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Objective Serratia marcescens is a Gram-negative bacterium that is found in hospital environments and commonly associated with outbreaks in neonatal units. One S. marcescens isolate was detected from a bloodstream culture from a neonate in our hospital that was followed by an outbreak. The aim of this study was to describe the molecular epidemiology of a S. marcescens outbreak in the neonatal unit. Methods In order to investigate the outbreak, weekly surveillance rectal swabs were submitted for culture from all patients admitted in this unit from August to September 2018. Environmental samples were obtained from potential sources in September 2018. Typing of isolates was performed by pulsed field gel electrophoresis (PFGE). In addition, we studied the in vitro activity of chlorhexidine against S. marcescens. Results During this period, 146 infants were hospitalised in our neonatal unit, of which 16 patients had a S. marcescens-positive sample. A total of 36 environmental surveillance samples were collected, and one sample from a stethoscope from an incubator of a colonized baby was positive for S. marcescens. All the 18 isolates, including the isolate from the stethoscope, belonged to a single PFGE cluster. We found that very low concentrations of chlorhexidine, even with application times close to 0 achieved significant reductions in the amount of S. marcescens. Conclusion A unique clone of S. marcescens caused this outbreak, including isolates from patients and from one stethoscope. The outbreak was controlled with the early implementation of specific control measures.

Background Dysbiosis in lung cancer has been underexplored. The aim of this study was to define the bacterial and fungal microbiota of the bronchi in central lung cancer and to compare it with that of the oral and intestinal compartments. Methods Twenty-five patients with central lung cancer and sixteen controls without antimicrobial intake during the previous month were recruited. Bacterial and fungal distribution was determined by massive sequencing of bronchial biopsies and saliva and faecal samples. Complex computational analysis was performed to define the core lung microbiota. Results Affected and contralateral bronchi of patients have almost identical microbiota dominated by Streptococcus, whereas Pseudomonas was the dominant genera in controls. Oral and pulmonary ecosystems were significantly more similar in patients, probably due to microaspirations. Streptococcal abundance in the bronchi differentiated patients from controls according to a ROC curve analysis (90.9% sensitivity, 83.3% specificity, AUC = 0.897). The saliva of patients characteristically showed a greater abundance of Streptococcus, Rothia, Gemella and Lactobacillus. The mycobiome of controls (Candida) was significantly different from that of patients (Malassezia). Cancer patients’ bronchial mycobiome was similar to their saliva, but different from their contralateral bronchi. Conclusions The central lung cancer microbiome shows high levels of Streptococcus, and differs significantly in its composition from that of control subjects. Changes are not restricted to tumour tissue, and seem to be the consequence of microaspirations from the oral cavity. These findings could be useful in the screening and even diagnosis of this disease.

2 Figuras, 1 Tabla, Microbiota have emerged as key modulators of both the carcinogenic process and the immune response against cancer cells, and, thus, it seems to influence the efficacy of immunotherapy. While most studies have focused on analyzing the influence of gut microbiota, its composition substantially differs from that in the lung. Here, we describe how microbial life in the lungs is associated with host immune status in the lungs and, thus, how the identification of the microbial populations in the lower respiratory tract rather than in the gut might be key to understanding the lung carcinogenic process and to predict the efficacy of different treatments. Understanding the influence of lung microbiota on host immunity may identify new therapeutic targets and help to design new immunotherapy approaches to treat lung cancer.

Objectives To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible MSSA (MSSA-PENS) isolates. Methods A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018–19 (6–12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type. Results The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton–Valentine leucocidin (‘PVL’) positive. Conclusions A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis.

7 figures, 2 tables., Multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired and community infections and pose a challenge to the human health care system. Therefore, it is important to find new drugs that show activity against these bacteria, both in monotherapy and in combination with other antimicrobial drugs. Gliotoxin (GT) is a mycotoxin produced by Aspergillus fumigatus and other fungi of the Aspergillus genus. Some evidence suggests that GT shows antimicrobial activity against S. aureus in vitro, albeit its efficacy against multidrug-resistant strains such as MRSA or vancomycin-intermediate S. aureus (VISA) strainsis not known. This work aimed to evaluate the antibiotic efficacy of GT as monotherapy or in combination with other therapeutics against MRSA in vitro and in vivo using a Caenorhabditis elegans infection model., This research was funded by FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón (Group B29_17R), Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R), ASPANOA, GILEAD (GLD18/00100), Donación Javier Saura Carceller, and Carrera de la Mujer de Monzón. J.P. was supported by Fundacion Aragon I+D (ARAID). P.E. was supported by Fondo Garantıa Empleo Juvenil/INAEM. L.C. and S.R. were granted funding by a DGA predoctoral contract. M.A. was granted funding by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (FJCI-2017-31629).

Introduction We characterized AmpC β-lactamase mutations that resulted in ceftolozane/tazobactam resistance in extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates recovered from patients treated with this agent from June 2016 to December 2018. Methods Five pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa XDR isolates were included among a total of 49 patients treated. Clonal relationship among isolates was first evaluated by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was further performed. AmpC mutations were investigated by PCR amplification of the blaPDC gene followed by sequencing. Results The ST175 high-risk clone was detected in four of the pairs of isolates and the ST1182 in the remaining one. All resistant isolates showed a mutation in AmpC: T96I in two of the isolates, and E247K, G183V, and a deletion of 19 amino acids (G229–E247) in the other three. The G183V mutation had not been described before. The five isolates resistant to ceftolozane/tazobactam showed cross-resistance to ceftazidime/avibactam and lower MICs of imipenem and piperacillin/tazobactam than the susceptible isolates. Conclusions Ceftolozane/tazobactam resistance was associated in all of the cases with AmpC mutations, including a novel mutation (G183V) not previously described. There is a vital need for surveillance and characterization of emerging ceftolozane/tazobactam resistance, in order to preserve this valuable antipseudomonal agent.

Objectives: To calculate the turnaround time of results to be able in the electronic medical record. Postulate some available tools regarding laboratory management to assume full response in an increasing demand scenario. Materials and methods: Retrospective analysis of all samples reaching the lab since February 17 until May 10 was performed using LIS. Records of personnel management and equipment delivery were consulted. Time to results was measured as the difference in hours between time of analytic request and the date of result upload to the electronic medical record. Results: Time to result started at 24 hours and continually decreased over time reaching stability on week 10 around 6.5 hours. Active measurements taken fall into groups: personnel management, Laboratory schedules and technical capacity. Conclusion: Adoption of an uninterrupted sample processing method (24/7) and the implementation of high throughput systems are the best options for increasing results performance, where other measurements like redistributing and re train personnel would be more successfully implemented. Keywords: clinical laboratory services, SARS-CoV-2, medical laboratory personnel, clinical laboratory techniques, health policy, COVID-19

Introducción La relación entre las infecciones nosocomiales en pacientes quirúrgicos y la vitamina D ha sido estudiada por algunos autores. Sin embargo, hasta la fecha no existe ningún estudio realizado sobre pacientes de cirugía hepatobiliar. El objetivo de nuestro trabajo es estudiar la infección del sitio quirúrgico (ISQ) en la unidad de cirugía hepatobiliar, y valorar su relación con la concentración sérica de vitamina D. Métodos Se llevó a cabo un estudio analítico observacional de pacientes sucesivos intervenidos en la unidad de cirugía hepatobiliar de nuestro centro durante un año. Se incluyeron las intervenciones relativas a enfermedad biliar, pancreática y hepática. Se determinaron los niveles de vitamina D al ingreso, así como las ISQ de tipo superficial, profunda y órgano-cavitaria diagnosticadas durante el estudio. El seguimiento del paciente se realizó durante al menos un mes tras la cirugía, dependiendo de la enfermedad. La estadística se realizó mediante el programa estadístico R v.3.1.3. Resultados La muestra quedó constituida por 321 pacientes, de los cuales el 25, 8% presentó ISQ a expensas fundamentalmente de las infecciones órgano-cavitarias que presentaron una incidencia del 24, 3%. Concentraciones séricas superiores a 33, 5 nmol/l demostraron reducir en un 50% el riesgo de ISQ. Conclusiones Las concentraciones elevadas de vitamina D en sangre demostraron ser un factor protector frente a las ISQ (OR: 0, 99). Nuestros resultados sugieren una relación directa entre la concentración sérica de vitamina D y la ISQ, justificando la realización de nuevos estudios prospectivos. Introduction: While several studies have examined the correlation between vitamin D concentrations and post-surgical nosocomial infections, this relationship has yet to be characterized in hepatobiliary surgery patients. We investigated the relationship between serum vitamin D concentration and the incidence of surgical site infection (SSI) in patients in our hepatobiliary surgery unit. Methods: Participants in this observational study were 321 successive patients who underwent the following types of interventions in the hepatobiliary surgery unit of our center over a 1-year period: cholecystectomy, pancreaticoduodenectomy, total pancreatectomy, segmentectomy, hepatectomy, hepaticojejunostomy and exploratory laparotomy. Serum vitamin D levels were measured upon admission and patients were followed up for 1 month. Mean group values were compared using a Student's T-test or Chi-squared test. Statistical analyses were performed using the Student's T-test, the Chi-squared test, or logistic regression models. Results: Serum concentrations >33.5 nmol/l reduced the risk of SSI by 50%. Out of the 321 patients analyzed, 25.8% developed SSI, mainly due to organ-cavity infections (incidence, 24.3%). Serum concentrations of over 33.5 nmol/l reduced the risk of SSI by 50%. Conclusions: High serum levels of vitamin D are a protective factor against SSI (OR, 0.99). Our results suggest a direct relationship between serum vitamin D concentrations and SSI, underscoring the need for prospective studies to assess the potential benefits of vitamin D in SSI prevention.

Introduction While several studies have examined the correlation between vitamin D concentrations and post-surgical nosocomial infections, this relationship has yet to be characterized in hepatobiliary surgery patients. We investigated the relationship between serum vitamin D concentration and the incidence of surgical site infection (SSI) in patients in our hepatobiliary surgery unit. Methods Participants in this observational study were 321 successive patients who underwent the following types of interventions in the hepatobiliary surgery unit of our center over a 1-year period: cholecystectomy, pancreaticoduodenectomy, total pancreatectomy, segmentectomy, hepatectomy, hepaticojejunostomy and exploratory laparotomy. Serum vitamin D levels were measured upon admission and patients were followed up for 1 month. Mean group values were compared using a Student's T-test or Chi-squared test. Statistical analyses were performed using the Student's T-test, the Chi-squared test, or logistic regression models. Results Serum concentrations >33.5 nmol/l reduced the risk of SSI by 50%. Out of the 321 patients analyzed, 25.8% developed SSI, mainly due to organ-cavity infections (incidence, 24.3%). Serum concentrations of over 33.5 nmol/l reduced the risk of SSI by 50%. Conclusions High serum levels of vitamin D are a protective factor against SSI (OR, 0.99). Our results suggest a direct relationship between serum vitamin D concentrations and SSI, underscoring the need for prospective studies to assess the potential benefits of vitamin D in SSI prevention.

Synergistic antimicrobial effects were observed for copper sulfide (CuS) nanoparticles together with indocyanine green (ICG) in the elimination of wild type pathogenic bacteria (Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853) and also opportunistic fungal infective yeast (Candida albicans ATCC 10231). Furthermore, large antibacterial effects were observed for clinical isolates of Methicillin-resistant S. aureus (MRSA) PFGE strain-type USA300. This efficient antimicrobial action was attributed to the combined extra- and intracellular generation of reactive oxygen species upon light irradiation. Instead of the use of visible-light for the activation of common photosensitizers, both ICG and CuS nanoparticles can be activated in the near infrared (NIR)-region of the electromagnetic spectrum and therefore, superior tissue penetration would be expected in a potential elimination of pathogenic microorganisms not only on the skin but also in the soft tissue. In the different bacteria studied a 3-log reduction in the bacterial counts was achieved after only 6 min of NIR irradiation and treatment with ICG or CuS alone at concentrations of 40 and 160 µg mL−1, respectively. A maximum bactericidal effect against S. aureus and USA300 strains was obtained for the combination of both photosensitizers at the same concentration. Regarding P. aeruginosa, a 4-log reduction in the CFU was observed for the combination of CuS and ICG at various concentrations. In Candida albicans the combination of both ICG and CuS and light irradiation showed an antimicrobial dose-dependent effect with the reduction of at least 3-log in the cell counts for the combination of ICG + CuS at reduced concentrations. The observed antimicrobial effect was solely attributed to a photodynamic effect and any photothermal effect was avoided to discard any potential thermal injury in a potential clinical application. The generation of reactive oxygen species upon near infrared-light irradiation for those photosensitizers used was measured either alone or in combination. The cytocompatibility of the proposed materials at the doses used in photodynamic therapy was also demonstrated in human dermal fibroblasts and keratinocytes by cell culturing and flow cytometry studies., Financial support from Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00 is gratefully acknowledged. This research was also funded by the Spanish Ministry of Economy and Competitiveness (grant number CTQ2017-84473-R). CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III (Spain) with assistance from the European Regional Development Fund. We acknowledge the LMA-INA (University of Zaragoza, Spain). G. M. acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III).

Candidiasis is very common and complicated to treat in some cases due to increased resistance to antifungals. Antimicrobial photodynamic therapy (aPDT) is a promising alternative treatment. It is based on the principle that light of a specific wavelength activates a photosensitizer molecule resulting in the generation of reactive oxygen species that are able to kill pathogens. The aim here is the in vitro photoinactivation of three strains of Candida spp., Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, using aPDT with different sources of irradiation and the photosensitizer methylene blue (MB), alone or in combination with chlorhexidine (CHX). Irradiation was carried out at a fluence of 18 J/cm2 with a light-emitting diode (LED) lamp emitting in red (625 nm) or a white metal halide lamp (WMH) that emits at broad-spectrum white light (420–700 nm). After the photodynamic treatment, the antimicrobial effect is evaluated by counting colony forming units (CFU). MB-aPDT produces a 6 log10 reduction in the number of CFU/100 μL of Candida spp., and the combination with CHX enhances the effect of photoinactivation (effect achieved with lower concentration of MB). Both lamps have similar efficiencies, but the WMH lamp is slightly more efficient. This work opens the doors to a possible clinical application of the combination for resistant or persistent forms of Candida infections.

Background: Antibiotic resistance and impaired wound healing are major concerns in S. aureus superficial skin infections, and new therapies are needed. Antimicrobial photodynamic therapy (aPDT) is a new therapeutic approach for infections, but it also improves healing in many wound models.Objective: To compare the antimicrobial activity and the effects on wound healing of aPDT based on Methylene Blue (MB-aPDT) with mupirocin treatment, either alone or in combination, in superficial skin wounds of S. aureus-infected mice. Additionally, to evaluate the clinical, microbiological, and cosmetic effects on wound healing.Materials and Methods: A superficial skin infection model of S. aureus was established in SKH-1 mice. Infected wounds were treated with MB-aPDT, MB-aPDT with a daily topical mupirocin or only with mupirocin. No treatment was carried out in control animals. Daily clinical and microbiological examinations were performed until complete clinical wound healing. Histopathological studies and statistical analysis were performed at the end of the study.Results: MB-aPDT treatment induced the best wound healing compared to mupirocin alone or to mupirocin plus MB-aPDT. Superficial contraction at 24 h and a greater reduction in size at 48 h, quicker detachment of the crust, less scaling, and absence of scars were observed. Histopathological studies correlated with clinical and gross findings. By contrast, mupirocin showed the highest logaritmic reduction of S. aureus.Conclusions: MB-aPDT and mupirocin treatments are effective in a murine superficial skin infection model of S. aureus. One session of MB-aPDT was the best option for clinical wound healing and cosmetic results. The addition of mupirocin to MB-aPDT treatment improved antimicrobial activity; however, it did not enhance wound healing. No synergistic antibacterial effects were detected.

[EN]: [Introduction]: Methicillin-resistant Staphylococcus aureus (MRSA) is considered a major cause of healthcare-associated (HA) and community-acquired (CA) infections. Considering non-β-lactam susceptibility as a potential marker for mecC-MRSA and CA-MRSA, the aim of this study was to determine the frequency and the associated genetic lineages of non-beta-lactam-antibiotic susceptible MRSA (NBLS-MRSA) strains in a multicenter study in Spain. [Methods]: A collection of 45 NBLS-MRSA strains recovered in the period from January to June 2016 from 12 Spanish hospitals was analyzed. Molecular typing through spa-type characterization, agr group and multi-locus-sequence typing was performed. Methicillin-resistant genes (mecA and mecC) as well as immune evasion cluster (scn-chp-sak-sea-sep, considering scn gene as the marker of IEC system) and Panton-Valentine leukocidin (PVL) genes were determined with PCR/sequencing. [Results]: The NBLS-MRSA phenotype was uncommon in the 12 hospitals analyzed (NBLS-MRSA/MRSA frequency: 0.3%-7.7%). All strains contained the mecA gene (and none contained mecC). Twenty-two different spa-types were detected among NBLS-MRSA strains, with spa-t008/agr-I the most prevalent (27%). The main clonal complexes were (CC/%): CC8/42.2%, CC5/33.3% and CC30/4.4%, with ST8 and ST5 as the main sequence types. The PVL toxin was present in 38% of strains (with spa-types t008, t024, t019, t044, t068, t318 and t3060). The IEC genes were detected in 78% of strains: IEC type-B (n = 17), type-F (n = 16), type-A (n = 1) and type-E (n = 1); 10 MRSA isolates were scn-negative. [Conclusion]: The NBLS-MRSA phenotype is uncommon in the analyzed hospitals; although no mecC-positive strains were detected, it could be a good marker for MRSA PVL-positive isolates (38%), frequently associated with CA-MRSA infections. [ES]: [Introducción]: Staphylococcus aureus resistente a meticilina (SARM) es una de las principales causas de infecciones tanto relacionadas con la asistencia sanitaria como asociadas a la comunidad (AC). Considerando la sensibilidad a antibióticos no-β-lactámicos como marcador potencial de SARM-mecC y SARM-AC, el objetivo de este estudio fue determinar la frecuencia y líneas genéticas de cepas SARM sensibles a antibióticos no-β-lactámicos (SARM-SNBL) en un estudio multicéntrico en España. [Métodos]: Se analizaron 45 cepas SARM-SNBL procedentes de 12 hospitales obtenidas durante enero-junio de 2016. El tipado molecular se realizó mediante caracterización del spa-tipo, grupo agr y multi-locus-sequence typing. Mediante PCR/secuenciación se determinaron los genes: de resistencia a meticilina (mecA y mecC), del sistema de evasión inmune humano (scn-chp-sak-sea-sep, usando scn como marcador del sistema IEC) y de la leucocidina de Panton-Valentine (LPV). [Resultados]: El fenotipo SARM-SNBL fue infrecuente en los 12 hospitales analizados (frecuencia SARM-SNBL/SARM: 0,3-7,7%). Todas las cepas fueron mecA-positivas (ninguna mecC). Se detectaron 22 spa-tipos diferentes, siendo el spa-t008/agr-I el prevalente (27%). Los principales complejos clonales fueron (CC/%): CC8/42,2%, CC5/33,3% y CC30/4,4%, destacando las secuencias tipo ST8 y ST5 como mayoritarias. El 38% de las cepas fue LPV-positiva (spa-tipos t008, t024, t019, t044, t068, t318 y t3060). El 78% de las cepas fue IEC-positivo: tipo-B (n = 17), tipo-F (n = 16), tipo-A (n = 1) y tipo-E (n = 1); 10 aislados fueron scn-negativos. [Conclusión]: El fenotipo SARM-SNBL es poco frecuente en los hospitales analizados; aunque no se detectaron cepas mecC-positivas, este fenotipo puede ser un buen marcador de aislados SARM LPV-positivos, frecuentemente asociados a infecciones por SARM-AC.

Study Group of clinical LA-MRSA, [Background] Tetracycline resistance (TetR) is a marker of livestock-associated MRSA of lineage CC398., [Objectives] To determine the MRSA CC398 prevalence among TetR-MRSA recovered in Spanish hospitals located in regions with different pig-farming densities, and the influence of pig density as a key risk factor for its acquisition., [Methods] TetR-MRSA isolates (n = 232) recovered from clinical and epidemiological samples during January–June 2016 in 20 hospitals in 13 regions with different pig-farming densities were analysed. MRSA CC398 identification, detection of spa types, methicillin resistance genes and immune evasion cluster (IEC) genes were performed by PCR/sequencing. Statistical analyses were performed to establish the relationships between MRSA CC398 prevalence and pig density., [Results] The global MRSA prevalence was 29.7% (6.9% TetR-MRSA/MRSA), with 137 CC398 isolates recovered, representing 4.1% of total MRSA and 59.1% of TetR-MRSA. Among MRSA CC398, 16 different spa types were recorded (t011: 72.3%), and all but two strains were IEC negative. Higher pig-density regions were associated with significant MRSA CC398 increases in hospitals located in adjacent regions (P, [Conclusions] High pig density leads to a significant increase in MRSA CC398 in hospitals in Spain, and its combination with a high human population could help its dissemination. In Spain, the prevalence of the zoonotic CC398 lineage is closely related to pig-farming density; therefore, specific tools could be implemented in order to detect its dissemination., This work was supported by the Sociedad de Enfermedades Infecciosas del Norte (SEINORTE) and by the Agencia Estatal de Investigación (AEI) of Spain (project SAF2016-76571-R) and by the Fondo Europeo de Desarrollo Regional (FEDER). Sara Ceballos and Laura Ruiz-Ripa have predoctoral fellowships of the University of La Rioja, Spain.

Background: Antimicrobial photodynamic therapy (aPDT) is a growing approach to treat skin and mucosal infections. Despite its effectiveness, investigators have explored whether aPDT can be further combined with antibiotics and antifungal drugs. Objective: To systematically assess the in vivo studies on the effectiveness of combinations of aPTD plus antimicrobials in the treatment of cutaneous and mucosal infections. Materials and methods: Searches were performed in four databases (PubMed, EMBASE, Cochrane library databases, ClinicaTrials.gov) until July 2018. The pooled information was evaluated according to the PRISMA guidelines. Results: 11 full-text articles were finally evaluated and included. The best aPDT combinations involved 5-aminolevulinic acid or phenothiazinium dye-based aPDT. In general, the combination shows benefits such as reducing treatment times, lowering drug dosages, decreasing drug toxicity, improving patient compliance and diminishing the risk of developing resistance. The mechanism of action may be that first aPDT damages the microbial cell wall or membrane, which allows better penetration of the antimicrobial drug. Limitations: The number of studies was low, the protocols used were heterogeneous, and there was a lack of clinical trials. Conclusions: The additive or synergistic effect of aPDT combined with antimicrobials could be promising to manage skin and mucosal infections, helping to overcome the microbial drug resistance.

Invasive fusariosis has a global increasing incidence and has emerged as a severe infection in nonneutropenic patients., Invasive fusariosis (IF) is associated with severe neutropenia in patients with concurrent hematologic conditions. We conducted a retrospective observational study to characterize the epidemiology of IF in 18 Spanish hospitals during 2000–2015. In that time, the frequency of IF in nonneutropenic patients increased from 0.08 cases per 100,000 admissions in 2000–2009 to 0.22 cases per 100,000 admissions in 2010–2015. Nonneutropenic IF patients often had nonhematologic conditions, such as chronic cardiac or lung disease, rheumatoid arthritis, history of solid organ transplantation, or localized fusariosis. The 90-day death rate among nonneutropenic patients (28.6%) and patients with resolved neutropenia (38.1%) was similar. However, the death rate among patients with persistent neutropenia (91.3%) was significantly higher. We used a multivariate Cox regression analysis to characterize risk factors for death: persistent neutropenia was the only risk factor for death, regardless of antifungal therapy.

The present review covers combination approaches of antimicrobial photodynamic therapy (aPDT) plus antibiotics or antifungals to attack bacteria and fungi in vitro (both planktonic and biofilm forms) focused on those microorganisms that cause infections in skin and soft tissues. The combination can prevent failure in the fight against these microorganisms: antimicrobial drugs can increase the susceptibility of microorganisms to aPDT and prevent the possibility of regrowth of those that were not inactivated during the irradiation; meanwhile, aPDT is effective regardless of the resistance pattern of the strain and their use does not contribute to the selection of antimicrobial resistance. Additive or synergistic antimicrobial effects in vitro are evaluated and the best combinations are presented. The use of combined treatment of aPDT with antimicrobials could help overcome the difficulty of fighting high level of resistance microorganisms and, as it is a multi-target approach, it could make the selection of resistant microorganisms more difficult.

REIPI-SEIMC COVID-19 group and COVID@HULP group., [Background] The clinical presentation of COVID-19 in patients admitted to hospital is heterogeneous. We aimed to determine whether clinical phenotypes of patients with COVID-19 can be derived from clinical data, to assess the reproducibility of these phenotypes and correlation with prognosis, and to derive and validate a simplified probabilistic model for phenotype assignment. Phenotype identification was not primarily intended as a predictive tool for mortality., [Methods] In this study, we used data from two cohorts: the COVID-19@Spain cohort, a retrospective cohort including 4035 consecutive adult patients admitted to 127 hospitals in Spain with COVID-19 between Feb 2 and March 17, 2020, and the COVID-19@HULP cohort, including 2226 consecutive adult patients admitted to a teaching hospital in Madrid between Feb 25 and April 19, 2020. The COVID-19@Spain cohort was divided into a derivation cohort, comprising 2667 randomly selected patients, and an internal validation cohort, comprising the remaining 1368 patients. The COVID-19@HULP cohort was used as an external validation cohort. A probabilistic model for phenotype assignment was derived in the derivation cohort using multinomial logistic regression and validated in the internal validation cohort. The model was also applied to the external validation cohort. 30-day mortality and other prognostic variables were assessed in the derived phenotypes and in the phenotypes assigned by the probabilistic model., [Findings] Three distinct phenotypes were derived in the derivation cohort (n=2667)—phenotype A (516 [19%] patients), phenotype B (1955 [73%]) and phenotype C (196 [7%])—and reproduced in the internal validation cohort (n=1368)—phenotype A (233 [17%] patients), phenotype B (1019 [74%]), and phenotype C (116 [8%]). Patients with phenotype A were younger, were less frequently male, had mild viral symptoms, and had normal inflammatory parameters. Patients with phenotype B included more patients with obesity, lymphocytopenia, and moderately elevated inflammatory parameters. Patients with phenotype C included older patients with more comorbidities and even higher inflammatory parameters than phenotype B. We developed a simplified probabilistic model (validated in the internal validation cohort) for phenotype assignment, including 16 variables. In the derivation cohort, 30-day mortality rates were 2·5% (95% CI 1·4–4·3) for patients with phenotype A, 30·5% (28·5–32·6) for patients with phenotype B, and 60·7% (53·7–67·2) for patients with phenotype C (log-rank test p, [Interpretation] Patients admitted to hospital with COVID-19 can be classified into three phenotypes that correlate with mortality. We developed and validated a simplified tool for the probabilistic assignment of patients into phenotypes. These results might help to better classify patients for clinical management, but the pathophysiological mechanisms of the phenotypes must be investigated., [Funding] Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA., Funding: Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA.

BackgroundLivestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported.ObjectivesTo determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates.MethodsOne thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6–12 months in 17 Spanish hospitals (2018–2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing.ResultsForty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin–inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p < 0.005).ConclusionCC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.

The EUCAST EDef 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against Aspergillus spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare visual and spectrophotometric MIC readings of azoles and amphotericin B against Aspergillus fumigatus sensu lato isolates. A total of 847 A. fumigatus sensu lato isolates (A. fumigatus sensu stricto [n = 828] and cryptic species [n = 19]) were tested against amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole using the EUCAST EDef 9.3.2 procedure. Isolates were classified as susceptible or resistant/non-wild type according to the 2020 updated breakpoints. The area of technical uncertainty for the azoles was defined in the updated breakpoints. Visual and spectrophotometric (fungal growth reduction of >95% compared to the control, read at 540 nm) MICs were compared. Essential (±1 2-fold dilution) and categorical agreements were calculated. Overall, high essential (97.1%) and categorical (99.6%) agreements were found. We obtained 100% categorical agreements for amphotericin B, itraconazole, and posaconazole, and consequently, no errors were found. Categorical agreements were 98.7 and 99.3% for voriconazole and isavuconazole, respectively. Most of the misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or within one 2-fold dilution above the breakpoint. The resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant cyp51A mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against A. fumigatus sensu lato isolates may be a convenient alternative to visual endpoint readings.

1 Figura.- 1 Tabla, Pulmonary aspergillosis is a severe infectious disease caused by some members of the Aspergillus genus, that affects immunocompetent as well as immunocompromised patients. Among the different disease forms, Invasive Aspergillosis is the one causing the highest mortality, mainly, although not exclusively, affecting neutropenic patients. This genus is very well known by humans, since different sectors like pharmaceutical or food industry have taken advantage of the biological activity of some molecules synthetized by the fungus, known as secondary metabolites, including statins, antibiotics, fermentative compounds or colorants among others. However, during infection, in response to a hostile host environment, the fungal secondary metabolism is activated, producing different virulence factors to increase its survival chances. Some of these factors also contribute to fungal dissemination and invasion of adjacent and distant organs. Among the different secondary metabolites produced by Aspergillus spp. Gliotoxin (GT) is the best known and better characterized virulence factor. It is able to generate reactive oxygen species (ROS) due to the disulfide bridge present in its structure. It also presents immunosuppressive activity related with its ability to killmammalian cells and/or inactivate critical immune signaling pathways like NFkB. In this comprehensive review, we will briefly give an overview of the lung immune response against Aspergillus as a preface to analyse the effect of different secondary metabolites on the host immune response, with a special attention to GT. We will discuss the results reported in the literature on the context of the animal models employed to analyse the role of GT as virulence factor, which is expected to greatly depend on the immune status of the host: why should you hide when nobody is seeking for you? Finally, GT immunosuppressive activity will be related with different human diseases predisposing to invasive aspergillosis in order to have a global view on the potential of GT to be used as a target to treat IA., This work was supported by Fondo Social Europeo (FSE; Gobierno de Aragón), ASPANOA and by grants SAF2017-83120-C2-1-R, SAF2014- 54763-C2-1-R, SAF2014-54763-C2-2-R from Spanish Ministry of Economy and Competitivenes. MV-G was granted by a Río Hortega contract of National Institute of Health Carlos III (CM16/00236) and MA by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (FJCI-2017-31629).

Introduction Antimicrobial resistance is a global health threat. It has been studied in humans and domestic animals, but there is a lack of data on wild animals. The objective of this study is the elucidation of its patterns in Staphylococcus spp. isolated from wild mammals of the Autonomous Community of Aragón (Spain). Material and Methods A total of 103 mammals (Artiodactyla, Carnivora, Chiroptera, Erinaceomorpha, and Lagomorpha) were studied. A recovery centre provided 32 and hunting 71. Nasal and faecal samples yielded 111 staphylococci, which were identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. A susceptibility test to 11 antibiotics was carried out, and statistical analysis was performed. Results Some differences were detected in bacterial prevalence depending on how the mammal fed. Artiodactyla, mainly hunted, were predisposed to carry coagulase-positive staphylococci. The staphylococci species recovered were resistant to at least two classes of antibiotics, and were disseminated in all of the geographical areas studied. Conclusion Resistant staphylococci are widely distributed in the wild mammals in the areas of the study, but the resistance quantified in them is lower than that to be expected if the use of antibiotics in farms had a direct influence on the wildlife and its environment. On the other hand, resistance to antibiotics restricted to human use was widely disseminated in various wild animal species.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of lineage CC398 is an emerging clone causing human infections but is mostly found in pigs. The aim of this study was to characterize the antimicrobial resistance phenotypes/genotypes of a collection of 137 MRSA CC398 isolates obtained in a previous study from 17 Spanish hospitals, using tetracycline resistance as marker for selection. A multidrug-resistant (MDR) phenotype was present in 79% of analysed isolates, with 17% of them resistant to at least six different antimicrobial families. All tetracycline-resistant isolates (n=137) carried the tetM gene and 75% also carried the tetK gene. Almost 50% of MRSA CC398 isolates showed macrolide and/or lincosamide resistance: a) 39% of isolates were ERYR-CLIR (all with constitutive phenotype), with 87% of them carrying the ermC gene, followed by msrA (25%), ermB (21%), vgaA (17%), ermA (6%), lsaB (4%), linA (2%), linB (2%), and ermT (2%, this isolate with the new spa-type t18071); and b) 9% of MRSA CC398 isolates showed the dissociated ERYS-CLIR phenotype carrying the linA, linB, lsaB and vgaA genes. Other antimicrobial resistance phenotypes in these MRSA CC398 isolates included resistance to ciprofloxacin (67%), aminoglycosides (21%), mupirocin (6%), chloramphenicol (4%) or fusidic acid (2%). The more common resistance genes detected for some of these antimicrobials were: aac(6’)-Ie-aph(2’’)-Ia (16%) and ant(4’)-Ia (12%) for aminoglycosides, and fexA (3%) for chloramphenicol. The high rate of MDR phenotypes with a wide range of antimicrobial resistance genes shown in this study reduce the potential therapeutic options in case of infections., This work was supported by the Agencia Estatal de Investigación (AEI) of Spain (project SAF2016-76571-R), the Fondo Europeo de Desarrollo Regional (FEDER) of the EU, and the Sociedad de Enfermedades Infecciosas del Norte (SEINORTE). Sara Ceballos and Laura Ruiz-Ripa had predoctoral fellowships of the University of La Rioja (Spain) during the experimental part of the study.

Purpose. The genus Arcobacter includes bacteria that are considered emergent pathogens because they can produce infections in humans and animals. The most common symptoms are bloody and non-bloody persistent diarrhea but cases with abdominal cramps without diarrhea or asymptomatic cases have also been described as well as cases with bacteremia. The objective was to characterize Arcobacter clinical strains isolated from the faeces of patients from three Spanish hospitals. Methodology. We have characterized 28 clinical strains (27 of A. butzleri and one of A. cryaerophilus) isolated from faeces, analysing their epidemiological relationship using the multilocus sequence typing (MLST) approach and screening them for their antibiotic susceptibility and for the presence of virulence genes. Results/Key findings. Typing results showed that only one of the 28 identified sequence types (i.e. ST 2) was already present in the MLST database. The other 27 STs constituted new records because they included new alleles for five of the seven genes or new combinations of known alleles of the seven genes. All strains were positive for the ciaB virulence gene and sensitive to tetracycline. However, 7.4 % of the A. butzleri and A. cryaerophilus strains showed resistance to ciprofloxacin. Conclusion. The fact that epidemiological unrelated strains show the same ST indicates that other techniques with higher resolution should be developed to effectively recognize the infection source. Resistance to ciprofloxacin, one of the antibiotics recommended for the treatment of Arcobacter intestinal infections, demonstrated in 10.7 % of the strains, indicates the importance of selecting the most appropriate effective treatment.

ECMM ISHAM EFISG Astellas MSD Pfizer Christian Doppler Laboratory for invasive fungal infections Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods : A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results : The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions : Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole. Med Univ Innsbruck, Div Hyg & Med Microbiol, Innsbruck, Austria Hosp Santa Creu & Sant Pau, Serv Microbiol, Barcelona, Spain Univ Zaragoza, Hosp Univ Miguel Servet, IIS Aragon, Microbiol, Zaragoza, Spain Univ Delhi, Vallabhbhai Patel Chest Inst, Dept Med Mycol, New Delhi, India Hamad Med Corp, Dept Lab Med & Pathol, Microbiol Div, Doha, Qatar Univ Copenhagen, Rigshosp, Dept Clin Microbiol, Unit Mycol,Statens Serum Inst, Copenhagen, Denmark Univ Catania, Dept Biomed & Biotechnol Sci, Catania, Italy Univ Texas MD Anderson Canc Ctr, Houston, TX USA Inst Salud Carlos III, Natl Ctr Microbiol, Madrid, Spain Katholieke Univ Leuven, Dept Microbiol & Immunol, Leuven, Belgium Univ Integrata, Azienda Osped, Dipartimento Patol Diagnost, Unita Operat Complessa Microbiol & Virol, Verona, Italy Canisius Wilhelmina Hosp, Dept Med Microbiol & Infect Dis, Nijmegen, Netherlands Med Univ Graz, Inst Hyg Microbiol & Environm Med, Graz, Austria ASST Papa Giovanni XXIII, Microbiol Inst, Bergamo, Italy Univ Athens, ATTIKON Univ Hosp, Dept Internal Med 4, Infect Dis Res Lab, Athens, Greece Ca Granda Osped Maggiore Policlin, IRCCS Fdn, Lab Cent Analisi Chim Clin & Microbiol, Milan, Italy Univ Milan, Dept Biomed Sci Hlth, Milan, Italy Med Univ Vienna, Dept Lab Med, Div Clin Microbiol, Vienna, Austria Univ Cologne, Inst Med Microbiol Immunol & Hyg, Cologne, Germany Publ Hlth England, Mycol Reference Lab, Bristol, Avon, England Karolinska Univ Hosp, Karolinska Inst, Dept Lab Med, Stockholm, Sweden Univ Belgrade, Inst Microbiol & Immunol, Fac Med, Natl Reference Med Mycol Lab, Belgrade, Serbia Univ Cologne, German Ctr Infect Res, Ctr Integrated Oncol CIO Koln Bonn,Clin Trials Ct, Dept Internal Med,Cologne Excellence Cluster Cell, Cologne, Germany ATTIKON Univ, Hosp Athens, Natl Kapodistrian Univ Athens, Clin Microbiol Lab, Athens, Greece Klinikum Wels Grieskirchen, Dept Hyg & Med Microbiol, Wels, Austria Div Microbiol, Dept Publ Hlth & Pediat, Turin, Italy Univ Hosp Cologne, Dept Internal Med, Cologne, Germany Partner Site Bonn Cologne, German Ctr Infect Res, Cologne, Germany Biometec Univ Catania, AOU Policlin Vittorio Emanuele Catania, Catania, Italy Univ Bologna, S Orsola Malpighi, Dept Med & Surg Sci, Infect Dis Unit, Bologna, Italy Tel Aviv Univ, Sackler Sch Med, Dept Clin Microbiol & Immunol, Tel Aviv, Israel Univ Duisburg Essen, Univ Hosp Essen, Inst Med Microbiol, Essen, Germany Palacky Univ Olomouc, Fac Med & Dent, Dept Microbiol, Olomouc, Czech Republic Univ Hosp Olomouc, Olomouc, Czech Republic Univ Cadiz, Puerta Mar Univ Hosp, Clin Microbiol, Cadiz, Spain Hippokrateion Hosp, Aristotle Univ Sch Hlth Sci, Fac Med, Infect Dis Unit,Dept Pediat 3, Thessaloniki, Greece Hacettepe Univ Med Sch, Dept Med Microbiol, Ankara, Turkey Postgrad Inst Med Educ & Res, Dept Medial Microbiol, Div Mycol, Chandigarh, India Univ Fed Sao Paulo, Escola Paulista Med, Sao Paulo, Brazil Univ Nacl Nordeste, CONICET, Inst Medicina Reg, Dept Micol, Resistencia, Argentina Vall Hebron Univ Hosp, Dept Microbiol, Div Clin Mycol, Barcelona, Spain Mazandaran Univ Med Sci, Dept Med Mycol & Parasitol, Invas Fungi Res Ctr, Sari, Iran European Univ Cyprus, Sch Med, Nicosia, Cyprus North Western State Med Univ, Dept Clin Mycol Allergy & Immunol, St Petersburg, Russia Univ Hosp Cologne, Dept Internal Med, Cologne, Germany Hacettepe Univ, Sch Med, Dept Infect Dis & Clin Microbiol, Ankara, Turkey Iran Univ Med Sci, Sch Med, Dept Med Mycol & Parasitol, Tehran, Iran Hosp Ernest Lluch Martin, Dept Dermatol, Zaragoza, Spain Westerdijk Fungal Biodivers Inst, Dept Appl & Ind Mycol, Utrecht, Netherlands Univ Fed Sao Paulo, Escola Paulista Med, Sao Paulo, Brazil Web of Science

Antimicrobial photodynamic therapy (aPDT) has shown to exert a bactericidal effect against Streptococcus sanguinis and Streptococcus mutans. However, this efficacy has been reported for either type of bacteria separately. Bacterial suspensions of both strains, separately or together, were treated with concentrations of methylene blue (MB) and rose bengal (RB). Suspensions were irradiated with a light-emitting diode lamp (λ center at 625nm for MB and λ center at 515nm for RB) using a fluence of 18J/cm2. RB-aPDT at concentrations of 0.16-0.62 and 0.16-0.31μg/mL, and MB-aPDT at concentrations of 0.62-1.25 and 0.31-1.25μg/mL inhibited the growth of S. mutans and S. sanguinis respectively by 6 log10. In suspensions of both strains together, the same 6 log10 reduction in bacterial growth was achieved using the same concentrations of each photosensiziser. In conclusion, RB-aPDT and MB-aPDT appear to exert the same bactericidal effect against suspensions of S. sanguinis and S. mutans either for single strain treatment or for samples constituted by both bacteria mixed together. RB shows to be slightly more efficient than MB.

The aim of the present work was to compare the impact on the gut microbiota composition and in the systemic inflammation of two types of breads denominated as industrial and celta with different composition (wheat flour vs . complex flour and seeds) and fermentation process (2 h vs. 24 h). Faecal and blood samples of 10 mice were collected at: (1) - basal, (2) - after 21 days of industrial bread intake and (3) - after another 21 days of celta bread exposure. Significant differences in the gut microbiota composition were detected after the consumption of both breads, whereas the celta bread increased the concentration of Akkermansia and Mucispirillum , the industrial bread favour the Bacteroidetes proliferation and additionally systemic inflammation. In conclusion, the more consumed industrial processed bread might provoke systemic inflammation, probably in relation with its composition and manufacture process.

Background: Despite the bacterial microbiota has been studied in the lung cancer context, the micobiome has not been explored yet. The aim of this study was to compare the bronchial and oral microbiome from patients with lung cancer with healthy controls Methods: A total of 25 lung cancer patients [non-small cell(18) and small cell lung cancer(7)], and 16 healthy controls were recruited. None had taken antibiotics in the previous month. Bronchial biopsies were obtained by broncoscopy from tumoral and contralateral non involved bronchi in cancer patients and from healthy bronchi in controls. Patients and controls also contributed with a saliva sample. Total DNA was obtained using the QiaAmp kit (Qiagen), determining the microbiome composition (16S rDNA V3-V4) and micobiome (ITS2) by massive sequencing in MiSeq platform (Illumina), statistical analysis in Qiime 2 and taxonomy assignation in the Silva19 database Results: Patients and controls had similar microbiome and micobiome in the saliva samples, which differ significantly from those obtained in the bronchial tract. The bronchial micobiome of patients and controls were also similar, whereas significant differences were observed for microbiota. The bronchi of lung cancer patients presented a higher bacterial charge (10^4vs10^3) than healthy controls, and a considerable increase of Firmicutes, mostly due to Streptococcus. Comparing the affected bronchi versus the contralateral, the differences were minor Conclusions: Despite the microbiome of saliva and bronchial tract were similar, ecological particularities of each anatomic localizations were observed. Micobiome seems not to be affected by the tumoral process, whereas the bacterial populations suffer several alterations in composition and density

Aims To determine the Staphylococcus aureus carriage rate in wild mammals in Aragon, northern Spain, to analyse their antimicrobial resistance phenotype/genotype and to characterize the recovered isolates. Methods and results Nasal and rectal swabs of 103 mammals were collected in Aragon during the period 2012-2015. Antimicrobial susceptibility, the presence of antimicrobial resistance genes and virulence factors were investigated. Molecular characterization was carried out by spa, MLST, agr and SCCmec. Staphylococcus aureus were recovered from 23 animals (22%). Four of the 23 S. aureus were methicillin-resistant S. aureus (MRSA). Three MRSA were mecC-positive and were isolated from European rabbits and were typed as t843 (ascribed to CC130). The remaining MRSA was a mecA-carrying isolate from European hedgehog, typed as ST1-t386-SCCmecIVa-agrIII and it harboured the blaZ, erm(C), ant(6)-Ia and aph(3´)-IIIa resistance genes. A high diversity of spa-types was detected among the 19 methicillin-susceptible S. aureus isolates, which showed high susceptibility to the antimicrobials tested. The tst gene and different combinations of staphylococcal enterotoxins were found. Conclusions Staphylococcus aureus were detected in nasal and rectal samples of wild mammals. Wild rabbits could be a reservoir of mecC-MRSA. Significance and impact of the study This work provides information on the presence and characteristics of S. aureus from mammals in a defined geographic region in Spain.

Urinary tract infection is the most common human infection with a high morbidity. In primary care and hospital services, conventional urine culture is a key part of infection diagnosis but results take at least 24 h. Therefore, a rapid and reliable screening method is still needed to discard negative samples as quickly as possible and to reduce the laboratory workload. In this aspect, this study aims to compare the diagnostic performance between Sysmex UF-1000i and FUS200 systems in comparison to urine culture as the gold standard. From March to June 2016, 1,220 urine samples collected at the clinical microbiology laboratory of the “Miguel Servet” hospital were studied in parallel with both analysers, and some technical features were evaluated to select the ideal equipment. The most balanced cut-off values taking into account bacteria or leukocyte counts were 138 bacteria/μL or 119.8 leukocyte/μL for the UF-1000i (95.3% SE and 70.4% SP), and 5.7 bacteria/μL or 4.3 leukocyte/μL for the FUS200 (95.8% SE and 44.4% SP). The reduction of cultured plates was 37.4% with the FUS200 and 58.3% with the UF-1000i. This study shows that both techniques improve the workflow in the laboratory, but the UF-1000i has the highest specificity at any sensitivity and the FUS200 needs a shorter processing time.

The microorganisms that cause infections are increasing their resistance to antibiotics. In this context, alternative treatments are necessary. The antimicrobial photodynamic therapy (aPDT) is a therapeutic modality based on photosensitizing molecules that end up generating reactive oxygen species that induce the destruction of the target cells when are irradiated with light of a suitable wavelength and at a proper dose. The cells targeted by aPDT are all types of microorganisms (bacteria, fungi and parasites) including viruses and has been proven effective against representative members of all of them. In the field of dermatology, aPDT has been tested with promising results in different infections such as chronic ulcers, acne, onychomycosis and other cutaneous mycoses, as well as in leishmaniasis. Therefore, it is presented as a possible treatment option against the agents that cause skin and/or mucous infections.

Background Onychomycosis is a common fungal nail infection that responds poorly to antifungals. Objective To investigate the efficacy and safety of methyl aminolevulinate (MAL) photodynamic therapy (PDT) in the treatment of onychomycosis. Methods A multicentre (3), randomized, placebo-controlled clinical trial compared the effects of three sessions of urea (40%) plus conventional MAL-PDT with urea (40%) plus placebo (red light) photodynamic therapy (pPDT) in onychomycosis patients. Efficacy, both clinical (onychomycosis severity index, OSI) and microbiological, was blindly evaluated after 36 weeks of follow-up. Results Forty patients were analysed in the trial. Twenty-two received MAL-PDT and 18 pPDT. A complete response (OSI = 0) was observed for four patients (18.18%) in the MAL-PDT group and one (5.56%) in the pPDT group (NTT 7.92, 95% CI: 2.98–9.69, P = 0.23). A decrease in OSI score of over 75% (OSI75) was achieved by 40.91% of the patients in the MAL-PDT group and 16.67% in the pPDT group (P = 0.096). Microbiological cure was achieved by seven patients (31.82%) in the MAL-PDT group and two (11.11%) in the pPDT group (P = 0.178). MAL-PDT resulted in better rates of clinical response [OSI >75%: 53.85% vs. 18.75% (P =0.048)] and microbiological cure [41.56% vs. 7.14% (P = 0.037)] in non-dystrophic vs. dystrophic onychomycosis patients. No significant side-effects were reported. The limitations of the study were the reduced sample size and the unexpected efficacy of the control treatment, which was attributed to the 40% urea pre-treatment. Conclusion This study did not show significant differences between urea 40% + MAL-PDT and urea 40% + pPDT in the treatment of onychomycosis. However, some results suggest that this treatment may constitute an alternative for dermatophyte and non-dermatophyte mould onychomycosis in patients not eligible for systemic treatment, particularly in the absence of total nail dystrophy.

Resumen La onicomicosis, o infeccion fungica de las unas, constituye una de las enfermedades micoticas mas prevalentes en la poblacion. Su tratamiento tiene una efectividad limitada, ademas de ser largo y tedioso y, en el caso de los antifungicos sistemicos, no esta exento de efectos adversos. La terapia fotodinamica (TFD) podria ser una buena alternativa para las infecciones cutaneas superficiales, entre ellas la onicomicosis. El presente articulo revisa la experiencia publicada, tanto in vitro como in vivo , acerca de la utilidad de la TFD en las onicomicosis, mostrando el potencial de diversos fotosensibilizantes, asi como sus limitaciones. Desde el punto de vista clinico el azul de metileno y el acido 5-aminolevulinico muestran tasas de curacion del 80% y el 43% respectivamente al ano de seguimiento. Finalmente, basado en la bibliografia y en la propia experiencia, se propone un protocolo de 3 sesiones de TFD, usando metil-aminolevulinato 16% como fotosensibilizante y luz roja (λ = 630 nm, 37 J.cm −2 ), separadas por 1 o 2 semanas. Estas iran precedidas de la aplicacion de urea 40% durante unos dias. Nuevos ensayos clinicos deben optimizar los protocolos y establecer que pacientes se benefician especialmente de recibir este tratamiento.

PURPOSE To compare the effects of 12-week training periods (2 d·wk) involving resistance training only with the effects of 12-week training periods involving combined resistance (once weekly) and endurance (once weekly) training on strength, endurance performance, and quality of life. METHODS Thirty-six patients with moderate-to-severe chronic obstructive pulmonary disease were randomized to combined training (REG), resistance training alone (RG), or control (CG) groups. Patients were tested for maximal strength of the upper and lower extremities, power output of the lower extremities, maximal (Wmax) and submaximal exercise capacity, performance on a 6-minute walk test (6MWT), and quality of life. RESULTS REG and RG induced similar maximal strength gains. Muscle power increased 19% (P ≤ .01) and Wmax improved 13% (P < .05) in REG. Reductions (P < .05) in the heart rate and blood lactate at a given submaximal workload were observed in REG. Improvements in 6MWT and quality of life were similar in both training groups. CONCLUSIONS Compared with a twice-weekly resistance training program, the combination of once-weekly resistance and once-weekly endurance training not only produced similar gains in maximal strength, 6MWT performance, and quality of life but also produced improvements in muscle power and endurance performance. These findings may have implications for the prescription of resistance and endurance exercise for patients with chronic obstructive pulmonary disease.

Staphylococcus pseudintermedius is an opportunistic pathogen that has been identified as infectious agent or colonizer mainly in dogs. S. pseudintermedius has been also detected in humans and more specifically in people in contact with dogs. In this study, the possible S. pseudintermedius pet-to-human transmission was analyzed in four clinical human cases. Two patients were dog owners and S. pseudintermedius was also detected as colonizer in these healthy animals. S. pseudintermedius isolates from patients and dogs of the same household showed identical pulsed-field gel electrophoresis patterns, sequence types (STs), and antimicrobial resistance phenotypes and genotypes, and were methicillin susceptible. Resistance to erythromycin, clindamycin, tetracycline, trimetoprim-sulfamethoxazole, and/or ciprofloxacin was identified among S. pseudintermedius strains. The lineages ST241 and the new ST521 were detected in the strains of the two dog-owner patients, respectively. The strains from the other two patients presented two new STs, ST719 and ST720. To our knowledge, this is the first description of human infections caused by S. pseudintermedius in Spain.

Antimicrobial photodynamic therapy (a-PDT), combined or not with antibiotics, constitutes a promising therapy for superficial infections caused by bacteria implicated in multidrug resistance processes. We compared the efficacy of aPDT using the photosensitizer methylene blue (MB), combined or not with the antibiotic gentamicin (GN), against Staphylococcus aureus and Pseudomonas aeruginosa. Different concentrations of MB (0.03-7000 μg/mL), with or without GN (1-20 μg/mL), were added to planktonic cultures or biofilms and the samples irradiated with a LED lamp (λ 625 nm, 7 mW/cm2, 18 J/cm2). The number of viable bacteria in the samples and in corresponding nonirradiated controls was quantified by counting colony-forming units to evaluate the individual effects of MB, GN, and irradiation. MB-aPDT resulted in significant bacterial photoinactivation. The combination of GN and MB-aPDT exerted a synergistic bactericidal effect against planktonic cultures of S. aureus and P. aeruginosa. This combination did not significantly alter the photoinactivating effect of MB against S. aureus biofilms, but exerted a positive bactericidal effect against P. aeruginosa biofilms. These results underscore the need for further clinical studies of this therapeutic combination for the management of difficult-to-treat skin and mucous infections, especially those caused by P. aeruginosa.

Antifungal resistance is increasing by the emergence of intrinsically resistant species and by the development of secondary resistance in susceptible species. A previous study performed in Spain revealed levels of azole resistance in molds of between 10 and 12.7%, but secondary resistance in Aspergillus fumigatus was not detected. We used itraconazole (ITZ)-supplemented medium to select resistant strains. A total of 500 plates supplemented with 2 mg/liter of ITZ were sent to 10 Spanish tertiary hospitals, and molecular identification and antifungal susceptibility testing were performed. In addition, the cyp51A gene in those A. fumigatus strains showing azole resistance was sequenced. A total of 493 isolates were included in the study. Sixteen strains were isolated from patients with an infection classified as proven, 104 were isolated from patients with an infection classified as probable, and 373 were isolated from patients with an infection classified as colonization. Aspergillus was the most frequent genus isolated, at 80.3%, followed by Scedosporium-Lomentospora (7.9%), Penicillium-Talaromyces (4.5%), Fusarium (2.6%), and the order Mucorales (1%). Antifungal resistance was detected in Scedosporium-Lomentospora species, Fusarium, Talaromyces, and Mucorales Three strains of A. fumigatus sensu stricto were resistant to azoles; two of them harbored the TR34+L98H mechanism of resistance, and the other one had no mutations in cyp51A The level of azole resistance in A. fumigatus remains low, but cryptic species represent over 10% of the isolates and have a broader but overall higher range of antifungal resistance. Gilead Sciences (IN-ES-131-1600) Fondo de Investigaciones Sanitarias (PI16CIII/00035, PI13/02783, FI14CIII/00025, PI13/02145) Sí

Urinary tract infections (UTI) are one of the most prevalent infections. A rapid and reliable screening method is useful to screen out negative samples. The objective of this study was to validate the Sysmex flow cytometer UF-1000i by evaluating its accuracy, linearity and carry-over; and define an optimal cut-off value to be used in routine practice in our hospital. For the validation of the UF-1000i cytometer, precision, linearity and carry-over were studied in samples with different counts of bacteria, leukocytes and erythrocytes. Between March and June 2016, urine samples were tested in the Clinical Microbiology Laboratory at University Miguel Servet Hospital, in Spain. Samples were analyzed with the Sysmex UF-1000i cytometer, and cultured. Growth of ≥ 105 CFUs/mL was considered positive. The validation study reveals that the precision in all the variables is acceptable; that there is a good linearity in the dilutions performed, obtaining values almost identical to those theoretically expected; and for the carry-over has practically null values. A total of 1,220 urine specimens were included, of which 213 (17.4%) were culture positive. The optimal cut-off point of the bacteria-leukocyte combination was 138.8 Bacteria or 119.8 leukocytes with an S and E of 95.3% and 70.4%, respectively. The UF-1000i cytometer is a valuable method to screen urine samples to effectively rule out UTI and, may contribute to the reduction of unnecessary urine cultures.

Gliotoxin (GT) is a fungal secondary metabolite that has attracted great interest due to its high biological activity since it was discovered by the 1930s. An inactive derivative of this molecule, bis(methylthio)gliotoxin (bmGT), has been proposed as an invasive aspergillosis (IA) biomarker. Nevertheless, studies regarding bmGT production among common opportunistic fungi, including the Aspergillus genus, are scarce and sometimes discordant. As previously reported, bmGT is produced from GT by a methyl-transferase, named as GtmA, as a negative feedback regulatory system of GT production. In order to analyze the potential of bmGT detection to enable identification of infections caused by different members of the Aspergillus genus we have assessed bmGT production within the genus Aspergillus, including A, fumigatus, A. niger, A. nidulans, and A. flavus, and its correlation with gtmA presence. In order to validate the relevance of our in vitro findings, we compared bmGT during in vitro culture with the presence of bmGT in sera of patients from whom the Aspergillus spp. were isolated. Our results indicate that most A. fumigatus isolates produce GT and bmGT both in vitro and in vivo. In contrast, A. niger and A. nidulans were not able to produce GT or bmGT, although A. niger produced bmGT from a exogenous GT source. The frequency and amount of bmGT production in A. terreus and A. flavus isolates in vitro was lower than in A. fumigatus. Our results suggest that this defect could be related to the in vitro culture conditions, since isolates that did not produce bmGT in vitro were able to synthetize it in vivo. In summary, our study indicates that bmGT could be very useful to specifically detect the presence of A. fumigatus, the most prevalent agent causing IA. Concerning A. terreus and A. flavus a higher number of analyses from sera from infected patients will be required to reach a useful conclusion., This work was supported by Fondo Social Europeo (FSE; Gobierno de Aragón) and by grants MAT2011-26851-C02-02,SAF2014- 54763-C2-1-R, SAF2014-54763-C2-2-R from Spanish Ministry of Economy and Competitiveness. JP was supported by Aragón ICD (ARAID). MV-G was granted by a Río Hortega contract of National Institute of Health Carlos III (CM16/00236), We kindly acknowledge Dr. J. F. Meis from Canisius-Wilhelmina Hospital, Nijmegen (Netherlands) and Dr. E. Bouza from Gregorio Marañón University Hospital, Madrid (Spain) for providing clinical isolates from several Aspergillus species and from cryptic species, respectively. We would like to acknowledge ASPANOA (Asociacion de Padres de Niños con Cáncer de Aragón) for its support to develop this work.