Bacterial infections: antimicrobial therapies


The Bacterial infections: antimicrobial therapies group is a junior group under IBEC’s Tenure Track scheme.


Eduard Torrents Serra | Group Leader
Maria del Mar Cendra Gascón | Postdoctoral Researcher
Marija Vukomanovic | Postdoctoral Researcher
Aida Baelo Álvarez | PhD Student
Núria Blanco Cabra | PhD Student
Laura Moya Andérico | PhD Student
Lucas Pedraz López | PhD Student
Maria Zimina | PhD Student
Paula Bellés Sancho | Masters Student
Núria Buxons Vives | Masters Student
Laura De Cubas Landaluce | Masters Student
Neus Sanfeliu Cerdán | Masters Student

Former Members
Maria del Mar Cendra | PhD Student

About

Infectious diseases constitute a tenacious and major public health problem all over the world. The emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics demand the discovery of new therapeutic approaches.

Pseudomonas aeruginosa cells treated with the antibiotic carbenicillin

In addition, there is an urgent need for reliable and rapid detection of infecting bacteria and its pattern of resistance to antibiotics.

Bacterial DNA synthesis open new horizons in the discovery of new antibacterial targets due to remarkably differences to the eukaryotic system. The enzyme ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides (dNTP) and thereby provides the building blocks for DNA synthesis and repair. The balance of the different dNTPs has to be carefully regulated and the RNR enzymes as well as its expression play important roles. In the bacterial world it is not known which transcriptional regulators are required to control the expression of the different RNR genes, their role in virulence and during bacterial biofilm formation. RNRs could be considered a good antimicrobial target candidate to inhibit bacterial growth because they present substantial differences relative to their eukaryote counterparts. The discoveries of new molecules against the activity of this system is crucial to be explored.

Detailed microscopy observations of structured biofilms from Pseudomonas aeruginosa PAO1 wild-type and different ribonucleotide reductase mutants. On the left side, a scheme of the longitudinal structure of P. aeruginosa biofilm is represented, labeled with indications of the oxygen concentration along the biofilm. On the right side, confocal laser scanning microscopy images are shown, which were taken from the aerobic region of the biofilm (top part, superficial biofilm) and from the anaerobic region (bottom part, deeper in the biofilm structure). The corresponding average thickness of each strain is representative of three independent experiments.

Our lab aims to investigate new antimicrobial therapies to combat bacterial infections with different objectives:
First, to establish the molecular basis for the regulation of RNR genes, their importance in virulence and biofilm formation;
Second, the identification and screening of new molecules for the highly selective inhibition of bacterial RNR;
Third, by using nanomedicine techniques the development of novel and specific nanoparticles to deliver existing antibiotics or new identify antimicrobial drugs, especially when the bacteria are growing in biofilm, close to the physiological conditions of the disease and where the current chemotherapy fails;
Fourth, we are studying new methodologies to threat bacterial chronic infections in patients suffering cystic fibrosis;
Finally, we will use lab-on-a-chip technology to deeply elucidate mechanisms to combat bacterial forming biofilm as well as new approaches to identify multiresistant bacteria to different antibiotics.

We believe these projects will be beneficial to society since we explore the use of different bioengineering approaches to elucidate ways to diagnose and eradicate multi-drug resistant bacteria.


Related links

https://sites.google.com/view/torrentslabwebpage

News/Jobs

IBEC-mentored student wins prize for research project
03/04/17

One of IBEC’s mentees under the BATX2LAB programme was a prize winner last Wednesday at the initiative’s annual ceremony to award the best research projects.


A new therapeutic target that could slow the progression of Parkinson’s disease
28/02/17

Researchers at IBEC have identified a potential way to slow down the neurodegenerative progression of Parkinson’s disease.


IBEC researchers win EIT Health awards
19/12/16

IBEC group leaders Eduard Torrents and Elisabeth Engel both received awards at last week’s EIT Health Spain event at the PCB.


Mycobacterium in olive oil for cancer treatment
23/06/16

Researchers at the Autonomous University of Barcelona (UAB) and IBEC have revealed a way to effectively deliver a mycobacterium needed for the treatment of bladder cancer in humans –using a formulation based on olive oil.


The enzymes that enable bacteria to make themselves at home
09/05/16

IBEC researchers have come a step closer to understanding how bacteria can cause chronic infections by identifying the key enzymes that allow them to create the right conditions for infection.


Telenoticias Hospitalet: “El present i el futur de la Fibrosi Quística”
04/05/16

The news programme L’Informatiu in L’Hospitalet featured the Cystic Fibrosis event, “El present i el futur de la Fibrosi Quística”, that was organised by IBEC and the ACFQ on 27th April.


Possible new treatment for bladder cancer using a mycobacterium
12/10/15

Collaborators at the UAB and IBEC have found a mycobacterium that is more effective in treating superficial bladder cancer and does not cause infections, unlike those used up to now.


IBEC groups join forces to combat chronic bacterial infections
28/04/2015

A study published today in the Journal of Controlled Release describes a new nanoparticle strategy able to target hard-to tackle infections caused by biofilm-forming bacteria.


New hope against growing threat of antibiotic resistance
18/03/2015

The increasing prevalence of bacteria that are resistant to current antibiotics pose an escalating threat to human health. Now, researchers in Barcelona have identified a molecule with huge potential as a new type of antibacterial agent.


Key player identified in bacterial infections
21/01/2015

A group at IBEC has a identified a important factor in E. coli infection, opening the way to developing targeted drugs against the potentially deadly condition.


“Suport a les unitats de FQ de Sant Joan de Déu, Parc Taulí i Vall d’Hebron, l’IDIBELL i l’IBEC”
23/12/2014

Eduard Torrents and his group appear a couple of times in the December 2014 edition of the newspaper of the Associació Catalana de Fibrosi Quística, Per a Vèncer la Fibrosi Quística. One article is about their contribution to a research and clinical trials day, and the other is about the funding they receive from the association for basic research into CF.

Download the issue here.


“Desenvolupament d’una nova família de compostos antimicrobians”
29/11/2014

Junior group leader Eduard Torrents gave a talk at the Jornada Formativa Anual 2014 of the Associació Catalana de Fibrosi Quística.


Further Pablo Motos support for IBEC research into bacterial infections
11/02/2013

Junior group leader Eduard Torrents is the recipient of a third “Pablo Motos” award from the Federación Española de Fibrosis Quística (FEFQ) to continue his research into identifying new antimicrobial molecules to fight the bacteria that cause complications and even death in many people with cystic fibrosis.


“El combate contra la pseudomona en manos del Dr. Torrents”
27/09/2012

Senior researcher Eduard Torrents appears several times in the July edition of the newspaper of the Associació Catalana de Fibrosi Quística, Per a Vèncer la Fibrosi Quística.


Continuing the fight against cystic fibrosis
03/05/2012

IBEC Senior Researcher Eduard Torrents participated in a conference to mark the National Day for Cystic Fibrosis last Wednesday 25 April. This event, which took place at the Spanish Society of Pneumology and Thoracic Surgery (SEPAR), also included the official presentation of funds from the Associació Catalana de Fibrosis Quística (Catalan Association of Cystic Fibrosis) to research groups specializing in the disease.


Opening new doors to combat bacterial infections
26/04/2011

We may be several steps closer to understanding one of the major pathologies that affects sufferers of cystic fibrosis, thanks to Senior researcher Eduard Torrents of IBEC’s Microbial biotechnology and host-pathogen interaction group


IBEC researcher receives Pablo Motos award
12/01/2011

Eduard Torrents, senior researcher in IBEC’s Microbial Biotechnology and Host-pathogen Interaction group, has been announced as a recipient of the 2010 Pablo Motos award from the Federación Española de Fibrosis Quística (Spanish Association of Cystic Fibrosis) this week.

Projects

International projects
BACTSHOT Novel antimicrobial therapy (2016-2017) EIT Health Head Start – Proof of Concept Eduard Torrents
National projects
Redes reguladoras de la expresión génica de las distintas ribonucleotidil reductasas en bacterias MINECO, I+D-Investigación fundamental no orientada Eduard Torrents
inhibitRNR Las ribonucleotido reductasas como una nueva diana terapéutica frente a patógenos bacterianos (2016-2018) MINECO, Retos investigación: Proyectos I+D Eduard Torrents
Privately-funded projects
Desarrollo de una nueva familia de compuestos antimicrobianos Asociación Catalana de Fibrosis Quística Eduard Torrents
Identificación y administración de nuevas moléculas antimicrobianas contra Pseudomonas aeruginosa creciendo en biofilm Asociación Española Fibrosis Quística, Becas de Investigación “Pablo Motos” Eduard Torrents
Ribonucleotide reductasas: una nueva diana terapéutica contra organismos patógenos en enfermos de fibrosis quística (2010-2017) Asociación Española Fibrosis Quística, Becas de Investigación “Pablo Motos” Eduard Torrents
RNRbiotic New strategy to combat bacterial infections (2015-2017) Obra Social La Caixa, Caixaimpulse Eduard Torrents
Novel strategies to combat bacterial chronic infections by the development of microfluidics platforms to analyse and treat bacterial growing in biofilms (2016) Obra Social La Caixa Eduard Torrents

Publications


Urrea, L., Segura-Feliu, M., Masuda-Suzukake, M., Hervera, A., Pedraz, L., Aznar, J. M. G., Vila, M., Samitier, J., Torrents, E., Ferrer, I., Gavín, R., Hagesawa, M., Del Río, J. A., (2017). Involvement of cellular prion protein in Molecular Neurobiology online, 1-14

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of

Keywords: Amyloid spreading, Microfluidic devices, Prnp, Synuclein


D'Auria, Giuseppe, Torrents, Eduard, Luquin, Marina, Comas, Iñaki, Julián, Esther, (2016). Draft genome sequence of Mycobacterium brumae ATCC 51384 Genome Announcements 4, (2), e00237-16

Here, we report the draft genome sequence of Mycobacterium brumae type strain ATCC 51384. This is the first draft genome sequence of M. brumae, a nonpathogenic, rapidly growing, nonchromogenic mycobacterium, with immunotherapeutic capacities.


Noguera-Ortega, Estela, Secanella-Fandos, Silvia, Eraña, Hasier, Gasión, Jofre, Rabanal, Rosa M., Luquin, Marina, Torrents, Eduard, Julián, Esther, (2016). Nonpathogenic Mycobacterium brumae inhibits bladder cancer growth in vitro, ex vivo, and in vivo European Urology Focus 2, (1), 67-76

Background Bacillus Calmette-Guérin (BCG) prevents tumour recurrence and progression in non–muscle-invasive bladder cancer (BC). However, common adverse events occur, including BCG infections. Objective To find a mycobacterium with similar or superior antitumour activity to BCG but with greater safety. Design In vitro, ex vivo, and in vivo comparisons of the antitumour efficacy of nonpathogenic mycobacteria and BCG. Intervention The in vitro antitumour activity of a broad set of mycobacteria was studied in seven different BC cell lines. The most efficacious was selected and its ex vivo capacity to activate immune cells and its in vivo antitumour activity in an orthotopic murine model of BC were investigated. Outcome measurements and statistical analysis Growth inhibition of BC cells was the primary outcome measurement. Parametric and nonparametric tests were use to analyse the in vitro results, and a Kaplan-Meier test was applied to measure survival in mycobacteria-treated tumour-bearing mice. Results and limitations Mycobacterium brumae is superior to BCG in inhibiting low-grade BC cell growth, and has similar effects to BCG against high-grade cells. M. brumae triggers an indirect antitumour response by activating macrophages and the cytotoxic activity of peripheral blood cells against BC cells. Although no significant differences were observed between BCG and M. brumae treatments in mice, M. brumae treatment prolonged survival in comparison to BCG treatment in tumour-bearing mice. In contrast to BCG, M. brumae does not persist intracellularly or in tumour-bearing mice, so the risk of infection is lower. Conclusions Our preclinical data suggest that M. brumae represents a safe and efficacious candidate as a therapeutic agent for non–muscle-invasive BC. Patient summary We investigated the antitumour activity of nonpathogenic mycobacteria in in vitro and in vivo models of non–muscle-invasive bladder cancer. We found that Mycobacterium brumae effectively inhibits bladder cancer growth and helps the host immune system to eradicate cancer cells, and is a promising agent for antitumour immunotherapy.

Keywords: Animal models, Bacillus Calmette-Guérin, Cytokines, Immunomodulation, Immunotherapy, Mycobacteria, Urothelial cell line


Noguera-Ortega, E., Rabanal, R. M., Secanella-Fandos, S., Torrents, E., Luquin, M., Julián, E., (2016). Gamma-irradiated mycobacteria enhance survival in bladder tumor bearing mice although less efficaciously than live mycobacteria Journal of Urology 195, (1), 198-205

Basas, J., Morer, A., Ratia, C., Martín, M.T., del Pozo, J.L., Gomis, X., Rojo-Molinero, E., Torrents, E., Almirante, B., Gavaldà, J., (2016). Efficacy of anidulafungin in the treatment of experimental Candida parapsolosis catheter infection Journal of Antimicrobial Chemotherapy 71, (10), 2895-2901

Objectives The effectiveness of anidulafungin versus liposomal amphotericin B (LAmB) for treating experimental Candida parapsilosis catheter-related infection by an antifungal-lock technique was assessed. Methods Two clinical strains of C. parapsilosis (CP12 and CP54) were studied. In vitro studies were used to determine the biofilm MICs (MBIC50 and MBIC90) by XTT reduction assay and LIVE/DEAD biofilm viability for anidulafungin and LAmB on 96-well microtitre polystyrene plates and silicone discs. An intravenous catheter was implanted in New Zealand white rabbits. Infection was induced by locking the catheter for 48 h with the inoculum. The 48 h antifungal-lock treatment groups included control, 3.3 mg/mL anidulafungin and 5.5 mg/mL LAmB. Results Anidulafungin showed better in vitro activity than LAmB against C. parapsilosis growing in biofilm on silicone discs. MBIC90 of LAmB: CP12, >1024 mg/L; CP54, >1024 mg/L. MBIC90 of anidulafungin: CP12, 1 mg/L; CP54, 1 mg/L (P ≤ 0.05). Moreover, only anidulafungin (1 mg/L) showed >90% non-viable cells in the LIVE/DEAD biofilm viability assay on silicone discs. No differences were observed between the in vitro susceptibility of anidulafungin or LAmB when 96-well plates were used. Anidulafungin achieved significant reductions relative to LAmB in log10 cfu recovered from the catheter tips for both strains (P ≤ 0.05). Only anidulafungin achieved negative catheter tip cultures (CP12 63%, CP54 73%, P ≤ 0.05). Conclusions Silicone discs may be a more reliable substrate for the study of in vitro biofilm susceptibility of C. parapsilosis. Anidulafungin-lock therapy showed the highest activity for experimental catheter-related infection with C. parapsilosis.


Noguera-Ortega, E., Blanco-Cabra, N., Rabanal, R.M., Sanchez-Chardi, A., Roldán, M., Torrents, E., Luquin, M., Julián, E., (2016). Mycobacteria emulsified in olive oil-in-water trigger a robust immune response in bladder cancer treatment Scientific Reports 6, 27232

The hydrophobic composition of mycobacterial cell walls leads to the formation of clumps when attempting to resuspend mycobacteria in aqueous solutions. Such aggregation may interfere in the mycobacteria-host cells interaction and, consequently, influence their antitumor effect. To improve the immunotherapeutic activity of Mycobacterium brumae, we designed different emulsions and demonstrated their efficacy. The best formulation was initially selected based on homogeneity and stability. Both olive oil (OO)- and mineral oil-in-water emulsions better preserved the mycobacteria viability and provided higher disaggregation rates compared to the others. But, among both emulsions, the OO emulsion increased the mycobacteria capacity to induce cytokines’ production in bladder tumor cell cultures. The OO-mycobacteria emulsion properties: less hydrophobic, lower pH, more neutralized zeta potential, and increased affinity to fibronectin than non-emulsified mycobacteria, indicated favorable conditions for reaching the bladder epithelium in vivo. Finally, intravesical OO-M. brumae-treated mice showed a significantly higher systemic immune response, together with a trend toward increased tumor-bearing mouse survival rates compared to the rest of the treated mice. The physicochemical characteristics and the induction of a robust immune response in vitro and in vivo highlight the potential of the OO emulsion as a good delivery vehicle for the mycobacterial treatment of bladder cancer.


Crespo, Anna, Pedraz, Lucas, Astola, Josep, Torrents, Eduard, (2016). Pseudomonas aeruginosa exhibits deficient biofilm formation in the absence of class II and III ribonucleotide reductases due to hindered anaerobic growth Frontiers in Microbiology 7, Article 688

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.

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


Baelo, Aida, Julián, Esther, Torrents, Eduard, (2016). Methyl-hydroxylamine specifically inhibits ribonucleotide reductase activity in pathogenic bacteria New Biotechnology Biotech Annual Congress (BAC 2015) , Elsevier (Salamanca, Spain) 33, (3), 417

Infectious diseases constitute a tenacious and major public-health problem all over the world; the emergence and increasing prevalence of multi-drug resistant bacteria demand the discovery of new therapeutic approaches. Bacterial DNA synthesis opens new horizons in the discovery of new antibacterial targets due to remarkable differences to the eukaryotic system. During the course of an infection, a great number of bacteria need to multiply inside the body and, for that, active DNA synthesis with a balanced supply of deoxyribonucleotides (dNTPs) is required. RiboNucleotide Reductase (RNR) is the key enzyme that provides the nucleotide precursors for DNA replication and repair. This enzyme is a suitable target candidate for bacterial growth inhibition. In this work we have firstly identified the radical scavenger methyl-hydroxylamine (M-HA) as an efficacious antimicrobial agent that inhibits gram-negative and gram-positive pathogenic bacteria, targeting the RNR enzyme. Later, we have focused our work studying the ability of M-HA to inhibit the intracellular growth of Mycobacteria in macrophages, and the formation of Pseudomonas aeruginosa biofilms.


Pedraz, Lucas, Crespo, Anna, Torrents, Eduard, (2016). A single transcription factor behind all bacterial dNTP synthesis revealed as a novel antimicrobial target New Biotechnology Biotech Annual Congress (BAC 2015) , Elsevier (Salamanca, Spain) 33, (3), 410

Nowadays, the fear of infectious diseases is again increasing. Antibiotic-resistant bacterial strains are appearing worldwide, and so there is an urgent need to develop new antimicrobial drugs. Ribonucleotide Reductases (RNRs) are essential enzymes that catalyse the reduction of ribonucleotides (NTPs) to their corresponding deoxyribonucleotides (dNTPs), thereby forming the building blocks for DNA synthesis and repair. A drug able to inhibit bacterial Ribonucleotide Reductase activity would completely inhibit bacterial growth. Behind bacterial Ribonucleotide Reductase activity there is a complex regulon; although eukaryotic cells codify only for one RNR enzyme, bacteria can use three different RNR classes, granting them a huge adaptability. Pseudomonas aeruginosa is a major human opportunistic pathogen, causing severe lung chronic infections in cystic fibrosis and COPD patients. It codifies for all three RNR classes, in a complex regulon necessary for its adaptability and virulence. The main focus of this work is a transcription factor, called NrdR, which is present in almost all bacterial species, and completely absent in eukaryotic organisms. This factor acts as a central regulator of all RNR enzymes in bacteria, hence being behind all dNTP synthesis. We have studied how NrdR regulates RNR activity in P. aeruginosa, being able to this point to propose a first model of the NrdR regulon, and being a step closer to new antimicrobial therapies.


Julián, E., Rabanal, R. M., Secanella-Fandos, S., Torrents, E., Luquin, M., Noguera-Ortega, E., (2016). Eficacia de micobacterias Gamma-irradiadas en el tratamiento de cancer vesical no-músculo invasivo Enfermedades Infecciosas y Microbiología Clínica XX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Barcelona, Spain) 34, (SE1), 229

Las micobacterias son las únicas bacterias utilizadas en el tratamiento del cáncer. Concretamente Mycobacterium bovis BCG se instila intravesicalmente en pacientes de cáncer vesical no-músculo invasivo, tras la resección del tumor, con el fin de evitar recidivas. A pesar de su eficacia, BCG presenta numerosos efectos adversos, entre ellos casos de infección por BCG. En nuestro laboratorio hemos desarrollado dos estrategias para evitar el riesgo de infección. Por un lado, hemos mostrado la capacidad antitumoral in vitro de BCG muerta mediante gamma-irradiación. Por otro lado, hemos demostrado la capacidad antitumoral de Mycobacterium brumae viva, tanto en modelos in vitro como en el modelo animal de la enfermedad. Aun así, se desconoce el potencial antitumoral de estas micobacterias irradiadas in vivo, y si su eficacia es comparable. El objetivo fue evaluar la capacidad antitumoral de BCG y M. brumae gamma-irradiadas en el modelo murino de cáncer vesical.


Torrents, E., Baelo, Aida, Levato, R., Julián, E., Crespo, Anna, Astola, Josep, Gavaldà, J., Engel, E., Mateos-Timoneda, M.A., (2016). Mejora en la administración antibiotic para el tratamiento de infecciones en forma de biofilm con el uso de nanopartículas que disgregan la matriz extracellular Enfermedades Infecciosas y Microbiología Clínica XX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Barcelona, Spain) 34, (SE1), 31

Las infecciones causadas por bacterias formadoras de biopelículas o biofilms son una amenaza importante para los pacientes hospitalizados y suponen la principal causa de infecciones crónicas, como las producidas en la enfermedad pulmonar obstructiva crónica (EPOC) y la fibrosis quística. Existe una necesidad urgente de desarrollar nuevos antibióticos o nuevos enfoques terapéuticos que permitan el tratamiento de este tipo de infecciones ya que los antibióticos convencionales no logran eliminar las bacterias que están formando biofilms


Baelo, Aida, Levato, Riccardo, Julián, Esther, Crespo, Anna, Astola, José, Gavaldà, Joan, Engel, Elisabeth, Mateos-Timoneda, Miguel Angel, Torrents, Eduard, (2015). Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections Journal of Controlled Release 209, 150-158

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.

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


Dreux, Nicolas, Cendra, Maria del Mar, Massier, Sébastien, Darfeuille-Michaud, Arlette, Barnich, Nicolas, Torrents, Eduard, (2015). Ribonucleotide reductase NrdR as a novel regulator for motility and chemotaxis during adherent-invasive Escherichia coli infection Infection and Immunity 83, (4), 1305-1317

A critical step in the life cycle of all organisms is the duplication of the genetic material during cell division. Ribonucleotide reductases (RNRs) are essential enzymes for this step because they control the de novo production of the deoxyribonucleotides required for DNA synthesis and repair. Enterobacteriaceae have three functional classes of RNRs (Ia, Ib and III), which are transcribed from separate operons and encoded, respectively by the genes nrdAB, nrdHIEF and nrdDG. Here, we investigated the role of RNRs in the virulence of adherent-invasive E. coli (AIEC) isolated from Crohn's disease (CD) patients. Interestingly, the LF82 strain of AIEC harbors four different RNRs (two class Ia, one class Ib and one class III). Although the E. coli RNR enzymes have been extensively characterized both biochemically and enzymatically, little is known about their roles during bacterial infection. We found that RNR expression was modified in AIEC LF82 bacteria during cell infection, suggesting that RNRs play an important role in AIEC virulence. Knockout of the nrdR and nrdD genes, which encodes a transcriptional regulator of RNRs and class III anaerobic RNR respectively, decreased AIEC LF82's ability to colonize the gut mucosa of transgenic mice that express human CEACAM6 (carcinoembryonic antigen-related cell-adhesion molecule 6). Microarray experiments demonstrated that NrdR plays an indirect role in AIEC virulence by interfering with bacterial motility and chemotaxis. Thus, the development of drugs targeting RNR classes, in particular NrdR and NrdD, could be a promising new strategy to control gut colonization by AIEC bacteria in CD patients.


Barreiros dos Santos, M., Azevedo, S., Agusil, J. P., Prieto-Simón, B., Sporer, C., Torrents, E., Juárez, A., Teixeira, V., Samitier, J., (2015). Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria Bioelectrochemistry 101, 146-152

Abstract Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm− 2 (0.08 pmol cm− 2). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL− 1) over a large linear working range (10–106 CFU mL− 1). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Keywords: E. coli O157:H7, Electrochemical Impedance Spectroscopy, Immunosensor, Indium tin oxide, Label-free detection


Crespo, A., Pedraz, L., Torrents, E., (2015). Function of the Pseudomonas aeruginosa NrdR transcription factor: Global transcriptomic analysis and its role on ribonucleotide reductase gene expression PLoS ONE 10, (4), e0123571

Ribonucleotide reductases (RNRs) are a family of sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides (dNTPs), the building blocks for DNA synthesis and repair. Although any living cell must contain one RNR activity to continue living, bacteria have the capacity to encode different RNR classes in the same genome, allowing them to adapt to different environments and growing conditions. Pseudomonas aeruginosa is well known for its adaptability and surprisingly encodes all three known RNR classes (Ia, II and III). There must be a complex transcriptional regulation network behind this RNR activity, dictating which RNR class will be expressed according to specific growing conditions. In this work, we aim to uncover the role of the transcriptional regulator NrdR in P. aeruginosa. We demonstrate that NrdR regulates all three RNR classes, being involved in differential control depending on whether the growth conditions are aerobic or anaerobic. Moreover, we also identify for the first time that NrdR is not only involved in controlling RNR expression but also regulates topoisomerase I (topA) transcription. Finally, to obtain the entire picture of NrdR regulon, we performed a global transcriptomic analysis comparing the transcription profile of wild-type and nrdR mutant strains. The results provide many new data about the regulatory network that controls P. aeruginosa RNR transcription, bringing us a step closer to the understanding of this complex system.


Julián, E., Baelo, A., Gavaldà, J., Torrents, E., (2015). Methyl-hydroxylamine as an efficacious antibacterial agent that targets the ribonucleotide reductase enzyme PLoS ONE 10, (3), e0122049

The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound's activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RNR enzyme.


Barniol-Xicota, M., Escandell, A., Valverde, E., Julián, E., Torrents, E., Vázquez, S., (2015). Antibacterial activity of novel benzopolycyclic amines Bioorganic and Medicinal Chemistry 23, (2), 290-296

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.

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


Jaramillo, Maria del Carmen, Huttener, Mario, Alvarez, Juan Manuel, Homs-Corbera, Antoni, Samitier, Josep, Torrents, Eduard, Juárez, Antonio, (2015). Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice Electrophoresis 36, (13), 1471-1478

DEP manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed and recovered in a small-volume fraction which, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.

Keywords: Dielectrophoresis, PCR, Saccharomyces, Yeast


Basas, J., Rojo, E., Gomis, X., Sierra, J.M., Torrents, E., Almirante, B., Gavaldà, J., (2015). Actividad de anidulafungia vs. anfotericina b liposomal frente a C. Parapsilopsis creciendo en biopelículas en distintos materiales Enfermedades Infecciosas y Microbiología Clínica XIX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Sevilla, Spain) 33, 37

La infección del catéter venoso central está estrechamente relacionada con la capacidad de los microorganismos para producir biopelículas. Existen metodologías distintas para el estudio in vitro de la sensibilidad antibiótica de microorganismos creciendo en biopelículas; con placas de microtitulación (poliestireno) y con diferentes materiales (discos silicona, placas titanio...). Se ha descrito que las especies de Candida crecen con morfologías diferentes dependiendo del sustrato donde se implantan. Por tanto, la elección del material donde crecen las biopelículas podría tener su importancia en los estudios de sensibilidad in vitro. Previamente, en un modelo experimental de infección de catéter por C. parapsilopsis observamos que el sellado con anidulafungina (And) era más eficaz que con anfotericina B liposomal (LAmB). Teniendo en cuenta estas consideraciones el mejor sustrato para valorar la eficacia in vitro sería en discos de silicona (utilizado para la fabricación de catéteres) y no en placas de microtitulación (técnica estándar).


Torrents, E., (2015). Tratamientos antimicrobianos dirigidos. ¿Es posible la nanomedicina en las enfermedades infecciosas? Enfermedades Infecciosas y Microbiología Clínica XIX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Sevilla, Spain) 33, 6

A lo largo del siglo XX, los avances en el desarrollo de los antibióticos han jugado un papel de gran importancia en la lucha contra las enfermedades infecciosas. Sin embargo, el uso inadecuado de éstos está conduciendo a la aparición de resistencias a múltiples fármacos (multidrug resistance, MDR) en diversos patógenos. El gran costo y complejidad asociados al descubrimiento de nuevos fármacos agrava la situación, propiciando que un número muy reducido de nuevos antibióticos se haya descubierto en los últimos 40 años. Esta situación provocará, si no se toman acciones para evitarlo, un gran problema de salud pública global durante el siglo XXI. En la última década se ha producido, en cambio, un gran avance en el campo de la nanotecnología, permitiendo el diseño de nanopartículas con propiedades fisicoquímicas deseables para su uso en microbiología. La escala de estas partículas ofrece un gran incremento en su relación superficie/volumen respecto a otras formas de liberación de fármacos, lo que ha permitido reconsiderar el uso de antiguas sustancias antimicrobianas, como la plata, el cobre o el zinc. Las nanopartículas se están así proyectando como una nueva línea de defensa contra los patógenos bacterianos, en especial los multirresistentes. Se comentarán los avances recientes en el diseño de nanopartículas, demostrando el potencial de éstas en la lucha contra las infecciones bacterianas. Igualmente, se comentarán nuevas estrategias, como la combinación diferentes fármacos y/o antibióticos encapsulados en nanopartículas (nanoantibióticos). Por último, explicaremos nuestra propia experiencia en el uso de nanopartículas de PLGA, o ácido poli(láctico-co-glicólico) modificadas, para la lucha contra la bacteria Pseudomonas aeruginosa creciendo en forma de biofilm.


Torrents, E., Albericio, F., Miret, L., (2014). Primary hydroxylamines and uses thereof Fundació Institut de Bioenginyeria de Catalunya; Fundació Institut de Recerca Biomédica , (EP14382032.2)

Cendra, M. M., Torrents, E., (2014). Enzims essencials per a la vida Treballs de la Societat Catalana de Biologia 65, 64-67

Les ribonucleòtid-reductases (RNR) són enzims essencials per a tota cèllula, perquè fan la transformació dels ribonucleòtids a desoxiribonucleòtids, els quals són necessaris per a la síntesi de l’àcid desoxiribonucleic (DNA). És evident que les RNR són enzims ancestrals i clau en l’evolució del material genètic que hi ha actualment, i són essencials per a l’evolució de tots els organismes que hi ha sobre la Terra. A causa de l’essencialitat de la reacció que fan aquests enzims, es poden considerar una diana ideal per al disseny de compostos que inhibeixen la replicació cel·lular, ja sigui en cèl·lules eucariòtiques (incloent-hi cèl·lules cancerígenes), com agents bacterians infecciosos.


Oliva, A. M., Homs-Corbera, A., Torrents, E., Juarez, A., Samitier, J., (2014). Synergystic effect of temperature and electric field intensity in Escherichia coli inactivation Micro and Nanosystems 6, (2), 79-86

Electric Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present a study on the effects of electric field intensity and temperature to bacterial cultures. Escherichia coli colonies have been exposed to different electric field intensities at 1MHz during 5 minutes by means of a microfluidic device specially designed for the experiment. From the analysis of the results it is possible to see that Escherichia coli survival rate diminishes when applying field intensities as low as 220V during 5 minutes. Death rates also increase when stronger fields are applied. However, viability of survived bacteria is maintained. Additionally, temperature shows a synergistic effect with voltage. When temperature was increased, results showed a stronger sensitivity of cells to the electric field. Moreover, the expression patterns of Outer Membrane Protein A and Ribosomal Proteins differ in control and treated samples, suggesting changes in bacterial metabolism and structure.

Keywords: E. coli, Electric field, Temperature, Viability


Torrents, Eduard, (2014). Ribonucleotide reductases: Essential Enzymes for bacterial life Frontiers in Cellular and Infection Microbiology 4, 1-9

Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria.

Keywords: Anaerobiosis, Transcription Factors, Evolution, Gene regulation, Ribonucleotide reductase, DNA Synthesis, NrdR,nrd


Oliva, A. M., Homs, A., Torrents, E., Juarez, A., Samitier, J., (2014). Effect of electric field and temperature in E.Coli viability IFMBE Proceedings XIII Mediterranean Conference on Medical and Biological Engineering and Computing 2013 (ed. Roa Romero, Laura M.), Springer (Seville, Spain) 41, 1833-1836

Electromagnetic Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present preliminary data on the effect of electric field intensity and temperature applica- tion. E. Coli colonies have been exposed to different voltages at 1MHz during 5 minutes by means of a custom-made micro- fluidic device. Results show that E.Coli survival rate is already reduced by applying field intensities as low as 220V/cm during 5 minutes. The use of stronger fields resulted in death rates increase also. Viability of survived bacteria was maintained. On the other hand, temperature has shown a synergistic effect with voltage. When temperature is increased results seem to indicate stronger sensitivity of cells to the electric field. It is necessary to continue studying the contribution of other para- meters as intensity, time, frequency or concentration, to study further synergies.

Keywords: E. Coli, Electromagnetic Field, Temperature, Viability


Jaramillo, M. C., Martínez-Duarte, R., Hüttener, M., Renaud, P., Torrents, E., Juárez, A., (2013). Increasing PCR sensitivity by removal of polymerase inhibitors in environmental samples by using dielectrophoresis Biosensors and Bioelectronics 43, (1), 297-303

Dielectrophoresis (DEP) is a powerful tool to manipulate cells and molecules in microfluidic chips. However, few practical applications using DEP exist. An immediate practical application of a carbon-electrode DEP system, in removing PCR inhibitors from a sample, is reported in this work. We use a high throughput carbon-electrode DEP system to trap yeast cells from a natural sample (fermented grape must) and then in situ remove contaminants that interfere with PCR analysis. Retrieval of this enriched and purified yeast population from the DEP system then allows for a significant increase of sensitivity during PCR analysis. Furthermore, the fact that DEP can discriminate between viable and non-viable cells minimizes the number of false positives commonly obtained when using PCR alone. Experimental results provide clear evidence that the carbon-electrode DEP-based sample preparation step can readily and effectively clean environmental samples from natural contaminants and improve PCR sensitivity.


Otero, J., Baños, R., González, L., Torrents, E., Juárez, A., Puig-Vidal, M., (2013). Quartz tuning fork studies on the surface properties of Pseudomonas aeruginosa during early stages of biofilm formation Colloids and Surfaces B: Biointerfaces 102, 117-123

Scanning probe microscopy techniques are powerful tools for studying the nanoscale surface properties of biofilms, such as their morphology and mechanical behavior. Typically, these studies are conducted using atomic force microscopy probes, which are force nanosensors based on microfabricated cantilevers. In recent years, quartz tuning fork (QTF) probes have been used in morphological studies due to their better performance in certain experiments with respect to standard AFM probes. In the present work QTF probes were used to measure not only the morphology but also the nanomechanical properties of Pseudomonas aeruginosa during early stages of biofilm formation. Changes in bacterium size and the membrane spring constant were determined in biofilms grown for 20, 24 and 28. h on gold with and without glucose in the culture media. The results obtained using the standard AFM and QTF probes were compared. Both probes showed that the bacteria forming the biofilm increased in size over time, but that there was no dependence on the presence of glucose in the culture media. On the other hand, the spring constant increased over time and there was a clear difference between biofilms grown with and without glucose. This is the first time that QTF probes have been used to measure the nanomechanical properties of microbial cell surfaces and the results obtained highlight their potential for studying biological samples beyond topographic measurements.


Cendra, M. M., Juárez, A., Madrid, C., Torrents, E., (2013). H-NS is a novel transcriptional modulator of the ribonucleotide reductase genes in escherichia coli Journal of Bacteriology 195, (18), 4255-4263

Ribonucleotide reductases (RNRs) are essential enzymes for DNA synthesis because they are responsible for the production of the four deoxyribonucleotides (dNTPs) from their corresponding ribonucleotides. Escherichia coli contains two classes of aerobic RNRs, encoded by the nrdAB (class Ia) and nrdHIEF (class Ib) operons, and a third RNR class, which is functional under anaerobic conditions and is encoded by the nrdDG (class III) operon. Because cellular imbalances in the amounts of the four dNTPs cause an increase in the rate of mutagenesis, the activity and the expression of RNRs must be tightly regulated during bacterial chromosome replication. The transcriptional regulation of these genes requires several transcription factors (including DnaA, IciA, FIS [factor for inversion stimulation], Fnr, Fur, and NrdR), depending on the RNR class; however, the factors that dictate the expression of some RNR genes in response to different environmental conditions are not known. We show that H-NS modulates the expression of the nrdAB and nrdDG operons. H-NS represses expression both in aerobically and in anaerobically growing cells. Under aerobic conditions, repression occurs at the exponential phase of growth as well as at the transition from the exponential to the stationary phase, a period when no dNTPs are needed. Under anoxic conditions, repression occurs mainly in exponentially growing cells. Electrophoretic mobility assays performed with two DNA fragments from the regulatory region of the nrdAB operon demonstrated the direct interaction of H-NS with these sequences.



(See full publication list in ORCID)


Equipment

  • Zeiss LSM 800 Confocal Laser Scanning Microscope
  • Nikon Inverted Fluorescent microscope ECLIPSE Ti-S/L100
  • Cell culture facilities for microbial infections
  • Biological safety cabinet (class II)
  • Characterization of nanoparticles/biomaterial antibacterial activity
  • Continuous flow system model for bacterial biofilm development
  • Single Channel Fiber-Optic Oxygen Meter with microsensor
  • Gradient thermocycler (PCR)
  • Molecular biology facilities
  • Protein and DNA electrophoresis
  • Bacterial expression systems for heterologous protein production
  • Protein purification systems (FPLC; Biologic DuoFlow System From Bio-Rad)
  • Technology of microbial culture facilities
  • Pressure microinjection system
  • Drosophila melanogaster as a model host for bacterial infections

Collaborations

  • Prof. Fernando Albericio
    Institut de Recerca Biomèdica (IRB), Barcelona, Spain
  • Dr. Elisabeth Engel
    IBEC
  • Dr. Esther Julián
    Dept. de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Spain
  • Dr. Nicolas Barnich
    Pathogénie Bactérienne Intestinale, Université Clermont 1, Clermont-Ferrand, France
  • Dr. Joan Gavaldà
    Infectious diseases, Vall d’Hebrón Hospital and Research Institute, Barcelona, Spain
  • Prof. Víctor Puntes
    Inorganic nanoparticles group, Institut Català de Nanociència i Nanotecnología, Barcelona, Spain
  • Prof. Josep Samitier
    IBEC
  • Prof. Santiago Vazquez
    Laboratori de química farmacèutica, Pharmacy Faculty, Barcelona University

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