEM.00546-06
Abstract: In the ubiquitous marine bacterium Pseudoalteromonas tunicata , subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic ersification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a ΔAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC 4 (3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from ΔAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the ΔAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically erse subpopulation of dispersal cells.
Publisher: Oxford University Press (OUP)
Date: 02-1998
DOI: 10.1046/J.1365-2672.1998.00337.X
Abstract: The use of wetlands is a promising technology to treat acid mine drainage, yet there is little understanding of the fundamental biological processes involved. They are considered to centre on the complex anaerobic ecology within sediments and involve the removal of metals by sulphate-reducing bacteria (SRB). These bacteria generate hydrogen sulphide and cause precipitation of metals from solution as the insoluble metal sulphide. Sulphate-reducing bacteria have been isolated from natural and constructed wetlands receiving acid mine drainage. Sulphide production by isolates and removal of the metals iron, manganese and zinc were measured, as well as utilization of a range of carbon sources. Marked ecological differences between the wetlands were reflected in population composition of SRB enrichments, and these consortia displayed significant differences in sulphide generation and rates of metal removal from solution. Rates of metal removal did not correlate with sulphide generation in all cultures, suggesting the involvement of other biological mechanisms of metal removal. Differences in substrate utilization have highlighted the need for further investigation of carbon flow and potential carbon sources within constructed wetlands.
Publisher: Informa UK Limited
Date: 09-1996
DOI: 10.1080/08927019609386273
Abstract: Sulphidogenic bacteria in oil reservoirs are of great economic importance in terms of souring, fouling and corrosion. Mixed cultures containing these bacteria were isolated from chalk formations in North Sea oil reservoirs. These were thermophilic cultures, growing optimally at 60°C. Oil formations are porous matrices, providing a very large surface area and ideal conditions for bacterial attachment, survival and growth. This study included assessments of sulphide production rates of thermophilic (t-)sulphidogen consortia with and without additional surfaces. The availability of a surface contributed significantly to the rate and extent of sulphide generation. Surfaces were offered in varying amounts to growing planktonic cultures: significantly more sulphide was produced from cultures in contact with a surface than from identical cultures in the absence of a surface. In another series of experiments, t-sulphidogens were added to chalk rock chips in the presence of nutrients and incubated for several months. This resulted in rapid sulphide generation, the final concentration being related to the initial nutrient concentration. Subsequent nutrient addition resulted in renewed sulphide generation. It is suggested that bacteria in reservoirs can withstand long periods of nutrient deprivation while attached within the porous rock matrix and opportunistically utilise nutrients when they become available.
Publisher: Springer Science and Business Media LLC
Date: 31-08-2020
DOI: 10.1007/S00253-020-10859-7
Abstract: Pseudomonas aeruginosa biofilms contribute heavily to chronic lung infection in cystic fibrosis patients, leading to morbidity and mortality. Nitric oxide (NO) has been shown to disperse P. aeruginosa biofilms in vitro, ex vivo and in clinical trials as a promising anti-biofilm agent. Traditional NO donors such as sodium nitroprusside (SNP) have been extensively employed in different studies. However, the dosage of SNP in different studies was not consistent, ranging from 500 nM to 500 μM. SNP is light sensitive and produces cyanide, which may lead to data misinterpretation and inaccurate predictions of dispersal responses in clinical settings. New NO donors and NO delivery methods have therefore been explored. Here we assessed 7 NO donors using P. aeruginosa PAO1 and determined that SNP and Spermine NONOate (S150) successfully reduced 60% biomass within 24 and 2 h, respectively. While neither dosage posed toxicity towards bacterial cells, chemiluminescence assays showed that SNP only released NO upon light exposure in M9 media and S150 delivered much higher performance spontaneously. S150 was then tested on 13 different cystic fibrosis P. aeruginosa (CF-PA) isolates most CF-PA biofilms were significantly dispersed by 250 μM S150. Our work therefore discovered a commercially available NO donor S150, which disperses CF-PA biofilms efficiently within a short period of time and without releasing cyanide, as an alternative of SNP in clinical trials in the future. • S150 performs the best in dispersing P. aeruginosa biofilms among 7 NO donors. • SNP only releases NO in the presence of light, while S150 releases NO spontaneously. • S150 successfully disperses biofilms formed by P. aeruginosa cystic fibrosis clinical isolates.
Publisher: American Society for Microbiology
Date: 15-04-2008
DOI: 10.1128/JB.01683-07
Abstract: Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of ∼6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant “anaerobiome” or “proteogenome” of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO 3 − and NO 2 − are those encoding Pf1 bacteriophage. With anaerobic NO 2 − , the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO 3 − and NO 2 − respiration. In addition to genes anticipated to play important roles in the anaerobiome ( anr , dnr , nar , nir , and nuo ), the cysG and dksA genes were found to be required for both anaerobic NO 3 − and NO 2 − respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO 3 − and NO 2 − respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.
Publisher: Elsevier BV
Date: 10-2017
DOI: 10.1016/J.JHIN.2017.06.005
Abstract: Pseudomonas aeruginosa infections have been linked to contaminated hospital taps, highlighting the potential for tap outlet fittings (OF) to harbour biofilm. P. aeruginosa may be transferred to OFs via contaminated cleaning cloths. Suggested interventions include flushing regimens and alternative OF designs. To investigate the transfer of P. aeruginosa from a contaminated cleaning cloth to conventional and 'antimicrobial/antibiofilm' OFs and to determine whether this contamination persists and/or leads to contamination of tap water. Microfibre cloths contaminated with P. aeruginosa (10 The median number of P. aeruginosa transferred from cloth to OF was 5.7 × 10 Contaminated cleaning cloths may transfer P. aeruginosa to OFs, leading to contamination of tap water. Although not removing the potential for contamination, 'antimicrobial/antibiofilm' OFs may prevent P. aeruginosa from continually contaminating water delivered from the outlet.
Publisher: American Chemical Society (ACS)
Date: 03-01-2022
DOI: 10.1021/ACS.ANALCHEM.1C02501
Abstract: The current methods for diagnosis of acute and chronic infections are complex and skill-intensive. For complex clinical biofilm infections, it can take days from collecting and processing a patient's s le to achieving a result. These aspects place a significant burden on healthcare providers, delay treatment, and can lead to adverse patient outcomes. We report the development and application of a novel multi-excitation Raman spectroscopy-based methodology for the label-free and non-invasive detection of microbial pathogens that can be used with unprocessed clinical s les directly and provide rapid data to inform diagnosis by a medical professional. The method relies on the differential excitation of non-resonant and resonant molecular components in bacterial cells to enhance the molecular finger-printing capability to obtain strain-level distinction in bacterial species. Here, we use this strategy to detect and characterize the respiratory pathogens
Publisher: Springer International Publishing
Date: 2020
Publisher: American Society for Microbiology
Date: 08-2000
DOI: 10.1128/AEM.66.8.3194-3200.2000
Abstract: Significant substratum damage can occur when plasticized PVC (pPVC) is colonized by microorganisms. We investigated microbial colonization of pPVC in an in situ, longitudinal study. Pieces of pPVC containing the plasticizers dioctyl phthalate and dioctyl adipate (DOA) were exposed to the atmosphere for up to 2 years. Fungal and bacterial populations were quantified, and colonizing fungi were identified by rRNA gene sequencing and morphological characteristics. Aureobasidium pullulans was the principal colonizing fungus, establishing itself on the pPVC between 25 and 40 weeks of exposure. A group of yeasts and yeast-like fungi, including Rhodotorula aurantiaca and Kluyveromyces spp., established themselves on the pPVC much later (after 80 weeks of exposure). Numerically, these organisms dominated A. pullulans after 95 weeks, with a mean viable count ± standard error of 1,000 ± 200 yeast CFU cm −2 , compared to 390 ± 50 A. pullulans CFU cm −2 . No bacterial colonization was observed. We also used in vitro tests to characterize the deteriogenic properties of fungi isolated from the pPVC. All strains of A. pullulans tested could grow with the intact pPVC formulation as the sole source of carbon, degrade the plasticizer DOA, produce extracellular esterase, and cause weight loss of the substratum during growth in vitro. In contrast, several yeast isolates could not grow on pPVC or degrade DOA. These results suggest that microbial succession may occur during the colonization of pPVC and that A. pullulans is critical to the establishment of a microbial community on pPVC.
Publisher: Elsevier BV
Date: 02-2023
Publisher: American Society for Microbiology
Date: 08-2003
DOI: 10.1128/JB.185.15.4585-4592.2003
Abstract: Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa . We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.
Publisher: American Society for Microbiology
Date: 06-2004
DOI: 10.1128/AEM.70.6.3232-3238.2004
Abstract: The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata , similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185: 4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata , designated AlpP, was found to be involved in this biofilm killing and detachment. A Δ alpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.
Publisher: Wiley
Date: 17-04-2009
Publisher: Public Library of Science (PLoS)
Date: 04-09-2014
Publisher: Springer Science and Business Media LLC
Date: 30-10-2020
DOI: 10.1038/S41522-020-00154-8
Abstract: Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.
Publisher: Elsevier BV
Date: 08-2011
Publisher: American Chemical Society (ACS)
Date: 24-04-2020
Publisher: American Society for Microbiology
Date: 12-2009
DOI: 10.1128/JB.00975-09
Abstract: Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa . Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO.
Publisher: Wiley
Date: 2006
DOI: 10.1002/PPUL.20420
Publisher: Microbiology Society
Date: 10-2019
DOI: 10.1099/JMM.0.001046
Publisher: BMJ
Date: 04-2014
Publisher: Oxford University Press (OUP)
Date: 23-06-2010
Publisher: International Union of Crystallography (IUCr)
Date: 28-08-2016
Publisher: Wiley
Date: 13-07-2022
Abstract: Coordination states of metal‐organic materials are known to dictate their physicochemical properties and applications in various fields. However, understanding and controlling coordination sites in metal‐organic systems is challenging. Herein, we report the synthesis of site‐selective coordinated metal‐phenolic networks (MPNs) using flavonoids as coordination modulators. The site‐selective coordination was systematically investigated experimentally and computationally using ligands with one, two, and multiple different coordination sites. Tuning the multimodal Fe coordination with catechol, carbonyl, and hydroxyl groups within the MPNs enabled the facile engineering of erse physicochemical properties including size, selective permeability (20–2000 kDa), and pH‐dependent degradability. This study expands our understanding of metal‐phenolic chemistry and provides new routes for the rational design of structurally tailorable coordination‐based materials.
Publisher: Elsevier BV
Date: 12-2020
Publisher: Oxford University Press (OUP)
Date: 24-03-2016
DOI: 10.1093/GBE/EVW066
Publisher: Springer Science and Business Media LLC
Date: 02-01-2020
DOI: 10.1186/S12866-019-1672-7
Abstract: Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode erse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein–DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas c estris pv. c estris ( Xcc ). The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner. We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.
Publisher: Cold Spring Harbor Laboratory
Date: 14-02-2012
Abstract: Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI + inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI + cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK ( O -acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK + cells arrests cell growth, whereas the growth of Δ cysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.
Publisher: Wiley
Date: 13-08-2012
Publisher: Proceedings of the National Academy of Sciences
Date: 28-03-2014
Abstract: Within-population genetic ersity is an essential evolutionary prerequisite for processes ranging from antibiotic resistance to niche adaptation, but its generation is poorly understood, with most studies focusing on fixed substitutions at the end point of long-term evolution. Using deep sequencing, we analyzed short-term, within-population genetic ersification occurring during biofilm formation of the model bacterium Pseudomonas aeruginosa . We discovered extensive parallel evolution between biological replicates at the level of pathways, genes, and even in idual nucleotides. Short-term ersification featured positive selection of relatively few nonsynonymous mutations, with the majority of the genome being conserved by negative selection. This result is broadly consistent with observations of long-term evolution and suggests ersifying selection may underlie genetic ersification of Pseudomonas aeruginosa biofilms.
Publisher: Springer Science and Business Media LLC
Date: 13-11-2008
Publisher: Oxford University Press (OUP)
Date: 03-2009
DOI: 10.1111/J.1574-6968.2008.01472.X
Abstract: Epidemic Pseudomonas aeruginosa have been identified in cystic fibrosis (CF) patients worldwide. The Australian Epidemic Strain-2 (AES-2) infects up to 40% of patients in three eastern Australian CF clinics. To investigate whether AES-2 isolates from chronically infected CF adults differentially express well-conserved genes potentially associated with transmissibility, we compared the transcriptomes of planktonic and biofilm-grown AES-2, infrequent P. aeruginosa clones and the reference P. aeruginosa PAO1 using the Affymetrix PAO1 array. The most interesting findings emerged from comparisons of planktonic and biofilm AES-2. AES-2 biofilms upregulated Type III secretion system genes, but downregulated quorum-sensing (QS)-regulatory genes, except lasR, QS-regulated, oxidative-stress and iron-storage genes. QS-regulated and iron-storage genes were downregulated to a greater extent in AES-2 biofilms compared with infrequent clone and PAO1 biofilms, suggesting enhanced anaerobic respiration in AES-2. Chitinase and chitin-binding protein maintained high expression in AES-2 biofilms compared with infrequent clone and PAO1 biofilms. Planktonic AES-2 upregulated QS regulators and QS-regulated genes, iron acquisition and aerobic respiration genes, and had high expression of Group III Type IV pilA compared with low expression of Group I Type IV pilA in infrequent clones. Together, these properties may enhance long-term survival of AES-2 in CF lung and contribute to its transmissibility.
Publisher: Springer Science and Business Media LLC
Date: 12-12-2019
DOI: 10.1038/S41598-019-55567-Z
Abstract: Non-surface attached bacterial aggregates are frequently found in clinical settings associated with chronic infections. Current methods quantifying the extent to which a suspended bacterial population is aggregated mainly rely on: (1) cell size distribution curves that are difficult to be compared numerically among large-scale s les (2) the average size roportion of aggregates in a population that do not specify the aggregation patterns. Here we introduce a novel application of Gini coefficient, herein named Aggregation Coefficient (AC), to quantify the aggregation levels of cystic fibrosis Pseudomonas aeruginosa (CF-PA) isolates in vitro using 3D micrographs, Fiji and MATLAB. Different aggregation patterns of five strains were compared statistically using the numerical AC indexes, which correlated well with the size distribution curves plotted by different biovolumes of aggregates. To test the sensitivity of AC, aggregates of the same strains were treated with nitric oxide (NO), a dispersal agent that reduces the biomass of surface attached biofilms. Strains unresponsive to NO were reflected by comparable AC indexes, while those undergoing dispersal showed a significant reduction in AC index, mirroring the changes in average aggregate sizes and proportions. Therefore, AC provides simpler and more descriptive numerical outputs for measuring different aggregation patterns compared to current approaches.
Publisher: Elsevier BV
Date: 04-2017
DOI: 10.1016/J.JSE.2016.09.042
Abstract: The role of Propionibacterium acnes in shoulder arthroplasty and broadly in orthopedic prosthetic infections has historically been underestimated, with biofilm formation identified as a key virulence factor attributed to invasive isolates. With an often indolent clinical course, P acnes infection can be difficult to detect and treat. This study investigates absorbable cements loaded with a broad-spectrum antibiotic combination as an effective preventive strategy to combat P acnes biofilms. P acnes biofilm formation on an unloaded synthetic calcium sulfate (CaSO P acnes surface colonization and biofilm formation on unloaded CaSO This study demonstrates that antibiotic-loaded CaSO
Publisher: Public Library of Science (PLoS)
Date: 12-2011
Publisher: American Society for Microbiology
Date: 02-2006
DOI: 10.1128/AEM.72.2.1639-1644.2006
Abstract: Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 μm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight) −1 day −1 with a 90% molar yield over a 45-h production period.
Publisher: Cold Spring Harbor Laboratory
Date: 23-05-2023
DOI: 10.1101/2023.05.23.541926
Abstract: The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind ferric (Fe 3+ ), while cytosolic FutA1 binds ferrous (Fe 2+ ). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a solvent molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell was consistent with an overall charge-neutral ferric binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the ferrous binding state here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral ferrous binding site. Room temperature dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between ferric and ferrous states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Oceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a switch in the FutA protein that allows it to accommodate binding of iron in either the ferric (Fe 3+ ) or ferrous (Fe 2+ ) state using structural biology techniques at room temperature and provide a plausible mechanism for FutA as a bifunctional redox state sensing protein.
Publisher: American Society for Microbiology
Date: 02-2017
DOI: 10.1128/AAC.02086-16
Abstract: PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability ( P 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed ( P 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.
Publisher: Cold Spring Harbor Laboratory
Date: 07-02-2020
DOI: 10.1101/2020.02.06.936690
Abstract: Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and missed genes by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq datasets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth. Experiments conducted across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated both known and novel genes involved in biofilm growth and antibiotic resistance identified in screens of the mutant collection. Differential protein expression data from planktonic cells versus biofilm confirmed upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type six secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance and resistance evolution.
Publisher: Springer Science and Business Media LLC
Date: 10-04-2020
DOI: 10.1038/S41598-020-63008-5
Abstract: In Pseudomonas aeruginosa , the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes Δ rbdA and Δ pa2072 that encode proteins with identical domain structure: while Δ rbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δ pa2072 , and biofilm formation was inhibited, compared to wild type. A second pair of genes, Δ fimX and Δ dipA , were selected on the basis of predicted impaired c-di-GMP turnover function: Δ fimX showed increased, Δ dipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for Δ fimX and reduced swimming for Δ dipA . For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2009
End Date: 2011
Funder: Bill and Melinda Gates Foundation
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Funder: National Institute for Health Research
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Funder: Australian Cystic Fibrosis Research Trust
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Funder: Royal Society
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Funder: Australian Research Council
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Funder: Leverhulme Trust
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Funder: University of New South Wales
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Funder: Biotechnology and Biological Sciences Research Council
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Funder: Wessex Medical Research
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Funder: Matoke Holdings Ltd, UK
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Funder: Unilever
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Funder: Rosetrees Trust
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Funder: Biotechnology and Biological Sciences Research Council
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Funder: Biotechnology and Biological Sciences Research Council
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Funder: Biotechnology and Biological Sciences Research Council
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Funder: Medical Research Council
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End Date: 2011
Funder: Biotechnology and Biological Sciences Research Council
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End Date: 2020
Funder: Swiss National Science Foundation
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End Date: 2008
Funder: Australian Research Council
View Funded ActivityStart Date: 2019
End Date: 2021
Funder: National Institute of Health Research (NIHR), UK and Department of Health and Social Care (DOHSC), UK
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End Date: 2022
Funder: Biotechnology and Biological Sciences Research Council
View Funded ActivityStart Date: 2018
End Date: 2021
Funder: Cystic Fibrosis Trust
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End Date: 2017
Funder: Wellcome Trust
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End Date: 2018
Funder: Cystic Fibrosis Trust
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End Date: 2015
Funder: National Institute for Health Research
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End Date: 2017
Funder: Healthcare Infection Society
View Funded ActivityStart Date: 2011
End Date: 2012
Funder: Engineering and Physical Sciences Research Council
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