A 21-Year Survey of Escherichia coli from Bloodstream Infections (BSI) in a Tertiary Hospital Reveals How Community-Hospital Dynamics of B2 Phylogroup Clones Influence Local BSI Rates.

This is a longitudinal study comprising 649 Escherichia coli isolates representing all 7,165 E. coli bloodstream infection (BSI) episodes recorded in a hospital (1996 to 2016). Strain analysis included clonal identification (phylogenetic groups/subgroups, STc131 subclades, pulsed-field gel electrophoresis [PFGE], and whole-genome sequencing [WGS]), antibiotic susceptibility (13 antibiotics), and virulence-associated genes (VAGs; 29 genes). The incidence of E. coli BSI increased from 1996 to 2016 (5.5 to 10.8 BSI episodes/1,000 hospitalizations, average 7 to 8/1,000). B2 isolates predominate (53%), with subgroups B2-I (STc131), B2-II, B2-IX, and B2-VI representing 25%, 25%, 14%, and 9%, respectively. Intertwined waves of community-acquired (CA) plus health care-associated and community-onset health care-associated (HCA) and hospital-acquired (HA) episodes of both B2 and non-B2 phylogroups occurred. A remarkable increase was observed only for B2-I-STc131 (C1/C2 subclades), with oscillations for other B2 subgroups and phylogroups throughout the years. Epidemic and persistent clones (comprising isolates with highly similar/identical PFGE types and genomes differing in 6 to 173 single nucleotide polymorphisms [SNPs]) of B2-I (STc131), B2-II (STc73), B2-III (STc127), B2-IX (STc95), and B2-VI (STc12) were recovered from different patients, most at hospital admission, for long periods (2 to 17 years), and extended-spectrum beta-lactamase (ESBL) producers or resistance to ciprofloxacin in B2 isolates was almost restricted to B2-I (STc131) subclade C. STc131 contributed to increasing the B2 rates but only transiently altered the E. coli population structure. The increase of E. coli BSI was determined by waves of CA+HCA BSI episodes that predate the waves of HA BSI. Besides the risk of hospital transmission that led to temporal increases in BSI, this study suggests that E. coli populations/clones from community-based healthy individuals may occasionally have an epidemic structure and provide a source of transmissible strains influencing the HA BSI incidence. IMPORTANCE Sepsis is the third leading cause of mortality in Western countries and one of the Global Health Threats recognized by the WHO since 2017. Despite Escherichia coli constituting the most common cause of bloodstream infections (BSI), its epidemiology is not fully understood, in part due to the scarcity of local and longitudinal studies. Our work analyzes the long-term dynamics of E. coli causing bacteremia in a single institution and reveals waves of different clonal lineages that emerge periodically and successfully spread afterward in both the community and hospitals. Because the origin of E. coli bloodstream infections is the gut, the microbiota of healthy individuals might occasionally have an epidemic structure, providing a source of E. coli strains to influence the incidence of hospital BSI. The study complements previous fractionated observations focusing on specific E. coli lineages or antibiotic-resistant isolates in the last decades and helps to understand the epidemiology of E. coli BSI and the dynamics of pandemic clones.

Real Life Clinical Impact of Antimicrobial Stewardship Actions on the Blood Culture Workflow from a Microbiology Laboratory.

BACKGROUND: Accelerating the diagnosis of bacteremia is one of the biggest challenges in clinical microbiology departments. The fast establishment of a correct treatment is determinant on bacteremic patients' outcomes. Our objective was to evaluate the impact of antimicrobial therapy and clinical outcomes of a rapid blood culture workflow protocol in positive blood cultures with Gram-negative bacilli (GNB). METHODS: A quasi-experimental before-after study was performed with two groups: (i) control group (conventional work-protocol) and (ii) intervention group (rapid workflow-protocol: rapid identification by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) and antimicrobial susceptibility testing (AST) from bacterial pellet without overnight incubation). Patients were divided into different categories according to the type of intervention over treatment. Outcomes were compared between both groups. RESULTS: A total of 313 patients with GNB-bacteremia were included: 125 patients in the control group and 188 in the intervention. The time from positive blood culture to intervention on antibiotic treatment decreased from 2.0 days in the control group to 1.0 in the intervention group (p < 0.001). On the maintenance of correct empirical treatment, the control group reported 2.0 median days until the clinical decision, while in the intervention group was 1.0 (p < 0.001). In the case of treatment de-escalation, a significant difference between both groups (4.0 vs. 2.0, p < 0.001) was found. A decreasing trend on the change from inappropriate treatments to appropriate ones was observed: 3.5 vs. 1.5; p = 0.12. No significant differences were found between both groups on 7-days mortality or on readmissions in the first 30-days. CONCLUSIONS: Routine implementation of a rapid workflow protocol anticipates the report of antimicrobial susceptibility testing results in patients with GNB-bacteremia, decreasing the time to effective and optimal antibiotic therapy.

Monitoring the antimicrobial susceptibility of Gram-negative organisms involved in intraabdominal and urinary tract infections recovered during the SMART study (Spain, 2016 and 2017) / Seguimiento de la sensibilidad antimicrobiana de microorganismos gramnegativos procedentes de infecciones intraabdominales y urinarias del estudio SMART (España, 2016 y 2017)

Introduction: Continuous antimicrobial resistance surveillance is recommended by Public Health authorities. We up-dated data from the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. Material and methods: The antimicrobial susceptibility data and extended-spectrum beta-lactamase (ESBL) production in isolates recovered from intra-abdominal (IAI) (n=1,429) and urinary tract (UTI) (n=937) infections during the 2016- 2017 SMART study in 10 Spanish hospitals were analysed. Results: Escherichia coli was the most frequently microorganism isolated (48.3% and 53.7%) followed by Klebsiella spp. (11.5% and 21.9%) in IAIs and UTIs, respectively. Figures for Pseudomonas aeruginosa were 9.0% and 6.1%, being more frequently recovered from patients with nosocomial infections. Overall, 9.9% (IAI) and 14.0% (UTI) of E. coli, Klebsiella spp. and Proteus mirabilis isolates were ESBL-producers, being Klebsiella pneumoniae (34.5%) from UTI of nosocomial origin the most frequent. ESBL-producers were higher in patients >60 years in both IAIs and UTIs. As in previous years, amikacin (96.3%-100% susceptibility), ertapenem (84.2%-100%) and imipenem (70.3%- 100%) were the most active antimicrobials tested among Enterobacterales species. The activity of amoxicillin-clavulanic, piperacillin-tazobactam, and ciprofloxacin susceptibility was lower, particularly among ESBL-producers. Ertapenem susceptibility (88.9%-100%) was retained in ESBL-E. coli isolates that were resistant to these antimicrobials but decreased (28.6%-100%) in similar isolates of K. pneumoniae. Conclusions: Continuous antimicrobial resistance surveillance from the SMART study reveals overall maintenance of ESBL-producers in Spain, although with higher presence in isolates from UTIs than from IAIs. Moreover, ertapenem activity was high in E. coli irrespective of ESBL production but decreased in K. pneumoniae, particularly among ESBL-producers

Introducción: Las autoridades de Salud Pública recomiendan la vigilancia continua de la resistencia a los antimicrobianos. Se actualizan los datos del estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) en España. Material y métodos: Se analizaron los datos de sensibilidad antimicrobiana y la producción de betalactamasas de espectro extendido (BLEE) en aislamientos obtenidos en el estudio SMART de infecciones intraabdominales (IIA) (n=1.429) y del tracto urinario (ITU) (n=937) durante 2016-2017 en 10 hospitales españoles. Resultados: Escherichia coli fue el microorganismo más frecuente (54,5% y 57,5%, respectivamente), seguido de Klebsiella spp. (18,4% y 25,4%) en IIA y en ITU. En Pseudomonas aeruginosa estas cifras fueron 9% y 6%, siendo más frecuente en la infección nosocomial. El 9,9% (IIA) y el 14% (ITU) del to-tal de los aislados de E. coli, Klebsiella spp. y Proteus mirabilis producían BLEE, obteniéndose la tasa más alta en Klebsiella pneumoniae (34.5%) en ITU nosocomial. El mayor porcentaje de aislados con BLEE se observó en pacientes >60 años, tanto en IIA como en ITU. Como en años anteriores, amikacina (sensibilidad 96,3%-100%), ertapenem (84,2%-100%) e imipenem (70,3%-100%) fueron los antimicrobianos más activos en Enterobacterales. La sensibilidad a amoxicilina-ácido clavulánico, piperacilina-tazobactam y ciprofloxacino fue menor, en particular en los productores de BLEE. La sensibilidad a ertapenem (88,9%-100%) se mantuvo en E. coli con BLEE resistente a estos antimicrobianos, pero disminuyó (28,6%-100%) en aislados similares de K. pneumoniae. Conclusiones: La vigilancia continua de la resistencia a los antimicrobianos en el estudio SMART revela el mantenimiento de la frecuencia de aislados productores de BLEE en España, pero con mayor presencia en las ITUs que en las IIAs. Además, la sensibilidad a ertapenem fue alta en E. coli con independencia de la producción de BLEE, pero disminuyó en K. pneumoniae, sobre todo en los productores de BLEE

A multicentre study investigating parameters which influence direct bacterial identification from urine.

Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification.

Antimicrobial susceptibility trends and evolution of isolates with extended spectrum Beta-lactamases among Gram-negative organisms recovered during the SMART study in Spain (2011-2015) / Evolución de la sensibilidad y de los aislados productores de Beta-lactamasas de espectro extendido en microorganismos gramnegativos en el estudio SMART en España (2011-2015)

Introduction. The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study monitors antimicrobial susceptibility and extended spectrum β-lactamases (ESBLs) in Gram-negative bacilli recovered from intra-abdominal infections (IAI). Material and methods. Antimicrobial susceptibility of 5,343 isolates from IAI recovered in 11 centres during the 2011-2015 SMART-Spain program was analysed by standard microdilution (EUCAST criteria) and compared with that from 2002-2010. ESBLs were phenotypically detected. Results. Escherichia coli, the most common isolate, significantly decreased in community acquired IAI (60.9% 2002-2010 vs. 56.1% 2011-2015, P=0.0003). It was followed in prevalence by Klebsiella pneumoniae that increased both in the community (8.9% vs. 10.8%, P=0.016) and nosocomial (9.2% vs. 10.8%, P=0.029) IAI and P. aeruginosa, which significantly increased in community acquired IAI (5.6% vs. 8.0%, P=0.0003). ESBLs were more prevalent in K. pneumoniae (16.3%) than in E. coli(9.5%) of nosocomial origin and were more frequently isolated from elderly patients (>60 years). Considering all Enterobacteriaceae, ertapenem (92.3-100%) and amikacin (95.5%-100%) were the most active antimicrobials. Ertapenem activity, unlike amoxicillin-clavulanate or piperacillin-tazobactam, remained virtually unchanged in ESBL (100%) and non-ESBL (98.8%) E. coli producers. Its activity decreased in ESBL-K. pneumonia (74.7%) but was higher than that of amoxicillin-clavulanate (14.0%) and piperacillin-tazobactam (24.0%). Interestingly, ertapenem susceptibility was maintained in >60% of ESBL isolates that were resistant to amoxicillin-clavulanate, piperacillin-tazobactam or luoroquinolones. Conclusions. SMART-Spain results support current guidelines which include ertapenem as empiric treatment in mild-moderate community-acquired IAI, particularly with ESBL producers. These recommendations will need to be updated with the recently introduction of new antimicrobials

Introducción. El estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) monitoriza la sensibilidad antimicrobiana y las β-lactamasas de espectro extendido (BLEE) en bacilos gramnegativos obtenidos de infecciones intraabdominales (IIA). Material y Métodos. Se ha analizado la sensibilidad antimicrobiana (microdilución estándar, criterios EUCAST) y las BLEE (detección fenotípica) de 5.343 aislados de IIA en 11 centros del programa SMART-España durante 2011-2015 en comparación con 2002-2010. Resultados. Escherichia coli, el microorganismo más prevalente, disminuyó significativamente en las IIA de origen comunitario (60,9% 2002-2010 vs. 56,1% 2011-2015, P=0,0003). Fue seguido en prevalencia por Klebsiella pneumoniae que aumentó tanto en IIA comunitaria (8,9% vs. 10,8%, P=0,016) como nosocomial (9,2% vs. 10,8%, P=0,029) y por P. aeruginosa que aumentó en la IIA comunitaria (5,6% vs. 8,0%, P=0,0003). Las BLEE fueron más prevalentes en la IIA nosocomial por K. pneumoniae(16,3%) que por E. coli(9,5%), siendo más frecuentes en pacientes de mayor edad (>60 años). Considerando todas las Enterobacteriaceae, ertapenem (92,3-100%) y amikacina (95,5%-100%) fueron los antimicrobianos más activos. La sensibilidad a ertapenem, al contrario que a amoxicilina-clavulánico o piperacilina-tazobactam, se mantuvo sin cambios en E. coli con (98,8%) y sin BLEE (100%). Su sensibilidad disminuyó en BLEE-K. pneumoniae (74,7%) pero fue mayor que la de amoxicilina-clavulánico (14,0%) o piperacilina-tazobactam (24,0%). Es de resaltar que esta actividad se mantuvo >60% en los aislados con BLEE resistentes a amoxicilina-clavulánico, piperacilina-tazobactam o fluoroquinolonas.Conclusiones. El estudio SMART-España sustenta las guías actuales que incluyen al ertapenem como tratamiento empírico en la IIA leve-moderada comunitaria, en particular con BLEE. Estas recomendaciones precisaran actualizarse con la reciente introducción de nuevos antimicrobianos

Long-term clonal dynamics of Enterococcus faecium strains causing bloodstream infections (1995-2015) in Spain.

OBJECTIVES: To investigate the population structure of Enterococcus faecium causing bloodstream infections (BSIs) in a tertiary Spanish hospital with low glycopeptide resistance, and to enhance our knowledge of the dynamics of emergence and spread of high-risk clonal complexes. METHODS: All available E. faecium causing BSIs (n = 413) in our hospital (January 1995-May 2015) were analysed for antibiotic susceptibility (CLSI), putative virulence traits (PCR, esp, hylEfm) and clonal relationship (SmaI-PFGE, MLST evaluated by goeBURST and BAPS). RESULTS: The increased incidence of BSIs caused by enterococci [2.3‰ of attended patients (inpatients and outpatients) in 1996 to 3.0‰ in 2014] significantly correlated with the increase in BSIs caused by E. faecium (0.33‰ of attended patients in 1996 to 1.3‰ in 2014). The BSIs Enterococcus faecalis:E. faecium ratio changed from 5:1 in 1996 to 1:1 in 2014. During the last decade an increase in E. faecium BSIs episodes in cancer patients (10.9% in 1995-2005 and 37.1% in 2006-15) was detected. Ampicillin-susceptible E. faecium (ASEfm; different STs/BAPS) and ampicillin-resistant E. faecium (AREfm; ST18/ST17-BAPS 3.3a) isolates were recovered throughout the study. Successive waves of BAPS 2.1a-AREfm (ST117, ST203 and ST80) partially replaced ASEfm and ST18-AREfm since 2006. CONCLUSIONS: Different AREfm clones (belonging to BAPS 2.1a and BAPS 3.3a) consistently isolated during the last decade from BSIs might be explained by a continuous and dense colonization (favouring both invasion and cross-transmission) of hospitalized patients. High-density colonization by these clones is probably enhanced in elderly patients by heavy and prolonged antibiotic exposure, particularly in oncological patients.

High MICs for Vancomycin and Daptomycin and Complicated Catheter-Related Bloodstream Infections with Methicillin-Sensitive Staphylococcus aureus.

We investigated the prognostic role of high MICs for antistaphylococcal agents in patients with methicillin-sensitive Staphylococcus aureus catheter-related bloodstream infection (MSSA CRBSI). We prospectively reviewed 83 episodes from 5 centers in Spain during April 2011-June 2014 that had optimized clinical management and analyzed the relationship between E-test MICs for vancomycin, daptomycin, oxacillin, and linezolid and development of complicated bacteremia by using multivariate analysis. Complicated MSSA CRBSI occurred in 26 (31.3%) patients; MICs for vancomycin and daptomycin were higher in these patients (optimal cutoff values for predictive accuracy = 1.5 µg/mL and 0.5 µg/mL). High MICs for vancomycin (hazard ratio 2.4, 95% CI 1.2-5.5) and daptomycin (hazard ratio 2.4, 95% CI 1.1-5.9) were independent risk factors for development of complicated MSSA CRBSI. Our data suggest that patients with MSSA CRBSI caused by strains that have high MICs for vancomycin or daptomycin are at increased risk for complications.

[Applicability of new diagnostic techniques in microbiology; technological innovation]. / Aplicabilidad de las nuevas técnicas de diagnóstico microbiológico; innovación tecnológica.

Different new techniques have been introduced in microbiology laboratories during the last years, including mass spectrometry and next generation sequencing. These techniques, in addition to automation, microfludics, nanotechnology and informatics, have impelled innovation in the prevention and management of patients with infectious diseases. These approaches are relevant for revitalization and consolidation Clinical Microbiology laboratories.

Aplicabilidad de las nuevas técnicas de diagnóstico microbiológico; innovación tecnológica / Applicability of new diagnostic techniques in microbiology; technological innovation

En los últimos años se han introducido nuevas técnicas en los laboratorios de microbiología, incluyendo la espectrometría de masas y los sistemas de secuenciación masiva de próxima generación. Estas técnicas, así como la automatización, la microfluídica, la nanotecnología y la informática, han impulsado la innovación en la prevención y el manejo de las enfermedades infecciosas. Esta aproximación es relevante en el proceso de revitalización y consolidación de los Servicios de Microbiología Clínica (AU)

Different new techniques have been introduced in microbiology laboratories during the last years, including mass spectrometry and next generation sequencing. These techniques, in addition to automation, microfludics, nanotechnology and informatics, have impelled innovation in the prevention and management of patients with infectious diseases. These approaches are relevant for revitalization and consolidation Clinical Microbiology laboratories (AU)

Antimicrobial susceptibility of Gram-negative organisms from intraabdominal infections and evolution of isolates with extended spectrum ß-lactamases in the SMART study in Spain (2002-2010).

INTRODUCTION: The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study records the antimicrobial susceptibility of Gram-negative bacilli obtain from intraabdominal infections with special focus in isolates with extended spectrum ß-lactamases (ESBLs). MATERIAL AND METHODS: The antimicrobial susceptibility of 8,869 isolates was analyzed by microdilution during the SMART study performed in Spain from 2002 to 2010. Isolates were recovered in 16 centres. RESULTS: Escherichia coli was the most prevalent pathogen (60.9% from intraabdominal infections acquired in the community and 49.9% in those from nosocomial origin) followed by Klebsiella pneumoniae (8.9% vs 9.2%). Pseudomonas aeruginosa was more common in intraabdominal infections from nosocomial origin (5.6% community and 8.6% nosocomial). Frequency of ESBL-producing isolates was: E. coli, 8.7%; K. pneumoniae, 8.4%; Klebsiella oxytoca, 1.4%; and Proteus mirabilis, 1.6%. Overall, ESBL-producing isolates were more frequently isolated from elderly patients (6.8% >60 years). Ertapenem and meropenem were the most active antimicrobials (susceptibility range with EUCAST criteria, 89.0-100%) when considering all Enterobacteriaceae isolates and also against ESBL producers (95.5-100%). Susceptibility of amoxicillin/clavulanic acid and piperacillin/tazobactam was lower, particularly among ESBL-producing isolates. Nevertheless, ertapenem maintained a good activity (susceptibility >95%) in ESBL-producers that were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam or fluoroquinolones. CONCLUSIONS: Antimicrobial susceptibility data from the SMART-Spain study reinforce current therapeutic guidelines of intraabdominal infections that include ertapenem as the empirical choice for treatment. This is also supported by the high frequency of ESBL-producers in our geographic area.

Antimicrobial susceptibility of Gram-negative organisms from intra abdominal infections and evolution of isolates with extended spectrum Beta-lactamases in the SMART study in Spain (2002-2010)

Introducción. El estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) tiene como objetivo monitorizar la sensibilidad a los antimicrobianos de los microorganismos gramnegativos aislados en la infección intraabdominal, con especial seguimiento de los que producen Beta-lactamasas de espectro extendido (BLEE). Material y métodos. Se han analizado por microdulución los datos de sensibilidad de 8.869 aislados recogidos en el estudio SMART en España entre 2002 y 2010 en el que han participado 16 centros. Resultados. Escherichia coli fue el patógeno más frecuente (60,9% en la infección intraabdominal adquirida en la comunidad y 49,9% en la nosocomial) seguido de Klebsiella pneumoniae (8,9% vs 9,2%). Pseudomonas aeruginosa fue más habitual en la infección nosocomial (5,6% comunitaria y 8,6% nosocomial). La frecuencia de aislados con BLEE fue: E. coli 8,7%, K. pneumoniae 8,4%, Klebsiella oxytoca 1,4% y Proteus mirabilis 1,6%. En los pacientes de mayor edad aumentó la proporción global de aislados con BLEE (6,8% en pacientes >60 años). Ertapenem y meropenem fueron los antimicrobianos más activos en el conjunto de las enterobacterias (rango de sensibilidad con criterios EUCAST, 89- 100%) y también entre los aislados con BLEE (95,5-100%). La actividad de amoxicilina/ácido clavulánico y piperacilina/tazobactam fue considerablemente inferior, en particular en los aislados con BLEE. Ertapenem mantuvo una buena actividad (sensibilidad >95%) en los productores de BLEE resistentes a amoxicilina/ácido clavulánico, piperacilina/tazobactam o fluoroquinolonas. Conclusiones. Los datos de sensibilidad del estudio SMART en España avalan las guías terapéuticas actuales de infecciónintraabdominal que sitúan al ertapenem como tratamiento empírico de elección, teniendo en cuenta sobre todo la elevada frecuencia de aislados con BLEE en nuestro medio(AU)

Introduction. The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study records the antimicrobial susceptibility of Gram-negative bacilli obtain from intraabdominal infections with special focus in isolates with extended spectrum Beta-lactamases (ESBLs). Material and Methods. The antimicrobial susceptibility of 8,869 isolates was analyzed by microdilution during the SMART study performed in Spain from 2002 to 2010. Isolates were recovered in 16 centres. Results. Escherichia coli was the most prevalent pathogen (60.9% from intraabdominal infections acquired in the community and 49.9% in those from nosocomial origin) followed by Klebsiella pneumoniae (8.9% vs 9.2%). Pseudomonas aeruginosa was more common in intraabdominal infections from nosocomial origin (5.6% community and 8.6% nosocomial). Frequency of ESBL-producing isolates was: E. coli, 8.7%; K. pneumoniae, 8.4%; Klebsiella oxytoca, 1.4%; and Proteus mirabilis, 1.6%. Overall, ESBL-producing isolates were more frequently isolated from elderly patients (6.8% >60 years). Ertapenem and meropenem were the most active antimicrobials (susceptibility range with EUCAST criteria, 89.0-100%) when considering all Enterobacteriaceae isolates and also against ESBL producers (95.5-100%). Susceptibility of amoxicillin/clavulanic acid and piperacillin/tazobactam was lower, particularly among ESBL-producing isolates. Nevertheless, ertapenem maintained a good activity (susceptibility >95%) in ESBL-producers that were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam or fluoroquinolones. Conclusions. Antimicrobial susceptibility data from the SMART-Spain study reinforce current therapeutic guidelines of intraabdominal infections that include ertapenem as the empirical choice for treatment. This is also supported by the high frequency of ESBL-producers in our geographic area(AU)

Reidentification of Streptococcus bovis isolates causing bacteremia according to the new taxonomy criteria: still an issue?

All Streptococcus bovis blood culture isolates recovered from January 2003 to January 2010 (n = 52) at the Hospital Universitario Ramón y Cajal were reidentified on the basis of their genetic traits using new taxonomic criteria. Initial identification was performed by the semiautomatic Wider system (Fco. Soria-Melguizo, Spain) and the API 20 Strep system (bioMérieux, France). All isolates were reidentified by PCR amplification and sequencing of both the 16S rRNA and sodA genes and by mass spectrometry using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Germany). Results of 16S rRNA/sodA gene sequencing were as follows: Streptococcus gallolyticus subsp. gallolyticus, 14/14 (number of isolates identified by 16S rRNA/number of isolates identified by sodA gene sequencing); Streptococcus gallolyticus subsp. pasteurianus, 24/24; Streptococcus spp., 7/0; Streptococcus infantarius subsp. infantarius, 0/2; Streptococcus lutetiensis, 0/5; Leuconostoc mesenteroides, 4/0; and Lactococcus lactis, 3/3. MALDI-TOF MS identified 27 S. gallolyticus isolates but not at the subspecies level, 4 L. mesenteroides isolates, 3 L. lactis isolates, and 6 S. lutetiensis isolates, whereas 12 isolates rendered a nonreliable identification result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. gallolyticus isolates into 3 major clusters clearly different from those of the S. gallolyticus subsp. pasteurianus isolates, which, in turn, exhibited no clonal relationship. The percentages of resistance to the tested antimicrobials were 38% for erythromycin, 23% for fosfomycin, 10% for levofloxacin, 6% for tetracycline, and 4% for co-trimoxazole. The most frequent underlying diseases were hepatobiliary disorders (53%), endocarditis (17%), and malignancies (12%). We conclude that sequencing of the sodA gene was the most discriminatory method and that S. gallolyticus subsp. pasteurianus appears to have a higher genetic diversity than S. gallolyticus subsp. gallolyticus.

High clonal diversity in erythromycin-resistant Streptococcus pneumoniae invasive isolates in Madrid, Spain (2000-07).

OBJECTIVES: Erythromycin resistance in Streptococcus pneumoniae is still increasing worldwide. All 78 erythromycin-resistant S. pneumoniae isolates collected from blood cultures in our hospital (2000-07) were studied and the population structure was analysed by using different mathematical diversity indexes. METHODS: Erythromycin resistance determinants were screened by PCR. The population structure, including multilocus sequence typing, was analysed by using quantitative clonal diversity (diversity ratio, Simpson, Selander-Levin and Shannon mathematical indexes). RESULTS: The leading resistance gene was erm(B) (74.3% of the isolates), followed by the erm(B) plus mef(A) combination (17.9%) and mef(A) alone (7.7%). The most frequent serotypes were 14 (18%), 19A (15.4%) and 6B (11.5%). A polyclonal structure was detected in resistant strains, including the Spain(9V)-3, Spain(6B)-2 and Denmark(14)-32 international clones. Both genetic diversity and genetic distribution were high, particularly among clones containing erm(B) and erm(B) plus mef(A) determinants. CONCLUSIONS: The resistance determinants erm(B) and the combination of erm(B) plus mef(A) were observed within multiple S. pneumoniae bacteraemic clones. The preservation of a polyclonal structure might provide a suitable background for further evolution of antibiotic resistance.

[Comparative in vitro activity of daptomycin against gram-positive microorganisms: SENTRY surveillance program, Spain (2002-2006)]. / Actividad comparativa de daptomicina frente a microorganismos grampositivos: programa SENTRY España (2002-2006).

INTRODUCTION AND OBJECTIVE: Daptomycin is a bactericidal lipopeptide antibiotic, active against gram-positive bacteria. In this study, the comparative in vitro activity of daptomycin and other antimicrobial agents against isolates recovered in 3 Spanish hospitals from 2002 to 2006 was determined as part of the international SENTRY antimicrobial resistance surveillance program. The possible therapeutic role of daptomycin is addressed in the light of Spanish susceptibility data. METHODS: Antimicrobial susceptibility testing was performed by the microdilution method with 1398 consecutively recovered gram-positive isolates. RESULTS: All the staphylococci (n = 1024), enterococci (n = 228), and streptococci (n = 146) studied were susceptible to daptomycin. The highest MIC values were 1, 4, and 0.5 microg/mL, respectively, regardless of methicillin, vancomycin, or penicillin resistance status. All Staphylococcus aureus isolates were also susceptible to vancomycin, teicoplanin, linezolid, and quinupristin-dalfopristin. Coagulase-negative staphylococci were susceptible to vancomycin, linezolid, and quinupristin-dalfopristin. Only daptomycin and linezolid were active against all enterococcal isolates. In addition, vancomycin, teicoplanin, and ampicillin were fully active against Enterococcus faecalis. Daptomycin, vancomycin, teicoplanin, linezolid, and quinupristin-dalfopristin were active against all Streptococcus pyogenes, S. agalactiae, and S. viridans group isolates. The distribution of daptomycin MIC values in S. aureus and enterococci was homogeneously sustained along the 5-year study period. CONCLUSION: The sustained antimicrobial activity of daptomycin against staphylococci, enterococci, and streptococci in Spain makes this antibiotic an excellent therapeutic option in severe infection caused by gram-positive microorganisms, including those with multiresistance.

Actividad comparativa de daptomicina frente a microorganismos grampositivos: programa SENTRY España (2002-2006) / Comparative in vitro activity of daptomycin against gram-positive microorganisms: SENTRY surveillance program, Spain (2002-2006)

Introducción y objetivo. La daptomicina es un antibiótico lipopeptídico bactericida frente a microorganismos grampositivos. En este trabajo estudiamos la actividad in vitro de daptomicina comparativamente con otros antibióticos frente a aislados de tres hospitales españoles recogidos durante el período 2002-2006, en el marco del programa internacional de vigilancia de la resistencia, SENTRY, para establecer sus posibilidades terapéuticas con datos específicos de España. Métodos. Los antibióticos se estudiaron por la técnica de microdilución frente a 1.398 microorganismos grampositivos aislados consecutivamente. Resultados. Todos los estafilococos (n = 1.024), enterococos (n = 228) y estreptococos (n = 146) fueron sensibles a daptomicina y presentaban valores máximos de concentraciones inhibitorias mínimas (CIM) de 1, 4 y 0,5 mg/ml, respectivamente, con independencia de la resistencia a meticilina, vancomicina o penicilina. Staphylococcus aureus fue también sensible a vancomicina, teicoplanina, linezolid y quinupristina-dalfopristina y los estafilococos coagulasa negativa (SCN) a vancomicina, linezolid y quinupristina-dalfopristina. Frente a enterococos, sólo daptomicina y linezolid fueron activos frente a la totalidad de éstos. Además, vancomicina, teicoplanina y ampicilina fueron totalmente efectivos frente a Enterococcus faecalis. Frente a Streptococcus pyogenes, Streptococcus agalactiae y estreptococos del grupo viridans, daptomicina, vancomicina, teicoplanina, linezolid y quinupristina dalfopristina fueron plenamente activos. La distribución de las CIM de daptomicina a través de los 5 años fue homogénea en S. aureus y enterococos. Conclusión. La actividad de daptomicina frente a estafilococos, enterococos y estreptococos, mantenida a lo largo de los años en España, la convierte en una excelente opción terapéutica en infecciones graves por microorganismos grampositivos, incluyendo los multirresistentes (AU)

INTRODUCTION AND OBJECTIVE. Daptomycin is a bactericidal lipopeptide antibiotic, active against gram-positive bacteria. In this study, the comparative in vitro activity of daptomycin and other antimicrobial agents against isolates recovered in 3 Spanish hospitals from 2002 to 2006 was determined as part of the international SENTRY antimicrobial resistance surveillance program. The possible therapeutic role of daptomycin is addressed in the light of Spanish susceptibility data. METHODS. Antimicrobial susceptibility testing was performed by the microdilution method with 1398 consecutively recovered gram-positive isolates. RESULTS. All the staphylococci (n 1024), enterococci (n 228), and streptococci (n 146) studied were susceptible to daptomycin. The highest MIC values were 1, 4, and 0.5 g/mL, respectively, regardless of methicillin, vancomycin, or penicillin resistance status. All Staphylococcus aureus isolates were also susceptible to vancomycin, teicoplanin, linezolid, and quinupristin-dalfopristin. Coagulase-negative staphylococci were susceptible to vancomycin, linezolid, and quinupristin-dalfopristin. Only daptomycin and linezolid were active against all enterococcal isolates. In addition, vancomycin, teicoplanin, and ampicillin were fully active against Enterococcus faecalis. Daptomycin, vancomycin, teicoplanin, linezolid, and quinupristin-dalfopristin were active against all Streptococcus pyogenes, S. agalactiae, and S. viridans group isolates. The distribution of daptomycin MIC values in S. aureus and enterococci was homogeneously sustained along the 5-year study period CONCLUSION. The sustained antimicrobial activity of daptomycin against staphylococci, enterococci, and streptococci in Spain makes this antibiotic an excellent therapeutic option in severe infection caused by gram-positive microorganisms, including those with multiresistance (AU)

Evaluation of 4 swab transport systems for the recovery of ATCC and clinical strains with characterized resistance mechanisms.

Despite limitations of swab transport systems (STS), many clinical samples and bacterial isolates are sent on these devices. We evaluated the performance of 4 commercial STS: M40 Transystem (S1, Copan Italia, Bovezzo, Italy), UNI-TER (S2, MEUS, Piove di Sacco, Italy), Euromed (S3, LAB SERVICE, Surbo, Italy), and Eurotubo (S4, Deltalab, Rubí, Spain). Survival of ATCC and clinical strains with characterized resistance mechanisms, stored at room temperature and using the roll-plate method (NCCLS M40-A) after holding times of 0, 6, 24, 48, and 72 h, and 7 days, was assessed. After 24 h, only S1 was able to maintain Streptococcus pneumoniae (ATCC 6305), Haemophilus influenzae (10211), and Neisseria meningitidis (13090) viability with a percentage range of recovery with respect to the baseline count at zero time of 104-184%. Neisseria gonorrhoeae (43069) was uncultivable after 6 h with all STS except S1 (46% recovery). Eighteen percent recovery after 24 h and 7% after 6 h was observed for Fusobacterium nucleatum (25586) and Peptostreptococcus anaerobius (27337), respectively, but only with S1. Resistance mechanisms in clinical isolates do not affect survival with any of the STS.

Antibiotic coresistance in extended-spectrum-beta-lactamase-producing Enterobacteriaceae and in vitro activity of tigecycline.

The spread of extended-spectrum-beta-lactamase (ESBL)-producing organisms, particularly those harboring the CTX-M-type enzymes, both in the hospital and in the community, is difficult to discontinue due to the successful mobilization and evolution of the genetic elements harboring ESBL genes and coresistance rates in these isolates. The activities of tigecycline against 285 non-clonally related isolates (172 from Escherichia coli, 84 from Klebsiella spp., 20 from Enterobacter spp., 5 from Salmonella spp., and 4 from Citrobacter spp.) expressing well-characterized ESBLs and recovered in our hospital and its community area of influence were comparatively assessed (CLSI microdilution). Susceptibility rates for meropenem, imipenem, tigecycline, amikacin, and piperacillin-tazobactam were 100%, 100%, 97.5%, 93.3%, and 93%, respectively. Tigecycline (mode MIC, 0.5 microg/ml; MIC(90), 1 microg/ml) was 4- to 256-fold more active than doxycycline and minocycline (mode MIC range, 2 to 128 microg/ml). CTX-Ms were the most frequent ESBLs (61.4%), 65.8% in community and 58.6% in nosocomial isolates. CTX-M-9 (22%), CTX-M-14 (15.8%), and CTX-M-10 (14%) were the most represented derivatives. SHV and TEM variants constituted 22.8% and 15.8% of the ESBLs, respectively. Overall coresistance rates were as follows: gentamicin, 27.4%; tobramycin, 27.4%; amikacin, 6.7%; and chloramphenicol, 29.1%. Sulfonamide (61.7%), trimethoprim (52.3%), streptomycin (50.5%), and ciprofloxacin (37.2%) resistance levels were significantly (P < 0.001) associated with CTX-M-9 producers. No tigecycline resistance was observed, although seven Klebsiella pneumoniae isolates exhibited intermediate MICs (4 mug/ml). Tigecycline, lacking cross-resistance with other compounds, could represent an opportunity to reduce the intensity of selection for ESBL-producing organisms derived from the use of other antimicrobial agents. However, this in vitro promise requires support from clinical studies.

Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips.

Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against Pseudomonas aeruginosa. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing P. aeruginosa isolates from cystic fibrosis (CF) patients (susceptible, < or =64 microg/ml; resistant, > or =128 microg/ml). The antimicrobial susceptibilities of 206 prospectively collected CF P. aeruginosa isolates were determined by the reference agar dilution method. For tobramycin, the performance of high range tobramycin Etest strips (AB Biodisk, Solna, Sweden) and conventional tobramycin disks were assessed with the same collection. Applying MENSURA proposed breakpoints, 95.1% of the strains were categorized as susceptible to tobramycin, either using agar dilution or Etest high-range strips (99% categorical agreement between both methods). With CLSI breakpoints, susceptibility rates decreased to 79.1 and 81.1% for agar dilution and Etest strips, respectively (83.5% categorical agreement). Minor, major, and very major errors for Etest strips (CLSI criteria) were 13.6, 1.2, and 14.8%, respectively. Upon applying the new proposed criteria for inhaled tobramycin, only one major and one very major error were observed with Etest strips. Whenever inhaled tobramycin is considered for therapy, we suggest that P. aeruginosa strains from CF patients categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be retested with high-range Etest strips and recategorized using MENSURA interpretive criteria. CLSI breakpoints should still be followed when intravenous tobramycin is used in CF patients, particularly during the course of exacerbations.

Population structure of Enterococcus faecium causing bacteremia in a Spanish university hospital: setting the scene for a future increase in vancomycin resistance?

Over an 8-year period (1995 to 2002), 86 Enterococcus faecium blood isolates from 84 patients, of which 54 were ampicillin resistant (AREF) and 32 were ampicillin susceptible (ASEF), were studied in a university hospital (1,200 beds; serving a population of 600,000) in Spain, a country characterized by a near-absence of resistance to vancomycin and very high rates of ampicillin resistance among enterococci. Clonal relatedness by pulsed-field gel electrophoresis (PFGE), antibiotic susceptibility, presence of the virulence/epidemicity genes esp(Efm) and hyl(Efm), and identification of purK alleles were studied. A group of isolates was also analyzed by amplified fragment length polymorphism (AFLP) and multilocus sequence typing. Medical charts (30 variables collected) were reviewed for 60/84 patients. ASEF showed high clonal diversity (32 PFGE types, 11 purK alleles, 4 AFLP genogroups), did not harbor putative virulence genes, and had no specific association with hospital acquisition. AREF isolates belonged to a clonal complex (CC) of genetically related strains (purK-1, AFLP genogroup C), occasionally harboring putative virulence traits, and were from patients with particular risk factors. Within this CC, previously associated with vancomycin-resistant E. faecium isolates causing outbreaks worldwide (W. L. Homan et al., J. Clin. Microbiol. 40:1963-1971, 2002), a great genetic diversity of antibiotic resistance and virulence/epidemicity profiles was found. Associations between esp and a >7-day hospital stay and between purK-1, hospital location, and nosocomial acquisition were noted (P < 0.001). These findings reflect the importance of local environmental differences in the evolution of this CC, suggesting that the emergence of vancomycin resistance among AREF strains in Spain may be a question of time.