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.     

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.     

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。     

解脲脲原体Parvo和T960生物群msr基因的检测

目的 检测解脲脲原体(Uu)是否携带介导对红霉素耐药的msr基因,并分析其在Uu两生物群间分布的差异.方法 采用微量肉汤稀释法测定72株Uu临床株对红霉素的体外耐性,PCR检测msrA、msrB、msrG、msrD基因,并对Uu进行PCR分群.结果 72株Uu的最低抑菌浓度(MIC)范围是≤0.125 μg/ml≥128 μg/ml,MIC_(50)为32 μg/ml,MIC_(90)≥128μg/ml.分群结果示Parvo生物群51株,占70.83%,T960生物群21株,占29.17%.共检测到msrD基因的Uu24株,msrB基因12株,msrA基因1株,没有发现Uu菌株携带msrC基因.5株Uu同时检测到msrB和msrD基因,1株Uu同时检测到msrA、msrB和msrD基因.以MIC≥8μg/ml为耐药判定值时,两生物群对红霉素耐药性无显著差异,msrB基因主要分布在T960生物群.结论 Uu临床菌株携带对大环内酯类耐药的msr基因(包括msrA、msrB、msrP),msrB基因主要分布在T960生物群.     

解脲脲原体生物膜胞外多糖的检测

目的 研究解脲脲原体(Ureaplasma urealyticum,Uu)4个型别标准株在体外形成的生物膜之胞外多糖的分布及结构成分.方法 将Uu标准株Parvo群中4、8血清型和T960群中3、14血清型进行体外生物膜培养后,扫描电镜下观察生物膜组成及结构,并在FITC-ConA/PI及ECA/PI双荧光染色后进行激光共聚焦显微镜观察及测定平均荧光强度.秩和检验及t检验分别比较两种荧光标记物、两生物群的总体平均荧光强度的差异.结果 4个型别Uu标准株均可在体外形成生物膜,生物膜结构主要呈网格状,胞外物质占大部分比例.在激光共聚焦显微镜下,Uu生物膜胞外多糖均可被FITC-ConA和ECA染色,FTTC-ConA呈网格状分布,ECA小片状聚集分布.FITC-ConA的总体平均荧光强度较ECA高,差异有统计学意义(P＜0.001).结论 Uu体外培养生物膜主要呈网格状结构,胞外多糖中含有葡萄糖、甘露糖、半乳糖、N-乙酰葡聚糖残基,并以葡萄糖、甘露糖残基为主.     

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。     

42株形成生物被膜的嗜麦芽窄食单胞菌的药物敏感性研究

目的 研究单独及联合使用不同种类抗生素对嗜麦芽窄食单胞菌(Stenotrophomonasmahophilia,SML)体外生物被膜的抗菌活性. 方法 收集从患者分离的非重复SML 42株,分别采用微量接种针和硅胶膜片在水解酪蛋白肉汤中构建细菌生物被膜(bacterial biofilm)的体外模型,经过抗生素(左氧氟沙星、环丙沙星、头孢哌酮/舒巴坦、头孢他啶、哌拉西林、红霉素、磺胺甲噁唑、庆大霉素)作用20 h,超声振荡并测定孵育前后6 h吸光度(A)值的变化,计算相应的细菌生物被膜抑制浓度,进而测定红霉素与左氧氟沙星、头孢哌酮/舒巴坦、哌拉西林对细菌生物被膜的联合作用. 结果 SML对左氧氟沙星、磺胺甲噁唑和哌拉西林敏感率分别为83.33%、66.67%和54.76%,其他抗生素的耐药率均在50%以上.形成生物被膜后,细菌对抗生素的生物被膜抑制浓度大于相应的最小抑菌浓度(MIC)值. 结论 42株SML呈现多重耐药现象,形成生物被膜后耐药性增强.左氧氟沙星的抗菌活性在形成生物被膜前后均优于其他抗生素,联合红霉素与左氧氟沙星有助于增强杀菌效果.     

87例培养阳性解脲脲原体的药敏分析

目的了解泌尿生殖道标本中解脲脲原体对不同抗生素的敏感性差异.方法用法国生物梅里埃生产的支原体IST试剂盒对泌尿生殖道分泌物等标本进行培养鉴定和药敏试验.结果其中有87例解脲支原体培养阳性,药敏结果分析发现解脲脲原体分别对6种抗生素敏感如下:四环素60例(69.00%)、强力霉素68例(78.16%)、交沙霉素82例(94.25)、原始霉素全部敏感率(100%)、红霉素只有7例(8.05%)、氧氟沙星28例(32.18%).结论应将原始霉素、交沙霉素、强力霉素、四环素列为临床治疗解脲支原体感染首先药物.     

Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials

Failure to treat and eradicate prosthetic hip infection with systemic antibiotic regimens is usually due to the fact that the infection is associated with biofilm formation and that bacterial cells growing within a biofilm exhibit increased resistance to antimicrobial agents. In this in vitro study, we investigated the susceptibility of prosthetic hip Propionibacterium acnes and Staphylococcus spp. isolates growing within biofilms on polymethylmethacrylate (PMMA) bone cement to a range of antibiotics. All P. acnes isolates in the biofilm mode of growth demonstrated considerably greater resistance to cefamandole, ciprofloxacin and vancomycin. In contrast, only four of the eight P. acnes isolates demonstrated an increase in resistance to gentamicin. All ten Staphylococcus spp. isolates in the biofilm mode of growth exhibited large increases in resistance to gentamicin and cefamandole with eight of the ten isolates also exhibiting an increase in resistance to vancomycin. However, only three of the ten Staphylococcus spp. isolates exhibited an increase in resistance to ciprofloxacin. Biofilms were also formed on three different titanium alloys and on PMMA bone cement using P. acnes, Staphylococcus epidermidis and Staphylococcus aureus strains to determine if the underlying biomaterial surface had an effect on biofilm formation and the antimicrobial susceptibility of the bacteria growing within biofilms. Although differences in the rate at which the three strains adhered to the different biomaterials were apparent, no differences in biofilm antibiotic resistance between the biomaterials were observed. In the light of these results, it is important that the efficacy of other antibiotics against P. acnes and Staphylococcus spp. prosthetic hip isolates growing within biofilms on orthopaedic biomaterials be determined to ensure optimal treatment of orthopaedic implant infection.     

Comparative antimicrobial susceptibility of biofilm versus planktonic forms of Salmonella enterica strains isolated from children with gastroenteritis

In the present study, 194 Salmonella enterica strains, isolated from infected children and belonging to various serotypes, were investigated for their ability to form biofilms and the biofilm forms of the isolated strains were compared to their corresponding planktonic forms with respect to the antimicrobial susceptibility. For the biofilm-forming strains, the minimum inhibitory concentration for bacterial regrowth (MICBR) from the biofilm of nine clinically applicable antimicrobial agents was determined, and the results were compared to the respective MIC values of the planktonic forms. One hundred and nine S. enterica strains out of 194 (56%) belonging to 13 serotypes were biofilm-forming. The biofilm forms showed increased antimicrobial resistance compared to the planktonic bacteria. The highest resistance rates of the biofilm bacteria were observed with respect to gentamicin (89.9%) and ampicillin (84.4%), and the lowest rates with respect to ciprofloxacin and moxifloxacin (2.8% for both). A remarkable shift of the MICBR₅₀ and MICBR₉₀ toward resistance was observed in the biofilm forms as compared to the respective planktonic forms. The development of new consensus methods for the determination of the antimicrobial susceptibility of biofilm forms seems to be a major research challenge. Further studies are required in order to elucidate the biofilm antimicrobial resistance mechanisms of the bacterial biofilms and their contribution to therapeutic failure in infections with in vitro susceptible bacteria.     

Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors

Propionibacterium acnes plays an important role in the pathogenesis of acne vulgaris, a common disorder of the pilosebaceous follicles. Recently, it was suggested that P. acnes cells residing within the follicles grow as a biofilm. In the present study, we tested the biofilm-forming ability of several P. acnes strains in a microtiter plate model. We also evaluated the resistance of biofilm-grown P. acnes towards antimicrobial agents commonly used in the treatment of acne and the production of putative virulence factors. Our results indicate that P. acnes can form biofilms in vitro. The results also show that sessile P. acnes cells are more resistant to various commonly used antimicrobial agents than planktonic cells. In addition, sessile cells produce more extracellular lipases as well as significant amounts of the quorum-sensing molecule autoinducer-2.     

Interleukin-1beta-induced growth enhancement of Staphylococcus aureus occurs in biofilm but not planktonic cultures

Staphylococcus aureus causes recalcitrant infections and forms resistant biofilms. Mechanisms of biofilm resistance to host defenses may include changes in gene expression that confer responsiveness to chemical mediators. In earlier studies fresh clinical isolates responded to inflammatory cytokines, but responsiveness was lost after multiple in vitro passages [Meduri et al. Cytokines IL-1beta, IL-6, and TNF-alpha enhance the In vitro growth of bacteria. Am J Respir Crit Care Med 1999;160:961-7]. Since biofilms more closely resemble in vivo growth and are implicated in recalcitrant infections, we hypothesized that biofilms, but not planktonic cells, would respond to cytokines. Biofilms were induced by ethanol in S. aureus ATCC 12600. Biofilms treated with 2 ng/mL interleukin-1beta (IL-1beta) for 6 h contained 2.5-fold more cells than untreated biofilms, but no growth-enhancement occurred in planktonic cultures. As determined by flow cytometry, IL-beta bound to 63.1% of biofilm cells, but only 11.2% of planktonic cells. Our results provide evidence of a differential response of biofilm and planktonic bacteria to chemical mediators, and suggest that biofilm bacteria may evade host defenses by growing more rapidly in response to the inflammatory mediators released by activated host defense cells.     

gyrA和parE基因检测在脲原体基因分型中的应用

目的 研究gyrA和parE基因检测在脲原体基因分型中的作用.方法 脲原体培养与药敏分析用Mycoplasma IST检测试剂盒;在脲原体培养阳性者中选取对喹诺酬耐药标本60份,PCR扩增gyrA和parE基因,扩增产物经测序分析后与基因库中的脲原体各血清型进行比对.结果 gyrA扩增片段在血清型1、3、6、14之间核苷酸序列相似性为100%,在血清型2,4、5、7～13之间核苷酸序列相似性100%,两组之间核苷酸序列相似性91%.parE扩增片段在血清型1、3、6、14之间的核苷酸序列相似性为98%～99%;pare扩增片段在血清犁2、5、7、8、11之间核苷酸序列相似性100%,在血清型4、12、13之间核苷酸序列相似性100%,两组之间核苷酸序列相似性为90%.60份标本中微小脲原体(Ureaplasma parvum,Up)占68.3%(41/60),解脲脲原体(Ureaplasma urealyticum,Uu)占21.7%(13/60),两型混合感染占10%(6/60).在Up中,血清型3占48.8%(20/41).结论 gyrA检测可把脲原体分为微小脲原体和解脲脲原体两个基因型,parE检测可把微小脲原体分为4个亚型,分别与血清型1、3、6、14完全一致,其中血清型3感染最常见.     

Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade‐offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well‐defined collection of 42 CF Pseudomonas aeruginosa isolates. MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10^?8) than in biofilm (9.9 × 10^?9) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10^?9 in the planktonic mode to 6 × 10^?8 in biofilm, whereas normomutators (from 9.4 × 10^?8 to 5.3 × 10^?8) and hypermutators (from 1.6 × 10^?6 to 7.7 × 10^?7) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa, leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.     

Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and ATCC 10145 biofilms

Pseudomonas aeruginosa is a relevant opportunistic pathogen involved in nosocomial infections that frequently shows low antibiotic susceptibility. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the isolation and characterization of lytic phages capable of infecting antibiotic-resistant P. aeruginosa strains. In addition, characterization of P. aeruginosa biofilms and the potential of newly isolated phages for planktonic and biofilm control was accessed. According to the results, the isolated phages showed different spectra of activity and efficiency of lysis. Four broad lytic phages were selected for infection of planktonic cells; however, despite their broad range of activity, two of the selected phages failed to efficiently control planktonic cultures. Therefore, only two phages (phiIBB-PAA2 and phiIBB-PAP21), highly capable of causing strong biomass reduction of planktonic cells, were tested against 24 h biofilms using a m.o.i. of 1. Both phages reduced approximately 1-2 log the biofilm population after 2 h of infection and reduction was further enhanced after 6 h of biofilm infection. However, biofilm cells of P. aeruginosa PAO1 acquired resistance to phiIBB-PAP21; consequently, an increase in the number of cells after 24 h of treatment was observed. Conversely, phage phiIB-PAA2 for P. aeruginosa ATCC10145 continued to destroy biofilm cells, even after 24 h of infection. In these biofilms, phages caused a 3 log reduction in the number of viable counts of biofilm cells.