Development of in vitro co-culture models and characterization of biofilm inhibitors for bio-fabrication of medical devices and drug discovery applications

Nosocomial infections imply a great risk for hospitalized patients, causing around 90 000 deaths per year just in the European Union. Bacterial biofilms- microaggregates of bacteria attached to a surface and embedded in a self-produced matrix-are often responsible of this kind of infections, especially those related to medical device use. Medical devices offer an ideal surface for bacteria to attach and form a biofilm and, by switching into this state, bacteria can withstand antibiotic chemotherapy and evade the immune system of the host. One of the current problems to tackle biofilm-associated infections is the limited number of molecules able to act on them at sufficiently low concentrations. For this reason, there is an intense search of biofilm inhibitors, which is often performed via the screening of compound libraries utilizing microplate assays. However, the conditions in which these assays are carried out often differ from those found in clinical environments, and for compounds to be truly effective in translational applications, they ought to be tested in relevant experimental models. This doctoral project has been focused on the development of new in vitro models that better resemble the conditions in which bacterial biofilms are formed and function in medical device-related infections (i.e. orthopedic infections and ventilator-associated pneumonia). Moreover, the anti-biofilm capacity of three previously identified biofilm inhibitors was re-assessed using the newly developed models. These biofilm inhibitors included two dehydroabietic acid derivatives, DHA1 and DHA2 and one flavan derivative, FLA1. In the clinical environment, biofilms can often be composed of multiple species, whose tolerance to antibiotic treatments may be different from those composed of single species. Because of this, in the first study, a co-culture model of Staphylococcus aureus and Pseudomonas aeruginosa was optimized to create a dual species biofilm. These species were chosen because they have been often co-isolated from biofilms related to numerous infections, including medical device-related ones. To be able to develop efficient treatments against multi-species biofilms, it is essential to understand the different biofilm dynamics. Because of this, as part of this study, we exhaustively characterized the variations of the proteomes (specifically the surfaceome and the exoproteome) of S. aureus and P. aeruginosa when co-existing in the same biofilm in comparison to the proteomes of these bacteria in monoculture. Moreover, the hints of several phenotypical changes derived from the proteome analysis were confirmed by a series of follow up studies, which included the analysis of their susceptibility to conventional antibiotics and to the studied compounds (DHA1, DHA2 and FLA1). Out of the three compounds tested, DHA1 was the only one preserving its activity against the S. aureus biofilm formed in co-culture with P. aeruginosa. The second and third studies aimed at assessing the applicability of these three biofilm inhibitors as part of anti-infective materials, specifically bone implanted devices and endotracheal tubes. In the specific case of implantable devices, the prevention of the infection and correct tissue integration are equally important for their success. Therefore, we first developed in the second study a model based on the co-culture of osteogenic cells (SaOS-2), and bacterial cells, specifically, S. aureus, on titanium surface. This model allowed assessing the effects of the newly identified biofilm inhibitors on their anti-biofilm capability on clinically relevant surfaces while simultaneously assessing their effects on tissue integration, apart from providing information on their effects on the interactions between these two cell types. The compound DHA1 was shown to display the best results in terms of prevention of bacterial adherence and cytocompatibility. Nevertheless, when inserting a medical device, not only the cells of the tissue (and possibly bacterial cells) are present, but also immune cells of the host. The insertion procedure often triggers an inflammatory response, which might cause an inefficient clear out of planktonic bacteria due to the efforts of immune cells being directed to degrading the foreign material. Because of this, a third model was developed based on the co-culture of bacterial and host immune cells, specifically neutrophils. This assay aimed at predicting the effects of biofilm inhibitors on the antimicrobial capacity of neutrophils. From the three compounds tested, DHA1 seemed to be the only one increasing the capacity of neutrophils in preventing bacterial adherence to the surfaces. Based upon the results obtained in the three newly developed models, DHA1 was selected to be integrated as part of 3D-printed antimicrobial coating for titanium surfaces. The functionality tests performed on the developed material confirmed its capacity in preventing S. aureus biofilm formation and its cytocompatibility, as well as its activity favoring the antimicrobial capacity of neutrophils. This doctoral research attempts to provide the science community dedicated to anti-biofilm research with new protocols able to shorten the gap between the in vitro testing and the clinical scenario. The results presented here shed some light on the dynamics of a clinically relevant dual-species biofilm, which we hope will contribute to the identification and development of new therapeutic strategies. Finally, it provides novel biofilm inhibitor candidates to be incorporated as part of medical devices, in particular a promising dehydroabietic acid derivative (DHA1) that was successfully incorporated in proof-of-concept studies, within a 3D-printed antimicrobial coating for titanium implants.Sairaalainfektiot muodostavat suuren riskin sairaalapotilaille, ja ne aiheuttavat vuosittain noin 90 000 kuolemaa yksin Euroopan Unionin alueella. Bakteeribiofilmit eli pintaan kiinnittyneet ja itse tuottamassaan matriksissa elävät bakteerien mikroaggregaatit ovat usein näiden taustalla, erityisesti lääkinnällisten laitteiden käyttöön liittyvissä infektioissa. Lääketieteelliset laitteet tarjoavat bakteereille ihanteellisen pinnan kiinnittymiseen ja biofilmin muodostamiseen, ja tähän tilaan siirtyessään bakteerit kestävät mikrobilääkehoitoa ja kykenevät välttämään isännän immuunipuolustusta. Yksi biofilmi-infektioiden torjuntaan liittyvistä haasteista on rajallinen määrä molekyylejä, jotka pystyvät vaikuttamaan biofilmeihin riittävän pieninä pitoisuuksina. Tämän vuoksi käynnissä on intensiivinen biofilmiä estävien yhdisteiden etsintä, joka toteutetaan usein seulomalla yhdistekirjastoja mikrokuoppalevymenetelmin. Näiden määritysten olosuhteet eroavat kuitenkin usein kliinisissä ympäristöissä olevista, ja jotta yhdisteet olisivat tehokkaita käytännön sovelluksissa, niitä tulisi testata asiaankuuluvissa kokeellisissa malleissa. Tässä väitöskirjatyössä kehitettiin uusia in vitro -malleja, jotka muistuttavat paremmin olosuhteita, joissa bakteeribiofilmit muodostuvat ja elävät lääkinnällisiin laitteisiin liittyvissä infektioissa (ortopedisissa infektioissa ja hengityskonehoidosta johtuvassa keuhkokuumeessa). Lisäksi kolmen aiemmin tunnistetun biofilmiestäjän tehoa arvioitiin uudelleen käyttämällä äskettäin kehitettyjä malleja. Nämä estäjät sisälsivät kaksi dehydroabietiinihappojohdannaista, DHA1:n ja DHA2:n, sekä yhden flavaanijohdannaisen, FLA1:n. Kliinisessä ympäristössä biofilmit voivat usein koostua useista lajeista, joiden sietokyky mikrobilääkehoidolle voi yhdessä olla erilainen kuin lajien esiintyessä yksin. Tämän vuoksi ensimmäisessä tutkimuksessa optimoitiin Staphylococcus aureus- ja Pseudomonas aeruginosa -bakteerien yhteisviljelymalli kahden lajin biofilmin luomiseksi. Nämä lajit valittiin, koska niitä on usein eristetty lukuisiin eri infektioihin liittyvistä biofilmeistä, mukaan lukien lääkinnällisiin laitteisiin liittyvistä infektioista. Jotta monilajisia biofilmejä vastaan voidaan kehittää tehokkaita hoitoja, on välttämätöntä ymmärtää biofilmien dynamiikkaa. Tämän vuoksi osana tutkimusta verrattiin S. aureus- ja P. aeruginosa -bakteerien proteomeja (erityisesti pinta- ja eksoproteomeja) niiden esiintyessä yksin ja rinnakkain samassa biofilmissä. Proteomianalyysistä saadut vihjeet useista fenotyyppisistä muutoksista vahvistettiin sarjalla jatkotutkimuksia, jotka sisälsivät selvityksen patogeenien herkkyydestä tavanomaisille antibiooteille ja tutkimusyhdisteille (DHA1, DHA2 ja FLA1). Kolmesta testatusta yhdisteestä DHA1 oli ainoa, joka säilytti aktiivisuutensa S. aureus -biofilmiä vastaan sen muodostettua yhteisviljelmän P. aeruginosa -bakteerin kanssa. Toisessa ja kolmannessa tutkimuksessa pyrittiin arvioimaan kolmen biofilmi-inhibiittorin soveltuvuutta käytettäväksi infektioita ehkäisevissä materiaaleissa, erityisesti luuhun implantoiduissa laitteissa ja endotrakeaalisissa putkissa. Implantoitavien laitteiden kohdalla infektion ehkäisy ja oikea kudosintegraatio ovat yhtä tärkeitä hoidon onnistumisen kannalta. Siksi kehitimme ensin mallin, joka perustuu osteogeenisten solujen (SaOS-2) ja S. aureus -bakteerisolujen yhteisviljelyyn titaanin pinnalla. Tämä malli mahdollisti äskettäin tunnistettujen biofilmiestäjien tehon arvioinnin kliinisesti merkityksellisillä pinnoilla samalla, kun tutkittiin niiden vaikutuksia kudosten integraatioon. Lisäksi saatiin tietoa yhdisteiden vaikutuksista näiden kahden solutyypin välisiin vuorovaikutuksiin. Yhdisteen DHA1 havaittiin toimivan parhaiten sekä bakteerien kiinnittymisen ehkäisyn että soluyhteensopivuuden kannalta. Lääkinnällistä laitetta asetettaessa läsnä ei kuitenkaan ole vain kudoksen soluja ja mahdollisia bakteerisoluja, vaan myös isännän immuunisoluja. Implantaatio laukaisee usein tulehdusvasteen, joka saattaa heikentää irrallisten bakteerisolujen tuhoamista, sillä immuunisolujen toiminta kohdistuu patogeenin sijaan vieraan materiaalin hajottamiseen. Tämän vuoksi kehitettiin kolmas malli, joka perustuu bakteerien sekä isännän immuunisolujen, erityisesti neutrofiilien, yhteisviljelyyn. Tämän määrityksen tarkoituksena oli ennustaa biofilmiestäjien vaikutuksia neutrofiilien antimikrobiseen kapasiteettiin. Kolmesta testatusta yhdisteestä DHA1 näytti olevan ainoa, joka lisäsi neutrofiilien kykyä estää bakteerien kiinnittymistä pintoihin. Kolmessa uudessa mallissa saatujen tulosten perusteella yhdiste DHA1 valittiin sisällytettäväksi 3D-tulostettuun antimikrobiseen pinnoitteeseen titaanipinnoille. Kehitetylle materiaalille suoritetut toiminnallisuustestit vahvistivat sen soluyhteensopivuuden sekä kyvyn estää S. aureus -biofilmin muodostumista ja edistää neutrofiilien antimikrobikapasiteettia. Tämä väitöstutkimus pyrkii tarjoamaan biofilmitutkimusyhteisölle uusia menetelmiä, jotka pienentävät in vitro -kokeiden ja kliinisten olosuhteiden välistä kuilua. Työssä esitetyt tulokset myös valottavat kliinisesti merkittävän kahden lajin sekabiofilmin dynamiikkaa, minkä toivomme edistävän uusien hoitostrategioiden tunnistamista ja kehittämistä. Tutkimus esittelee uusia ehdokkaita lääkinnällisiin laitteisiin inkorporoitaviksi biofilmiestäjäyhdisteiksi, joista erityisen lupaava dehydroabietiinihappojohdannainen (DHA1) sisällytettiin koetoteutuksessa onnistuneesti 3D-tulostettuun titaani-implanttien antimikrobipäällysteeseen

Screening of natural compounds identifies ferutinin as an antibacterial and anti-biofilm compound

Antibacterial screenings are most commonly targeted at planktonic bacteria but less effort is dedicated to the exploration of agents acting on biofilms. Here, a natural compounds library was screened against Staphylococcus aureus using a 384-well plate platform to identify compounds preventing biofilm formation. Five structurally diverse hits were selected for follow-up studies: honokiol, tschimganidin, ferutinin, oridonin and deoxyshikonin. The compounds were evaluated against different bacterial species for their capacity to prevent and disrupt biofilms. The development of resistance and cytotoxicity were also investigated. Ferutinin displayed the best antibacterial activity, with a minimum inhibitory, bactericidal and biofilm preventive concentration of 25 mu M against S. aureus. It efficiently disrupted pre-formed biofilms (over 5-log reduction of viable cells) and reduced biofilm formation on a catheter in the presence of neutrophils. This work provides new information on the antibacterial activity of five natural compounds and identified ferutinin as a promising candidate against S. aureus biofilms.Peer reviewe

Chlamydia pneumoniae Interferes with Macrophage Differentiation and Cell Cycle Regulation to Promote Its Replication

Chlamydia pneumoniae is a ubiquitous intracellular bacterium which infects humans via the respiratory route. The tendency of C. pneumoniae to persist in monocytes and macrophages is well known, but the underlying host-chlamydial interactions remain elusive. In this work, we have described changes in macrophage intracellular signaling pathways induced by C. pneumoniae infection. Label-free quantitative proteome analysis and pathway analysis tools were used to identify changes in human THP-1-derived macrophages upon C. pneumoniae CV6 infection. At 48-h postinfection, pathways associated to nuclear factor kappa B (NF-kappa B) regulation were stressed, while negative regulation on cell cycle control was prominent at both 48 h and 72 h. Upregulation of S100A8 and S100A9 calcium binding proteins, osteopontin, and purine nucleoside hydrolase, laccase domain containing protein 1 (LACC1) underlined the proinflammatory consequences of the infection, while elevated NF-kappa B2 levels in infected macrophages indicates interaction with the noncanonical NF-kappa B pathway. Infection-induced alteration of cell cycle control was obvious by the downregulation of mini chromosome maintenance (MCM) proteins MCM2-7, and the significance of host cell cycle regulation for C. pneumoniae replication was demonstrated by the ability of a cyclin-dependent kinase (CDK) 4/6 inhibitor Palbociclib to promote C. pneumoniae replication and infectious progeny production. The infection was found to suppress retinoblastoma expression in the macrophages in both protein and mRNA levels, and this change was reverted by treatment with a histone deacetylase inhibitor. The epigenetic suppression of retinoblastoma, along with upregulation of S100A8 and S100A9, indicate host cell changes associated with myeloid-derived suppressor cell (MDSC) phenotype.Peer reviewe

Alterations in Bacterial Metabolism Contribute to the Lifespan Extension Exerted by Guarana in Caenorhabditis elegans

Guarana (Paullinia cupana) is a widely consumed nutraceutical with various health benefits supported by scientific evidence. However, its indirect health impacts through the gut microbiota have not been studied. Caenorhabditis elegans is a useful model to study both the direct and indirect effects of nutraceuticals, as the intimate association of the worm with the metabolites produced by Escherichia coli is a prototypic simplified model of our gut microbiota. We prepared an ethanoic extract of guarana seeds and assessed its antioxidant capacity in vitro, with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and in vivo, utilizing C. elegans. Additionally, we studied the impact of this extract on C. elegans lifespan, utilizing both viable and non-viable E. coli, and assessed the impact of guarana on E. coli folate production. The extract showed high antioxidant capacity, and it extended worm lifespan. However, the antioxidant and life-extending effects did not correlate in terms of the extract concentration. The extract-induced life extension was also less significant when utilizing dead E. coli, which may indicate that the effects of guarana on the worms work partly through modifications on E. coli metabolism. Following this observation, guarana was found to decrease E. coli folate production, revealing one possible route for its beneficial effects

Strategies to Prevent Biofilm Infections on Biomaterials: Effect of Novel Naturally-Derived Biofilm Inhibitors on a Competitive Colonization Model of Titanium by Staphylococcus aureus and SaOS-2 Cells

Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications

Alterations in Bacterial Metabolism Contribute to the Lifespan Extension Exerted by Guarana in Caenorhabditis elegans

Guarana (Paullinia cupana) is a widely consumed nutraceutical with various health benefits supported by scientific evidence. However, its indirect health impacts through the gut microbiota have not been studied. Caenorhabditis elegans is a useful model to study both the direct and indirect effects of nutraceuticals, as the intimate association of the worm with the metabolites produced by Escherichia coli is a prototypic simplified model of our gut microbiota. We prepared an ethanoic extract of guarana seeds and assessed its antioxidant capacity in vitro, with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and in vivo, utilizing C. elegans. Additionally, we studied the impact of this extract on C. elegans lifespan, utilizing both viable and non-viable E. coli, and assessed the impact of guarana on E. coli folate production. The extract showed high antioxidant capacity, and it extended worm lifespan. However, the antioxidant and life-extending effects did not correlate in terms of the extract concentration. The extract-induced life extension was also less significant when utilizing dead E. coli, which may indicate that the effects of guarana on the worms work partly through modifications on E. coli metabolism. Following this observation, guarana was found to decrease E. coli folate production, revealing one possible route for its beneficial effects

3D-Printed Drug Delivery Systems: The Effects of Drug Incorporation Methods on Their Release and Antibacterial Efficiency

Additive manufacturing technologies have been widely used in the medical field. More specifically, fused filament fabrication (FFF) 3D-printing technology has been thoroughly investigated to produce drug delivery systems. Recently, few researchers have explored the possibility of directly 3D printing such systems without the need for producing a filament which is usually the feedstock material for the printer. This was possible via direct feeding of a mixture consisting of the carrier polymer and the required drug. However, as this direct feeding approach shows limited homogenizing abilities, it is vital to investigate the effect of the pre-mixing step on the quality of the 3D printed products. Our study investigates the two commonly used mixing approaches—solvent casting and powder mixing. For this purpose, polycaprolactone (PCL) was used as the main polymer under investigation and gentamicin sulfate (GS) was selected as a reference. The produced systems’ efficacy was investigated for bacterial and biofilm prevention. Our data show that the solvent casting approach offers improved drug distribution within the polymeric matrix, as was observed from micro-computed topography and scanning electron microscopy visualization. Moreover, this approach shows a higher drug release rate and thus improved antibacterial efficacy. However, there were no differences among the tested approaches in terms of thermal and mechanical properties

3D-Printed Drug Delivery Systems: The Effects of Drug Incorporation Methods on Their Release and Antibacterial Efficiency

Additive manufacturing technologies have been widely used in the medical field. More specifically, fused filament fabrication (FFF) 3D-printing technology has been thoroughly investigated to produce drug delivery systems. Recently, few researchers have explored the possibility of directly 3D printing such systems without the need for producing a filament which is usually the feedstock material for the printer. This was possible via direct feeding of a mixture consisting of the carrier polymer and the required drug. However, as this direct feeding approach shows limited homogenizing abilities, it is vital to investigate the effect of the pre-mixing step on the quality of the 3D printed products. Our study investigates the two commonly used mixing approaches—solvent casting and powder mixing. For this purpose, polycaprolactone (PCL) was used as the main polymer under investigation and gentamicin sulfate (GS) was selected as a reference. The produced systems’ efficacy was investigated for bacterial and biofilm prevention. Our data show that the solvent casting approach offers improved drug distribution within the polymeric matrix, as was observed from micro-computed topography and scanning electron microscopy visualization. Moreover, this approach shows a higher drug release rate and thus improved antibacterial efficacy. However, there were no differences among the tested approaches in terms of thermal and mechanical properties

Strategies to Prevent Biofilm Infections on Biomaterials: Effect of Novel Naturally-Derived Biofilm Inhibitors on a Competitive Colonization Model of Titanium by Staphylococcus aureus and SaOS-2 Cells

Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications

Viola cornuta and Viola x wittrockiana: phenolic compounds, antioxidant and neuroprotective activities on Caenorhabditis elegans

Different Viola species are known for their traditional use as analgesic, antitussive, febrifuge, hipnotic, analgesic and anti-inflammatory medicinal agents. Additionally, they are considered edible flowers in certain cultures. Thus, the aim of this work was to characterize the phenolic composition and to assess the neuroprotective properties of Viola cornuta and Viola x wittrockiana using in vitro and in vivo methodologies with Caenorhabditis elegans as model. The identification of the phenolic compounds was carried out with a LCDAD- ESI/MSn. The antioxidant activity of the extracts was determined in vitro using Folin- Ciocalteu, DPPH and FRAP assays and in vivo with a juglone-induced oxidative stress in C. elegans. The neuroprotective properties were evaluated measuring the ability to inhibit CNS enzymes (MAO A, AChE), and the capability to avoid paralyzing the C. elegans CL4176, an Alzheimer disease model. The phenolic content was higher in V. x wittrockiana, being quercetin-3-O-(6-O-rhamnosylglucoside)-7-O-rhamnoside the predominant compound in the extract, which also exhibited a stronger antioxidant capacity in vitro and a higher response to lethal oxidative stress on C. elegans than V. cornuta. Only V. x wittrockiana showed inhibitory effect on CNS enzymes, such as acetylcholinesterase and monoamine oxidase A, but both had protective effect against the paralysis of C. elegans. These findings suggest that the studied V. cornuta and V. x wittrockiana could be interesting candidates for age related neurodegenerative disorder associated with oxidative stress.Universidad San Jorge is acknowledged for financial support and providing Cristina Moliner with a PhD scholarship. Innoflower S.L. is thanked for supplying fresh flowers. The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013) and L. Barros contract; to FEDER-Interreg Espa~na-Portugal programme for financial support through the project 0377_Iberphenol_6_E.info:eu-repo/semantics/publishedVersio