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Colorectal cancer (CRC) is a leading cause of mortality worldwide. We conducted a genome-wide association study meta-analysis of 100,204 CRC cases and 154,587 controls of European and east Asian ancestry, identifying 205 independent risk associations, of which 50 were unreported. We performed integrative genomic, transcriptomic and methylomic analyses across large bowel mucosa and other tissues. Transcriptome- and methylome-wide association studies revealed an additional 53 risk associations. We identified 155 high-confidence effector genes functionally linked to CRC risk, many of which had no previously established role in CRC. These have multiple different functions and specifically indicate that variation in normal colorectal homeostasis, proliferation, cell adhesion, migration, immunity and microbial interactions determines CRC risk. Crosstissue analyses indicated that over a third of effector genes most probably act outside the colonic mucosa. Our findings provide insights into colorectal oncogenesis and highlight potential targets across tissues for new CRC treatment and chemoprevention strategies.

Automated antimicrobial susceptibility testing devices are widely implemented in clinical microbiology laboratories in Spain, mainly using EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published recommendations for including antimicrobial agents and selecting concentrations in these systems. Under the patronage of the Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish National Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this document. The main modifications from the previous version comprise the inclusion of new antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new resistance mechanisms. This proposal should be considered by different manufacturers and users when designing new panels or cards. In addition, recommendations for selective reporting are also included. With this approach, the implementation of EUCAST breakpoints will be easier, increasing the quality of antimicrobial susceptibility testing data and their microbiological interpretation. It will also benefit epidemiological surveillance studies as well as the clinical use of antimicrobials aligned with antimicrobial stewardship programs.

To characterize OXA-48 carbapenemase-producing Klebsiella pneumoniaestrains isolated after an increase in carbapenem resistance in Catalonia.

Ascitic neutrophils from cirrhotic patients with spontaneous bacterial peritonitis (SBP) exhibit an impaired oxidative burst that could facilitate bacterial infection. However, the influence of the cell‐free ascitic fluid of these patients on neutrophil function has not been investigated. To analyze this influence, we determined the ascitic levels of cytokines, resistin, and lactoferrin and their association with neutrophil function, disease severity score, and SBP resolution. We analyzed NETosis induction by microscopy and oxidative burst by the flow cytometry of healthy neutrophils cultured in ascitic fluid from cirrhotic patients with sterile ascites (SA) and with SBP before and after antibiotic treatment. Resistin, IL‐6, IL‐1 receptor antagonist, IL‐1β, and lactoferrin levels were measured in ascitic fluids and supernatants of cultured neutrophils and PBMCs by ELISA. Upon stimulation, healthy neutrophils cultured in SBP ascitic fluid produced lower NETosis and oxidative burst than those cultured in SA. Ascitic resistin levels were negatively correlated with NETosis, oxidative burst, and ascitic glucose levels; and positively correlated with the model for end‐stage liver disease score. After an E. colior TNF‐α stimulus, neutrophils were the major resistin producers. Resistin indirectly reduced the oxidative burst of neutrophils and directly reduced the inflammatory phenotype of monocytes and TNF‐α production. Bacterial‐induced resistin production can down‐regulate the inflammatory response of macrophages and neutrophil function in ascitic fluid. Consequently, this down‐regulation may jeopardize the elimination of bacteria that translocate to ascitic fluid in patients with cirrhosis. Downregulation of the immune response of ascitic cells from cirrhotic patients can be induced by the content of ascitic fluid.

En los últimos años se ha observado un incremento de la resistencia a fluoroquinolonas en enterobacterias, estando asociado significativamente a la resistencia a betalactámicos. Nuestro objetivo fue conocer la prevalencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas en aislados productores de betalactamasas de claseC adquiridas y/o carbapenemasas.

ABSTRACTThe 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 Enterobacteriaceaeusing 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 pneumoniaeand Escherichia coli. A total of 702 Enterobacteriaceaeisolates 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. pneumoniaeand 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. coliis 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.

In recent years, Enterobacteriaceae isolates have increased their potential to become highly drug resistant by acquiring resistance to carbapenems, primarily due to the production of acquired carbapenemases. The carbapenemases detected in Enterobacteriaceae are largely of the KPC, VIM, NDM, IMP and OXA-48 types. Although the epidemiological origin and geographic distribution of carbapenemases are clearly different, they all first appeared in the late 20th Century. Only a decade later, these enzymes have already become established and have expanded globally.

ABSTRACTDuring a Spanish surveillance study, two natural variants of DHA β-lactamases, DHA-6 and DHA-7, were found, with the replacements Ala226Thr and Phe322Ser, respectively, with respect to DHA-1. The DHA-6 and DHA-7 enzymes were isolated from Escherichia coliand Enterobacter cloacaeclinical isolates, respectively. The aim of this study was to genetically, microbiologically, and biochemically characterize the DHA-6 and DHA-7 β-lactamases. The blaDHA-6and blaDHA-7genes were located in the I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The genetic contexts of blaDHA-6and blaDHA-7were similar to that already described for the blaDHA-1gene and included the qnrB4and aadAgenes. The MICs for cephalothin, aztreonam, cefotaxime, and ceftazidime were 8- to 32-fold lower for DHA-6 than for DHA-1 or DHA-7 expressed in the same isogenic E. coliTG1 strain. Interestingly, the MIC for cefoxitin was higher in the DHA-6-expressing transformant than in DHA-1 or DHA-7. Biochemical studies with pure β-lactamases revealed slightly lower catalytic efficiencies of DHA-6 against cephalothin, ceftazidime, and cefotaxime than those of DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed significantly higher stability than the DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N terminus in the tertiary protein structure in DHA-6 may promote this stabilization and, consequently, may induce a slight reduction in the dynamic of this enzyme that primarily affects the hydrolysis of some of the bulkiest antibiotics.

ABSTRACTIn a previous prospective multicenter study in Spain, we found that OXA-1 and inhibitor-resistant TEM (IRT) β-lactamases constitute the most common plasmid-borne mechanisms of genuine amoxicillin-clavulanate (AMC) resistance in Escherichia coli. In the present study, we investigated the population structure and virulence traits of clinical AMC-resistant E. colistrains expressing OXA-1 or IRT and compared these traits to those in a control group of clinical AMC-susceptible E. coliisolates. All OXA-1-producing (n= 67) and IRT-producing (n= 45) isolates were matched by geographical and temporal origin to the AMC-susceptible control set (n= 56). We performed multilocus sequence typing and phylogenetic group characterization for each isolate and then studied the isolates for the presence of 49 virulence factors (VFs) by PCR and sequencing. The most prevalent clone detected was distinct for each group: group C isolates of sequence type (ST) 88 (C/ST88) were the most common in OXA-1 producers, B2/ST131 isolates were the most common in IRT producers, and B2/ST73 isolates were the most common in AMC-susceptible isolates. The median numbers of isolates per ST were 3.72 in OXA-1 producers, 2.04 in IRT producers, and 1.69 in AMC-susceptible isolates; the proportions of STs represented by one unique isolate in each group were 19.4%, 31.1%, and 48.2%, respectively. The sum of all VFs detected, calculated as a virulence score, was significantly higher in AMC-susceptible isolates than OXA-1 and IRT producers (means, 12.5 versus 8.3 and 8.2, respectively). Our findings suggest that IRT- and OXA-1-producing E. coliisolates resistant to AMC have a different and less diverse population structure than AMC-susceptible clinical E. coliisolates. The AMC-susceptible population also contains more VFs than AMC-resistant isolates.

Los β-lactámicos son los antimicrobianos más utilizados para el tratamiento de las infecciones producidas por enterobacterias, y el principal mecanismo de resistencia frente a ellos es la producción de β-lactamasas. Las β-lactamasas de mayor relevancia clínica, debido a su perfil de sustrato y a sus repercusiones epidemiológicas, son las de espectro extendido, las de clase Cy las carbapenemasas. La detección fenotípica de estas β-lactamasas puede ser complicada y se basa, generalmente, en la utilización de inhibidores específicos y en la demostración de la pérdida de actividad frente a β-lactámicos indicadores. A pesar de que diferentes comités internacionales no consideran necesaria la determinación del mecanismo de resistencia ni la interpretación del antibiograma, son varias las voces críticas que estiman imprescindible esta detección y alertan de la conveniencia de interpretar el antibiograma mientras no se disponga de más datos sobre la eficacia clínica de los β-lactámicos en estos casos.

ABSTRACTA group of antibiotic resistance genes (ARGs) (blaTEM, blaCTX-M-1, mecA, armA, qnrA, and qnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs. blaTEM, qnrA, and, blaCTX-M-1were the most abundant, and armA, qnrS, and mecAwere less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra- and extraintestinal environments.

Disk diffusion is a well standardized method that provides reliable categorical results to guide antimicrobial therapy in numerous types of infections. Based on the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which are widely implemented in Spain, the Spanish Antibiogram Committee (COESANT) has drawn up recommendations for antimicrobial selection by the disk diffusion technique, including selective reporting and its use for the detection of resistance mechanisms. Factors affecting disk diffusion results, along with advantages and shortcomings of the method, are also discussed.

El Comité Español del Antibiograma (COESANT) presenta en este documento una serie de recomendaciones cuya finalidad es unificar la forma en la que los Servicios y Unidades de Microbiología Clínica españoles realizan los informes de sensibilidad acumulada de las bacterias, aisladas en muestras clínicas, frente a los antimicrobianos. Las recomendaciones se fundamentan en las recogidas en el Procedimiento de Microbiología Clínica n° 51, «Preparación de informes acumulados de sensibilidad a los antimicrobianos» de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), publicado en 2014, y recoge las modificaciones en las definiciones de las interpretaciones de las categorías clínicas publicadas en el año 2019 por el European Committee on Antimicrobial Susceptibility Testing(EUCAST). Su objetivo final es establecer una forma homogénea de elaborar estos resúmenes para poder comparar resultados de diferentes centros o sumar su información y así realizar una adecuada vigilancia local o incluso nacional de la evolución de la sensibilidad a los antimicrobianos.

ABSTRACTA novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. colistrains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Kmfor ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. colistrains carrying this variant.

ABSTRACTProspective hospital-based surveillance for Clostridium difficile-associated disease (CDAD) was conducted in Barcelona (Spain) to describe the epidemiology of this condition and investigate the risk factors for an unfavorable outcome. All patients diagnosed with CDAD during 2009 were included. Using logistic regression modeling, we analyzed the potential risk factors associated with recurrent and complicated CDAD, defined as a need for colectomy or death within 30 days. There were 365 episodes of CDAD, yielding an incidence of 22.5 cases/105person-years, 1.22 cases/103hospital discharges, and 1.93 cases/104patient-days. The main PCR ribotypes identified were 241 (26%), 126 (18%), 078 (7%), and 020 (5%). PCR ribotype 027 was not detected. Among the 348 cases analyzed, 232 (67%) patients were cured, 63 (18%) had a recurrence of CDAD, and 53 (15%) developed complicated CDAD. Predictors of complicated CDAD were continued use of antibiotics following CDAD diagnosis (odds ratio [OR], 2.009; 95% confidence interval [CI], 1.012 to 3.988; P= 0.046), Charlson comorbidity index score (OR, 1.265; 95% CI, 1.105 to 1.449; P= 0.001), and age (OR, 1.028; 95% CI, 1.005 to 1.053; P= 0.019). A leukocyte count of >15 × 103cells/ml (OR, 2.277; 95% CI, 1.189 to 4.362; P= 0.013), continuation of proton pump inhibitor (PPI) use after CDAD diagnosis (OR, 2.168; 95% CI, 1.081 to 4.347; P= 0.029), and age (OR, 1.021; 95% CI, 1.001 to 1.041; P= 0.036) were independently associated with higher odds of recurrence. The incidence of CDAD in Barcelona during 2009 was on the lower end of the previously described range for all of Europe. Our analysis suggests that the continuation of non-C. difficileantibiotics and use of PPIs in patients diagnosed with CDAD are associated with unfavorable clinical outcomes.

ABSTRACTAn isolate of Klebsiella oxytocacarrying a novel IMP metallo-β-lactamase was discovered in Madrid, Spain. The blaIMP-28gene is part of a chromosomally located class I integron. The IMP-28 kcat/Kmvalues for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior.

ABSTRACTWe conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coliisolates (MIC ≥ 32/16 μg/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC (18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30 (28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79 (2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88 [ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance in E. coliis widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance.

En los últimos años han aumentado los laboratorios de microbiología clínica que han incorporado sistemas automáticos para la determinación de la sensibilidad de los microorganismos a los antimicrobianos. La mayoría, mediante paneles o tarjetas de microdilución, obtienen valores de concentración inhibitoria mínima (CIM) que traducen en categorías clínicas (sensible, intermedio o resistente) e incorporan sistemas expertos para inferir mecanismos de resistencia. Este documento recoge las recomendaciones de un grupo de expertos, designados por GEMARA (Grupo de Estudio de los Mecanismos de Acción y Resistencia a los Antimicrobianos) y MENSURA (Mesa Española de Normalización de la Sensibilidad y Resistencia a los Antimicrobianos), para la inclusión de antimicrobianos y selección de concentraciones en los paneles de sensibilidad de los sistemas automáticos. Se definen diferentes categorías de antimicrobianos (A: deben incluirse en el panel de estudio; B: es recomendable su inclusión, y C: su inclusión es secundaria pero facilita la lectura interpretada) y grupos de antimicrobianos (0: no utilizados en clínica pero útiles para la detección de mecanismos de resistencia; 1: deben estudiarse e informarse como norma; 2: deben estudiarse de manera habitual e informarse selectivamente; 3: deben estudiarse en un segundo nivel e informarse selectivamente, y 4: deben estudiarse en patógenos urinarios aislados en orina y muestras relacionadas). Las concentraciones recomendadas cubren las concentraciones críticas o puntos de corte establecidos por EUCAST (European Committee on Antimicrobial Susceptibility Testing), CLSI (Clinical and Laboratory Standards Institute) y MENSURA. Estos criterios redundarán en una mejor calidad de los resultados, una mejor detección de los mecanismos de resistencia y un cumplimiento del objetivo clínico del antibiograma.

Objectives: This study aimed to determine the presence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in different environments.Methods: Clinical samples and stool samples from animal farms, sewage, human faecal carriers attending the emergency room and faecal carriers in the context of food-borne disease outbreaks were subcultured onto MacConkey agar supplemented with cefotaxime for the detection of ESBL-producing Enterobacteriaceae. Identification, susceptibility pattern and ERIC–PCR were used for clone delineation in each sample. Community consumption of antibiotics was also recorded.Results: An ESBL-producing Enterobacteriaceae prevalence of 1.9% was observed in human infections. A cross-sectional survey of human faecal carriers in the community showed a general prevalence of 6.6% with a temporal distribution. High use of antibiotics in winter coincided with a lower prevalence in carriers. ESBL-producing Enterobacteriaceae were detected in the five samples of human sewage, in samples from 8 of 10 pig farms, 2 of 10 rabbit farms, from all 10 poultry farms and in 3 of 738 food samples studied. Faecal carriage of ESBL-producing Enterobacteriaceae was detected in samples from 19 of 61 food-borne outbreaks evaluated. All food-borne outbreaks were due to enteropathogens. The prevalence of carriers in these outbreaks ranged from 4.4% to 66.6%.Conclusions: This widespread occurrence of ESBL-producing Enterobacteriaceae suggests that the community could act as a reservoir and that food could contribute to the spread of these strains.

Background: A new, fast ‘low cost and density’ DNA microarray (LCD array), designed for the detection of mutations that confer isoniazid or rifampicin resistance in Mycobacterium tuberculosis isolates, has been developed and was evaluated using 46 resistant clinical isolates from Barcelona.Methods: LCD chips are pre-structured polymer supports using a non-fluorescent detection principle based on the precipitation of a clearly visible dark substrate. One LCD chip consists of eight identical microarrays, designed to detect mutations within the 90 bp rpoB region, codon 315 in the katG gene and the mabA-inhA regulatory region. A total of 22 strains with a katG 315 mutation, 19 strains with alterations in the mabA-inhA regulatory region and 16 strains with mutations in the rpoB region, characterized previously, were studied.Results: The identification of S315T and S315N mutations using the LCD was 100% concordant with the sequencing data. A strain with the S315R mutation, which is not tiled on the LCD array, was detected by the absence of hybridization using the wild-type probe. Of 19 strains with low-level isoniazid resistance related to the mabA-inhA regulatory region, 18 were identified correctly. The detection of mutations in the rpoB region was 93.8% concordant with the sequencing data. One mabA-inhA and rpoB mutated strain showed a cross-hybridization.Conclusions: The LCD array protocol takes 45 min (15 min ‘hands-on’ time) after prior PCR amplification. Only minimal laboratory equipment is required. LCD arrays provide a rapid and economical method to characterize mutations in codon 315 of the katG gene, in the mabA-inhA regulatory region and in the rpoB gene.

ABSTRACTAbout 7% of 7,252 nonduplicated clinical Escherichia colistrains from a Spanish hospital showed reduced susceptibility to amoxicillin-clavulanate. Of these, 0.37% produced the IRTs TEM-30, TEM-31, TEM-33, TEM-34, TEM-37, TEM-40, TEM-51, and TEM-54; 5.3% were probable class C β-lactamase overproducers; 0.8% were probable TEM-1 hyperproducers; 0.18% produced OXA-30; 0.15% overexpressed SHV-1; and 0.03% produced a PSE-1 enzyme.

ABSTRACTFor the present report, a novel complex class 1 integron, In60, was characterized. Part of this integron includes the blaCTX-M-9gene and its downstream nucleotide sequence, which shares 81% and 78% nucleotide identity with those of kluA-1β-lactamase and orf3of K. ascorbata, respectively. Furthermore, a new insertion sequence, IS3000, has been found in In60. PCR analysis indicates that integron In60 is present in 33 of 34 nonclonal enterobacterial isolates carrying the putative β-lactamase CTX-M-9.

The aim of this study was to evaluate the incidence of decreased susceptibility to broad-spectrum cephalosporins in Enterobacteriaceae that lack inducible chromosomal bla genes, and to determine the enzymes responsible for resistance. From all clinically relevant Enterobacteriaceae strains isolated between 1994 and 1996, 88 of 7054 Escherichia coli, seven of 581 Klebsiella pneumoniae and 23 of 166 Klebsiella oxytoca strains were studied because of their decreased susceptibilities to broad-spectrum cephalosporins (as reflected in intermediate susceptibilities and/or positive synergy tests and/or irregular crenellated inhibition zones). The most frequent mechanism implicated in decreased susceptibility to broad-spectrum cephalosporins displayed by E. coli and K. oxytoca was hyperproduction of chromosomal β-lactamase, followed by plasmid-mediated SHV-1 hyperproduction in E. coli. In our hospital, the incidence of plasmid-mediated extended-spectrum β-lactamases (ESBLs) between 1994 and 1996 was low. ESBLs were found in only 10 (0.14%) E. coli strains (six CTX-M-9, two TEM-12 and two SHV-2), in one (0.17%) K. pneumoniae strain (SHV-2) and in no K. oxytoca strains. The relatively wide variety of β-lactamases that were detected among these common bacteria isolated from a single medical centre, including non-TEM- and non-SHV-derived ESBLs, appears epidemiologically remarkable.

The aim of this study was to evaluate the incidence of decreased susceptibility to broad-spectrum cephalosporins in Enterobacteriaceae that lack inducible chromosomal bla genes, and to determine the enzymes responsible for resistance. From all clinically relevant Enterobacteriaceae strains isolated between 1994 and 1996, 88 of 7054 Escherichia coli, seven of 581 Klebsiella pneumoniae and 23 of 166 Klebsiella oxytoca strains were studied because of their decreased susceptibilities to broad-spectrum cephalosporins (as reflected in intermediate susceptibilities and/or positive synergy tests and/or irregular crenellated inhibition zones). The most frequent mechanism implicated in decreased susceptibility to broad-spectrum cephalosporins displayed by E. coli and K. oxytoca was hyperproduction of chromosomal beta-lactamase, followed by plasmid-mediated SHV-1 hyperproduction in E. coli. In our hospital, the incidence of plasmid-mediated extended-spectrum beta-lactamases (ESBLs) between 1994 and 1996 was low. ESBLs were found in only 10 (0.14%) E. coli strains (six CTX-M-9, two TEM-12 and two SHV-2), in one (0.17%) K. pneumoniae strain (SHV-2) and in no K. oxytoca strains. The relatively wide variety of beta-lactamases that were detected among these common bacteria isolated from a single medical centre, including non-TEM- and non-SHV-derived ESBLs, appears epidemiologically remarkable.

In the past 10 years an increasing number of members of a family of class A extended-spectrum β-lactamases (ESBL), the CTX-M-type, which are not closely related to TEM, SHV or OXA enzymes, have been detected. The extended-spectrum activity of CTX-M enzymes seems to be an ‘intrinsic’ enzymatic property of these ESBL, due mainly to the hydrogen-binding pattern and the formation of the hydrophobic core observed near the Ω loop. When we compared CTX-M-type enzymes with other class A β-lactamases, the degree of amino acid identity was found to be lower than 40 . Among all CTX-M β-lactamases, four different clusters were observed. The homologies among these clusters were quite low, suggesting that they branched off early from an unknown protein or, as was found more recently, from different proteins. The CTX-M-1 cluster could have originated from KLUC-1 of Kluyvera cryocrescens, whereas the CTX-M-2 cluster could have originated from KLUA-1 or KLUA-2 of Kluyvera ascorbata. It has been observed that the insertion sequence IS Ecp1, IS 903and complex class 1 integron In60 may be implicated in jumping DNA fragments (in these cases containing genes that code for β-lactamases) from chromosomes to plasmids, which could contribute to the rapid spread of these β-lactamases in various species of the family Enterobacteriaceae and in geographically widely distant areas.

The aim of this study was to evaluate the incidence of decreased susceptibility to broad-spectrum cephalosporins in Enterobacteriaceae that lack inducible chromosomal bla genes, and to determine the enzymes responsible for resistance. From all clinically relevant Enterobacteriaceae strains isolated between 1994 and 1996, 88 of 7054 Escherichia coli, seven of 581 Klebsiella pneumoniae and 23 of 166 Klebsiella oxytoca strains were studied because of their decreased susceptibilities to broad-spectrum cephalosporins (as reflected in intermediate susceptibilities and/or positive synergy tests and/or irregular crenellated inhibition zones). The most frequent mechanism implicated in decreased susceptibility to broad-spectrum cephalosporins displayed by E. coli and K. oxytoca was hyperproduction of chromosomal β-lactamase, followed by plasmid-mediated SHV-1 hyperproduction in E. coli. In our hospital, the incidence of plasmid-mediated extended-spectrum β-lactamases (ESBLs) between 1994 and 1996 was low. ESBLs were found in only 10 (0.14%) E. coli strains (six CTX-M-9, two TEM-12 and two SHV-2), in one (0.17%) K. pneumoniae strain (SHV-2) and in no K. oxytoca strains. The relatively wide variety of β-lactamases that were detected among these common bacteria isolated from a single medical centre, including non-TEM- and non-SHV-derived ESBLs, appears epidemiologically remarkable.

ABSTRACTA new CTX-M-type β-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia colistrain. Despite the close identity that exists between the CTX-M-9 and Toho-2 β-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.

ABSTRACTTrends in resistance to antimicrobial agents used for therapy have been evaluated with 3,797 enteropathogenic bacteria,Campylobacter, Salmonella,Shigella, and Yersinia, between 1985–1987 and 1995–1998. The greater increase in the rate of resistance was observed in Campylobacter jejunifor quinolones (from 1 to 82%) and tetracycline (from 23 to 72%) and in gastroenteric salmonellae for ampicillin (from 8 to 44%), chloramphenicol (from 1.7 to 26%), and trimethoprim-sulfamethoxazole and nalidixic acid (from less than 0.5 to 11%). Multidrug resistance was detected in several Salmonellaserotypes. In the 1995–1998 period, 76% of Shigellastrains were resistant to trimethoprim-sulfamethoxazole, 43% were resistant to ampicillin, and 39% were resistant to chloramphenicol. Seventy-two percent ofYersinia enterocoliticaO3 strains were resistant to streptomycin, 45% were resistant to sulfonamides, 28% were resistant to trimethoprim-sulfamethoxazole, and 20% were resistant to chloramphenicol.

ABSTRACTTo clarify the clinical and bacteriological correlates of urinary-tract infection (UTI) due to Escherichia coliO15:K52:H1, during a 1-year surveillance period we prospectively screened all 1,871 significant E. coliurine isolates at the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, for this serotype and assessed the epidemiological features of community-acquired UTI due to E. coliO15:K52:H1 versus other E. coliserotypes. We also compared the 25 O15:K52:H1 UTI isolates from the present study with 22 O15:K52:H1 isolates from other, diverse geographic locales and with 23 standard control strains (8 strains from the ECOR reference collection and 15 strains of nonpathogenic O:K:H serotypes) with respect to multiple phenotypic and genotypic traits. Although E. coliO15:K52:H1 caused only 1.4% of community-acquired E. coliUTIs during the surveillance period, these UTIs were more likely to present as pyelonephritis and to occur in younger hosts, with similar risk factors, than were UTIs due to other E. coliserotypes. Irrespective of geographic origin, E. coliO15:K52:H1 strains exhibited a comparatively restricted repertoire of distinctive virulence factor profiles (typically, they were positive forpapGallele II, papAallele F16, andaerand negative for sfa, afa,hly, and cnf1), biotypes, ribotypes, and amplotypes, consistent with a common clonal origin. In contrast, their antimicrobial resistance profiles were more extensive and more diverse than those of control strains. These findings indicate that E. coliO15:K52:H1 constitutes a broadly distributed and clinically significant uropathogenic clone with fluid antimicrobial resistance capabilities.

ABSTRACTWe report the case of a 3-year-old girl with lymphadenopathy caused by the recently described species Mycobacterium colombiense. M. colombienseis a nonpigmented slow grower that is included in the Mycobacterium aviumcomplex. Partial sequencing of the 16S rRNA gene was used for species identification.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with blaCMY-2in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.

ABSTRACTTwo strains of Enterobacteriaceae, Escherichia coliand Klebsiella pneumoniae, producing VIM-1 were isolated for the first time in Spain. In both strains, blaVIM-1was found to be carried on a gene cassette inserted into a class 1 integron. The blaVIM-1-containing integron was located on a transferable plasmid.

ABSTRACTWe studied in parallel the population structure of 90 carbapenemase-producing and 88 carbapenemase-susceptible Klebsiella pneumoniaeisolates collected in 20 Spanish hospitals, in the context of the EuSCAPE project. Fourteen and 50 multilocus sequence types (MLSTs) were detected among the carbapenemase-producing and carbapenem-susceptible isolates, respectively. ST11 and ST15 clones were more frequent in the carbapenemase-producing group than in the carbapenemase-susceptible group (P< 0.0001). Among the members of the carbapenem-suceptible group, the cefotaxime-resistant population showed population parameters that differed between the populations of the wild-type strains and the carbapenemase producers.

ABSTRACTThe increase in the level of quinolone resistance ofHaemophilus influenzaeclinical isolates during ofloxacin therapy of a patient with recurrent respiratory infections was investigated. The first isolate (MIC of ciprofloxacin of 2 μg/ml) and the second isolate (MIC of 32 μg/ml) belonged to the same clone, as shown by pulsed-field gel electrophoresis, and the increase in the resistance level was associated with a substitution in Ser-84 to Arg in the ParC protein. These results emphasize the potential risk of development of quinolone-resistant H. influenzaeduring fluoroquinolone therapy in patients with recurrent respiratory infection.

&lt;it>Objectives&lt;/it>: The aim of the present study was to characterize and compare the extended-spectrum β-lactamase (ESBL)-producing organisms isolated from clinical samples and faecal carriers in 2001 and 2002. &lt;it>Methods&lt;/it>: A total of 5251 Enterobacteriaceae isolated from clinical samples and 1321 stool samples were evaluated for the presence of ESBLs. The stool samples were spread onto plates of MacConkey agar containing 2 mg&sol;L cefotaxime for selection of ESBL-producing strains. These strains were defined as those showing synergism between amoxicillin&sol;clavulanic acid and third-generation cephalosporins. The β-lactamases involved were characterized by isoelectric focusing, PCR assays and DNA sequencing. &lt;it>Results&lt;/it>: The prevalence of ESBL-producing strains among clinical Enterobacteriaceae was 1.7%. Of these, 87.6% produced CTX-M, 25.8% produced SHV and 2.2% were TEM-type-producing strains. All clinical ESBL-producing strains were &lt;it>Escherichia coli&lt;/it>, with the exception of four &lt;it>Klebsiella pneumoniae&lt;/it> and one &lt;it>Citrobacter freundii&lt;/it>. The prevalence of faecal carriage of ESBL-producing organisms was 3.3%. Of these, 75% produced CTX-M-type enzymes followed by 22.7% SHV-producing strains. All faecal ESBL-producing strains were &lt;it>E. coli&lt;/it> except for one &lt;it>Enterobacter cloacae&lt;/it> and one &lt;it>Proteus mirabilis&lt;/it>. This latter strain produced the PER-1 enzyme reported for the first time in Spain. &lt;it>Conclusions&lt;/it>: The prevalence of ESBL-producing strains in stool samples was higher than that observed in clinical samples from the same period. The different types of ESBLs found were similar in both contexts. The most prevalent ESBLs were the CTX-M-related enzymes, with nine different types, followed by SHV-12.

&lt;it>Objectives&lt;/it>: The dispersion of a clone, a plasmid or a mobile element carrying the &lt;it>bla&lt;/it>&lt;inf>CTX-M-9&lt;/inf> gene was evaluated in 30 &lt;it>Escherichia coli&lt;/it> strains isolated in Barcelona between 1996 and 1999. The presence of the previously described &lt;it>orf513&lt;/it>-bearing class 1 integron, In60, carrying the &lt;it>bla&lt;/it>&lt;inf>CTX-M-9&lt;/inf> gene, was also studied. &lt;it>Methods&lt;/it>: The clonality was analysed by pulsed-field gel electrophoresis. Plasmid analysis was performed by S1 digestion and hybridization with the CTX-M-9 probe. PCR mapping using specific designed primers was used to study the presence of In60 and In60-like structures. &lt;it>Results&lt;/it>: The clonality between the 30 strains was minor. The size of &lt;it>bla&lt;/it>&lt;inf>CTX-M-9&lt;/inf> carrying plasmids ranged between ∼80 and 430 kb. One strain produced only a chromosome-encoded CTX-M-9 β-lactamase. Thirty-six per cent of the strains showed differences with respect to the In60 structure due to an insertion or deletion events. &lt;it>Conclusions&lt;/it>: These findings suggest that the &lt;it>bla&lt;/it> &lt;inf>CTX-M-9&lt;/inf> gene may be carried by a mobile element that disperses it between plasmids. The fast dispersion of the CTX-M-9 enzyme could therefore be due to both diffusion of plasmids and mobile elements.