解脲脲原体生物被膜形成与耐药性的相关性研究

目的 研究解脲脲原体(Ureaplasma urealyticum,Uu)标准株及临床分离株体外形成生物被膜的能力及游离状态与形成生物被膜后药物敏感性的差异.方法 对Uu标准株3、8血清型(Uu3、Uu8)及从女性患者宫颈中分离鉴定的21株Uu临床株进行体外培养后,扫描电镜、激光共聚焦显微镜鉴定生物被膜形成,并在生物被膜形成前后进行约敏测定(四环素、红霉素、环丙沙星).配对秩和检验及x2检验分别比较Uu游离状态及形成生物被膜后最低抑菌浓度间及耐药率间的差异.结果 Uu3、Uu8及21株Uu临床株均具有体外形成生物被膜的能力.Uu形成牛物被膜后对四环素、红霉素及环丙沙星的最低抑菌浓度较游离状态明显增高(P<0.001).Uu形成生物被膜后对红霉素及环丙沙星的耐药率增高具有统计学意义(P值分别为<0.001及0.035),但对四环素的耐药率增高无统计学意义(P=0.293).结论 Uu标准株及临床株均具有体外形成生物被膜的能力,Uu形成生物被膜后对抗菌素的抵抗力增加,出现了多重耐药现象.

Abstract：

Objective To study the ability of standard strain and clinical isolates of Ureaplasma spp. to form biofilms in vitro and to compare the antibiotic susceptibility of sessile cells and their planktonic counterparts. Methods A total of 21 Ureaplasma wealyticum(Uu) isolates recovered from female patients diagnosed with cervicitis and Uu serovar 3 and Uu serovar 8( Uu3, Uu8) were included. Scanning electron microscope and confocal scanning laser microscopy were used to identify biofilm formation. Conventional antibiotic susceptibility tests and biofilm susceptibility assays for tetracycline, erythromycin and ciprofloxacin were carried out. The paired rank sum test and was applied to analyze the statistical differences between the MIC and the minimal biofilm inhibitory concentration. The x2 test was applied to analyze the statistical differences of global resistance percentages between planktonic cells and sessile cells. Results Uu3, Uu8 and 21 Uu isolates all can form biofilms in vitro. Minimal inhibitory concentration of sessile cells compared with planktonic cells were obviously higher for tetracycline, erythromycin and ciprofloxacin (P <0.001). Global resistance percentages between planktonic cells and sessile cells were different for erythromycin (9.52% vs 61.90% , P < 0. 001), ciprofloxacin ( 80. 95% vs 100% , P = 0. 035 ) and tetracycline (4. 76% vs 14.29% , P =0.293). Conclusion Uu isolates and Uu1, Uu8 all can form biofilms in vitro, and biofilm formation can strengthen resistance of Uu to antibiotics, even multidrug resistance was observed.     

解脲脲原体生物被膜形成与耐药性的相关性研究

目的 研究解脲脲原体(Ureaplasma urealyticum,Uu)标准株及临床分离株体外形成生物被膜的能力及游离状态与形成生物被膜后药物敏感性的差异.方法 对Uu标准株3、8血清型(Uu3、Uu8)及从女性患者宫颈中分离鉴定的21株Uu临床株进行体外培养后,扫描电镜、激光共聚焦显微镜鉴定生物被膜形成,并在生物被膜形成前后进行约敏测定(四环素、红霉素、环丙沙星).配对秩和检验及x2检验分别比较Uu游离状态及形成生物被膜后最低抑菌浓度间及耐药率间的差异.结果 Uu3、Uu8及21株Uu临床株均具有体外形成生物被膜的能力.Uu形成牛物被膜后对四环素、红霉素及环丙沙星的最低抑菌浓度较游离状态明显增高(P<0.001).Uu形成生物被膜后对红霉素及环丙沙星的耐药率增高具有统计学意义(P值分别为<0.001及0.035),但对四环素的耐药率增高无统计学意义(P=0.293).结论 Uu标准株及临床株均具有体外形成生物被膜的能力,Uu形成生物被膜后对抗菌素的抵抗力增加,出现了多重耐药现象.     

80株解脲脲原体的药敏及耐药机制分析

目的　分析解脲脲原体(Ureaplasmaurealyticm ,Uu)的耐药情况,并探讨Uu可能的耐药机制。方法　对80株临床上分离到的Uu进行了药敏分析、PCR生物分群、tetM基因的检测和PCR扩增喹诺酮类药物耐药区(QRDR ,gyrA、gyrB、parC及parE)基因并分析其核苷酸序列。结果　80株临床分离的Uu有6 6份为生物1群,占82 .5 % ;对所测的9种药物全敏感的Uu比例仅为10 % (8 80 ) ;7株耐四环素Uu中有3株出现了tetM基因阳性条带;对6株临床分离Uu的QRDR(gyrA、gyrB、parC及parE)进行了突变分析,未发现环丙沙星、氧氟沙星均敏感Uu株有gyrA、gyrB、parC及parE突变,但耐喹诺酮Uu株有gyrA、parC和parE基因的点突变,并导致其编码的氨基酸改变。在这些QRDR的改变中,gyrA 137C→A和parC基因10 0C→T的误义突变导致其编码的相应氨基酸的改变,但parC基因的2 2 5G→A和parE基因4 0G→A ,4 1T→C的误义突变导致其编码的相应氨基酸的改变。对Uu耐药率及生物群分型结果进行分析发现,虽然两群Uu的耐药率不完全一样,但差异无统计学意义(P均>0 .0 5 )。结论　UuQRDR的改变是导致耐喹诺酮类药物的原因,单独parE基因突变引起其酶蛋白的氨基酸改变也可导致Uu耐喹诺酮类药物。     

PCR法解脲脲原体分群和四环素耐药检测

目的 建立解脲脲原体(Ureaplasma urealyxicum，Uu)的分群和四环素耐药PCR检测方法。方法 1．根据Uu的多带抗原(multi—banded antigen，MBA)基因序列自行设计引物(P15′-ATT，TGC，AAT，CTT，TAT，ATG，TT；P25′-TTC，AGC，TGA，TGT，AAG，TGC，AGC，ATT，AAA，T)，并建立分群PCR反应体系。2．根据TetM基因序列自行设计引物(P15′-TTA，TCA，ACG，GTT，TAT，CAG，G；P25′-GCT，ATA，TAT，GCA，AGA，CG)，并建立四环素耐药反应体系。结果 1．MBA基因PCR能将Uul4个血清型正确分为二个生物群，其中生物一群(1、3、6、14等4个血清型)出现404bp的产物带，生物二群(2、4、5、7、8、9、10、11、12、13等10个血清型)出现448bp的产物带。60株Uu临床分离株经MBA基因PCR检测，生物一群53例、生物二群6例、生物一／二群同时存在1例。2.Uu典型株14个血清型，TetM基因PcR扩增的不能出现任何条带；60株Uu临床分离株经TetM基因PCR检测41例出现目的条带。结论 PCR法进行Uu生物分群和四环素耐药检测简单、快速，为相关研究提供了手段。     

解脲脲原体对红霉素体外耐药性与ermB基因相关性研究

目的 探讨解脲脲原体(Uu)临床株对红霉素体外耐药性与ermB耐药基因之间的关系.方法 采用微量肉汤稀释法体外测定143株临床分离的Uu对红霉素的最低抑菌浓度(MIC),以MIC≥8μg/ml为耐药判读标准;设计引物扩增ermB基因,并以多条带抗原基因为靶位设计引物对Uu进行生物学分群.结果 Uu对红霉素MIC范围为≤0.125μg/至≥128μg/ml,MIC50为16 μg/ml,MIC90≥128 μg/ml,耐药率为64.38%.ermB基凶总的阳性率为27.97%,主要分布在MIC≥8 μg/ml的菌株.两生物群之间不存在红霉素耐药性及ermB基因阳性率的差异.结论 ermB基因可能是介导Uu对红霉素耐药的基因,两生物群在对红霉素耐药性机制的差异需进一步研究.     

解脲脲原体感染的检测与体外耐药性分析

目的探讨本地区解脲脲原体(Uu)感染及耐药情况,指导临床合理用药。方法对临床送检的531份标本进行Uu培养、计数、鉴定和药敏试验;对775份标本进行荧光定量PCR(FQ-PCR)法和培养法检测Uu。结果FQ-PCR法检出Uu阳性339例,阳性率43.74%。培养法检出Uu206例,阳性率38.79%。Uu对9种抗菌药物的耐药率最高为环丙沙星(CIP)80.10%,最低为交沙霉素(JOS)0.00%,敏感率最高为原始霉素(PRI)98.54%。结论Uu用两种方法检测均可有效检出。培养法能提供药敏结果,更有利于临床治疗。药敏结果提示本地区Uu感染经验用药可选择交沙霉素和强力霉素。     

解脲脲原体的实验室检测方法的研究现状

解脲脲原体是一种条件致病菌，多与宿主共存，仅在某些条件下引起机会性感染，感染部位常为男女泌尿道，其中更易致病的为U．urealyticum型。近年来检测解脲脲原体的方法主要有液体培养法、固体培养法、免疫学方法和分子生物学方法等等，这些检测方法各有优劣。本文就临床使用较多的液体培养法、固体培养法和分子生物学方法进行重点分析，希望能给临床提供更多的帮助。     

女性NGU与单纯解脲脲原体感染的分群分型初步研究

目的探讨女性非淋菌性尿道炎或宫颈炎（NGU）与解脲脲原体（Uu）分群和分型的关系。方法采用液体培养,A7固体分离,液体纯化的方法获得Uu纯菌株,分别对114例女性单纯Uu感染的NGU和113例单纯Uu感染的女性携带者的Uu做PCR分群和生长抑制试验分型。结果NGU组1群56例（49.12%）,2群58例（50.88%）。正常组1群51例（45.13%）,2群62例（54.87%）;NGU组Uu分型阳性率最高依次为2型、7型、6型,正常人组最高依次为7型、2型、14型。结论Uu感染所致NGU与Uu分群关系不大。     

免疫组化法检测解脲脲原体的研究

目的　建立检测解脲脲原体 (UU)的实验方法。方法　应用亲和素 生物素 酶复合物技术 (ABC法 )观察UU感染者 ,并与培养法和PCR法相比较。结果　ABC法对 75例临床标本的检出率为 5 2 % ,与培养法和PCR法比较 ,在统计学上差异无显著性 (P >0 .0 5 )。结论　ABC法可作为诊断UU感染的快速检测方法 ,在临床应用中有一定实用价值。     

Investigation of Ureaplasma urealyticum biovars and their relationship with antimicrobial resistance

Purpose: To develop Taqman fluorescence quantitative polymerase chain reaction (PCR) method for investigating the characteristics of the distributions of Ureaplasma urealyticum (UU) biovars and to explore the relationship between UU biovars and antimicrobial resistance. Materials and Methods: By the method of culture, Ureaplasma species were detected. Taqman fluorescence quantitative PCR for detecting UU biovars were developed and UU clinical isolates were detected to distinguish biovars. The broth micro-dilution susceptibility testing methods were used to determine UU susceptibility. Results: By Taqman PCR method, UU biovars was successfully detected. Of 126 samples, biovar 1 was found in 73 (57.94%). There was a statistical difference between genital-urinary tract infection group and asymptomatic group (P<0.05). In the region, UU biovar 1 to 9 kinds of agents kept higher susceptibility rates, but biovar 2 maintained higher susceptibility rates only to tetracyclines. Compared with biovar 1, UU biovar 2 resistance rates to 7 kinds of agents were higher (P<0.05). Conclusions: (1) Our new established Taqman PCR method is a useful tool for screening UU biovars. (2) UU biovar 1 predominated in asymptomatic population; whereas in genital-urinary tract infection population UU biovar 2 had a higher proportion. (3) The characteristics of drug resistance were different between UU biovars. Overall, both two biovars remained higher susceptibility rates to tetracyclines. A majority of biovor 1 strains were sensitive to macrolides and quinolones; while only a small number of biovar 2 strains kept sensitive to roxithromycin and quinolones, a large proportion of biovar 2 strains were found in intermediate ranges.     

Ureaplasma urealyticum binds mannose-binding lectin

Mannose-binding C-type lectin (MBL) is an important component of innate immunity in mammals. Mannose-binding lectin (MBL), an acute phase protein, acts as an opsonin for phagocytosis and also activates the mannan-binding lectin complement pathway. It may play a particularly significant role during infancy before adequate specific protection can be provided by the adaptive immune system. Ureaplasma urealyticum has been linked to several diseases including pneumonia and chronic lung disease (CLD) in premature infants. We therefore investigated the ability of U. urealyticum to bind MBL. A guinea pig IgG anti-rabbit-MBL antiserum was produced. An immunoblot (dot-blot) assay done on nitrocellulose membrane determined that the anti-MBL antibody had specificity against both rabbit and human MBL. Pure cultures of U. urealyticum, serotype 3, were used to make slide preparations. The slides containing the organisms were then incubated with nonimmune rabbit serum containing MBL. Ureaplasma was shown to bind rabbit MBL with an immunocytochemical assay using the guinea pig IgG anti-rabbit MBL antiserum. Horseradish peroxidase (HRP)-labeled anti-guinea pig IgG was used to localize the reaction. The anti-MBL antiserum was also used in an immunocytochemical assay to localize U. urealyticum in histological sections of lungs from mice specifically infected with this organism. The same method also indicated binding of MBL by ureaplasma in human lung tissue obtained at autopsy from culture positive infants. Our results demonstrate that ureaplasma has the capacity to bind MBL. The absence of MBL may play a role in the predisposition of diseases related to this organism.     

继发不育男性泌尿生殖道支原体感染状况与耐药性分析及其临床应用研究

目的探讨继发不育男性患者泌尿生殖系支原体的感染状况及其耐药性。方法采用支原体分离培养鉴定与药敏试剂盒，对继发不育男性患者泌尿生殖系分泌物进行支原体分离培养鉴定与药敏测试。结果572例继发不育男性患者，泌尿生殖系支原体培养阳性278例，阳性率为48．6％，其中Uu占90．3％（251／278）；Mh占1．4％（4／278）；Uu合并Mh占8．3％（23／278）。药敏试验表明：喹诺酮类耐药率最高，四环素类和大环内酯类耐药率最低。结论支原体感染是男性继发不育的重要危险因素，在支原体感染的治疗过程中，我们应根据药敏测试结果选择敏感抗生素。     

荧光定量聚合酶链反应检测解脲支原体感染的临床意义

目的　采用荧光定量聚合酶链反应 (FQ -PCR)技术 ,准确定量检测待检标本中解脲支原体 (UU)的感染状况。方法　采用FQ -PCR技术 ,检测男女生殖道标本 76 5份 ,并同时用常规PCR技术 ,对比检测了 85例。结果　FQ -PCR检测76 5份标本 ,UU -DNA阳性 2 79份 ,阳性率 36 .5 % ,其DNA平均拷贝数为 2 .4× 10 4。 38例阳性患者治疗两周内复查 ,转阴率仅为 4 4 .7% ,治疗 3w后复查 ,转阴率达 76 % (P <0 .0 0 5 )。常规PCR结果重复检测时 ,2 5例阳性中 2例变为阴性 ,而 6 0例阴性中 4例变为阳性。FQ -PCR结果重复时完全吻合。结论　FQ -PCR检测UU ,具有快速、特异性高、定量准确等优点 ,并可为临床评价疗效提供依据。     

解脲支原体的PCR快速检测技术在临床上的应用

采用聚合酶链反应（ＰＣＲ）技术扩增解脲支原体４２９ｂｐＤＮＡ片段靶基因，检测２０９６例临床疑为非淋菌感染，阴道炎病人宫颈分泌物，检出阳性７８４例，阳性率为３７．４０％。     

解脲脲原体套式（Nested）PCR检测研究

本文报告解脲脲原体（ＵＵ）的套式（ＮＥＳＴＥＤ）ＰＣＲ检测方法。经方法学考核表明，本法的特异性、灵敏度以及试剂的稳定性和对临检标本的顺应性均较好。９３份各种生殖道炎症患者之宫颈拭子标本套式ＰＣＲ检出２１份阳性（阳性率２２．６％），而市售ＰＣＲ试剂盒仅检出１份阳性（阳性率１．１％）。前者阳性检出率明显高于后者（Ｐ＜０．０１）。