Carbapenem-resistant Citrobacter spp. isolated in Spain from 2013 to 2015 produced a variety of carbapenemases including VIM-1, OXA-48, KPC-2, NDM-1 and VIM-2.

OBJECTIVES: There is little information about carbapenemase-producing (CP) Citrobacter spp. We studied the molecular epidemiology and microbiological features of CP Citrobacter spp. isolates collected in Spain (2013-15). METHODS: In total, 119 isolates suspected of being CP by the EUCAST screening cut-off values were analysed. Carbapenemases and ESBLs were characterized using PCR and sequencing. The genetic relationship among Citrobacter freundii isolates was studied by PFGE. RESULTS: Of the 119 isolates, 63 (52.9%) produced carbapenemases, of which 37 (58.7%) produced VIM-1, 20 (31.7%) produced OXA-48, 12 (19%) produced KPC-2, 2 (3.2%) produced NDM-1 and 1 (1.6%) produced VIM-2; 9 C. freundii isolates co-produced VIM-1 plus OXA-48. Fourteen isolates (22.2%) also carried ESBLs: 8 CTX-M-9 plus SHV-12, 2 CTX-M-9, 2 SHV-12 and 2 CTX-M-15. Fifty-seven isolates (90.5%) were C. freundii, 4 (6.3%) were Citrobacter koseri, 1 (1.6%) was Citrobacter amalonaticus and 1 (1.6%) was Citrobacter braakii. By EUCAST breakpoints, eight (12.7%) of the CP isolates were susceptible to the four carbapenems tested. In the 53 CP C. freundii analysed by PFGE, a total of 44 different band patterns were observed. Four PFGE clusters were identified: cluster 1 included eight isolates co-producing VIM-1 and OXA-48; blaVIM-1 was carried in a class 1 integron (intI-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1) and blaOXA-48 was carried in a Tn1999.2 transposon. CONCLUSIONS: We observed the clonal and polyclonal spread of CP Citrobacter spp. across several Spanish geographical areas. Four species of Citrobacter spp. produced up to five carbapenemase types, including co-production of VIM-1 plus OXA-48. Some CP Citrobacter spp. isolates were susceptible to the four carbapenems tested, a finding with potential clinical implications.

Characterization of mobile genetic elements carrying VIM-1 and KPC-2 carbapenemases in Citrobacter freundii isolates in Madrid.

Carbapenemase producing Citrobacter freundii (CPCF) infections are still uncommon in European countries. Here we report a molecular study conducted in a tertiary care facility in southern Madrid, Spain, from 2009 to 2014 to investigate the epidemiology of CPCF. The blaIMP-1,blaIMP-2,blaKPC,blaNDM,blaOXA-48,blaVIM-1 and blaVIM-2 genes were screened by PCR. Molecular typing was carried out by Pulsed-field gel electrophoresis analysis (PFGE) and multilocus sequence typing (MLST). Whole genome sequencing (WGS) was performed to characterize the resistome and the mobile genetic elements associated with the carbapenems resistance of CPCF. A total of 11/521 (2.1%) isolates had reduced susceptibility to carbapenems. PCR amplification revealed the presence of blaVIM-1 in 10 isolates and blaKPC-2 in 2 isolates. One C. freundii isolate co-harbored blaVIM-1 and blaKPC-2 genes. PFGE and MLST assigned 10 different clonal, 4 previously reported (ST11, ST18, ST22 and ST64) and 6 new STs (ST89, ST90, ST91, ST92, ST92 and ST94). The blaVIM-1 gene was part of In624 (intI1-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1). In 3 of these isolates, plasmid-mediated quinolone resistance genes (qnrA1 and qnrB4) were present in its downstream region, taking part of a complex class 1 integron ([In624:ISCR1:qnrB4-blaDHA-1] and [In624:ISCR1:qnrA1]). On the other hand, the blaKPC-2 gene was associated with a Tn3-based transposon. The dissemination of the blaVIM-1 gene among various clones suggests a successful horizontal transfer of integron carrying elements that play a dominant role in the development of multidrug resistance in Enterobacteriaceae.

Evolution of antibiotic multiresistance in Escherichia coli and Klebsiella pneumoniae isolates from urinary tract infections: A 12-year analysis (2003-2014).

INTRODUCTION: The aim of this study was to identify multi-drug resistance (MDR) in the main enterobacteriaceae implicated in urinary tract infections (Escherichia coli and Klebsiella pneumoniae) from both, community and hospitalized patients and to analyze the evolution over a 12-year period. METHODS: Microb Dynamic software was used to analyze the microbiology laboratory database and a chi square test was applied to compare differences in group proportions and to determine the linear trend over 12 years in three different periods: 2003-2006, 2007-2010, 2011-2014. We chose amoxicillin, gentamicin, ciprofloxacin and trimethoprim-sulphamethoxazole as MDR markers. RESULTS: A total of 39,980 positive urine samples were analyzed, 34,564 (3786 from hospitalized patients and 30,778 from non-hospitalized patients) E. coli isolates, and 5,422 (774 from hospitalized patients and 4,648 from non-hospitalized patients) K. pneumoniae isolates. The prevalence of UTI due to MDR E. coli and MDR K. pneumoniae significantly increased in the period studied, both in hospitalized and outpatients. A higher percentage of MDR E. coli (5.89% in 2007-2010 versus 8.18% in 2011-2014) and MDR K. pneumoniae (2.38% in 2007-2010 versus 9.35% in 2011-2014) was evident and maintained constant over time in hospitalized patients in comparison to non-hospitalized ones. Infection due to MDR ESBL-producing E. coli and K. pneumoniae increased significantly during the last 8 years in both, hospitalized (20% versus 38% and 66.8% versus 82.6%, respectively) and non-hospitalized patients (18.2% versus 23.6% and 51% versus 74.6%, respectively). CONCLUSIONS: This study includes data of a large sample size of urinary strains isolated over a 12 year period and demonstrates that MDR is an increasing phenomenon of particular importance in the main UTI-causing species.

[Susceptibility to azithromycin and other antibiotics in recent isolates of Salmonella, Shigella and Yersinia]. / Sensibilidad a azitromicina y otros antibióticos en aislados recientes de Salmonella, Shigella y Yersinia.

INTRODUCTION: Azithromycin represents an alternative option to treat bacterial diarrhea when the antibiotic therapy is indicated. Little is known regarding the susceptibility to azithromycin in enteropathogens in Spain. METHODS: The MICs of azithromycin were determined by E-test against Salmonella non-typhi (SNT), Shigella and Yersinia isolates collected over the last three years (2010-2012). In addition, the susceptibility to other antibiotics usually used to treat gastrointestinal diseases was determined in these isolates by using a microdilution method. RESULTS: A total of 139 strains of SNT, Shigella and Yersinia were studied. All of them, except one strain, had a MIC≤16mg/L of azithromycin. In the adult population, 14.7% and 40.6% of SNT and Shigella isolates, respectively, were resistant to at least 2 of following antibiotics: amoxicillin, trimethoprim-sulfamethoxazole and ciprofloxacin. In the pediatric population, 10% of SNT clinical isolates and 28.6% (2/7) of Shigella isolates were resistant to amoxicillin and trimethoprim-sulfamethoxazole. CONCLUSIONS: In our experience, azithromycin would be a useful antibiotic alternative to treat bacterial diarrhea.

Rapid antibiotic susceptibility testing (RAST) of blood cultures in enterobacteria with inducible chromosomal AmpC-type ß-lactamase.

INTRODUCTION: Early and adequate treatment of bloodstream infections decreases patient morbidity and mortality. The objective is to develop a preliminary method for rapid antibiotic susceptibility testing (RAST) in enterobacteria with inducible chromosomal AmpC. METHODS: RAST was performed directly on spiked blood cultures of 49 enterobacteria with inducible chromosomal AmpC. Results were read at 4, 6 and 8h of incubation. Commercial broth microdilution was considered the reference method. Disks of 10 antibiotics were evaluated. RESULTS: The proportion of readable tests at 4h was 85%. All RAST could be read at 6 and 8h. For most antibiotics, the S or R result at 4, 6 and 8h was greater than 80% after tentative breakpoints were established and Area of Technical Uncertainty was defined. CONCLUSIONS: This preliminary method seems to be of practical use, although it should be extended to adjust the breakpoints and differentiate them by species.

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures.

INTRODUCTION: This study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. METHODS: Fifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. RESULTS: A total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. CONCLUSION: This method provides rapid bacterial identification and AST, offering definitive results 24-48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.

Identification of Clinical Isolates of Candida albicans with Increased Fitness in Colonization of the Murine Gut.

The commensal and opportunistic pathogen Candida albicans is an important cause of fungal diseases in humans, with the gastrointestinal tract being an important reservoir for its infections. The study of the mechanisms promoting the C. albicans commensal state has attracted considerable attention over the last few years, and several studies have focused on the identification of the intestinal human mycobiota and the characterization of Candida genes involved in its establishment as a commensal. In this work, we have barcoded 114 clinical C. albicans isolates to identify strains with an enhanced fitness in a murine gastrointestinal commensalism model. The 114 barcoded clinical isolates were pooled in four groups of 28 to 30 strains that were inoculated by gavage in mice previously treated with antibacterial therapy. Eight strains that either exhibited higher colonization load and/or remained in the gut after antibiotic removal were selected. The phenotypic analysis of these strains compared to an RFP-tagged SC5314 wild type strain did not reveal any specific trait associated with its increased colonization; all strains were able to filament and six of the eight strains displayed invasive growth on Spider medium. Analysis of one of these strains, CaORAL3, revealed that although mice required previous bacterial microbiota reduction with antibiotics to be able to be colonized, removal of this procedure could take place the same day (or even before) Candida inoculation. This strain was able to colonize the intestine of mice already colonized with Candida without antibiotic treatment in co-housing experiments. CaORAL3 was also able to be established as a commensal in mice previously colonized by another (CaHG43) or the same (CaORAL3) C. albicans strain. Therefore, we have identified C. albicans isolates that display higher colonization load than the standard strain SC5314 which will surely facilitate the analysis of the factors that regulate fungal colonization.

Candida albicans beta-glucan exposure is controlled by the fungal CEK1-mediated mitogen-activated protein kinase pathway that modulates immune responses triggered through dectin-1.

Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as beta-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall beta-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IkappaB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in beta-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.

The Sko1 protein represses the yeast-to-hypha transition and regulates the oxidative stress response in Candida albicans.

Cells respond to environmental changes triggering adaptive responses which are, in part, mediated by a transcriptional response. These responses are complex and are dependent on different transcription factors. The present work reports the implication of the Sko1 protein in several processes relevant to the physiology of Candida albicans. First, Sko1 acts as transcriptional repressor of genes involved in pathogenesis and hyphal formation, which results in increased expression of the hyphal related genes ECE1 and HWP1 without significant changes in the virulence using a mouse model of systemic infection. Second Sko1 is involved in the response to oxidative stress and sko1 mutants increase the sensitivity of hog1 to the myelomonocytic cell line HL-60. Genome-wide transcriptional analysis after hydrogen peroxide treatment revealed that sko1 mutants were able to generate an adaptive response similar to wild type strains, although important differences were detected in the magnitude of the transcriptional response. Collectively, these results implicate Sko1 as an important mediator of the oxidative stress response in C. albicans.

Fluconazole at subinhibitory concentrations induces the oxidative- and nitrosative-responsive genes TRR1, GRE2 and YHB1, and enhances the resistance of Candida albicans to phagocytes.

OBJECTIVES: To analyse the oxidative and nitrosative stress response in Candida albicans generated by fluconazole at subinhibitory concentrations, and the functional consequences of such a response for the interaction with phagocytic cells. METHODS: The C. albicans CAI-4 strain carrying transcriptional fusions of the TRR1p, YHB1p and GRE2p genes to the Renilla reniformis luciferase LUC gene was pre-treated with subinhibitory concentrations of fluconazole and incubated with oxidants (diamide and hydrogen peroxide) or with the myelomonocytic cell line HL-60. RESULTS: Fluconazole induced oxidative and nitrosative stress in a time- and dose-dependent manner as determined using oxidative- and nitrosative-specific gene reporters. At subinhibitory concentrations, fluconazole was able to induce protection in vitro to subsequent challenges with oxidants in both liquid and solid media, and also induced partial protection against the oxidative-mediated killing mechanisms of the myelocytic HL-60 cells. CONCLUSIONS: Subinhibitory concentrations of fluconazole protect against oxidants and killing mediated by phagocytes.

ESBL-producing-multidrug resistant E. coli population from urinary tract infections is less diverse than non-ESBL-multidrug resistant population.

OBJECTIVE: The aim of this study was to compare the population structure of three different representative groups of E. coli isolates causing urinary tract infections in a large area of Madrid, Spain: two groups of multidrug resistant isolates (MDR), ESBL- and non-ESBL producers, and one of fully-susceptible isolates (35 isolates in each group). METHODS: Epidemiological relatedness was studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The presence of genes encoding ESBL was determined by using PCR and sequencing. Antimicrobial susceptibility testing was performed by broth microdilution. RESULTS: PFGE analysis revealed a high degree of genetic diversity in susceptible and non-ESBL-MDR groups. However, the ESBL-MDR E. coli population was less diverse and a large cluster consisting of ST131 and CTX-M-15-producing isolates was detected. CONCLUSIONS: The present study revealed that ESBL-producing-MDR E. coli population was less diverse than the non-ESBL MDR group and that ST131 was dominant among CTX-M-15-producing isolates that reflects the spread of this successful MDR lineage.

MAP kinase pathways as regulators of fungal virulence.

MAP kinases are dual phosphorylated protein kinases, present in eukaryotes, which mediate differentiation programs and immune responses in mammalian cells. In pathogenic fungi, MAP kinases are key elements that control adaptation to environmental stress. Recent studies have shown that these pathways have an essential role in the control of essential virulence factors such as capsule biogenesis in Cryptococcus neoformans or morphogenesis, invasion and oxidative stress in Candida albicans. Although MAP kinases sense different activating signals, there is a considerable degree of crosstalk and/or overlap, which enables them to integrate, amplify and modulate the appropriate protective and adaptive response. MAP kinases behave as a 'functional nervous system' that controls virulence and influences the progression of the disease.

Prediction of infection caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae: development of a clinical decision-making nomogram.

OBJECTIVE: This study aimed to assess the population at risk of infection by extended-spectrum beta-lactamase (ESBL)-producing organisms, using clinical criteria. MATERIALS AND METHODS: All urine cultures positive for Enterobacteriaceae in a Spanish hospital department from January 2010 to 2014 were reviewed. All isolates with ESBL-positive strains were collected, and isolates received during the first week of each month with ESBL-negative strains from symptomatic patients hospitalized or admitted to the emergency room. Multivariate analysis of the factors involved was undertaken and a nomogram developed to predict the probability of infection by ESBL-producing microorganisms. RESULTS: The study included 1524 patients with urinary tract infection (UTI): 416 ESBL-positive and 1108 ESBL-negative. In univariate analysis, risk factors were: male gender (p = 0.036), age (p < 0.0001), nursing home (p < 0.0001), previous antimicrobial therapy (p < 0.0001) or hospitalization (p < 0.0001), diabetes (p < 0.0001), chronic renal insufficiency (p < 0.0001), severe underlying disease (p < 0.0001), neoplasia (p = 0.0005), urological (p < 0.0001) and non-urological invasive procedure (p = 0.0003), recurrent UTI (p < 0.0001), urological (p < 0.0001) or abdominal surgery (p < 0.0001) and permanent urethral catheter (p < 0.0001). In multivariate analysis, the data set was split into a development cohort of 1067 patients and a validation cohort of 457 cases. A nomogram was developed to predict the probability of infection by ESBL-producing bacteria, which included seven variables: age (p < 0.0001), gender (p = 0.004), nursing home (p < 0.0001), previous antimicrobial therapy (p = 0.04) or hospitalization (p < 0.0001), recurrent UTI (p < 0.0001) and non-urological invasive procedure (p = 0.005). The discriminative accuracy was 0.79 (95% confidence interval 0.77-0.83). CONCLUSIONS: A nomogram was developed that predicts the risk of infection by ESBL-producing Enterobacteriaceae with reasonable accuracy. It could improve clinical decision making and enable more efficient empirical treatment.

PARP-1/PARP-2 double deficiency in mouse T cells results in faulty immune responses and T lymphomas.

The maintenance of T-cell homeostasis must be tightly regulated. Here, we have identified a coordinated role of Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function. Mice bearing a T-cell specific deficiency of PARP-2 in a PARP-1-deficient background showed defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T-cells. Meanwhile, peripheral T-cell number was not affected in single PARP-1 or PARP-2-deficient mice. T-cell lymphopenia was associated with dampened in vivo immune responses to synthetic T-dependent antigens and virus, increased DNA damage and T-cell death. Moreover, double-deficiency in PARP-1/PARP-2 in T-cells led to highly aggressive T-cell lymphomas with long latency. Our findings establish a coordinated role of PARP-1 and PARP-2 in T-cell homeostasis that might impact on the development of PARP-centred therapies.

First clinical isolate in Europe of clindamycin-resistant group B Streptococcus mediated by the lnu(B) gene.

We characterize the mechanisms implicated in an unusual phenotype of resistance to macrolides-lincosamides (no halos of inhibition around clindamycin and lincomycin discs, and a 15 mm halo around erythromycin disc) in a Streptococcus agalactiae isolate recovered in Spain. The presence of macrolide or lincosamide resistance genes [erm(A), erm(B), erm(C), erm(T), mef(A), mrs(A), lnu(A), lnu(B), lsa(B), lsa(C) and vga(C)] was investigated by PCR and sequencing. The strain showed a resistant phenotype to erythromycin and clindamycin (MIC = 2 mg/L and MIC = 8 mg/L, respectively) and the presence of lnu(B) and mef(A) genes was demonstrated. Clinical microbiology laboratories should be aware of this unusual phenotype due to the association of two mechanisms mediated by lnu(B) and mef(A) genes. This constitute, to our knowledge, the first report of lnu(B) in S. agalactiae in human isolates in Europe.

Rapid antibiotic susceptibility testing (RAST) of blood cultures in enterobacteria with inducible chromosomal AmpC-type B-lactamase / Pruebas rápidas de sensibilidad antibiótica de hemocultivos con enterobacterias portadoras de B-lactamasa tipo AmpC cromosómica inducible

Introduction: Early and adequate treatment of bloodstream infections decreases patient morbidity and mortality. The objective is to develop a preliminary method for rapid antibiotic susceptibility testing (RAST) in enterobacteria with inducible chromosomal AmpC. Methods: RAST was performed directly on spiked blood cultures of 49 enterobacteria with inducible chromosomal AmpC. Results were read at 4, 6 and 8h of incubation. Commercial broth microdilution was considered the reference method. Disks of 10 antibiotics were evaluated. Results: The proportion of readable tests at 4h was 85%. All RAST could be read at 6 and 8h. For most antibiotics, the S or R result at 4, 6 and 8h was greater than 80% after tentative breakpoints were established and Area of Technical Uncertainty was defined. Conclusions: This preliminary method seems to be of practical use, although it should be extended to adjust the breakpoints and differentiate them by species.(AU)

Introducción: El tratamiento precoz y adecuado de las bacteriemias disminuye la morbilidad y mortalidad de los pacientes. El objetivo es desarrollar un método preliminar de pruebas rápidas de sensibilidad antibiótica (PRSA) en enterobacterias con AmpC cromosómica inducible. Métodos: Las PRSA se realizaron directamente de hemocultivos simulados positivos para 49 enterobacterias con AmpC cromosómica inducible. Los resultados se leyeron a las 4, 6 y 8 horas de incubación. La microdilución en caldo comercial se consideró el método de referencia. Se evaluaron discos de 10 antibióticos. Resultados: La proporción de pruebas legibles a las 4 horas fue del 85%. Todas las PRSA pudieron leerse a las 6 y 8 horas. Para la mayoría de los antibióticos, el resultado S o R a las 4, 6 y 8 horas fue superior al 80%, después de que se establecieran puntos de corte provisionales y se definiera el área de incertidumbre técnica. Conclusiones: Este método preliminar parece ser de utilidad práctica, aunque debería ampliarse para ajustar los puntos de corte y diferenciar por especies.(AU)

ESBL-producing-multidrug resistant E. coli population from urinary tract infections is less diverse than non-ESBL-multidrug resistant population / La población de E. coli multirresistente productora de BLEE presente en infecciones del tracto urinario es menos diversa que la población multirresistente no productora de BLEE

OBJECTIVE: The aim of this study was to compare the population structure of three different representative groups of E. coli isolates causing urinary tract infections in a large area of Madrid, Spain: two groups of multidrug resistant isolates (MDR), ESBL- and non-ESBL producers, and one of fully-susceptible isolates (35 isolates in each group). METHODS: Epidemiological relatedness was studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The presence of genes encoding ESBL was determined by using PCR and sequencing. Antimicrobial susceptibility testing was performed by broth microdilution. RESULTS: PFGE analysis revealed a high degree of genetic diversity in susceptible and non-ESBL-MDR groups. However, the ESBL-MDR E. coli population was less diverse and a large cluster consisting of ST131 and CTX-M-15-producing isolates was detected. CONCLUSIONS: The present study revealed that ESBL-producing-MDR E. coli population was less diverse than the non-ESBL MDR group and that ST131 was dominant among CTX-M-15-producing isolates that reflects the spread of this successful MDR lineage

OBJETIVO: El objetivo del presente estudio fue comparar la estructura poblacional de tres grupos representativos diferentes de aislados de E. coli que producen infecciones del tracto urinario en una gran área de Madrid, España: dos grupos de aislados multirresistentes (multidrug resistant, MDR), productores y no productores de BLEE (β-lactamasas de espectro extendido), y uno de aislados totalmente sensibles (35 aislamientos en cada grupo). MÉTODOS: La relación epidemiológica se estudió mediante electroforesis en gel de campo pulsado (pulsed-field gel electrophoresis, PFGE) y tipificación de secuencias multilocus (multilocus sequence typing, MLST). La presencia de genes que codifican para BLEE se determinó mediante el uso de PCR y secuenciación. La prueba de sensibilidad a los antimicrobianos se realizó por microdilución del medio. RESULTADOS: El análisis de PFGE reveló un alto grado de diversidad genética en el grupo de sensibles y de MDR no productores de BLEE. Sin embargo, la población de E. coli MDR productora de BLEE era menos diversa y se detectó un grupo grande de aislados formado por productores de CTX-M-15 y ST131. CONCLUSIONES: El presente estudio reveló que la población de E. coli MDR productora de BLEE era menos diversa que el grupo MDR no productor de BLEE, y que ST131 era dominante en los aislados de productores de CTX-M-15, lo que refleja la propagación de esta exitosa línea de MDR

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures / Método sencillo basado en una doble centrifugación diferencial para obtener una identificación bacteriana y sensibilidad a antibióticos rápida a partir de hemocultivos positivos

Introduction: This study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. Methods: Fifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. Results : A total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. Conclusion: This method provides rapid bacterial identification and AST, offering definitive results 24–48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.(AU)

Introducción: Este trabajo propone un método sencillo, rápido y barato de identificación bacteriana y sensibilidad antibiótica utilizando MALDI-TOF y una doble centrifugación diferencial a partir de hemocultivo positivo. Métodos: Se estudiaron 52 hemocultivos positivos (37 bacilos gramnegativos y 15 cocos grampositivos). Se compararon 2 métodos: un método convencional de identificación y determinación de sensibilidad a antibióticos automatizada partiendo de colonia crecida, y un método rápido utilizando MALDI-TOF, caracterizado por la obtención de un pellet purificado procedente de un hemocultivo positivo, mediante un procedimiento basado en una doble centrifugación diferencial. Resultados: Se analizaron y compararon 1.101 valores de CMI (mg/l) de acuerdo con los criterios establecidos por EUCAST y obtenidos por ambos métodos. Se detectaron discrepancias en 81 valores de CMI (7,35%). Considerando todos los aislados, la concordancia esencial fue del 98,28% y la concordancia categórica del 92,65%. Conclusión: Este método proporciona resultados de identificación y sensibilidad a antibióticos definitivos 24-48h antes que un método convencional (p<0,001), mejorando el tiempo de respuesta en el diagnóstico microbiológico de bacteriemias, especialmente en laboratorios sin servicio de guardias de 24h.(AU)

N-ras couples antigen receptor signaling to Eomesodermin and to functional CD8+ T cell memory but not to effector differentiation.

Signals from the TCR that specifically contribute to effector versus memory CD8⁺ T cell differentiation are poorly understood. Using mice and adoptively transferred T lymphocytes lacking the small GTPase N-ras, we found that N-ras-deficient CD8⁺ T cells differentiate efficiently into antiviral primary effectors but have a severe defect in generating protective memory cells. This defect was rescued, although only partly, by rapamycin-mediated inhibition of mammalian target of rapamycin (mTOR) in vivo. The memory defect correlated with a marked impairment in vitro and in vivo of the antigen-mediated early induction of T-box transcription factor Eomesodermin (Eomes), whereas T-bet was unaffected. Besides N-ras, early Eomes induction in vitro required phosphoinositide 3-kinase (PI3K)-AKT but not extracellular signal-regulated kinase (ERK) activation, and it was largely insensitive to rapamycin. Consistent with N-ras coupling Eomes to T cell memory, retrovirally enforced expression of Eomes in N-ras-deficient CD8⁺ T cells effectively rescued their memory differentiation. Thus, our study identifies a critical role for N-ras as a TCR-proximal regulator of Eomes for early determination of the CD8⁺ T cell memory fate.

Evolution of antibiotic multiresistance in Escherichia coli and Klebsiella pneumoniae isolats from urinary traerichia coli and Klebsiella pneumoniae isolates from urinary trct infections: A 12-year analysis (2003-2014) / Evolución de la multirresistencia a los antibióticos en Escherichia coli y Klebsiella pneumoniae aislados de infecciones del tracto urinario. Un análisis de 12 años (2003-2014)

INTRODUCTION: The aim of this study was to identify multi-drug resistance (MDR) in the main enterobacteriaceae implicated in urinary tract infections (Escherichia coli and Klebsiella pneumoniae) from both, community and hospitalized patients and to analyze the evolution over a 12-year period. METHODS: Microb Dynamic software was used to analyze the microbiology laboratory database and a chi square test was applied to compare differences in group proportions and to determine the linear trend over 12 years in three different periods: 2003-2006, 2007-2010, 2011-2014. We chose amoxicillin, gentamicin, ciprofloxacin and trimethoprim-sulphamethoxazole as MDR markers. RESULTS: A total of 39,980 positive urine samples were analyzed, 34,564 (3786 from hospitalized patients and 30,778 from non-hospitalized patients) E. coli isolates, and 5,422 (774 from hospitalized patients and 4,648 from non-hospitalized patients) K. pneumoniae isolates. The prevalence of UTI due to MDR E. coli and MDR K. pneumoniae significantly increased in the period studied, both in hospitalized and outpatients. A higher percentage of MDR E. coli (5.89% in 2007-2010 versus 8.18% in 2011-2014) and MDR K. pneumoniae (2.38% in 2007-2010 versus 9.35% in 2011-2014) was evident and maintained constant over time in hospitalized patients in comparison to non-hospitalized ones. Infection due to MDR ESBL-producing E. coli and K. pneumoniae increased significantly during the last 8 years in both, hospitalized (20% versus 38% and 66.8% versus 82.6%, respectively) and non-hospitalized patients (18.2% versus 23.6% and 51% versus 74.6%, respectively). CONCLUSIONS: This study includes data of a large sample size of urinary strains isolated over a 12 year period and demonstrates that MDR is an increasing phenomenon of particular importance in the main UTI-causing species

INTRODUCCIÓN: El objetivo principal de este estudio fue identificar multirresistencia a antibióticos (multi-drug resistance [MDR]) en las principales enterobacterias implicadas en infecciones del tracto urinario (ITU) (Escherichia coli y Klebsiella pneumoniae) procedentes de pacientes hospitalizados y ambulatorios, y analizar su evolución durante un periodo de 12años. MÉTODOS: Se eligieron como marcadores de MDR amoxicilina, gentamicina, ciprofloxacino y trimetoprim-sulfametoxazol. Se realizó un tratamiento estadístico por chi cuadrado de los resultados obtenidos de nuestra base de datos y se analizó la tendencia lineal de la MDR en 3 periodos de 4 años: 2003-2006, 2007-2010 y 2011-2014. RESULTADOS: Se analizaron un total de 39.980 muestras de orina con cultivo positivo para E.coli (3.786 de pacientes hospitalizados y 30.778 de pacientes ambulatorios) y 5.422 con cultivo positivo para K.pneumoniae (774 de pacientes hospitalizados y 4.648 de pacientes ambulatorios). La prevalencia de ITU debida a MDR E.coli y MDR K.pneumoniae aumentó significativamente en el periodo estudiado, tanto en pacientes hospitalizados como en pacientes ambulatorios, observándose un mayor porcentaje de MDR E.coli (5,89% en 2007-2010 versus 8,18% en 2011-2014) y MDR K.pneumoniae (2,38% in 2007-2010 versus 9,35% en 2011-2014) en pacientes hospitalizados. La infección debida a MDR E.coli y K.pneumoniae productoras de β-lactamasas de espectro extendido (BLEA) aumentó también de forma significativa durante los últimos 8años, tanto en pacientes hospitalizados (20% versus 38% y 66,8% versus 82,6%, respectivamente) como en los no hospitalizados (18,2% versus 23,6% y 51% versus 74,6%, respectivamente). CONCLUSIONES: En este estudio se demuestra que la MDR es un fenómeno en aumento de particular importancia en las principales enterobacterias implicadas en ITU