J Korean Med Sci.  2004 Dec;19(6):779-782. 10.3346/jkms.2004.19.6.779.

Phase Variation of Biofilm Formation in Staphylococcus aureus by IS 256 Insertion and Its Impact on the Capacity Adhering to Polyurethane Surface

• Kiem S
• Oh WS
• Peck KR
• Lee NY
• Lee JY
• Song JH
• Hwang ES
• Kim EC
• Cha CY
• Choe KW

• KMID: 1778556
• DOI: http://doi.org/10.3346/jkms.2004.19.6.779

Abstract

While ica gene of Staphylococcus epidermidis is known to undergo phase variation by insertion of IS256, the phenomenon in Staphylococcus aureus has not been evaluated. Six biofilm-positive strains were tested for the presence of biofilm-nega-tive phase-variant strains by Congo red agar test. For potential phase-variant strains, pulsed-field gel electrophoresis was done to exclude the possibility of contamination. To investigate the mechanism of the biofilm-negative phase variation, PCR for each ica genes were done. Changes of ica genes detected by PCR were confirmed by southern hybridization, and their nucleotides were analyzed by DNA sequencing. Influence of ica genes and biofilm formation on capacity for adherence to biomedical material was evaluated by comparing the ability of adhering to polyurethane sur-face among a biofilm-negative phase-variant strain and its parent strain. A biofilm-negative phase-variant S. aureus strain was detected from 6 strains tested. icaC gene of the phase-variant strain was found to be inactivated by insertion of additional gene segment, IS256. The biofilm-negative phase-variant strain showed lower adher-ing capacity to polyurethane than its parent strain. This study shows that phase variation of ica gene occurs in S. aureus by insertion of IS256 also, and this biofilm-neg-ative phase variation reduces adhering capacity of the bacteria.

Keyword

Bacterial Adhesion; Biofilms; Cell Adhesion Molecules; Staphylococcus aureus

MeSH Terms

Bacterial Adhesion/*physiology
Biofilms/*growth & development
Cell Adhesion Molecules/genetics/*metabolism
Comparative Study
Equipment Contamination/prevention & control
Mutagenesis, Insertional/methods
Mutagenesis, Site-Directed/genetics
Phase Transition
Polysaccharides, Bacterial/genetics/*metabolism
*Polyurethanes
Species Specificity
Staphylococcus aureus/cytology/*physiology
Structure-Activity Relationship

Figure

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  Fig. 1 Congo red agar tests of 2 biofilm-negative phase variants (SA204/v-5, SA204/v-6), the revertant strain (SA204/r-6), and their parent strain (SA204). Biofilm forming strains show dark-red color, whereas biofilm-ngative strains show light-red color. Left upper quadrant, SA204; right upper quadrant, SA204/v-5; left lower quadrant, SA204/v-6; right lower quadrant, SA204/r-6.

• 

  Fig. 2 Biofilm assay of 2 biofilm-negative phase variants (SA204/v-5, SA204/v-6), the revertant strain (SA204/r-6), and their parent strain (SA204). Biofilm forming strains show red color. From left; SA204, SA204/v-5, SA204/v-6, and SA204/r-6.

• 

  Fig. 3 Pulse field gel electrophoresis (PFGE) analysis of biofilm-negative phase-variant strains, compared to parent strain and the revertant strain. Chromosomal DNA fragments, generated by SmaI digestion, were analyzed by PFGE. Lane 1 refers to Lambda ladder PFGE marker. All strains show same PFGE pattern. WT, SA204; V5, SA204/v-5; V6, A204/v-6; R5, SA204/r-6.

• 

  Fig. 4 PCR amplification of icaC gene in biofilm-negative phase-variant strains, compared to parent strain and the revertant strain. Lane 1 refers to Lambda/HindIII marker. Biofilm-negative phase-variant strains show shifted icaC-specific PCR product, the increased size of which is about 1.3 kb. WT, SA204; V5, SA204/v-5; V6, A204/v-6; R5, SA204/r-6.

• 

  Fig. 5 Southern blot with icaC-specific DNA probe in EcoRI-digested chromosomal DNA of biofilm-negative phase-variant strains, compared to parent strain and the revertant strain. Biofilm-negative phase-variant strains show shifted icaC-specific PCR product of increased size. WT, SA204; V5, SA204/v-5; V6, A204/v-6; R5, SA204/r-6.

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