Detection of infectious agents in heart valves during endocarditis using PCR technique

Infectious endocarditis can be caused by various microorganisms. Diagnostics of local infection by microbiological methods is not always effective. For that reason we performed a study aimed for direct detection of potential infectious agents by polymerase chain reaction in patients' heart valve tissue. DNA of infectious agents was revealed in 72% of heart valve tissue samples from patients with septic endocarditis; in studied samples, along with bacterial DNA, herpesviruses' DNA was detected. Obtained results confirm the presence of infection, which allows to perform specific diagnostics of infectious complications after implantation of prosthetic cardiac valves.     

Finegoldia magna,an early post-operative cause of infectious endocarditis: Report of two cases and review of the literature

We report two cases of infectious endocarditis (IE) on prosthetic valves caused by Finegoldia magna. The diagnosis was obtained by detection of the bacterium in valvular biopsies using 16S rRNA PCR amplication and sequencing, and prolonged culture. Five other cases were previously published in the literature. Following analysis of these seven cases, F. magna endocarditis presented as a subacute endocarditis, developing early (60 days) following valvular replacement (85%), with an elevated mortality (28%). Our report highlights the potential role of F. magna in early post-surgical endocarditis on prosthetic valves.     

Propionibacterium acnes in the etiology of endocarditis]

Propionibacterium acnes is the gram positive anaerobic bacteria belongs to the normal skin and oral microbial flora. The participation of this microorganism in the infective endocarditis is still controversial. The aim of the study was to perform the diagnostic and therapeutic difficulties in 5 patients with infective endocarditis caused by Propionibacterium acnes. In 3 out of 5 patients the infective endocarditis developed after prosthesis valve replacement, in 2 others on the native valves. The inserted prostheses were mechanical ones, propionibacterium acnes was identified as causative organisms in all of the causes (two positive blood and/or valve culture). The bacterial strains were sensitive to the antibiotics as: penicillins, cephalosporins, clindamycin, and vancomycin, however cephalosporins used at the beginning of the treatment in 3 patients and clindamycin in 1 patient had limited clinical efficacy. Later treatment with timentin, augmentin and tienamycin was successful in 3 patients; one patient was cured with vancomycin. One patient died because of septic, embolic complication in early stage of illness. We conclude the effectiveness of penicillins in combination with clavulanic acid and tienamycin in therapy of infective endocarditis due to Propionibacterium acnes. The treatment should be lasted during 4-6 weeks.     

Course and outcomes of infectious endocarditis in HIV-infected patients

39 patients with acute infectious endocarditis were observed; of these, 28 patients had HIV infection at different stages of the disease. The specific features of the course of acute infectious endocarditis in HIV-infected patients were established. The severe course of acute septic endocarditis was observed in those patients whose parameters of the cell-mediated immune system (cells CD4+) were in the state of compensation or subcompensation. At different stages of HIV infection different clinical syndromes of infectious endocarditis prevailed. In patients with HIV infection the combined lesions of the heart valve apparatus were observed and mixed microflora was isolated from the blood more frequently. The development of acute septic endocarditis negatively affected the course of HIV infection and was manifested by a rapid decrease in the amount of CD4 lymphocytes.     

New insight into the diagnosis of fastidious bacterial endocarditis

Sterile blood cultures are noted in one third of patients with infectious endocarditis. Although in half of cases this is due to previous antibiotic therapy, in the other half, the aetiology of culture-negative endocarditis is intracellular bacteria such as Coxiella burnetii or fastidious growing bacteria. Although it was previously considered that the prevalence of such organisms was identical throughout the world, recent investigations on Bartonella endocarditis clearly showed that the aetiology of culture-negative endocarditis is likely to be strongly related to epidemiology of the agent in each country. During the past decade the use of molecular techniques such as PCR with subsequent sequencing to detect or to identify bacteria in valves from patients with infectious endocarditis have considerably improved the aetiological diagnosis. This is especially true in the case of culture-negative endocarditis following earlier antibiotic therapy. However, the fact that DNA remnants of past endocarditis can be detected some time after the acute episode, when the patient has been cured, suggests that the predictive value of these techniques along with the traditional histology and culture need to be evaluated closely.     

DNA提取过程中混入的实验室空气微生物DNA定量

元基因组测序方法为微生物研究提供了有力的工具。但其中的DNA提取过程,会不可避免地混入实验室中的空气微生物。这些微生物DNA,是否会对一些极微量的元基因组检测(如皮肤样本等)结果造成影响,有多大影响,仍没有明确结论。本研究首先收集了实验室空气样品,用16S rRNA引物建立了基于qPCR的标准曲线,并检测了在开放环境下提取DNA过程中可掺杂的环境微生物DNA量。然后在开放环境下提取纯水DNA样品并进行元基因组分析,以确定掺杂环境微生物的种类。最后分别在生物安全柜和实验室开放环境下提取皮肤样本,并用鸟枪测序方法对样本的微生物组成进行分析,以评估掺杂环境微生物对元基因组检测结果的影响。结果显示,在实验室开放环境的DNA提取过程中,环境微生物的DNA残留可达28.9 pg,可达某些极微量样本DNA总量的30%。元基因组分析显示,样品中掺杂的环境微生物主要是痤疮杆菌Cutibacteriumacnes、大肠杆菌Escherichia coli等皮肤常见细菌。与洁净皮肤样本的信息相比,开放环境下提取掺杂了数十种环境微生物,并导致主要菌种的丰度大幅降低,从而影响结果的真实性。因此,微量样品的DNA...     

MRI Visualization of Staphyloccocus aureus-Induced Infective Endocarditis in Mice

Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 10⁵ S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic strategy.     

Rapid method of determining the dry mass of whooping cough microbes

A colorimetric method for the rapid determination of the quantitative content of microbial mass in B. pertussis suspensions has been developed. The method is based on the indirect determination of carbon in microbial suspensions by its oxidation with the mixture consisting of potassium bichromate in concentrated sulfuric acid and the subsequent colorimetric analysis of the products of this reaction. The method ensures sufficient accuracy, the determination procedure is simple, takes not more than 2 hours and requires no complex reagents. The results thus obtained are well comparable with those obtained by the classical gravimetric method. The new method permits the determination of microbial mass in B. pertussis suspensions with a minimum concentration of 0.5 mg/ml. The method is recommended for the determination of dry microbial mass in B. pertussis suspensions.     

Experimental endocarditis model of methicillin resistant Staphylococcus aureus (MRSA) in rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.     

体内生物膜研究进展

生物膜是指由黏附于生命体或非生命体表面的微生物和其胞外聚合物所构成的复杂、多维的空间结构,在自然界中普遍存在。微生物在人类体内许多部位均可形成生物膜结构,如肺、心脏瓣膜、泌尿生殖道、肝胆系统、耳、鼻、皮肤等。越来越多的感染性疾病患者或动物模型体内均可检测出生物膜,如囊性纤维化肺炎、慢性中耳炎、细菌性心内膜炎、皮肤外伤后慢性感染、慢性鼻窦炎等。生物膜的形成与感染性疾病的关系及其在疾病的发生、发展、转归过程中的作用成为近年来研究的热点。目前体内生物膜检测技术主要有扫描电镜、激光共聚焦显微镜、荧光原位杂交技术等。就临床常见感染性疾病与生物膜的关系及体内生物膜检测方法展开综述。     

Minor fatty acids of biological fluids of urogenital organs and their significance in the diagnosis of inflammatory processes

Minor lipid components (less than 1%), not occurring in the cells of mammals, have been detected in the profile of fatty acids (FA) in urine and vaginal fluid in females, as well as in ejaculate in males, by the method of gas chromatography and mass spectrometry. Their origin is supposedly attributed to microorganisms colonizing urogenital organs under normal conditions and in infectious pathology. Minor FA in the biological fluids of the urogenital tract (UGT) in man are compared with the FA composition of pure microbial cultures. The statistical analysis of FA concentrations in 500 specimens of UGT biological fluids indicates that lipid markers of microbial origin (in UGT inflammatory diseases) have a range of quantitative changes exceeding the limits of values characteristic of healthy persons and in a number of cases correlate with the diagnosis of the disease or the results of the bacteriological study. The reliability of the interpretation of the results of the marker analysis is confirmed by the difference in the composition of UGT microorganisms under normal conditions and in pathology, correlation with the clinical picture of pathological deviations, a decrease (to zero or normal values) in the concentration of the markers of pathogens as the result of treatment with antibiotics, as well as by the adequate composition of UGT microorganisms, determined by the marker method in comparison with the cultural biochemical method.     

Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10(+) and Asc10(-)E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2-4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis.     

Characteristics of determining the microbial contamination of penicillin series antibiotic powders and tablets]

Optimal conditions for determination of microbial contamination of drugs were studied on artificially contaminated powders and tablets of phenoxymethylpenicillin, ampicillin, oxacillin and dicloxacillin. The method of membrane filtration was the best for determination of the microbial contamination of the powders. However, it was not possible to wash out completely the antibiotic from the membrane filter. To prevent this it was necessary to add penicillinase into the nutrient medium onto which the filter was put for providing the microbial growth. For determination of microbial contamination of tablets direct plating of 3 per cent suspension of the tablet mass onto the surface of the nutrient medium with penicillinase was the best.     

感染性心内膜炎与肠道菌群的相关性研究

目的 探讨感染性心内膜炎与肠道乳杆菌、双歧杆菌和大肠埃希菌的临床相关性。方法 选择2008年4月至2016年4月湖州市第一人民医院心内科病房收治的单纯感染性心内膜炎患者67例为观察组,选择同期来院体检结果正常者65例为对照组。分别收集观察组患者治疗前1天、治疗第3天和治疗1周后3个不同时间节点的粪便标本,同时收集对照组的粪便标本,通过FISH实验检测粪便中双歧杆菌和乳杆菌的含量差异,同时通过16SrRNA/DNA荧光定量PCR技术进一步明确观察组患者3个时期肠道双歧杆菌、大肠埃希菌和乳杆菌与对照组的数量差异。结果 观察组患者治疗前1天双歧杆菌值为6.2%±0.56%,治疗第3天为4.3%±0.34%,治疗1周后为8.7%±0.56%,对照组双歧杆菌值为9.4%±0.98%。观察组患者治疗前1天乳杆菌值为5.4%±0.86%,治疗第3天为4.9%±0.24%,治疗1周后为6.1%±0.72%,对照组乳杆菌值为8.3%±0.51%。治疗前1天与治疗1周后,观察组患者双歧杆菌和乳杆菌值与对照组相比,差异均有统计学意义（P<0.05）。16SrRNA/DNA荧光定量PCR检测表明,观察组患者在3个治疗时期中肠道乳杆菌、双歧杆菌和大肠埃希菌数量的对数值与对照组比较差异均有统计学意义（P<0.05）。其中观察组患者双歧杆菌和乳杆菌数量在治疗第3天较低,1周后有所回升;大肠埃希菌数量在治疗第3天亦较低, 但1周后没有明显回升。结论 感染性心内膜炎患者肠道菌群数量与健康人相比有所下降,肠道乳杆菌、大肠埃希菌和双歧杆菌失调可能影响其发病和转归。     

物种与科属的数量关系——以中国鸟类为例

许多研究表明,物种与高级分类阶元在数量上密切相关,这种关系在地区物种数量预测中具有很高的应用价值。应用中国不同地理类型(保护区、行政区和动物地理亚区)的鸟类物种名录,研究物种与科属的数量相关关系和频次分布规律,结果表明(1)物种与高级分类阶元在数量上有着极显著相关关系(P<0.01),其中物种—属的相关关系要显著于物种—科,且在对数、乘幂、指数和线数4种模型中,用乘幂模型拟合最好;(2)物种在科属间的频次分布具有规律性(P<0.05),随着物种数/科属的增加,频次迅速减少,在4种模型中,乘幂模型拟合最好,属的规律性较科显著。根据以上结果,物种与科属间的数量强相关关系与物种数量在科属间有规律的频次分布有关。     

Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis

Aims

Infective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization.

Methods and Results

IE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO). The presence of neutrophil extracellular traps (NETs) associating MPO and externalized nucleosomes, was shown by immunostaining in the VG. Both MPO and cell-free DNA were released in larger amounts by VG than N samples, suggesting bacterial activation of PMNs within the vegetation. Finally, evidence of proteolytic tissue damage was obtained by the release of fragments of extracellular matrix components such as fibrinogen and fibronectin, as well as protease-sensitive receptors such as the uPA receptor.

Conclusion

Our data obtained using human IE valves suggest that septic vegetations represent an important source of proteases originating from massive leukocyte recruitment and activation of the host plasminergic system. The latter forms a potential therapeutic target to minimize valvular tissue degradation independently from that induced by bacterial proteases.     

Fibrinous Endocarditis in Dogs

Fibrinous endocarditis was observed in 67 (0.58 per cent) of 11,574 dog autopsies. The lesions were valvular in 62 animals and parietal in five. Valvular endocarditis mainly involved the left side of the heart, particularly the mitral valve. The pulmonary valves were never involved. The endocardial deposits in 42 animals were examined bacteriologically. Infection with streptococci could be demonstrated in more than half the dogs. Infections with staphylococci or Escherichia coli were also encountered. There was no evidence of sex predisposition, but a distinct age predisposition; fibrinous endocarditis was most common among dogs seven years old and over. A breed predisposition was seen for German Shepherds. There are reasons for believing that fibrous endocarditis is generally not a sequel to fibrinous endocarditis.     

Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves

The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for alpha-smooth muscle actin (alpha-SMA, a myofibroblast marker) were rare (0.69 +/- 0.48%), but in sclerotic valves of animals fed an atherogenic diet, myofibroblasts were spatially clustered and abundant (31.2 +/- 6.3%). In cultured VIC populations from normal valves, SMA-positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage), and stained for collagen type I and vimentin but not desmin. For an analysis of stem cells, two-color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated approximately 85% SMA-positive cells, indicating that some myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Exogenous tensile force also significantly increased SMA expression in VICs. Isotope-coded affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the nine proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with small-interfering RNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by the SMA incorporation into stress fibers. When compared with normal valves, diseased valves showed strong immunostaining for cofilin that colocalized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.     

The role of endothelial cell biology in endocarditis

The treatment of endocarditis remains a challenge for physicians, even in times of modern antibiotic treatment. Depending on its cause, endocarditis can either be of infectious or non-infectious origin. Infective endocarditis is caused by bacterial (or fungal) pathogens, and the clinical course is critically dependent on the virulence factors of the specific microorganisms involved. Therefore, the clinical type of endocarditis can be divided into an acute and more aggressive form and a subacute form (endocarditis lenta). Much of our knowledge regarding the pathogenesis of infective endocarditis is based on studies of the virulence of Staphylococcus aureus, which has become the most frequent cause of infective endocarditis nowadays. However, independently of the underlying cause of endocarditis (infectious or noninfectious), the pathogenesis involves the damage and disturbance of endothelial function and the formation of associated “vegetation”. Surprisingly little is known about the specific role of the endothelium in the pathogenesis of endocarditis. This review will thus give insights into current knowledge of the pathogenesis of endocarditis with a focus on the role of the endothelium.     

Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis

The avascularity of cardiac valves is abrogated in several valvular heart diseases (VHDs). This study investigated the molecular mechanisms underlying valvular avascularity and its correlation with VHD. Chondromodulin-I, an antiangiogenic factor isolated from cartilage, is abundantly expressed in cardiac valves. Gene targeting of chondromodulin-I resulted in enhanced Vegf-A expression, angiogenesis, lipid deposition and calcification in the cardiac valves of aged mice. Echocardiography showed aortic valve thickening, calcification and turbulent flow, indicative of early changes in aortic stenosis. Conditioned medium obtained from cultured valvular interstitial cells strongly inhibited tube formation and mobilization of endothelial cells and induced their apoptosis; these effects were partially inhibited by chondromodulin-I small interfering RNA. In human VHD, including cases associated with infective endocarditis, rheumatic heart disease and atherosclerosis, VEGF-A expression, neovascularization and calcification were observed in areas of chondromodulin-I downregulation. These findings provide evidence that chondromodulin-I has a pivotal role in maintaining valvular normal function by preventing angiogenesis that may lead to VHD.