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The switch between planktonic and biofilm lifestyle correlates with intracellular concentration of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). While bacteria possess cyclase and phosphodiesterase enzymes to catalyse formation or hydrolysis of c-di-GMP, both enzymatic domains often occur in a single protein. It is tacitly assumed that one of the two enzymatic activities is dominant, and that additional domains and protein interactions enable responses to environmental conditions and control activity. Here we report the structure of the phosphodiesterase domain of the membrane protein RbdA (regulator of biofilm dispersal) in a dimeric, activated state and show that phosphodiesterase activity is controlled by the linked cyclase. The phosphodiesterase region around helices α5/α6 forms the dimer interface, providing a rationale for activation, as this region was seen in contact with the cyclase domain in an auto-inhibited structure previously described. Kinetic analysis supports this model, as the activity of the phosphodiesterase alone is lower when linked to the cyclase. Analysis of a computed model of the RbdA periplasmatic domain reveals an all-helical architecture with a large binding pocket that could accommodate putative ligands. Unravelling the regulatory circuits in multi-domain phosphodiesterases like RbdA is important to develop strategies to manipulate or disperse bacterial biofilms., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Priority question exercises are increasingly used to frame and set future research, innovation and development agendas. They can provide an important bridge between the discoveries, data and outputs generated by researchers, and the information required by policy makers and funders. Microbial biofilms present huge scientific, societal and economic opportunities and challenges. In order to identify key priorities that will help to advance the field, here we review questions from a pool submitted by the international biofilm research community and from practitioners working across industry, the environment and medicine. To avoid bias we used computational approaches to group questions and manage a voting and selection process. The outcome of the exercise is a set of 78 unique questions, categorized in six themes: (i) Biofilm control, disruption, prevention, management, treatment (13 questions); (ii) Resistance, persistence, tolerance, role of aggregation, immune interaction, relevance to infection (10 questions); (iii) Model systems, standards, regulatory, policy education, interdisciplinary approaches (15 questions); (iv) Polymicrobial, interactions, ecology, microbiome, phage (13 questions); (v) Clinical focus, chronic infection, detection, diagnostics (13 questions); and (vi) Matrix, lipids, capsule, metabolism, development, physiology, ecology, evolution environment, microbiome, community engineering (14 questions). The questions presented are intended to highlight opportunities, stimulate discussion and provide focus for researchers, funders and policy makers, informing future research, innovation and development strategy for biofilms and microbial communities., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Given his role as Co-Editor in Chief, TOM COENYE had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to BIRTHE KJELLERUP. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). 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 neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) 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 Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

The rapid emergence of bacterial resistance to antibiotics in current use is occurring worldwide and poses a significant threat to global healthcare systems. Recent research to identify new effective anti-bacterial agents has focused on regulatory pathways as targets for interference. Regulatory mechanisms employing intracellular Bis-(3',5') cyclic di-guanylate (c-di-GMP) as a secondary messenger represent a distinct category of subjects. This molecule, c-di-GMP, is present in nearly all bacterial species and plays a pivotal role in governing various biological processes, encompassing antibiotic resistance, biofilm formation, and virulence. Alteration of the cellular concentrations of the nucleotide through modulation of associated signaling pathways has the potential to reduce biofilm formation or increase susceptibility of the biofilm bacteria to antibiotics. Here, we have developed a screen for compounds that alter c-di-GMP levels in Pseudomonas aeruginosa in co-culture with bronchial epithelial cells. Through the assay of 200 natural compounds, we were able to identify several substances showing promising effects on P. aeruginosa in a host biofilm infection model. Importantly, we detected compounds that inhibit c-di-GMP levels and showed significant influence on biofilm formation and virulence in P. aeruginosa in vitro and in vivo . Consequently, we offer proof-of-concept information regarding swift and practical drug screening assays, suitable for medium- to high-throughput applications, which target the c-di-GMP signaling pathways in this significant Gram-negative pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hu, Webb and An.)

Pseudomonas aeruginosa is an opportunistic pathogen of considerable medical importance, owing to its pronounced antibiotic tolerance and association with cystic fibrosis and other life-threatening diseases. The aim of this study was to highlight the genes responsible for P. aeruginosa biofilm tolerance to antibiotics and thereby identify potential new targets for the development of drugs against biofilm-related infections. By developing a novel screening approach and utilizing a public P. aeruginosa transposon insertion library, several biofilm-relevant genes were identified. The Pf phage gene ( PA0720 ) and flagellin gene ( fliC ) conferred biofilm-specific tolerance to gentamicin. Compared with the reference biofilms, the biofilms formed by PA0720 and fliC mutants were completely eliminated with a 4-fold-lower gentamicin concentration. Furthermore, the mreC , pprB , coxC , and PA3785 genes were demonstrated to play major roles in enhancing biofilm tolerance to gentamicin. The analysis of biofilm-relevant genes performed in this study provides important novel insights into the understanding of P. aeruginosa antibiotic tolerance, which will facilitate the detection of antibiotic resistance and the development of antibiofilm strategies against P. aeruginosa. IMPORTANCE 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. Biofilms protect bacteria from antibiotic treatments and represent a major reason for antibiotic failure in the treatment of chronic infections caused by cystic fibrosis. Therefore, it is crucial to develop new therapeutic strategies aimed at specifically eradicating biofilms. The aim of this study was to generalize a novel screening method for biofilm research and to identify the possible genes involved in P. aeruginosa biofilm tolerance to antibiotics, both of which could improve the understanding of biofilm-related infections and allow for the identification of relevant therapeutic targets for drug development.

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 divided 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 example, in chronic wounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 LuTheryn, Hind, Campbell, Crowther, Wu, Keller, Glynne-Jones, Sutton, Webb, Gray, Wilks, Stride and Carugo.)

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., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

The human oral cavity is host to a diverse microbiota. Much of what is known about the behaviour of oral microbes derives from studies of individual 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., (© 2021 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

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 sample 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 samples 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 Pseudomonas aeruginosa and Staphylococcus aureus as typical infectious agents associated with cystic fibrosis. Planktonic specimens were analyzed both in isolation and in artificial sputum media. The resonance Raman components, excited at different wavelengths, were characterized as carotenoids and porphyrins. By combining the more informative multi-excitation Raman spectra with multivariate analysis (support vector machine) the accuracy was found to be 99.75% for both species (across all strains), including 100% accuracy for drug-sensitive and drug-resistant S. aureus . The results demonstrate that our methodology based on multi-excitation Raman spectroscopy can underpin the development of a powerful platform for the rapid and reagentless detection of clinical pathogens to support diagnosis by a medical expert, in this case relevant to cystic fibrosis. Such a platform could provide translatable diagnostic solutions in a variety of disease areas and also be utilized for the rapid detection of anti-microbial resistance.

PAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico . The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture. IMPORTANCE 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 affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.

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.

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. KEY POINTS: • 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.

The formation of biofilms provides a formidable defense for many bacteria against antibiotics and host immune responses. As a consequence, biofilms are thought to be the root cause of most chronic infections, including those occurring on medical indwelling devices, endocarditis, urinary tract infections, diabetic and burn wounds, and bone and joint infections. In cystic fibrosis (CF), chronic Pseudomonas aeruginosa ( P. aeruginosa ) respiratory infections are the leading cause of morbidity and mortality in adults. Previous studies have shown that many bacteria can undergo a coordinated dispersal event in the presence of low concentrations of nitric oxide (NO), suggesting that NO could be used to initiate biofilm dispersal in chronic infections, enabling clearance of the more vulnerable planktonic cells. In this study, we describe efforts to create "all-in-one" cephalosporin-based NO donor prodrugs (cephalosporin-3'-diazeniumdiolates, C3Ds) that show both direct β-lactam mediated antibacterial activity and antibiofilm effects. Twelve novel C3Ds were synthesized and screened against a panel of P. aeruginosa CF clinical isolates and other human pathogens. The most active compound, AMINOPIP2 (( Z )-1-(4-(2-aminoethyl)piperidin-1-yl)-2-(((6 R ,7 R )-7-(( Z )-2-(2-aminothiazol-4-yl)-2-(((2-carboxypropan-2-yl)oxy)imino)acetamido)-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methoxy)diazene 1-oxide)-ceftazidime 12 , showed higher antibacterial potency than its parent cephalosporin and front-line antipseudomonal antibiotic ceftazidime, good stability against β-lactamases, activity against ceftazidime-resistant P. aeruginosa in vitro biofilms, and efficacy equivalent to ceftazidime in a murine P. aeruginosa respiratory infection model. The results support further evaluation of AMINOPIP2-ceftazidime 12 for P. aeruginosa lung infections in CF and a broader study of "all-in-one" C3Ds for other chronic infections.

Bacterial biofilms are an ever-growing concern for public health, featuring both inherited genetic resistance and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing interest in novel methods of drug delivery, in order to increase the efficacy of antimicrobial agents. One such method is the use of acoustically activated microbubbles, which undergo volumetric oscillations and collapse upon exposure to an ultrasound field. This facilitates physical perturbation of the biofilm and provides the means to control drug delivery both temporally and spatially. In line with current literature in this area, this review offers a rounded argument for why ultrasound-responsive agents could be an integral part of advancing wound care. To achieve this, we will outline the development and clinical significance of biofilms in the context of chronic infections. We will then discuss current practices used in combating biofilms in chronic wounds and then critically evaluate the use of acoustically activated gas microbubbles as an emerging treatment modality. Moreover, we will introduce the novel concept of microbubbles carrying biologically active gases that may facilitate biofilm dispersal., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections., (Copyright © 2020 Frapwell et al.)

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 ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. 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.

Background: Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse 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 campestris pv. campestris (Xcc)., Results: 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., Conclusion: 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.

Objectives: 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., Methods: 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., Results: 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., Conclusions: 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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

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 samples; (2) the average size/proportion 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.

The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays., Competing Interests: The authors declare no competing interests., (© 2019 The Author(s).)

Respiratory tract infections are responsible for over 2.8 million deaths per year worldwide. Colonization is the first step in the process of microbes occupying the respiratory tract, which may lead to subsequent infection. Carriage, in contrast, is defined as the occupation of microbial species in the respiratory tract. The duration of carriage may be affected by host immunity, the composition and interactions between members of the microbial community, and the characteristics of colonizing bacteria, including physiology associated with being present in a bacterial biofilm. Numerous vaccines have been implemented to control infections caused by bacteria that can colonize and be subsequently carried. Such vaccines are often species-specific and may target a limited number of strains thereby creating a vacant niche in the upper respiratory tract. Epidemiological changes of bacteria found in both carriage and disease have therefore been widely reported, since the vacant niche is filled by other strains or species. In this review, we discuss the use of carriage-prevalence studies in vaccine evaluation and argue that such studies are essential for (1) examining the epidemiology of carriage before and after the introduction of new vaccines, (2) understanding the dynamics of the respiratory tract flora and (3) identifying the disease potential of emerging strains. In an era of increasing antibiotic resistance, bacterial carriage-prevalence studies are essential for monitoring the impact of vaccination programmes.

Purpose of Review: Biofilm-associated infections cause difficulties in the management of childhood chronic infections and other diseases, due to the invasive nature of interventions which are often necessary for definitive management. Despite their importance, there are challenges in diagnosing biofilm infections and gaps in clinicians' understanding regarding the significance of biofilms., Recent Findings: Many chronic infections associated with biofilms remain difficult or impossible to eradicate with conventional therapy. Surgical intervention, implant removal or long-term intermittent or suppressive antimicrobial therapy may be required. There are still significant challenges in detecting biofilms which presents a barrier in clinical practice and research. Novel therapies to disrupt biofilms are currently under investigation, which may help reduce the impact of antimicrobial resistance., Summary: Biofilm-associated infection should be considered wherever there is clinical concern for an infection affecting prosthetic material, where there is a predisposing condition such as suppurative lung disease; or in the setting of chronic or relapsing infections which may be culture negative. New diagnostic methods for detecting biofilms are a research priority for both clinical diagnosis and the ability to conduct high quality clinical trials of novel antibiofilm interventions.

Rhizosphere microbial communities are known to be highly diverse and strongly dependent on various attributes of the host plant, such as species, nutritional status, and growth stage. High-throughput 16S rRNA gene amplicon sequencing has been used to characterize the rhizosphere bacterial community of many important crop species, but this is the first study to date to characterize the bacterial and archaeal community of Brassica oleracea var. capitata . The study also tested the response of the bacterial community to fertilizer type (organic or synthetic) and N dosage (high or low), in addition to plant age (9 or 12 weeks) and aphid ( Myzus persicae ) herbivory (present/absent). The impact of aboveground herbivory on belowground microbial communities has received little attention in the literature, and since the type (organic or mineral) and amount of fertilizer applications are known to affect M. percicae populations, these treatments were applied at agricultural rates to test for synergistic effects on the soil bacterial community. Fertilizer type and plant growth were found to result in significantly different rhizosphere bacterial communities, while there was no effect of aphid herbivory. Several operational taxonomic units were identified as varying significantly in abundance between the treatment groups and age cohorts. These included members of the S-oxidizing genus Thiobacillus , which was significantly more abundant in organically fertilized 12-week-old cabbages, and the N-fixing cyanobacteria Phormidium , which appeared to decline in synthetically fertilized soils relative to controls. These responses may be an effect of accumulating root-derived glucosinolates in the B. oleracea rhizosphere and increased N-availability, respectively.

Pseudomonas aeruginosa opportunistically infects the airways of patients with cystic fibrosis and causes significant morbidity and mortality. Initial infection can often be eradicated though requires prompt detection and adequate treatment. Intermittent and then chronic infection occurs in the majority of patients. Better detection of P. aeruginosa infection using biomarkers may enable more successful eradication before chronic infection is established. In chronic infection P. aeruginosa adapts to avoid immune clearance and resist antibiotics via efflux pumps, β-lactamase expression, reduced porins and switching to a biofilm lifestyle. The optimal treatment strategies for P. aeruginosa infection are still being established, and new antibiotic formulations such as liposomal amikacin, fosfomycin in combination with tobramycin and inhaled levofloxacin are being explored. Novel agents such as the alginate oligosaccharide OligoG, cysteamine, bacteriophage, nitric oxide, garlic oil and gallium may be useful as anti-pseudomonal strategies, and immunotherapy to prevent infection may have a role in the future. New treatments that target the primary defect in cystic fibrosis, recently licensed for use, have been associated with a fall in P. aeruginosa infection prevalence. Understanding the mechanisms for this could add further strategies for treating P. aeruginosa in future., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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/penicillin-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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: 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., Methods: P acnes biofilm formation on an unloaded synthetic calcium sulfate (CaSO 4 ) bone void filler cement bead was evaluated by scanning electron microscopy over a period of 14 days. Beads loaded with tobramycin alone or vancomycin alone (as comparative controls) and beads loaded with a vancomycin-tobramycin dual treatment were assessed for their ability to eradicate planktonic P acnes, prevent biofilm formation, and eradicate preformed biofilms using a combination of viable-cell counts, confocal microscopy, and scanning electron microscopy., Results: P acnes surface colonization and biofilm formation on unloaded CaSO 4 beads was slow. Beads loaded with antibiotics were able to kill planktonic cultures of 10 6  colony-forming units/mL, prevent bacterial colonization, and significantly reduce biofilm formation over periods of weeks. Complete eradication of established biofilms was achieved with a contact time of 1 week., Conclusions: This study demonstrates that antibiotic-loaded CaSO 4 beads may represent an effective antibacterial and antibiofilm strategy to combat prosthetic infections in which P acnes is involved., (Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Background: Since 2000, the widespread adoption of pneumococcal conjugate vaccines (PCVs) has had a major impact in the prevention of pneumonia. Limited access to international financial support means some middle-income countries (MICs) are trailing in the widespread use of PCVs. We review the status of PCV implementation, and discuss any needs and gaps related to low levels of PCV implementation in MICs, with analysis of possible solutions to strengthen the PCV implementation process in MICs., Main Body: We searched PubMed, PubMed Central, Ovid MEDLINE, and SCOPUS databases using search terms related to pneumococcal immunization, governmental health policy or programmes, and MICs. Two authors independently reviewed the full text of the references, which were assessed for eligibility using pre-defined inclusion and exclusion criteria. The search terms identified 1,165 articles and the full texts of 21 were assessed for suitability, with eight articles included in the systematic review. MICs are implementing PCVs at a slower rate than donor-funded low-income countries and wealthier developed countries. A significant difference in the uptake of PCV in lower middle-income countries (LMICs) (71%) and upper middle-income countries (UMICs) (48%) is largely due to an unsuccessful process of "graduation" of MICs from GAVI assistance, an issue that arises as countries cross the income eligibility threshold and are no longer eligible to receive the same levels of financial assistance. A lack of country-specific data on disease burden, a lack of local expertise in economic evaluation, and the cost of PCV were identified as the leading causes of the slow uptake of PCVs in MICs. Potential solutions mentioned in the reviewed papers include the use of vaccine cost-effectiveness analysis and the provision of economic evidence to strengthen decision-making, the evaluation of the burden of disease, and post-introduction surveillance to monitor vaccine impact., Conclusion: The global community needs to recognise the impediments to vaccine introduction into MICs. Improving PCV access could help decrease the incidence of pneumonia and reduce the selection pressure for pneumococcal antimicrobial resistance.

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., Competing Interests: The authors declare no competing financial interests.

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., (Copyright © 2017 American Society for Microbiology.)

Biofilm formation represents a key stage in the pathogenesis of prosthetic infections (PIs). More tolerant to antibiotics than their planktonic counterparts, biofilm bacteria are difficult to eradicate using conventional therapeutic regimes. A common approach in PI management is the adjunctive use of localised antibiotics in addition to systemic administration in an attempt to protect the implant from colonisation by infiltrating bacteria. This study evaluates the antibacterial and antibiofilm efficacy of antibiotic-loaded dissolvable calcium sulphate, previously shown to be effective against key gram-positive pathogens, against gram-negative species important in the establishment of chronic infection in PIs. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin and both antibiotics in combination were assessed for their ability to eradicate planktonic Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae strains. The efficacy of the beads in preventing biofilm formation and eliminating established biofilms over multiple days was evaluated using confocal laser scanning microscopy (CSLM) imaging combined with image analysis and viable cell counts. Beads loaded with antibiotics demonstrated effective eluting concentrations for up to 37 d depending on the bacterial strain. In the presence of repeated bacterial challenges, antibiotic-loaded beads prevented bacterial colonisation and significantly reduce biofilm formation for the duration of the assay (7 d). Complete eradication of established biofilms was more difficult with evidence of biofilm regrowth after 1 week of contact with antibiotic-loaded beads, despite data suggesting a complete kill was achieved at earlier timepoints of 24 h and 72 h in the case of K. pneumoniae and P. aeruginosa. This study provides further evidence that calcium sulfate beads loaded with vancomycin and tobramycin may be a useful adjunctive component to the successful management of PIs.

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Streptococcus pneumoniae is a major cause of meningitis, sepsis, and pneumonia worldwide. Pneumococcal conjugate vaccines have been part of the United Kingdom's childhood immunization program since 2006 and have significantly reduced the incidence of disease due to vaccine efficacy in reducing carriage in the population. Here we isolated two clones of 22F (an emerging serotype of clinical concern, multilocus sequence types 433 and 698) and conducted comparative genomic analysis on four isolates, paired by Sequence Type (ST) with one of each pair being derived from carriage and the other disease (sepsis). The most compelling observation was of nonsynonymous mutations in pgdA, encoding peptidoglycan N-acetylglucosamine deacetylase A, which was found in the carriage isolates of both ST433 and 698. Deacetylation of pneumococcal peptidoglycan is known to enable resistance to lysozyme upon invasion. Althought no other clear genotypic signatures related to disease or carriage could be determined, additional intriguing comparisons between the two STs were possible. These include the presence of an intact prophage, in addition to numerous additional phage insertions, within the carriage isolate of ST433. Contrasting gene repertoires related to virulence and colonization, including bacteriocins, lantibiotics, and toxin--antitoxin systems, were also observed., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Streptococcus pneumoniaeis 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 ofS. pneumoniaein 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 inS. pneumoniaebiofilm dispersal. Instead, treatment of bothin vitrobiofilms andex vivoadenoid tissue samples (a reservoir forS. pneumoniaebiofilms) 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (© International & American Associations for Dental Research 2015.)

The ergot alkaloid biosynthesis system has become an excellent model to study evolutionary diversification of specialized (secondary) metabolites. This is a very diverse class of alkaloids with various neurotropic activities, produced by fungi in several orders of the phylum Ascomycota, including plant pathogens and protective plant symbionts in the family Clavicipitaceae. Results of comparative genomics and phylogenomic analyses reveal multiple examples of three evolutionary processes that have generated ergot-alkaloid diversity: gene gains, gene losses, and gene sequence changes that have led to altered substrates or product specificities of the enzymes that they encode (neofunctionalization). The chromosome ends appear to be particularly effective engines for gene gains, losses and rearrangements, but not necessarily for neofunctionalization. Changes in gene expression could lead to accumulation of various pathway intermediates and affect levels of different ergot alkaloids. Genetic alterations associated with interspecific hybrids of Epichloë species suggest that such variation is also selectively favored. The huge structural diversity of ergot alkaloids probably represents adaptations to a wide variety of ecological situations by affecting the biological spectra and mechanisms of defense against herbivores, as evidenced by the diverse pharmacological effects of ergot alkaloids used in medicine.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC-PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections., (Copyright © 2014. Published by Elsevier B.V.)

Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10(-3) substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern short-term, within-population bacterial diversification.

Clonally derived bacterial populations exhibit significant genotypic and phenotypic diversity that contribute to fitness in rapidly changing environments. Here, we show that serial passage of Salmonella enterica serovar Typhimurium LT2 (StLT2) in broth, or within a mouse host, results in selection of an evolved population that inhibits the growth of ancestral cells by direct contact. Cells within each evolved population gain the ability to express and deploy a cryptic "orphan" toxin encoded within the rearrangement hotspot (rhs) locus. The Rhs orphan toxin is encoded by a gene fragment located downstream of the "main" rhs gene in the ancestral strain StLT2. The Rhs orphan coding sequence is linked to an immunity gene, which encodes an immunity protein that specifically blocks Rhs orphan toxin activity. Expression of the Rhs orphan immunity protein protects ancestral cells from the evolved lineages, indicating that orphan toxin activity is responsible for the observed growth inhibition. Because the Rhs orphan toxin is encoded by a fragmented reading frame, it lacks translation initiation and protein export signals. We provide evidence that evolved cells undergo recombination between the main rhs gene and the rhs orphan toxin gene fragment, yielding a fusion that enables expression and delivery of the orphan toxin. In this manner, rhs locus rearrangement provides a selective advantage to a subpopulation of cells. These observations suggest that rhs genes play important roles in intra-species competition and bacterial evolution.