Influence of Tn917 Insertion on Transcription of the icaADBC Operon in Six Biofilm-Negative Transposon Mutants of Staphylococcus epidermidis

Plasmid ◽  
2002 ◽  
Vol 47 (1) ◽  
pp. 10-17 ◽  
Author(s):  
Sabine Dobinsky ◽  
Katrin Bartscht ◽  
Dietrich Mack
Keyword(s):  
Staphylococcus Epidermidis ◽  
Transposon Mutants ◽  
Tn917 Insertion

scholarly journals The ica Locus of Staphylococcus epidermidis Encodes Production of the Capsular Polysaccharide/Adhesin

1998 ◽  
Vol 66 (10) ◽  
pp. 4711-4720 ◽  
Author(s):  
David McKenney ◽  
Johannes Hübner ◽  
Eugene Muller ◽  
Ying Wang ◽  
Donald A. Goldmann ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Staphylococcus Epidermidis ◽  
Capsular Polysaccharide ◽  
High Titer ◽  
Immune Serum ◽  
Coagulase Negative Staphylococci ◽  
Transposon Mutants ◽  
Bacterial Accumulation ◽  
Cellular Aggregates

ABSTRACT Clinical isolates of coagulase-negative staphylococci often elaborate a biofilm involved in adherence to medical devices and resistance to host defenses. The biofilm contains the capsular polysaccharide/adhesin (PS/A), which mediates cell adherence to biomaterials, and another antigen, termed polysaccharide intercellular adhesin (PIA), which is thought to mediate bacterial accumulation into cellular aggregates. PIA is a polymer of β-1,6-linkedN-acetyl glucosamine residues with a molecular mass of <30,000 kDa. We found that recombinant Staphylococcus carnosus and Staphylococcus aureus carrying a plasmid with genes of the ica locus, which was reported to encode the biosynthetic proteins for production of PIA, were also able to synthesize PS/A. PS/A and a chemically and immunologically identical polysaccharide isolated from S. carnosus carrying theica genes on plasmid pCN27 were found to be high-molecular-mass (>250,000 kDa), acid-stable polymers of β-1,6-linked glucosamine substituted on the amino group primarily with succinate, although some preparations also contained acetate. Moreover, all recombinant staphylococcal strains with theica genes had the biologic properties previously attributed to PS/A. ica-positive strains readily formed an in vitro biofilm on plastic, adhered 3- to 10-fold more to catheters during a 30-min assay compared with control strains carrying only the cloning vector, adsorbed out antibodies to PS/A from immune serum, and elaborated a capsule visualized by immunoelectron microscopy with antisera to PS/A. These properties were also seen with PS/A-producing strains of Staphylococcus epidermidis, but not with transposon mutants lacking PS/A. An antiserum raised to PIA contained high-titer antibody to PS/A that was readily adsorbed out by PS/A-positive strains of S. epidermidis and recombinant strains of staphylococci carrying the ica genes. We conclude that the ica locus encodes production of PS/A and that the properties of S. epidermidis associated with initial bacterial adherence, biofilm formation, and intercellular adhesion can be correlated with elaboration of PS/A.

scholarly journals Establishment of an Arbitrary PCR for Rapid Identification of Tn917 Insertion Sites in Staphylococcus epidermidis: Characterization of Biofilm-Negative and Nonmucoid Mutants

2003 ◽  
Vol 69 (10) ◽  
pp. 5812-5818 ◽  
Author(s):  
Johannes K.-M. Knobloch ◽  
Max Nedelmann ◽  
Kathrin Kiel ◽  
Katrin Bartscht ◽  
Matthias A. Horstkotte ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Regulatory Gene ◽  
Rapid Identification ◽  
Chromosomal Dna ◽  
Phosphotransferase System ◽  
Gram Positive Bacteria ◽  
Staphylococcus Carnosus ◽  
Transposon Mutants ◽  
Insertion Sites

ABSTRACT Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.

Scanning Electron Microscopy of Staphylococcus Epidermidis Adherence to Continuous Ambulatory Peritoneal Dialysis Catheters

1985 ◽  
Vol 43 ◽  
pp. 520-521
Author(s):  
William J. Lamoreaux ◽  
David L. Smalley ◽  
Larry M. Baddour ◽  
Alfred P. Kraus
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Peritoneal Dialysis ◽  
Continuous Ambulatory Peritoneal Dialysis ◽  
Staphylococcus Epidermidis ◽  
Sterile Saline ◽  
Intravascular Devices ◽  
Capd Patient ◽  
Scanning Electron

Infections associated with the use of intravascular devices have been documented and have been reported to be related to duration of catheter usage. Recently, Eaton et al. reported that Staphylococcus epidermidis may attach to silastic catheters used in continuous ambulatory peritoneal dialysis (CAPD) treatment. The following study presents findings using scanning electron microscopy (SEM) of S. epidermidis adherence to silastic catheters in an in vitro model. In addition, sections of polyvinyl chloride (PVC) dialysis bags were also evaluated by SEM.The S. epidermidis strain RP62A which had been obtained in a previous outbreak of coagulase-negative staphylococcal sepsis at local hospitals was used in these experiments. The strain produced surface slime on exposure to glucose, whereas a nonadherent variant RP62A-NA, which was also used in these studies, failed to produce slime. Strains were grown overnight on blood agar plates at 37°C, harvested from the surface and resuspended in sterile saline (0.85%), centrifuged (3,000 rpm for 10 minutes) and then washed twice in 0.1 M phosphate-buffered saline at pH 7.0. Organisms were resuspended at a concentration of ca. 106 CFU/ml in: a) sterile unused dianeal at 4.25% dextrose, b) sterile unused dianeal at 1.5% dextrose, c) sterile used dialysate previously containing 4.25% dextrose taken from a CAPD patient, and d) sterile used dialysate previously containing 1.5% dextrose taken from a CAPD patient.

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