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Identification, susceptibility, and detection of integron-gene cassettes of Arcanobacterium pyogenes in bovine endometritis

M.-C. Liu^*,, C.-M. Wu^*, Y.-C. Liu, J.-C. Zhao, Y.-L. Yang and J.-Z. Shen^*,1

^* Department of Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, P. R. China
College of Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110161, P. R. China

¹ Corresponding author: sjz{at}cau.edu.cn

The present study aimed to identify, determine the susceptibility, and detect gene cassettes of Arcanobacterium (Actinomyces) pyogenes isolates from cows with endometritis. Arcanobacterium pyogenes isolates were identified first by using the API Coryne Vit system test, and further through PCR. Minimum inhibitory concentrations of 23 antimicrobial agents against A. pyogenes were tested using standard broth microdilution assays according to the protocols of the Clinical and Laboratory Standards Institute. The genes of integrons I and II were amplified by PCR using specific primers. Thirty-two A. pyogenes isolates were isolated from 136 endometritic cows in the Hohhot region. Antibiotic susceptibility tests revealed that all isolates were highly sensitive to fluoroquinolones (100%), macrolides (~81.2 to 100%) and florfenicol (90.6%), aminoglycosides (~15.6 to 81.2%), and tetracyclines (~43.7 to 68.7%). However, 53.1% were resistant to clindamycin, ~50 to 65.6% were resistant to penicillins, and ~37.5 to 71.9% were resistant to cephalosporins. One hundred percent were resistant to sulfonamides and bacitracin zinc. The integrons were further confirmed by sequencing. No class II integrons were detected, whereas 50% (n = 16) of the A. pyogenes isolates were positive for the presence of the intI I gene, but only 13 contained gene cassettes. Sequence analysis of gene cassettes revealed 6 gene cassettes, 4 of which encode resistant determinants of aminoglycosides (aadA1, aadA5, aadA24, and aadB) and 1 of which encodes the resistance gene of chloramphenicol (cmlA6). The function of the sixth identified cassette, designated ORF1, is unknown. The gene cassette arrays aadA24-ORF1, aadA5, and aadA1-addB-cmlA6 were found in 46.13% (6/13), 38.46% (5/13), and 38.46% (5/13) of the isolates, respectively. These cassettes segregated according to a consistent pattern, with aadA5 always alone, ORF1 always with aadA24, and aadA1-aadB and cmlA6 always together. Most of the positive integrons existed in the multiresistant isolates (n = ~3 to 7), indicating that the integrons played an important role in the dissemination and spread of antimicrobial resistance. This is the first report of A. pyogenes infections in dairy cows in China and of detection of gene cassettes and integrons in A. pyogenes.

Key Words: Arcanobacterium (Actinomyces) pyogenes • antimicrobial susceptibility • integron • gene cassette