Characterization of antibiotic resistance and pathotypes of avian pathogenic Escherichia coli isolates in Korea

Abstract

Antimicrobial therapy has been an important tool in reducing the enormous losses caused by avian pathogenic Escherichia coli (APEC) infections in the poultry industry. However, it has been less effective due to antibiotic resistance in APEC which has also been increased dramatically. 101 APEC Korean isolates from 1985 to 2005 were tested for fundamental information of antibiotic resistance in Korea. Antibiograms and relevant genotypes of these isolates were assessed via disc diffusion test, polymerase chain reactions (PCR), restriction enzyme analysis (REA), and sequencing. Significant increases of resistances to several antibiotics were observed during these periods. Resistance to streptomycin and tetracycline (both at 84.2%) was highest, followed by enrofloxacin (71.3%), ampicillin (67.3%), trimethoprim/sulfamethoxazole (37.6%), gentamicin (26.7%). Relevant resistance genes (tetA, tetB, aadA, strA-strB and TEM) and mutations in certain region (gyrA and parC) were increased during same period. As shown in this test results, the tendency of antibiotic resistance has been increased and another protection tool will be needed urgently. One hundred-one APEC Korean isolates were examined for the characteristics that affect pathogenicity. The serotypes and virulence genes of isolates were determined and they were classified into molecular pathotypes (MPs) on the basis of virulence gene content. Only twenty-eight isolates (27.8%) were serotyped and the frequencies of virulence genes were various. The frequency of iroN (100%) was highest, followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), irp2 (43.6%), and vat (10.9%). All isolates were classified into 27 MPs on the basis of virulence gene content. The virulence gene profiles of MPs showed a cumulative pattern. Therefore, the molecular pathotyping can be used for identifying higher pathogenic APEC isolates. Eleven APEC isolates were selected to investigate correlation between MPs and pathogenicity. Seven-day-old chickens were inoculated subcutaneously with 10-fold serial dilutions of each isolates (109 to 106 CFU/0.2 ml) to improve reproducibility of virulence assay and obtain statistical significance. Necropsy, gross pathological examinations, and re¬¬-isolation in infected chickens were conducted. Based on LD50 of infected chickens, 11 APEC isolates were classified into lethality classes (LC) 1 to 3 as follows: LC1 (LD50 ≤ 5 X 106 CFU), LC2 (5 X 106 to 108 CFU), and LC3 (≥ 5 X 108 CFU). The ratio LC1, LC2, and LC3 were 18.2% (2/11), 27.3% (3/11), and 54.5% (6/11), respectively. Chickens inoculated with LC1 isolates showed clinical signs of illness as early as 24 hours, and some of chickens were found dead 24 hours after infection. All samples collected from chickens inoculated APEC isolates showed positive re-isolation result. The MPs of higher pathogenic isolates (LC1

lower 50% lethal dose), E64 (MP26

iroN-fimC-ompT-hlyF-iucD-iss-fyuA-irp2-vat) and E89 (MP25

iroN-fimC-ompT-hlyF-iucD-iss-fyuA-irp2-tsh) were more abundant possession of virulence genes than others, which suggests the correlation between MPs and pathogenicity of APEC isolates. The present study supplied fundamental information of antibiotic resistance in Korean APEC isolates and showed molecular pathotyping might be a powerful tool for identifying higher pathogenic APEC isolates. The efficacy of higher pathogenic isolates (LC 1) as a vaccine candidate may be needed in further study.