Summary. Introduction

Transcription

1 MRSA biofilm ( ) Summary (MRSA) MRSA 2 MRSA MSSA 16 Stap ylococcus aureus ica P CR ica ica Introduction Staphylococcus aureus(2,3) (1) 1990 MRSA (7) (8) 90 MRSA (9,10) MRSA surgical-site infection(ssi) (11) SSI (13) SSI 50 S.aureus 59 MRSA (12) biofilm MRSA Biofilm Polysaccharide intercellular adhesion; PIA quorum sensing (14) biofilm in vitro (5,6) biofilm device S.aureus Staphylococcus biofilm biofilm Polymeric N-acetyl glucosamine(pnag)

2 intercellular adhesion(ica) gene (4) Figure1 Coagulase negative Staphylococcus (CoNS) biofilm atle icaadb (10) S.aureus icaadb biofilm Surgical site infection MRSA(12) Heilmann PCR icaadb genotype biofilm Phenotype icaa, D genotype biofilm Phenotype Figure1 Model of PIA biosynthesis.. icaa and icad synthesize oligomers derived from UDP-N-acetylglucosamine(4). Materials and Methods Bacterial samples MRSA(SAGA1001~1013) 2 MRSA(UOEH6,45) MSSA(UOEH79) 16 S.aureus Assay of biofilm on polystyrene MRSA TBS tryptosoya broth +1% glucose 37 24

3 TSB 1% glucose 200 l 96 plate over night culture full growth 1 l well triplicates well well PBS - 3 well biofilm 0.1% 1 well Polymerase chain reaction (PCR) boiling DNA rpm 2 DNA PCR 50 dose Primer forward 0.5 µ Primer reverse 0.5 µ Taq (Takara Ex Taq ) 0.5 µ 10xBuffer 5.0 µ MgCl2 25mM 4.0 µ H O 25.5 µ Sample 10 µ PCR 30 cycle Step initial denaturation at 94 for 2min Step2 denaturation at 94 for 1min Step3 annealing at 55 for 1min Step4 amplification at 72 for 2min Step5 Step2 30 cycle TAE+Ethyldium bromide DNAsize marker 100bp ladder PCR primer Table1 position icaadb 5 -TTATCAATGCCGAGTTGTC-3' S.epidermides specific (10) 5 -AGTTTAACGCGAGTGCGCTAT-3'

4 icaa 5 -ACACTTGCTGGCGCAGTCAA S.aureus specific (16) 5 -TGTTGGATGTTGGTTCCAGA icaa 5 -TCTCTTGCAGGAGCAATCAA S.epidermidis specific (15) 5 -TCAGGCACTAACATCCAGCA icad 5 -ATGGTCAAGCCCAGACAGAG S.aureus specific (16) 5 -TTGCTTTAAACATTGAAAATACT icad 5 -ATGGTCAAGCCCAGACAGAG S.epidermidis specific (15) 5 -CGTGTTTTCAACATTTAATGCAA Table1. primer 15,17,18 S.aureus ica gene Results Phenotype Figure2 biofilm (Phenotype) sample 2 5 biofilm sample biofilm biofilm biofilm 1 sample 2 No No control

5 Figure2 sample triplicates,biofilm sample No.1~10, No.11~16 control No.15 biofilm Negative control Figure2 biofilm sample No.1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 16 No.5,6,14,16 biofilm Genotype (PCR) icaabd, icaa, icad primer PCR genotype (Figure 3~7, band DNA size marker, No.1~16 ) Figure3. icaadb Sample No.1,3,5,16 546bp PCR Figure4. icaa Sample No.13,15, bp PCR

6 Figure5. icaa sample non-specific pattern Figure6. icad sample PCR Figure7. icad Figure6 sample PCR icaadb primer PCR No.1,5, bp PCR size non specific PCR 100bp primer dimer biofilm phenotype PCR (genotype) Table2 Table3 Table2 Table2 biofilm ± 4 sample15 10

7 sample biofilm negative control Materials and Methods

8 Biofilm formation Biofilm formation Presence of icaadb Presence of icaa positive negative total positive 4(100%) 9(75.0%) 13(81.3%) negative 0(0%) 3(25.0%) 3(18.7%) total 4(25.0%) 12(75.0%) 16(100%) Presence of icad positive negative total positive 4(100%) 9(75.0%) 13(81.3%) negative 0(0%) 3(25.0%) 3(18.7%) total 4(25.0%) 12(75.0%) 16(100%) Biofilm formation positive negative total positive 4(100%) 9(75.0%) 13(81.3%) negative 0(0%) 3(25.0%) 3(18.7%) total 4(25.0%) 12(75.0%) 16(100%) Table3 biofilm formation ica gene icaadb, A, D positive or negative biofilm Discussion icaadb gene primer Miyamoto H et al (10) primer S.epdermidis icaadb gene 546bp primer 10 sample S.epidermides S.aureus ica gene primer (15) primer Table2 icaadb sample biofilm No.1, 3, 5, 16 icaadb sample 75.0 biofilm No.2, 4, 6, 7, 8, 9, 10, 14 (17) biofilm icaadb biofile ica gene icaa S.aureus primer icaa icaa positive 3samples biofilm 1sample(33.3 icaa negative 13samples biofilm 12samples(92.3%) icad S.aureus primer icad icad positive 10samples biofilm samples 80 icad negative 6samples biofilm 5samples(83.3%) S.aureus primer icaa, icad negative sample biofilm icaa, icad biofilm Rohde et al 16 icaa, icad biofilm S.aureus icaa, icad

9 biofilm S.epidermidis primer(icaa icad ) 15 PCR sample icaa, icad Arciola et al 15 icaa, icad S.aureus S.epidermidis Arciolara et al (15) 23 S. aureus icaa gene Horstkotte 80 S.aureus icaa gene (Horstkotte MA, Knobloch JKM, Rohde H, Mack D, Unpublished data) (16) Cramton (17) icaa gene biofilm S.aureus biofilm ica gene sample sample primer References 1. Arakawa Y, Ike Y, Nagasawa M, Shibata N, Doi Y, Shibayama K, Yagi T and Kurata T: Trends in antimicrobial-drug resistance in Japan. Emerg Infect Dis(2000)6: Tenover FC and Gaynes RP: The epidemiology of Staphylococcus infections; in Gram-Positive Pathogens, Fischetti VA, Novick RP, Ferretti JJ, Portnoy DAand Rood JI eds, American Society for Microbiology, Washington DC(2000)pp Konno M: Nosocomial infections caused by methicillin-resistant StaphylococcuS.aureus in Japan. J Infect Chemother(1995)1: Gotz F: Staphylococcus and biofilms. Mol Microbiol(2002)43: Donlan RM and Costerton JW: Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev(2002)15: Hall-Stoodley, L, Costerton JW and Stoodley P: Bacterial biofilms: From the natural environment to infectious disease. Nature Rev Microbiol(2004)2: Witte W, Braulke C, Heuck D, Cuny C: Analysis of nosocominal outbreaks with multiply and mathicillin-resistant StaphylococcuS.aureus (MRSA) in Germany: implications for hospital hygiene. Infection (1994)22: The Hospital Infection Control Practices Advisory Committee, Centers for Disease Control and Prevention: Guideline for isolation precautions in hospitals: Part. Recommendations for isolation

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