Journal List > J Bacteriol Virol > v.38(4) > 1033906

Cho, Jin, Cui, Yoon, and Ryu: Effects of Biofilm Formation on The Antimicrobial Susceptibility of Staphylococcus aureus


Staphylococcus aureus induces chronic infection in form of biofilm that exists in the host cells and arthroplastic prosthesis surface. In this study, the biofilm formation ability of S. aureus clinically isolated from bacteremia patients, biofilm processing and relationship of resistance to antibiotics, and difference of biofilm formation ability on different prosthetic material surfaces were studied. All of them formed biofilm and especially 6 strains of S. aureus had high ability of biofilm formation. In addition, it was found that some strains with higher biofilm formation ability make more higher polysaccharide layer production. When S. aureus ATCC 25923 forms biofilm, minimal bactericidal concentration (MBC) of biofilm bacteria is more increased than that of the planktonic state bacteria about one thousand folds. Especially, after 6 hours from starting on biofilm formation, the resistance to antibiotics was increased by more than 256 μg/ml of MBC to every antibiotics and after 8 hours prominent increase (more than 4096 μg/ml) was noted. Biofilm formation after bacterial adherence to plastic cover-slip was increased with time-dependent manner. Microcolonies were formed after 5 hours from a point that bacteria adhere to plastic cover-slip surface and after 6 hours biofilm was diffusely formed on entire surface, and then after 8 hours very thick biofilm was formed. Thicker biofilm was found on cobalt-chromium than titanium surface. These results suggest that titanium alloy materials are better than cobalt-chromium to minimize S. aureus biofilm formation on the arthroplastic material surface. Also, when microcolonies are formed after adherence of S. aureus to the arthroplastic material surface, resistance to antibiotics is starting.


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Figure 1.
Detection of glycocalyx formation ability of Staphylococcus aureus isolated from bacteremia patients. S. aureus strains were cultured on the congo red plates for 24 hrs, and colony morphology was observed. (A) Black colonies of highly-slime-producing S. aureus H05-749. (B) Bordeaux colonies of moderate-slime-producing S. aureus H05-736. (C) Red colonies of nonslime-producing S. aureus H05-735.
Figure 2.
Biofilm formation kinetics of S. aureus ATCC 25923 in tryptic soy broth with 0.5% glucose. A photograph of typical crystal violet-stained biofilms is shown at top of the panel and spectrophotometric results are charted with respect to time at the bottom of the panel.
Figure 3.
Microscopic photographs of S. aureus ATCC 25923 incubated on the plastic cover-slips for 4, 5, 6, and 8 hrs (100 × magnification). Arrow indicates the microcolony of S. aureus.
Figure 4.
Microscopic photographs of biofilm of S. aures ATCC 25923 incubated on the artificial bone surfaces for 24 hrs without shaking. Ti-ELi; titanium, Co-Cr; cobalt-chromium.
Figure 5.
Microscopic photographs of biofilm of S. aures ATCC 25923 incubated on the artificial bone surface for 1, 2, 4, and 6 days with shaking (3 × magnification).
Table 1.
Association of biofilm production with slime production in Staphylococcus aureus isolates from bacteremia patients
Tested strains OD at 570 nm Colony color on the CRA
H05-727 1.276 Bordeaux
H05-735 1.093 Red
H05-736 1.152 Bordeaux
H05-749 2.489 Black
H05-756 1.007 Bordeaux
H05-764 1.367 Bordeaux
H05-774 2.671 Black
H05-877 1.499 Bordeaux
H05-887 1.361 Bordeaux
H05-896 3.019 Black
H05-914 1.472 Bordeaux
H05-915 2.303 Black
H05-916 3.919 Black
H05-918 2.305 Black
H05-929 1.347 Bordeaux
H05-931 1.559 Bordeaux
H05-932 1.105 Bordeaux
ATCC14458 1.622 Bordeaux
ATCC25923 2.725 Black
ATCC29213 1.562 Bordeaux

: congo red agar

Table 2.
Minimum bactericidal concentration (MBC, μg/ml) of planktonic bacteria (PB) and biofilm-forming bacteria (BB) of S. aureus ATCC 25923
Antiicrobial agents PB BB
Clarithromycin 1 >2080
Cefotaxime 4 >2080
Cefmetazole 2 >2080
Erythromycin 4 >2080
Benzylpenicillin 2 >2080
Vancomycin 4 >2080
Table 3.
Change of minimum bactericidal concentration (MIC, μg/ml) of biofilm-forming sessile S. aureus ATCC 25923 with the biofilm ages
Biofilm age (hours) Antimicrobial agents
0 1 4 2 4 2 4
2 <256 <256 <256 <256 <256 <256
4 <256 <256 <256 <256 <256 <256
6 512 512 256 1024 512 512
8 2048 >4096 >4096 >4096 >4096 4096
10 >4096 >4096 >4096 >4096 >4096 >4096
24 >4096 >4096 >4096 >4096 >4096 >4096
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