Aim: To explore the possibility of repression of chloromycetin (Cm) acyl transferase by using external guided sequence (EGS) in order to converse the clinical E coli isolates from Cm- resistant to Cm- sensitive.
Methods: EGS directed against chloromycetin acetyl transferase gene (cat) was cloned to vector pEGFP-C1 which contains the kanamycin (Km) resistance gene. The recombinant plasmid pEGFP-C1+EGScat1+cat2 was constructed and the blank vector without EGS fragment was used as control plasmids. By using the CaCl(2) transformation method, the recombinant plasmids were introduced into the clinically isolated Cm resistant but Km sensitive E coli strains. Transformants were screened on LB agar plates containing Km. Extraction of plasmids and PCR were applied to identify the positive clones. The growth curve of EGS transformed bacteria cultured in broth with Cm resistance was determined by using spectrophotometer at A(600). Drug sensitivity was tested in solid culture containing Cm by using KB method.
Results: Transformation studies were carried out on 16 clinically isolated Cm-resistant (250 microg/mL of Cm) E coli strains by using pEGFP-C1-EGScat1cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after the transformation using EGS. Of the 16 tested strains, 4 strains were transformed successfully. Transformants with EGS plasmid showed growth inhibition when grown in liquid broth culture containing 200 microg/mL of Cm. In drug sensitivity test, these strains were sensitive to Cm on LB-agar plates containing 200 microg/mL of Cm. Extraction of plasmids and PCR amplification showed the existence of EGS plasmids in these four transformed strains. These results indicated that the Cat of the four clinical isolates had been suppressed and the four strains were converted to Cm sensitive ones.
Conclusion: The EGS directed against Cat is able to inhibit the expression of Cat, and hence convert Cm-resistant bacteria to Cm-sensitive ones. Thus, the EGS has the capability of converting the phenotype of clinical drug-resistant isolates strains to drug-sensitive ones.