Taketoshi Arai

Genetic Transformation of Vibrio parahaemolyticus, Vibrio alginolyticus , and Vibrio cholerae Non O-1 with Plasmid DNA by Electroporation

An electroporation procedure for the plasmid‐mediated transformation of the genus Vibrio was performed, as part of an effort to develop recombinant DNA techniques for genetic manipulation of the genus Vibrio. Vibrio parahaemolyticus, V. alginolyticus, and V. cholerae non O‐1 (9 different strains) were transformed with 3 vector plasmids (pACYC184, pHSG398, and pBR325). The efficiency of transformation was highly dependent on three parameters: the concentration of plasmid DNA; the strength of the electric field; and the combination of plasmid DNA and recipient strain. The drug‐resistance genes on the vector plasmid were expressed in the Vibrio strains.

Isolation of a New Drug-Resistance Plasmid from a Strain of Vibrio parahaemolyticus

A new R plasmid, pSA55, with a molecular weight of 112 mcgadaltons (Md), was isolated from a strain of Vibrio parahaemolyticus with multiple drug resistance. The pSA55 plasmid conferred on its host resistance to chloramphenicol, tetracycline, streptomycin, kanamycin, ampicillin, trimethoprim and 2,4‐diamino‐6,7‐diisopropyl pteridine, and belongs to incompatibility group C. The plasmid was transferable to Escherichia coli, V. parahaemolyticus, V. alginolyticus and NAG bivrio at a frequency of 10−3~‐7, and was stably inherited by the transconjugants of these species. The conjugal transfer of pSA55 plasmid was significantly affected by the growth culture phase. The resistance pattern and resistance levels of transconjugants were the same as those of the donor strain. We did not observe fluctuations in minimal inhibitory concentrations with transfer, unlike the case of V. cholerae. The relationship between the pSA55 plasmid and the Kanagawa phenomenon was not clarified in the present study.

Mechanisms of Plasmid-Mediated Antibiotic Resistances in Vibrio parahaemolytcus

The mechanisms of drug resistance of clinical isolate, Vibrio (V.) parahaemolyticus ST550, resistant to chloramphenicol (CP), aminoglycoside antibiotics (AGs) and β‐lactam antibiotics were investigated. The mechanisms of resistance to CP, AGs and β‐lactam antibiotics were dependent on chloramphenicol acetyltransferase (CAT), aminoglycoside‐3″‐adenylyltransferase AAD(3″) and aminoglycosides‐3′‐phosphotransferase APH(3′) and TEM type penicillinase, respectively.

Pyomelanin Production by Pseudomonas aeruginosa

Pseudomonas aeruginosa was successfully transformed from a pyomelanin‐producing strain to a non‐pyomelanin‐producing strain by genetic transformation, with an average frequency of 1.17 × 1−3/recipient. Although the transformation frequency was not affected by doses of DNA between 17 and 51 μg/ml, it was influenced by the growth phase of the recipient bacteria, i.e., it was highest in the late logarithmic phase. Biochemical functions of the transformants were the same as those of the recipient strain except for pyomelanin production. Some of them, however, showed an intermediate growth behavior and cell arrangement between the donor and recipient. The serological type of the donor strain was sometimes contransduced although a few transformants became nonagglutinable with either donor or recipient type antiserum. The pyomelanin producing activity and serological type gained of some transformants were eliminated by either subculturing in nutrient broth or acridine treatment. The results obtained suggested that the pyomelanin productivity of P. aeruginosa is controlled by a plasmid.