Epidemic Strain Shigella dysenteriae Type 1 Dt66 Encodes Several Drug Resistances by Chromosome

Abstract

Multiple antibiotic-resistant strains of Shigella dysenteriae type 1 were isolated from an epidemic in West Bengal, India (1984). During the past two decades, much attention was given to reevaluation of treatment recommendations. However, there are no useful data on drug resistance encoded by chromosome. A total of 300 strains of Shigella dysenteriae type 1 were isolated from an epidemic. Strains were biochemically identified by API 20E system and further confirmed serologically. Antibiotic susceptibility was determined by disk diffusion method and plasmid DNA was prepared by alkaline lysis procedure. Elimination of plasmids was achieved by curing with acridine orange from a representative epidemic strain S. dysenteriae 1 Dt66. PFGE was performed for typing of wild-type and plasmid-cured strains. Southern blot of PFGE separated XbaI digested chromosomal DNA was done onto positively charged nylon membrane. For Southern hybridization, plasmid DNA was used as probe. All isolates showed identical drug resistance patterns and plasmid profiles. All these isolates contained six plasmids ranging in sizes from 3 to 145 kb. We have eliminated all the plasmids from a representative strain of S. dysenteriae 1 Dt66 by using acridine orange as curing agent. All epidemic Shigella isolates were resistant to amoxycillin, ampicillin, bacitracin, carbenicillin, cefixime, ceftazidime, chloramphenicol, clarithromycin, erythromycin, fusidic acid, methicillin, penicillin G, polymixin B, streptomycin, rifampicin, tetracycline and vancomycin, among 29 antibiotics used. Out of 17 resistant antibiotics, 12 were encoded by chromosome. Resistance to ampicillin, chloramphenicol, streptomycin, tetracycline and ceftazidime was plasmid encoded. Southern blot hybridization showed the recognition of two clear sites in the chromosome used plasmid DNA of Dt66 strain as probe, which reveled some sequential genetic homology between chromosome and plasmids. Pulsed-field gel electrophoresis (PFGE) was performed for typing of the chromosome of plasmidless strains of Dt66 and wild-type strain Dt66 (having plasmids) that remain unaltered. Seventy percent drug-resistant loci of Shigella dysenteriae 1 Dt66 are present in chromosome and the remaining are plasmid mediated.