Conclusion The present results suggest that TSST-1 production is not directly associated with the agr structure, but is instead controlled by unknown transcriptional/translational regulatory systems, or synthesized by multiple regulatory mechanisms that are interlinked in a complex manner. Methods Bacterial strains Of 152 clinical MRSA isolates that we analyzed, 66 were randomly selected from the nationwide MRSA collection representing various regions of Japan in 2003, and the remainder was isolated from the bloodstream of patients in different wards at a university hospital between 1996 and check details 2003. Detection of the tst gene and agr-genotyping by
PCR Bacterial chromosomal DNA was extracted after overnight growth on Luria Bertani agar as described . We detected
the tst gene by PCR amplification using the specific primers, TGT AGA TCT ACA AAC GAT AAT ATA AAG GAT (forward) and ATT AAG CTT AAT TAA TTT CTG CTT CTA TAG TT (reverse). Genes were amplified by denaturation for 5 min Selleckchem Ku0059436 at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 52°C, 60 s at 72°C and a final extension at 72°C for 5 min in a 25-μl mixture, comprising 1 μl template DNA, 0.2 mM dNTP mix, 1.5 mM 10× Ex buffer (Takara, Tokyo, Japan), 1.25 U Ex Taq (Takara) and 0.5 μM each of the forward and reverse primers. The agr class was determined by PCR amplification of the hypervariable domain of the agr locus using specific oligonucleotide primers as described . Preparation of recombinant partial TSST-1 and anti TSST-1 antibody Fragments of the tst gene
DNA were amplified by PCR using primers with BglII-HindIII restriction sites (Table 3). Amplified 280-bp DNA fragments were subcloned into the pBluescriptII plasmid, digested with EcoRV and transformed into Escherichia coli DH5α, which was then digested with BglII and HindIII. The BglII -HindIII fragment of E. coli DH5α was subcloned into the BamHI-HindIII site of pQE30 (Qiagen, Hilden, Germany) and transformed Avelestat (AZD9668) into E. coli JM109. His-tagged recombinant partial TSST-1 protein (rTSST-1) was expressed in E. coli JM109 and the cells were lysed using a French press (SLM Instruments, Inc., IL, USA). Recombinant TSST-1 was purified from the cell lysate using Ni-NTA agarose (Qiagen) according to the manufacturer’s instructions. Purified rTSST-1 (100 μg/ml) was emulsified with an equal volume of Freund’s complete adjuvant (Difco, NJ, USA) and subcutaneously injected into Japanese white rabbits to generate anti-TSST-1 antiserum. A second antibody response was elicited by immunization with the antigen alone and serum was collected. Table 3 Primers used in this study.