Dissecting cell heterogeneities in bacterial biofilms and their implications for antibiotic tolerance.

Bacterial biofilms consist of large, self-formed aggregates where resident bacteria can exhibit very different physiological states and phenotypes. This heterogeneity of cell types is crucial for many structural and functional emergent properties of biofilms. Consequently, it becomes essential to understand what drives cells to differentiate and how they achieve it within the three-dimensional landscape of the biofilms. Here, we discuss recent advances in comprehending two forms of cell heterogeneity that, while recognized to coexist within biofilms, have proven challenging to distinguish. These two forms include cell heterogeneity arising as a consequence of bacteria physiologically responding to resource gradients formed across the biofilms and cell-to-cell phenotypic heterogeneity, which emerges locally within biofilm subzones among neighboring bacteria due to stochastic variations in gene expression. We describe the defining features and concepts related to both forms of cell heterogeneity and discuss their implications, with a particular focus on antibiotic tolerance.

Selective inhibition of the amyloid matrix of Escherichia coli biofilms by a bifunctional microbial metabolite.

The propensity of bacteria to grow collectively in communities known as biofilms and their ability to overcome clinical treatments in this condition has become a major medical problem, emphasizing the need for anti-biofilm strategies. Antagonistic microbial interactions have extensively served as searching platforms for antibiotics, but their potential as sources for anti-biofilm compounds has barely been exploited. By screening for microorganisms that in agar-set pairwise interactions could antagonize Escherichia coli's ability to form macrocolony biofilms, we found that the soil bacterium Bacillus subtilis strongly inhibits the synthesis of amyloid fibers -known as curli-, which are the primary extracellular matrix (ECM) components of E. coli biofilms. We identified bacillaene, a B. subtilis hybrid non-ribosomal peptide/polyketide metabolite, previously described as a bacteriostatic antibiotic, as the effector molecule. We found that bacillaene combines both antibiotic and anti-curli functions in a concentration-dependent order that potentiates the ecological competitiveness of B. subtilis, highlighting bacillaene as a metabolite naturally optimized for microbial inhibition. Our studies revealed that bacillaene inhibits curli by directly impeding the assembly of the CsgB and CsgA curli subunits into amyloid fibers. Moreover, we found that curli inhibition occurs despite E. coli attempts to reinforce its protective ECM by inducing curli genes via a RpoS-mediated competition sensing response trigged by the threatening presence of B. subtilis. Overall, our findings illustrate the relevance of exploring microbial interactions not only for finding compounds with unknown and unique activities, but for uncovering additional functions of compounds previously categorized as antibiotics.

Care of patients on home parenteral nutrition during the first year of the COVID-19 pandemic: Management of central line-associated bloodstream infections.

BACKGROUND AND AIM: The aim of this study was to analyze central line-associated bloodstream infections (CLABSI) in home parenteral nutrition (HPN) patients assisted by an interdisciplinary team during the first year of the COVID-19 pandemic in Argentina. METHODS: Longitudinal, retrospective and analytical study of patients on HPN for ≥90 days during 2020. Data collection included age (adults >18 years, pediatric ≤18 years), gender, diagnosis, type of catheter, number of lumens, venous access, days on HPN, infusion modality and number of CLABSI-associated events. In COVID-19 cases, number of patients, disease progression, mortality rate and microorganisms involved were analyzed. RESULTS: A total of 380 patients were included, 120 (31.6%) pediatric and 260 (68.4%) adult patients. Median age was 44.50 years (10; 62.25). Twelve patients (3.15% of the total) had COVID-19; of these, two pediatric and seven adult patients had no complications, and three adults died of COVID-19 pneumonia. The diagnoses observed were benign chronic intestinal failure (CIF, n = 311), grouped into short bowel (n = 214, 56.3%), intestinal dysmotility (n = 56, 14.7%), intestinal fistula (n = 20, 5.3%), and extensive small bowel mucosal disease (n = 21, 5.5%); malignant tumors (n = 52, 13.7%); other (n = 17, 4.4%). Total catheter days were 103,702. Median days of PN duration per patient were 366 (176.2, 366). The types of catheters used were tunneled (317 patients, 83.4%); peripherally inserted central (PICC) line (55 patients, 14.5%) and ports (8 patients; 2.1%). A total of 111 CLABSI was registered, with a prevalence of 1.09/1000 catheter days (adult, 0.86/1000 days; pediatric, 1.51/1000 days). The microorganisms identified in infectious events were Gram + bacteria (38, 34.5%); Gram-bacteria (36, 32%); mycotic (10, 9%); polymicrobial (4, 3.6%); negative culture and signs/symptoms of CLABSI (23, 20.3%). The odds ratio between pediatric and adult patients was 2.29 (1.35, 3.90). CONCLUSION: The rate of CLABSI during the COVID-19 pandemic was within the ranges reported by international scientific societies. The risk of CLABSI was higher in pediatric patients, and mortality rate in COVID-19 infected patients was higher than in the general population.

Cyclic di-GMP Signaling Links Biofilm Formation and Mn(II) Oxidation in Pseudomonas resinovorans.

Bioaugmentation of biological sand filters with Mn(II)-oxidizing bacteria (MOB) is used to increase the efficiency of Mn removal from groundwater. While the biofilm-forming ability of MOB is important to achieve optimal Mn filtration, the regulatory link between biofilm formation and Mn(II) oxidation remains unclear. Here, an environmental isolate of Pseudomonas resinovorans strain MOB-513 was used as a model to investigate the role of c-di-GMP, a second messenger crucially involved in the regulation of biofilm formation by Pseudomonas, in the oxidation of Mn(II). A novel role for c-di-GMP in the upregulation of Mn(II) oxidation through induction of the expression of manganese-oxidizing peroxidase enzymes was revealed. MOB-513 macrocolony biofilms showed a strikingly stratified pattern of biogenic Mn oxide (BMnOx) accumulation in a localized top layer. Remarkably, elevated cellular levels of c-di-GMP correlated not only with increased accumulation of BMnOx in the same top layer but also with the appearance of a second BMnOx stratum in the bottom region of macrocolony biofilms, and the expression of mop genes correlated with this pattern. Proteomic analysis under Mn(II) conditions revealed changes in the abundance of a PilZ domain protein. Subsequent analyses supported a model in which this protein sensed c-di-GMP and affected a regulatory cascade that ultimately inhibited mop gene expression, providing a molecular link between c-di-GMP signaling and Mn(II) oxidation. Finally, we observed that high c-di-GMP levels were correlated with higher lyophilization efficiencies and higher groundwater Mn(II) oxidation capacities of freeze-dried bacterial cells, named lyophiles, showing the biotechnological relevance of understanding the role of c-di-GMP in MOB-513. IMPORTANCE The presence of Mn(II) in groundwater, a common source of drinking water, is a cause of water quality impairment, interfering with its disinfection, causing operation problems, and affecting human health. Purification of groundwater containing Mn(II) plays an important role in environmental and social safety. The typical method for Mn(II) removal is based on bacterial oxidation of metals to form insoluble oxides that can be filtered out of the water. Evidence of reducing the start-up periods and enhancing Mn removal efficiencies through bioaugmentation with appropriate biofilm-forming and MOB has emerged. As preliminary data suggest a link between these two phenotypes in Pseudomonas strains, the need to investigate the underlying regulatory mechanisms is apparent. The significance of our research lies in determining the role of c-di-GMP for increased biofilm formation and Mn(II)-oxidizing capabilities in MOB, which will allow the generation of super-biofilm-elaborating and Mn-oxidizing strains, enabling their implementation in biotechnological applications.

Adaptation of Escherichia coli Biofilm Growth, Morphology, and Mechanical Properties to Substrate Water Content.

Biofilms are complex living materials that form as bacteria become embedded in a matrix of self-produced protein and polysaccharide fibers. In addition to their traditional association with chronic infections or clogging of pipelines, biofilms currently gain interest as a potential source of functional material. On nutritive hydrogels, micron-sized Escherichia coli cells can build centimeter-large biofilms. During this process, bacterial proliferation, matrix production, and water uptake introduce mechanical stresses in the biofilm that are released through the formation of macroscopic delaminated buckles in the third dimension. To clarify how substrate water content could be used to tune biofilm material properties, we quantified E. coli biofilm growth, delamination dynamics, and rigidity as a function of water content of the nutritive substrates. Time-lapse microscopy and computational image analysis revealed that softer substrates with high water content promote biofilm spreading kinetics, while stiffer substrates with low water content promote biofilm delamination. The delaminated buckles observed on biofilm cross sections appeared more bent on substrates with high water content, while they tended to be more vertical on substrates with low water content. Both wet and dry biomass, accumulated over 4 days of culture, were larger in biofilms cultured on substrates with high water content, despite extra porosity within the matrix layer. Finally, microindentation analysis revealed that substrates with low water content supported the formation of stiffer biofilms. This study shows that E. coli biofilms respond to substrate water content, which might be used for tuning their material properties in view of further applications.

Bacterial Multicellularity: The Biology of Escherichia coli Building Large-Scale Biofilm Communities.

Biofilms are a widespread multicellular form of bacterial life. The spatial structure and emergent properties of these communities depend on a polymeric extracellular matrix architecture that is orders of magnitude larger than the cells that build it. Using as a model the wrinkly macrocolony biofilms of Escherichia coli, which contain amyloid curli fibers and phosphoethanolamine (pEtN)-modified cellulose as matrix components, we summarize here the structure, building, and function of this large-scale matrix architecture. Based on different sigma and other transcription factors as well as second messengers, the underlying regulatory network reflects the fundamental trade-off between growth and survival. It controls matrix production spatially in response to long-range chemical gradients, but it also generates distinct patterns of short-range matrix heterogeneity that are crucial for tissue-like elasticity and macroscopic morphogenesis. Overall, these biofilms confer protection and a potential for homeostasis, thereby reducing maintenance energy, which makes multicellularity an emergent property of life itself.

Staphylococcus aureus Potentiates the Hemolytic Activity of Burkholderia cepacia Complex (Bcc) Bacteria.

Polymicrobial lung infections in individuals with Cystic Fibrosis (CF) contribute to the complexity of this disease and are a major cause of morbidity and mortality in the CF community. The microorganisms most commonly associated with severe airway infections in individuals with CF are the opportunistic pathogens S. aureus, P. aeruginosa and bacteria from the Burkholderia cepacia complex (Bcc), particularly B. cenocepacia and B. multivorans. Three Bcc strains, two S. aureus wild-type strains, and two derivative mutants were used to investigate the interplay between S. aureus and Bcc with a focus on the hemolytic activity of Bcc. Our results revealed that extracellular products from S. aureus potentiated the hemolysis of Bcc strains. Moreover, this effect was influenced by the composition of the medium in which S. aureus is grown. These findings contribute towards the understanding of the impact of interactions between S. aureus and Bcc and their possible implications in the context of co-infections by these pathogens in individuals with CF.

A c-di-GMP-Based Switch Controls Local Heterogeneity of Extracellular Matrix Synthesis which Is Crucial for Integrity and Morphogenesis of Escherichia coli Macrocolony Biofilms.

The extracellular matrix in macrocolony biofilms of Escherichia coli is arranged in a complex large-scale architecture, with homogenic matrix production close to the surface, whereas zones further below display pronounced local heterogeneity of matrix production, which results in distinct three-dimensional architectural structures. Combining genetics, cryosectioning and fluorescence microscopy of macrocolony biofilms, we demonstrate here in situ that this local matrix heterogeneity is generated by a c-di-GMP-dependent molecular switch characterized by several nested positive and negative feedback loops. In this switch, the trigger phosphodiesterase PdeR is the key component for establishing local heterogeneity in the activation of the transcription factor MlrA, which in turn activates expression of the major matrix regulator CsgD. Upon its release of direct inhibition by PdeR, the second switch component, the diguanylate cyclase DgcM, activates MlrA by direct interaction. Antagonistically acting PdeH and DgcE provide for a PdeR-sensed c-di-GMP input into this switch and-via their spatially differentially controlled expression-generate the long-range vertical asymmetry of the matrix architecture. Using flow cytometry, we show heterogeneity of CsgD expression to also occur in spatially unstructured planktonic cultures, where it is controlled by the same c-di-GMP circuitry as in macrocolony biofilms. Quantification by flow cytometry also showed CsgDON subpopulations with distinct CsgD expression levels and revealed an additional fine-tuning feedback within the PdeR/DgcM-mediated switch that depends on c-di-GMP synthesis by DgcM. Finally, local heterogeneity of matrix production was found to be crucial for the tissue-like elasticity that allows for large-scale wrinkling and folding of macrocolony biofilms.

Una concepción integradora de la motivación humana / Uma concepção integradora da motivação humana / An integrating conception of human motivation

RESUMEN Las teorías de la motivación desarrolladas en el siglo XX son opuestas. Se requiere una que, sobre la base de los hechos, integre armónicamente los aportes logrados. Destacamos el aprendizaje de las necesidades, basado en determinantes orgánicos y adquiridos, mediante el reforzamiento afectivo de los objetos del mundo exterior y de las palabras en que se canalizan. Los requerimientos orgánicos se concretan en los objetos y en las palabras. Las necesidades superiores surgen por la asimilación del lenguaje y se constituyen en determinantes independientes de los requerimientos orgánicos y entre ellos y con el desarrollo psíquico, los deberes asumidos predominan por lo general en los conflictos. En su conducta, inicialmente reactiva y adaptativa, el sujeto va elaborando proyectos creativos dirigidos al futuro que conducen a su auto realización. Así existe la unidad y determinación recíprocas de la adaptación y la auto realización, como explicación de la conducta humana. En las contradicciones de la vida se imponen dos dinamismos: la motivación específica, dirigida a obtener el objeto meta de la necesidad, y la motivación inespecífica ante la frustración y el conflicto, dirigida a reducir por otras vías la tensión insatisfactoria. Ambos dinamismos se encuentran en unidad, pero la específica es típica de la motivación normal en la cual predominan, en forma relativamente armónica e integradora, los proyectos y fines asumidos, mientras que la inespecífica es predominante en la motivación anormal, en la cual los determinantes inconscientes y externos asumen un rol principal. Solo la verdad más plena será lo más útil para el ser humano.

RESUMO As teorias da motivação desenvolvidas no século XX são opostas. Requer-se uma que, sobre a base dos fatos, integre harmonicamente os resultados alcançados. Destacamos a aprendizagem das necessidades, baseado em determinantes orgânicos e adquiridos, mediante o reforço afetivo dos objetos do mundo exterior e das palavras em que se canalizam. Os requerimentos orgânicos se concretizam nos objetos e nas palavras. As necessidades superiores surgem pela assimilação da linguagem e se constituem em determinantes independentes dos requerimentos orgânicos e entre eles e com o desenvolvimento psíquico, os deveres assumidos predominam pelo geral nos conflitos. Em sua conduta, inicialmente reativa e adaptativa, o sujeito vai elaborando projetos criativos dirigidos ao futuro que conduzem à auto realização. Assim, existe a unidade e determinação recíprocas da adaptação e da auto realização, como explicação da conduta humana. Nas contradições da vida se impõem dois dinamismos: a motivação específica, dirigida a obter o objeto meta da necessidade, e a motivação inespecífica ante a frustração e o conflito, dirigida a reduzir por outras vias a tensão insatisfatória. Ambos dinamismos se encontram em unidade, mas a específica é típica da motivação normal na qual predominam, em forma relativamente harmônica e integradora, os projetos e fins assumidos, enquanto que a inespecífica é predominante na motivação anormal, na qual os determinantes inconscientes e externos assumem um rol principal. Só a verdade mais plena será o mais útil para o ser humano.

ABSTRACT The theories of motivation developed in the twentieth century are opposite. A theory that harmoniously integrates the contributions based on the facts is needed. The learning of needs is highlighted, based on organic and acquired determinants, through the affective reinforcement of the objects of the external world and the words in which they are channeled. The organic requirements are specified in the objects and words. The higher needs come from the assimilation of the language and they are independent determinants of the organic requirements and between them and the assumed duties usually predominate in the conflicts with the psychic development. The subject in his initially reactive and adaptive behavior develops creative projects aimed at the future that lead to self-realization. Thus, there is the reciprocal unity and determination of adaptation and self-realization, as an explanation of human behavior. In the contradictions of life, two dynamisms are imposed: the specific motivation, aimed at obtaining the goal of necessity, and the non-specific motivation for the frustration and conflict, aimed at reducing the unsatisfactory tension in other ways. Both dynamics are in unity, but the specific one is typical of the normal motivation in which the projects and goals predominated, in a relatively harmonic and integrating way, while the nonspecific one is predominant in the abnormal motivation, in which the determinants and external unconscious assume a leading role. Only the fullest truth will be the most useful for the human being.

Corrigendum: The Intestinal Roundworm Ascaris suum Releases Antimicrobial Factors Which Interfere With Bacterial Growth and Biofilm Formation.

[This corrects the article DOI: 10.3389/fcimb.2018.00271.].

Spatial organization of different sigma factor activities and c-di-GMP signalling within the three-dimensional landscape of a bacterial biofilm.

Bacterial biofilms are large aggregates of cells embedded in an extracellular matrix of self-produced polymers. In macrocolony biofilms of Escherichia coli, this matrix is generated in the upper biofilm layer only and shows a surprisingly complex supracellular architecture. Stratified matrix production follows the vertical nutrient gradient and requires the stationary phase σS (RpoS) subunit of RNA polymerase and the second messenger c-di-GMP. By visualizing global gene expression patterns with a newly designed fingerprint set of Gfp reporter fusions, our study reveals the spatial order of differential sigma factor activities, stringent control of ribosomal gene expression and c-di-GMP signalling in vertically cryosectioned macrocolony biofilms. Long-range physiological stratification shows a duplication of the growth-to-stationary phase pattern that integrates nutrient and oxygen gradients. In addition, distinct short-range heterogeneity occurs within specific biofilm strata and correlates with visually different zones of the refined matrix architecture. These results introduce a new conceptual framework for the control of biofilm formation and demonstrate that the intriguing extracellular matrix architecture, which determines the emergent physiological and biomechanical properties of biofilms, results from the spatial interplay of global gene regulation and microenvironmental conditions. Overall, mature bacterial macrocolony biofilms thus resemble the highly organized tissues of multicellular organisms.

The Intestinal Roundworm Ascaris suum Releases Antimicrobial Factors Which Interfere With Bacterial Growth and Biofilm Formation.

Ascariasis is a widespread soil-transmitted helminth infection caused by the intestinal roundworm Ascaris lumbricoides in humans, and the closely related Ascaris suum in pigs. Progress has been made in understanding interactions between helminths and host immune cells, but less is known concerning the interactions of parasitic nematodes and the host microbiota. As the host microbiota represents the direct environment for intestinal helminths and thus a considerable challenge, we studied nematode products, including excretory-secretory products (ESP) and body fluid (BF), of A. suum to determine their antimicrobial activities. Antimicrobial activities against gram-positive and gram-negative bacterial strains were assessed by the radial diffusion assay, while effects on biofilm formation were assessed using the crystal violet static biofilm and macrocolony assays. In addition, bacterial neutralizing activity was studied by an agglutination assay. ESP from different A. suum life stages (in vitro-hatched L3, lung-stage L3, L4, and adult) as well as BF from adult males were analyzed by mass spectrometry. Several proteins and peptides with known and predicted roles in nematode immune defense were detected in ESP and BF samples, including members of A. suum antibacterial factors (ASABF) and cecropin antimicrobial peptide families, glycosyl hydrolase enzymes such as lysozyme, as well as c-type lectin domain-containing proteins. Native, unconcentrated nematode products from intestine-dwelling L4-stage larvae and adults displayed broad-spectrum antibacterial activity. Additionally, adult A. suum ESP interfered with biofilm formation by Escherichia coli, and caused bacterial agglutination. These results indicate that A. suum uses a variety of factors with broad-spectrum antibacterial activity to affirm itself within its microbe-rich environment in the gut.

Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose.

Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.

Experimental Detection and Visualization of the Extracellular Matrix in Macrocolony Biofilms.

By adopting elaborate three-dimensional morphologies that vary according to their extracellular matrix composition, macrocolony biofilms offer a unique opportunity to interrogate about the roles of specific matrix components in shaping biofilm architecture. Here, we describe two methods optimized for Escherichia coli that profit from morphology and the high level of structural organization of macrocolonies to gain insight into the production and assembly of amyloid curli and cellulose-the two major biofilm matrix elements of E. coli-in biofilms. The first method, the macrocolony morphology assay, is based on the ability of curli and cellulose-either alone or in combination-to generate specific morphological and Congo Red-staining patterns in E. coli macrocolonies, which can then be used as a direct visual readout for the production of these matrix components. The second method involves thin sectioning of macrocolonies, which along with in situ staining of amyloid curli and cellulose and microscopic imaging allows gaining fine details of the spatial arrangement of both matrix elements inside macrocolonies. Beyond their current use with E. coli and related curli and cellulose-producing Enterobacteriaceae, both the methods offer the potential to be adapted to other bacterial species.

The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σ(E) -dependent sRNA RybB.

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self-produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ(E) cell envelope stress response and thereby reducing the expression of CsgD - a crucial activator of curli and cellulose biosynthesis - due to csgD mRNA targeting by the σ(E) -dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm-associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.

Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition.

Bordetella pertussis Isolates from Argentinean Whooping Cough Patients Display Enhanced Biofilm Formation Capacity Compared to Tohama I Reference Strain.

Pertussis is a highly contagious disease mainly caused by Bordetella pertussis. Despite the massive use of vaccines, since the 1950s the disease has become re-emergent in 2000 with a shift in incidence from infants to adolescents and adults. Clearly, the efficacy of current cellular or acellular vaccines, formulated from bacteria grown in stirred bioreactors is limited, presenting a challenge for future vaccine development. For gaining insights into the role of B. pertussis biofilm development for host colonization and persistence within the host, we examined the biofilm forming capacity of eight argentinean clinical isolates recovered from 2001 to 2007. All clinical isolates showed an enhanced potential for biofilm formation compared to the reference strain Tohama I. We further selected the clinical isolate B. pertussis 2723, exhibiting the highest biofilm biomass production, for quantitative proteomic profiling by means of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, which was accompanied by targeted transcriptional analysis. Results revealed an elevated expression of several virulence factors, including adhesins involved in biofilm development. In addition, we observed a higher expression of energy metabolism enzymes in the clinical isolate compared to the Tohama I strain. Furthermore, all clinical isolates carried a polymorphism in the bvgS gene. This mutation was associated to an increased sensitivity to modulation and a faster rate of adhesion to abiotic surfaces. Thus, the phenotypic biofilm characteristics shown by the clinical isolates might represent an important, hitherto underestimated, adaptive strategy for host colonization and long time persistence within the host.

Vertical stratification of matrix production is essential for physical integrity and architecture of macrocolony biofilms of Escherichia coli.

Bacterial macrocolony biofilms grow into intricate three-dimensional structures that depend on self-produced extracellular polymers conferring protection, cohesion and elasticity to the biofilm. In Escherichia coli, synthesis of this matrix - consisting of amyloid curli fibres and cellulose - requires CsgD, a transcription factor regulated by the stationary phase sigma factor RpoS, and occurs in the nutrient-deprived cells of the upper layer of macrocolonies. Is this asymmetric matrix distribution functionally important or is it just a fortuitous by-product of an unavoidable nutrient gradient? In order to address this question, the RpoS-dependent csgD promoter was replaced by a vegetative promoter. This re-wiring of csgD led to CsgD and matrix production in both strata of macrocolonies, with the lower layer transforming into a rigid 'base plate' of growing yet curli-connected cells. As a result, the two strata broke apart followed by desiccation and exfoliation of the top layer. By contrast, matrix-free cells at the bottom of wild-type macrocolonies maintain colony contact with the humid agar support by flexibly filling the space that opens up under buckling areas of the macrocolony. Precisely regulated stratification in matrix-free and matrix-producing cell layers is thus essential for the physical integrity and architecture of E. coli macrocolony biofilms.

Stress responses go three dimensional - the spatial order of physiological differentiation in bacterial macrocolony biofilms.

In natural habitats, bacteria often occur in multicellular communities characterized by a robust extracellular matrix of proteins, amyloid fibres, exopolysaccharides and extracellular DNA. These biofilms show pronounced stress resistance including a resilience against antibiotics that causes serious medical and technical problems. This review summarizes recent studies that have revealed clear spatial physiological differentiation, complex supracellular architecture and striking morphology in macrocolony biofilms. By responding to gradients of nutrients, oxygen, waste products and signalling compounds that build up in growing biofilms, various stress responses determine whether bacteria grow and proliferate or whether they enter into stationary phase and use their remaining resources for maintenance and survival. As a consequence, biofilms differentiate into at least two distinct layers of vegetatively growing and stationary phase cells that exhibit very different cellular physiology. This includes a stratification of matrix production with a major impact on microscopic architecture, biophysical properties and directly visible morphology of macrocolony biofilms. Using Escherichia coli as a model system, this review also describes our detailed current knowledge about the underlying molecular control networks - prominently featuring sigma factors, transcriptional cascades and second messengers - that drive this spatial differentiation and points out directions for future research.

Ipsilateral jugular access to treat an otherwise inaccessible puncture-related arteriovenous fistula pseudoaneurysm: a technical note.

BACKGROUND: The standard approach for the endovascular treatment of a dysfunctional or occluded hemodialysis access in the upper limbs includes a direct intervention through the access itself or alternatively, when not feasible, through the brachial or radial artery access. Nonetheless, there are certain circumstances in which these approaches are not easily achieved. METHODS: An 89-year-old male with end-stage renal disease developed a pseudoaneurysm after an hemorrhagic complication of a recently transposed native basilic arteriovenous fistula secondary to a needle puncture. Dehiscence of the injured access with spontaneous arterial bleeding evolved as a consequence of the upper limb swelling, rendering therapeutic intervention of the access through a conventional route impossible. A fistulogram through puncture of the common femoral artery was performed to obtain an accurate diagnosis. However, this approach was insufficient to advance the covered stent with the intention of excluding the pseudoaneurysm, as the stent delivery system could not reach the desired site. Given that the covered stent insertion required a 9 Fr introducer, the radial artery approach was ruled out. Therefore, we chose a venous access via the ipsilateral internal jugular vein, which was punctured under ultrasound guidance. RESULTS: This strategy was useful to advance the stent and exclude the pseudoaneurysm successfully. CONCLUSIONS: This technique should be considered for those individuals in whom conventional routes of approach for repairing dialysis accesses are not feasible or are extremely risky.