Publications

Candida albicans and Staphylococcus aureus have been co-isolated from several biofilm-associated diseases, including those related to medical devices. This association confers advantages to both microorganisms, resulting in detrimental effects on the host. To elucidate this phenomenon, the present study investigated colony changes derived from non-physical interactions between C. albicans and S. aureus. We performed proximity assays by confronting colonies of the yeast and the bacteria on agar plates at six different distances for 9-10 days. We found that colony variants of S. aureus originated progressively after prolonged exposure to C. albicans proximity, specifically in response to pH neutralization of the media by the fungi. The new phenotypes of S. aureus were more virulent in a Galleria mellonella larvae model compared to colonies grown without C. albicans influence. This event was associated with an upregulation of RNA III and agrA expression, suggesting a role for alpha-toxin. Our findings indicate that C. albicans enhances S. aureus virulence by inducing the formation of more aggressive colonies. This highlights the importance of understanding the intricate connection between environmental responses, virulence and, fitness in S. aureus pathogenesis.

JTD Keywords: Agr system, Aureus, Biofil, Expression, Galleria mellonella, Gene regulator agr, Interkingdom interactions, Pathogenicit, Polymicrobial interactions, Proximity assay, Synergistic effects

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombi nant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.

JTD Keywords: Antimicrobial peptide, Antimicrobial peptides, Binding, Biofilm, Galleria mellonella, Host-defense, Peptid, Protein, Proteins, Toxicity, Treatment

Objective: Most endodontic diseases are bacterium-mediated inflammatory or necrotic process induced by contaminated dental pulp. Although great advances are being performed to obtain more efficient antibacterial strategies for persistent infections, most studies lack of representative models to test their antibacterial effects and their outcomes cannot be promptly translated to clinical practice. Therefore, this study aimed to refine an ex vivo endodontic biofilm model combining human tooth, computer guided design and 3D printing to obtain a more reproducible and predictable model. Methods: Monoradicular teeth were cut using three different methods: hand-held (HCC), mechanical precision (MPC) and computer aid guided cutting (CGC). Then, blocks were reassembled. The different model preparations were assessed in terms of dimensional tolerance, surface analysis, liquid tightness and Enterococcus faecalis biofilm development for 21 days, which was studied by metabolic assays and confocal microscopy. Then, the proposed model was validated using different commercial disinfecting treatments. Results: CGC exhibited significantly lower deviation and surface without defects compared to HHC and MPC, leading to superior liquid tightness. Similarly, mature biofilms with high metabolic activity and vitality were observed in all conditions, CGC showing the lowest variation. Regarding the model validation, all antibacterial treatments resulted in the complete eradication of bacteria in the standard 2D model, whereas commercial treatments exhibited varying levels of efficacy in the proposed ex vivo model, from moderately reduction of metabolic activity to complete elimination of biofilm. Conclusions: The novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies. Clinical Significance: During antibacterial treatment development, challenging 3D models using teeth substrates to test antibacterial treatments novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies.

JTD Keywords: 3d printin, Bacteria, Biofilm, Computer-aided manufacturing, Dental model, Dentin, Efficacy, Endodontics, Enterococcus faecalis, Enterococcus-faecalis, Irrigation, Protocols, Removal, Resistance, Susceptibility, Syste

The Boston Keratoprosthesis (BKPro) serves as a medical solution for restoring vision in complex cases of corneal blindness. Comprising a front plate made of polymethylmethacrylate (PMMA) and a back plate of titanium (Ti), this device utilizes the beneficial biomaterial properties of Ti. While BKPro demonstrates promising retention rates, infection emerges as a significant concern that impacts its long-term efficacy. However, limited research exists on enhancement of BKPros through intrinsic infection-preventing mechanisms. In this regard, metal ions, especially the well-known Ag+ ions, are a promising alternative to obtain implants with innate antibacterial properties. However, little information is available about the effects of Ag in corneal tissue, especially within human corneal keratocytes (HCKs). In this work, an electrodeposition treatment using a constant pulse is proposed to attach Ag complexes onto rough Ti surfaces, thus providing antibacterial properties without inducing cytotoxicity. Complete physicochemical characterization and ion release studies were carried out with both control and Ag-treated samples. The possible cytotoxic effects in the short and long term were evaluated in vitro with HCKs. Moreover, the antibacterial properties of the silver-treated surfaces were tested against the gram-negative bacterial strain Pseudomonas aeruginosa and the gram-positive strain Staphylococcus epidermidis, that are common contributors to infections in BKPros. Physicochemical characterization confirmed the presence of silver, predominantly in oxide form, with low release of Ag+ ions. Ag-treated surfaces demonstrated no cytotoxicity and promoted long-term proliferation of HCKs. Furthermore, the silver-treated surfaces exhibited a potent antibacterial effect, causing a reduction in bacterial adhesion and evident damage to the bacterial cell walls of P. aeruginosa and S. epidermidis. The low release of Ag+ ions suggested reactive oxygen species (ROS)-mediated oxidative stress imbalance as the bactericidal mechanism of the silver deposits. In conclusion, the proposed electrodeposition technique confers antibacterial protection to the Ti backplate of BKPro, mitigating implant-threatening infections while ensuring non-cytotoxicity within the corneal tissue.

JTD Keywords: Antibacterial properties, Biofilm, Boston keratoprosthesis (bkpro), Corneal blindness, Cytotoxicicity, Cytotoxicit, Electrodeposition, I keratoprosthesis, Infection, Infectious endophthalmitis, Ion, Long-term outcomes, Nanoparticles, Silver depositio, Surface, Titanium (ti)

Biofilm formation by the pathobiont Haemophilus influenzae is associated with human nasopharynx colonization, otitis media in children, and chronic respiratory infections in adults suffering from chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). beta-lactam and quinolone antibiotics are commonly used to treat these infections. However, considering the resistance of biofilm-resident bacteria to antibiotic -mediated killing, the use of antibiotics may be insufficient and require being replaced or complemented with novel strategies. Moreover, unlike the standard minimal inhibitory concentration assay used to assess antibacterial activity against planktonic cells, standardization of methods to evaluate anti-biofilm drug activity is limited. In this work, we detail a panel of protocols for systematic analysis of drug antimicrobial effect on bacterial biofilms, customized to evaluate drug effects against H. influenzae biofilms. Testing of two cinnamaldehyde analogs, (E)- trans-2-nonenal and (E)-3-decen-2-one, demonstrated their effectiveness in both H. influenzae inhibition of biofilm formation and eradication or preformed biofilms. Assay complementarity allowed quantifying the dynamics and extent of the inhibitory effects, also observed for ampicillin resistant clinical strains forming biofilms refractory to this antibiotic. Moreover, cinnamaldehyde analog encapsulation into poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles allowed drug vehiculization while maintaining efficacy. Overall, we demonstrate the usefulness of cinnamaldehyde analogs against H. influenzae biofilms, present a test panel that can be easily adapted to a wide range of pathogens and drugs, and highlight the benefits of drug nanoencapsulation towards safe controlled release.

JTD Keywords: Anti-biofilm drugs, Antibodies, Biofilm, Cinnamaldehyde-analogs, Haemophilus influenzae, In-vitro, Maturation, Multimodal methods, Nanoformulation

Viability and vitality assays play a crucial role in assessing the effectiveness of novel therapeutic approaches, with stain-based methods providing speed and objectivity. However, their application in yeast research lacks consensus. This study aimed to assess the performance of four common dyes on C. parapsilosis planktonic cells as well as sessile cells that form well-structured biofilms (treated and not treated with amphotericin B). Viability assessment employed Syto-9 (S9), thiazole orange (TO), and propidium iodide (PI). Metabolic activity was determined using fluorescein diacetate (FDA) and FUN-1. Calcofluor white (CW) served as the cell visualization control. Viability/vitality percentage of treated samples were calculated for each dye from confocal images and compared to crystal violet and PrestoBlue results. Heterogeneity in fluorescence intensity and permeability issues were observed with S9, TO, and FDA in planktonic cells and biofilms. This variability, influenced by cell morphology, resulted in dye-dependent viability/vitality percentages. Notably, PI and FUN-1 exhibited robust C. parapsilosis staining, with FUN-1 vitality results comparable to PrestoBlue. Our finding emphasizes the importance of evaluating dye permeability in yeast species beforehand, incorporating cell visualization controls. An improper dye selection may lead to misinterpreting treatment efficacy.

JTD Keywords: Albicans,quantification,biomass,image,aci, Biofilms,microscopy,imaging,amphotericin b,stain-based methods,yeast staining,fluorescence,live and dea

Microbial biofilms are complex three-dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm-related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set-backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

JTD Keywords: Anti-bacterial agents, Anti-infective agents, Antiinfective agent, Antimicrobial, Antimicrobials, Antimicrobials,bacteria,biofilm,infectious diseases,microorganism, Bacteria, Biofilm, Biofilm infections, Biofilms, Complex three dimensional structures, Diseases, Diverse range, Drug-delivery systems,in-vitro,cellular toxicity,nanoparticles,penetration,model,biocompatibility,perspectives,hyperthermia,diagnosi, Extracellular matrices, Global public health, Health risks, Infectious disease, Infectious diseases, Medical nanotechnology, Microbial biofilm, Microorganisms, Nanomedicine, Polymer, Polymers, Regulatory issues, Roadmap

Laser surface micropatterning of dental-grade zirconia (3Y-TZP) was explored with the objective of providing defined linear patterns capable of guiding bone-cell response.A nanosecond (ns-) laser was employed to fabricate microgrooves on the surface of 3Y-TZP discs, yielding three different groove periodicities (i.e., 30, 50 and 100 µm). The resulting topography and surface damage were characterized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). X-Ray diffraction (XRD) and Raman spectroscopy techniques were employed to assess the hydrothermal degradation resistance of the modified topographies. Preliminary biological studies were conducted to evaluate adhesion (6 h) of human mesenchymal stem cells (hMSC) to the patterns in terms of cell number and morphology. Finally, Staphylococcus aureus adhesion (4 h) to the microgrooves was investigated.The surface analysis showed grooves of approximately 1.8 µm height that exhibited surface damage in the form of pile-up at the edge of the microgrooves, microcracks and cavities. Accelerated aging tests revealed a slight decrease of the hydrothermal degradation resistance after laser patterning, and the Raman mapping showed the presence of monoclinic phase heterogeneously distributed along the patterned surfaces. An increase of the hMSC area was identified on all the microgrooved surfaces, although only the 50 µm periodicity, which is closer to the cell size, significantly favored cell elongation and alignment along the grooves. A decrease in Staphylococcus aureus adhesion was observed on the investigated micropatterns.The study suggests that linear microgrooves of 50 µm periodicity may help in promoting hMSC adhesion and alignment, while reducing bacterial cell attachment.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

JTD Keywords: abutment material, alumina toughened zirconia, antibacterial, bacterial adhesion, biofilm growth, cell adhesion, dental implants, hydrothermal degradation, implant surfaces, in-vitro, laser patterning, osseointegration, osteogenic differentiation, part 1, surface topography, y-tzp ceramics, Antibacterial, Antibacterials, Bacteria, Bone, Cell adhesion, Cell culture, Cells adhesion, Ceramics, Chemistry, Degradation resistance, Dental implants, Dental material, Dental materials, Dental prostheses, Human, Human mesenchymal stem cells, Humans, Hydrothermal degradation, Laser patterning, Laser surface, Lasers, Low-temperature degradation, Materials testing, Microscopy, electron, scanning, Nanosecond lasers, Osseointegration, Piles, Scanning electron microscopy, Staphylococcus aureus, Stem cells, Surface analysis, Surface damages, Surface properties, Surface property, Surface topography, Topography, Yttrium, Zirconia, Zirconium

Introduction: Candida parapsilosis, a pathogenic yeast associated with systemic infections, exhibits metabolic adaptability in response to nutrient availability.Methods: We investigated the impact of RPMI glucose supplemented (RPMId), TSB, BHI and YPD media on C. parapsilosis growth, morphology, susceptibility (caspofungin and amphotericin B), and in vivo virulence (Galleria mellonella) in planktonic and biofilm states.Results: High-glucose media favors growth but hinders metabolic activity and filamentation. Media promoting carbohydrate production reduces biofilm susceptibility. Virulence differences between planktonic cells and biofilm suspensions from the same media shows that biofilm-related factors influence infection outcome depending on nutrient availability. Pseudohyphal growth occurred in biofilms under low oxygen and shear stress, but its presence is not exclusively correlated with virulence.Discussion: This study provides valuable insights into the intricate interplay between nutrient availability and C. parapsilosis pathogenicity. It emphasizes the importance of considering pathogen behavior in diverse conditions when designing research protocols and therapeutic strategies.

JTD Keywords: Amphotericin b, Antifungal, Biofilm, Candida, Caspofungin, Galleria mellonella, Pathogenicity, Pseudohyphal growth

Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that Pseudomonas aeruginosa and Burkholderia cenocepacia strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins.

JTD Keywords: biomass quantification, burkholderia cenocepacia, burkholderia-cepacia, crystal violet, cystic-fibrosis, dual-species biofilms, pseudomonas aeruginosa, quantification, Biomass quantification, Burkholderia cenocepacia, Crystal violet, Dual-species biofilms, Pseudomonas aeruginosa, Pseudomonas-aeruginosa

JTD Keywords: Bacterial biofilms, Device, Growth, In-vitro, Infections, Laboratory method, Lung model, Responses, Temperature, Time

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.Copyright © 2023 Liang, Nilsson, Kragh, Hedal, Alcàcer-Almansa, Kiilerich, Andersen and Tolker-Nielsen.

JTD Keywords: aggregates, antibiotic tolerance, biofilm formation, extracellular matrix, genome, growth, lungs, molecular-mechanisms, mutations, polysaccharide, pseudomonas aeruginosa, psl, system, Aggregates, Antibiotic tolerance, Biofilm, Extracellular matrix, Pseudomonas aeruginosa, Small-colony variants

Virtually, all implantable medical devices are susceptible to infection. As the main healthcare issue concerning implantable devices is the elevated risk of infection, different strategies based on the coating or functionalization of biomedical devices with antiseptic agents or antibiotics are proposed. In this work, an alternative approach is presented, which consists of the functionalization of implantable medical devices with sensors capable of detecting infection at very early stages through continuous monitoring of the bacteria metabolism. This approach, which is implemented in surgical sutures as a representative case of implantable devices susceptible to bacteria colonization, is expected to minimize the risk of worsening the patient's clinical condition. More specifically, non-absorbable polypropylene/polyethylene (PP/PE) surgical sutures are functionalized with conducting polymers using a combination of low-pressure oxygen plasma, chemical oxidative polymerization, and anodic polymerization, to detect metabolites coming from bacteria respiration. Functionalized suture yarns are used for real-time monitoring of bacteria growth, demonstrating the potential of this strategy to fight against infections.© 2023 Wiley-VCH GmbH.

JTD Keywords: adhesion, biofilm, conducting polymers, contamination, derivatives, detections, functionalized sutures, nadh, poly(3,4-ethylenedioxythiophene), Bacteria growth, Conducting polymers, Detections, Functionalized sutures, Monofilament, Nadh

The emergence of multidrug-resistant bacteria is a serious problem worldwide. Pseudomonas aeruginosa is a pathogen that causes severe infections because it can form a biofilm that protects it from immune system mechanisms such as the production of oxidative stress. Ribonucleotide reductases are essential enzymes which synthesize deoxyribonucleotides used in the replication of DNA.

JTD Keywords: algr, biofilm, galleria mellonella, nrdj, oxidative stress, Gene-expression, Ribonucleotide reductase

Escherichia coli is one of the most common members of the intestinal microbiota. Many of its strains are associated with various inflammatory infections, including urinary or gut infections, especially when displaying antibiotic resistance or in patients with suppressed immune systems. According to recent reports, the biofilm-forming potential of E. coli is a crucial factor for its increased resistance against antibiotics. To overcome the limitations of using antibiotics against resistant E. coli strains, the world is turning once more towards bacteriophage therapy, which is becoming a promising candidate amongst the current personalized approaches to target different bacterial infections. Although matured and persistent biofilms pose a serious challenge to phage therapy, they can still become an effective alternative to antibiotic treatment. Here, we assess the efficiency of clinically isolated phages in phage therapy against representative clinical uropathogenic and invasive biofilm-forming E. coli strains. Our results demonstrate that irrespective of host specificity, bacteriophages producing clear plaques with a high burst size, and exhibiting depolymerizing activity, are good candidates against biofilm-producing E. coli pathogens as verified from our in vitro and in vivo experiments using Galleria mellonella where survival was significantly increased for phage-therapy-treated larvae.

JTD Keywords: antibiotic resistance, assay, bacteriophage, bacteriophages, biofilm-forming potential, infection, inflammatory infections, mechanisms, Galleria-mellonella, Intestinal microflora

The etiology of dental caries remains poorly understood. With the advent of next-generation sequencing, a number of studies have focused on the microbial ecology of the disease. However, taxonomic associations with caries have not been consistent. Researchers have also pursued function-centric studies of the caries microbial communities aiming to identify consistently conserved functional pathways. A major question is whether changes in microbiome are a cause or a consequence of the disease. Thus, there is a critical need to define conserved functional signatures at the onset of dental caries.Since it is unethical to induce carious lesions clinically, we developed an innovative longitudinal ex-vivo model integrated with the advanced non-invasive multiphoton second harmonic generation bioimaging to spot the very early signs of dental caries, combined with 16S rRNA short amplicon sequencing and liquid chromatography-mass spectrometry-based targeted metabolomics.For the first time, we induced longitudinally monitored caries lesions validated with the scanning electron microscope. Consequently, we spotted the caries onset and, associated with it, distinguished five differentiating metabolites - Lactate, Pyruvate, Dihydroxyacetone phosphate, Glyceraldehyde 3-phosphate (upregulated) and Fumarate (downregulated). Those metabolites co-occurred with certain bacterial taxa; Streptococcus, Veillonella, Actinomyces, Porphyromonas, Fusobacterium, and Granulicatella, regardless of the abundance of other taxa.These findings are crucial for understanding the etiology and dynamics of dental caries, and devising targeted interventions to prevent disease progression.© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

JTD Keywords: bacteria, biofilms, children, dental caries, generation, genomics, longitudinal model, metabolism, metabolomics, microscopy, non-invasive bioimaging, oral microbiome, plaque, restorations, signatures, Dental caries, Field-emission sem, Signatures

Chronic wounds infected by Pseudomonas aeruginosa and Staphylococcus aureus are a relevant health problem worldwide because these pathogens grow embedded in a network of polysaccharides, proteins, lipids, and extracellular DNA, named biofilm, that hinders the transport of antibiotics and increases their antimicrobial tolerance. It is necessary to investigate therapies that improve the penetrability and efficacy of antibiotics. In this context, our main objectives were to study the relationship between P. aeruginosa and S. aureus and how their relationship can affect the antimicrobial treatment and investigate whether functionalized silver nanoparticles can improve the antibiotic therapy. We used an optimized in vitro wound model that mimics an in vivo wound to co-culture P. aeruginosa and S. aureus biofilm. The in vitro wound biofilm was treated with antimicrobial combinatory therapies composed of antibiotics (gentamycin and ciprofloxacin) and biofilm-dispersing free or silver nanoparticles functionalized with enzymes (alpha-amylase, cellulase, DNase I, or proteinase K) to study their antibiofilm efficacy. The interaction and colocalization of P. aeruginosa and S. aureus in a wound-like biofilm were examined and detailed characterized by confocal and electronic microscopy. We demonstrated that antibiotic monotherapy is inefficient as it differentially affects the two bacterial species in the mixed biofilm, driving P. aeruginosa to overcome S. aureus when using ciprofloxacin and the contrary when using gentamicin. In contrast, dual-antibiotic therapy efficiently reduces both species while maintaining a balanced population. In addition, DNase I nanoparticle treatment had a potent antibiofilm effect, decreasing P. aeruginosa and S. aureus viability to 0.017 and 7.7%, respectively, in combined antibiotics. The results showed that using nanoparticles functionalized with DNase I enhanced the antimicrobial treatment, decreasing the bacterial viability more than using the antibiotics alone. The enzymes alpha-amylase and cellulase showed some antibiofilm effect but were less effective compared to the DNase I treatment. Proteinase K showed insignificant antibiofilm effect. Finally, we proposed a three-dimensional colocalization model consisting of S. aureus aggregates within the biofilm structure, which could be associated with the low efficacy of antibiofilm treatments on bacteria. Thus, designing a clinical treatment that combines antibiofilm enzymes and antibiotics may be essential to eliminating chronic wound infections.

JTD Keywords: antimicrobial therapies, biofilm, chronic infection, nanoparticle, Antimicrobial therapies, Biofilm, Chronic infection, In-vitro, Matrix, Model, Nanoparticle, Wound healing

Quantification of odor emissions in wastewater treatment plants (WWTPs) is key to minimize odor impact to surrounding communities. Odor measurements in WWTPs are usually performed via either expensive and discontinuous olfactometry hydrogen sulfide detectors or via fixed electronic noses. We propose a portable lightweight electronic nose specially designed for real-time odor monitoring in WWTPs using small drones. The so-called RHINOS e-nose allows odor measurements with high spatial resolution, and its accuracy is only slightly worse than that of dynamic olfactometry. The device has been calibrated using odor samples collected in a WWTP in Spain over a period of six months and validated in the same WWTP three weeks after calibration. The promising results obtained support the suitability of the proposed instrument to identify the odor sources having the highest emissions, which may give a useful indication to the plant managers as regards odor control and abatement.© 2021 The Author(s).

JTD Keywords: biofiltration, calibration transfer, chemical sensor arrays, chemistry, drift compensation, engineering, environmental chemical engineering, h2s, model, oxide gas sensors, removal, sensor, system, Chemistry, Engineering, Environmental chemical engineering, Sensor, Sensor system, Variable selection methods

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3?UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3?UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3?UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

JTD Keywords: aggregative adherence, arginine metabolism, biofilm formation, escherichia-coli, gene-expression, messenger-rna, operon, persistent diarrhea, untranslated region, Fimbria-i expression

Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and interact with a broad range of microorganisms have a substantial clinical impact. This review focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and regulatory networks involved, placing particular emphasis on the clinical challenges they represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, the ability of P. aeruginosa to grow together with other microorganisms is a significant pathogenic attribute with clinical relevance; hence, the main microbial interactions of Pseudomonas are especially highlighted and detailed throughout this review. This article also explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the current models used to recreate them under laboratory conditions. Finally, the antimicrobial and antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are detailed at the end of this review.

JTD Keywords: aeruginosa models, antibiotic-resistance, antimicrobials, bacterial biofilms, biofilms, c-di-gmp, chronic infections, enterococcus-faecalis, extracellular dna, in-vitro, lectin pa-iil, p, p. aeruginosa models, polymicrobial, polymicrobial interactions, staphylococcus-aureus, Antimicrobials, Biofilms, Chronic infections, P. aeruginosa models, Polymicrobial, Pseudomonas aeruginosa, Urinary-tract-infection

The low efficacy of current conventional treatments for bacterial infections increases mortality rates worldwide. To alleviate this global health problem, we propose drug-free enzyme-based nanomotors for the treatment of bacterial urinary-tract infections. We develop nanomotors consisting of mesoporous silica nanoparticles (MSNPs) that were functionalized with either urease (U-MSNPs), lysozyme (L-MSNPs), or urease and lysozyme (M-MSNPs), and use them against nonpathogenic planktonic Escherichia coli. U-MSNPs exhibited the highest bactericidal activity due to biocatalysis of urea into NaHCO3 and NH3, which also propels U-MSNPs. In addition, U-MSNPs in concentrations above 200 μg/mL were capable of successfully reducing 60% of the biofilm biomass of a uropathogenic E. coli strain. This study thus provides a proof-of-concept, demonstrating that enzyme-based nanomotors are capable of fighting infectious diseases. This approach could potentially be extended to other kinds of diseases by selecting appropriate biomolecules.

JTD Keywords: biofilms, carbonate, e. coli, enzymatic nanomotors, infections, lysozyme, micromotors, nanomachines, proteins, self-propulsion, Anti-bacterial agents, Biocatalysis, Biofilms, Canavalia, Drug carriers, E. coli, Eliminate escherichia-coli, Enzymatic nanomotors, Escherichia coli, Escherichia coli infections, Humans, Infections, Muramidase, Nanomachines, Nanoparticles, Self-propulsion, Silicon dioxide, Urease, Urinary tract infections

Oleanolic acid (OA) and maslinic acid (MA) are pentacyclic triterpenic compounds that abound in industrial olive oil waste. These compounds have renowned antimicrobial properties and lack cytotoxicity in eukaryotic cells as well as resistance mechanisms in bacteria. Despite these advantages, their antimicrobial activity has only been tested in vitro, and derivatives improving this activity have not been reported. In this work, a set of 14 OA and MA C-28 amide derivatives have been synthesized. Two of these derivatives, MA-HDA and OA-HDA, increase the in vitro antimicrobial activity of the parent compounds while reducing their toxicity in most of the Gram-positive bacteria tested, including a methicillin-resistant Staphylococcus aureus-MRSA. MA-HDA also shows an enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model of infection. A preliminary attempt to elucidate their mechanism of action revealed that these compounds are able to penetrate and damage the bacterial cell membrane. More significantly, their capacity to reduce antibiofilm formation in catheters has also been demonstrated in two sets of conditions: a static and a more challenged continuous-flow S. aureus biofilm.

JTD Keywords: Antibiofilm, Galleria mellonella, In vitro and in vivo antimicrobials, Maslinic and oleanolic acids, Natural products, Staphylococcus aureus

The coexistence between species that occurs in some infections remains hard to achieve in vitro since bacterial fitness differences eventually lead to a single organism dominating the mixed culture. Pseudomonas aeruginosa and Staphylococcus aureus are major pathogens found growing together in biofilms in disease-affected lungs or wounds. Herein, we tested and analyzed different culture media, additives and environmental conditions to support P. aeruginosa and S. aureus coexistence in vitro. We have unraveled the potential of DMEM to support the growth of these two organisms in mature cocultured biofilms (three days old) in an environment that dampens the pH rise. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate S. aureus microcolonies that grow embedded in a P. aeruginosa biofilm, as well as S. aureus biofilm overgrowth when bovine serum albumin is added to the system. Remarkably, we also found that S. aureus survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether P. aeruginosa and S. aureus were growing in mono- or coculture biofilms, was used to validate our in vitro coculture conditions.

JTD Keywords: Applied microbiology, Biofilms

P. aeruginosa is a major pathogenic bacterium in chronic infections and is a model organism for studying biofilms. P. aeruginosa is considered an aerobic bacterium, but in the presence of nitrate, it also grows in anaerobic conditions. Oxygen diffusion through the biofilm generates metabolic and genetic diversity in P. aeruginosa growth, such as in ribonucleotide reductase activity. These essential enzymes are necessary for DNA synthesis and repair. Oxygen availability determines the activity of the three-ribonucleotide reductase (RNR) classes. Class II and III RNRs are active in the absence of oxygen; however, class II RNRs, which are important in P. aeruginosa biofilm growth, require a vitamin B12 cofactor for their enzymatic activity. In this work, we elucidated the conditions in which class II RNRs are active due to vitamin B12 concentration constraints (biosynthesis or environmental availability). We demonstrated that increased vitamin B12 levels during aerobic, stationary and biofilm growth activate class II RNR activity. We also established that the cobN gene is essentially responsible for B12 biosynthesis under planktonic and biofilm growth. Our results unravel the mechanisms of dNTP synthesis by P. aeruginosa during biofilm growth, which appear to depend on the bacterial strain (laboratory-type or clinical isolate).

JTD Keywords: Vitamin B12, Adenosylcobalamin, Ribonucleotide Reductases, Pseudomonas aeruginosa, NrdJ, Bacterial growth, Biofilm,Anaerobiosis

Virulence expression in the enteroaggregative E. coli strain 042 requires the transcriptional activator AggR. We show in this report that, as reported for other virulence factors, the nucleotide second messenger (p)ppGpp is needed for a high expression level of AggR. As expected from these findings, expression of AggR-activated genes such as the AafA pilin subunit is downregulated in the absence of (p)ppGpp. Considering the fact that biofilm formation in strain 042 requires the AafA protein, biofilm development in strain 042 is impaired in derivatives that lack either the AggR protein, the virulence plasmid that encodes AggR (pAA2) or the ability to synthesize (p)ppGpp. These results show a direct correlation between (p)ppGpp, expression of AggR and biofilm development in strain 042.

JTD Keywords: (p)ppGpp, AggR, EAEC, Biofilm, AAF/II

Chronic lung infections by the ubiquitous and extremely adaptable opportunistic pathogen Pseudomonas aeruginosa correlate with the formation of a biofilm, where bacteria grow in association with an extracellular matrix and display a wide range of changes in gene expression and metabolism. This leads to increased resistance to physical stress and antibiotic therapies, while enhancing cell-to-cell communication. Oxygen diffusion through the complex biofilm structure generates an oxygen concentration gradient, leading to the appearance of anaerobic microenvironments. Ribonucleotide reductases (RNRs) are a family of highly sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides, and they constitute the only de novo pathway for the formation of the building blocks needed for DNA synthesis and repair. P. aeruginosa is one of the few bacteria encoding all three known RNR classes (Ia, II, and III). Class Ia RNRs are oxygen dependent, class II are oxygen independent, and class III are oxygen sensitive. A tight control of RNR activity is essential for anaerobic growth and therefore for biofilm development. In this work we explored the role of the different RNR classes in biofilm formation under aerobic and anaerobic initial conditions and using static and continuous-flow biofilm models. We demonstrated the importance of class II and III RNR for proper cell division in biofilm development and maturation. We also determined that these classes are transcriptionally induced during biofilm formation and under anaerobic conditions. The molecular mechanism of their anaerobic regulation was also studied, finding that the Anr/Dnr system is responsible for class II RNR induction. These data can be integrated with previous knowledge about biofilms in a model where these structures are understood as a set of layers determined by oxygen concentration and contain cells with different RNR expression profiles, bringing us a step closer to the understanding of this complex growth pattern, essential for P. aeruginosa chronic infections.

JTD Keywords: Pseudomonas aeruginosa, Ribonucleotide Reductases, Vitamin B 12, Anaerobic metabolism, Biofilm formation, DNA Synthesis, Oxygen diffusion, nrd genes.

Abstract Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections.

JTD Keywords: Pseudomonas aeruginosa, Biofilm, Ciprofloxacin, DNase I, Nanoparticles

Staphylococcus aureus, especially strains resistant to multiple antibiotics, is a major pathogen for humans and animals. In this paper we have synthesized and evaluated the antibacterial activity of a new series of benzopolycyclic amines. Some of them exhibited μM MIC values against Staphylococcus aureus and other bacteria, including methicillin-resistant S. aureus MRSA. Compound 8 that displayed a good selectivity index, showed to be active in eliminating bacterial cells forming a preexisting biofilm.

JTD Keywords: Antibacterials, Minimal biofilm inhibitory concentration, Polycyclic compounds, Staphylococcus aureus

The roles of different ribonucleotide reductases (RNRs) in bacterial pathogenesis have not been studied systematically. In this work we analyzed the importance of the different Pseudomonas aeruginosa RNRs in pathogenesis using the Drosophila melanogaster host-pathogen interaction model. P. aeruginosa codes for three different RNRs with different environmental requirements. Class II and III RNR chromosomal mutants exhibited reduced virulence in this model. Translational reporter fusions of RNR gene nrdA, nrdJ, or nrdD to the green fluorescent protein were constructed to measure the expression of each class during the infection process. Analysis of the P. aeruginosa infection by flow cytometry revealed increased expression of nrdJ and nrdD and decreased nrdA expression during the infection process. Expression of each RNR class fits with the pathogenicities of the chromosomal deletion mutants. An extended understanding of the pathogenicity and physiology of P. aeruginosa will be important for the development of novel drugs against infections in cystic fibrosis patients.

JTD Keywords: Broad-host-range, Anaerobic growth, Drosophila-melanogaster, Bacterial biofilms, Escherichia-coli, Cystic-fibrosis, Model host, Virulence, Promoter, Vectors