To determine the epidemiologic features and clinical outcomes of bloodstream infections caused by extended-spectrum ␤-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae isolates, cases of bacteremia caused by these organisms in children were analyzed retrospectively. Among the 157 blood isolates recovered from 1993 to 1998 at the Seoul National University Children's Hospital, the prevalence of ESBL production was 17.9% among the E. coli isolates and 52.9% among the K. pneumoniae isolates. The commonest ESBLs were SHV-2a and TEM-52. A novel ESBL, TEM-88, was identified. Pulsed-field gel electrophoresis analysis of the ESBL-producing organisms showed extensive diversity in clonality. The medical records of 142 episodes were reviewed. The risk factors for bloodstream infection with ESBL-producing organisms were prior hospitalization, prior use of oxyimino-cephalosporins, and admission to an intensive care unit within the previous month. There was no difference in clinical severity between patients infected with ESBL-producing strains (the ESBL group) and those infected with ESBL-nonproducing strains (the non-ESBL group) at the time of presentation. However, the overall fatality rate for the ESBL group was significantly higher than that for the non-ESBL group: 12 of 45 (26.7%) versus 5 of 87 (5.7%) (P ‫؍‬ 0.001). In a subset analysis of patients treated with extended-spectrum cephalosporins with or without an aminoglycoside, favorable response rates were significantly higher in the non-ESBL group at the 3rd day (6 of 17 versus 33 of 51; P ‫؍‬ 0.035), the 5th day (6 of 17 versus 36 of 50; P < 0.05), and the end of therapy (9 of 17 versus 47 of 50; P < 0.001). In conclusion, the ESBL production of the infecting organisms has a significant impact on the clinical course and survival of pediatric patients with bacteremia caused by E. coli and K. pneumoniae.Escherichia coli and Klebsiella pneumoniae are leading causes of serious infections in neonates, neutropenic cancer patients, and other patients with underlying diseases. These bacteria had been uniformly susceptible to oxyimino-␤-lactam antimicrobials. However, since the initial description of extended-spectrum ␤-lactamase (ESBL) production by K. pneumoniae strains in 1983 (18) and E. coli strains in 1987 (3), strains of E. coli and K. pneumoniae that are resistant to broadspectrum cephalosporins are increasingly being recognized (6, 14). There have been many reports of outbreaks caused by these organisms in cancer centers, pediatric and geriatric wards, and hospitalized nursing home patients. However, epidemiologic descriptions of bloodstream infections caused by ESBL-producing E. coli and K. pneumoniae are limited (32,36), and clinical data regarding treatment are further limited (2,31,35,36). At present, carbapenems are recommended for the treatment of infections caused by ESBL-producing organisms. However, this recommendation is primarily based on the in vitro effect (12), the results of animal experiments (33), and only very limited clinical da...

Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by Gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

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Regression testing is the process of validating modi ed software to detect whether new errors have been introduced into previously tested code and to provide con dence that modi cations are correct. Since regression testing is an expensive process, researchers have proposed regression test selection techniques as a way to reduce some of this expense. These techniques attempt to reduce costs by selecting and running only a subset of the test cases in a program's existing test suite. Although there have been some analytical and empirical evaluations of individual techniques, to our knowledge only one comparative study, focusing on one aspect of two of these techniques, has been reported in the literature. We conducted an experiment to examine the relative costs and bene ts of several regression test selection techniques. The experiment examined ve techniques for reusing test cases, focusing on their relative abilities to reduce regression testing e ort and uncover faults in modi ed programs. Our results highlight several di erences between the techniques, and expose essential tradeo s that should be considered when choosing a technique for practical application.