In a 34-month prospective study to determine the prevalence of Staphylococcus aureus small colony variants (SCVs) in cystic fibrosis (CF) patients, S. aureus SCVs or SCVs plus normal S. aureus were recovered from 26 of 78 patients; 27 patients harbored only normal S. aureus. By pulsed-field gel electrophoresis, clonal identity was demonstrated of SCV and normal strains isolated at the same time and of multiple S. aureus SCV and normal strains in consecutive specimens from individual patients. All S. aureus SCVs were resistant to antifolate antibiotics, while the corresponding parent strains were susceptible, and in 11 of 12 SCV/normal pairs, gentamicin was less active against S. aureus with the SCV phenotype than against the normal isolate. Analysis of the underlying auxotrophism of SCVs revealed hemin, thymidine, and/or menadione dependencies. Thus, S. aureus SCVs are highly prevalent in respiratory secretions of CF patients, persist over extended periods, and may contribute to S. aureus persistence in CF patients.
MRP8 and MRP14 are specifically released during the interaction of monocytes with inflammatory activated endothelium, probably at sites of local inflammation. Their serum concentrations represent a useful marker for monitoring local inflammation in JRA.
Molecular typing of normal (n ؍ 456) and small-colony-variant (SCV; n ؍ 239) Staphylococcus aureus isolates cultured from the airways of 52 of 72 cystic fibrosis (CF) patients (72.2%) during a 6-year prospective study revealed a median long-term persistence of 37 months (range, 6 to 70). SCV persisted longer in the airways than the normal S. aureus (statistically not significant). Pulsed-field gel electrophoresis identified six prevalent clonal lineages, which were cultured from more than one patient (3 to 12 patients), and 39 individual clones, which were isolated only from single patients. The SCV phenotype was not restricted to a distinct clonal lineage but occurred in many different clones. Most patients (33 of 52, 63.46%) harbored single clones. This study provides a basis for improved understanding of S. aureus colonization and infection dynamics in CF patients.Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, leading to chronic respiratory infection with deteriorating lung function in spite of antibiotic treatment (13). Only a limited number of bacterial species have been found to be major pathogens chronically infecting the airways of CF patients: Staphylococcus aureus and Haemophilus influenzae are mainly present in children, and Pseudomonas aeruginosa is the leading pathogen in adolescents and adults (4, 5).Persistence of S. aureus in CF and other invasive infections has been associated with the isolation of a subpopulation of S. aureus with the small-colony-variant (SCV) phenotype (10,15,23,25). In contrast to normal S. aureus, with typical colony size, pigmentation, and hemolysis on Columbia blood agar, SCV S. aureus strains can be altered in their electron transport activity or thymidine synthesis, thus growing as nonhemolytic, nonpigmented, very small colonies or as colonies with a fried-egg appearance (11). Decreased expression of ␣-hemolysin allows SCV isolates to persist intracellularly in in vitro systems (1, 24).Enhanced understanding of both bronchopulmonary pathophysiology and consequent microbial colonization and adaptivity in CF has been hampered by limited longitudinal studies on S. aureus infection as a function of colonization and infection dynamics of the pathogen (3, 16). Therefore, the goals of our study were to determine the duration and dynamics of S. aureus (normal and SCV) persistence in the airways of individual CF patients (same clone or new infection) over an extended period and concurrently to assess the population structure of chronic S. aureus colonization and infection in this defined patient group. Specimens were obtained from CF patients attending our institution from April 1994 to January 2000. Only patients with an observation period of more than 12 months were included into the study (n ϭ 72). The study group consisted of 41 male and 31 female patients with a median age of 9.5 years (range, 1 to 33) at the beginning of the study period and 14.5 years at the end (range, 1 to 36). Sputum or deep throat swabs...
Abstract. Endosomes are prelysosomal organdies that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organdies. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postuuclear superuatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP-dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 lxg protein) sufficient for biochemical, immunological, and functional analysis.
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