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Staphylococcus aureus; Biofilm; Infection Staphylococcus aureus; Biopelícula; Infección Staphylococcus aureus; Biopel·lícula; Infecció The aim of this study was to quantify in vitro biofilm formation by methicillin-susceptible Staphylococcus aureus (MSSA) on the surfaces of different types of commonly used vascular grafts. We performed an in vitro study with two clinical strains of MSSA (MSSA2 and MSSA6) and nine vascular grafts: Dacron (Hemagard), Dacron-heparin (Intergard heparin), Dacron-silver (Intergard Silver), Dacron-silver-triclosan (Intergard Synergy), Dacron-gelatin (Gelsoft Plus), Dacron plus polytetrafluoroethylene (Fusion), polytetrafluoroethylene (Propaten; Gore), Omniflow II, and bovine pericardium (XenoSure). Biofilm formation was induced in two phases: an initial 90-minute adherence phase and a 24-hour growth phase. Quantitative cultures were performed, and the results were expressed as log10 CFU per milliliter. The Dacron-silver-triclosan graft and Omniflow II were associated with the least biofilm formation by both MSSA2 and MSSA6. MSSA2 did not form a biofilm on the Dacron-silver-triclosan graft (0 CFU/mL), and the mean count on the Omniflow II graft was 3.89 CFU/mL (standard deviation [SD] 2.10). The mean count for the other grafts was 7.01 CFU/mL (SD 0.82). MSSA6 formed a biofilm on both grafts, with 2.42 CFU/mL (SD 2.44) on the Dacron-silver-triclosan graft and 3.62 CFU/mL (SD 2.21) on the Omniflow II. The mean biofilm growth on the remaining grafts was 7.33 CFU/mL (SD 0.28). The differences in biofilm formation on the Dacron-silver-triclosan and Omniflow II grafts compared to the other tested grafts were statistically significant. Our findings suggest that of the vascular grafts we studied, the Dacron-silver-triclosan and Omniflow II grafts might prevent biofilm formation by MSSA. Although further studies are needed, these grafts seem to be good candidates for clinical use in vascular surgeries at high risk of infections due to this microorganism. IMPORTANCE The Dacron silver-triclosan and Omniflow II vascular grafts showed the greatest resistance to in vitro methicillin-susceptible Staphylococcus aureus biofilm formation compared to other vascular grafts. These findings could allow us to choose the most resistant to infection prosthetic graft.

Listeria monocytogenes; Malaltia bacteriana; Aliments Listería monocytogenes; Enfermedad bacteriana; Alimentos Listeria monocytogenes; Bacterial disease; Food L’objectiu d’aquest informe és descriure l’epidemiologia dels casos confirmats de listeriosi invasiva declarats en el sistema de notificació microbiològica de Catalunya (SNMC) de la Subdirecció General de Vigilància i Resposta a Emergències de Salut Pública (SGVRESP) en el període 2015 – 2021.

© 2021 The Authors., Bacterial infection of implanted scaffolds may have fatal consequences and, in combination with the emergence of multidrug bacterial resistance, the development of advanced antibacterial biomaterials and constructs is of great interest. Since decades ago, metals and their ions had been used to minimize bacterial infection risk and, more recently, metal-based nanomaterials, with improved antimicrobial properties, have been advocated as a novel and tunable alternative. A comprehensive review is provided on how metal ions and ion nanoparticles have the potential to decrease or eliminate unwanted bacteria. Antibacterial mechanisms such as oxidative stress induction, ion release and disruption of biomolecules are currently well accepted. However, the exact antimicrobial mechanisms of the discussed metal compounds remain poorly understood. The combination of different metal ions and surface decorations of nanoparticles will lead to synergistic effects and improved microbial killing, and allow to mitigate potential side effects to the host. Starting with a general overview of antibacterial mechanisms, we subsequently focus on specific metal ions such as silver, zinc, copper, iron and gold, and outline their distinct modes of action. Finally, we discuss the use of these metal ions and nanoparticles in tissue engineering to prevent implant failure., M.G-G and U.E have received funding from the postdoctoral fellowship programme Beatriu de Pinós, funded by the Secretary of Universities and Research (Government of Catalonia) and by the Horizon 2020 programme of research and innovation of the European Union under the Marie Sklodowska-Curie grant agreement No 801370. R.A.P is supported by the Spanish Ministry by the Ramón y Cajal Program (RYC2018-025977-I). Additional financial support was provided by the Government of Catalonia (2017 SGR 708) and MINECO/FEDER project (RTI2018-096088-J-100).

Fecal microbiota transplantation (FMT) is one of the recommended treatments for recurrent Clostridioides difficile infection, but endoscopy and available oral formulations still have several limitations in their preparation, storage, and administration. The need for a viable oral formulation that facilitates the implementation of this highly effective therapy in different settings has led us to test the microcrystalline cellulose particles as an adsorbent of concentrated filtered fresh feces in comparison to lyophilized feces. This free-flowing material can provide protection to bacteria and results in a dried product able to maintain the viability of the microbiota for a long time. Adsorbate formulation showed a stabilizing effect in gut microbiota, maintaining bacteria viability and preserving its diversity, and is a competitive option for lyophilized capsules. Keywords: FMT; adsorbate; capsules; gut microbiota; lyophilization.

Listeria monocytogenes; Aliments preenvasats; Comerç minorista Listería monocytogenes; Alimentos preenvasados; Comercio minorista Listeria monocytogenes; Prepackaged foods; Retail commerce Aquest informe tracta sobre quina és la vida útil segura en relació amb Listeria monocytogenes de determinats productes (formatges, carnis cuits, inclosos patés, i carnis curats) llescats i preenvasats abans de la venda en comerç minorista. This document is an opinion on the safe shelf life of Listeria monocytogenes in certain products (cheeses, cooked meats, including pâtés, and cured meats) sliced and prepackaged prior to retail sale.

Targeted bactericide nanosystems hold significant promise to improve the efficacy of existing antimicrobials for treatment of severe bacterial infections, minimizing the side effects and lowering the risk of the development of antibiotic resistance. In this work, we developed antibody-functionalized nanocapsules (NCs) containing antibacterial essential oil (EO) for selective and effective eradication of Staphylococcus aureus. Antibacterial EO NCs were produced via self-assembly nanoencapsulation in the plant-derived protein zein. The obtained EO NCs were decorated with aminocellulose to provide more reactive surface groups for carboxyl-to-amine immobilization of a antibody that is specific against S. aureus. The antibody-enabled EO NCs (Ab@EO NCs) demonstrated 2-fold higher bactericidal efficacy against the targeted bacterium compared to the pristine EO NCs at the same concentrations. The improved antibacterial effect of the Ab@EO NCs toward S. aureus was also confirmed in a real-time assay by monitoring bacterial cells elimination using a quartz crystal microbalance. Furthermore, the Ab@EO NCs selectively decreased the load and changed the cell morphology of the targeted S. aureus in a mixed inoculum with nontargeted Pseudomonas aeruginosa. Applying the nanoformulated antibacterial actives to an in vitro coculture model of the bacteria and skin fibroblasts resulted in suppression of S. aureus growth while preserving the human cells viability. The novel antibody-enabled antibacterial NCs showed potential for improving the treatment efficacy of staphylococcal infections, minimally affecting the beneficial microbial and human cells.

Funder: European society of Intensive Care Medicine, Funder: European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Funder: Norva Dahlia foundation and the Redcliffe Hospital Private Practice Trust Fund, Background The study aimed to describe the epidemiology and outcomes of hospital-acquired bloodstream infections (HABSIs) between COVID-19 and non-COVID-19 critically ill patients. Methods We used data from the Eurobact II study, a prospective observational multicontinental cohort study on HABSI treated in ICU. For the current analysis, we selected centers that included both COVID-19 and non-COVID-19 critically ill patients. We performed descriptive statistics between COVID-19 and non-COVID-19 in terms of patients’ characteristics, source of infection and microorganism distribution. We studied the association between COVID-19 status and mortality using multivariable fragility Cox models. Results A total of 53 centers from 19 countries over the 5 continents were eligible. Overall, 829 patients (median age 65 years [IQR 55; 74]; male, n = 538 [64.9%]) were treated for a HABSI. Included patients comprised 252 (30.4%) COVID-19 and 577 (69.6%) non-COVID-19 patients. The time interval between hospital admission and HABSI was similar between both groups. Respiratory sources (40.1 vs. 26.0%, p < 0.0001) and primary HABSI (25.4% vs. 17.2%, p = 0.006) were more frequent in COVID-19 patients. COVID-19 patients had more often enterococcal (20.5% vs. 9%) and Acinetobacter spp. (18.8% vs. 13.6%) HABSIs. Bacteremic COVID-19 patients had an increased mortality hazard ratio (HR) versus non-COVID-19 patients (HR 1.91, 95% CI 1.49–2.45). Conclusions We showed that the epidemiology of HABSI differed between COVID-19 and non-COVID-19 patients. Enterococcal HABSI predominated in COVID-19 patients. COVID-19 patients with HABSI had elevated risk of mortality. Trial registration ClinicalTrials.org number NCT03937245. Registered 3 May 2019.

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanopar-ticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaf-folds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concen-trations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N1-107) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant–pathogen co-evolution., This work was funded by the Nature Science Basic Research Plan of Shaanxi Province (2020JM-160), the Special Research project from Shaanxi province (F2020221001), the Program of Introducing Talents of Innovative Discipline to Universities (Project 111) from the State administration of Foreign Experts Affairs (B18042) and the External Science and Technology Cooperation Program of Ningxia Academy of Agriculture and Forestry Sciences (DW-X-2018012), the Start-up Funds of Northwest A&F University (Z111021601), the Fundamental Research Fund for the Central Universities of China (Z109021706), the Key Research and Development plan of Ningxia Province (2021BEF02033), and the National Natural Science Foundation of China for C.Z. (31901573).

This work was supported by the Spanish Ministerio de Economia y Competitividad (PID2019-106832RB-100, and SAF2017-82261-P grant cofounded by the European Regional Development Fund) and the Generalitat de Catalunya (2017SGR1720). J. A. M. Xavier acknowledges DOC-FAM program under the Marie Sklodowska-Curie grant agreement N degrees 754397. A. B. Buades, M. Nuez and J. A. M. Xavier are enrolled in the PhD program of the UAB., The Na[3,3’-Fe(8-I-1,2-C2B9H10)2] and Na[2,2’-M(1,7-C2B9H11)] (M = Co3+, Fe3+) small molecules are synthesized and the X-ray structures of [(H3O)(H2O)5][2,2’-Co(1,7-C2B9H11)2] and [Cs(MeCN)][8,8’-I2-Fe(1,2 C2B9H10)2], both displaying a transoid conformation of the [M(C2B9)2]− framework, are reported. Importantly, the supramolecular structure of [(H3O)(H2O)5][2,2’-Co(1,7-C2B9H11)2] presents 2D layers leading to a lamellar arrangement of the anions while the cation layers form polymeric water rings made of six- and fourmembered rings of water molecules connected via OH⋯H hydrogen bonds; B–H⋯O contacts connect the cationic and anionic layers. Herein, we highlight the influence of the ligand isomers (ortho-/meta-), the metal effect (Co3+/Fe3+) on the same isomer, as well as the influence of the presence of the iodine atoms on the physical–chemical and biological properties of these molecules as antimicrobial agents to tackle antibioticresistant bacteria, which were tested with four Gram-positive bacteria, five Gram-negative bacteria, and three Candida albicans strains that have been responsible for human infections. We have demonstrated an antimicrobial effect against Candida species (MIC of 2 and 3 nM for Na[3,3’-Co(8-I-1,2-C2B9H10)2] and Na[2,2’-Co (1,7-C2B9H11)2], respectively), and against Gram-positive and Gram-negative bacteria, including multiresistant MRSA strains (MIC of 6 nM for Na[3,3’-Co(8-I-1,2-C2B9H10)2]). The selectivity index for antimicrobial activity of Na[3,3’-Co(1,2-C2B9H11)2] and Na[3,3’-Co(8-I-1,2-C2B9H10)2] compounds is very high (165 and 1180, respectively), which reveals that these small anionic metallacarborane molecules may be useful to tackle antibioticresistant bacteria. Moreover, we have demonstrated that the outer membrane of Gram-negative bacteria constitutes an impermeable barrier for the majority of these compounds. Nonetheless, the addition of two iodine groups in the structure of the parent Na[3,3’-Co(1,2-C2B9H11)2] had an improved effect (3–7 times) against Gram-negative bacteria. Possibly the changes in their physical–chemical properties make the meta-isomers and the ortho-di-iodinated small molecules more permeable for crossing this barrier. It should be emphasized that the most active metallabis(dicarbollide) small molecules are both transoid conformers in contrast to the ortho- [3,3’-Co(1,2-C2B9H11)2]− that is cisoid. The fact that these small molecules cross the mammalian membrane and have antimicrobial properties but low toxicity for mammalian cells (high selectivity index, SI) represents a promising tool to treat infectious intracellular bacteria. Since there is an urgent need for antibiotic discovery and development, this study represents a relevant advance in the field., Spanish Government PID2019-106832RB-100 SAF2017-82261-P, Generalitat de Catalunya, General Electric 2017SGR1720, DOC-FAM program under the Marie Sklodowska-Curie grant 754397

Background Vancomycin-resistant enterococci (VRE) are major therapeutic challenges. Prospective contemporary data characterizing the clinical and molecular epidemiology of VRE bloodstream infections (BSIs) are lacking. Methods The Vancomycin-Resistant Enterococcal BSI Outcomes Study (VENOUS I) is a prospective observational cohort of adult patients with enterococcal BSI in 11 US hospitals. We included patients with Enterococcus faecalis or Enterococcus faecium BSI with ≥1 follow-up blood culture(s) within 7 days and availability of isolate(s) for further characterization. The primary study outcome was in-hospital mortality. Secondary outcomes were mortality at days 4, 7, 10, 12, and 15 after index blood culture. A desirability of outcome ranking was constructed to assess the association of vancomycin resistance with outcomes. All index isolates were subjected to whole genome sequencing. Results Forty-two of 232 (18%) patients died in hospital and 39 (17%) exhibited microbiological failure (lack of clearance in the first 4 days). Neutropenia (hazard ratio [HR], 3.13), microbiological failure (HR, 2.4), VRE BSI (HR, 2.13), use of urinary catheter (HR, 1.85), and Pitt BSI score ≥2 (HR, 1.83) were significant predictors of in-hospital mortality. Microbiological failure was the strongest predictor of in-hospital mortality in patients with E faecium bacteremia (HR, 5.03). The impact of vancomycin resistance on mortality in our cohort changed throughout the course of hospitalization. Enterococcus faecalis sequence type 6 was a predominant multidrug-resistant lineage, whereas a heterogeneous genomic population of E faecium was identified. Conclusions Failure of early eradication of VRE from the bloodstream is a major factor associated with poor outcomes.

Objectives: We described the current incidence and risk factors of bacterial co-infection in hospitalized patients with COVID-19. Methods: Observational cohort study was performed at the Hospital Clinic of Barcelona (February 2020-February 2021). All patients with COVID-19 who were admitted for >48 hours with microbiological sample collection and procalcitonin (PCT) determination within the first 48 hours were included. Results: A total of 1125 consecutive adults met inclusion criteria. Co-infections were microbiologically documented in 102 (9.1%) patients. Most frequent microorganisms were Streptococcus pneumoniae (79%), Staphylococcus aureus (6.8%), and Haemophilus influenzae (6.8%). Test positivity was 1% (8/803) for blood cultures, 10.1% (79/780) for pneumococcal urinary antigen test, and 11.4% (15/132) for sputum culture. Patients with PCT higher than 0.2, 0.5, 1, and 2 ng/mL had significantly more co-infections than those with lower levels (p=0.017, p=0.031, p94%.

Ralstonia solanacearum causes bacterial wilt, a devastating plant disease, responsible for serious losses on many crop plants. R. solanacearum phylotype II-B1 strains have caused important outbreaks in temperate regions, where the pathogen has been identified inside asymptomatic bittersweet (Solanum dulcamara) plants near rivers and in potato fields. S. dulcamara is a perennial species described as a reservoir host where R. solanacearum can overwinter, but their interaction remains uncharacterised. In this study, we have systematically analysed R. solanacearum infection in S. dulcamara, dissecting the behaviour of this plant compared with susceptible hosts such as tomato cv. Marmande, for which the interaction is well described. Compared with susceptible tomatoes, S. dulcamara plants (i) show delayed symptomatology and bacterial progression, (ii) restrict bacterial movement inside and between xylem vessels, (iii) limit bacterial root colonisation, and (iv) show constitutively higher lignification in the stem. Taken together, these results demonstrate that S. dulcamara behaves as partially resistant to bacterial wilt, a property that is enhanced at lower temperatures. This study proves that tolerance (i.e., the capacity to reduce the negative effects of infection) is not required for a wild plant to act as a reservoir host. We propose that inherent resistance (impediment to colonisation) and a perennial habit enable bittersweet plants to behave as reservoirs for R. solanacearum., This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2016-78002-R) and the Ministry of Science and Innovation PID2019-108595RB-I00/AEI/10.13039/501100011033. We also acknowledge financial support from the “Severo Ochoa Program for Centers of Excellence in R&D” (SEV-2015-0533 and CEX2019-000902-S), and the CERCA Program from the Catalan Government (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PS held an INPHINIT Programme fellowship from Fundació “La Caixa” and AK was a recipient of a Netaji Subhas - Indian Council of Agricultural Research (ICAR) International Fellowship. RP-J received FI and FPU fellowships from Generalitat de Catalunya and the Ministry of Education and Professional Formation, respectively. BD have received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement No. 945043.

Currently, three major circumstances threaten the management of bacterial infections: increasing antimicrobial resistance, expansion of chronic biofilm-associated infections, and lack of an appropriate approach to treat them. To date, the development of accelerated drug susceptibility testing of biofilms and of new antibiofouling systems has not been achieved despite the availability of different methodologies. There is a need for easy-to-use methods of testing the antibiotic susceptibility of bacteria that form biofilms and for screening new possible antibiofilm strategies. Herein, we present a microfluidic platform with an integrated interdigitated sensor (BiofilmChip). This new device allows an irreversible and homogeneous attachment of bacterial cells of clinical origin, even directly from clinical specimens, and the biofilms grown can be monitored by confocal microscopy or electrical impedance spectroscopy. The device proved to be suitable to study polymicrobial communities, as well as to measure the effect of antimicrobials on biofilms without introducing disturbances due to manipulation, thus better mimicking real-life clinical situations. Our results demonstrate that BiofilmChip is a straightforward tool for antimicrobial biofilm susceptibility testing that could be easily implemented in routine clinical laboratories.

Les lents de contacte (LC) són dispositius mèdics àmpliament utilitzats per a la correcció visual o per raons cosmètiques o terapèutiques. Tot i això, les LC són un factor en la predisposició a tenir infeccions oculars difícils de tractar com poden ser la queratitis microbiana i l’úlcera perifèrica, les quals poden causar deficiència visual i fins i tot ceguesa. Els principals factors en aquestes infeccions són l’adhesió bacteriana i la formació de biofilms a la superfície de les LC degut a la resistència de les cèl·lules sèssils als antibiòtics convencionals. En virtut d’això, hi ha una necessitat urgent de desenvolupar unes noves superfícies antibacterianes i antibiofilm per a LC que siguin capaces de prevenir les infeccions bacterianes oculars. En aquest estudi, nanopartícules bimetàl·liques de plata/or (AgAuNPs) i gelatina modificada amb DOPA sintetitzades mitjançant mètodes amb “química verda” s’han utilitzat per a dissenyar nous nanocompostos amb diferents mètodes d’acció per a minimitzar les infeccions bacterianes relacionades amb LC mitjançant un procés ultrasonoquímic d’un sol pas. El recobriment híbrid desenvolupat va portar a un augment de la humectabilitat del material de silicona i va demostrar una gran capacitat per impedir als bacteris Staphylococcus aureus i Pseudomona aeruginosa la formació del biofilm en un 80% i 60% respectivament. Els nous recobriments nano-actius tenen un gran potencial per eliminar els patògens en humans, sense afectar a la viabilitat ni a la morfologia de les cèl·lules dels mamífers convertint-se en una estratègia molt prometedora per a la prevenció d’infeccions bacterianes relacionades amb les LC. Las lentes de contacto (LC) son dispositivos médicos ampliamente utilizados para la corrección visual y para fines cosméticos o terapéuticos. Sin embargo, las LC son un factor en la predisposición a tener infecciones oculares difíciles de tratar como pueden ser la queratitis microbiana y la úlcera periférica, las cuales pueden causar deficiencia visual e incluso ceguera. Los principales factores en estas infecciones son la adhesión bacteriana y la formación de biofilm en la superficie de las LC debido a la resistencia de las células sésiles a los antibióticos convencionales. En virtud de ello, hay una necesidad urgente de desarrollar unas nuevas superficies antibacterianas y antibiofilm para LC que sean capaces de prevenir las infecciones bacterianas oculares. En este estudio, nanopartículas bimetálicas de plata/oro (AgAuNPs) y gelatina modificada con DOPA sintetizadas mediante métodos con “química verde” se han utilizado para diseñar nuevos nanocompuestos con diferentes métodos de acción para minimizar las infecciones bacterianas relacionadas con las LC mediante un proceso ultraosonoquímico de un solo paso. El recubrimiento híbrido desarrollado llevó a una augmento de la humectabilidad del material de silicona y demostró una gran capacidad para impedir a las bacterias Staphylococcus aureus y Pseudomona aeruginosa la formación del biofilm en un 80% y 60% respectivamente. Los nuevos recubrimientos nano-activos tienen un gran potencial para eliminar a los patógenos en humanos, sin afectar a la viabilidad ni a la morfología de las células de los mamíferos convirtiéndose en una estrategia muy prometedora para la prevención de infecciones bacterianas relacionadas con las LC. The contact lenses (CLs) are widely used medical devices for visual correction or therapeutic and cosmetic reasons. However, the CLs are a predisposing factor for difficult-to-treat ocular infections such as microbial keratitis and peripheral ulcer, which can cause visual impairment and blindness. The main significant impact on these infections is the bacterial adhesion and biofilm formation on the CLs surfaces due to drug resistance of the sessile cells to the conventional antibiotics. In this line, there is an urgent need for development of novel antibacterial and antibiofilm CLs surfaces for prevention of ocular bacterial infections. Herein, in the present work, green synthetized bimetallic silver/gold nanoparticles (AgAu NPs) and dopamine-modified gelatin have been employed for engineering of novel nanostructured composites with complimentary modes of action to minimize the CLs-associated infections occurrence in a one-step high-intensity sonochemical process. The developed hybrid coating led to enhanced wettability of the silicone material and demonstrated very strong capacity to impede the Staphylococcus aureus and the Pseudomonas aeruginosa biofilm formation by 80% and 60% respectively. The novel nano-enabled coatings had a great potential to eliminate the human pathogens, without affecting the mammalian cells viability and morphology, showing to be a promising strategy for prevention of CL-related bacterial infections.

Infeccions cutànies bacterianes; Tractament empíric; Atenció primària Infecciones cutáneas bacterianas; Tratamiento empírico; Atención primaria Bacterial skin infections; Empirical treatment; Primary care Les infeccions cutànies bacterianes són un dels motius de consulta per causa infecciosa més freqüents a l’atenció primària, darrere de les infeccions respiratòries i urinàries. El tipus i la gravetat de la infecció depèn de la profunditat i del compartiment de la pell afectat, la qual pot ser primària si es produeix sobre pell prèviament sana o secundària si es produeix sobre lesions prèvies. En aquest Butlletí s’inclou el tractament de les infeccions més freqüents, com l’impetigen, l’erisipela, la cel·lulitis, la fol·liculitis, la hidrosadenitis supurativa, l’acne i les infeccions per mossegades. Per a l’abordatge de les infeccions cutànies es recomana fer un tractament empíric i només davant una evolució dolenta es pot sol·licitar un estudi microbiològic. Les infeccions superficials habitualment només requereixen un bon rentat, utilització d’antisèptics i limitar l’ús d’antibiòtics tòpics perquè generen resistències de forma ràpida. El tractament amb antibiòtics via oral s’ha de reservar per a formes més greus o per a pacients d’alt risc. La durada del tractament ha de ser el més curta possible, habitualment no més de 5 dies. Las infecciones cutáneas bacterianas son uno de los motivos de consulta más frecuentes por causa infecciosa en la Atención Primaria, seguidas de las infecciones respiratorias y urinarias. El tipo y la gravedad de la infección depende de la profundidad y del compartimento de la piel afectada. Puede ser una infección primaria si se produce sobre piel sana, o secundaria si se produce sobre lesiones previas. Este Boletín revisa el tratamiento de las infecciones cutáneas más frecuentes como el impétigo, la erisipela, la celulitis, la foliculitis, la hidrosadenitis supurativa, el acné y las infecciones por mordeduras. En el abordaje de las infecciones cutáneas se recomienda tratamiento empírico, y solo en los casos en los que no evoluciona favorablemente se recomienda solicitar un estudio microbiológico. Las infecciones superficiales generalmente solo requieren un buen lavado, la utilización de antisépticos y limitar el uso de antibióticos tópicos ya que generan resistencias de forma rápida. El tratamiento con antibióticos por vía oral se debe reservar para las formas más graves o para pacientes de alto riesgo. La duración de los tratamientos deberá ser lo más corta posible, habitualmente no más de 5 días.

[eng] Bacterial infections are a major health concern worldwide due to the mortality and comorbidity associated levels. Besides the development of drug-resistance mechanisms, most recalcitrant infections are caused by bacteria forming biofilms, which are bacterial communities that grow within an extracellular polymeric substance (EPS) matrix that protects bacterial cells from the action of antimicrobials and immune mechanisms. The frequent appearance and spreading of bacterial drug-resistant strains, together with the recalcitrance of infections caused by biofilms, has pointed out the urgent need to develop novel antibacterial agents that target essential bacterial processes and biofilm-forming bacteria. This thesis investigated the development of new antibacterial strategies by both targeting bacterial essential processes and biofilm-forming bacteria. Ribonucleotide reductase (RNR) are essential enzymes required by any cellular organism, since they catalyze the synthesis of deoxyribonucleotides, critical for both DNA synthesis and repair processes. Due to its key role in DNA replication, several RNR inhibitors have been developed as antiproliferative drugs in cancer and infectious diseases. Amongst RNR inhibitors, radical scavenger molecules have proved for long its inhibitory activity against the RNR enzyme, including some cytotoxic hydroxylamine derivatives such as hydroxyurea (HU). Here, the use of hydroxylamine derivative compounds as antibacterial agents specifically targeting the bacterial RNR enzyme was investigated. The results provided show that N-methyl-hydroxylamine (M-HA) molecule and some newly synthesized Nhydroxylamine derivative molecules (N-HA) inhibit bacterial RNR, displaying a wide range of antibacterial activity against different bacterial pathogens, together with low cytotoxicity to eukaryotic cells. Also, M-HA molecule shows intracellular antimycobacterial activity during macrophages infection, together with Pseudomonas aeruginosa in vitro antibiofilm activity. Several of the N-HA molecules display antibiofilm activity against P. aeruginosa, Staphylococcus aureus, and Escherichia coli biofilms, and we demonstrate the ability of such molecules to inhibit bacterial growth by radical scavenging of the RNR enzyme. Further, this thesis explores the use of a drug delivery system based on poly(lactic-co-glycolic) (PLGA) nanoparticles (NPs) to remove P. aeruginosa biofilms by combining the action of the antibiotic ciprofloxacin and the DNA-hydrolytic activity of the deoxyribonuclease I enzyme (DNase I). The synthesized biodegradable PLGA NPs, loaded with ciprofloxacin and functionalized with DNase I through poly(lysine) (PL) coating, synergistically improve the ciprofloxacin antibacterial efficacy by disassembling the extracellular DNA, one of the main components of the EPS matrix.

[eng] INTRODUCTION: Antimicrobial resistance is one of the world’s major challenges in both microbiology and public health since infections caused by multidrug-resistant bacteria are reaching alarming levels. The world is currently facing a global antibiotics crisis and some new strategies need to be explored to tackle these resistant infections. The outer membrane is a differential structure of Gram-negative bacteria that works as a highly effective selective permeability barrier. Thus, the permeation through the Gram- negative cell envelope is a challenge for drug compounds to reach their targets. One path to open new antimicrobial perspectives is the chemical modification of old antimicrobial compounds that may result in optimized drugs with improved antimicrobial properties. In this context, the activity exploration of the derivatives of Microcin J25 and Trimethoprim was carried out. HYPOTHESIS: The main hypotheses of this thesis are that chemical modifications of current antibiotics may significantly contribute to overcoming the problems arising from the increase and spread of antibiotic resistance in bacteria. Moreover, the combination of these modified compounds with old-rescued antibiotics may contribute to the solution bases of the problem caused by resistance. Objectives: The main objective of this thesis is to determine the antimicrobial properties of chemically modified drug compounds. The secondary objectives of this thesis are divided in two parts: Microcin J25: Study of the antimicrobial activity of the modified Microcin J25 as well as determination of its toxicity. Trimethoprim: Study of the antimicrobial activity and cytotoxicity of the Trimethoprim derivatives, exploration of the effect of the new derivatives on the Dihydrofolate Reductase (DHFR) enzyme and start a computational approach to decipher the intimal mechanisms of action. METHODOLOGY: The antimicrobial activity of the derived compounds was explored against planktonic bacteria (studying the Minimum Inhibitory Concentration and the FIC index) and against sessile bacteria (exploring the Minimum Biofilm Eradication Concentration and the Biofilm Prevention Concentration). The growth curves of several microorganisms in contact with these compounds and in combination with colistin were also studied. The cytotoxicity of all the compounds was tested. An enzymatic assay with E. coli DHFR as well as a docking modelling were carried out to investigate the mechanism of action of TMP derivatives. RESULTS: With respect to Microcin MccJ25, it has been detected that the chemical modification of the compound resulted in a new peptide without antimicrobial activity. It acted synergistically with sublethal concentrations of colistin. When referring to Trimethoprim (TMP), some of the new derivatives showed antibacterial similar to that of TMP. Moreover, almost all the new TMP-like compounds acted synergistically with SMX. P. aeruginosa PAO1 was fully resistant to TMP and all its derivatives as well as to the combination of TMP-SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhanced their antimicrobial efficacy against E. coli, P. aeruginosa and S. marcescens. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR. Additionally, it was detected that the heterocyclic ring of the compound 1a fills the pocket occupied by the nicotinamide ring of NADPH. CONCLUSIONS: The present PhD thesis has led to some relevant conclusions. With respect to Microcin MccJ25, the chemical modification of the compound avoided its detection by the membrane receptor FhuA. Moreover, it did not affect the interaction with the target and, as the polymyxin facilitated the microcin entrance across the membrane, once inside the cell, the new compound retained its ability to inhibit the growth of bacteria. When referring to Trimethoprim (TMP), the derivatives showed interesting antimicrobial activities acting synergistically with SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhances their antimicrobial efficacy by permeabilizing the cells. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR and it was suggested that both molecules interact with the analogues during inhibition. The search of new antimicrobial compounds is one of the main pathways to overtake bacterial resistance to antibiotics. All putative compounds should be tested in conditions in which outer membrane role as permeability barrier is inactivated. Their assay together with sublethal concentrations of colistin is proposed as one of the methods of election., [spa] INTRODUCCIÓN: La resistencia a los antimicrobianos es uno de los principales desafíos del mundo tanto en microbiología como en salud pública, ya que las infecciones causadas por bacterias multirresistentes están alcanzando niveles alarmantes. Una opción para abrir nuevas perspectivas antimicrobianas es la modificación química de compuestos antimicrobianos ya existentes que pueden resultar medicamentos optimizados con propiedades antimicrobianas mejoradas. En este contexto, en esta tesis se llevó a cabo la exploración de la actividad de los derivados de Microcina J25 y Trimethoprim. HIPÓTESIS: Las principales hipótesis de esta tesis son que las modificaciones químicas de los antibióticos actuales y la combinación de estos compuestos modificados con antibióticos antiguos rescatados pueden contribuir significativamente a superar los problemas que surgen del aumento y la propagación de la resistencia a los antibióticos en las bacterias. Objetivos: El principal objetivo de esta tesis es determinar las propiedades antimicrobianas de compuestos químicamente modificados. Metodología: Se exploró la actividad antimicrobiana de los compuestos derivados frente bacterias planctónicas y frente bacterias sésiles. También se estudiaron las curvas de crecimiento de varios microorganismos en contacto con estos compuestos y en combinación con colistina. Se ensayó la citotoxicidad de todos los compuestos. Se llevó a cabo un ensayo enzimático con E. coli DHFR así como un modelo de docking. RESULTADOS Y CONCLUSIONES: Con respecto a la Microcina MccJ25, se detectó que la modificación química del compuesto dio como resultado un nuevo péptido sin actividad antimicrobiana ya que se evitó su detección por el receptor de membrana FhuA. Esta actuó de forma sinérgica con concentraciones subletales de colistina debido a que la polimixina facilitó la entrada de la microcina a través de la membrana y, una vez dentro de la célula, el nuevo compuesto conservó su capacidad para inhibir el crecimiento de las bacterias. En cuanto a Trimethoprim (TMP), algunos de los nuevos derivados mostraron un efecto antibacteriano similar al de TMP. Además, casi todos los nuevos compuestos actuaron sinérgicamente con SMX. P. aeruginosa PAO1 fue totalmente resistente a TMP y todos sus derivados, así como a la combinación de TMP-SMX. La combinación de TMP, análogos de TMP y SMX con colistina mejoró su eficacia antimicrobiana contra E. coli, P. aeruginosa y S. marcescens ya que ésta permeabilizó las células. Los compuestos 1a y 1b, como TMP, inhibieron la actividad de la DHFR de E. coli. Además, se observó que el anillo heterocíclico del compuesto 1a llena el bolsillo ocupado por el anillo de nicotinamida de NADPH y se sugirió que ambas moléculas interactúan con los análogos durante la inhibición.

Trial enrichment using gut microbiota derived biomarkers by high-risk individuals can improve the feasibility of randomized controlled trials for prevention of Clostridioides difficile infection (CDI). Here, we report in a prospective observational cohort study the incidence of CDI and assess potential clinical characteristics and biomarkers to predict CDI in 1,007 patients ≥ 50 years receiving newly initiated antibiotic treatment with penicillins plus a beta-lactamase inhibitor, 3rd/4th generation cephalosporins, carbapenems, fluoroquinolones or clindamycin from 34 European hospitals. The estimated 90-day cumulative incidences of a first CDI episode is 1.9% (95% CI 1.1-3.0). Carbapenem treatment (Hazard Ratio (95% CI): 5.3 (1.7-16.6)), toxigenic C. difficile rectal carriage (10.3 (3.2-33.1)), high intestinal abundance of Enterococcus spp. relative to Ruminococcus spp. (5.4 (2.1-18.7)), and low Shannon alpha diversity index as determined by 16 S rRNA gene profiling (9.7 (3.2-29.7)), but not normalized urinary 3-indoxyl sulfate levels, predicts an increased CDI risk., Here, the authors report the incidence of Clostridioides difficile infection (CDI) and assess potential clinical characteristics and biomarkers to predict CDI in 1,007 patients of 50 years and above receiving newly initiated antibiotic treatment.

Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs.

Catheter-associated urinary tract infections (CAUTIs), caused by biofilms, are the most frequent health-care associated infections. Novel antibiofilm coatings are needed to increase the urinary catheters' life-span, decrease the prevalence of CAUTIs and reduce the development of antimicrobial resistance. Herein, antibacterial zinc oxide nanoparticles (ZnO NPs) were decorated with a biofilm matrix-degrading enzyme amylase (AM) and simultaneously deposited onto silicone urinary catheters in a one-step sonochemical process. The obtained nano-enabled coatings inhibited the biofilm formation of Escherichia coli and Staphylococcus aureus by 80% and 60%, respectively, for up to 7 days in vitro in a model of catheterized bladder with recirculation of artificial urine due to the complementary mode of antibacterial and antibiofilm action provided by the NPs and the enzyme. Over this period, the coatings did not induce toxicity to mammalian cell lines. In vivo, the nano-engineered ZnO@AM coated catheters demonstrated lower incidence of bacteriuria and prevent the early onset of CAUTIs in a rabbit model, compared to the animals treated with pristine silicone devices. The nano-functionalization of catheters with hybrid ZnO@AM coatings appears as a promising strategy for prevention and control of CAUTIs in the clinic

To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller-Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution. This study has been funded by Instituto de Salud Carlos III through the Projects from the Fondo de Investigaciones Sanitarias “PI16/00977” to SM, and CIBER de Enfermedades Respiratorias (CIBERES - CB06/06/0037), co-funded by the European Regional Development Fund/European Social Fund (ERDF/ESF, “Investing in your future”), and the Ministerio de Ciencia, Innovación y Universidades through the Projects SAF2015-66520-R and RTI2018-096369-B-I00 to JG. info:eu-repo/semantics/publishedVersion

Microbial infections are still among the major public health concerns since several yeasts and fungi, and other pathogenic microorganisms, are responsible for continuous growth of infections and drug resistance against bacteria. Antimicrobial resistance rate is fostering the need to develop new strategies against drug-resistant superbugs. Antimicrobial peptides (AMPs) are small peptide-based molecules of 5–100 amino acids in length, with potent and broad-spectrum antimicrobial properties. They are part of the innate immune system, which can represent a minimal risk of resistance development. These characteristics contribute to the description of these molecules as promising new molecules in the development of new antimicrobial drugs. However, efforts in developing new medicines have not resulted in any decrease of drug resistance yet. Thus, a technological approach on improving existing drugs is gaining special interest. Nanomedicine provides easy access to innovative carriers, which ultimately enable the design and development of targeted delivery systems of the most efficient drugs with increased efficacy and reduced toxicity. Based on performance, successful experiments, and considerable market prospects, nanotechnology will undoubtedly lead a breakthrough in biomedical field also for infectious diseases, as there are several nanotechnological approaches that exhibit important roles in restoring antibiotic activity against resistant bacteria., Elena Sanchez-Lopez belongs to 2017SGR-1477. Elena Sanchez-Lopez, Marta Espina and Maria L. GarciaacknowledgethesupportfromtheInstituteofNanoscienceandNanotechnology(ART2018project). Eliana B. Souto acknowledges the Portuguese Science and Technology Foundation (FCT/MCT) and European Funds (PRODER/COMPETE) for the projects M-ERA-NET-0004/2015-PAIRED and UIDB/04469/2020 (strategic fund), co-funded by FEDER, under the partnership Agreement PT2020. Maria C. Teixeira wishes to acknowledge FCT for the individual fellowship (PD/BDE/135086/2017)., info:eu-repo/semantics/publishedVersion

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterized by acute decompensation of cirrhosis (AD), organ failure(s) and high risk of short-term mortality with bacterial infection frequently as precipitating event. Innate immune pattern recognition receptors and members of the lectin pathway of complement activation are crucial to the innate immune response to pathogens. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) of innate immune components are associated with the occurrence of bacterial infections or mortality in patients with cirrhosis hospitalized for AD or ACLF. METHODS: Twenty-one innate immunity SNPs with known functional implications were genotyped in 826 AD/ACLF patients included in the CANONIC study. Associations between baseline characteristics of the patients, the occurrence of bacterial infections and survival rate at 90 days of follow-up in relation to the innate immunity genetic variants were analysed. RESULTS: The NOD2-G908R genetic variant was associated with mortality (HR 2.25, P = .004) independently of age and MELD Score. This association was also found in a predefined subgroup analysis in patients with bacterial infections (HR 2.78, P

Bacteria use protein-protein interactions to infect their hosts and hijack fundamental pathways, which ensures their survival and proliferation. Hence, the infectious capacity of the pathogen is closely related to its ability to interact with host proteins. Here, we show that hubs in the host-pathogen interactome are isolated in the pathogen network by adapting the geometry of the interacting interfaces. An imperfect mimicry of the eukaryotic interfaces allows pathogen proteins to actively bind to the host’s target while preventing deleterious effects on the pathogen interactome. Understanding how bacteria recognize eukaryotic proteins may pave the way for the rational design of new antibiotic molecules., Author summary To fulfil their function, proteins need to interact with each other forming complexes. Understanding how pathogen proteins bind their host counterparts is important to explain how bacteria can infect, survive and proliferate inside cells. To achieve that, pathogen proteins mimic eukaryote interfaces to interact with the host. However, we discovered that such mimicry is imperfect, and pathogen proteins display particular features that are not found in eukaryotic complexes. This imperfect mimicry would allow pathogen proteins to actively bind to the host targets while preventing deleterious effects on the pathogen network. Indeed, we show that highly connected proteins (hubs) in the host-pathogen networks are mostly isolated in the pathogen network. The existence of imperfect mimicry opens the door to the design of new molecules aimed to target host-pathogen complexes with reduced side effects. Hence, in the long term, our results may lay the foundation of a new class of antimicrobials.

Bacterial infections have been on the rise world-wide in recent years and have a considerable impact on human well-being in terms of attributable deaths and disability-adjusted life years. Yet many mechanisms underlying bacterial pathogenesis are still poorly understood. Here, we introduce the BacFITBase database for the systematic characterization of bacterial proteins relevant for host infection aimed to enable the identification of new antibiotic targets. BacFITBase is manually curated and contains more than 90 000 entries with information on the contribution of individual genes to bacterial fitness under in vivo infection conditions in a range of host species. The data were collected from 15 different studies in which transposon mutagenesis was performed, including top-priority pathogens such as Acinetobacter baumannii and Campylobacter jejuni, for both of which increasing antibiotic resistance has been reported. Overall, BacFITBase includes information on 15 pathogenic bacteria and 5 host vertebrates across 10 different tissues. It is freely available at www.tartaglialab.com/bacfitbase. This study was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades [SAF2015-72518-EXP, SAF2017-82158-R, RYC-2012-09999]; European Research Council [RIBOMYLOME_309545]; European Union's Horizon 2020 Research and Innovation Programme [727658, IASIS]; Spanish Ministry of Economy and Competitiveness [BFU2014-55054-P, BFU2017-86970-P]; Spanish Ministry of Economy and Competitiveness; ‘Centro de Excelencia Severo Ochoa 2013–2017’; CERCA Programme of the Generalitat de Catalunya; European Union's Horizon 2020 Research and Innovation Programme, Marie Sklodowska-Curie Individual Fellowship [793135, ‘DeepRNA’ to B.L.]; European Society of Clinical Microbiology and Infectious Diseases, Research Grant 2016 (ESCMID). Funding for open access charge: Spanish Ministerio de Ciencia, Innovación y Universidades [SAF2017-82158-R]

Bacteriúria sense símptomes; Tractament; Recomanacions per a pacients Bacteriuria sin síntomas; Tratamiento; Recomendaciones para pacientes Asymptomatic bacteria; Treatment; Recommendations for patients Essencial és una iniciativa que identifica pràctiques clíniques de poc valor i promou recomanacions per tal d'evitar-ne la realització. En aquest document es tracta la bacteriúria sense símptomes (presència elevada de bactèries a l’orina sense que la persona tingui cap símptoma d’infecció d’orina).

Infectious diseases kill over 17 million people a year, among which bacterial infections stand out. From all the bacterial infections, tuberculosis, diarrhoea, meningitis, pneumonia, sexual transmission diseases and nosocomial infections are the most severe bacterial infections, which affect millions of people worldwide. Moreover, the indiscriminate use of antibiotic drugs in the last decades has triggered an increasing multiple resistance towards these drugs, which represent a serious global socioeconomic and public health risk. It is estimated that 33,000 and 35,000 people die yearly in Europe and the United States, respectively, as a direct result of antimicrobial resistance. For all these reasons, there is an emerging need to find novel alternatives to overcome these issues and reduced the morbidity and mortality associated to bacterial infectious diseases. In that sense, nanotechnological approaches, especially smart polymeric nanoparticles, has wrought a revolution in this field, providing an innovative therapeutic alternative able to improve the limitations encountered in available treatments and capable to be effective by theirselves. In this review, we examine the current status of most dangerous human infections, together with an in-depth discussion of the role of nanomedicine to overcome the current disadvantages, and specifically the most recent and innovative studies involving polymeric nanoparticles against most common bacterial infections of the human body., Authors acknowledge the support of the Spanish Ministry of Economy and Competitiveness (SAF2017-84283-R and RTI2018-098641-B-I00), Biomedical Research Networking Centre in Neurodegenerative Diseases (CIBERNED, CB06/05/0024), Scientifc Project Marató TV3 (ref 201829-10) and European Regional Development Founds. Authors also acknowledge the Portuguese Science and Technology Foundation (FCT) for the strategic fund (UIDB/04469/2020)., info:eu-repo/semantics/publishedVersion

Urban wild boars (Sus scrofa) from Barcelona, Spain, harbor great diversity of Streptococcus suis strains, including strains with the cps2 gene and with the same molecular profile as local human cases. The increasing trend of potential effective contacts for S. suis transmission is of public health concern. info:eu-repo/semantics/publishedVersion

Aim: To compare the in vitro activity of the anti-impetigo agent, ozenoxacin, and other antimicrobial agents against Gram-positive clinical isolates from skin and soft tissue infections. Materials & methods: Isolates were collected in two studies: 1097 isolates from 49 centers during 2009–2010 and 1031 isolates from ten centers during 2014. Minimum inhibitory concentrations were determined for 18 and 11 antimicrobials in these studies, respectively, using standard broth microdilution methods. Isolates were stratified by species and methicillin susceptibility/resistance and/or levofloxacin susceptibility/nonsusceptibility status. Results: Ozenoxacin exhibited high in vitro activity against Staphylococcus aureus and coagulase-negative staphylococci isolates in both studies. Ozenoxacin was also highly active against Streptococcus pyogenes and Streptococcus agalactiae isolates. Conclusion: Ozenoxacin is a potent antimicrobial agent against staphylococci and streptococci.

Altres ajuts: The authors want to thank the Dr. Vilallonga Foundation for the financial support in the preparation of this article (http://www.fundacioramonvilallonga.org). Necrotizing fasciitis (NF) is a complicated soft tissue infection frequently associated with severe sepsis if an early medical and surgical treatment is not performed. We report two postoperative cases of severe NF after oophorectomy and colorectal resection. Because of the similarity with more benign skin infections at the early steps, clinical suspicion is crucial. Surgical exploration and resection will provide both the diagnosis confirming necrotizing infection of the fascia with vessels and treatment. Also, empirical broad-spectrum antibiotics must be initiated as soon as possible. Regardless of the presence of risk factors, NF is a condition with a high mortality rate and only an expeditious and undelayed treatment may improve the patient's outcome. Surgical focus control requires wide and repeated resections, and planned reconstructive plastic surgery might be necessary.

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.

Nutrient iron sequestration is the most significant form of nutritional immunity and causes bacterial pathogens to evolve strategies of host iron scavenging. Cigarette smoking contains iron particulates altering lung and systemic iron homeostasis, which may enhance colonization in the lungs of patients suffering chronic obstructive pulmonary disease (COPD) by opportunistic pathogens such as nontypeable. NTHi is a heme auxotroph, and the NTHi genome contains multiple heme acquisition systems whose role in pulmonary infection requires a global understanding. In this study, we determined the relative contribution to NTHi airway infection of the four heme-acquisition systems HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF that are located at the bacterial outer membrane or the periplasm. Our computational studies provided plausible 3D models for HbpA, SapA, PE, and HxuA interactions with heme. Generation and characterization of single mutants in the hxuCBA, hpe, sapA, and hbpA genes provided evidence for participation in heme binding-storage and inter-bacterial donation. The hxuA, sapA, hbpA, and hpe genes showed differential expression and responded to heme. Moreover, HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF presented moonlighting properties related to resistance to antimicrobial peptides or glutathione import, together likely contributing to the NTHi-host airway interplay, as observed upon cultured airway epithelia and in vivo lung infection. The observed multi-functionality was shown to be system-specific, thus limiting redundancy. Together, we provide evidence for heme uptake systems as bacterial factors that act in a coordinated and multi-functional manner to subvert nutritional- and other sources of host innate immunity during NTHi airway infection., I.R.A. was funded by a PhD studentship from Universidad Pública de Navarra, Spain; S.M. is funded by a postdoctoral contract from CIBERES. This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520- R, from Health Department, Regional Govern from Navarra, Spain, reference 03/2016, and from SEPAR 31/2015 to J.G. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain. This work was also supported by grants from Foundations of Anna and Edwin Berger (KR), the Swedish Medical Research Council (KR: grant number K2015-57X-03163-43-4, www.vr.se), the Cancer Foundation at the University Hospital in Malmö (KR), the Royal Physiographical Society (Forssman’s Foundation) (TAJ), Skåne County Council’s research and development foundation (KR), the Heart Lung Foundation (KR: grant number 20150697, www.hjart-lungfonden.se). The FP7 WeNMR (project# 261572), H2020 West-Life (project# 675858) and the EOSC-hub (project# 777536) European e-Infrastructure projects are acknowledged for the use of their web portals, which make use of the EGI infrastructure with the dedicated support of CESNET-MetaCloud, INFN-PADOVA, NCGINGRID-PT, TW-NCHC, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, Taiwan and the US Open Science Grid

Introduction: Impetigo is the most common bacterial skin infection in children. Treatment is becoming complicated due to the development of antimicrobial resistance, especially in the main pathogen, Staphylococcus aureus. Ozenoxacin, a novel non-fluorinated topical quinolone antimicrobial, has demonstrated efficacy in impetigo. Areas covered: This article reviews the microbiology, pharmacodynamic and pharmacokinetic properties of ozenoxacin, and its clinical and microbiological efficacy in impetigo. Expert opinion: In an environment of increasing antimicrobial resistance and concurrent slowdown in antimicrobial development, the introduction of a new agent is a major event. Ozenoxacin is characterized by simultaneous affinity for DNA gyrase and topoisomerase IV, appears to be impervious to certain efflux pumps that confer bacterial resistance to other quinolones, shows low selection of resistant mutants, and has a mutant prevention concentration below its concentration in skin. These mechanisms protect ozenoxacin against development of resistance, while the absence of a fluorine atom in its structure confers a better safety profile versus fluoroquinolones. In vitro studies have demonstrated high potency of ozenoxacin against staphylococci and streptococci including resistant strains of S. aureus. Clinical trials of ozenoxacin in patients with impetigo reported high clinical and microbiological success rates. Preserving the activity and availability of ozenoxacin through antimicrobial stewardship is paramount.

Phage therapy is an abandoned antimicrobial therapy that has been resumed in recent years. In this study, we mutated a lysogenic phage from Acinetobacter baumannii into a lytic phage (Ab105-2phi&Delta, CI) that displayed antimicrobial activity against A. baumannii clinical strain Ab177_GEIH-2000 (isolated in the GEIH-REIPI Spanish Multicenter A. baumannii Study II 2000/2010, Umbrella Genbank Bioproject PRJNA422585, and for which meropenem and imipenem MICs of respectively, 32 &micro, g/mL, and 16 &micro, g/mL were obtained). We observed an in vitro synergistic antimicrobial effect (reduction of 4 log&ndash, 7 log CFU/mL) between meropenem and the lytic phage in all combinations analyzed (Ab105-2phi&Delta, CI mutant at 0.1, 1 and 10 MOI and meropenem at 1/4 and 1/8 MIC). Moreover, bacterial growth was reduced by 8 log CFU/mL for the combination of imipenem at 1/4 MIC plus lytic phage (Ab105-2phi&Delta, CI mutant) and by 4 log CFU/mL for the combination of imipenem at 1/8 MIC plus lytic phage (Ab105-2phi&Delta, CI mutant) at both MOI 1 and 10. These results were confirmed in an in vivo model (G. mellonella), and the combination of imipenem and mutant Ab105-2phi&Delta, CI was most effective (p &lt, 0.05). This approach could help to reduce the emergence of phage resistant bacteria and restore sensitivity to antibiotics used to combat multi-resistant strains of Acinetobacter baumannii.

Background: We aimed to analyze the impact of cefotaxime non-susceptibility on the 30-day mortality rate in patients receiving a third-generation cephalosporin for pneumococcal bacteremic pneumonia. Methods: We conducted a retrospective observational study of prospectively collected data from the Hospital Clinic of Barcelona. All adult patients with monomicrobial bacteremic pneumonia due to Streptococcus pneumoniae and treated with a third-generation cephalosporin from January 1991 to December 2016 were included. Risk factors associated with 30-day mortality were evaluated by univariate and multivariate analyses. Results: During the study period, 721 eligible episodes were identified, and data on the susceptibility to cefotaxime was obtainable for 690 episodes. Sixty six (10%) cases were due to a cefotaxime non-susceptible strain with a 30-day mortality rate of 8%. Variables associated with 30-day mortality were age, chronic liver disease, septic shock, and the McCabe score. Infection by a cefotaxime non-susceptible S. pneumoniae did not increase the mortality rate. Conclusion: Despite the prevalence of cefotaxime non-susceptible S. pneumoniae has increased in recent years. We found no evidence to suggest that patients hospitalized with bacteremic pneumonia due to these strains had worse clinical outcomes than patients with susceptible strains.

[eng] Bacteria and their sub-products are the main cause of the occurrence and perpetuation of endodontic infection. However, only a few bacterial species are seen as responsible of persistent endodontic infections, among them Enterococcus faecalis is the most frequently isolated species. It has several virulence factors, such as aggregation substances, enterococcal surface protein (Esp), “endocarditis and biofilm-associated pili” (ebp) and cytolysin. Moreover, its high resistance to antibacterial agents is emhanced by its ability to form biofilm. Due to the complex and unpredictable root canal morphology, the complete removal of smear layer and bacterial biofilm is difficult. Recently, laser-activated irrigation (LAI) has been introduced as an alternative to achieve a deeper cleaning and disinfection of the root canal system. Its mechanism of action is based on the generation of cavitation bubbles, through the absorption of laser energy by the irrigant. The most used lasers are from the Erbium family, Er, Cr: YSGG (2780nm) and Er: YAG (2980nm). Sodium hypochlorite (NaOCl) is considered the "gold standard" of endodontic irrigators. It has a broad antibacterial spectrum and is capable of dissolving organic tissue. It is used in a range between 0.5% and 6%, varying its degree of effectiveness. Nevertheless, at high concentrations it is toxic, causing damage to endothelial cells and the periodontal ligament cells, which generates an acute inflammatory reaction and pain. Hence, the aim of this thesis was to explore the bactericidal effect of low concentration of NaOCl activated by Er, Cr: YSGG laser-activated irrigation against E. faecalis biofilms in root canals in order to decipher if it may be similar to the one achieved by high concentrations of NaoCl. IN IN VITRO ROOT CANAL MODEL: The main objective of the first stage of this thesis was to build a laboratory model to simulate the conditions inside a single-tooth root canal. The Pasteur pipettes were inoculated with E. faecalis ATCC 29212 for 24 hours bacterial colonization and the subsequent formation of biofilm in the proposed in vitro model was demonstrated by atomic force microscope. The second essential point of this part was to determine antimicrobial capacity of Er,Cr:YSGG laser against E.faecalis. In addition, passive ultrasonic irrigation was also tested. Laser-activated irrigation demonstrated higher antimicrobial activity than passive ultrasonic irrigation. The final stage of this first part consisted in the analysis and measurement of the nano-roughness by atomic force microscopy of the cells treated and its comparison with that of untreated cells. EXTRACTED TEETH: This part was focused on the endodontic preparation of extracted human teeth. The root canals were instrumented by a crown-down / step-back technique using conventional sequence of 0.02 taper files up to the master # 55. The teeth were inoculated with E. faecalis ATCC 29212 for 10 days. The following stage was focused on the study of the antibacterial action of low concentration of NaOCl activated by Er,Cr: YSGG laser in extracted human teeth. The antimicrobial effectiveness of laser-activated irrigation was compared with passive ultrasonic irrigation activation and conventional manual irrigation. Er,Cr:YSGG laser and 0.5% NaOCl showed a considerable synergistic action. Finally, the last part was focused on the microscopic visualization of the samples, to complement the results of the microbiological count. The scanning electron microscope was used to determine the degree of effectiveness of bacterial biofilm and smear layer removal both on the dentin surface and inside the dentinal tubules. Additionally, the confocal scanning electron microscope was used to visualize the proportion of alive and dead bacteria after treatment. The results obtained in this thesis show that laser-activated irrigation is a therapeutic alternative for infections caused by E. faecalis inside of root canals., [spa] Las bacterias y sus subproductos son la principal causa de la infección endodóntica y su perpetuación. E. faecalis es la especie más frecuentemente aislada de los fracasos endodónticos. Su alta resistencia a los agentes antibacterianos viene incrementada por la capacidad para formar biofilms. Recientemente, el riego activado por láser (LAI) se ha propuesto como una alternativa para lograr una limpieza y desinfección más profunda del sistema de canales radiculares. Su mecanismo de acción se basa en la generación de burbujas de cavitación, a través de la absorción de energía láser por parte del irrigante. El objetivo de esta tesis fue explorar si el efecto bactericida de una baja concentración de NaOCl activado por el láser Er, Cr: YSGG, puede ser similar al alzanzado por altas concentraciones de NaOCl contra biofilms de E. faecalis en canales radiculares. MODELO DE CANAL RADICULAR IN VITRO: El objetivo principal de la primera etapa de esta tesis fue construir un modelo de laboratorio para simular las condiciones al interior de un canal radicular. Las pipetas Pasteur fueron inoculadas con E. faecalis ATCC 29212 durante 24 horas. Luego se determinó la capacidad antimicrobiana del láser Er, Cr: YSGG contra E.faecalis. La irrigación activada por láser demostró una mayor actividad antimicrobiana que la irrigación ultrasónica pasiva. Finalmente se realizó el análisis y la medición de la nano rugosidad mediante microscopía de fuerza atómica de las células tratadas y no tratadas. DIENTES EXTRAÍDOS: Los dientes fueron inoculados con E. faecalis ATCC 29212 durante 10 días. El láser Er, Cr: YSGG y 0,5% de NaOCl mostraron una acción sinérgica considerable. Finalmente, el microscopio electrónico de barrido se utilizó para determinar el grado de efectividad en la remoción de biofilms bacterianos y de barro dentinario. Además, se utilizó el microscopio electrónico de barrido confocal para visualizar la proporción de bacterias vivas y muertas después del tratamiento. Los resultados obtenidos en esta tesis muestran que la irrigación activada por láser es una alternativa terapéutica para las infecciones causadas por E. faecalis dentro de los canales radiculares.

E Armengol,1 O Domenech,2 E Fusté1,3 I Pérez-Guillén,1 JH Borrell,2 JM Sierra,1 M Vinas11Laboratory of Molecular Microbiology and Antimicrobials, Department of Pathology and Experimental Therapeutics, School of Medicine, University of Barcelona, Barcelona, Spain; 2Department of Pharmacy, Pharmaceutical Technology and Physical-Chemistry, University of Barcelona, Barcelona, Spain; 3Department of Public Health, Mental Health and Perinatal Nursing. School of Nursing, University of Barcelona, Barcelona, SpainObjective: Despite its use was abandoned several decades ago, the polycationic peptide colistin has become the last hope to treat severe infections caused by multidrug-resistant Gram-negative bacteria. Thus, the development of colistin resistance may seriously compromise the efficacy of treatment. Moreover, colistin has high toxicity being dose dependent. A potentially effective strategy to avoid resistance may be to combine colistin with other antimicrobials. This may help in the rescue of old antimicrobials and in reducing toxic undesired effects.Methods: Antimicrobial susceptibility determination, efflux machinery function measurements in different conditions and measurement of inhibition of the extrusion by colistin were performed. Moreover, modifications of anisotropy of the membranes by using fluorescent dyes was accomplished.Results: Sub-inhibitory concentrations of colistin have a synergistic effect with several antimicrobials that act intracellularly (targeting protein synthesis and DNA replication). This effect was demonstrated through the uptake increases of acridine orange. in Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumanii but also in an intrinsically colistin-resistant species as Serratia marcescens. Measurements of the anisotropy of bacterial membranes, as a measure of membrane fluidity, showed significant changes indicative of colistin activity.Conclusion: The alterations in the cellular efflux machinery that resulted in higher intracellular concentrations of acridine orange, and likely of other antimicrobials combined with data of membrane fluidity and measured synergism in vitro allow us to envisage the use of these combinations to fight infections caused by multidrug–resistant bacteria.Keywords: synergism, cationic antimicrobial peptides, efflux pumps, membrane fluidity

Typhoid fever is a bacterial infection caused by the Gram-negative bacterium Salmonella enterica subspecies enterica serovar Typhi (S. Typhi), prevalent in many low- and middle-income countries. In high-income territories, typhoid fever is predominantly travel-related, consequent to travel in typhoid-endemic regions; however, data show that the level of typhoid vaccination in travellers is low. Successful management of typhoid fever using antibiotics is becoming increasingly difficult due to drug resistance; emerging resistance has spread geographically due to factors such as increasing travel connectivity, affecting those in endemic regions and travellers alike. This review provides an overview of: the epidemiology and diagnosis of typhoid fever; the emergence of drug-resistant typhoid strains in the endemic setting; drug resistance observed in travellers; vaccines currently available to prevent typhoid fever; vaccine recommendations for people living in typhoid-endemic regions; strategies for the introduction of typhoid vaccines and stakeholders in vaccination programmes; and travel recommendations for a selection of destinations with a medium or high incidence of typhoid fever.

Over the past decade, unusual mortality outbreaks have decimated echinoderm populations over broad geographic regions, raising awareness globally of the importance of investigating such events. Echinoderms are key components of marine benthos for top-down and bottom-up regulations of plants and animals; population declines of these individuals can have significant ecosystem-wide effects. Here we describe the first case study of an outbreak affecting Antarctic echinoderms and consisting of an ulcerative epidermal disease affecting ~10% of the population of the keystone asteroid predator Odontaster validus at Deception Island, Antarctica. This event was first detected in the Austral summer 2012–2013, coinciding with unprecedented high seawater temperatures and increased seismicity. Histological analyses revealed epidermal ulceration, inflammation, and necrosis in diseased animals. Bacterial and fungal alpha diversity was consistently lower and of different composition in lesioned versus unaffected tissues (32.87% and 16.94% shared bacterial and fungal operational taxonomic units OTUs respectively). The microbiome of healthy stars was more consistent across individuals than in diseased specimens suggesting microbial dysbiosis, especially in the lesion fronts. Because these microbes were not associated with tissue damage at the microscopic level, their contribution to the development of epidermal lesions remains unclear. Our study reveals that disease events are reaching echinoderms as far as the polar regions thereby highlighting the need to develop a greater understanding of the microbiology and physiology of marine diseases and ecosystems health, especially in the era of global warming.

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum., Spanish Ministry of Economy and Competitiveness, MINECO http://dx.doi.org/10.13039/501100003329AGL2013–46898-RRyC 2014–16158EU-Marie Curie Actions http://dx.doi.org/10.13039/100010665PIIF-331392ERC starting grant http://dx.doi.org/10.13039/100010663258413ERC consolidator grant http://dx.doi.org/10.13039/100010663616449 'GreenProteases'EuroTransBio project 'PredicSeed'COST Action SUSTAIN http://dx.doi.org/10.13039/501100000921FA1208

Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification. Sysmex España S.L. financially supported the study, providingreagents and equipment. Additionally, Yuliya Zboromyrska receivedtravel grants from Sysmex España S.L. The funder had no role in data analysis and interpretation, decision to publish, or preparation of the manuscript.