Chronological study of antibiotic resistances and their relevant genes in Korean avian pathogenic Escherichia coli isolates

Abstract

Antibiograms and relevant genotypes of Korean avian pathogenic Escherichia coli (APEC) isolates (n = 101) recovered between 1985 and 2005 were assessed via disc diffusion test, PCR, restriction enzyme analysis, and sequencing. These isolates were highly resistant to tetracycline (84.2%), streptomycin (84.2%), enrofloxacin (71.3%), and ampicillin (67.3%), and most of the tetracycline, streptomycin, enrofloxacin, and ampicillin resistances were associated with tetA and/or tetB, aadA and/or strA-strB, mutations in gyrA and/or parC, and TEM, respectively. Class 1 integrons were detected in 40 isolates (39.6%), and a variety of gene cassettes conferring streptomycin (aadA), gentamicin (aadB), and trimethoprim (dfr) resistances were identified: aadA1a (27.5%), dfrV-orfD (2.5%), aadB-aadA1a (2.5%), dfrI-aadA1a (47.5%), dfrXVII-aadA5 (12.5%), and dfrXII-orfF-aadA2 (7.5%). In addition, several types of common promoters (P(ant)) of the gene cassettes (hybrid P1, weak P1, or weak P1 plus P2) and single-nucleotide polymorphisms in aadA1a were identified. The results of a chronological analysis demonstrated significant and continuous increases in the frequencies of resistances to several antibiotics (tetracycline, streptomycin, enrofloxacin, ampicillin, and trimethoprim-sulfamethoxazole) and of the relevant resistance genes (tetA, strA-strB, and TEM), mutations in gyrA and parC, and multidrug-resistant APEC strains during the period 2000 to 2005.

FIG. 1.

Strategy for the differentiation of common promoters (P1 and P2) of class 1 integron gene cassettes via REA. Arrowheads indicate enzymatic cleavage sites.

FIG. 2.

Amplified class 1 integron gene cassettes of APEC strains isolated from Korean chickens. Lanes: M, 1-kb molecular weight marker (iNtRON Biotechnology, Seoul, Republic of Korea); 1, 1,000 bp; 2, 1,478 bp; 3, 1,662 bp; 4, 1,680 bp; 5, 2,000 bp.

FIG. 3.

Molecular typing of P1 by REA. Each lane represents an individual isolate. (A) REA with AluI. Lanes 1 to 4, no cut by AluI; lanes 5 to 8, cut by AluI; M, 100-bp molecular weight marker (iNtRON Biotechnology, Seoul, Korea). (B) REA with HincII. Lanes 1 to 8, individual isolates showing no cut by HincII.

FIG. 4.

Molecular typing of P2. Each lane represents an individual isolate. (A) Molecular typing by PAGE. A 3-bp (GGG) insertion (85 bp) resulted in the creation of a new promoter, P2, which could be distinguished from the 82-bp promoter by an upward shift of the amplicon. (B) Molecular typing by REA (BsrgI). The 3-bp insertion destroys the enzyme site of BsrgI, and the enzyme does not recognize the P2 promoter.