Stanton Thaddeus B, Humphrey Samuel B
Pre-Harvest Food Safety and Enteric Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, PO Box 70, Ames, IA 50010, USA.
Appl Environ Microbiol. 2003 Jul;69(7):3874-82. doi: 10.1128/AEM.69.7.3874-3882.2003.
Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.
从喂食或未喂食金霉素的猪的盲肠内容物和盲肠组织中分离出对金霉素不敏感（64至256微克/毫升）的厌氧菌。使用一种营养复杂、基于瘤胃液的培养基培养这些细菌。根据其大球菌形态、在乳酸上的快速生长以及与埃氏巨球型菌LC-1(T)的16S核糖体DNA序列相似性，从7只不同动物的84株分离物中鉴定出8株为埃氏巨球型菌菌株。所有8株菌株的四环素最低抑菌浓度在128至256微克/毫升之间。基于区分14种四环素类别的PCR检测，这些菌株对tet(O)基因呈阳性反应。相比之下，从30年的培养物中获得的3株反刍动物埃氏巨球型菌菌株的四环素最低抑菌浓度为4微克/毫升，且不含四环素基因。扩增并克隆了两株四环素抗性埃氏巨球型菌菌株的四环素基因。这两个基因赋予重组大肠杆菌菌株四环素抗性（最低抑菌浓度 = 32至64微克/毫升）。序列分析表明，埃氏巨球型菌的基因代表由涉及tet(O)和tet(W)的类间（双交换）重组事件形成的两种不同的嵌合基因。在这些研究中分离出的8株四环素抗性埃氏巨球型菌菌株中的每一株都存在一种或另一种基因型。这些发现表明共生细菌不仅在肠道抗生素抗性的保存和传播中起作用，而且在抗性的进化中也起作用。
