Viable Chlamydia Research Articles

In vitro susceptibility and eradication of Chlamydia pneumoniae cardiovascular strains from coronary artery endothelium and smooth muscle cells.

Recovery of viable Chlamydia pneumoniae from atheromas of coronary heart diseases patients has initiated pilot studies to eradicate C. pneumoniae from vascular tissue by antibiotic treatment. To provide data for the selection of effective antibiotics, we investigated the in vitro activity of anti-chlamydial antibiotics to eliminate vascular strains of C. pneumoniae from coronary endothelial and smooth muscle cells, celltypes that are involved in the pathogenesis of atherosclerosis. The susceptibility of the obligate intracellular chlamydiae was studied in primary coronary endothelial cells, smooth muscle cells and immortalized epithelial cells. Minimal inhibitory concentrations (MICs) were determined for ofloxacin, levofloxacin, trovafloxacin, moxifloxacin, erythromycin, azithromycin, roxithromycin, rifapentin and rifampin. In vitro, rifampin was the most effective drug overall. Moxifloxacin and trovafloxacin were as effective as the macrolides of which roxithromycin was the most active one. Actively replicating C. pneumoniae can be eliminated in vitro from cell types, involved in the pathogenesis of atherosclerosis by various antibiotics. These data provide an experimental background for the selection of antibiotics in clinical eradication studies and will help to assess the potential success of prevention and eradication therapies.

Detection of Viable Chlamydia pneumoniae inAbdominal Aortic Aneurysms

Objectives to investigate the presence of Chlamydia pneumoniae in the wall of abdominal aortic aneurysms (AAAs) and in the aortas of patients without a history of cardiovascular disease. Design case–control study. Materials twenty-six consecutive patients operated for AAA were compared to 17 controls.Methods aorta was obtained at surgery or autopsy (controls) and prepared for immunohistochemical (IHC) analysis and culture for C. pneumoniae. Throat swabs from 14/26 patients were analysed by PCR for C. pneumoniae. Blood was obtained from 24/26 patients and from 178 70-year-old males. ResultsC. pneumoniae was detected in the aortic aneurysms of 20/26 patients by IHC. C. pneumoniae was cultured from 10 of the 20 IHC-positive patients. Only 1/17 controls was positive for C. pneumoniae by IHC (p=0.0001). PCR was positive for C. pneumoniae in 5/14 patients. Serological analysis by microimmunofluoresence (MIF) showed significantly more high titres of the specific antibodies to C. pneumoniae in patients than in age-matched male controls. Conclusions we conclude that C. pneumoniae is often present in AAAs in a viable form and that C. pneumoniae is linked to the pathogenesis of AAA.

Isolation and continuous growth of Chlamydia pneumoniae from arterectomy specimens.

For the purpose of collecting Chlamydia pneumoniae strains of vascular origin that could be grown continuously in vitro, a cell culture system has been established. Using different types of vascular specimens obtained from 38 patients, Chlamydia pneumoniae could be isolated in three (7.9%) cases. The strains were obtained from specimens of the carotid artery, the femoral artery and an infrarenal aneurysm of the abdominal aorta of three male atherosclerosis patients. Thus, viable Chlamydia pneumoniae strains are also present in vascular regions other than those hitherto described.

Demonstration of Viable Chlamydia pneumoniae in Atherosclerotic Plaques of Carotid Arteries by Reverse Transcriptase Polymerase Chain Reaction

The presence of Chlamydia pneumoniae in atheromas has been demonstrated in several studies. Culture of the organism from arterial tissue has been difficult. We report the use of a reverse transcriptase polymerase chain reaction to detect viable Chlamydia pneumoniae in carotid atheromas. We analyzed 30 patients (14 females, mean age 69.6 +/- 8.8 years) who underwent surgery for the removal of atherosclerotic plaques from carotid arteries. During surgery, samples of lingual vein and superior thyroideal artery were also taken. We applied two molecular biology techniques to the carotid plaques on lingual vein or thyroideal artery samples: 1) polymerase chain reaction (PCR) and 2) reverse transcriptase-PCR (RT-PCR) for the detection of bacterial mRNA, employing PCR primers designed to detect a fragment of the 16S rRNA gene. Blood samples were obtained from the patients for determination of Chlamydia pneumoniae IgG, IgA, and IgM antibody titers by a microimmunofluorescence technique. The results of the present study confirmed the presence of viable Chlamydia pneumoniae in atheromas and support the hypothesis that the organism may be an active factor in the pathogenesis of atherosclerosis.

Synoviocyte-packaged Chlamydia trachomatis induces a chronic aseptic arthritis.

The basic mechanisms underlying reactive arthritis and specifically the joint injury that follows intra-articular Chlamydia trachomatis infection have not been defined. The present study addresses this question through the development of an experimental model. Stable cell lines were generated from synoviocytes harvested from the knee joints of Lewis rats. The synoviocytes were cocultivated with C. trachomatis to allow invasion by the microbe and were then transferred by intra-articular injection into the knee joints of Lewis rats. The ensuing arthritis could be subdivided into an early phase (</= 14 d) and a late phase. The early phase was characterized by intense, primarily neutrophilic, synovitis; accelerated cartilage injury; dissemination of Chlamydia to liver and spleen; and viable Chlamydia in the joints. The late phase was marked by mixed mononuclear lymphocyte infiltration in the joint; dysplastic cartilage injury and repair; absence of viable organisms; and development of a distinctive humoral response. Western blot analysis comparing reactive arthritis patients to the experimental model indicates that candidate arthritogenic chlamydial antigens are comparable between the two. This model demonstrates that an intense synovitis can be induced by this intracellular pathogen, and that chronic inflammation can persist well beyond the culture-positive phase. Furthermore, these data show that the synoviocyte is a suitable host cell for C. trachomatis and can function as a reservoir of microbial antigens sufficient to perpetuate joint injury.

Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease

The common respiratory pathogen Chlamydia pneumoniae has been implicated in the pathogenesis of coronary artery disease and acute myocardial infarction. In order to verify the endovascular presence of potentially viable chlamydia by detection of genomic DNA, we examined atherosclerotic arteries from various vascular regions using a C. pneumoniae specific nested polymerase chain reaction (PCR). The samples were obtained during surgical revascularization procedures or at autopsy. Chlamydial DNA was detected in 51/238 (21%) atherosclerotic samples. A total of 17 non-atherosclerotic control samples were PCR-negative. Chlamydial presence was detected in 36/140 (26%) vascular samples obtained at coronary revascularization procedures, in 9/61 (15%) samples from carotid artery stenosis, 3/17 (18%) samples from the aorta, and 3/20 (15%) iliac artery samples. Histomorphological discrimination of infected and non-infected arterial samples was not possible. Antichlamydial IgG and IgM response as examined by microimmunofluorescence assay did not aid identification of individual endovascular infection. C. pneumoniae is present in a significant proportion of atherosclerotic arteries. Its occurrence in atheromatous plaques is not limited to coronary arteries and may be considered indicative of an infectious component in atherosclerosis. However, it remains unclear whether chlamydia actually initiates atherosclerotic injury, facilitates its progression, or merely colonizes pre-existing atheromata.

Does Chlamydia pneumoniae cause coronary heart disease?

Evidence continues to accumulate to support a role for Chlamydia pneumoniae in the pathogenesis of atherosclerosis, and in particular, coronary heart disease. In an analysis of 18 seroepidemiological studies, individuals with high-titer antibodies to Chlamydia pneumoniae have at least a twofold or larger odds ratio for coronary heart disease compared with seronegative individuals. In addition, viable Chlamydia pneumoniae has been cultured directly from atherosclerotic tissue, and has been demonstrated by polymerase chain reaction, immunocytochemistry, and electron microscopy in nearly 50% of atheromatous lesions, but in only 5% of control samples. Chlamydia pneumoniae has been shown to infect and reproduce in vitro in human smooth muscle cells, coronary artery endothelial cells, and macrophages, and has been shown to induce procoagulant activity and platelet adhesion factors known to contribute to the pathogenesis of atherosclerosis. Furthermore, Chlamydia pneumoniae has been identified in atherosclerotic tissue in two experimental animal models. Finally, two pilot antibiotic trials of post-myocardial infarction patients receiving antibiotics effective against Chlamydia pneumoniae have demonstrated a fivefold reduction in cardiovascular events compared with those patients receiving placebo. Although a causal effect has not been proved, the relationship between Chlamydia pneumoniae and atherosclerosis continues to grow and to stimulate more definitive studies.

Endovascular Presence of Viable Chlamydia pneumoniaeIs a Common Phenomenon in Coronary Artery Disease

Objectives. We sought to examine coronary arteries for the presence of viable bacteria of the fastidious species Chlamydia pneumoniae.Background. The respiratory pathogen C. pneumoniaehas been implicated in the pathogenesis of coronary artery disease (CAD). Previous studies have demonstrated an antichlamydial seroresponse to be a cardiovascular risk factor and coronary atheromata to contain chlamydial components in varying proportions. Endovascular demonstration of replicating bacteria is required to provide evidence for an infectious component in CAD and a rationale to discuss antimicrobial therapy.Methods. Myocardial revascularization was performed in 70 patients. Atherosclerotic lesions from 53 coronary endarterectomy and 17 restenotic bypass samples were cultured and subjected to nested polymerase chain reaction (PCR) for C. pneumoniae.Antichlamydial immunoglobulin G (IgG), IgA and IgM was examined by microimmunofluorescence.Results. Viable C. pneumoniaewas recovered from 11 (16%) of 70 atheromata, and chlamydial deoxyribonucleic acid (DNA) was detected in 21 (30%) of 70 atheromata; 17 nonatherosclerotic control samples were PCR-negative (p < 0.01). Fifteen (28%) of 53 endarterectomy and 6 (35%) of 17 bypass samples were PCR-positive. DNA sequencing of six different PCR products did not reveal differences between coronary isolates and respiratory reference strains, suggesting that common respiratory strains gain access to the systemic circulation. Serologic results did not correlate with direct detection results and did not identify individual endovascular infection.Conclusions. A significant proportion of atherosclerotic coronary arteries harbor viable C. pneumoniae. This finding supports the hypothesis of a chlamydial contribution to atherogenesis. Whether chlamydiae initiate atherosclerotic injury, facilitate its progression or colonize atheromata is unknown. However, the endovascular presence of viable bacteria justifies a controlled clinical investigation of antimicrobial treatment benefit in the therapy and prevention of CAD.

Chlamydia trachomatis Can Persist in Joint Tissue After Antibiotic Treatment in Chronic Reiterʼs Syndrome / Reactive Arthritis

Considerable evidence suggests that viable Chlamydia trachomatis are present in joint tissues of patients with Reiter's syndrome/reactive arthritis (RS/ReA), but the use of antibiotics to treat such patients remains controversial. We investigated the continued presence of chlamydia in synovial tissues of patients with RS/ReA; these patients had been treated with antibiotics for relatively extended periods, had shown clinical improvement, but had persistent active disease. Knee synovial tissue was obtained from two patients with RS/ReA and two controls with osteoarthritis (OA). Each sample was screened for chlamydia by culture, direct fluorescent antibody assay (DFA), in situ hybridization (ISH), and polymerase chain reaction (PCR).Synovial tissues from antibiotic-treated RS/ReA patients were negative for chlamydia when analyzed by culture and DFA, but positive when analyzed by ISH for chlamydial RNA and by PCR for chlamydial DNA. Samples from OA patients were negative by all screening methods. Thus, antibiotic treatment does not appear to easily eradicate chlamydia from the joints of RS/ReA patients. Rather, the organism can persist in synovial tissue in a form not detectable by routine laboratory screening methods. Further studies are needed to determine whether antibiotic regimens other than those used here can eradicate synovial chlamydia and to determine how this relates to disease activity. Optimal therapy for patients with RS/ ReA is therefore not yet clear.

A microcarrier culture method for the production of large quantities of viable Chlamydia pneumoniae.

We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on microcarrier beads. Infection of the cells in microcarrier culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption. The yield in microcarrier culture was similar to that of microtitre-plate culture using centrifugation-assisted infection (120 x 10(6) and 225 x 10(6) bacteria/10(6) HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively). However, up to 64% savings in labour time and 27% saving in culture medium were achieved if the microcarrier culture method was used instead of the microtitre-plate culture method. The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24 - 1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection of PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35 degrees C or 37 degrees C). The yield of microcarrier cultures was the same at an incubation temperature of 35 degrees C or 37 degrees C in contrast to an increased production at 35 degrees C in the microtitre-plate culture method using centrifugation-assisted infection. In conclusion, the microcarrier culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically.

Differential modulation of lymphocyte proliferative responses and lymphokine secretion in mice during development of immunity to Chlamydia psittaci

A murine model was utilized to study immune responses occurring during the period of acquisition of immunity to chlamydial infection. C3H (H-2k) mice were immunized by intramuscular injection of 5 x 10(3) viable Chlamydia psittaci elementary bodies (EBs) by a protocol which permits animals to survive an otherwise lethal intraperitoneal challenge 10 days later with the homologous chlamydial strain. Spleen cells assayed during the 10-day period of development to immunity showed depressed proliferative responses in vitro to the T-cell mitogen, concanavalin A, and also exhibited suppressor cell activity. Spleen cell mitogen responses returned to normal levels by 30 days postimmunization, concomitant with the detectable development in vitro of responses to chlamydia-specific antigen. In marked contrast to the reduced proliferative responses, mitogen-stimulated production of the T-cell-derived lymphokines interleukin-2 and gamma interferon by spleen cells from immunized animals was within the normal range at 10 days postimmunization, and supernatant fluids containing these products had both microbicidal and microbistatic effects on chlamydial organisms in vitro. These results demonstrate that independent regulation of T-cell proliferation and lymphokine production occurs in vivo as part of the development of an antigen-specific protective immune response. These results also suggest that such differential modulation of T-cell responses may contribute to the development of protective immunity to chlamydiae in mice, perhaps through limited T-cell clonal expansion coupled with early or preferential maturation of cytokine-secreting helper T cells.

Transmission of chlamydiae by the housefly.

The ability of the housefly to carry viable Chlamydia trachomatis and to transmit a chlamydial ocular infection was studied under laboratory conditions. After feeding flies (Musca domestica) on suspensions of egg yolk sac infected with C. trachomatis serotypes A or B (responsible for hyperendemic trachoma) the agents were reisolated from flies' intestines for up to 6 hours and from their legs and/or proboscises for up to 2 hours. It was found that the viability of chlamydiae is dependent on the protective effect of yolk concentration in the original inoculum. Results of experiments with guinea-pig inclusion conjunctivitis as an animal model show that under laboratory conditions flies can readily transmit this chlamydial ocular infection from one animal to another. These results suggest that under field conditions flies can play an important role in the transmission of trachoma, particularly in areas with favourable conditions such as a large reservoir of infection among children with severe trachoma, copious eye discharge caused by trachoma and associated bacterial infections, a large fly population, and close proximity of children in large family groups.