关于泌尿生殖道解脲支原体抗药性与生殖支原体分离检测的临床与实验研究
摘要
全文分上、下两部分，就解脲支原体的抗药性及我国有无生殖支原体感染作一初步探讨。
上篇重点阐述解脲支原体对四环素类药的耐药性、耐药种类、程度以及耐药机理问题。通过采用96孔板微量培基稀释法测定三类七种抗菌药物的敏感性发现：临床Uu株对四环素耐药频率及耐药性增高，同时对美满霉素及强力霉素敏感性有所下降。大环内酯类的红霉素及阿奇霉素抑菌效果最好，其次为喹诺酮类。未发现对三类药同时耐药的Uu菌株，提示在临床上对四环素发生抵抗，Uu难以阴转且有症状的患者应换用其他种类抗生素。
Uu耐四环素药的机理基于四环素抗性决定子(tet M)的存在，故本文首次建立了tet M基因的PCR检测方法，此法可敏感、快速地检测10~(-8)μg级的模板DNA。Uu分离株中tet M阳性检出率为71.4％。广东、上海、昆明及南京高危人群的tet M检出率分别为47.14％、30.83％，33.6％及26.04％。将标准株的tet M-PCR产物标记成探针，可与93.3％的阳性PCR产物反应出现杂交信号，另外，此探针尚可直接与MIC≥16μg／ml的Uu-DNA提取物产生杂交反应。经Taq-1酶切消化，6例PCR产物可产生不同的酶切图谱带型，提示Uu的tet M可能有核苷酸序列上的差异，此变异可成为有用的流行病学研究工具，可为耐药菌株监测提供参考依据。
下篇重点阐述在我国性病高危人群中首次分离出生殖支原体及采用PCR技术对部分地区Mg感染率的检测结果。研究表明采用SP-4改良培基，从227例性病高危人群中培养分离出8株Mg，总分离率为3.52％，性病门诊患者与性罪错人群间以及男女两性别间的分离率无显著差异。本文首次分离的Mg符合Tully所描述的特征：1．只在SP-4培基中生长；2．初代生长时间大于1个月；3．分解葡萄糖；4．可滤性(0.45μM微孔滤膜)；5．对青霉素不敏感；6．电镜下呈现与标准株类似结构；7．PCR技术可同时扩增出培养物及分泌物标本中的特异性DNA片段。
应用PCR技术并采用2对以上引物，包括支原体种属间保守序列(16S-rRNA)引物及Mg-粘附蛋白(Mg-Pa)基因引物，对昆明、广东、上海、常州及南京等地的部分分泌物标本进行检测。结果表明，正常人群检出率极低(1.79％)，广东性病门诊的检出率及昆明性乱人群的检出率均较其他地区为高，STD门诊人群与性乱人群间、高危人群两性别间的检出率差异均无显著性。NG~+与NG~-、Ct~+与Ct~-、Uu~+与Uu~-间检出率的差异无统计学意义。NG、Ct、Uu均阴性的182份标本可检出Mg，来自有尿道炎症状的分泌物标本有较高的Mg阳性率，这些均可说明Mg在STD中特别是在NGU、NSU中可能具有致病作用。
The paper is divided into 2 sections.
The first section elaborated the study on the drug resistance, the specific kind, the de-gree and the mechanism in Ureaplasma urealyticum to tetracyclines. By means of micro -volume of broth dilution, MIC of 7 kinds from 3 classes of antimicrobial agents were determined . There was increasing rate of Uu strains resistant to tetracycline. These strains also showed lower susceptibility to minocycline and doxycycline. The inhibitory efficacy of Azithromycine and erythromycine in macrolides are the best ones,and fluoroquinolones are next to them. No Uu strain simultaneously resistant to the above 3 classes of antibitics was found in our study, which suggested other anti - mycoplasma agents should be substituted for the patients with clinical symptoms and the drug resistant strains.
The mechanism to tetracycline resistance in Uu bases on the presence of drug resistant determinant(tet M), so PCR technique for tet M detection was developed,by which tet M determinant with 10~(-8)μg of template DNA can be detected sensitively, rapidely and on large scale .The positive detection rate of tet M was 71.4% in Uu isolates.The detection rates in high - risk population from Guangdong, Shanghai, Kunming and Nanjing were 47.14.%, 30. 83% ,33.60% and 26.04%, respectively.The PCR product of standard strains was labelled to the probe and 93.3% positive hybridized signals were obtained. Uu - DNA extracts from Uu strains with MIC≥16μg/ml could also be detected on the dot blot by the probe. After digestion with Taq - 1 restriction endonuclease, fragments with 190 bp, 130 bp and 77 bp were produced, but some different fragment patterns were observed, which suggested that there may be some variation of tet M nucleotide sequences in Uu strains. The variation of tet M gene sequences may be helpful for epidemiologic study and may provide the information and reference for surveillanc of drug resistant strains in our country.
上篇重点阐述解脲支原体对四环素类药的耐药性、耐药种类、程度以及耐药机理问题。通过采用96孔板微量培基稀释法测定三类七种抗菌药物的敏感性发现：临床Uu株对四环素耐药频率及耐药性增高，同时对美满霉素及强力霉素敏感性有所下降。大环内酯类的红霉素及阿奇霉素抑菌效果最好，其次为喹诺酮类。未发现对三类药同时耐药的Uu菌株，提示在临床上对四环素发生抵抗，Uu难以阴转且有症状的患者应换用其他种类抗生素。
Uu耐四环素药的机理基于四环素抗性决定子(tet M)的存在，故本文首次建立了tet M基因的PCR检测方法，此法可敏感、快速地检测10~(-8)μg级的模板DNA。Uu分离株中tet M阳性检出率为71.4％。广东、上海、昆明及南京高危人群的tet M检出率分别为47.14％、30.83％，33.6％及26.04％。将标准株的tet M-PCR产物标记成探针，可与93.3％的阳性PCR产物反应出现杂交信号，另外，此探针尚可直接与MIC≥16μg／ml的Uu-DNA提取物产生杂交反应。经Taq-1酶切消化，6例PCR产物可产生不同的酶切图谱带型，提示Uu的tet M可能有核苷酸序列上的差异，此变异可成为有用的流行病学研究工具，可为耐药菌株监测提供参考依据。
下篇重点阐述在我国性病高危人群中首次分离出生殖支原体及采用PCR技术对部分地区Mg感染率的检测结果。研究表明采用SP-4改良培基，从227例性病高危人群中培养分离出8株Mg，总分离率为3.52％，性病门诊患者与性罪错人群间以及男女两性别间的分离率无显著差异。本文首次分离的Mg符合Tully所描述的特征：1．只在SP-4培基中生长；2．初代生长时间大于1个月；3．分解葡萄糖；4．可滤性(0.45μM微孔滤膜)；5．对青霉素不敏感；6．电镜下呈现与标准株类似结构；7．PCR技术可同时扩增出培养物及分泌物标本中的特异性DNA片段。
应用PCR技术并采用2对以上引物，包括支原体种属间保守序列(16S-rRNA)引物及Mg-粘附蛋白(Mg-Pa)基因引物，对昆明、广东、上海、常州及南京等地的部分分泌物标本进行检测。结果表明，正常人群检出率极低(1.79％)，广东性病门诊的检出率及昆明性乱人群的检出率均较其他地区为高，STD门诊人群与性乱人群间、高危人群两性别间的检出率差异均无显著性。NG~+与NG~-、Ct~+与Ct~-、Uu~+与Uu~-间检出率的差异无统计学意义。NG、Ct、Uu均阴性的182份标本可检出Mg，来自有尿道炎症状的分泌物标本有较高的Mg阳性率，这些均可说明Mg在STD中特别是在NGU、NSU中可能具有致病作用。
The paper is divided into 2 sections.
The first section elaborated the study on the drug resistance, the specific kind, the de-gree and the mechanism in Ureaplasma urealyticum to tetracyclines. By means of micro -volume of broth dilution, MIC of 7 kinds from 3 classes of antimicrobial agents were determined . There was increasing rate of Uu strains resistant to tetracycline. These strains also showed lower susceptibility to minocycline and doxycycline. The inhibitory efficacy of Azithromycine and erythromycine in macrolides are the best ones,and fluoroquinolones are next to them. No Uu strain simultaneously resistant to the above 3 classes of antibitics was found in our study, which suggested other anti - mycoplasma agents should be substituted for the patients with clinical symptoms and the drug resistant strains.
The mechanism to tetracycline resistance in Uu bases on the presence of drug resistant determinant(tet M), so PCR technique for tet M detection was developed,by which tet M determinant with 10~(-8)μg of template DNA can be detected sensitively, rapidely and on large scale .The positive detection rate of tet M was 71.4% in Uu isolates.The detection rates in high - risk population from Guangdong, Shanghai, Kunming and Nanjing were 47.14.%, 30. 83% ,33.60% and 26.04%, respectively.The PCR product of standard strains was labelled to the probe and 93.3% positive hybridized signals were obtained. Uu - DNA extracts from Uu strains with MIC≥16μg/ml could also be detected on the dot blot by the probe. After digestion with Taq - 1 restriction endonuclease, fragments with 190 bp, 130 bp and 77 bp were produced, but some different fragment patterns were observed, which suggested that there may be some variation of tet M nucleotide sequences in Uu strains. The variation of tet M gene sequences may be helpful for epidemiologic study and may provide the information and reference for surveillanc of drug resistant strains in our country.
引文
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31 Wang RYH, Shih JIVk, Grandinetti T. High frequency of antibodies to penetrans in HIV infected ps-tients. Lancet, 1992, 340: 1312-16
32 Hannan PCT. The in vitro susceptabilities of AIDS-associated and buman urogenital tract Mycoplas-mas to 10 classes of antinicrobial agent. IOM Letters, July 1996, Programmes and Abstracts of the11th International Congress of IOM U.S., 1996, 4: 396-881 Tully JG et al. Int J Syst Bacteriol, 1983; 33:(2): 387-396
2 Taylor-Robinson D et al. Genitourin Med, 1985; 61: 319-324
3 Hooton TM et al. Laneet, 1988; 1: 266-268
4 Taylor-Robimson D et al. Clin Infect Dis, 1993; 17(Suppl 1): 66-68
5 Horner PJ et al. Lancet, 1993; 342(8871): 582-585
6 Jensen JS et al. Genitourin Med, 1993; 69: 265-269
7 Peterson SN et al. J Rocteriol, 1993; 175(24)7918-7930
8 Tully JD et al. Isr J Med Sci, 1987; 23: 561-564
9 Dello SF et al. Infect Immun, 1989; 57: 1059-1065
10 Homer PG et al. Lancet, 1994; 343: 790
11 Hyman HC et al. J Clin Microbiol, 1987; 25(4): 726-728
12 Palmer HM et al. FEMS Mierobiol Lett, 1991; 77: 199-204
13 Jensen JS et al. J Clin Miembiol, 1991; 29: 46-50
14 Tully JD et al. J Infect Dis, 1986; 153: 1046-1054
15 Jensen JS et al. IOM Letters, Boreaux, July 1994. Pro-grammes and Abstacts of the 10th International Congress of IOM, France, 1994; 3: 329-331
16 Taylor-Robinson D. Curr Opin Infect Dis, 1995; 8: 16-21
17 Hooton TM et al. Lancet, 1994; 343(8889): 69
18 Moiler BR et al. Lancet, 1984; 1: 1102-1103
19 Mernaagh GR et al. Clin Infect Dis, 1993; 17(Suppl): 69-78
20 Taylor-Robinson D et al. Genitoirn Med, 1995; 71: 1-8
21 Lueier TS et al. Gene, 1994; 150: 27-34
22 Blanchard A et al. Mol Cell Biol of STD, 1st ed, London: David wright and Leonard Archard, 1992: 55-71
25 Jensen JS et al. IOM Letters, Boreaux, July 1994, Programmes and Abstracts of the 10th International Coagress of IOM, France, 1994; 3: 339-M0
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30 Grau O, Koracic R, Griffais R et al. Development of as elective polymerase chain reac-tion assay for the detection of Mycoplasma pirum, FEMS Microbiol lett, 1993, 106: 327-34
31 Wang RYH, Shih JIVk, Grandinetti T. High frequency of antibodies to penetrans in HIV infected ps-tients. Lancet, 1992, 340: 1312-16
32 Hannan PCT. The in vitro susceptabilities of AIDS-associated and buman urogenital tract Mycoplas-mas to 10 classes of antinicrobial agent. IOM Letters, July 1996, Programmes and Abstracts of the11th International Congress of IOM U.S., 1996, 4: 396-881 Tully JG et al. Int J Syst Bacteriol, 1983; 33:(2): 387-396
2 Taylor-Robinson D et al. Genitourin Med, 1985; 61: 319-324
3 Hooton TM et al. Laneet, 1988; 1: 266-268
4 Taylor-Robimson D et al. Clin Infect Dis, 1993; 17(Suppl 1): 66-68
5 Horner PJ et al. Lancet, 1993; 342(8871): 582-585
6 Jensen JS et al. Genitourin Med, 1993; 69: 265-269
7 Peterson SN et al. J Rocteriol, 1993; 175(24)7918-7930
8 Tully JD et al. Isr J Med Sci, 1987; 23: 561-564
9 Dello SF et al. Infect Immun, 1989; 57: 1059-1065
10 Homer PG et al. Lancet, 1994; 343: 790
11 Hyman HC et al. J Clin Microbiol, 1987; 25(4): 726-728
12 Palmer HM et al. FEMS Mierobiol Lett, 1991; 77: 199-204
13 Jensen JS et al. J Clin Miembiol, 1991; 29: 46-50
14 Tully JD et al. J Infect Dis, 1986; 153: 1046-1054
15 Jensen JS et al. IOM Letters, Boreaux, July 1994. Pro-grammes and Abstacts of the 10th International Congress of IOM, France, 1994; 3: 329-331
16 Taylor-Robinson D. Curr Opin Infect Dis, 1995; 8: 16-21
17 Hooton TM et al. Lancet, 1994; 343(8889): 69
18 Moiler BR et al. Lancet, 1984; 1: 1102-1103
19 Mernaagh GR et al. Clin Infect Dis, 1993; 17(Suppl): 69-78
20 Taylor-Robinson D et al. Genitoirn Med, 1995; 71: 1-8
21 Lueier TS et al. Gene, 1994; 150: 27-34
22 Blanchard A et al. Mol Cell Biol of STD, 1st ed, London: David wright and Leonard Archard, 1992: 55-71
25 Jensen JS et al. IOM Letters, Boreaux, July 1994, Programmes and Abstracts of the 10th International Coagress of IOM, France, 1994; 3: 339-M0