微生物学通报 MAR 20, 2008, 35(3): 402~407 Microbiology tongbao@im.ac.cn 研究报告 2008 by Institute of Microbiology, CAS 不同人群肠道携带耐万古霉素肠球菌的研究 * 于艳华许淑珍 ( 100050) 摘要 : 调查住院病人 门诊病人和正常人粪便中耐万古霉素肠球菌的携带率及其耐药表型 基因型和同源性, 为临床预防和治疗选药提供依据 根据 CLSI 指南做药敏试验, 用 PCR 法检测万古霉素耐药基因, 用重复基因外回文序列 - 聚合酶链反应 (REP-PCR) 进行同源性分析 从住院病人肠道中分离出 31 株 (20.67 %) 耐万古霉素肠球菌, 其中 22 株 VRE 为 VanA 基因型, 9 株 VIE 为 VanC1 基因型 22 株 VRE 同源性分析, A 型有 19 株, 其中 A1 型 4 株, A2 型 6 株, A3 型 4 株, A4 型 3 株, A5 型 2 株 B 型 C 型 D 型各 1 株 9 株 VIE 中有 3 对菌株分别有 100% 同源性 门诊病人分离出 4 株 VIE(4%), 为 VanC1 型, 其中两株有 100% 同源性 正常人中分离出 11 株 (27.5%)VIE, 8 株为 VanC1 型, 3 株为 VanC2 型 ; 11 株 VIE 同源性分析, 以 A 型为主, A1 型有 1 株, A2 型有 2 株, A3 型有 1 株, A4 型有 1 株, D1 D2 D3 型各有 1 株, B C E 型各有 1 株 2 株住院病人 VIE 与 2 株门诊病人 VIE 有 100% 同源性, 另外 2 株住院病人 VIE 与 1 株门诊病人 VIE 有 100% 同源性 他们与正常人分离的 VIE 同源性低 22 株 VRE 对万古霉素的 MIC 值 >512 µg/ml, 16 株 VIE 对万古霉素的 MIC 值为 16 µg/ml 8 株 VIE 对万古霉素 MIC 值为 8 µg/ml 可见住院病人肠道中 VRE 携带率高, 是医院感染的危险因素, 46 株耐万古霉素肠球菌的耐药表型与耐药基因型一致 ; 耐万古霉素肠球菌对多种抗菌药物耐药 ; 部分菌株有较高的同源性 关键词 : 万古霉素, 耐药, 肠球菌, 同源性, 肠道 Study on Vancomycin-resistant Enterococci Carried by Intestinal Tract in Different People Groups YU Yan-Hua XU Shu-Zhen * (Beijing Friendship Hospital-affiliate of the Capital University of Medical Sciences, Beijing 100050) Abstract: To investigate the rate of carriage, the phenotype, the genotype and homology in stool specimen from outpatients, inpatients and health adult for prevention and guiding the clinical treatment. According to CLSI s guideline, antimicrobial susceptibility tests were performed; To detect vancomycin resistant gene by PCR; To analyze the homology by REP-PCR method. The vancomycin resistant carriage rate in intestinal tract of inpatients was 20.67%. All 22 isolates harbored VanA genotype; 9 isolates harbored VanC1 genotype. The homology in 22 vancomycin resistant enterococci was mostly type A, which was divided into A1-A5 subtypes, and they had high homology. Type A had 19 isolates. Of all, type A1 had 4 isolates; type A2 had 6 * 通讯作者 : : xushuzhen123@sohu.com 收稿日期 :2007-07-18; 接受日期 : 2007-11-28
: 403 isolates; type A3 had 4 isolates; type A4 had 3 isolates; type A5 had 2 isolates. Type B C D had no subtypes. Three pairs of isolates had 100% homologies. There were 4 isolates (4%) separated from outpatients. All were vanc1 types. Two were likely to be the same isolate. There were 11 isolates (27.5%) separated from health adults. Of all, 8 isolates were VanC1, and the others were vanc2. The homology in 11 VIE was mostly type A, which was divided into A1-A4 subtypes. Type A1 had 1 isolate; type A2 had 2 isolates; type A3 had 1 isolate; type A4 had 1 isolate. Type D1 D2 D3 was 1 isolate separately. Type B C E had no subtypes. Two VIE from inpatients and two from outpatients had 100% homologies. The other two VIE from inpatients and two from outpatients had 100 homologies, too. They had low homology with the isolates from health adults.minimum inhibitory concentration (MIC) of 22 VRE to vancomycin were > 512µg/mL; MIC of 16 VIE to vancomycin were 16µg/mL; MIC of 8 VIE to vancomycin were 8µg/mL. It is a risk factor for hospital infection that VRE carriage of inpatients in intestinal tract is high. There is 100% agreement between phenotypes and genotypes in 46 vancomycin resistance enterococci. VRE are multiresistant. Part isolates have high homology. Keywords: Vancomycin, Resistance, Enterococci, Homology, Intestinal tract,, [1],,,, 1 材料与方法 1.1 材料 1.1.1 粪便标本与质控菌株 :2006 9 2007 8 150 ( ICU,, ) 100 ( ), 40 ( 3 ) ATCC29212 ATCC51299 1.1.2 抗菌药物 : (30 μg/ ) (15 μg/ ) (10 μg/ ) (300 μg/ )(300 μg/ ) (120 μg/ )(30 μg/ )(5 μg/ ) (5 μg/ ) (30 μg/ ) (30 μg/ ) (30 μg/ ) 1.1.3 主要试剂和仪器 :PCR M-HBBL,, KH 2 PO 4,,, NaN 3, TTC.VITEK-2 compact VITEK-32 AG-9600 PCR Biotronic 1.1.4 PCR 引物 :vana 5 -GCTATCA GCTGTACTC-3 5 -CAGCGGCCATCATACGG- 3 ; vanc1 5 -AGCTGACTTTTTCTAG CCAAG-3 5 -CAGTGCAATCAATTGCTCTAC- 3 ; vanc2/3 5 -CAGCAGCCATTGGCG TACAA-3 5 -CAAGCAGTTTTTGTAGTTC-3 REP-PCR 5 -IIICGICGICATCIGGC-3 5 - ICGICTTATCIGGCCTAC-3 1.2 方法 1.2.1 筛选耐万古霉素肠球菌 :, 35 24 h 48 h, 1.2.2 菌株鉴定 : VITEK-2 compact VITEK-32 1.2.3 药敏试验 : 2006 (CLSI) 1.2.4 基因组 DNA 的提取 :[2] 1.2.5 质粒 DNA 的提取 :[3] 1.2.6 PCR 法检测万古霉素耐药基因 : 93 2 min; 93 1 min, 55 1 min,
404 微生物学通报 2008, Vol.35, No.3 72 1 min, 35 ; 72 2 min 20 μl Taq DNA 1 U, 10 buffer 2 μl, dntps 0.2 mmol/l, 4 μl, 1 μl,vana 783 bp, vanc1 800 bp, vanc2/3 577 bp 1.2.7 扩增产物检测 : ( 0.5 μg/ml )100V 20 min ~ 30 min, PCR DNA GenBank 1.2.8 REP-PCR 法检测万古霉素耐药菌株同源性 : 95 2 min; 94 3 s, 92 30 s, 35 1 min, 65 8 min, 35 ; 65 8 min 20 μl 10 buffer 2.5 μl, 25 mmol/l MgCl 2 1 μl, 25 mmol/l dntps 0.25 mmol/l, 100 μmol/l 0.75 μl, TaqDNA 2.5 U/μL 0.4 μl, DNA, 5 μl 0.8% EB, 90 V 1 h,, : 3, 1~2, 2 结果 2.1 住院病人 门诊病人和正常人肠道分离菌株情况 2.1.1 住院病人肠道分离菌株情况 : 31 (20.67 %), 22 (70.97%)VRE VanA, 9 (29.03%)VIE VanC1, 1 表 1 不同病区病人耐万古霉素肠球菌的携带率及使用万古霉素情况的调查结果 Table 1 The investigation results of VRE s carriage and vancomycin usage from different area patients (Division) ICU (ICU ward) (Emergency ward) (NS ward) (The Medical Department of Neurology ward) (The others) (Sum) (E.faecium) (%) (E.gallinarum) (%) (Have eaten vancomycin) (%) (Haven t eaten vancomycin) (%) 7 4.7 2 1.3 5 16.1 4 12.9 7 4.7 1 0.7 6 19.4 2 6.5 3 2.0 0 0 2 6.5 1 3.2 3 2.0 0 0 1 3.2 2 6.5 2 1.3 6 4.0 6 19.4 2 6.5 22 14.7 9 6.0 20 64.5 11 35.5 2.1.2 门诊病人肠道分离菌株情况 : 4 VIE, 4%, 2.1.3 正常人肠道分离菌株情况 : 11 VIE, 27.5%, 8, 3 2.2 46 株万古霉素耐药菌株 (22 株 VRE 和 24 株 VIE) 对 12 种抗菌药物的药敏结果 2, 22 VRE MIC >512 μg/ml, 16 VIE MIC 16 μg/ml 8 VIE MIC 8 μg/ml 2.3 耐药基因检测结果 46 22 VanA, 21 VanC1, 3 VanC2 3 VanA 4 VanC1 2 VanC2 (PCR 1 2), VanA EF206284.1 VanA 97%
: 405 (Antibacterials) 表 2 46 株耐万古霉素肠球菌对 12 种抗生素药敏率 (%) Table 2 The drug resistance to 12 antibiotics in 46 VRE (Resistant) (Intermediate) (Susceptible) (%) (Resistant (%) (Intermediate rate ) (%) (Susceptible (Vancomycin) 22 47.8 24 52.2 0 0.0 (Teicoplanin) 22 47.8 0 0.0 24 52.2 (Erythromycin) 36 78.3 9 19.6 1 0.1 (Ampicillin) 35 76.1 0 0.0 11 23.9 (Furadantin) 27 58.7 4 8.7 15 32.6 (High level Streptomycin) 20 43.5 3 6.5 23 50.0 (Chlormycetin) 1 2.2 2 4.3 43 93.5 (High level Gentmycin) 32 69.6 0 0.0 14 30.4 (Levofloxacin) 34 73.9 6 13.0 6 13.0 (Ciprofloxacin) 36 78.3 7 15.2 3 6.5 (Doxycycline) 9 19.6 4 8.7 33 71.7 (Minocycline) 12 26.1 3 6.5 31 67.4 图 1 万古霉素耐药基因 PCR 电泳图 Fig. 1 The PCR results of vancomycin resistance genes M: DNA marker; P1: VanA ; P2: VanC1 ; N ; 1,2,3 VanA ; 4,5 VanC1 M: DNA marker; P1: PCR positive control of vana sequced; P2: PCR positive control of vanc1 sequced; N negtive control; 1,2,3 PCR positive products of vana; 4,5 PCR positive products of vanc1., VanC1 3 AF162694.1 99%, 1 100%VanC2 L29638.1 99% 2.4 46 株万古霉素耐药肠球菌的同源性比较 2.4.1 22 株住院病人分离的 VRE 同源性比较 : 22 VRE 3-9, 100 bp 2000 bp 4, A-D A A1-A5, B C D 4 A A 19,, A1 图 2 万古霉素耐药基因 PCR 电泳图 Fig.2 The PCR results of vancomycin resistance genes M: DNA marker; P1: VanC2 ; P2: VanC1 ; N1 VanC2 ; N2 VanC1 ; 1,2 VanC2 ; 3,4 VanC1 M: DNA marker; P1:PCR positive control of vanc2 sequced; P2: PCR positive control of vanc1 sequced; N1 negtive control of vanc2; N2 negtive control of vanc1; 1,2 PCR positive products of vanc2; 3,4 PCR positive products of vanc1. 4, A2 6, A3 4, A4 3, A5 2 B C D 1 3, A3 B C D A1 A1 A2 A5 A3 A2 A3 A4 3, 5 6, 1 9 11, 7 10, 100% 5 6, ; 1 9 11 ; 7 10, ICU VRE 100%, VRE 1 9 11,,
406 微生物学通报 2008, Vol.35, No.3 图 3 12 株粪便分离的耐万古霉素肠球菌 REP-PCR 电泳图谱 Fig. 3 The REP-PCR electrophoregram of 12 VRE isolated from feces M: DNA Marker; 1-12: Strain 1-12. C D A2 A3 A3 A4 A1 A2 A1 3, 5, 4, 2 4 11 13 100% ; 1 7 12 100% ; 8 9 100%2 4 11 13 ; 1 7 12 ; 8 9 2.4.3 11 株正常人分离的 VIE 同源性比较 : 11 VIE 4-10, 200 bp 2000 bp, 5 A-E A A1-A4 D D1-D3 B C E 4 A1 A1 1, A2 2, A3 1, A4 1 D1 D2 D3 1 B C E 1 5, D1 E A4 A3 D3 A1 A2 D2 A2 B C 1 5 8, 8 图 4 13 株粪便分离的 VIE 的 REP-PCR 电泳图谱 Fig. 4 The REP-PCR electrophoregram of 13 VIE isolated from feces M: DNA Marker; 1-13: Strain 1-13. 图 5 11 株粪便分离的 VIE 的 REP-PCR 电泳图谱 Fig. 5 The REP-PCR electrophoregram of 11 VIE isolated from feces M: DNA Marker; 1-11: Strain 1-11. 2.4.2 13 株 VIE(9 株为住院病人分离, 4 株为门诊病人分离 ) 同源性比较 :13 VIE 4-10, 100 bp 2000 bp, 4 A-D A A1-A4 B C D 4 A1 A1 4, A2 3, A3 2, A4 1 B C D 1 4, A2 A1 B A1 3 讨论,, [4],,, 46, VRE 20.67%, VRE, VIE VIE,,, VRE, VRE VIE ICUVRE 12.2% [5] 33% [6] VRE
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