Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein

Abstract The ability of Mycoplasma penetrans to invade eukaryotic cells was studied using a HeLa cell line. The bactericidal antibiotic, gentamicin, in combination with low concentrations of Triton X-100, was utilized to kill mycoplasmas that had not entered the cells, allowing the quantitation of internalized organisms. The intracellular location of the mycoplasma was also documented by transmission electron microscopy. The actin polymerization inhibitor cytochalasin-D markedly inhibited the internalization process, whereas the tyrosine phosphorylation inhibitors, staurosporin and genistein had only a slight effect. As against the invasion of enteropathogenic Escherichia coli which depends on tyrosine phosphorylation of a 90-kDa (Hp90) HeLa cell protein, internalization of M. penetrans by HeLa cells was independent of the phosphorylation of Hp90. Nonetheless, tyrosine phosphorylation of a 145-kDa HeLa cell protein was found to be associated with the interaction of M. penetrans with HeLa cells.     

肺炎支原体肺炎患儿口咽部菌群的改变

目的 比较健康儿童与肺炎支原体肺炎患儿口咽部菌群的差异,分析肺炎支原体感染对儿童口咽部菌群变化的可能影响。方法 采用高通量测序技术,对30例肺炎支原体肺炎患儿及30例健康儿童16S rDNA进行测序分析,比较两组间的菌群多样性及在门、属水平上菌群结构差异。结果 健康儿童与肺炎支原体肺炎患儿在性别、年龄、抗生素使用等方面差异无统计学意义。肺炎支原体肺炎患儿口咽部菌群多样性比健康儿童明显降低。在门水平上,Proteobacteria（变形菌门）、Bacteroidetes（拟杆菌门）和Fusobacteria（梭杆菌门）在健康儿童中相对丰度显著高于肺炎支原体肺炎患儿组,而Firmicutes（厚壁菌门）、Tenericutes（柔膜菌门）和Actinobacteria（放线菌门）在肺炎支原体肺炎患儿组中相对丰度均显著高于健康儿童组;在属水平上,两组排在相对丰度前10位的属差异具有统计学意义,共有6个属在肺炎支原体肺炎患儿中显著增加,分别为Staphylococcus（葡萄球菌）、Actinomyces（放线菌属）、Acinetobacter（不动细菌属）、Atopobium（阿托波菌属）、Corynebacterium（棒状杆菌）和Abiotrophia（营养缺陷菌属）。结论 肺炎支原体肺炎患儿口咽部菌群存在明显的改变,其口咽部菌群多样性比健康儿童明显降低,在门、属水平肺炎支原体肺炎患儿与健康儿童差异均有统计学意义。     

11788例儿童血清肺炎支原体抗体检测结果分析

目的了解宁波儿童肺炎支原体（MP）感染情况,为儿童MP感染的临床诊断与防治提供依据。方法采用被动凝集法测定11788例14岁以下有呼吸道感染症状儿童的血清MP抗体,并根据患儿性别、年龄和不同季节对MP的阳性率进行统计学分析。结果11788例有呼吸道感染症状患儿中,MP阳性3081例,阳性率为26．14％,不同性别、年龄和季节MP抗体的阳性率差异有统计学意义（P〈0．05）。女性儿童阳性率高于男性儿童,并随着年龄的增加阳性率有上升趋势。结论MP在呼吸道感染中发病率较高,春冬季节、女性儿童和学龄期儿童易感。     

Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells

In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluorescent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.     

肺炎支原体抗体阳性对咳嗽变异性哮喘患儿肺功能的影响

目的探索肺炎支原体（Mycoplasma pneumoniae,MP）抗体阳性对咳嗽变异性哮喘（cough variant asthmaCVA）患儿肺功能的影响。方法收集72例初次诊断CVA的患儿,根据血清MP抗体结果分为CVA合并MP抗体阳性（以下记CVA合并MP）组,CVA组。分别对两组进行支原体抗体及肺功能检测。结果纳入本研究的CVA患儿,MP抗体阳性占29％,且这部分患儿中女孩比男孩多。CVA患儿肺功能FVC、FEV1／FVC、PEF、MMEF75／25各项指标均下降（P〈0．05）,合并MP组FVC、FEV1／FVC、PEF值变化差异无统计学意义（P〉0．05）,而MMEF75／25下降差异有统计学意义（P〈0．05）;CVA合并MP组支气管激发试验以极轻度、轻度为主（P〈0．05）,而CVA组以中重度为主（P〈0．05）;CVA合并MP组,在治疗一个月时FEV1％升高,差异有统计学意义（P〈0．05）;治疗相同时间两组间FEV1％差异无统计学差异（P〉0．05）。结论呼吸道的多种微生物间形成复杂而又互相联系的群落,MP使气道黏膜受到损害,影响呼吸系统局部微生态,这与哮喘的形成有某种联系。MP抗体阳性的CVA患儿气道高反应性较低,相比之下,小气道阻塞加重,且其对CVA治疗过程中肺通气功能的变化没有影响。     

中西医结合治疗成人支原体肺炎的临床评价

为评价中西医结合治疗成人支原体肺炎的疗效。对我院2009~2010年住院的60例支原体肺炎患者的临床资料进行回顾性分析,通过患者体温、临床表现及平均住院日变化比较中西医结合治疗与单用阿奇霉素治疗对支原体肺炎的疗效。用药后治疗组的退热时间、咳嗽消失时间、肺部啰音消失时间均较对照组明显缩短,差异显著（P〈0.01或P〈0.05）。平均住院日也明显缩短,但无统计学差异（P〉0.05）。治疗组的治愈及显效病例高于单纯西药组,胃肠道不良反应低。中西医结合治疗成人支原体肺炎疗效较好,较单纯应用阿奇霉素治疗能更快改善症状。     

肺炎支原体肺炎合并Epstein-Barr病毒感染患儿的血清因子及免疫分析

为了解肺炎支原体(Mycoplasma pneumoniae)合并Epstein-Barr 病毒(Epstein-Barr virus,EBV)感染患儿的血清因子水平和免疫功能,选取2016年9月-2017年3月于上海交通大学附属第六人民医院儿科住院的肺炎支原体肺炎(Mycoplasma pneumoniae pneumonia,MPP)合并EBV感染患儿(A组)、单纯MPP患儿(B组)和小儿骨科择期行六指切除手术的患儿(C组),采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测3组患儿血清白细胞介素6(interleukin 6,IL-6)、IL-4、γ干扰素(interferon γ,IFN-γ)、IL-10水平。结果显示,A和B组IL-6、IL-4、FIN-γ、IL-10水平高于C组(P＜0.05),A组IL-6、IL-10水平高于B组(P＜0.05),且A组中重症肺炎患儿血清IL-6、IL-10、IL-4水平较非重症肺炎患儿高(P＜0.05)。结果提示,MPP合并EBV感染患儿体内存在细胞免疫紊乱,MPP合并EBV感染后可通过诱导机体分泌 IL-6、IL-4、IFN-γ、IL-10而造成组织损伤。因此, IL-6、IL-4、IFN-γ、IL-10的检测对MPP合并EBV感染的诊断及评估病情具有重要意义。     

Pathogenesis and association of Mycoplasma pneumoniae infection with cardiac and hepatic damage

The aim of this study was to investigate the pathogenesis of Mycoplasma pneumoniae (MP) infection and its association with cardiac and hepatic damage. Between March 2013 and March 2014, 59 children with MP pneumonia (MPP) and 30 healthy children were enrolled. Serum titers of TLR4, T cell immunoglobulin and mucin‐domain (TIM) 3, IL‐10, TNF‐α, and IFN‐γ were measured both in children with MPP and healthy children. Additionally, MP‐specific antibody titer and creatine kinase‐MB (CK‐MB), and alanine transaminase (ALT) titers were measured in patients with MPP. There were significant differences between the MPP patients and healthy controls in titers of TIM1 (P < 0.01), TLR2 (P = 0.028), TLR4 (P = 0.019), IL‐10 (P < 0.01), TNF‐α (P < 0.01) and IFN‐γ (P < 0.01); however, no significant difference was found in TIM3 titers (P = 0.8181). TIM1 was correlated with CK‐MB (P = 0.025), whereas both TIM1 and TLR2 titers were correlated with MP‐specific antibody titers (P < 0.001; P = 0.003, respectively). Additionally, there were correlations between ALT, TIM3, and TLR2 titers (P = 0.025; P = 0.037, respectively). The titers of TIM1 were significantly higher in patients with cardiac damage (P = 0.007) than in those without it, whereas the titers of TLR2 were significantly higher in patients with hepatic damage (P = 0.026) than in those without it. TLR2, TLR4 and TIM1 may be involved in the process of MP infection. Additionally, TLR2, TLR4, TIM1 and TIM3 may play particular roles in the pathogenesis of MPP‐associated cardiac and hepatic damage.     

肺炎支原体IgG类抗体亲和力检测的实验研究与临床应用

目的探讨肺炎支原体IgG类抗体亲和力检测在肺炎支原体感染诊断中的意义。方法用被动颗粒凝集试验(PPA)和间接免疫荧光法(IFA)检测儿童上呼吸道感染者血清IgM类抗体水平,同时用IFA法检测其IgG类抗体的亲和力,并对检测结果进行相关性分析和一致性检验。结果在IgM类抗体检测上PPA法检出阳性率(60/120)高于IFA法(49/120),两法检测结果缺乏一致性。而IFA法检测IgG类抗体阳性的97例中,有22例检出低亲和力IgG类抗体,其中20例同时检出IgM类抗体,两法检测结果具有显著的相关性(P<0.001)和较好的一致性(0.7>Kappa>0.4)。结论肺炎支原体低亲和力IgG类抗体检测有与IgM类抗体检测类似的早期诊断价值,可与IgM类抗体联合检测用于区分近期感染、感染后复发或再次感染。     

儿童哮喘与肺炎支原体感染关系的探讨

目的:探讨儿童哮喘发作与肺炎支原体(MP)感染之间的关系,并分析合并MP感染的患儿的临床表现。方法:将79例2-14岁急性哮喘发作的患儿依据病史分做两组:第一次哮喘发作的35人(始发哮喘组),已经有哮喘病史的44人(复发哮喘组)。采用被动冷凝集法检测两组患儿肺炎支原体抗体(MP-IgM)。结果:始发哮喘组和复发哮喘组分别有16例(45.7%)和10例(22.7%)患儿MP-IgM阳性(P0.05)。始发哮喘组与复发哮喘组MP-IgM阳性的患儿发热和肺部啰音发生率明显高于MP-IgM阴性的患儿(P0.05),血清IgE水平也明显高于MP-IgM阴性的患儿(P0.05)。结论:MP感染与儿童哮喘发作关系密切,合并MP感染的哮喘患儿发热或肺部啰音发生率明显高于未合并MP感染的哮喘患儿。     

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium

Abstract Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium , are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn 4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn 4001 was not localized in or near the hmw 1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw 1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn 4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn 4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.     

儿科呼吸道疾病中肺炎支原体感染的结果分析

目的探讨儿科肺炎支原体（MP）感染特点,辅助临床医师早期诊断,合理用药。方法测定我院一年来2013例儿科呼吸道疾病患儿的肺炎支原体抗体（IgM）。结果2013例呼吸道感染患儿,检出肺炎支原体抗体（IgM）阳性者769例,占38.2%,769例阳性分别表现为肺炎369例（48%）,支气管炎238例（31%）,咽炎92例（12%）,哮喘70例（9%）。其中,肺炎组与各组相比较,具有统计学意义。MP IgM感染的检出率明显高于其他各组（P〈0.01）。结论MP感染是患儿不可忽视的病原体,检测患儿血清MP抗体能够及早诊断,指导治疗。     

Defective strain of Mycoplasma pneumoniae isolated from a patient

Abstract The isolation from a patient, for the first time, of a defective Mycoplasma pneumoniae strain (146-2) is herewith reported. The strain was isolated 28 days after onset of the disease. It is devoid of two characteristic properties: haemadsorption and adherence to glass. The absence of these two characteristics coincides with significant physiological modifications of the microbial cell. Cell multiplication of strain 146-2 in the logarithmic phase is more rapid and is followed by the culture's rapid decline and death. The sera obtained from the immunized rabbits showed that the strain is immunogenic.     

儿童副流感病毒、肺炎支原体感染情况研究

摘要 目的：通过检测及分析住院患儿血清中副流感病毒、肺炎支原体IgM抗体,探讨两种病原体感染的流行病学特征。方法：应用酶联免疫分析的方法定性检测患儿血清中的副流感病毒、肺炎支原体IgM抗体,采用卡方检验对不同年份、性别、年龄及季节的数据进行分析。结果：副流感病毒、肺炎支原体阳性率分别为3.6%、22.4%,混合感染的阳性率为1.3%。PIV每年女性患儿比男性患儿阳性率高,每一年中6-14岁感染阳性率最高;MP每年女性患儿比男性患儿阳性率高,随着年龄增长感染的阳性率也升高,6-14岁阳性率最高。PIV与MP在每年的四季中,感染的阳性率各不相同,并且没有相对的规律可循,但是在冬季阳性率还是较高,其次是春季、秋季,最低的是夏季。结论：掌握儿童副流感病毒和肺炎支原体感染流行病学特点,有助于了解疾病感染特点,从而在疾病的不同时期及时快速地采取相应措施。     

Invasion and intracellular survival of Hafnia alvei strains in human epithelial cells

Aims: The aim of this study was to investigate the invasion and intracellular survival of different Hafnia alvei strains in HeLa cells. Methods and Results: We performed different experiments on the bacterial invasion of different strains of H. alvei into the HeLa cell line using gentamicin protection assays and immunofluorescence. We also report the time course of cell internalization and the effects of inhibitors on the invasion of H. alvei. Levels of invasion varied depending on the conditions (strain, time and inoculum size) used. Conclusions: This study revealed that H. alvei strains were able to enter and persist in a human epithelial cell line. Significance and Impact of the Study: Our in vitro findings highlight the possibility that some H. alvei strains may exploit nonprofessional phagocytes or nonphagocytic cells to spread in vivo, which may be important for the persistence and establishment of an asymptomatic carrier state.     

The reducing antioxidant capacity of Mycoplasma fermentans

Mycoplasma fermentans is an extracellular microorganism capable of adhering to the surface of host cells. It has been recently shown that plasminogen binding to M. fermentans in the presence of the urokinase-type plasminogen activator promotes the invasion of host cells by this organism. In this report, we show that viable mycoplasmas persist within the infected HeLa cells for prolonged periods of time despite the expectation that within host cells the organism may be exposed to oxidative stress. Using cyclic voltammetry and luminol-enhanced chemiluminescence assays, we detected a potent reducing antioxidant activity in M. fermentans. The reducing antioxidant activity was heat stable, not affected by proteolysis and was almost totally lost upon dialysis suggesting that the activity is due to a nonproteinaceus low molecular weight antioxidant. This antioxidant was partially purified by Bio-Gel column chromatography followed by high-pressure liquid chromatographic analysis. We suggest that the high reducing antioxidant capacity in M. fermentans is a principal defense mechanism playing a major role in the battle of the organism against oxidative stress within the host cells.     

肺炎支原体感染对川崎病的影响及机制探讨

目的 分析肺炎支原体感染对川崎病的影响及其机制,以期指导临床诊疗。 方法 回顾性分析2013年8月至2018年8月我科住院的496例川崎病患儿临床资料,其中合并肺炎支原体感染组193例,未合并肺炎支原体感染组303例。应用倾向评分匹配法1∶1校正2组的年龄、性别、是否为不完全型川崎病、是否使用糖皮质激素、丙种球蛋白使用时间、丙种球蛋白使用方法和阿司匹林初始剂量,比较2组患儿的总发热天数、冠状动脉直径、冠状动脉扩张发生率、冠状动脉瘤发生率和丙种球蛋白无反应发生率。 结果 川崎病合并肺炎支原体感染组男性患儿多,更易发生颈部淋巴结肿大,年龄、中性粒细胞百分比、血沉和低密度脂蛋白高于川崎病未合并肺炎支原体感染组,血钾、血钙、高密度脂蛋白和白蛋白低于川崎病未合并肺炎支原体感染组,差异有统计学意义(均P2=5.516,P结论 肺炎支原体感染可能引起川崎病患儿脂质代谢及电解质的紊乱,其炎症反应更强,持续时间更长,对冠状动脉损伤更大。     

儿童肺炎支原体咽拭子快速液体培养结果分析

目的分析儿童肺炎支原体（MP）咽拭子快速液体培养的结果及与患儿年龄以及季节的关系,为临床诊断、治疗肺炎支原体感染提供依据。方法选取深圳市宝安区人民医院1217例急性呼吸道感染的患儿,采用咽拭子快速液体培养基进行筛查,观察其阳性率。结果在1217例患儿中,共检出阳性281例,阳性率为23．09％。〈1岁、1～3岁、3～6岁和6～14岁各年龄组的阳性率分别为15．13％、26．52％、28．31％和18．07％;不同季节MP感染率分别为春23．75％,夏20．11％,秋18．61％,冬29．72％。1～3岁,3—6岁小儿感染率明显高于6—14和〈1岁儿童（P〈0．01）;冬春季阳性率较夏秋季高。结论MP快速液体培养鉴定对MP诊断具有重要的临床诊断价值。肺炎支原体感染全年均可发病,好发于冬季,以3—6岁小儿多见。     

肺炎支原体分型与肺炎患儿临床症状相关性

目的 研究肺炎支原体分型与肺炎患儿临床症状的相关性。方法 选择2017年11月至2018年5月在郑州大学附属儿童医院儿科治疗的确诊为肺炎支原体（MP）肺炎的患儿86例。采用自行设计的调查表研究患儿基本资料,包括性别、年龄、既往史、入院24 h临床症状、实验室指标、影像学症状等。利用实时定量PCR技术分析样本中肺炎支原体基因拷贝数,并设计引物分析MP基因分型。结果 共检出MP低拷贝量患儿15例（17.44%）,中拷贝量患儿24例（27.91%）,高拷贝量患儿47例（54.65%）;P1-I型MP 80例（93.02%）,P1-II型MP 6例（6.98%）。患儿MP基因拷贝数与病程≥10 d,体温＞39℃,畏寒或寒战,GRA%,GRA,CRP,PCT,大片状实变/不张,胸腔积液呈正相关,且为弱或中等相关。MP基因拷贝数与LYM%,LYM呈负相关,且为弱相关。患儿MP分型与体温＞39℃呈负相关,且为弱相关。结论 MP肺炎患儿上呼吸道MP数量与患儿临床表现,实验室指标和影像学特征存在一定相关性,因此上呼吸道MP数量可成为MP肺炎病情评估的指标之一。     

新型p1 基因型肺炎支原体分型方法的建立与应用

【目的】针对肺炎支原体新型p1基因型(V2c型)菌株检测工作的需要,建立相应PCR检测方法并进行评价。【方法】针对新型V2c型肺炎支原体菌株p1基因变异区域序列设计特异性扩增引物,建立对V2c型肺炎支原体菌株进行PCR检测的检测方法并用相关基因测序进行验证。使用所建立的巢式多重PCR对北京地区2008-2011年分离到的214株临床肺炎支原体进行分型分析。【结果】特异引物可有效检测出V2c菌株,在其它型别菌株均无阳性扩增。214株肺炎支原体临床分离株中1型菌株占90.2%(193/214),V2a型菌株占0.9%(2/214),V2c型菌株占8.9%(19/214);未检出2型菌株。【结论】针对V2c型肺炎支原体所建立的基于p1基因的PCR检测方法,能有效区分以往方法无法检测出的新型V2c型肺炎支原体菌株,对开展肺炎支原体流行病学调查和病原分析有重要意义。