Colistin conditioning surfaces combined with antimicrobial treatment to prevent ventilator-associated infections.

Biofilm formation on endotracheal tubes (ETT) is an important factor in the development of ventilator-associated pneumonia (VAP). This work aimed to investigate the effectiveness of colistin (COL) against the early stages of biofilm formation by Pseudomonas aeruginosa. Two strategies were used: pre-conditioning the adhesion surfaces with COL before biofilm formation and growing biofilms in its presence. The combined effect of treating P. aeruginosa 24-hours old biofilms with Ciprofloxacin (CIP) or colistin (COL) on clean and COL-conditioned surfaces was also assessed. Random deposition of COL residues altered the physico-chemical properties of the adhesion surfaces and impaired biofilm formation. Moreover, as a consequence of the reduced amount of biofilms attached to COL conditioned surfaces, adhered cells became more exposed to the subsequent action of CIP or COL, suggesting a combined outcome of prophylactic and therapeutic COL-based strategies. Results highlighted the promising use of COL to prevent the establishment of biofilms on ETT.

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.

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.

Pseudomonas aeruginosa and Staphylococcus aureus communication in biofilm infections: insights through network and database construction.

The polymicrobial nature of most infections is often characterized by complex biofilm communities, where pathogen interactions promote infection progression and severity. Quorum-sensing, the major regulator of virulence and inter-species communication, is a promising target for new anti-infective strategies. This study aimed at collecting and analysing experimental information on the molecular basis of Pseudomonas aeruginosa and Staphylococcus aureus interactions in biofilms. Data were systematically annotated from relevant full-text papers optimally retrieved from PubMed, reconstructed as networks and integrated with specialized databases to identify promising antimicrobial targets. Network analysis revealed key entities regulating P. aeruginosa/S. aureus interactions, for instance the PqsABCDE/PqsR quorum-sensing system, which affects S. aureus growth and biofilm formation. By identifying the most reported P. aeruginosa virulence factors affecting S. aureus, for example, HQNO and siderophores, a list of experimentally validated agents affecting those factors, ranging from synthetic drugs to natural plant extracts, was constructed. The complex experimental data on P. aeruginosa/S. aureus interactions were for the first time systematically organized and made publically available in the new Inter-Species CrossTalk Database (www.ceb.uminho.pt/ISCTD).

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.

Exploring anti-quorum sensing and anti-virulence based strategies to fight Candida albicans infections: an in silico approach.

The complex virulence attributes of Candida albicans are an attractive target to exploit in the development of new antifungals and anti-virulence strategies to combat C. albicans infections. Particularly, quorum sensing (QS) has been reported as critical for virulence regulation in C. albicans. This work presents two knowledge networks with up-to-date information about QS regulation and experimentally tested anti-QS and anti-virulence agents for C. albicans. A semi-automatic bioinformatics workflow that combines literature mining and expert curation was used to retrieve otherwise scattered information from the scientific literature. The network representation offers an innovative and continuously updatable means for the Candida research community to query QS and virulence data systematically and in a user-friendly way. Notably, the reconstructed networks show the complexity of QS regulation and the impact that some molecules have on the inhibition of virulence mechanisms responsible for infection establishment (e.g. hyphal development) and perseverance (e.g. biofilm formation). In the future, the compiled knowledge may be used to build decision-making models that help infer new knowledge of practical significance. The knowledge networks are publicly available at http://pcquorum.org/. This Web platform enables the exploration of fungal virulence cues as well as reported inhibitors in a user-friendly fashion.

Pathogenicity phenomena in three model systems: from network mining to emerging system-level properties.

Understanding the interconnections of microbial pathogenicity phenomena, such as biofilm formation, quorum sensing and antimicrobial resistance, is a tremendous open challenge for biomedical research. Progress made by wet-lab researchers and bioinformaticians in understanding the underlying regulatory phenomena has been significant, with converging evidence from multiple high-throughput technologies. Notably, network reconstructions are already of considerable size and quality, tackling both intracellular regulation and signal mediation in microbial infection. Therefore, it stands to reason that in silico investigations would play a more active part in this research. Drug target identification and drug repurposing could take much advantage of the ability to simulate pathogen regulatory systems, host-pathogen interactions and pathogen cross-talking. Here, we review the bioinformatics resources and tools available for the study of the gram-negative bacterium Pseudomonas aeruginosa, the gram-positive bacterium Staphylococcus aureus and the fungal species Candida albicans. The choice of these three microorganisms fits the rationale of the review converging into pathogens of great clinical importance, which thrive in biofilm consortia and manifest growing antimicrobial resistance.

Developing a model for cystic fibrosis sociomicrobiology based on antibiotic and environmental stress.

Cystic fibrosis (CF) infections are invariably biofilm-mediated and polymicrobial, being safe to assume that a myriad of factors affects the sociomicrobiology within the CF infection site and modulate the CF community dynamics, by shaping their social activities, overall functions, virulence, ultimately affecting disease outcome. This work aimed to assess changes in the dynamics (particularly on the microbial composition) of dual-/three-species biofilms involving CF-classical (Pseudomonas aeruginosa) and unusual species (Inquilinus limosus and Dolosigranulum pigrum), according to variable oxygen conditions and antibiotic exposure. Low fluctuations in biofilm compositions were observed across distinct oxygen environments, with dual-species biofilms exhibiting similar relative proportions and P. aeruginosa and/or D. pigrum populations dominating three-species consortia. Once exposed to antibiotics, biofilms displayed high resistance profiles, and microbial compositions, distributions, and microbial interactions significantly challenged. The antibiotic/oxygen environment supported such fluctuations, which enhanced for three-species communities. In conclusion, antibiotic therapy hugely disturbed CF communities' dynamics, inducing significant compositional changes on multispecies consortia. Clearly, multiple perturbations may disturb this dynamic, giving rise to various microbiological scenarios in vivo, and affecting disease phenotype. Therefore, an appreciation of the ecological/evolutionary nature within CF communities will be useful for the optimal use of current therapies and for newer breakthroughs on CF antibiotherapy.

Quorum sensing inhibition in Pseudomonas aeruginosa biofilms: new insights through network mining.

Quorum sensing plays a pivotal role in Pseudomonas aeruginosa's virulence. This paper reviews experimental results on antimicrobial strategies based on quorum sensing inhibition and discusses current targets in the regulatory network that determines P. aeruginosa biofilm formation and virulence. A bioinformatics framework combining literature mining with information from biomedical ontologies and curated databases was used to create a knowledge network of potential anti-quorum sensing agents for P. aeruginosa. A total of 110 scientific articles, corresponding to 1,004 annotations, were so far included in the network and are analysed in this work. Information on the most studied agents, QS targets and methods is detailed. This knowledge network offers a unique view of existing strategies for quorum sensing inhibition and their main regulatory targets and may be used to readily access otherwise scattered information and to help generate new testable hypotheses. This knowledge network is publicly available at http://pcquorum.org/ .

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.

Data Quality in Biofilm High-Throughput Routine Analysis: Intralaboratory Protocol Adaptation and Experiment Reproducibility.

Biofilm research is growing more diverse and dependent on high-throughput technologies, and the large-scale production of results aggravates data substantiation. In particular, experimental protocols are often adapted to meet the needs of a particular laboratory, and no statistical validation of the modified method is provided. This paper discusses the impact of intralaboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non- rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intralaboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories.

Use of an aerobic selector to overcome filamentous bulking in an activated sludge wastewater treatment plant.

An urban wastewater treatment plant (WWTP) with two different parallel lines of treatment and located in the north of Portugal was studied during four months, following recurrent episodes of filamentous bulking caused by Sphaerotilus natans and eventual occurrences of Nocardioforms and Type 1863. An aerobic selector was introduced in both lines in the beginning of the studied period, suppressed in one of the lines during six weeks, and then put into operation again until the end of the study. A total of 14 filamentous bacteria morphotypes were identified. The results showed that the presence of an aerobic biological selector in continuous operation prevented the overgrowth of the filamentous Type 1863, of Nocardioforms and, in particular, of S. natans. Simultaneously, it allowed lowering the oxygen levels in the aeration tanks without negative consequences in the overall performance of the WWTP, namely bulking occurrence. Dissolved oxygen (DO) in the aeration tank varied initially between 2 and 3 mg/L, but the introduction of the selector enabled to lower it to 1-1.5 mg/L in the aeration tank and around 0.5 mg/L in the selector. A significant energy save was allowed, even considering the aeration of the selector. The results are more relevant if one considers the fact that the main cause of the bulking problems in this WWTP was the overgrowth of S. natans, a filamentous bacterium known to be stimulated by low DO levels.

Co-immobilization of Ciprofloxacin and Chlorhexidine as a Broad-Spectrum Antimicrobial Dual-Drug Coating for Poly(vinyl chloride) (PVC)-Based Endotracheal Tubes.

The endotracheal tube (ETT) affords support for intubated patients, but the increasing incidence of ventilator-associated pneumonia (VAP) is jeopardizing its application. ETT surfaces promote (poly)microbial colonization and biofilm formation, with a heavy burden for VAP. Devising safe, broad-spectrum antimicrobial materials to tackle the ETT bioburden is needful. Herein, we immobilized ciprofloxacin (CIP) and/or chlorhexidine (CHX), through polydopamine (pDA)-based functionalization, onto poly(vinyl chloride) (PVC) surfaces. These surfaces were characterized regarding physicochemical properties and challenged with single and polymicrobial cultures of VAP-relevant bacteria (Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis) and fungi (Candida albicans). The coatings imparted PVC surfaces with a homogeneous morphology, varied wettability, and low roughness. The antimicrobial immobilization via pDA chemistry was still evidenced by infrared spectroscopy. Coated surfaces exhibited sustained CIP/CHX release, retaining prolonged (10 days) activity. CIP/CHX-coated surfaces evidencing no A549 lung cell toxicity displayed better antibiofilm outcomes than CIP or CHX coatings, preventing bacterial attachment by 4.1-7.2 Log10 CFU/mL and modestly distressingC. albicans. Their antibiofilm effectiveness was endured toward polymicrobial consortia, substantially inhibiting the adhesion of the bacterial populations (up to 8 Log10 CFU/mL) within the consortia in dual- and even inP. aeruginosa/S. aureus/C. albicans triple-species biofilms while affecting fungal adhesion by 2.7 Log10 CFU/mL (dual consortia) and 1 Log10 CFU/mL (triple consortia). The potential of the dual-drug coating strategy in preventing triple-species adhesion and impairing bacterial viability was still strengthened by live/dead microscopy. The pDA-assisted CIP/CHX co-immobilization holds a safe and robust broad-spectrum antimicrobial coating strategy for PVC-ETTs, with the promise laying in reducing VAP incidence.

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.

Antimicrobial materials for endotracheal tubes: A review on the last two decades of technological progress.

Ventilator-associated pneumonia (VAP) is an unresolved problem in nosocomial settings, remaining consistently associated with a lack of treatment, high mortality, and prolonged hospital stay. The endotracheal tube (ETT) is the major culprit for VAP development owing to its early surface microbial colonization and biofilm formation by multiple pathogens, both critical events for VAP pathogenesis and relapses. To combat this matter, gradual research on antimicrobial ETT surface coating/modification approaches has been made. This review provides an overview of the relevance and implications of the ETT bioburden for VAP pathogenesis and how technological research on antimicrobial materials for ETTs has evolved. Firstly, certain main VAP attributes (definition/categorization; outcomes; economic impact) were outlined, highlighting the issues in defining/diagnosing VAP that often difficult VAP early- and late-onset differentiation, and that generate misinterpretations in VAP surveillance and discrepant outcomes. The central role of the ETT microbial colonization and subsequent biofilm formation as fundamental contributors to VAP pathogenesis was then underscored, in parallel with the uncovering of the polymicrobial ecosystem of VAP-related infections. Secondly, the latest technological developments (reported since 2002) on materials able to endow the ETT surface with active antimicrobial and/or passive antifouling properties were annotated, being further subject to critical scrutiny concerning their potentialities and/or constraints in reducing ETT bioburden and the risk of VAP while retaining/improving the safety of use. Taking those gaps/challenges into consideration, we discussed potential avenues that may assist upcoming advances in the field to tackle VAP rampant rates and improve patient care. STATEMENT OF SIGNIFICANCE: The use of the endotracheal tube (ETT) in patients requiring mechanical ventilation is associated with the development of ventilator-associated pneumonia (VAP). Its rapid surface colonization and biofilm formation are critical events for VAP pathogenesis and relapses. This review provides a comprehensive overview on the relevance/implications of the ETT biofilm in VAP, and on how research on antimicrobial ETT surface coating/modification technology has evolved over the last two decades. Despite significant technological advances, the limited number of gathered reports (46), highlights difficulty in overcoming certain hurdles associated with VAP (e.g., persistent colonization/biofilm formation; mechanical ventilation duration; hospital length of stay; VAP occurrence), which makes this an evolving, complex, and challenging matter. Challenges and opportunities in the field are discussed.

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.

Effect of ginger essential oil and 6-gingerol on a multispecies biofilm of Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas aeruginosa.

The objective of this study was to evaluate the potential antimicrobial and antibiofilm effect of ginger essential oil (GEO) and 6-gingerol on a multispecies biofilm formed by Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas aeruginosa on a polypropylene surface. The minimum inhibitory concentration concentrations obtained for GEO were 100 and 50 mg/mL and for 6-gingerol 1.25 mg/mL. Sessile cell counts ranged within 5.35-7.35 log CFU/cm2 in the control biofilm, with the highest sessile growth at 72 h. GEO treatments acted on the total population regardless of concentration at 1 and 48 h. L. monocytogenes behaved similarly to the total population, showing GEO action at 1 h and keeping the same pattern at 48, 72, and 96 h. Better action on S. Typhimurium was obtained at times of 1, 72, and 96 h. P. aeruginosa showed logarithmic reduction only when treated with GEO 50 mg at 24 h. As for 6-gingerol, in general, there was no significant action (p > 0.05) on the evaluated sessile cells. GEO showed antimicrobial activity against L. monocytogenes, S. Typhimurium, and P. aeruginosa, acting as an inhibitor of biofilm formation. As for 6-gingerol, it was considered a possible antimicrobial agent but without efficacy during biofilm formation.

New trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches.

Antimicrobial peptides (AMPs) have a broad spectrum of activity and unspecific mechanisms of action. Therefore, they are seen as valid alternatives to overcome clinically relevant biofilms and reduce the chance of acquired resistance. This paper reviews AMPs and anti-biofilm AMP-based strategies and discusses ongoing and future work. Recent studies report successful AMP-based prophylactic and therapeutic strategies, several databases catalogue AMP information and analysis tools, and novel bioinformatics tools are supporting AMP discovery and design. However, most AMP studies are performed with planktonic cultures, and most studies on sessile cells test AMPs on growing rather than mature biofilms. Promising preliminary synergistic studies have to be consubstantiated and the study of functionalized coatings with AMPs must be further explored. Standardized operating protocols, to enforce the repeatability and reproducibility of AMP anti-biofilm tests, and automated means of screening and processing the ever-expanding literature are still missing.

Adaptive response of single and binary Pseudomonas aeruginosa and Escherichia coli biofilms to benzalkonium chloride.

The main goal of this work was to examine whether the continuous exposure of single and binary P. aeruginosa and E. coli biofilms to sub-lethal benzalkonium chloride (BC) doses can induce adaptive response of bacteria. Biofilms were formed during 24 h and then put continuously in contact with BC for more 5 days. The six-day-old adapted biofilms were then submitted to BC challenge, characterized and inspected by SEM. Both single and binary adapted biofilms have clearly more biomass, polysaccharides and proteins and less activity even though the number of cells was identical. After BC treatment, adapted biofilms maintained their mass and activity. SEM examination revealed that those adapted biofilms had a slimier and denser matrix that became thicker after BC treatment. Continuous exposure of bacteria to antimicrobials can lead to development of biofilms encompassing more virulent and tolerant bacteria. This adaptive resistance can be the result of a phenotypic adaptation, a genetic acquired resistance or both. Instead of eradicating biofilms and kill microorganisms, the use of a disinfectant can, favour biofilm formation and tolerance. This must be a genuine concern as it can happen in clinical environments, where the use of antimicrobials is unavoidable.

Effect of antimicrobial residues on early adhesion and biofilm formation by wild-type and benzalkonium chloride-adapted Pseudomonas aeruginosa.

Antimicrobial residue deposition can change the physico-chemical properties of bacteria and surfaces and thus promote or impair bacterial adhesion. This study focuses on benzalkonium chloride (BC) deposition on polystyrene (PS) surfaces and the influence of this conditioning film on the physico-chemical properties of PS and on early adhesion and biofilm formation by Pseudomonas aeruginosa wild-type and its laboratory BC-adapted strain. The latter readily acquired the ability to grow in BC, and also exhibited physico-chemical surface changes. The existence of residues on PS surfaces altered their hydrophobicity and favoured adhesion as determined by the free energy and early adhesion characterization. Adapted bacteria revealed a higher ability to adhere to surfaces and to develop biofilms, especially on BC-conditioned surfaces, which thereby could enhance resistance to sanitation attempts. These findings highlight the importance of investigations concerning the antimicrobial deposition effect after cleaning procedures, which may encourage bacterial adhesion, especially of bacteria that have been previously exposed to chemical stresses.