Liu M-C, Wu C-M, Liu Y-C, Zhao J-C, Yang Y-L, Shen J-Z
Department of Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China.
J Dairy Sci. 2009 Aug;92(8):3659-66. doi: 10.3168/jds.2008-1756.
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 (approximately 81.2 to 100%) and florfenicol (90.6%), aminoglycosides (approximately 15.6 to 81.2%), and tetracyclines (approximately 43.7 to 68.7%). However, 53.1% were resistant to clindamycin, approximately 50 to 65.6% were resistant to penicillins, and approximately 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 = approximately 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.
本研究旨在鉴定、测定化脓隐秘杆菌（放线菌属）分离株的药敏性，并检测来自患子宫内膜炎奶牛的该菌基因盒。首先使用API Coryne Vit系统试验鉴定化脓隐秘杆菌分离株，然后通过聚合酶链反应（PCR）进一步鉴定。根据临床和实验室标准协会的方案，采用标准肉汤微量稀释法检测23种抗菌药物对化脓隐秘杆菌的最低抑菌浓度。使用特异性引物通过PCR扩增整合子I和II的基因。从呼和浩特地区136头患子宫内膜炎的奶牛中分离出32株化脓隐秘杆菌。药敏试验表明，所有分离株对氟喹诺酮类药物（100%）、大环内酯类药物（约81.2%至100%）、氟苯尼考（90.6%）、氨基糖苷类药物（约15.6%至81.2%）和四环素类药物（约43.7%至68.7%）高度敏感。然而，53.1%的分离株对克林霉素耐药，约50%至65.6%对青霉素耐药，约37.5%至71.9%对头孢菌素耐药。100%的分离株对磺胺类药物和杆菌肽锌耐药。通过测序进一步确认整合子。未检测到II类整合子，而50%（n = 16）的化脓隐秘杆菌分离株intI I基因呈阳性，但只有13株含有基因盒。基因盒序列分析显示有6个基因盒，其中4个编码氨基糖苷类耐药决定簇（aadA1、aadA5、aadA24和aadB），1个编码氯霉素耐药基因（cmlA6）。第六个已鉴定的基因盒（命名为ORF1）的功能未知。分别在46.13%（6/13）、38.46%（5/13）和38.46%（5/13）的分离株中发现基因盒阵列aadA24 - ORF1、aadA5和aadA1 - addB - cmlA6。这些基因盒按一致模式分离，aadA5总是单独存在，ORF1总是与aadA有关A24，aadA而1 - aadB和cmlA6总是在一起。大多数阳性整合子存在于多重耐药分离株中（n =约3至7），表明整合子在抗菌药物耐药性的传播和扩散中起重要作用。这是中国关于奶牛化脓隐秘杆菌感染以及化脓隐秘杆菌中基因盒和整合子检测的首次报道。
