Generalized Transduction for Genetic Linkage Analysis and Transfer of Transposon Insertions in Different Staphylococcus epidermidis Strains

Nedelmann, Max; Sabottke, Axel; Laufs, Rainer; Mack, Dietrich

doi:10.1016/s0934-8840(98)80151-5

Cited by 34 publications

(31 citation statements)

References 15 publications

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“…The plasmid subsequently was transformed into chemically competent E. coli DH5␣ and then sequentially electroporated into S. aureus RN4220 (51) competent cells and S. aureus RN4220/pGO1-pC221 (52) competent cells. pNF220 was transferred by conjugative mobilization to S. epidermidis 1457NR and finally transduced into S. epidermidis 1457 (49) using phage 71 (61).…”

Section: Methodsmentioning

confidence: 99%

“…Colonies able to grow on TSA plus Tet but not TSA plus Erm were streaked onto TSA plus Tet, TSA plus Erm, and modified mannitol salt agar (53). Following allelic replacement, the aap mutation was backcrossed into strain 1457 using the transducing phage 71 as described previously (61). Mutant construction was confirmed by PCR, PFGE, and immunoblotting with antibodies to both the A and B domains of Aap (12,46).…”

Section: Methodsmentioning

confidence: 99%

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Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

et al. 2015

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Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

et al. 2015

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“…Transduction of the Tn917 insertion of mutant M15 into the independent and biofilm-producing wild-type strain 8400 was performed essentially as described previously using S. epidermidis phage 71 (25,37).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, the significance of PIA as a virulence factor could be demonstrated by comparison of isogenic PIA-negative transposon mutant 1457-M10 and the corresponding wild-type strain in a central venous catheter rat infection model and a subcutaneous foreign-body mice infection model (45,46). By transposon mutagenesis, four unlinked gene loci were identified whose mutation leads to a biofilm-negative phenotype and abolished PIA synthesis and produces mutants that are classified, according to genotypic and phenotypic differences, as class I to IV mutants (30,37). Class I mutants represent those in which icaADBC is inactivated.…”

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confidence: 99%

Biofilm Formation by Staphylococcus epidermidis Depends on Functional RsbU, an Activator of the sigB Operon: Differential Activation Mechanisms Due to Ethanol and Salt Stress

et al. 2001

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Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB NaCl ) or ethanol (TSB EtOH ), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor B , led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB EtOH , the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB NaCl , the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.Staphylococcus epidermidis, a normal inhabitant of human skin and mucous membranes, is the predominant cause of foreign-body-associated infections (43). In addition, S. epidermidis is isolated with increasing frequency as the causative pathogen of nosocomial sepsis and other nosocomial infections, ranking among the five most frequent nosocomial pathogens (43, 49). The pathogenesis of S. epidermidis infections is correlated with the ability to form biofilms on polymer surfaces (5, 58).Biofilm formation proceeds in two phases (23,24). Primary attachment of bacterial cells to a polymer surface is a complex process influenced by a variety of factors, including hydrophobic interactions, presence of host proteins, and specific bacterial proteins and polysaccharides like the capsular polysaccharide adhesin, the autolysin AtlE, and other staphylococcal surface proteins (15,17,33,38,39,50,51). This is followed by the second phase leading to accumulation of bacteria in a multilayered biofilm embedded in an amorphous glycocalyx. Synthesis of the polysaccharide intercellular adhesin (PIA) is essential for bacterial cell accumulation because it mediates cell-to-ce...

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“…Biofilm-producing S. epidermidis 1457, 8400, RP62A, and 9142 and the isogenic biofilm-negative transductant 1457-M11 have been described previously (36,37,40,45). Basic T A medium was assembled from the individual components and contained (wt/vol) 1.7% Tryptone (lot 208080; Oxoid, Basingstoke, England), 0.3% neutralized soya peptone (lot 14782150; Oxoid), 0.5% NaCl, and 0.25% dipotassium phosphate as recommended by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

Glucose-Related Dissociation between icaADBC Transcription and Biofilm Expression by Staphylococcus epidermidis : Evidence for an Additional Factor Required for Polysaccharide Intercellular Adhesin Synthesis

et al. 2003

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Biofilm formation in Staphylococcus epidermidis depends, in the majority of the strains, on the activity of the icaADBC locus. The expression of the operon that encodes the synthetic enzymes of the intercellular polysaccharide adhesin (PIA) depends on a variety of exogenic environmental conditions and is, at least in part, regulated by the alternative sigma factor B . We investigated the transcriptional regulation of the ica operon and the respective phenotypes expressed under growth conditions differing in the content of glucose in the growth medium. In the presence of glucose, S. epidermidis exhibited a PIA-and biofilm-positive phenotype whereas ica transcription was down-regulated in the postexponential and stationary phases of growth. Surprisingly, maximum transcription of ica was detectable in the stationary phase of growth in the absence of glucose despite the expression of a PIA-and biofilm-negative phenotype. In vitro enzymatic assays and phenotypic characterization showed that the abundant amount of ica mRNA was functionally active because induction of stationary-phase cells with glucose led to immediate PIA synthesis. Induction of biofilm formation could be completely inhibited by chloramphenicol, which, given at a later stage of biofilm accumulation, also inhibited further development of preformed biofilm, indicating that continuous translation of an additional, icaADBC-independent factor is required for the expression of a biofilm-positive phenotype.

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Generalized Transduction for Genetic Linkage Analysis and Transfer of Transposon Insertions in Different Staphylococcus epidermidis Strains

Cited by 34 publications

References 15 publications

Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

Biofilm Formation by Staphylococcus epidermidis Depends on Functional RsbU, an Activator of the sigB Operon: Differential Activation Mechanisms Due to Ethanol and Salt Stress

Glucose-Related Dissociation between icaADBC Transcription and Biofilm Expression by Staphylococcus epidermidis : Evidence for an Additional Factor Required for Polysaccharide Intercellular Adhesin Synthesis

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