Kasimanickam V R, Owen K, Kasimanickam R K
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA.
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA.
Theriogenology. 2016 Jan 15;85(2):173-9. doi: 10.1016/j.theriogenology.2015.10.014. Epub 2015 Oct 23.
Reckless use of antibiotics and/or development of biofilm are the rationale for the development of multidrug resistance (MDR) of pathogenic bacteria. The objective of the present study was to detect MDR genes in Trueperella pyogenes and to detect biofilm virulence factor (VF) genes in Escherichia coli isolated from the uterus of postpartum dairy cows. Uterine secretions from different parity postpartum Holstein cows (n = 40) were collected using cytobrush technique after a sterile procedure from cows with varying degree of uterine inflammatory conditions. The cytobrush was stored in a specimen collector, placed in a cooler with ice, and transported to the laboratory within 2 hours. The pathogens were isolated and were identified initially by their colony morphology and biochemical characteristics. To further identify and classify the single species, and to determine the presence of MDR and VF genes, the genes fragments were amplified using the respective primers by either singleplex or multiplex polymerase chain reaction protocol, and amplicons were detected by electrophoresis method. T pyogenes was isolated in 17 of 40 (42.5%) cows in the study population as recognized by the 16S rRNA gene. Of the positive T pyogenes samples, 8 of 17 (42.1%) were positive for integron type 1 (intI I), and none were positive for integron type 2 (intI II). Of those 8 positive for intI I, six of eight (66.7%) were positive for amplicons aadA5 and aadA24-ORF1 at 1048 and 1608 bp, respectively, associated with specific drug resistance. Presence of addA5 indicated resistance to sulfadiazine, bacitracin, florfenicol, and ceftiofur. Presence of addA24-ORF1 indicated resistant to sulfadiazine, bacitracin, penicillin, clindamycin, and erythromycin. E coli was isolated in 18 of 40 (45.0%) cows in the study population. The genes for VF, Agn43a, and Agn43 b, associated with biofilm production, were found in 6 of 18 (33.3%) of the positive isolates. Both T pyogenes MDR gene and E coli biofilm VF existed in more severe form of uterine diseases than subclinical endometritis. In conclusion, 35% of T pyogenes isolates found were positive for a gene cassette associated with antibiotic resistance, and 33% of the E coli isolates contained genes for the VF associated with biofilm production.
滥用抗生素和/或生物膜的形成是病原菌产生多重耐药性（MDR）的原因。本研究的目的是检测化脓放线杆菌中的多重耐药基因，并检测从产后奶牛子宫中分离出的大肠杆菌中的生物膜毒力因子（VF）基因。采用细胞刷技术，在无菌操作后，从不同程度子宫炎症的产后荷斯坦奶牛（n = 40）中收集子宫分泌物。细胞刷保存在标本收集器中，置于有冰的冷却器中，并在2小时内运至实验室。分离病原菌并首先根据其菌落形态和生化特征进行鉴定。为了进一步鉴定和分类单一菌种，并确定多重耐药基因和毒力因子基因的存在，使用各自的引物通过单重或多重聚合酶链反应方案扩增基因片段，并通过电泳法检测扩增产物。在研究群体的40头奶牛中，有17头（42.5%）分离出化脓放线杆菌，通过16S rRNA基因得以确认。在17份化脓放线杆菌阳性样本中，17份中有8份（42.1%）整合子1型（intI I）呈阳性，整合子2型（intI II）均为阴性。在这8份intI I阳性样本中，8份中有6份（66.7%）分别在1048 bp和1608 bp处的扩增产物aadA5和aadA24 - ORF1呈阳性，与特定耐药性相关。addA5的存在表明对磺胺嘧啶、杆菌肽、氟苯尼考和头孢噻呋耐药。addA24 - ORF1的存在表明对磺胺嘧啶、杆菌肽、青霉素、克林霉素和红霉素耐药。在研究群体的40头奶牛中，有18头（45.0%）分离出大肠杆菌。在18份阳性分离株中，有6份（33.3%）发现了与生物膜产生相关的毒力因子基因Agn43a和Agn43 b。化脓放线杆菌多重耐药基因和大肠杆菌生物膜毒力因子在子宫疾病较严重的形式中比亚临床子宫内膜炎中更为常见。总之，发现35%的化脓放线杆菌分离株对与抗生素耐药相关的基因盒呈阳性，33%的大肠杆菌分离株含有与生物膜产生相关的毒力因子基因。
