Aptamer-based therapy for fighting biofilm-associated infections.

Biofilms are key players in the pathogenesis of most of chronic infections associated with host tissue or fluids and indwelling medical devices. These chronic infections are hard to be treated due to the increased biofilms tolerance towards antibiotics in comparison to planktonic (or free living) cells. Despite the advanced understanding of their formation and physiology, biofilms continue to be a challenge and there is no standardized therapeutic approach in clinical practice to eradicate them. Aptamers offer distinctive properties, including excellent affinity, selectivity, stability, making them valuable tools for therapeutic purposes. This review explores the flexibility and designability of aptamers as antibiofilm drugs but, importantly, as targeting tools for diverse drug and delivery systems. It highlights specific examples of application of aptamers in biofilms of diverse species according to different modes of action including inhibition of motility and adhesion, blocking of quorum sensing molecules, and dispersal of biofilm-cells to planktonic state. Moreover, it discusses the limitations and challenges that impaired an increased success of the use of aptamers on biofilm management, as well as the opportunities related to aptamers modifications that can significantly expand their applicability on the biofilm field.

Evolving biofilm inhibition and eradication in clinical settings through plant-based antibiofilm agents.

BACKGROUND: After almost 100 years since evidence of biofilm mode of growth and decades of intensive investigation about their formation, regulatory pathways and mechanisms of antimicrobial tolerance, nowadays there are still no therapeutic solutions to eradicate bacterial biofilms and their biomedical related issues. PURPOSE: This review intends to provide a comprehensive summary of the recent and most relevant published studies on plant-based products, or their isolated compounds with antibiofilm activity mechanisms of action or identified molecular targets against bacterial biofilms. The objective is to offer a new perspective of most recent data for clinical researchers aiming to prevent or eliminate biofilm-associated infections caused by bacterial pathogens. METHODS: The search was performed considering original research articles published on PubMed, Web of Science and Scopus from 2015 to April 2023, using keywords such as "antibiofilm", "antivirulence", "phytochemicals" and "plant extracts". RESULTS: Over 180 articles were considered for this review with a focus on the priority human pathogens listed by World Health Organization, including Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. Inhibition and detachment or dismantling of biofilms formed by these pathogens were found using plant-based extract/products or derivative compounds. Although combination of plant-based products and antibiotics were recorded and discussed, this topic is currently poorly explored and only for a reduced number of bacterial species. CONCLUSIONS: This review clearly demonstrates that plant-based products or derivative compounds may be a promising therapeutic strategy to eliminate bacterial biofilms and their associated infections. After thoroughly reviewing the vast amount of research carried out over years, it was concluded that plant-based products are mostly able to prevent biofilm formation through inhibition of quorum sensing signals, but also to disrupt mature biofilms developed by multidrug resistant bacteria targeting the biofilm extracellular polymeric substance. Flavonoids and phenolic compounds seemed the most effective against bacterial biofilms.

Viable but non-cultivable state: a strategy for Staphylococcus aureus survivable in dual-species biofilms with Pseudomonas aeruginosa?

Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in cystic fibrosis patients. Both organisms often cause chronic and recalcitrant infections, in large part due to their ability to form biofilms, being these mixed-species infections correlated with poor clinical outcomes. In this study, the hypothesis that S. aureus adopts phenotypes allowing its coexistence with P. aeruginosa during biofilm growth was put forward. We noticed that S. aureus undergoes a viable but non-cultivable (VBNC) state in the dominated P. aeruginosa dual-species consortia, whatsoever the strains used to form the biofilms. Moreover, an increased expression of genes associated with S. aureus virulence was detected suggesting that the phenotypic switching to VBNC state might account for S. aureus pathogenicity and, in turn, influence the clinical outcome of the mixed-species infection. Thus, P. aeruginosa seems to induce both phenotypic and transcriptomic changes in S. aureus, helping its survival and coexistence in the dual-species biofilms. Overall, our findings illustrate how interspecies interactions can modulate bacterial virulence in vitro, contributing to a better understanding of the behaviour of P. aeruginosa-S. aureus dual-species biofilms.

Long-term coexistence of Pseudomonas aeruginosa and Staphylococcus aureus using an in vitro cystic fibrosis model.

Aim: To investigate the role of pre-established Staphylococcus aureus on Pseudomonas aeruginosa adaptation and antibiotic tolerance. Materials & methods: Bacteria were cultured mimicking the sequential pattern of lung colonization and exposure to ciprofloxacin. Results: In the absence of ciprofloxacin exposure, S. aureus and P. aeruginosa coexisted supported by the physicochemical characteristics of the artificial sputum medium. S. aureus had no role in P. aeruginosa tolerance against ciprofloxacin and did not select P. aeruginosa small-colony variants during antibiotic treatment. rhlR and psqE were downregulated after the contact with S. aureus indicating that P. aeruginosa attenuated its virulence potential. Conclusion:P. aeruginosa and S. aureus can cohabit in cystic fibrosis airway environment for long-term without significant impact on P. aeruginosa adaptation and antibiotic tolerance.

Discerning the role of polymicrobial biofilms in the ascent, prevalence, and extent of heteroresistance in clinical practice.

Antimicrobial therapy is facing a worrisome and underappreciated challenge, the phenomenon of heteroresistance (HR). HR has been gradually documented in clinically relevant pathogens (e.g. Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia spp., Acinetobacter baumannii, Klebsiella pneumoniae, Candida spp.) towards several drugs and is believed to complicate the clinical picture of chronic infections. This type of infections are typically mediated by polymicrobial biofilms, wherein microorganisms inherently display a wide range of physiological states, distinct metabolic pathways, diverging refractory levels of stress responses, and a complex network of chemical signals exchange. This review aims to provide an overview on the relevance, prevalence, and implications of HR in clinical settings. Firstly, related terminologies (e.g. resistance, tolerance, persistence), sometimes misunderstood and overlapped, were clarified. Factors generating misleading HR definitions were also uncovered. Secondly, the recent HR incidences reported in clinically relevant pathogens towards different antimicrobials were annotated. The potential mechanisms underlying such occurrences were further elucidated. Finally, the link between HR and biofilms was discussed. The focus was to recognize the presence of heterogeneous levels of resistance within most biofilms, as well as the relevance of polymicrobial biofilms in chronic infectious diseases and their role in resistance spreading. These topics were subject of a critical appraisal, gaining insights into the ascending clinical implications of HR in antimicrobial resistance spreading, which could ultimately help designing effective therapeutic options.

Further evidence for a late locus of holistic word processing: Exploring vertex effect in the word composite task.

Previous studies have shown a rather late and lexical level for holistic word processing. In the present study, we evaluated whether there are early effects in holistic processing of words, taking into consideration the role of lower-level visual processes that are critical in the hierarchy of visual word recognition: the extraction of viewpoint-invariant line junctions/vertices. We used contour-deleted words in two conditions: preservation of the vertices versus preservation of midsegments and an all-contour condition. We found evidence of a composite effect that was equivalent for all materials. Thus, we found no evidence of an early contribution of holistic processing to word recognition, and confirmed that holistic word processing is related to late lexical orthographic representations.

Fostering Innovation in the Treatment of Chronic Polymicrobial Cystic Fibrosis-Associated Infections Exploring Aspartic Acid and Succinic Acid as Ciprofloxacin Adjuvants.

Cystic fibrosis (CF) disease provokes the accumulation of thick and viscous sputum in the lungs, favoring the development of chronic and polymicrobial infections. Pseudomonas aeruginosa is the main bacterium responsible for these chronic infections, and much of the difficulty involved in eradicating it is due to biofilm formation. However, this could be mitigated using adjuvant compounds that help or potentiate the antibiotic action. Therefore, the main goal of this study was to search for substances that function as adjuvants and also as biofilm-controlling compounds, preventing or dismantling P. aeruginosa biofilms formed in an in vitro CF airway environment. Dual combinations of compounds with subinhibitory (1 and 2 mg/L) and inhibitory concentrations (4 mg/L) of ciprofloxacin were tested to inhibit the bacterial growth and biofilm formation (prophylactic approach) and to eradicate 24-h-old P. aeruginosa populations, including planktonic cells and biofilms (treatment approach). Our results revealed that aspartic acid (Asp) and succinic acid (Suc) restored ciprofloxacin action against P. aeruginosa. Suc combined with 2 mg/L of ciprofloxacin (Suc-Cip) was able to eradicate bacteria, and Asp combined with 4 mg/L of ciprofloxacin (Asp-Cip) seemed to eradicate the whole 24-h-old populations, including planktonic cells and biofilms. Based on biomass depletion data, we noted that Asp induced cell death and Suc seemed somehow to block or reduce the expression of ciprofloxacin resistance. As far as we know, this kind of action had not been reported up till now. The presence of Staphylococcus aureus and Burkholderia cenocepacia did not affect the efficacy of the Asp-Cip and Suc-Cip therapies against P. aeruginosa and, also important, P. aeruginosa depletion from polymicrobial communities did not create a window of opportunity for these species to thrive. Rather the contrary, Asp and Suc also improved ciprofloxacin action against B. cenocepacia. Further studies on the cytotoxicity using lung epithelial cells indicated toxicity of Suc-Cip caused by the Suc. In conclusion, we provided evidences that Asp and Suc could be potential ciprofloxacin adjuvants to eradicate P. aeruginosa living within polymicrobial communities. Asp-Cip and Suc-Cip could be promising therapeutic options to cope with CF treatment failures.

Antimicrobial resistance three ways: healthcare crisis, major concepts and the relevance of biofilms.

Worldwide, infections are resuming their role as highly effective killing diseases, as current treatments are failing to respond to the growing problem of antimicrobial resistance (AMR). The social and economic burden of AMR seems ever rising, with health- and research-related organizations rushing to collaborate on a worldwide scale to find effective solutions. Resistant bacteria are spreading even in first-world nations, being found not only in healthcare-related settings, but also in food and in the environment. In this minireview, the impact of AMR in healthcare systems and the major bacteria behind it are highlighted. Ecological aspects of AMR evolution and the complexity of its molecular mechanisms are explained. Major concepts, such as intrinsic, acquired and adaptive resistance, as well as tolerance and heteroresistance, are also clarified. More importantly, the problematic of biofilms and their role in AMR, namely their main resistance and tolerance mechanisms, are elucidated. Finally, some of the most promising anti-biofilm strategies being investigated are reviewed. Much is still to be done regarding the study of AMR and the discovery of new anti-biofilm strategies. Gladly, considerable research on this topic is generated every day and increasingly concerted actions are being engaged globally to try and tackle this problem.

Therapeutic reactance in adolescents: the psychometrics of the Therapeutic Reactance Scale in adolescents.

BACKGROUND: The Therapeutic Reactance Scale (TRS) is a classic measure of psychological reactance, yet only two studies have evaluated its factorial structure. Both proposed different multidimensional structures based on exploratory analyses. Not only is the factorial structure of the TRS unclear, but the scale has yet to be validated in adolescents. OBJECTIVE: This study aimed to test the factorial structure of the TRS in adolescents. METHODS: The authors conducted exploratory and confirmatory factor analyses, and analyses of reliability and validity, with a sample of 1,344 adolescents. RESULTS: A four-factor model fits well to the data. Three of the four TRS dimensions (not susceptibility to influence, SI) were correlated with the Hong Psychological Reactance Scale (HPRS). These three dimensions were also correlated with novelty seeking, cooperativeness and persistence components of personality (Cloninger's psychobiological model of personality), while SI showed a different pattern. CONCLUSIONS: Overall, this study demonstrates that the TRS is a suitable and potentially useful tool for measuring reactance in adolescents, but the authors propose that practitioners may wish to consider excluding items pertaining to the SI dimension.

Unveiling the early events of Pseudomonas aeruginosa adaptation in cystic fibrosis airway environment using a long-term in vitro maintenance.

Pseudomonas aeruginosa chronic infections are the major cause of high morbidity and mortality in cystic fibrosis (CF) patients due to the use of sophisticated mechanisms of adaptation, including clonal diversification into specialized CF-adapted phenotypes. In contrast to chronic infections, very little is known about what occurs after CF lungs colonization and at early infection stages. This study aims to investigate the early events of P. aeruginosa adaptation to CF environment, in particular, to inspect the occurrence of clonal diversification at early stages of infection development and its impact on antibiotherapy effectiveness. To mimic CF early infections, three P. aeruginosa strains were long-term grown in artificial sputum (ASM) over 10 days and phenotypic diversity verified through colony morphology characterization. Biofilm sub- and inhibitory concentrations of ciprofloxacin were applied to non- and diversified populations to evaluate antibiotic effectiveness on P. aeruginosa eradication. Our results demonstrated that clonal diversification might occur after ASM colonization and growth. However, this phenotypic diversification did not compromise ciprofloxacin efficacy in P. aeruginosa eradication since a biofilm minimal inhibitory dosage would be applied. The expected absence of mutators in P. aeruginosa populations led us to speculate that clonal diversification in the absence of ciprofloxacin treatments could be driven by niche specialization. Yet, biofilm sub-inhibitory concentrations of ciprofloxacin seemed to overlap niche specialization as "fitter" variants emerged, such as mucoid, small colony and pinpoint variants, known to be highly resistant to antibiotics. The pathogenic potential of all emergent colony morphotypes-associated bacteria, distinct from the wild-morphotypes, revealed that P. aeruginosa evolved to a non-swimming phenotype. Impaired swimming motility seemed to be one of the first evolutionary steps of P. aeruginosa in CF lungs that could pave the way for further adaptation steps including biofilm formation and progress to chronic infection. Based on our findings, impaired swimming motility seemed to be a candidate to disease marker of P. aeruginosa infection development. Despite our in vitro CF model represents a step forward towards in vivo scenario simulation and provided valuable insights about the early events, more and distinct P. aeruginosa strains should be studied to strengthen our results.

Electrospinning Pullulan Fibers from Salt Solutions.

There is an increasing interest in applying the technology of electrospinning for making ultrafine fibers from biopolymers for food-grade applications, and using pullulan (PUL) as a carrier to improve the electrospinnability of proteins and other naturally occurring polyelectrolytes. In this study, PUL solutions containing NaCl or Na3C6H5O7 at different concentrations were electrospun. The inclusion of salts interrupted the hydrogen bonding and altered solution properties, such as viscosity, electric conductivity, and surface tension, as well as physical properties of fibers thus obtained, such as appearance, size, and melting point. The exogenous Na⁺ associated to the oxygen in the C6 position of PUL as suggested by FTIR measurement and was maintained during electrospinning. Bead-free PUL fibers could be electrospun from PUL solution (8%, w/v) in the presence of a 0.20 M NaCl (124 ± 34 nm) or 0.05 M Na3C6H5O7 (154 ± 36 nm). The further increase of NaCl or Na3C6H5O7 resulted in fibers that were flat with larger diameter sizes and defects. SEM also showed excess salt adhering on the surfaces of PUL fibers. Since most food processing is not carried out in pure water, information obtained through the present research is useful for the development of electrospinning biopolymers for food-grade applications.

Role of bolA and rpoS genes in biofilm formation and adherence pattern by Escherichia coli K-12 MG1655 on polypropylene, stainless steel, and silicone surfaces.

Escherichia coli has developed sophisticated means to sense, respond, and adapt in stressed environment. It has served as a model organism for studies in molecular genetics and physiology since the 1960s. Stress response genes are induced whenever a cell needs to adapt and survive under unfavorable growth conditions. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to various stress factors as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stressed environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. This study has been undertaken to address the adherence pattern and formation of biofilms by E. coli on stainless steel, polypropylene, and silicone surfaces after 24 h of growth at 37 °C. Scanning electron microscopy was used for direct examination of the cell attachment and biofilm formation on various surfaces and it was found that, in the presence of bolA, E. coli cells were able to attach to the stainless steel and silicone very well. By contrast, polypropylene surface was not found to be attractive for E. coli cells. This indicates that bolA responded and can play a major role in the presence and absence of rpoS in cell attachment.

Heteroresistance to colistin in Klebsiella pneumoniae is triggered by small colony variants sub-populations within biofilms.

The emergence of Klebsiella pneumoniae multidrug-resistant strains paves the way to the re-introduction of colistin as a salvage therapy. However, recent planktonic studies have reported several cases of heteroresistance to this antimicrobial agent. The aim of this present work was to gain better understanding about the response of K. pneumoniae biofilms to colistin antibiotherapy and inspect the occurrence of heteroresistance in biofilm-derived cells. Biofilm formation and its susceptibility to colistin were evaluated through the determination of biofilm-cells viability. The profiling of planktonic and biofilm cell populations was conducted to assess the occurrence of heteroresistance. Colony morphology was further characterized in order to inspect the potential role of colistin in K. pneumoniae phenotypic differentiation. Results show that K. pneumoniae was susceptible to colistin in its planktonic form, but biofilms presented enhanced resistance. Population analysis profiles pointed out that K. pneumoniae manifest heteroresistance to colistin only when grown in biofilm arrangements, and it was possible to identify a resistant sub-population presenting a small colony morphology (diameter around 5 mm). To the best of our knowledge, this is the first report linking heteroresistance to biofilm formation and a morphological distinctive sub-population. Moreover, this is the first evidence that biofilm formation can trigger the emergence of heteroresistance in an apparently susceptible strain.

MorphoCol: An ontology-based knowledgebase for the characterisation of clinically significant bacterial colony morphologies.

BACKGROUND: One of the major concerns of the biomedical community is the increasing prevalence of antimicrobial resistant microorganisms. Recent findings show that the diversification of colony morphology may be indicative of the expression of virulence factors and increased resistance to antibiotic therapeutics. To transform these findings, and upcoming results, into a valuable clinical decision making tool, colony morphology characterisation should be standardised. Notably, it is important to establish the minimum experimental information necessary to contextualise the environment that originated the colony morphology, and describe the main morphological features associated unambiguously. RESULTS: This paper presents MorphoCol, a new ontology-based tool for the standardised, consistent and machine-interpretable description of the morphology of colonies formed by human pathogenic bacteria. The Colony Morphology Ontology (CMO) is the first controlled vocabulary addressing the specificities of the morphology of clinically significant bacteria, whereas the MorphoCol publicly Web-accessible knowledgebase is an end-user means to search and compare CMO annotated colony morphotypes. Its ultimate aim is to help correlate the morphological alterations manifested by colony-forming bacteria during infection with their response to the antimicrobial treatments administered. CONCLUSIONS: MorphoCol is the first tool to address bacterial colony morphotyping systematically and deliver a free of charge resource to the community. Hopefully, it may introduce interesting features of analysis on pathogenic behaviour and play a significant role in clinical decision making. DATABASE URL: http://morphocol.org.

Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review.

Pseudomonas aeruginosa is the most prevalent pathogen of cystic fibrosis (CF) lung disease. Its long persistence in CF airways is associated with sophisticated mechanisms of adaptation, including biofilm formation, resistance to antibiotics, hypermutability and customized pathogenicity in which virulence factors are expressed according the infection stage. CF adaptation is triggered by high selective pressure of inflamed CF lungs and by antibiotic treatments. Bacteria undergo genetic, phenotypic, and physiological variations that are fastened by the repeating interplay of mutation and selection. During CF infection development, P. aeruginosa gradually shifts from an acute virulent pathogen of early infection to a host-adapted pathogen of chronic infection. This paper reviews the most common changes undergone by P. aeruginosa at each stage of infection development in CF lungs. The comprehensive understanding of the adaptation process of P. aeruginosa may help to design more effective antimicrobial treatments and to identify new targets for future drugs to prevent the progression of infection to chronic stages.

An harmonised vocabulary for communicating and interchanging biofilms experimental results.

Biofilm studies are at the crossroads of Biology, Chemistry, Medicine, Material Science and Engineering, among other fields. Data harmonisation in Biofilms is therefore crucial to allow for researchers to collaborate, interchange, understand, and replicate studies at an inter-laboratory and inter-domain scale. The international Minimum Information About a Biofilms Experiment initiative has prepared a set of guidelines for documenting biofilms experiments and data, namely the minimum information checklist. This paper goes a step forward and describes a new ontology for the broad description of biofilm experiments and data. In such an interdisciplinary context we chose to rely on a common integration framework provided by a foundational ontology that facilitates the addition and extension of various sub-domain modules, and the consistent integration of terminology extracted from several existing ontologies, e.g. EXPO and ChEBI. The community is participating actively in the production of this resource, and it is already used by public biofilms-centred databases, such as BiofOmics, and bioinformatics tools, such as the Biofilms Experiment Workbench. This practical validation serves the purpose of disseminating the controlled vocabulary among researchers and identifying current limitations, glitches, and inconsistencies. Information branches will be added, extended or refactored according to user feedback and group discussions.

A new approach to bacterial colony morphotyping by matrix-assisted laser desorption ionization time of flight-based mass spectrometry.

Matrix assisted laser desorption ionization time of flight mass spectrometry has been explored as a tool to bacterial colony morphotyping. To this end, four colony morphotypes of Pseudomonas aeruginosa and four of Staphylococcus aureus were analysed using intact bacteria. Results suggest that mass spectrometry of intact bacteria could, in some extent, be used to complement the classical morphological classification of bacteria.

Improvements on colony morphology identification towards bacterial profiling.

Colony morphology may be an indicator of phenotypic variation, this being an important adaptive process adopted by bacteria to overcome environmental stressors. Furthermore, alterations in colony traits may reflect increased virulence and antimicrobial resistance. Despite the potential relevance of using colony morphological traits, the influence of experimental conditions on colony morphogenesis has been scarcely studied in detail. This study aims to clearly and systematically demonstrate the impact of some variables, such as colony growth time, plate colony density, culture medium, planktonic or biofilm mode of growth and strain genetic background, on bacterial colony morphology features using two Pseudomonas aeruginosa strains. Results, based on 5-replicate experiments, demonstrated that all variables influenced colony morphogenesis and 18 different morphotypes were identified, showing different sizes, forms, colours, textures and margins. Colony growth time and composition of the medium were the variables that caused the highest impact on colony differentiation both derived from planktonic and biofilm cultures. Colony morphology characterization before 45 h of incubation was considered inadequate and TSA, a non-selective medium, provided more colony diversity in contrast to P. aeruginosa selective media. In conclusion, data obtained emphasized the need to perform comparisons between colony morphologies in equivalent experimental conditions to avoid misinterpretation of microbial diagnostics and biomedical studies. Since colony morphotyping showed to be a reliable method to evaluate phenotypic switching and also to infer about bacterial diversity in biofilms, these unambiguous comparisons between morphotypes may offer a quite valuable input to clinical diagnosis, aiding the decision-making towards the selection of the most suitable antibiotic and supportive treatments.

Computational approaches to standard-compliant biofilm data for reliable analysis and integration.

The study of microorganism consortia, also known as biofilms, is associated to a number of applications in biotechnology, ecotechnology and clinical domains. Nowadays, biofilm studies are heterogeneous and data-intensive, encompassing different levels of analysis. Computational modelling of biofilm studies has become thus a requirement to make sense of these vast and ever-expanding biofilm data volumes. The rationale of the present work is a machine-readable format for representing biofilm studies and supporting biofilm data interchange and data integration. This format is supported by the Biofilm Science Ontology (BSO), the first ontology on biofilms information. The ontology is decomposed into a number of areas of interest, namely: the Experimental Procedure Ontology (EPO) which describes biofilm experimental procedures; the Colony Morphology Ontology (CMO) which characterises morphologically microorganism colonies; and other modules concerning biofilm phenotype, antimicrobial susceptibility and virulence traits. The overall objective behind BSO is to develop semantic resources to capture, represent and share data on biofilms and related experiments in a regularized fashion manner. Furthermore, the present work also introduces a framework in assistance of biofilm data interchange and analysis - BiofOmics (http://biofomics.org) - and a public repository on colony morphology signatures - MorphoCol (http://stardust.deb.uminho.pt/morphocol).