Detection of Mycoplasma genitalium using a wireless magnetoelastic immunosensor

A wireless immunosensor for the detection of Mycoplasma genitalium was fabricated by immobilizing polyclonal antibody onto the surface of a magnetostrictive strip. In response to a time-varying magnetic field, the immunosensor longitudinally vibrates at a resonance frequency, emitting magnetic flux that can be remotely detected by a pickup coil. No physical connections between the immunosensor and the detection system are required, facilitating wireless aseptic operation. The binding of M. genitalium to the immunosensor surface resulted in a decrease in the resonance frequency of the immunosensor. When solutions with varying concentrations of the bacteria were tested, the shift of the resonance frequency was proportional to the concentration of M. genitalium. Under the optimized conditions, the linear range for the determination of M. genitalium was 2.0 × 10³ to 2.9 × 10⁴ color change units (ccu)/ml with a detection limit of 3.4 × 10² ccu/ml. The immunosensor was successfully applied to real samples containing M. genitalium with results similar to those previously obtained by the color change unit method.     

Porcine circovirus type 2 (PCV2) vaccination is effective in reducing disease and PCV2 shedding in semen of boars concurrently infected with PCV2 and Mycoplasma hyopneumoniae

The objectives were to determine whether the amount of porcine circovirus type 2 (PCV2) shed in semen increased in boars experimentally coinfected with Mycoplasma hyopneumoniae (MHYO), and whether PCV2 vaccination of boars prior to PCV2 exposure reduced PCV2 viremia and virus shedding in semen. Twelve specific-pathogen-free PCV2- and MHYO-naïve boars were randomly and equally assigned to one of four groups. Six boars were vaccinated against PCV2 (VAC) on Day 0; three PCV2 vaccinated and three non-vaccinated boars were inoculated with MHYO on Day 21, and all boars were challenged with PCV2 on Day 35. The four treatment groups included PCV2-Infected (I), VAC-PCV2-I, MHYO-PCV2-Coinfected (CoI), and VAC-MHYO-PCV2-CoI. Semen, blood swabs, feces, and serum samples were collected weekly until Day 70. All vaccinated boars had seroconverted to PCV2 by Day 35. Between Days 28 and 35, MHYO boars developed moderate respiratory disease, characterized by coughing, respiratory distress, mucopurulent nasal discharge and loss of body condition. One MHYO-PCV2-CoI boar died on Day 50. Boars in the PCV2-I and MHYO-PCV2-CoI groups had significantly higher PCV2 DNA loads in blood swabs than the remaining boars. Moreover, PCV2 vaccination significantly reduced the incidence and amount of PCV2 shedding in semen and feces. In summary, although concurrent MHYO infection did not influence PCV2 shedding patterns, coinfection of boars with PCV2 and MHYO resulted in severe clinical disease and viral shedding was significantly decreased by PCV2 vaccination.     

Mycoplasma pneumoniae infection induces a neutrophil-derived antimicrobial peptide, cathelin-related antimicrobial peptide

In innate immunity, cationic antimicrobial peptides including cathelin-related antimicrobial peptide (CRAMP) are known to play critical roles in protecting the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between CRAMP and mycoplasmas. In the present study, the antimicrobial activity of CRAMP against M. pneumoniae and the expression of CRAMP in bronchoalveolar lavage fluid (BALF) of M. pneumoniae-infected mice was examined. CRAMP at 10-20 μg/mL reduced the growth of two strains of M. pneumoniae by 100 to 1000-fold. The amount of CRAMP in the BALF of M. pneumoniae-infected mice was 20～25 ng/mL by ELISA. The presence of mature CRAMP in BALF was observed by Western blotting. Neutrophils in BALF showed a fair amount of CRAMP in their cytoplasm by immunofluorescence. Furthermore, the addition of M. pneumoniae resulted in the release of a large amount of CRAMP from neutrophils induced by thioglycolate. These results suggest that CRAMP from neutrophils may play an important role in protection against M. pneumoniae infection.     

Expression and characterization of a Mycoplasma genitalium glycosyltransferase in membrane glycolipid biosynthesis: potential target against mycoplasma infections

Mycoplasmas contain glycoglycerolipids in their plasma membrane as key structural components involved in bilayer properties and stability. A membrane-associated glycosyltransferase (GT), GT MG517, has been identified in Mycoplasma genitalium, which sequentially produces monoglycosyl- and diglycosyldiacylglycerols. When recombinantly expressed in Escherichia coli, the enzyme was functional in vivo and yielded membrane glycolipids from which Glcβ1,6GlcβDAG was identified as the main product. A chaperone co-expression system and extraction with CHAPS detergent afforded soluble protein that was purified by affinity chromatography. GT MG517 transfers glucosyl and galactosyl residues from UDP-Glc and UDP-Gal to dioleoylglycerol (DOG) acceptor to form the corresponding β-glycosyl-DOG, which then acts as acceptor to give β-diglycosyl-DOG products. The enzyme (GT2 family) follows Michaelis-Menten kinetics. k(cat) is about 5-fold higher for UDP-Gal with either DOG or monoglucosyldioleoylglycerol acceptors, but it shows better binding for UDP-Glc than UDP-Gal, as reflected by the lower K(m), which results in similar k(cat)/K(m) values for both donors. Although sequentially adding glycosyl residues with β-1,6 connectivity, the first glycosyltransferase activity (to DOG) is about 1 order of magnitude higher than the second (to monoglucosyldioleoylglycerol). Because the ratio between the non-bilayer-forming monoglycosyldiacylglycerols and the bilayer-prone diglycosyldiacylglycerols contributes to regulate the properties of the plasma membrane, both synthase activities are probably regulated. Dioleoylphosphatidylglycerol (anionic phospholipid) activates the enzyme, k(cat) linearly increasing with dioleoylphosphatidylglycerol concentration. GT MG517 is shown to be encoded by an essential gene, and the addition of GT inhibitors results in cell growth inhibition. It is proposed that glycolipid synthases are potential targets for drug discovery against infections by mycoplasmas.     

Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes

Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma strain Aster Yellows-Witches' Broom (AY-WB) and Spiroplasma kunkelii are near completion. Complete genome sequences of seven obligate animal and human pathogenic mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii were compared to those of the completed genomes. This resulted in identification of at least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts. Phylogenetic analyses using parsimony were employed to study the origin of these genes, resulting in identification of one gene that may have undergone horizontal gene transfer. The possible involvement of these genes in plant pathogenicity is discussed.     

支原体肺炎患儿血清中相关炎性因子的表达变化情况及其临床意义探讨

目的：探讨支原体肺炎（Mycoplasma Pneumoniae Pneumonia,MPP）患儿血清中细胞因子IL-8,IL-12的表达水平及hs-CRP、IgG和血清补体（C）的变化及其临床意义。方法：收集MPP患儿50例,分为重症组、轻症组。健康儿童42例作为对照组;用ELISA法测定MPP患儿急性期、恢复期及对照组儿童血清IL-8、IL-12的水平,用血浆蛋白分析仪速率散射比浊法测定hs-CRP,Ig和C含量。结果：在急性期和恢复期MPP患儿血清IL-12含量明显低于正常对照组（P〈0.05）;而血清IL-8含量在急性期明显高于正常对照组（P〈0.01）。重症组患儿血清中IL-12明显低于轻症组,而血清中IL-8较轻症组高（P均〈0.01）。急性期MPP患儿血清IgM,IgG与对照组相比明显升高（P均〈0.01）;而IgA明显降低（P〈0.05）。急性期MPP患儿hs-CRP、C3、C4与对照组比较显著升高（分别为P〈0.01、P〈0.01、P〈0.05）。重症组患儿血清中IgM,IgG与轻症组相比明显升高（P均〈0.01）;IgA与轻症组相比明显降低（P〈0.05）;重症组患儿血清中hs-CRP、C3、C4与轻症组相比明显升高（P均〈0.01）。结论：检测相关血清炎性细胞因子对判定MPP患儿的病情和预后有较高的临床应用价值。     

肺炎支原体感染小鼠体内IL-6、IFN-γ、IL-5的水平及克拉霉素的影响

目的：研究肺炎支原体感染小鼠血清及支气管肺泡灌洗液中细胞因子的水平,了解克拉霉素治疗对细胞因子的影响。方法：将昆明小鼠分为感染组、正常组及药物治疗组,建立小鼠肺炎支原体感染模型成功后,药物组用克拉霉素（6g·kg^-1·d^-1）进行治疗,连续5天。在肺炎支原体感染模型建立后的第8天,检测三组小鼠的血清及支气管肺泡灌洗液中IL-6、IFN-γ、IL-5水平。结果-相比较于正常组,感染组小鼠支气管灌洗液和血清中的IL-6、IFN-γ、IL-5均显著升高（P〈0．05）。而克拉霉素的使用能使IL-6、IFN-γ降低（P〈0．05）,但对IL-5水平无影响。结论：肺炎支原体感染使小鼠IL-6、IFN-γ、IL-5水平增高,克拉霉素治疗有一定疗效。     

1724例女性生殖道支原体感染调查及耐药性分析

目的了解本地区女性生殖道支原体的感染现状及耐药情况,为临床用药提供指导。方法采用生殖道支原体检测试剂盒,对2012年8月至2013年4月解放军第44医院妇科门诊就诊的1 724例女性生殖道标本进行支原体培养及药敏检测,并对结果做统计学分析。结果 1 724例女性生殖道支原体感染率为43.6%,其中单纯Uu感染占85.1%;单纯Mh感染占3.7%;Uu合并Mh感染占11.2%。感染人群中21-40岁年龄段占85.2%。支原体对交沙霉素、强力霉素、美满霉素耐药率较低,对环丙沙星、左氧氟沙星、红霉素及司帕沙星的耐药率较高,单纯Mh及Uu合并Mh感染对抗菌药物的耐药率高于单纯Uu感染。结论女性生殖道支原体感染以单纯Uu为主,感染人群多见于21-40岁,支原体对常用抗菌药物出现了不同程度的耐药性,建议临床根据药敏结果合理用药,以减少耐药菌株的产生。     

肺炎支原体感染鼠P1特异抗原的动态检测

通过实验动物模型探讨肺炎支原体感染动物肺泡灌洗液中特异抗原检出率的动态变化,为肺炎支原体感染的临床诊断提供理论依据。小鼠经鼻自然感染肺炎支原体,分别采集感染后不同时间点小鼠的支气管灌洗液,应用量子点标记肺炎支原体P1蛋白抗体,直接免疫荧光法检测感染鼠肺泡灌洗液中肺炎支原体P1特异抗原,同时通过PCR检测肺组织肺炎支原体DNA及肺组织病理切片观察肺部炎性变化确定小鼠感染。结果显示,感染鼠肺炎支原体特异抗原在感染后第3天检出阳性率为75%,第7天达高峰为83%,之后随病程延长,抗原检测的阳性率逐渐下降,在感染后第14、21天检出阳性率分别为58%和25%。肺炎支原体特异抗原在感染早期检出率高。应用量子点标记肺炎支原体P1蛋白抗体,直接免疫荧光法检测肺炎支原体特异抗原可应用于肺炎支原体感染的早期诊断。     

1株多重耐药肺炎克雷伯菌耐药基因和整合子、转座子遗传标记研究

目的 了解多重耐药肺炎克雷伯菌的耐药基因存在状况和遗传学背景。方法 聚合酶链反应(RCR)法对多重耐药的肺炎克雷伯菌进行β-内酰胺酶基因、氨基糖苷类修饰酶基因、质粒AmpC酶基因、qacEΔ1-sull耐消毒剂和磺胺基因、整合子遗传标记(整合酶基因)、Tn21/Tn501转座子遗传标记(汞离子还原酶基因)检测。结果 TEM、SHV型β-内酰胺酶基因, DHA型质粒AmpC酶基因,aac(6′)-1型氮基糖苷类修饰酶基因,qacEΔ1-sul1耐消毒剂和磺胺基因,整合子遗传标记(intI1整合酶基因),Tn21/Tn501转座子遗传标记(merA汞离子还原酶基因)检测阳性。结论 多重耐药肺炎克雷伯菌存在多种耐药基因和Ⅰ类整合子、Tn21/Tn501转座子。