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.

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)

Evolución de los serotipos causantes de enfermedad neumocócica invasiva aislados durante 2008-2022 en un Hospital Público Madrileño de nivel dos, en relación con su inclusión en diferentes vacunas conjugadas / Evolution of the serotypes causing invasive pneumococcal disease along 2008-2022 in a Level 2 Public Hospital of the Madrid Region, in relation to their inclusion in different conjugate vaccines

Introducción. El uso de vacunas conjugadas frente a Streptococcus pneumoniae ocasiona cambios en la epidemio logía de la Enfermedad Neumocócica Invasiva (ENI). El objetivo de este estudio fue analizar la evolución de los serotipos de S. pneumoniae aislados en el Hospital Universitario de Getafe entre 2008 y 2022. Material y métodos. Se estudiaron 313 cepas de S. pneu moniae. El serotipado se realizó mediante el test de aglutina ción por látex (Pneumotest-latex) y la reacción de Quellung. Además, se determinó la concentración mínima inhibitoria (CMI) frente a penicilina, eritromicina y levofloxacino por el método de gradiente de concentración (E-test) según los cri terios de corte EUCAST. Resultados. Los serotipos más frecuentes en todo el pe riodo de estudio fueron 8, 3, 19A, 1, 11A y 22F correspondien do con el 46,6 % de los aislados. Durante los años 2008-2012, los serotipos 3, 1, 19A, 7F, 6C y 11A supusieron en conjunto el 53,6% de los aislamientos. Entre 2013 y 2017 los serotipos 3, 8, 12F, 19A, 22F y 19F representaron el 51% de los aislados. Entre 2018-2022 los serotipos 8, 3, 11A, 15A, 4 y 6C incluyeron al 55,5% de los casos. En total, 5 cepas (1,6%) se mostraron resistentes a penicilina, 64 (20,4%) resistentes a eritromicina y 11 (3,5%) resistentes a levofloxacino. Los niveles de CMI50 y CMI90 frente a los tres antibióticos se mantuvieron estables a lo largo del tiempo. Conclusiones. El uso de vacunas conjugadas condicionó un descenso de los serotipos cubiertos junto con un aumento de los no vacunales. Los patrones de sensibilidad a eritromicina y levofloxacino se mantuvieron relativamente estables. La re sistencia a penicilina fue muy baja, no encontrándose este tipo de cepas resistentes en el último periodo de estudio (AU)

Introduction. The use of conjugate vaccines against Streptococcus pneumoniae originates changes in the invasive pneumococcal disease (IPD). The aim of this study was to in vestigate the evolution of S. pneumoniae serotypes isolated in the Hospital Universitario de Getafe between 2008 and 2022. Material and Methods. 313 of S. pneumoniae strains were studied. Serotyping was carried out by latex agglutina tion (Pneumotest-latex) and the Quellung reaction. In addi tion, the minimal inhibitory concentration (MIC) was deter mined against penicillin, erythromycin and levofloxacin by the concentration gradient method (E-test) according the EUCAST breakpoints. Results. The most frequent serotypes throughout the study period were 8, 3, 19A, 1, 11A and 22F corresponding to 46.6% of the isolates. Along 2008-2012 the serotypes 3, 1, 19A, 7F, 6C and 11A represented altogether 53.6% of the isolates. Between 2013 and 2017 the serotypes 3, 8, 12F, 19A, 22F and 19F grouped 51% of the isolates. During 2018-2022 the serotypes 8, 3, 11A, 15A, 4 and 6C included the 55.5% of the cases. In total 5 strains (1.6%) were penicillin resistant, 64 (20.4%) erythromycin resistant and 11 (3.5%) levofloxacin re sistant. The MIC50 and MIC90 levels maintained stables along the time. Conclusion. The conjugate vaccines use with different se rotype coverage conditioned a decrease of the vaccine-includ ed and an increase of non-covered. Despite these changes, the global antimicrobial susceptibility patterns to erythromycin and levofloxacin maintained relatively stables. The resistance a penicillin was low, not finding this type of resistant strains in the last study period (AU)

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)

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.

A Prospective Study of the Serological, Clinical, and Epidemiological Features of a SARS-CoV-2 Positive Pediatric Cohort.

Background: SARS-CoV-2 was a global pandemic. Children develop a mild disease and may have a different rate of seroconversion compared to adults. The objective was to determine the number of seronegative patients in a pediatric cohort. We also reviewed the clinical−epidemiological features associated with seroconversion. Methods: A multicenter prospective observational study during September−November 2020, of COVID-19, confirmed by reverse transcription-polymerase chain reaction. Data were obtained 4−8 weeks after diagnosis. Blood samples were collected to investigate the humoral response, using three different serological methods. Results: A total of 111 patients were included (98 symptomatic), 8 were admitted to hospital, none required an Intensive Care Unit visit. Median age: 88 months (IQR: 24−149). Median time between diagnosis and serological test: 37 days (IQR: 34−44). A total of 19 patients were non-seroconverters when using three serological techniques (17.1%; 95% CI: 10.6−25.4); most were aged 2−10 years (35%, p < 0.05). Univariate analysis yielded a lower rate of seroconversion when COVID-19 confirmation was not present amongst household contacts (51.7%; p < 0.05). Conclusions: There was a high proportion of non-seroconverters. This is more commonly encountered in childhood than in adults. Most seronegative patients were in the group aged 2−10 years, and when COVID-19 was not documented in household contacts. Most developed a mild disease. Frequently, children were not the index case within the family.

Staphylococcus aureus Community-acquired Pneumonia in Children After 13-Valent Pneumococcal Vaccination (2008-2018): Epidemiology, Clinical Characteristics and Outcomes.

BACKGROUND: The epidemiology of community-acquired pneumonia (CAP) has changed, influenced by sociosanitary conditions and vaccination status. We aimed to analyze the recent epidemiology of bacterial CAP in hospitalized children in a setting with high pneumococcal vaccination coverage and to describe the clinical characteristics of pediatric Staphylococcus aureus CAP. METHODS: Children <17 years old hospitalized from 2008 to 2018 with bacterial CAP in 5 tertiary hospitals in Spain were included. Cases with pneumococcal CAP were randomly selected as comparative group following a case-control ratio of 2:1 with S. aureus CAP. RESULTS: A total of 313 bacterial CAP were diagnosed: Streptococcus pneumoniae CAP (n = 236, 75.4%), Streptococcus pyogenes CAP (n = 43, 13.7%) and S. aureus CAP (n = 34, 10.9%). Throughout the study period, the prevalence of S. pyogenes increased (annual percentage change: +16.1% [95% CI: 1.7-32.4], P = 0.031), S. pneumoniae decreased (annual percentage change: -4.4% [95 CI: -8.8 to 0.2], P = 0.057) and S. aureus remained stable. Nine isolates of S. aureus (26.5%) were methicillin-resistant. Seventeen cases (50%) with S. aureus CAP had some pulmonary complication and 21 (61.7%) required intensive care. S. pneumoniae CAP showed a trend toward higher prevalence of pulmonary complications compared with S. aureus CAP (69.1% vs. 50.0%, P = 0.060), including higher frequency of pulmonary necrosis (32.4% vs. 5.9%, P = 0.003). CONCLUSIONS: The incidence of S. aureus CAP in children remained stable, whereas the prevalence of pneumococcal CAP decreased and S. pyogenes CAP increased. Patients with S. aureus presented a high frequency of severe outcomes, but a lower risk of pulmonary complications than patients with S. pneumoniae.

Evaluation of the Chagas VirClia® and Chagas TESA VirClia® for the Diagnosis of Trypanosoma cruzi Infection.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is an important problem of public health even in regions where it is not endemic. Spain ranks second worldwide in terms of imported cases of T. cruzi infection in the chronic phase. The diagnosis in this stage is made via the detection of antibodies against T. cruzi. Therefore, we aimed to evaluate the sensitivity and specificity of two fully automated chemiluminescence immunoassays, Chagas VirClia® (CHR), which uses a mixture of recombinant antigens, and Chagas TESA VirClia® (TESA), the first chemiluminescence assay based on excretion-secretion antigens of trypomastigotes, both designed in monotest format. A retrospective case-control study was performed using 105 well-characterized samples: 49 from patients with CD, 22 from uninfected individuals, and 32 from patients with other pathologies. Sensitivity was 98% for CHR and 92% for TESA. In contrast, the specificity in both was 100%. Cross-reactivity was observed in leishmaniasis (2/10). CHR meets the criteria to become a tool for serological screening, while TESA has the potential for confirmation and cross-reaction discrimination. The monotest format allows its application in laboratories with a small number of samples. The high specificity of both assays is useful in areas where leishmaniasis is endemic.

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.

Diagnostic Accuracy of the Panbio Severe Acute Respiratory Syndrome Coronavirus 2 Antigen Rapid Test Compared with Reverse-Transcriptase Polymerase Chain Reaction Testing of Nasopharyngeal Samples in the Pediatric Population.

We conducted a multicenter clinical validity study of the Panbio coronavirus disease 2019 Antigen Rapid Test of nasopharyngeal samples in pediatric patients with coronavirus disease 2019-compatible symptoms of ≤5 days of evolution. Our study showed limited accuracy in nasopharyngeal antigen testing: overall sensitivity was 45.4%, and 99.8% of specificity, positive-predictive value was 92.5%.

Genotypic and Phenotypic Characteristics of Staphylococcus aureus Prosthetic Joint Infections: Insight on the Pathogenesis and Prognosis of a Multicenter Prospective Cohort.

BACKGROUND: Staphylococcus aureus is the leading cause of prosthetic joint infection (PJI). Beyond the antibiogram, little attention has been paid to the influence of deep microbiological characteristics on patient prognosis. Our aim was to investigate whether microbiological genotypic and phenotypic features have a significant influence on infection pathogenesis and patient outcome. METHODS: A prospective multicenter study was performed, including all S. aureus PJIs (2016-2017). Clinical data and phenotypic (agr functionality, ß-hemolysis, biofilm formation) and genotypic characteristics of the strains were collected. Biofilm susceptibility to antimicrobials was investigated (minimal biofilm eradication concentration [MBEC] assay). RESULTS: Eighty-eight patients (39.8% men, age 74.7 ±â€…14.1 years) were included. Forty-five had early postoperative infections (EPIs), 21 had chronic infections (CPIs), and 19 had hematogenous infections (HIs). Twenty (22.7%) were caused by methicillin-resistant S. aureus. High genotypic diversity was observed, including 16 clonal complexes (CCs), with CC5 being the most frequent (30.7%). agr activity was greater in EPI than CPI (55.6% vs 28.6%; P = .041). Strains causing EPI were phenotypically and genotypically similar, regardless of symptom duration. Treatment failure (36.5%) occurred less frequently among cases treated with implant removal. In cases treated with debridement and implant retention, there were fewer failures among those who received combination therapy with rifampin. No genotypic or phenotypic characteristics predicted failure, except vancomycin minimal inhibitory concentration ≥1.5 mg/L (23.1% failure vs 3.4%; P = .044). MBEC50 was >128 mg/L for all antibiotics tested and showed no association with prognosis. CONCLUSIONS: S. aureus with different genotypic backgrounds is capable of causing PJI, showing slight differences in clinical presentation and pathogenesis. No major microbiological characteristics were observed to influence the outcome, including MBEC.

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

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.

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.

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 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

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.

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.

Cryptosporidium baileyi in wild captive psittacines in Brazil.

Cryptosporidiosis in birds manifests as an acute or chronic disease of the respiratory or digestive tracts. The objective of our study was to perform the molecular characterization of Cryptosporidium spp. in wild psittacines kept in captivity at the Araçatuba Municipal Zoo, São Paulo, Brazil. A total of 47 fecal samples were collected from Amazona aestiva, Psittacara leucophthalma, and Ara ararauna. All samples were collected at the time the birds defecated. DNA extraction was performed using the ZR Fecal DNA MiniPrep™ kit (Zymo Research). Screening for Cryptosporidium spp. was accomplished using nested PCR targeting the 18S RNA gene and sequencing of amplified fragments. Positivity for Cryptosporidium spp. (10.64%; 5/47) was found in A. ararauna (4) and P. leucophthalma (1) samples. The amplified fragments were sequenced and showed 100% genetic similarity with Cryptosporidium baileyi.