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Cell-surface signalling (CSS) is a signal transfer system of Gram-negative bacteria used to detect extracellular signals and modulate gene transcription in response. These three-protein systems are formed by an outer membrane receptor, a cytoplasmic membrane-embedded anti-σ factor and a cytosolic extracytoplasmic function σ factor (σECF). In absence of an inducing signal, the anti-σ factor binds to and keeps the σECFfactor sequestered, thus preventing its interaction with the RNA polymerase and the transcription of signal response genes. Presence of the signal triggers a signalling cascade that extends from the outer membrane to the cytosol and results in σECFfactor activation. Recently, we and others have reported that CSS σECFfactor activation requires the regulated and sequential proteolysis of the cognate anti-σ factor, and the function of the Prc and RseP proteases. However, many features of this proteolytic cascade are still unclear. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. We show that both CtpA and the previously identified protease Prc control CSS activation by modulating the levels of the anti-σ factor. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-σ factor. Importantly, using zebrafish embryos and the A549 cell line as hosts, we show that mutants in thersePandctpAproteases of the human pathogenPseudomonas aeruginosaare considerably attenuated in virulence while theprcmutation increases virulence likely by enhancing the production of outer membrane vesicles. Because proteases are druggable proteins, the identification of regulatory proteases involved inP. aeruginosavirulence holds promise for the development of novel strategies to fight this clinically relevant pathogen.

Pseudomonas bacteria are widespread and are found in soil and water, as well as pathogens of both plants and animals. The ability of Pseudomonas to colonize many different environments is facilitated by the multiple signaling systems these bacteria contain that allow Pseudomonas to adapt to changing circumstances by generating specific responses. Among others, signaling through extracytoplasmic function σ (σ) factors is extensively present in Pseudomonas. σ factors trigger expression of functions required under particular conditions in response to specific signals. This manuscript reviews the phylogeny and biological roles of σ factors in Pseudomonas, and highlights the diversity of σ-signaling pathways of this genus in terms of function and activation. We show that Pseudomonas σ factors belong to 16 different phylogenetic groups. Most of them are included within the iron starvation group and are mainly involved in iron acquisition. The second most abundant group is formed by RpoE-like σ factors, which regulate the responses to cell envelope stress. Other groups controlling solvent tolerance, biofilm formation and the response to oxidative stress, among other functions, are present in lower frequency. The role of σ factors in the virulence of Pseudomonas pathogenic species is described., We thank W. Koudstaal for critical review of the manuscript. This work was funded by the European Regional Development Fund and the Spanish Ministry of Economy and Competitiveness (MINECO) with grant BIO2017‐83763‐P.

The extracytoplasmic function sigma factor σVreI of the human pathogen Pseudomonas aeruginosa promotes transcription of potential virulence determinants, including secretion systems and secreted proteins. Its activity is modulated by the VreR anti-σ factor that inhibits the binding of σVreI to the RNA polymerase in the absence of a (still unknown) inducing signal. The vreI-vreR genes are expressed under inorganic phosphate (Pi) starvation, a physiological condition often encountered in the host that increases P. aeruginosa pathogenicity. However, whether or not σVreI is active in vivo during infection and contributes to the Pi starvation-induced virulence of this pathogen has not been analyzed yet. Using zebrafish embryos and a human alveolar basal epithelial cell line as P. aeruginosa hosts, we demonstrate in this work that σVreI is active during infection and that lack of σVreI considerably reduces the Pi starvation-induced virulence of this pathogen. Surprisingly, lack of the σVreI inhibitor, the VreR anti-σ factor, also diminishes the virulence of P. aeruginosa. By transcriptomic analyses we show that VreR modulates gene expression not only in a σVreI-dependent but also in a σVreI-independent manner. This includes potential virulence determinants and transcriptional regulators that could be responsible for the reduced virulence of the ΔvreR mutant., We thank W. Koudstaal for critical review of the manuscript, A. van der Sar and T. Verboom (AUMC, Amsterdam) for facilitating the work on zebrafish embryos, F. Madrazo (Instituto de Investigación Sanitaria Valdecilla) for assistance with the confocal fluorescence imaging, and R. Tobes (Era7 Bioinformatics) for the RNA-seq bioinformatics analysis. This work was funded by FEDER and the Spanish Ministry of Economy with grants SAF2015-68873-P and BIO2017-83763-P. JOA was supported by the Spanish Ministry of Economy through a FPI fellowship (BES-2013-066301).

Pathogens have developed several strategies to obtain iron during infection, including the use of iron-containing molecules from the host. Haem accounts for the vast majority of the iron pool in vertebrates and thus represents an important source of iron for pathogens. Using a proteomic approach, we have identified in this work a previously uncharacterized system, which we name Hxu, that together with the known Has and Phu systems, is used by the human pathogen Pseudomonas aeruginosa to respond to haem. We show that the Has and Hxu systems are functional signal transduction pathways of the cell-surface signalling class and report the mechanism triggering the activation of these signalling systems. Both signalling cascades involve an outer membrane receptor (HasR and HxuA respectively) that upon sensing haem in the extracellular medium produces the activation of an σ factor in the cytosol. HxuA has a major role in signalling and a minor role in haem acquisition in conditions in which the HasR and PhuR receptors or other sources of iron are present. Remarkably, P. aeruginosa compensates the lack of the HasR receptor by increasing the production of HxuA, which underscores the importance of haem signalling for this pathogen., This work was funded by the European Regional Development Fund and the Spanish Ministry of Economy with grants SAF2015‐68873‐P and BIO2017‐83763‐P. JOA is supported by the Spanish Ministry of Economy through a FPI fellowship (BES‐2013‐066301) and MAL through a Ramon&Cajal grant (RYC2011‐08874).

Pseudomonas bacteria are widespread and are found in soil and water, as well as pathogens of both plants and animals. The ability of Pseudomonas to colonize many different environments is facilitated by the multiple signaling systems these bacteria contain that allow Pseudomonas to adapt to changing circumstances by generating specific responses. Among others, signaling through extracytoplasmic function σ (σ

Introduction Influenza viruses and respiratory syncytial virus (RSV) can cause an acute respiratory disease that occurs seasonally in epidemic waves. This retrospective study was conducted to evaluate the Sofia® Influenza A + B and the Sofia® RSV fluorescence immunoassays (FIAs), two novel rapid detection tests (RDTs) for influenza A and B and RSV. Methods Two hundred and nine breath samples were selected from patients with respiratory symptoms determined to be positive/negative for influenza A, influenza B or RSV using one of the reference diagnostic techniques, cell culture and/or RT-PCR (Simplexa™Flu A/B & RSV). The Sofia Influenza A + B FIA was tested on 123 samples (63 from children and 60 from adults) and the Sofia RSV FIA was tested on 86 pediatric samples. Sensitivity and specificity values of both assays were calculated assuming the reference techniques as the gold standard. Results Sensitivity and specificity values for the Sofia Influenza A + B FIA were 73.1% and 97.8%, respectively. Sensitivity and specificity values for the Sofia RSV FIA were 87.5% and 86.7%, respectively. Conclusion The sensitivity results obtained for the two assays were considerably higher than those reported for other RDTs. In conclusion, the Sofia Influenza A + B and the Sofia RSV FIAs are appropriate tools for the rapid diagnosis of these viruses.

Summary Cell-surface signalling (CSS) enables Gram-negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti-sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic func- tion (ECF) sigma factor. Recent studies have de- monstrated that RseP-mediated proteolysis of the anti-sigma factors is key to σ ECF activation. Using the Pseudomonas aeruginosa FoxR anti-sigma factor, we show here that RseP is responsible for the generation of an N-terminal tail that likely contains pro-sigma activity. Furthermore, it has been reported previously that this anti-sigma factor is processed in two sepa- rate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti-sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti-sigma factors.

Objectives: Limited data exist regarding the role of agr dysfunction in reducing susceptibility to vancomycin in methicillin-susceptibleStaphylococcus aureus (MSSA). This study investigated the clinical and molecular epidemiology of MSSA causing bacteraemia, with emphasis on the reduced susceptibility to vancomycin (RSV) phenotype (MIC ≥1.5 mg/L) and its relationship with agr dysfunction. Methods: All MSSA bloodstream isolates obtained at our hospital during 2010 were analysed. Antimicrobial susceptibility was determined and time –kill experiments were performed for oxacillin. Multilocus sequence type andagr genotype were determined and DNA microarray analysis of virulence factors was performed.agr dysfunction was assessed phenotypically and by RT–PCR quantification of RNAIII. Results: Of 84 MSSA, 55 (65.5%) exhibited the RSV phenotype, comprising 13 clonal complexes.agr II polymorphism was more prevalent in RSV than non-RSV isolates (41.8% versus 17.2%,P ¼ 0.023) and average levels of RNAIII gene expression were higher in RSV than non-RSV isolates (DCt 4.05+3.29 versus 1.5+2.11, P ¼ 0.005), implying greater agr dysfunction in RSV MSSA. Conclusions: We demonstrated a correlation between RSV phenotype in MSSA and reducedagr expression, particularly in association with theagr II genotype. These results may help to understand the role ofagr dysfunction in the increased mortality in MSSA infections.

a b s t r a c t Background: Staphylococcus aureus is the main pathogen causing nosocomial infections. Health profes- sionals, including medical students, could be a source of transmission. The aims of the study were to determine the rate of nasal carriage of S. aureus susceptible and resistant to methicillin (MRSA) and evaluate the knowledge and adherence that students had about hand hygiene. Methods: The study included medical students attached to the Hospital Universitario 12 de Octubre (Madrid, Spain). We collected samples from both nasal vestibules, and the antimicrobial susceptibility was determined on all isolates. Data collection was performed using a self-administered questionnaire that included risk factors for colonization, hygiene habits and knowledge of hand hygiene protocols. Results: Of the 140 students included, 55 (39.3%) were colonized by S. aureus, and 3 (2,1%) by MRSA. The exposure to antibiotics in the last 3 months was lower in colonized students (12.3% vs. 25.9%, P = .03).

Multilocus sequence typing and nrdA sequence analysis identified 6 different species or genogroups and 13 sequence types (STs) among 15 Achromobacter isolates from cystic fibrosis (CF) patients and 7 species or genogroups and 11 STs among 11 isolates from non-CF patients. Achromobacter xylosoxidans was the most frequently isolated species among CF patients.

Background Xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (MLV)-related virus are recently described human gammaretroviruses that have been associated with prostate cancer and chronic fatigue syndrome. These studies have been controversial because a number of laboratories have been unable to find evidence of XMRV in similar groups of patients or controls. Because the existence of XMRV raises many questions, we decided to study its presence in a group of patients infected with HIV-1 with a high proportion of intravenous drug use and coinfection by hepatitis C virus. Methods Forty HIV-1-infected patients under follow-up in our institution were screened for XMRV/MLV by nested polymerase chain reaction using primers targeting the gag and env region. Specific primers for mouse mitochondrial DNA were used to rule out contamination. Results No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls. Four samples yielded polymerase chain reaction bands whose sequence corresponded to murine endogenous retroviral sequences, however, contamination with mouse cell DNA was subsequently confirmed. Conclusions XMRV/MLV viruses do not seem to be associated with HIV-1 infection or intravenous drug use. Contamination of samples or reagents by genomic murine DNA or XMRV vectors could account for the sporadic detection of positive samples for XMRV and related agents.

A total of 183 patients were colonized or infected with multidrug-resistant Pseudomonas aeruginosa isolates at a hospital in Spain during 2007-2010; prevalence increased over this period from 2.8% to 15.3%. To characterize these isolates, we performed molecular epidemiologic and drug resistance analysis. Genotyping showed that 104 (56.8%) isolates belonged to a single major clone (clone B), which was identified by multilocus sequence typing as sequence type (ST) 175. This clone was initially isolated from 5 patients in 2008, and then isolated from 23 patients in 2009 and 76 patients in 2010. PCR analysis of clone B isolates identified the b/a(VIM-2) gene in all but 1 isolate, which harbored b/a(IMP-22). ST175 isolates were susceptible to only amikacin (75%) and colistin (100%). Emergence of the ST175 clone represents a major health problem because it compromises therapy for treatment of P aeruginosa nosocomial infections., This study was supported by Spanish Network for the Research in Infectious Diseases (RD06/0008) from the Instituto de Salud Carlos III, Spain.

Introduction Diagnosis of HSV-1 keratitis (HK) is frequently based on clinical findings. Invasive specimens (corneal scrapings, biopsies) are required for microbiological diagnosis. Methods Corneal scrapings and conjunctival swabs were collected on patients with/without clinical suspicion of HK from 2007 to 2012. Results The sensitivity, specificity, positive and negative predictive values for conjunctival swabs by PCR was 77.8, 92.1, 84.4 and 88.3, respectively. Discussion Conjunctival swabs by PCR may help in the diagnosis of HK, despite the limited sensitivity.

Background The influence of acute graft pyelonephritis (AGPN) on graft outcome in renal transplant recipients still remains controversial. Methods We retrospectively analysed 189 patients (113 males; mean age: 49.7 ± 13.1 years) undergoing renal transplantation at the University Hospital 12 de Octubre (Madrid, Spain) from January 2002 to December 2004, with a minimum follow-up of 36 months. Factors associated with AGPN were assessed by logistic regression analysis. Long-term graft function was compared according to the occurrence of this complication during follow-up. 'Decline in renal graft function' was defined as the increase in serum creatinine (SC) levels > 0.33 mg/dL between Month 3 and Year 1 after transplantation. Results Nineteen patients (10.0%) were diagnosed with 25 episodes of AGPN (incidence rate: 4.4 episodes per 100 patient-years). The presence of glomerulonephritis as the underlying disease [odds ratio (OR) 4.2; 95% confidence interval (95%CI): 1.3-14.1] and the previous occurrence of two to five (OR 9.4; 95%CI: 1.5-56.8) or more than five episodes of asymptomatic bacteriuria after transplantation (OR 19.8; 95%CI: 2.4-160.2) emerged as independent predictors for AGPN. A near-significant association was found for cytomegalovirus infection (OR 4.2; 95%CI: 0.9-18.4), whereas receiving a single-kidney transplant (vs. double-kidney) showed a protective effect (OR 0.2; 95%CI: 0.0-0.8). During the 36-month follow-up, levels of SC, creatinine clearance and 24-h proteinuria did not differ significantly between patients with or without AGPN, and this complication did not exert any effect on the risk for decline in renal graft function. Conclusions AGPN does not impair long-term graft function in renal transplant recipients.

Background. Approximately 25% of haemodialysis (HD) patients use catheters as vascular access. Catheter-related bloodstream infections (CRBSI) are a major risk in this population. The objective of our study was to determine whether endoluminal catheter colonization (ECC) predicts CRBSI. Methods. We followed up a cohort of HD patients in our institution who underwent HD with tunnelled cuffed central venous catheters (TCC) between December 2006 and June 2008. Colonization of the inner catheter lumen was assessed every 15 days immediately before HD by culture of blood―heparin mixture and the time to positivity (TTP) was recorded by the BacT/Alert automated system. CRBSI was confirmed by differential TTP (>2 h) between TCC and peripheral blood cultures. Results. We studied 51 patients who required 64 TCC. The incidence of CRBSI was 1.65 episodes per 1000 catheter-days, with Staphylococcus epidermidis being the most common cause of infection (76.2%). ECC was more frequent in the CRSBI group than in the non-CRBSI group (100 vs 5.4%, P < 0.001). For S. epidermidis CRBSIs, the median time from ECC to CRBSI was 31.5 days (interquartile range, 27.0―79.0). The sensitivity, specificity and negative and positive predictive values of arterial lumen cultures for S. epidermidis CRBSIs were 100, 96.3, 92.3 and 100%, respectively, while for venous culture, these values were 92.3, 96.3, 92.3 and 96.3%, respectively. For predicting S. epidermidis CRBSI, endoluminal cultures with a TTP of ≤ 14 h had sensitivity and specificity of 52.1 and 97.7%, respectively. Conclusions. This study shows that ECC may predict the risk of developing CRSBI. Surveillance cultures could, therefore, be used to triage individual HD patients who might benefit from specific intervention measures.

Most Staphylococcus aureus small-colony variants (SCVs) are auxotrophs for menadione, hemin, or thymidine but rarely for CO 2 . We conducted a prospective investigation of all clinical cases of CO 2 -dependent S. aureus during a 3-year period. We found 14 CO 2 -dependent isolates of S. aureus from 14 patients that fulfilled all requirements to be considered SCVs, 9 of which were methicillin resistant. The clinical presentations included four cases of catheter-related bacteremia, one complicated by endocarditis; two deep infections (mediastinitis and spondylodiscitis); four wound infections; two respiratory infections; and two cases of nasal colonization. Pulsed-field gel electrophoresis typing showed that the 14 isolates were distributed into 4 types corresponding to sequence types ST125- agr group II ( agr II), ST30- agr III, ST34- agr III, and ST45- agr I. An array hybridization technique performed on the 14 CO 2 -dependent isolates and 20 S. aureus isolates with normal phenotype and representing the same sequence types showed that all possessed the enterotoxin gene cluster egc , as well as the genes for α-hemolysin and δ-hemolysin; biofilm genes icaA , icaC , and icaD ; several microbial surface components recognizing adhesive matrix molecules (MSCRAMM) genes ( clfA , clfB , ebh , eno , fib , ebpS , sdrC , and vw ); and the isaB gene. Our study confirms the importance of CO 2 -dependent SCVs of S. aureus as significant pathogens. Clinical microbiologists should be aware of this kind of auxotrophy because recovery and identification are challenging and not routine. Further studies are necessary to determine the incidence of CO 2 auxotrophs of S. aureus , the factors that select these strains in the host, and the genetic basis of this type of auxotrophy.

The mechanisms responsible for the increasing prevalence of colistin-only-sensitive (COS) Pseudomonas aeruginosa isolates in a Spanish hospital were investigated. Pulsed-field gel electrophoresis revealed that 24 (50%) of the studied isolates belonged to the same clone, identified as the internationally spread sequence type 235 (ST235) through multilocus sequence typing. In addition to several mutational resistance mechanisms, an integron containing seven resistance determinants was detected. Remarkably, the extended-spectrum β-lactamase GES-1 and its Gly170Ser carbapenem-hydrolyzing derivative GES-5 were first documented to be encoded in a single integron. This work is the first to describe GES enzymes in Spain and adds them to the growing list of β-lactamases of concern (PER, VIM, and OXA) detected in ST235 clone isolates.

Durante estos ultimos anos hemos asistido a un incremento significativo de la resistencia de los microorganismos grampositivos, con un impacto epidemiologico y clinico importante en la practica diaria. Mientras que en los hospitales espanoles la prevalencia de Staphylococcus aureus resistente a la meticilina (SARM) se mantiene en unos valores elevados (alrededor del 25%), en la comunidad estan emergiendo nuevas cepas de SARM en pacientes sin factores de riesgo, a menudo productoras de toxinas como la leucocidina de Panton- Valentine. Por otra parte, los estafilococos coagulasanegativos (ECN) estan implicados en una gran variedad de infecciones hospitalarias y su resistencia a los antimicrobianos es globalmente superior (p. ej., > 60% a la meticilina) y de mas rapida adquisicion, en general, que la de S. aureus. Destaca un tercer grupo de microorganismos, Enterococcus sp., por el incremento significativo de la resistencia a ampicilina en E. faecium (el 75% en 2006), y por la emergencia de resistencia a la vancomicina, principalmente en E. faecium (el 3,9% en 2006). Los estudios revisados en este articulo muestran los cambios epidemiologicos experimentados recientemente y la emergencia y diseminacion de cepas multirresistentes. Esta diseminacion se esta produciendo dentro de los hospitales, entre hospitales y centros sociosanitarios, dentro la propia comunidad, e incluso entre paises y continentes. Por otra parte, la existencia de un reservorio nosocomial de genes de resistencia, en algunos casos potencialmente transmisibles mediante transferencia horizontal, puede contribuir a agravar el problema de la resistencia y a restringir las opciones terapeuticas.

Introduccion En los ultimos anos se ha observado un aumento de la incidencia de tos ferina. El objetivo de este estudio fue evaluar la aportacion de varios procedimientos, incluyendo una tecnica de reaccion en cadena de la polimerasa (PCR) en tiempo real, al diagnostico microbiologico de tos ferina, asi como conocer la relacion clonal de los aislamientos clinicos de Bordetella pertussis. Metodos Desde agosto de 2002 a octubre de 2003, se recogieron los exudados nasofaringeos de pacientes pediatricos y adultos con signos clinicos de tos ferina o como parte de estudios de contactos. Estas muestras fueron procesadas para la deteccion de B. pertussis mediante cultivo, inmunofluorescencia directa (IFD) y PCR en tiempo real. Los aislamientos disponibles se caracterizaron molecularmente mediante electroforesis en campo pulsante (ECP). Resultados De las 121 muestras recogidas de 117 pacientes, se detecto B. pertussis en cultivo en 17 muestras (14,1%), con IFD en 30 (24,8%) y con PCR en 41 (33,9%). Un total de 17 aislamientos clinicos, 14 obtenidos durante el periodo de estudio y 3 en los anos 1997, 2000 y 2001, estuvieron disponibles para ECP. Se identificaron 5 genotipos, dos de ellos (C y E) mayoritarios, con 8 y 6 aislados, respectivamente. Uno de ellos incluyo aislamientos de 1997 y 2001. Conclusion La PCR en tiempo real aplicada a la deteccion de B. pertussis proporciono mas resultados positivos que la IFD o el cultivo, pero su valor diagnostico debe ser aclarado. Entre los aislamientos conseguidos predominaron dos clones, uno de ellos en circulacion desde al menos 1997.

Introduccion Durante el ano 2003 hubo un incremento de casos de listeriosis diagnosticados en un hospital terciario de Madrid. Los objetivos del presente estudio fueron revisar las caracteristicas clinicas de los casos diagnosticados, y estudiar la relacion clonal entre los aislados clinicos de Listeria monocytogenes. Metodos Se realizo un estudio retrospectivo de todos los pacientes diagnosticados de listeriosis en el Hospital 12 de Octubre durante el periodo de 2001 a 2003. Se revisaron las historias clinicas de los pacientes, y en todos los aislados clinicos se realizo serotipificacion y electroforesis en campo pulsante (ECP) con las enzimas AscI y SmaI. Resultados Se diagnosticaron un total de 18 pacientes: cuatro en el ano 2001, dos en 2002 y 12 en el ano 2003. Las tasas estimadas fueron: 7,3 casos/1.000.000 habitantes para el ano 2001, 3,6 para el ano 2002 y 21,8 para el ano 2003. El serotipo mas frecuente fue el 4b (66,7%), al que pertenecian el 83,3% de los casos diagnosticados en el ano 2003. La ECP puso de manifiesto 12 patrones moleculares distintos, uno de los cuales fue comun a 5 casos del ano 2003. Conclusion La aparente expansion de un clon de L. monocytogenes durante el ano 2003 contribuyo en parte al incremento de la incidencia de listeriosis en ese ano. Las tecnicas de epidemiologia molecular son muy utiles para la deteccion de posibles brotes de origen alimentario, y deberian promover investigaciones complementarias sobre la naturaleza de los alimentos implicados.

Objectives:To describe the relative proportions of nosocomial and community-onsetStaphylococcus aureusbacteremia at our institution and the epidemiologic characteristics and clonal diversity ofS. aureusisolates, as determined by pulsed-field gel electrophoresis (PFGE) and antimicrobial resistance patterns.Design:Retrospective cohort study of all cases ofS. aureusbacteremia between October 2001 and October 2002.Setting:A 1,300-bed, tertiary-care hospital.Results:One hundred sixty-two unique episodes ofS. aureusbacteremia were identified. Forty-three cases (26.5%) were caused by methicillin-resistantS. aureus(MRSA). Most cases ofS. aureusbacteremia, whether MRSA or methicillin susceptible (MSSA), were nosocomial in origin (77.2%) or were otherwise associated with the healthcare system (16%). Only 11 (6.8%) of the cases (all MSSA) were strictly community acquired. Thirty-five unique macrorestriction patterns were identified among the 154 isolates that were typed by PFGE. Four major genotypes were defined among the isolates of MRSA, with 36 (85.7%) represented by a single PFGE type. Of the isolates within this major clone, all (100%) were ciprofloxacin resistant and 77.8% were erythromycin resistant. In contrast, the 112 isolates of MSSA comprised 31 different PFGE types, 3 of which represented 42.9% of all MSSA isolates and were associated with both nosocomial and community-onset bacteremia.Conclusions:Most cases ofS. aureusbacteremia in our healthcare region are nosocomial in origin or are acquired through contact with the healthcare system and are thus potentially preventable. To preclude dissemination of pathogenic clones, it is therefore necessary to redouble preventive measures in both the hospital and the community.

Fundamento y objetivo La infeccion por citomegalovirus (CMV) es la complicacion infecciosa mas frecuente en los receptores de un trasplante hepatico. Para disminuir la incidencia de esta infeccion uno de los metodos es el tratamiento anticipado con ganciclovir guiado por la determinacion seriada de antigenemia pp65 de CMV. Pacientes y metodo En los receptores de un trasplante hepatico seropositivos para CMV antes del trasplante, se realizaron determinaciones de antigenemia pp65 de CMV los dias 14, 28, 45, 60, 90 y 180 postrasplante y cuando se considero indicado clinicamente. Se realizo tratamiento anticipado con ganciclovir intravenoso (5 mg/kg de peso cada 12 h durante 14 dias) en todos los pacientes que tuvieron antigenemia superior a 50 celulas/200.000 leucocitos. Se analizaron los factores de riesgo para el desarrollo de enfermedad activa por CMV. Resultados Se incluyo en el estudio a 182 pacientes seropositivos para CMV. Se realizo tratamiento anticipado con ganciclovir en 16 pacientes con antigenemia superior a 50 celulas/200.000 leucocitos. Presentaron enfermedad activa por CMV 9 de 182 pacientes (4,9%): 2 de los 16 que recibieron tratamiento anticipado con ganciclovir y 7 de los 166 que no lo recibieron. El unico factor de riesgo asociado de manera significativa con enfermedad activa por CMV fue la no realizacion de alguna de las antigenemias previstas (p odds ratio = 8,17; intervalo de confianza del 95% 1,94–34,36). Conclusiones El tratamiento anticipado con ganciclovir se asocia a una baja incidencia de enfermedad por CMV. La mala adherencia al protocolo de realizacion seriada de antigenemia pp65 aumenta el riesgo de enfermedad por CMV.

The annual incidence of Serratia marcescens bacteremia in a neonatal intensive care unit increased significantly between 2002 and 2010. Molecular epidemiology studies revealed that 8 clones were responsible for 85.2% of cases. Given that these infections are potentially preventable, even the appearance of 1 case of bacteremia should be an indicator for outbreak management.

Cell-surface signalling (CSS) enables Gram-negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti-sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP-mediated proteolysis of the anti-sigma factors is key to σ(ECF) activation. Using the Pseudomonas aeruginosa FoxR anti-sigma factor, we show here that RseP is responsible for the generation of an N-terminal tail that likely contains pro-sigma activity. Furthermore, it has been reported previously that this anti-sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti-sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti-sigma factors.

Over a 6-year period (2007-2012), the emergence of Enterobacter cloacae isolates resistant to β-lactams and with reduced susceptibility to carbapenems was observed in Hospital Universitario 12 de Octubre (Madrid, Spain). To determine the possible role of metallo-β-lactamases (MBLs) in the resistance profile of these isolates, a molecular and clinical epidemiological study was performed, including determination of patients' clinical characteristics, genetic diversity of strains, resistance mechanisms to carbapenems, and the genetic environment of VIM-1. A total of 73 E. cloacae isolates showed resistance to extended-spectrum cephalosporins and reduced susceptibility to at least one carbapenem during 2007-2012. PCR amplification revealed the presence of bla(VIM-1) gene in 37 isolates, bla(VIM-2) in 1 isolate and bla(KPC) in 5 isolates. Molecular typing showed high clonal diversity of E. cloacae isolates carrying bla(VIM-1). The genetic environment of bla(VIM-1) was investigated and two integron structures were found: intI-bla(VIM-1)-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1 (In624); and intI-bla(VIM-1)-aacA4-aadA1-qacEΔ1/sul1 (In488). Isolates belonging to three clones (A, F and G) harboured different types of integron (In624 or In488) despite belonging to the same clone. Conjugal experiments showed an association with a conjugative plasmid of ca. 300 kb belonging to IncHI2 group, which is common in Spanish hospitals, suggesting that the widespread dissemination of bla(VIM-1) may be due to horizontal transfer of mobile genetic determinants rather than the result of spreading of a few clones. These results have implications for infection control programmes in the hospital.

We report recurrent bacteremia due to mixed infection with two clonally derived isolates of Staphylococcus aureus in a patient with Sezary syndrome. The two isolates, one gentamicin resistant and methicillin susceptible and the other gentamicin susceptible and methicillin resistant, developed by the deletion of the mecA, ant(4 ′ )Ia , and aacA-aphD genes from a common gentamicin-resistant and methicillin-susceptible ancestor.

Cytomegalovirus (CMV) infection is the most frequent complication in solid organ transplant recipients. Currently, the antigenemia assay is widely used to detect this infection, although its success is being questioned to a great extent nowadays. The aim of our study is to compare a quantitative real time PCR to measure CMV DNA to the antigenemia assay, for the diagnosis to CMV disease. For our research, we prospectively processed 1198 samples (plasma and peripheral blood leukocytes [PBMC]), which belonged to 158 transplant recipients. In every sample the detection of the pp65 antigen in PBMC was carried out, as well as the quantification of CMV DNA by PCR (Light Cycler, LC-PCR). For this process, FRET probes, which detect a 254-bp fragment from the CMV gB gene, were used. The dynamic range of the LC-PCR was 500 to 5.10(7) copies/mL plasma and from 62 to 6.10(6) copies/10(6) PBMC. Twenty-three episodes of cytomegalovirus (CMV) disease occurred in 22 out of 158 patients and PCR displayed levels of sensitivity and specificity of 100% and 67%, respectively. The antigenemia assay obtained values of 91% and 57%. We established a cutoff value of 10(3) copies/mL plasma and 315 copies/10(6) cells. According to these cutoff values, PCR showed levels of sensitivity, specificity, VPN and VPP of 95.6%, 81.6%, 99%, and 53% respectively. Moreover, the LC-PCR assay anticipated the antigenemia assay in 10 patients out of 22 who developed CMV disease and the appearance of any clinical symptoms in 12 out of 22 patients. In conclusion, we believe that the quantification of CMV DNA by LC-PCR is a superior assay to pp65 antigenemia test regarding the early diagnosis of CMV disease in solid organ transplant recipients.

From 2002 to 2003, 32 isolates of Staphylococcus hominis subsp. novobiosepticus (SHN) were recovered from 21 patients, 18 of whom were neonates, with 13 considered to have late-onset SHN sepsis. All isolates from neonates had an indistinguishable pulsed-field gel electrophoresis pattern. Our data support SHN as an important nosocomial pathogen in neonates.

The VIM-1-producing multidrug-resistant strain Enterobacter cloacae was isolated from blood culture. The strain showed multiple resistances to clinically used antibiotics, including all β-lactams, fluoroquinolones, aminoglycosides, and sulfonamides. Sequence analysis showed the presence of 14 genes associated with resistance to antibiotics, including the metallo-β-lactamase VIM-1 gene, which was located in a class 1 integron.

Here, we report the draft genome sequence of a methicillin-resistant Staphylococcus aureus (MRSA) strain with high-level mupirocin resistance (SA_ST125_MupR), isolated from a patient with recurrent bacteremia. This strain belonged to sequence type ST125, which was responsible for more than 50% of the health care-associated infections caused by MRSA in Spain.

The VIM-2-producing multidrug-resistant high-risk clone Pseudomonas aeruginosa sequence type (ST) 175 was isolated in the setting of a large outbreak in Hospital Universitario 12 de Octubre (Spain) from 2007 to 2010. This strain was resistant to all β-lactams, fluoroquinolones, and aminoglycosides, with the exception of amikacin, and has become an endemic clone in our institution.

3 páginas, 3 figuras., We have designed a glycodendritic structure, BH30sucMan, that blocks the interaction between dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and Ebola virus (EBOV) envelope. BH30sucMan inhibits DC-SIGN-mediated EBOV infection at nanomolar concentrations. BH30sucMan may counteract important steps of the infective process of EBOV and, potentially, of microorganisms shown to exploit DC-SIGN for cell entry and infection., This research was supported by DGI grant no. BQU2002-03734 to J.R. and grants FIS 01/1430 and FIPSE 3026/99 to R.D.

We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR. Six additional cell culture-negative samples were positive by PCR with both sets of primers. Total processing time was less than 3 h. Real-time PCR using SYBR Green I as detection signal is a sensitive procedure for the rapid diagnosis of HSV in genital lesions.

A common receptor for coxsackie B virus and adenovirus has been described recently in cells of human and murine origin. Since the established cell line A-549 is suitable for adenoviruses, the potential use of A-549 cells for the isolation of coxsackie B viruses from clinical samples was investigated. All throat swabs sent to the laboratory between April 1998 and June 1999 were inoculated onto monolayers of MRC-5 and A-549 cells in tubes, and the enterovirus isolates obtained were typed. From April to June 1999, A-549 cells were compared prospectively to Buffalo green monkey (BGM) cells, considered as the most susceptible cell line for isolating coxsackie B viruses. Fifty-six out of 171 enterovirus isolates (33%) displayed a cytopathic effect (CPE) in the A-549 monolayer only, 48 isolates (28%) in the MRC-5 monolayer only, and 67 isolates (39%) in both cell lines. Most isolates that showed CPE in A-549 cells only (48 out of 56, 86%) were coxsackie B viruses, belonging to four different serotypes (B1, B2, B4, and B6). When BGM and A-549 cells were inoculated in parallel, both recovered the same number of coxsackie B isolates (n = 20), and the CPE was noted on approximately the same day. In conclusion, growth in A-549 but not MRC-5 cells identified coxsackie B viruses in most cases. A-549 was comparable to BGM for primary isolation of coxsackie B viruses.

New commercial techniques for determination of the viral load (VL) in plasma are able to detect as few as 20 copies of HIV-1 RNA/ml. The relevance of this new technical threshold is uncertain. Upon multivariate analysis, factors associated with detection of VLs between 20 and 49 copies/ml by the Cobas TaqMan HIV-1 v2.0 assay in an HIV clinic were the basal VL and time on antiretroviral therapy.

This report documents the nosocomial spread for an 18-month period of a single clone of linezolid- and teicoplanin-resistant Staphylococcus haemolyticus associated primarily with catheter-related bacteremia in intensive care unit patients. All linezolid-resistant isolates had the same G2576T mutation in at least 1 copy of the 23S rRNA gene.

We reported a ganciclovir (GCV)-resistant cytomegalovirus (CMV) infection in a heart transplant recipient. Genotypic and phenotypic susceptibility assays demonstrated an A594V mutation in the UL97 phosphotransferase gene and GCV IC(50)>96 microM. Low GCV concentration exposure, immunosuppressive treatment, donor-positive/recipient-negative CMV serostatus, viral reactivations within antiviral prophylaxis or treatment, contributed to GCV-resistant strain selection.

Molecular epidemiology of Staphylococcus aureus strains causing bacteremia in neonates during 2002 to 2005 revealed seven clones, with four MSSA clones responsible for 80% of the cases. Some clones persisted or reappeared throughout the study. Three bacteremic clones were found colonizing health care workers (HCWs), particularly clone C, which was harbored by at least 15% of HCWs.

Most studies focusing on respiratory infections in immunocompromised children have been addressed to bacterial etiology. However, respiratory virus infections in this population can also lead to severe disease. The objective of this study is to evaluate the clinical significance of respiratory virus infections in children with cancer or human immunodeficiency virus (HIV) infection. Retrospective study conducted in a teaching hospital in Madrid. Medical records from children

During 2003, an increase in the number of cases of listeriosis was observed in a tertiary hospital in Madrid. The objectives of this study were to review the clinical characteristics of the cases diagnosed from 2001 to 2003 and to investigate clonal relationships among the clinical isolates of Listeria monocytogenes.A retrospective analysis was performed of all cases of listeriosis diagnosed in Hospital 12 de Octubre (Madrid) during 2001-2003. Clinical records for each patient were reviewed and all clinical isolates were compared using serotyping and pulsed-field gel electrophoresis (PFGE) with the AscI and SmaI restriction enzymes.A total of 18 patients were diagnosed: 4 in 2001, 2 in 2002, and 12 in 2003. The estimated incidence rates were 7.3 cases per 1,000,000 inhabitants in 2001, 3.6 in 2002 and 21.8 in 2003. The most frequent serotype during the study period was 4b (66.7%), and this serotype represented 83.3% of the cases diagnosed in 2003. PFGE yielded 12 different genotypes; one of them (PFGE type G) was common to 5 cases diagnosed in 2003.The expansion of a single clone of L. monocytogenes during 2003 partially contributed to increasing the incidence of listeriosis that year. Molecular epidemiology techniques are useful for detecting outbreaks of a possible foodborne origin and their results should promote epidemiological studies to investigate the food products involved.

Mupirocin (Mup) is a topical antibacterial agent that interferes with protein synthesis by competitively inhibiting bacterial isoleucyl-tRNA synthetase (11, 19). A 2% Mup calcium ointment (Bactroban Nasal; GlaxoSmithKline) applied topically to the anterior nares eradicates carriage of Staphylococcus aureus and prevents infection in certain settings (2, 7, 8, 13). An important concern, however, is the development of Mup resistance (Mupr) (14, 17), of which there are two types. Low-level Mupr (MIC, 8 to 256 mg/liter) is usually associated with a mutation in the gene for target enzyme, while high-level Mupr (Hi-Mupr) (MIC, >256 mg/liter) is mediated by a plasmid containing the ileS2 gene that encodes an additional isoleucyl-tRNA synthetase enzyme (3). Such transmissible resistance raises concern about the spread of Mupr as Mup usage becomes more widespread (9, 10, 17). The objectives of this study were to determine (i) the prevalence of Mupr in methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates from nasal samples; (ii) the location, if present, of the ileS2 gene in Mupr isolates; and (iii) the organism genotype, as defined by pulsed-field gel electrophoresis (PFGE). Nasal samples submitted to the Clinical Microbiology Laboratory of the Hospital Universitario Doce de Octubre for isolation of S. aureus between October 2001 and October 2002 were included in the study. Specimens were obtained from two groups. Group I comprised adults who underwent elective heart surgery, before which a sample from the anterior nares was taken to determine whether the patient was a carrier of S. aureus. Group II included all new cases of MRSA infection diagnosed at the hospital that were associated with concurrent nasal carriage. Since 1996, we have instituted the use of topical Mup ointment to reduce the prevalence of nasal MRSA. Samples were inoculated onto phenol-red mannitol salt agar plates that were incubated at 37°C for 48 h. Isolation and identification of S. aureus were based upon standard microbiological procedures. All isolates were screened for resistance to Mup on Mueller-Hinton agar with a 5-μg disk (Oxoid). A zone of inhibition ≤ 13 mm in diameter was considered to reflect Mupr (5). Mupr organisms underwent MIC analysis by the E-test strip method (AB Biodisk). Susceptibility testing with other antibiotics was performed by disk diffusion (12, 16). All isolates were confirmed as MRSA by PCR detection of the mecA gene (6). PCR was also performed on all Mupr isolates to detect the plasmid-associated ileS2 gene (1). Mupr isolates were typed by PFGE following DNA extraction and digestion with SmaI (4). Restriction fragments were separated in a CHEF DRIII PFGE system (Bio-Rad Laboratories). Migration of DNA fragments was normalized using an appropriate size marker. Computer-assisted analysis of PFGE patterns was carried out using GelCompar software (Applied Maths). PFGE types (designated by letters) differed by

In liver transplant recipients the most frequent infection is that produced by cytomegalovirus (CMV). One of the methods to reduce the incidence of that infection is the CMV pp65 antigenemia-guided preemptive therapy with intravenous ganciclovir.Liver transplant recipients were tested for CMV antigenemia at days 14, 28, 45, 60, 90 and 180 postransplantation and when clinically indicated. Patients showing50/200.000 leukocytes received ganciclovir 5 mg/kg/12 h for 14 days. Risk factors for active CMV disease where studied.182 CMV seropositive patients where included in the study. 16 patients with50/200.000 leukocytes received ganciclovir as preemptive therapy. CMV disease appeared in 9/182 patients (4.9%): 2/16 who received PT and 7/166 among those who did not receive preemptive therapy. The only factor associated with increased incidence of CMV disease was to have missing samples for CMV antigenemia during the follow up (p0.0042; OR = 8,17; 95% CI, 1.94-34.36).Ganciclovir antigenemia-guided preemptive therapy is associated with a low incidence of CMV disease. Bad adherence to the protocol of antigenemia samples increases the risk for CMV disease.

Cyanovirin-N (CV-N) has been shown to inhibit Ebola Zaire virus (EboZV) infection, both in vitro and in vivo, through its ability to bind to oligomannoses-8/9 on the EboZV surface glycoprotein (GP). Here, we report the in vitro potency of CV-N to inhibit EboZV GP- and Marburg virus GP-pseudotyped viruses (EC50 approximately 40-60 nmol/L and approximately 6-25 nmol/L, respectively) from mediating gene transduction into HeLa cells. In addition, we provide evidence that CV-N can effectively inhibit DC-SIGN-mediated EboZV infection. Our data emphasize both the utility of GP-pseudotyped vectors in the assessment of compounds that affect cell entry by filovirus and the use of CV-N as a reagent for the probing of carbohydrate-dependent interactions at viral entry.

Two new series of BTD derivatives have been synthesised allowing to explore the steric requirements for their biological activity. The N3-alkylBTD compounds have shown antiviral activity in the same order or lower than previously prepared compounds. However, the cytotoxicity values observed prevent this new series of BTD derivatives from its potential therapeutic application. Concerning BTD derivatives with the modified linker attached to N1 position, we have obtained new non-nucleoside anti-HCMV derivatives. The activity against HCMV is shown at concentrations that were 10-fold lower than the concentration that was toxic for the host cells, which confirm that these derivatives show a specific antiviral effect against HCMV. SAR conclusions derived from these last compounds have provided new knowledge about the structural requirements of BTD showing certain positions that could be modified for enhancing the anti-HCMV action. © 2003 Elsevier Science Ltd. All rights reserved.

Streptococcus pneumoniae is the major cause of bacterial meningitis. We report a case of meningitis due to a mixed infection with two distinct strains of S. pneumoniae : one penicillin-resistant strain of serotype 9V and one penicillin-susceptible strain of serotype 7. The two strains exhibited different pulsed-field gel electrophoresis profiles.

The benzothiadiazine dioxide derivatives are potent non-nucleoside human cytomegalovirus (HCMV) inhibitors. As part of our comprehensive structure-activity relationship (SAR) study of these compounds, we have now proposed structural modifications on the heterocyclic moiety both on the number and the nature of the fused heterocycle and on the kind of heteroatoms present on it. Synthesis of these new compounds (benzyl derivatives of thiadiazines, thienothiadiazines, benzothienothiadiazines and quinazolines) and the antiviral evaluation against HCMV has been performed. SAR investigation on this class of compounds has defined the structural requirements for potency and toxicity. They have revealed two important clues: i) a fused ring to the thiadiazine framework is necessary to maintain the anti-HCMV action, and ii) the sulfamido moiety in the main heterocycle is crucial to avoid cytotoxicity.