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[Background] Infections caused by multidrug-resistant gram-negative bacilli (MDR GNB), in particular extended-spectrum β-lactamase-producing (ESBL-E) and carbapenem-resistant Enterobacterales (CRE), pose a major threat in solid organ transplantation (SOT). Outcome prediction and therapy are challenging due to the scarcity of randomized clinical trials (RCTs) or well-designed observational studies focused on this population., [Methods] Narrative review with a focus on the contributions provided by the ongoing multinational INCREMENT-SOT consortium (ClinicalTrials identifier NCT02852902) in the fields of epidemiology and clinical management., [Results] The Spanish Society of Transplantation (SET), the Group for Study of Infection in Transplantation of the Spanish Society of Infectious Diseases and Clinical Microbiology (GESITRA-SEIMC), and the Spanish Network for Research in Infectious Diseases (REIPI) recently published their recommendations for the management of MDR GNB infections in SOT recipients. We revisit the SET/GESITRA-SEIMC/REIPI document taking into consideration new evidence that emerged on the molecular epidemiology, prognostic stratification, and treatment of post-transplant ESBL-E and CRE infections. Results derived from the INCREMENT-SOT consortium may support the therapeutic approach to post-transplant bloodstream infection (BSI). The initiatives devoted to sparing the use of carbapenems in low-risk ESBL-E BSI or to repurposing existing non-β-lactam antibiotics for CRE in both non-transplant and transplant patients are reviewed, as well as the eventual positioning in the specific SOT setting of recently approved antibiotics., [Conclusion] Due to the clinical complexity and relative rarity of ESBL-E and CRE infections in SOT recipients, multinational cooperative efforts such as the INCREMENT-SOT Project should be encouraged. In addition, RCTs focused on post-transplant serious infection remain urgently needed.

Spanish Network for Research in Infectious Diseases/Enterobacter spp. Bacteriemia Project Group., [Objectives] The genus Enterobacter is a common cause of nosocomial infections. Historically, the most frequent Enterobacter species were those of Enterobacter cloacae complex and Enterobacter aerogenes. In 2019, E. aerogenes was re-classified as Klebsiella aerogenes owing to its higher genotypic similarity with the genus Klebsiella. Our objective was to characterise and compare the clinical profiles of bacteraemia caused by E. cloacae and K. aerogenes., [Methods] This 3-year multicentre, prospective cohort study enrolled consecutive patients with bacteraemia by E. cloacae or K. aerogenes. Baseline characteristics, bacteraemia features (source, severity, treatment), antibiotic susceptibility, resistance mechanisms and mortality were analysed. bacteraemia had received more antibiotics., [Results] The study included 285 patients with bacteraemia [196 (68.8%) E. cloacae and 89 (31.2%) K. aerogenes]. The groups showed no differences in age, sex, previous use of invasive devices, place of acquisition, sources or severity at onset. The Charlson score was higher among patients with E. cloacae bacteraemia [2 (1–4) vs. 1 (0.5–3); P = 0.018], and previous antibiotic therapy was more common in patients with K. aerogenes bacteraemia (57.3% vs. 41.3%; P = 0.01). Mortality was 19.4% for E. cloacae and 20.2% for K. aerogenes (P = 0.869). Antibiotic susceptibility was similar for both species, and the incidence of multidrug resistance or ESBL production was low (6% and 5.3%, respectively), with no differences between species., [Conclusion] Bacteraemias caused by E. cloacae and K. aerogenes share similar patient profiles, presentation and prognosis. Patients with E. cloacae bacteraemia had more co-morbidities and those with K. aerogenes bacteraemia had received more antibiotics., This work was supported by the Instituto Carlos III [grant PS09/00916], Ministerio de Ciencia e Innovación, the Plan Nacional I+D+i 2013–2016 and Instituto Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases [REIPI, RD16/0016/0001; RD16/0016/0005; RD16/0016/0007; RD16/0016/0008; RD16/0016/0009], co-financed by the European Regional Development Fund ‘A way to achieve Europe’, Operative program Intelligent Growth 2014–2020.

Spanish Network for Research in Infectious Diseases/Enterobacter spp. Bacteriemia Project group., [Background] Enterobacter is among the main etiologies of hospital-acquired infections. This study aims to identify the risk factors of acquisition and attributable mortality of Enterobacter bacteremia., [Results] The study included 285 cases and 570 controls. E. cloacae was isolated in 198(68.8%) cases and E. aerogenes in 89(31.2%). Invasive procedures (hemodialysis, nasogastric tube, mechanical ventilation, surgical drainage tube) and previous antibiotics or corticosteroids were independently associated with Enterobacter bacteremia. Its attributable mortality was 7.8%(CI95%2.7–13.4%), being dissimilar according to a McCabe index: non-fatal=3.2%, ultimately fatal=12.9% and rapidly fatal=0.12%. Enterobacter bacteremia remained an independent risk factor for mortality among cases with severe sepsis or septic shock (OR 5.75 [CI95%2.57–12.87], p, [Conclusions] Invasive procedures, previous antibiotics and corticosteroids predispose to acquire Enterobacter bacteremia. This entity increases mortality among fragile patients and those with severe infections. Antibiotic resistances did not affect the outcome., This study was supported by the Instituto Carlos III, grant PS09/00916, Ministerio de Ciencia e Innovación, and the Plan Nacional I+D+i 2013-2016 and Instituto Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI, RD 16/0016/0001; RD 16/0016/0005; RD 16/0016/0007; RD 16/0016/0008; RD 16/0016/0009) – co – financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014-2020.

Correct and rapid identification of bacteria is essential for the correct diagnosis and treatment of infected patients. Until a few years ago, biochemical, colorimetric or even antibiotic sensitivity tests were used to identify genera and species. The main limitations of these methods were the time needed for their performance and the difficulty of distinguishing between microorganisms that were little reactive, highly similar, or difficult to culture. Many of these problems have been solved by the introduction of mass spectrometry (MS) in the laboratory with the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight). Knowledge of the strengths and weaknesses of this technology is essential to be able to take maximum advantage of this technique. Not all microorganisms can be identified with the same ease and reliability by MALDI-TOF and microbiologists need to know how to interpret the results obtained with this technique and the available alternatives in order to identify the microorganisms causing the most problems. This article aims to summarise the available information on the correct identification of the main human pathogenic bacteria through the use of MALDI-TOF MS, focusing on Gram-negative, Grampositive and anaerobic microorganisms. The main factors that must be taken into account for the reliable identification of any bacterium are the conditions for culture, sample preparation with the ideal extraction method and especially the use of a correct and updated database.

OBJECTIVES The objective of this study was to analyse whether there is an association between reduced susceptibility to biocides in Acinetobacter baumannii and (i) antimicrobial resistance (co-resistance), (ii) prevalent (epidemic) clones, (iii) changes in the fitness or (iv) expression of genes related to efflux pumps and porins. METHODS Susceptibility to biocides and antimicrobials was determined in 49 clonally unrelated isolates of A. baumannii. Biological cost, in terms of mean generation time, was determined by spectrophotometry. Quantitative real-time RT-PCR was used to determine the relative expression of genes encoding several efflux pumps and porins. RESULTS Reduced susceptibility to chlorhexidine digluconate, benzalkonium chloride and Irgasan(®) was associated with resistance to aminoglycosides, tetracycline and ciprofloxacin (P

The aim of this study was to determine the impact of carbapenemase-producing Enterobacteriaceae (CPE) in Spain in 2013 by describing the prevalence, dissemination, and geographic distribution of CPE clones, and their population structure and antibiotic susceptibility. From February 2013 to May 2013, 83 hospitals (about 40,000 hospital beds) prospectively collected nonduplicate Enterobacteriaceae using the screening cutoff recommended by EUCAST. Carbapenemase characterization was performed by phenotypic methods and confirmed by PCR and sequencing. Multilocus sequencing types (MLST) were determined for Klebsiella pneumoniae and Escherichia coli . A total of 702 Enterobacteriaceae isolates met the inclusion criteria; 379 (54%) were CPE. OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequent carbapenemases, and K. pneumoniae (74.4%), Enterobacter cloacae (10.3%), and E. coli (8.4%) were the species most affected. Susceptibility to colistin, amikacin, and meropenem was 95.5%, 81.3%, and 74.7%, respectively. The most prevalent sequence types (STs) were ST11 and ST405 for K. pneumoniae and ST131 for E. coli . Forty-five (54.1%) of the hospitals had at least one CPE case. For K. pneumoniae , ST11/OXA-48, ST15/OXA-48, ST405/OXA-48, and ST11/VIM-1 were detected in two or more Spanish provinces. ST11 isolates carried four carbapenemases (VIM-1, OXA-48, KPC-2, and OXA-245), but ST405 isolates carried OXA-48 only. A wide interregional spread of CPE in Spain was observed, mainly due to a few successful clones of OXA-48-producing K. pneumoniae (e.g., ST11 and ST405). The dissemination of OXA-48-producing E. coli is a new finding of public health concern. According to the susceptibilities determined in vitro , most of the CPE (94.5%) had three or more options for antibiotic treatment.

The in vitro activity of amikacin, gentamicin, kanamycin, tobramycin, neomycin, and netilmicin against 420 Escherichia coli producing extended spectrum β-lactamases (Ec-ESBLs) and 139 Klebsiella pneumoniae producing extended spectrum β-lactamase (Kp-ESBL) collected in two multicenter studies performed in Spain in 2000 and 2006 was determined. The presence of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methylases [aac(3)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6′)-Ib, ant(2")-Ia, ant(4′)-IIa, aph(3′)-Ia, aph(3′)-IIa, armA, rmtB, and rmtC] was also investigated. The resistance to (one or more) aminoglycosides was significantly higher in Kp-ESBL (104/139, 74.8%) than in Ec-ESBL (171/420, 40.7%; p

[Objectives] To evaluate the proficiency of Spanish microbiology laboratories with respect to the antimicrobial susceptibility testing (AST) of Acinetobacter spp., [Methods] Eight Acinetobacter spp. with different resistance mechanisms were sent to 48 Spanish centres which were asked to report: (i) the AST system used; (ii) MICs; (iii) breakpoints used (CLSI versus EUCAST); (iv) clinical category; and (v) resistance mechanisms inferred. Minor, major and very major errors (mE, ME and VME, respectively) were determined., [Results] The greatest percentages of discrepancies were: (i) by AST method: 18.5% Etest, 14.3% Vitek 2 and Sensititre; (ii) by breakpoints: 20.5% (CLSI) and 10.8% (EUCAST); and (iii) by antimicrobial agent: ampicillin/sulbactam (56.2% CLSI), minocycline (40.7% CLSI), tobramycin (38.7% CLSI, 16.8% EUCAST), imipenem (27.8% CLSI, 30.0% EUCAST) and meropenem (25.4% CLSI, 20.8% EUCAST). Categorical error rates: (i) by AST method ranged from 30.0% (Phoenix) to 100% (Sensititre and disc diffusion) for mE, 0.0% (Etest, Sensititre, disc diffusion) to 40% (Phoenix) for ME, and 0.0% (Sensititre and disc diffusion) to 30% (Phoenix) for VME; (ii) by breakpoints: mE (80.1% CLSI, 58.4% EUCAST), ME (3.5% CLSI, 12.4% EUCAST) and VME (16.4% CLSI, 29.2% EUCAST); and (iii) by antimicrobial agent: mE (100% levofloxacin/CLSI, 100% levofloxacin and meropenem/EUCAST), ME (35.3% colistin/CLSI, 25.0% colistin/EUCAST) and VME (64.7% colistin/CLSI, 86.7% gentamicin/EUCAST)., [Conclusions] Clinical microbiology laboratories must improve their ability to determine antimicrobial susceptibilities of Acinetobacter spp. isolates. Higher discrepancies using CLSI when compared with EUCAST are mainly due to mE and to a much lesser extent to ME or VME., This work was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (projects PI11-02046, PI10/00056 and PI12/00552) and the Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (P11-CTS-7730), Spain, by the Plan Nacional de I + D+i 2008–2011 and the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015)—co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF, and the Programa integral de prevención, control de las infecciones relacionadas con la asistencia sanitaria, y uso apropiado de los antimicrobianos (PIRASOA; Junta de Andalucía, Consejería de Salud, Junta de Andalucía).

Results: One hundred and one patients were included in the analysis; 68 (67.3%) received MT and 33 (32.7%) received CT. Pneumonia was the most common infection (50.5%), 68.6% of cases being associated with mechanical ventilation. Colistin (67.6%) and carbapenems (14.7%) were the most common drugs used in MT; colistin plus tigecycline (27.3%) and carbapenem plus tigecycline (12.1%) were the most frequent combinations. Crude 30 day mortality was 23.5% and 24.2% for the MT and CT groups, respectively (RR ¼ 1.03; 95% CI 0.49 – 2.16; P ¼ 0.94). Multivariate analysis of 30 day survival showed no trend towards reduced 30 day mortality with CT (HR ¼1.35; 95% CI 0.53–3.44; P ¼ 0.53). Subgroup analysis showed similar results. Conclusions: Our data do not support an association of CT with reduced mortality in MDRAB infections. More data for specific types of infection and combinations are needed.

Molecular typing methods are useful in the surveillance and control of nosocomial outbreaks because they can provide information on the clonal relatedness among isolates, identify reservoirs, and determine routes of transmission. The gold standard assay for molecular typing is pulsed-field gel electrophoresis (PFGE) due to its high discriminatory power. Some major disadvantages of PFGE include the high cost of the equipment, its labor intensiveness (the technique is not automated) and the time required to analyze the profiles of DNA bands (pulsotypes). Although there are many molecular typing methods based on polymerase-chain reaction (PCR), the most widely used is repetitive sequence-based PCR (REP-PCR). Most of the PCR techniques used for molecular typing have none of the limitations of PFGE as they are less expensive and labor intensive (some, such as bioMerieux's Diversilab system, are commercially available) and generate DNA profiles that are easier to interpret, depending on the microorganism. The discriminatory power of PCR is generally lower than or similar to that of PFGE. Both PFGE and PCR require optimal laboratory standardization to guarantee good reproducibility. PCR methods are preferable in the study of small, timelimited outbreaks. In more complex outbreaks of longer duration, in which clonal evolution and dynamics are studied, the use of PFGE is preferable. Molecular typing methods based on DNA sequencing, such as multilocus sequence typing, are applicable in global epidemiological studies or in analyses of the population structure of microorganisms.

REIPI/GEIH Study Group: A. Jover, F. Barcenilla, M. García (Hospital ‘Arnau de Vilanova’, Lleida, Spain); M. Pujol, O. Gasch, M.A. Domínguez, M. Camoez (Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain); C. Dueñas, E. Ojeda (Hospital Universitario de Burgos, Burgos, Spain); J. A. Martinez, F. Marco (Hospital Clinic, Barcelona, Spain); F. Chaves, M. Fernández-Ruiz, M. Lagarde, F. López-Medrano, J. M. Aguado (Hospital Universitario ‘12 de Octubre’, Madrid, Spain); J. M. Montejo, E. Bereciartua, J. L. Hernández (Hospital de Cruces, Barakaldo, Spain); M. A. Von Wichmann, A. Goenaga, J. M. García-Arenzana (Hospital de Donostia, Donostia, Spain); B. Padilla, C. Padilla, E. Cercenado (Hospital General Universitario ‘Gregorio Marañón’, Madrid, Spain); G. García-Pardo, J. Tapiol (Hospital Universitari ‘Joan XXIII’, Tarragona, Spain); J. P. Horcajada, M. Montero, M. Salvadó (Hospital Universitari del Mar, Barcelona, Spain); A. Arnáiz, C. Fernández (Hospital Universitario ‘Marqués de Valdecilla’, Santander, Spain); E. Calbo, M. Xercavins (Hospital Universitari Mútua de Terrassa, Terrassa, Spain); A. Granados, D. Fontanals (Corporació Sanitària Parc Taulí, Sabadell, Spain); V. Pintado, E. Loza (Hospital Universitario ‘Ramón y Cajal’, Madrid, Spain); J. Torre-Cisneros, R. Lara, F. Rodríguez-López, M. Rodríguez, C. Natera (Hospital Universitario ‘Reina Sofía’, Córdoba, Spain); J. R. Blanco, I. Olarte (Hospital Universitario ‘San Pedro’, Logroño, Spain); N. Benito, B. Mirelis (Hospital Universitari de la Santa Creu i Sant Pau, Barcelona, Spain); J. Murillas, E. Ruiz de Gopegui (Hospital Universitari Son Dureta, Palma de Mallorca, Spain); H. Espejo, M. A. Morera (Hospiyal de Terrassa, Terrassa, Spain); J. Rodríguez-Baño, E. López, A. Pascual (Hospital Universitario ‘Virgen Macarena’, Sevilla, Spain); C. Martín, J. A. Lepe, J. Molina (Hospital Universitario ‘Virgen del Rocío’, Sevilla, Spain); R. Sordé, B. Almirante, N. Larrosa (Hospital Universitari Vall d’Hebrón, Barcelona, Spain)., [Background] It has been suggested that there is an increased risk of treatment failure in episodes of MRSA bloodstream infection (BSI) caused by strains with high vancomycin MICs. However, it is unknown if this phenomenon may also act as a risk factor for the development of infective endocarditis (IE)., [Methods] We analysed 207 episodes of catheter-related (CR)-BSI recruited from June 2008 to December 2009 within a prospective study on MRSA BSI in 21 Spanish hospitals. Vancomycin susceptibility was centrally tested. The impact of high vancomycin MIC values (≥1.5 mg/L by Etest) on the subsequent development of IE was investigated by Cox regression., [Results] High vancomycin MIC values were observed in 46.9% of the isolates. Initial therapy consisted of vancomycin [99 episodes (44.7%)], daptomycin [25 (12.1%)], linezolid [18 (8.7%)] and other antistaphylococcal agents [16 (7.7%)]. Haematogenous complications occurred in 41 patients (19.8%), including 10 episodes complicated by IE. Early (48 h) and late (30 day) all-cause mortality were 3.4% and 25.1%, respectively. High vancomycin MIC isolates were more common among patients that developed IE compared with those free from this complication [90.9% (9/10) versus 44.7% (88/197); P = 0.007]. This association remained significant after adjusting for multiple confounders (including initial antibiotic therapy and catheter removal) in different models (minimum hazard ratio: 9.18; 95% CI: 1.16–72.78; P = 0.036). There were no differences in mortality according to vancomycin MIC values., [Conclusions] Decreased susceptibility to vancomycin acted as a predictor of the development of IE complicating MRSA CR-BSI., This study was co-funded by the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa [co-financed by the European Development Regional Fund (ERDF) ‘A way to achieve Europe’] and by the Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015). M. F. R. holds a clinical research contract ‘Juan Rodés’ (JR14/00036) from the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III.

18 p.-4 fig.-6 tab. López, María et al., Acinetobacter baumannii is an important pathogen that causes nosocomial infections generally associated with high mortality and morbidity in Intensive Care Units (ICUs). Currently, little is known about the Quorum Sensing (QS)/Quorum Quenching (QQ) systems of this pathogen. We analyzed these mechanisms in seven clinical isolates of A. baumannii. Microarray analysis of one of these clinical isolates, Ab1 (A. baumannii ST-2_clon_2010), previously cultured in the presence of 3-oxo-C12-HSL (a QS signalling molecule) revealed a putative QQ enzyme (α/ß hydrolase gene, AidA). This QQ enzyme was present in all non-motile clinical isolates (67% of which were isolated from the respiratory tract) cultured in nutrient depleted LB medium. Interestingly, this gene was not located in the genome of the only motile clinical strain growing in this medium (A. baumannii strain Ab421_GEIH-2010 [Ab7], isolated from a blood sample). The AidA protein expressed in E. coli showed QQ activity. Finally, we observed downregulation of the AidA protein (QQ system attenuation) in the presence of H2O2 (ROS stress). In conclusion, most of the A. baumannii clinical strains were not surface motile (84%) and were of respiratory origin (67%). Only the pilT gene was involved in surface motility and related to the QS system. Finally, a new QQ enzyme (α/ß hydrolase gene, AidA protein) was detected in these strains., This study was funded by grant PI13/02390 awarded to MT within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate of evaluation and Promotion of Research - European Regional Development Fund "A way of Making Europe" and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015).

Resumen Objetivos Determinar las tasas de resistencia antimicrobiana en Acinetobacter baumannii (A. baumannii) (proyecto GEIH-REIPI-Ab 2010) en Espana y compararlas con las obtenidas en el primer estudio realizado en 2000 (proyecto GEIH-Ab 2000). Metodos Se incluyen 446 aislados de A. baumannii obtenidos en 43 hospitales espanoles entre febrero y marzo de 2010. La identificacion de especie se realizo mediante ARDRA y MALDI-TOF. Se evaluaron 18 antimicrobianos mediante microdilucion en caldo (normativa del Clinical and Laboratory Standards Institute [CLSI]). Se aplicaron los puntos de corte establecidos por el CLSI, excepto para doripenem, rifampicina y sulbactam, en los que se utilizaron los puntos de corte establecidos por la Societe Francaise de Microbiologie (SFM), y tigeciclina (puntos de corte establecidos por el European Committee on Antimicrobial Susceptibility Testing [EUCAST]). Resultados Los porcentajes de aislados resistentes (se incluyeron tambien los de sensibilidad intermedia) fueron: > 94% (ceftacidima, piperacilina y ciprofloxacino), 82-86% (carbapenemicos y tetraciclina), 60-70% (tobramicina, sulbactam, gentamicina y doxiciclina), 49% (amicacina), 30% (minociclina, rifampicina), 24% (tigeciclina) y 3% (colistina). Estos aislados fueron, en comparacion con los del primer estudio, mas resistentes (p Conclusion Hay una elevada prevalencia de A. baumannii resistente a antimicrobianos, particularmente a los carbapenemicos. Se ha producido un incremento en la resistencia a los carbapenemicos, ceftacidima y sulbactam mientras que para los aminoglucosidos, tetraciclina y rifampicina las resistencias han disminuido. Colistina continua siendo el antimicrobiano con mayor actividad.

Resumen Introduccion Las variantes de colonia pequena de Staphylococcus aureus (VCPSA) constituyen una subpoblacion con caracteristicas especiales. Metodos Se estudio el comportamiento fenotipico y la sensibilidad antibiotica de 4 aislados clinicos de VCPSA. Resultados Las colonias crecieron en los medios de cultivo habituales excepto en Mueller Hinton. Todos los aislados fueron resistentes a ciprofloxacino y cotrimoxazol. Discusion Las VCPSA son aisladas con baja frecuencia, y es necesario conocer los metodos optimos para su identificacion y el estudio de su sensibilidad antibiotica.

Introduccion Durante el ano 2000 se llevo a cabo el primer estudio nacional de Escherichia coli y Klebsiella pneumoniae productoras de betalactamasas de espectro extendido (BLEE). En 2006, los autores de este articulo desarrollaron el segundo estudio nacional para conocer la evolucion de este problema en Espana. Metodo Estudio prospectivo multicentrico sobre aislados consecutivos de E. coli y K. pneumoniae productoras de BLEE en 44 hospitales espanoles entre febrero y marzo de 2006. En el centro coordinador se comprobo la identificacion y se confirmo la produccion de BLEE segun indicaciones del Clinical and Laboratory Standards Institute. Resultados Se recogieron 1.021 cepas de E. coli y 162 cepas de K. pneumoniae. Se aislaron cepas de E. coli productora de BLEE en los 44 hospitales participantes y cepas de K. pneumoniae productora de BLEE en 34 hospitales. El porcentaje de produccion de BLEE entre las cepas de E. coli y K. pneumoniae fue del 4,04% (rango de 0,4 a 20,3) y del 5,04% (rango de 0 a 30), respectivamente. Entre los casos de E. coli productora de BLEE, la adquisicion se considero como comunitaria estricta en el 32%, se considero relacionada con los cuidados sanitarios en el 37% y se considero nosocomial en el 29%. En los casos de K. pneumoniae productora de BLEE, la adquisicion se considero como comunitaria estricta en el 10%, se considero relacionada con los cuidados sanitarios en el 18% y se considero nosocomial en el 68% (p Conclusiones Desde el ano 2000, el porcentaje de aislados de E. coli y K. pneumoniae productoras de BLEE en Espana se ha multiplicado por 8 y por 2, respectivamente. El aumento de E. coli productora de BLEE en Espana se debe principalmente a cepas aisladas de pacientes no hospitalizados, en su mayoria de origen urinario.

The ability of 57 Spanish microbiology laboratories in detecting and reporting β-lactam resistance phenotypes in Escherichia coli and Klebsiella pneumoniae was evaluated. Laboratories received 6 well-characterized isolates expressing the most widespread extended-spectrum β-lactamases (ESBLs) in Spain (4 CTX-M type, 1 TEM type, and 1 SHV type), 3 isolates producing AmpC-type enzymes (2 plasmid mediated and 1 E. coli hyperproducing its chromosomal AmpC), and 3 quality control strains. Ninety-one percent of laboratories recognized all ESBL producers correctly, and therefore, low error rates were observed when testing cephalosporins and aztreonam. The highest error rates were observed with combinations of penicillin plus β-lactamase inhibitor, although more than 60% of cases were due to the interpretation made by the microbiologists. Correct recognition of all AmpC β-lactamase–producing strains occurred in only 47.4% of laboratories. These isolates were wrongly reported as ESBL producers and penicillinase hyperproducers in 7.6 % and 5.8% of cases, respectively. Detection of the AmpC-type phenotype by Spanish laboratories needs to be improved.

Introduccion Se analizo la distribucion epidemiological de los diferentes tipos de betalactamasas de espectro extendido (BLEE) en Escherichia coli y Klebsiella pneumoniae en distintos hospitales de Espana y se comparo con estudios previos. Metodos En 11 hospitales espanoles se recogieron los 15 primeros aislamientos de E. coli y los 5 primeros de K. pneumoniae con sospecha de ser portadores de BLEE, aislados en el primer trimestre de 2004. Los estudios de clonalidad se realizaron mediante electroforesis en gel de campos pulsantes (PFGE) tras digestion del ADN total con XbaI y mediante ERIC-PCR ( Enterobacterial Repetitive Intergenic Concensus Secuences-Polimerase Chain Reaction ). Las BLEE se caracterizaron mediante isoelectroenfoque, PCR y secuenciacion. Resultados Se estudiaron 124 aislamientos. El analisis de los patrones de restriccion obtenidos por PFGE mostro una gran diversidad clonal entre los aislamientos de E. coli , observandose cuatro agrupaciones de dos cepas en cada una de ellas. En las 92 cepas de E. coli , la caracterizacion de las BLEE mostro un predominio de CTX-M-14 (45,7%), CTX-M-9 (20,6%) y SHV-12 (21,7%). En las 32 cepas de K. pneumoniae se observo una menor diversidad clonal, detectandose tres agrupaciones que incluian el 53,1% de los aislamientos. Las BLEE detectadas en estas cepas fueron del tipo CTX-M en 20 casos (62,5%) (CTX-M-1, CTX-M-9, CTX-M-14 y CTX-M-15), de tipo SHV en 11 (34,4%) (SHV-12 y SHV-5) y TEM-4 (3,1%) en una. Conclusion Las cepas de E. coli y K. pneumoniae analizadas en ese periodo presentan una mayor diversidad de BLEE que la observada en estudios epidemiologicos realizados con anterioridad. Ademas, el analisis de la relacion clonal definio una gran diversidad en E. coli y menor en K. pneumoniae .

INTRODUCTION. Since 1986 we have carried out five nationwide point-prevalence studies in Spain analyzing Staphylococcus spp. The 2006 data, corresponding to the sixth study, are presented herein. METHODS. A total of 145 hospitals from all geographic areas of the country participated in the study. We investigated 866 staphylococcal isolates (463 S. aureus). Antimicrobial susceptibility testing was performed against 16 antimicrobials by an automated microdilution method. Susceptibility to tigecycline was determined by the E-test method. RESULTS. Resistance of S. aureus to oxacillin seemed to have stabilized (31.2% in 2002 vs. 29.2% in 2006), and the same was true for resistance to erythromycin, clindamycin and ciprofloxacin. In 2006, isolates were more susceptible to gentamicin (16.9% resistance in 2002 vs. 8.6% in 2006, P < 0.001). None of the isolates presented decreased susceptibility to vancomycin, and the resistance to cotrimoxazole (0.9%) and rifampin (0.6%) was minimal. One isolate showed linezolid resistance. Resistance of coagulase negative staphylococci to oxacillin (61.3% in 2002 vs. 66.7% in 2006) and erythromycin (63.0% in 2002 vs. 66.5% in 2006) remained stable, although resistance to gentamicin (27.8% in 2002 vs. 44.2% in 2006, P < 0.001), ciprofloxacin (44.9% in 2002 vs. 54.3% in 2006, P = 0.010) and clindamycin (33.8% in 2002 vs. 46.2% in 2006, P = 0.001) has increased. Two isolates presented decreased susceptibility to teicoplanin and one was linezolid-resistant. All Staphylococcus spp. were uniformly susceptible to quinupristin-dalfopristin and tigecycline. CONCLUSIONS. Resistance of Staphylococcus spp. to oxacillin remains high in Spain, but seems to have stabilized in the last years. Linezolid resistance is emerging.

Clonal dissemination of extended-spectrum β-lactamases (ESBL) in 170 Escherichia coli isolates and 70 Klebsiella pneumoniae isolates from a nationwide study of 40 Spanish centers in 2000 was not observed in most centers. The most prevalent ESBL were CTX-M-9 (27.3%), SHV-12 (23.9%), and CTX-M-14 (20.5%) for E. coli and TEM-3 (16.7%) and TEM-4 (25%) for K. pneumoniae . A new ESBL, TEM-133, with mutations L21F, E104K, and R164S, was identified.

A fastidious, slowly growing, strictly aerobic, gram-negative bacterium was isolated from a culture of blood from a 25-year-old man with common variable immunodeficiency. The man had been admitted to hospital with febrile progressive cerebellar ataxia. The use of standard phenotypic schemes did not lead to identification, but sequence analysis demonstrated that the 16S rRNA gene of the isolate was most similar to those of the environmental bacteria Duganella zoogloeoides (formerly Zoogloea ramigera 115) and Telluria mixta. Further characterization of the bacterium by biochemical analysis, electron microscopy, G+C content estimation, and fatty acid analysis demonstrated significant differences between the bacterium and D. zoogloeoides and Telluria species; thus, we propose it as a new taxon with the name Massilia timonae gen. nov., sp. nov.

Introduction To describe demographic aspects of a prospective multicentric nationwide study on Escherichia coli and Klebsiella pneumoniae producing extended spectrum b -lactamases (ESBL) in Spain, between March and June 2000. Methods 352 E. coli and K. pneumoniae strains showing a phenotype compatible to ESBL production were collected from 40 Spanish Hospitals. A form with demographic and clinical data was filled for every isolate. Identification and susceptibility testing (microdilution, NCCLS guidelines) were performed in a reference centre. RESULTS. 240 strains (170 E. coli and 70 K. pneumoniae) produced ESBL. E. coli ESBL (+) strains were isolated in 33 out of 40 participating hospitals, whereas K. pneumoniae were isolated in 17 hospitals. E. coli ESBL (+) prevalence ranged from 0% to 2.4% and K. pneumoniae ESBL (+) from 0% to 16.7%. Fifty-one percent of E. coli ESBL (+) were from outpatients, whereas 93% of K. pneumoniae ESBL (+) were from inpatients. E. coli ESBL (+) were mainly collected from Medicine and Surgery wards and K. pneumoniae from ICU and Paediatrics. ESBL (+) strains were isolated more frequently from urine samples. Patients' ages ranged from 0 to 93 years for E. coli (median 58 years) and 0 to 83 years for K. pneumoniae (median: 1 year). Paediatric cases excluded, these median values were 64 and 53 years, respectively. Conclusions E. coli and K. pneumoniae ESBL (+) were identificated in 90% of the participating hospitals. Although E. coli was more frequently isolated than K. pneumoniae, production of ESBL was more prevalent in K. pneumoniae. Fifty-one percent of ESBL producing E. coli were isolated from outpatients.

Acinetobacter baumannii is one of the most important antibiotic-resistant nosocomial bacteria. We investigated changes in the clinical and molecular epidemiology of A. baumannii over a 10-year period. We compared the data from 2 prospective multicenter cohort studies in Spain, one performed in 2000 (183 patients) and one in 2010 (246 patients), which included consecutive patients infected or colonized by A. baumannii. Molecular typing was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The incidence density of A. baumannii colonization or infection increased significantly from 0.14 in 2000 to 0.52 in 2010 in medical services (p

Molecular typing methods are useful in the surveillance and control of nosocomial outbreaks because they can provide information on the clonal relatedness among isolates, identify reservoirs, and determine routes of transmission. The gold standard assay for molecular typing is pulsed-field gel electrophoresis (PFGE) due to its high discriminatory power. Some major disadvantages of PFGE include the high cost of the equipment, its labor intensiveness (the technique is not automated) and the time required to analyze the profiles of DNA bands (pulsotypes). Although there are many molecular typing methods based on polymerase-chain reaction (PCR), the most widely used is repetitive sequence-based PCR (REP-PCR). Most of the PCR techniques used for molecular typing have none of the limitations of PFGE as they are less expensive and labor intensive (some, such as bioMérieux's Diversilab system, are commercially available) and generate DNA profiles that are easier to interpret, depending on the microorganism. The discriminatory power of PCR is generally lower than or similar to that of PFGE. Both PFGE and PCR require optimal laboratory standardization to guarantee good reproducibility. PCR methods are preferable in the study of small, timelimited outbreaks. In more complex outbreaks of longer duration, in which clonal evolution and dynamics are studied, the use of PFGE is preferable. Molecular typing methods based on DNA sequencing, such as multilocus sequence typing, are applicable in global epidemiological studies or in analyses of the population structure of microorganisms.

Objectives To determine the activity of oritavancin against methicillin-resistant staphylococci, vancomycin-resistant enterococci (VRE) and β-haemolytic streptococci recently isolated from acute bacterial skin and skin structure infections or bacteraemia in western Europe. Methods Forty-one centres in Spain (8), Italy (9), Germany (8), France (8) and the UK (8) submitted 866 isolates [204 methicillin-resistant Staphylococcus aureus (MRSA), 177 methicillin-resistant coagulase-negative staphylococci (MRCoNS), 101 VRE, 193 Streptococcus agalactiae and 191 Streptococcus pyogenes] that were collected during the first 6 months of 2011. These were re-identified and susceptibilities to oritavancin and comparators were determined. Results Oritavancin was very active against MRSA (MIC50/MIC90 0.03/0.06 mg/L), MRCoNS (0.06/0.12 mg/L), VRE (0.03/0.06 mg/L), S. agalactiae (0.03/0.06 mg/L) and S. pyogenes (0.06/0.25 mg/L). The highest oritavancin MIC observed was 0.25 mg/L (species were S. aureus, Staphylococcus epidermidis, Staphylococcus hominis, S. agalactiae, S. pyogenes and Enterococcus faecalis). Conclusions These data from recently collected Gram-positive bacteria in western Europe confirm the potent in vitro activity of oritavancin against a wide range of resistant MRSA, MRCoNS and VRE isolates, including ones resistant to newer agents.

Introduction: Control of Acinetobacter baumannii is a challenge. Methods: A survey was conducted on the control measures introduced against A baumannii in 30 Spanish hospitals. Results: We found significant differences in the application of contact precautions, active surveillance, hygiene of colonised patients, environmental cleaning, and educational activities. Hospitals with a written control program for A. baumannii had a lower incidence of colonisation/infection due to this organism. Conclusion: A multidisciplinary consensus document for the control of A. baumannii is needed in Spain.

The prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in Spain increased 8-fold from 2000 to 2006. ESBL type, clonal relationship, antimicrobial susceptibility, and clinical data about infections caused by ESBLEC are evaluated in a second nationwide study developed in 2006. From 1008 clinical isolates obtained over 2 months from 44 hospitals, 254 were used for further analysis. ESBL production was evaluated by synergy testing, PCR, and sequencing. Antimicrobial activity was evaluated by microdilution. The clonal relationship was evaluated by repetitive extragenic palindromic-PCR (REP-PCR). The O25b subtype and the new afa operon FM955459 were determined by triplex PCR in isolates producing CTX-M-15. Multilocus sequence typing was performed on these isolates. A total of 72% of all ESBLs were of the CTX-M type, 26.8% were of the SHV type, and 1.2% were of the TEM type. The most prevalent ESBLs were CTX-M-14 (119 isolates), SHV-12 (68 isolates), CTX-M-15 (37 isolates), and CTX-M-9 (21 isolates). By REP-PCR, 214 clones were detected. All but five CTX-M-15 ESBLEC isolates corresponded to the international O25b/ST131 clone. This clone had not been detected in the first study (published in 2000). Epidemiological and clinical features were studied in 304 representative patients. A total of 60% of the patients were older than 60 and had nonfatal underlying diseases, and 55% had recently received antibiotics. Urinary tract infections accounted for 71% of cases, and 9% were bacteremic. There has been a significant increase in the prevalence of ESBLEC in Spain, with most of these strains being CTX-M-producing isolates, including the pandemic O25b-ST131. SHV-12-producing E. coli remains an important cause of community-acquired infection.

En las últimas décadas, las mayores innovaciones en el campo de la terapéutica se han centrado en el desarrollo de elementos protésicos, terapia inmunosupresiva de enfermedades malignas y agentes antimicrobianos de amplio espectro para la terapia de las enfermedades infecciosas. Sin embargo, como respuesta negativa ante esta serie de avances terapéuticos, han surgido un número de patógenos nosocomiales, considerados previamente de escasa o nula virulencia en los humanos. A esta línea, que incluye entre otros Serratia marcescens, Aspergilus fumigatus, Mycobacterium fortuitum… tenemos que añadir estafilococo coagulasa negativo (ECN). Los tiempos en que el aislamiento de ECN en cultivo de exudados de heridas, sangre y catéteres era considerado automáticamente como contaminación, han terminado definitivamente y en la actualidad se le reconoce como un importante agente etiológico de infecciones en pacientes con catéteres intravenosos, bacteriemia en neutropénicos, endocarditis sobre válvulas protésicas e infecciones en shunts de derivación en hidrocefalias, entre otras. La taxonomía del género Staphylococcus ha sido renovada continuamente en los últimos años. En su inicio, el género Staphylococcus se dividió en función de la producción de la enzima coagulasa desde el momento en que se observó la relación entre la capacidad de producir este enzima y su poder patógeno en humanos. De esta manera, se diferenció una especie productora de coagulasa y por tanto patógena en humanos a la que se denominó S. aureus y el resto de especies que no poseían la capacidad de producirla y por lo tanto, sin aparente poder patógeno, fueron agrupadas como estafilococos coagulasa negativo, denominación que ha continuado hasta nuestros días. A partir del reconocimiento general de su papel en patología humana, la taxonomía de ECN, ha sufrido importantes modificaciones, de tal manera, que los excelentes estudios de Baird-Parker, que solamente reconocían dos especies, S. epidermidis y S. saprophyticus, han quedado relegados en la actualidad. Posteriormente, Kloos y Schleifer, en función de estudios genéticos y bioquímicos, distinguen nueve especies diferenciadas. En la actualidad se admite la existencia de hasta 15 especies de ECN, aunque continuamente se proponen nuevas especies. Clásicamente, sólo dos de ellas tenían importancia clínica: S. epidermidis y S. saprophyticus. El primero de ellos se considera el agente causal del 75% de las infecciones atribuidas a ECN. S. saprophyticus es productor de infecciones del tracto urinario, predominantemente en mujeres jóvenes. Otras especies incluyen: S. auricularis, S. capitis, S. cohnii, S. haemolyticus, S. hominis, S. lentus, S. sciuri, S. simulans, S. warneri, S. xylosus, S. intermedius y S. hyicus, que cada vez se van conociendo mejor. En las últimas décadas, ECN ha emergido como patógeno significativo, particularmente en pacientes que portan material protésico y catéteres intrasvasculares. Últimamente, las septicemias por ECN se reconocen como una complicación frecuente y problemática en recién nacidos prematuros, en los que se utilizan con frecuencia prótesis intravasculares para monitorización y nutrición. En adultos, las tromboflebitis y septicemias producidas por ECN, son complicaciones bien conocidas del uso de catéteres intravenosos, y aunque las consecuencias suelen ser menos graves que en neonatos, a veces también suponen una importante complicación. Christensen y cols. encontraron a S. epidermidis como agente causal de una epidemia de septicemia nosocomial de 20 meses de duración, relacionada con el uso de catéteres de nutrición parenteral en Estados Unidos. Otras epidemias relacionadas al uso de catéteres intravenosos han sido descritas en Estados Unidos, Canadá y países europeos. ECN y en particular S. epidermidis ha sido descrito como el agente etiológico más importante de peritonitis en pacientes que utilizan diálisis peritoneal crónica ambulatoria debido principalmente al uso de catéteres plásticos de diálisis, siendo el causante del 17 al 38% de los episodios. El incremento de las infecciones por ECN relacionadas con el uso d catéteres plásticos, viene determinada por una serie de circunstancias de las que detallaremos las más importantes. En primer lugar, hay una tendencia, cada vez más acentuada, a utilizar nutrición parenteral en pacientes graves y por largos periodos de tiempo. Para este tipo de alimentación, se utilizan vías plásticas que a veces se mantienen durante semanas. Paralelamente, cada vez es mayor el número de pacientes que se benefician de la diálisis peritoneal crónica ambulatoria y por periodos de tiempo más prolongados. En segundo lugar, al ser ECN flora residente de la piel y puesto que los catéteres se insertan a través de ella, serán los principales contaminantes involucrados en estos procedimientos. Una vez introducido, se forma alrededor del catéter un manguito de fibrina que favorece la emigración de las bacterias desde el exterior al interior. En tercer lugar, los sistemas de nutrición parenteral se utilizan frecuentemente en pacientes graves ingresados en zonas de fuerte presión antibiótica, lo que permite la colonización por cepas multirresistentes de ECN. Finalmente, es importante resaltar la especial afinidad de ECN por colonizar las superficies de catéteres. Diversos autores, han observado “in vitro” alteraciones erosivas en la superficie del catéter tras la interacción con ECN. Por otro lado, se ha descrito en cepas de ECN la producción de un polisacárido extracelular de naturaleza mucoide, al que se atribuyen propiedades de adherencia y protección frente a la fagocitosis por leucocitos polimorfonucleares. Estos hallazgos, podrían explicar parcialmente, la gran afinidad de ECN por el material protésico así como la gran dificultad para la erradicación de las infecciones producidas por estas bacterias, si no se retira previamente el cuerpo extraño. Respuesta del huésped frente a estafilococo coagulasa negativo. Con respecto a la interacción entre estafilococo y los factores de defensa del huésped, la piel y las membranas mucosas juegan un importantísimo papel de protección. Sin embargo, incluso en individuos sanos, estas barreras se rompen frecuentemente ofreciendo una puerta de entrada para estafilococos y otros habitantes de la piel que les permite alcanzar los tejidos subyacentes. En este momento, entra en funcionamiento la segunda línea de defensa, que está representada por el sistema de opsoninas y las células fagocíticas (leucocitos polimorfonucleares y monocitos). Una adecuada opsonización es un requisito indispensable para la ingestión por las células fagocíticas de estafilococo, demostrado por el hecho de que pacientes con defectos en el sistema de opsoninas sufren infecciones estafilocócicas recurrentes. El sistema de opsoninas está formado por unas opsoninas termoestables representadas por la Inmunoglobulina G (IgG) principalmente y unas opsoninas termolábiles representadas por la fracción C3b del complemento, que se unen a receptores específicos de la superficie bacteriana desencadenando el proceso de la fagocitosis una vez que entran en contacto con las células fagocíticas. En el suero de individuos sanos, existen anticuerpos tipo IgG con capacidad opsonizante dirigidas frente a componentes de peptidoglicano y ácidos teicoicos de estafilococo que se pueden detectar si se utiliza una técnica suficientemente sensible (ELISA, Radioimunoanálisis). En pacientes con infección estafilocócica severa, tal como endocarditis, se detectan frecuentemente, niveles elevados de estos anticuerpos, que pueden ayudar en el diagnóstico de estas infecciones cuando los cultivos permanecen negativos. Dentro de éste sistema de opsoninas termoestables, los anticuerpos tipo IgG dirigidos frente al peptidoglicano tienen mucha mayor capacidad opsonizante que aquellos dirigidos frente a los ácidos teicoicos. Sin embargo, actualmente existen evidencias de que el sistema de opsoninas termolábiles, representado por la fracción C3 del complemento, juega un papel de mucha relevancia que las inmunoglobulinas en la fagocitosis de estafilococo. En ese sentido, el peptidoglicano de estafilococo es capaz de activar el complemento a través de la vía alternativa, activación por lo tanto no dependiente de anticuerpos, que se traduce en la aposición de moléculas de C3b opsónicamente activas en la envuelta celular. Una vez opsonizado, estafilococo es fácilmente reconocido e ingerido por las células fagocíticas, especialmente por los leucocitos polimorgonucleares. En la muerte intracelular de estafilococo, actúan dos sistemas bactericidas de los leucocitos polimorfonucleares, un sistema independiente del oxígeno y representado por el contenido lisosomal (lisozima, proteínas catiónicas, lactoferrina...) y otro dependiente de oxígeno que se traduce en la producción de radicales tóxicos del mismo (radical superóxido (O-2), hidróxilo (OH) etc…). La importancia de los radicales tóxicos de oxígeno en la muerte intracelular de estafilococo se pone de manifiesto en los pacientes con Enfermedad Crónica Granulomatosa. Los granulocitos de estos pacientes son incapaces de generar estos radicales y su capacidad bactericida frente a estafilococo está muy reducida. Este fenómeno, demostrable fácilmente “in vitro”, queda demostrado “in vivo” por el hecho de que estos pacientes sufren frecuentemente infecciones estafilocócicas recurrentes, aunque la severidad y la tasa de recurrencia varía ampliamente entre los distintos pacientes. Llegado a este punto, debemos enfatizar el hecho de que para la mayoría de estos estudios sobre las defensas del huésped frente a estafilococo, se han utilizado cepas de S. aureus y sólo ocasionalmente algunas cepas de ECN. Anteriormente mencionábamos cómo numerosos investigadores han demostrado la importancia de las opsoninas para una adecuada fagocitosis de estafilococo. Sin embargo, en sus trabajos empleaban normalmente, bacterias suspendidas en un medio acuoso. Si tenemos en cuenta la especial afinidad de ECN por adherirse a material plástico como el utilizado en la fabricación de catéteres, sería interesante preguntarnos cómo actuarían los mecanismos de defensa del huésped ante estas bacterias adheridas a una superficie y qué papel desarrollaría el sistema de las opsoninas. El concepto de fagocitosis de “superficie”, de gran interés actual por los problemas mencionados anteriormente, fue descrito e introducido en 1946 por Woos y cols. Estos autores afirmaban que la fagocitosis de Streptococcus pneumoniae en los alveolos pulmonares era posible en ausencia de opsoninas cuando las bacterias estaban “atrapadas” en una superficie, lo que facilitaba la ingestión por parte de los macrófagos alveolares en una zona pobre de opsoninas. En la década actual, resurge el interés por la fagocitosis de superficie, pero se presta muy escasa atención al problema en relación con estafilococo. Solamente los estudios de Lee y cols. y Vandenbroucke y cols. han demostrado que la fagocitosis de S. aureus adheridos a superficie plástica. Sin embargo, no han sido realizados hasta la fecha estudios similares utilizando cepas de ECN, que es el agente etiológico más importante en infecciones asociadas a catéteres y material protésico. Otro problema al que se ha prestado escasa atención ha sido a la adherencia ECN al material plástico. Diferentes autores se atienen exclusivamente a estudios descriptivos utilizando microscopía electrónica. Otra serie de autores han demostrado que la adherencia a catéteres plásticos está asociada a la producción de flebitis infecciosa en modelos experimentales. Sin embargo, existe escasa información sobre la verdadera naturaleza de la unión de las bacterias a las superficies plásticas y los mecanismos inespecíficos que en ella actúan. Se postula la importancia de las fuerzas hidrofóbicas en la adherencia de estafilococo a superficies altamente hidrófobas como las utilizadas en la fabricación de biomateriales tales como el teflón…. Sin embargo, aunque la hidrofobicidad puede promover la adherencia, son necesarios un mayor número de estudios para confirmar tal aseveración. Valorando todos los problemas expuestos anteriormente y teniendo en cuenta la ausencia de estudios en este campo en el que se hayan utilizado gran número de cepas aisladas de muestras clínicas, fue por lo que decidimos la realización de este trabajo, con la intención de obtener una información concreta sobre la patogénesis de las infecciones producidas por ECN y especialmente aquellas relacionadas con el uso de catéteres plásticos. La hipótesis de trabajo se basa en el estudio de los siguientes puntos: 1. Identificación en especies de 38 cepas de ECN aisladas en procesos patológicos infecciosos en los que tenían un papel etiológico. 2. Estudio de las propiedades de superficie de estas cepas que se relacionan directamente con la colonización de catéteres: producción de sustancia mucoide e hidrofobicidad de superficie. 3. influencia de las propiedades de superficie de ECN en el metabolismo del leucocito polimorfonuclear durante la fagocitosis. Alteraciones cuantitativas en la emisión de quimioluminiscencia celular. 4. Modulación de la hidrofobicidad bacteriana por diferentes fuentes opsónicas y diversos tratamientos de las mismas. 5. Estudio de los requerimientos opsónicos de ECN suspendido en medio acuoso. Fagocitosis por leucocitos polimorfonucleares humanos utilizando bacterias marcadas por isótopos radioactivos. Papel de la hidrofobicidad y de la sustancia mucoide. 6. Estudio de los requerimientos opsónicos y fagocitosis de ECN utilizando bacterias adheridas a superficie plástica. Comparación con cepas de S. aureus. Papel de la hidrofobicidad y producción de sustancia mucoide. 7. Estudio cuantitativo de la adherencia de ECN a catéteres intravenosos de teflón y poliuretano. Papel que juegan las propiedades de superficie bacterianas. 8. Modulación de la adherencia bacteriana a catéteres. Papel de diferentes componentes del suero.

It has been evaluated the effect of norfloxacin, ofloxacin, levofloxacin, fleroxacin and lomefloxacin of the respiratory burst of human polymorphonuclear leukocytes and their intracellular activity against Staphylococcus aureus. None of these agents significantly affected the superoxide and hydrogen peroxide radicals production by the polymorphonuclear leukocytes. All the quinolones except norfloxacin showed significant intracellular activity against S. aureus.

A comparative evaluation of the intracellular penetration of norfloxacin, ofloxacin, levofloxacin, fleroxacin and lomefloxacin was carried out in human polymorphonuclear leucocytes (PMNs).The fluorometric method was used based on the natural fluorescence of some fluoroquinolones.All the fluoroquinolones accumulated within the human polymorphonuclear leucocytes achieving a quotient of between 4 to 6 in intracellular and extracellular concentration (I/C) with no significant differences being observed between the two. Penetration was rapid and was not saturable within the intervals of extracellular concentrations tested. The intracellular penetration of these fluoroquinolones was dependent on temperature and cell viability was altered in only levofloxacin and norfloxacin. The I/E values obtained significantly decreased when the cells were incubated at 4 degrees C. These values showed an important increase to pH 5 and decreased to pH 9, with this pH being statistically significant only in ofloxacin.All the fluoroquinolones evaluated penetrated rapidly into the phagocytic cells achieving intracellular concentrations which were several fold higher than the extracellular concentrations, with no notable differences being observed being the two, thus providing an additional advantage for their use in infections produced by microorganisms capable of surviving intracellularly.

The aim of this study was to evaluate the uptake of azythromycin at therapeutic concentrations by human polymorphonuclear leukocytes (PMN), as well as the effect of environmental temperature, pH, and cell viability on this uptake. The effect of azythromycin and other macrolides on hydrogen peroxide production by PMN was also assessed.Uptake of radiolabeled azythromycin by PMN was determined by a radiometric technique. Hydrogen peroxide production by PMN was measured by the phenol red method.The intracellular concentrations achieved by azythromycin in PMN were 20 to 60 fold the extracellular ones, even at extracellular concentration of 0.125 mg/l. The uptake was significantly affected by low temperature, acid pH, and cell viability. Hydrogen peroxide production was not affected by the macrolides studied.Azythromycin at therapeutical concentrations achieves high intracellular accumulation in human PMN. The effect of environmental temperature, pH and cell viability on uptake points out that a passive diffusion, with lysosome trapping, is the essential mechanism of azythromycin uptake by phagocytic cells. In spite of the high intracellular concentrations achieved by macrolides, hydrogen peroxide production by PMN is not affected by these compounds.