Kikuo Yamazoe

New Insertion Sequence Elements in the Upstream Region of cfiA in Imipenem-Resistant Bacteroides fragilis Strains

ABSTRACT The 747-bp cfiA gene, which encodes a metallo-β-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA1 to cfiA10) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-β-lactamases with full catalytic activities. PCR assay indicated that six metallo-β-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, one of the IS elements previously identified in the upstream region of cfiA. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 family. All the IS elements appear to have putative promoter motif sequences (the −7 regions TAnnTTTG motif and the −33 regions TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.

Simultaneous high-performance liquid Chromatographic determination of carboplatin, epirubicin hydrochloride and mitomycin C in a Lipiodol emulsion

A novel HPLC method for the simultaneous determination of carboplatin (CBDCA), epirubicin hydrochloride (EPI) and mitomycin C (MMC) was developed in order to determine these drugs in a Lipiodol emulsion for intra-arterial injection and in the medium of a drug-release test of the emulsion. The HPLC conditions included a reversed-phase column and a gradient programme of the mobile phase using 0.01 M phosphate buffer (pH 3.0)-acetonitrile. The accuracies of analysis for each drug added to the emulsion and the drug-release medium were in the ranges 96–104% and 98–108%, respectively. The R.S.D.s in the repeatability of the determination of each drug in the emulsion and in the drug-release medium were < 8% and < 2%, respectively. The method is simple and reliable enough to be utilized for the investigation of drug contents and drug release of the Lipiodol emulsion containing CBDCA, EPI and MMC.

Compatibility of Bricef Dry Syrup with Mixed Solutions

Compatibilty of Bricef Dry Syrup (cefatrizine oral suspension) with mixed solutions which were often pre scribed in our hospital was investigated in terms of changes in external appearance pH and residual potency of cefatrizine (CFT). These changes were checked immediately after and 1, 3, 5, 7 and 14 days after mixing. The results showed lowering, to some extent, of CFT potency in relatively high pH region. Especially, when dry syrup was mxed with an alkaline solution, CFT potency reduced significantly within 24 hours at room temperature. These results suggest that careful attention should be paid to the above combination since a risk of incompatibility may be involved.Color of suspensions of a few preparations changed during the storage. The change in color should be avoided to prevent anxiety in patients, though its correlation with stability of CFT is not clear. In combiantions with other solutions, the change in appearance, pH and potency of CFT was small, suggesting that such combinations be clinically recommendable.