Tetracycline-resistant Chlamydia suis isolates in Italy
Tetracycline-resistant Chlamydia suis isolates in Italy
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DOI：
10.1136/vr.163.8.251
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发表时间：
2008-08-23
影响因子：
2.2
通讯作者：
Cevenini, R.
中科院分区：
文献类型：
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作者：
Di Francesco, A.;Donati, M.;Cevenini, R.
抗菌剂常用于预防或治疗衣原体感染。四环素类抗生素以这种方式的广泛使用鼓励了对耐药微生物的选择。本研究的目的是评价在意大利分离的一些猪衣原体菌株对四环素的敏感性。在2004年至2007年期间，从北方和意大利南部的四个不同农场饲养的结膜和/或生殖疾病猪中收集了14个衣原体分离株。使用市售试剂盒（Tissue Kit; Qiagen）提取在LLC-MK 2细胞（一种源自恒河猴肾组织的连续细胞系）中生长的分离株的DNA用于分子分析，并用作扩增衣原体ompA基因的1050个碱基对（bp）片段的模板（沙耶达等，1995）。使用市售试剂盒（QIAquick PCR Purification Kit; Qiagen）纯化扩增子并测序。使用BLAST软件，将核苷酸序列与GenBank数据库中可获得的C. suis型菌株S45的相同区域进行比较。ompA扩增显示预期产物。将14个分离株的MOMP蛋白的推导氨基酸序列与参考CsuisS 45株的相同序列进行比对，发现氨基酸同源性为81%至91%。检测了14个分离株对强力霉素的敏感性，并与沙眼衣原体D血清型的意大利尿道分离株GO 86对强力霉素的敏感性进行了比较，鹦鹉热嗜衣原体的参考菌株6 BC，以及嗜衣原体、流产嗜衣原体、山羊嗜衣原体和猫嗜衣原体的分离株。如之前Donati等人（2002）所述，在24孔板中生长的LLC-MK2细胞中进行了敏感性试验。在用衣原体孵育细胞单层并离心后，除去培养基并用含有连续两倍稀释的多西环素的培养基代替。所有测试均重复三次。在35 ℃孵育48小时后，将培养物固定并染色，以使用对衣原体脂多糖属特异性抗原具有特异性的荧光素结合的单克隆抗体通过免疫荧光检测衣原体包涵体。多西环素的最低抑菌浓度（MIC）定义为与无药物对照相比，阻止检测到90%以上衣原体包涵体的最低浓度。通过抽吸含抗菌剂的培养基，用磷酸盐缓冲盐水洗涤威尔斯孔两次，加入不含抗菌剂的培养基，并在35 ℃下再孵育平板48小时，测定最小杀菌浓度（MBC）。MBC是最低的抗菌剂浓度，导致夹杂物减少90%以上。MIC值≥ 4 µg/ml和MBC值≥ 8 µg/ml定义为表明分离株耐药（勒纳特等，2001）。Dugan等人（2004）证明，猪衣原体四环素耐药（Tcr）表型与整合到衣原体染色体中的耐药基因泰特（C）相关。因此，通过PCR试验扩增泰特（C）基因编码区的525 bp产物，检测猪衣原体分离株是否存在泰特（C）抗性基因。根据Dugan等人（2004），使用以下正向和反向寡核苷酸引物进行反应：CS43 5 '-AGCACTGTCCGACCGCTTTG-3'和
ANTIMICROBIAL agents are often used to prevent or treat chlamydial infections. The widespread use of tetracycline antibiotics in this way has encouraged selection for resistant organisms. The aim of this study was to evaluate the sensitivity to tetracycline of some strains of Chlamydia suis isolated in Italy. In the period from 2004 to 2007, 14 chlamydial isolates were collected from pigs with conjunctival and/or reproductive disorders reared in four different farms in northern and southern Italy. DNA from the isolates grown in LLC-MK2 cells (a continuous cell line derived from rhesus monkey kidney tissue) was extracted for molecular analysis using a commercially available kit (Tissue Kit; Qiagen) and used as a template for amplification of a 1050 base pair (bp) fragment of the chlamydial ompA gene (Sayada and others 1995). The amplicons were purified using a commercially available kit (QIAquick PCR Purification Kit; Qiagen) and sequenced. Nucleotide sequences were compared with the same regions of the C suis type strain S45 available in the GenBank database, using BLAST software. The ompA amplification showed the expected products. Alignment of the deduced amino acid sequences of the MOMP protein of the 14 isolates with the same sequence of the reference C suisS45 strain revealed an amino acid homology of 81 to 91 per cent. The sensitivity of the 14 isolates to doxycycline was tested and compared with the susceptibility to doxycycline of the urethral Italian isolate GO86 of Chlamydia trachomatis serovar D, the reference strain 6BC of Chlamydophila psittaci, and isolates of Chlamydophila, abortus, Chlamydophila pecorum and Chlamydophila felis. The susceptibility tests were performed in LLC-MK2 cells grown in 24-well plates, as previously described by Donati and others (2002). After incubation of the cell monolayers with chlamydiae and centrifugation, the medium was removed and replaced with a medium containing serial twofold dilutions of doxycycline. All the tests were run in triplicate. After incubation at 35 C for 48 hours, the cultures were fixed and stained for the detection of chlamydial inclusions by immunofluorescence using a fluoresceinconjugated monoclonal antibody specific for the chlamydial lipopolysaccharide genus-specific antigen. The minimum inhibitory concentration (MIC) of doxycycline was defined as the lowest concentration preventing the detection of more than 90 per cent of the chlamydial inclusions compared with the drug-free control. The minimum bactericidal concentration (MBC) was determined by aspirating the antimicrobial-containing medium, washing the wells twice with phosphate-buffered saline, adding antimicrobial-free medium, and reincubating the plates for 48 hours at 35 C. The MBC was the lowest antimicrobial concentration resulting in more than a 90 per cent reduction of inclusions. MIC values of 4 µg/ml or greater and MBC values of 8 µg/ml or greater were defined as indicating resistance of the isolates (Lenart and others 2001). Dugan and others (2004) demonstrated that the C suis tetracycline resistance (Tcr) phenotype is associated with a resistance gene, tet (C), integrated into the chlamydial chromosome. Therefore, the C suis isolates were tested for the presence of the tet (C) resistance gene by a PCR assay amplifying a 525 bp product of the tet (C) gene-coding region. The reaction was performed according to Dugan and others (2004), using the following forward and reverse oligonucleotide primers: CS43 5’-AGCACTGTCCGACCGCTTTG-3’and