doi: 10.1161/CIRCULATIONAHA.105.579979
(Circulation. 2006;113:929-937.)
© 2006 American Heart Association, Inc.
Coronary Heart Disease |
Detection of Diverse Bacterial Signatures in Atherosclerotic Lesions of Patients With Coronary Heart Disease
From the Institute for Clinical Molecular Biology (S.J.O., N.E.E.M., M.M., A.R., J.H., S.S.), Department of Cardiology (N.E.E.M., A.R., D.K., M.L., G.H., R.S.), Department of General Internal Medicine (S.J.O., S.H., T.K., S.N., R.G., U.R.F., S.S.), Institute for Medical Informatics and Statistics (S.F.), Institute for Transfusion Medicine (A.R.), Institute for Microbiology (H.S.), and Department of Cardiovascular Surgery (N.H.), UKSH Campus Kiel, Kiel, and German Institute of Nutrition (P.N., M.B.), Department of Gastrointestinal Microbiology, Potsdam-Rehbrügge, Germany.
Correspondence to Stefan Schreiber, MD, Institute for Clinical Molecular Biology, Christian-Albrechts-University Kiel, Schittenhelmstr 12, 24105 Kiel, Germany. E-mail s.schreiber{at}mucosa.de
Received July 30, 2005; revision received November 5, 2005; accepted December 2, 2005.
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Abstract |
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Background— Bacterial infection has been discussed as a potential etiologic factor in the pathophysiology of coronary heart disease (CHD). This study analyzes molecular phylogenies to systematically explore the presence, frequency, and diversity of bacteria in atherosclerotic lesions in patients with CHD.
Methods and Results— We investigated 16S rDNA signatures in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients with CHD, control material from postmortem patients (n=15), and heart-beating organ donors (n=11) using clone libraries, denaturating gradient gel analysis, and fluorescence in situ hybridization. Bacterial DNA was found in all CHD patients by conserved PCR but not in control material or in any of the normal/unaffected coronary arteries. Presence of bacteria in atherosclerotic lesions was confirmed by fluorescence in situ hybridization. A high overall bacterial diversity of >50 different species, among them Staphylococcus species, Proteus vulgaris, Klebsiella pneumoniae, and Streptococcus species, was demonstrated in >1500 clones from a combined library and confirmed by denaturating gradient gel analysis. Mean bacterial diversity in atheromas was high, with a score of 12.33±3.81 (range, 5 to 22). A specific PCR detected Chlamydia species in 51.5% of CHD patients.
Conclusions— Detection of a broad variety of molecular signatures in all CHD specimens suggests that diverse bacterial colonization may be more important than a single pathogen. Our observation does not allow us to conclude that bacteria are the causative agent in the etiopathogenesis of CHD. However, bacterial agents could have secondarily colonized atheromatous lesions and could act as an additional factor accelerating disease progression.
Key Words: atherosclerosis • bacteria • coronary disease • infection • plaque
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Introduction |
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The pathophysiology of coronary heart disease (CHD) is characterized by degenerative and inflammatory mechanisms. The general importance of elevated C-reactive protein (CRP) as a marker for disease progression1 has raised interest in the role of bacterial infection as a pathogenic factor. Specific pathogens have been detected in the arterial vessel wall in subsets of the patient population, and an association of disease with serological responses to viral and bacterial pathogens such as cytomegalovirus, herpes simplex virus, Chlamydia pneumoniae, or Helicobacter pylori has been reported.2–8 Periodontitis, which would lead to frequent bacteremia from the oral lesions, has been implicated as a risk factor for the development of cardiovascular disease.9,10 A recent study by Lehtiniemi et al11 hypothesized that atheromas might act as mechanical sieves collecting bacteria from the circulation as they observed a diverse colonization of the plaques. However, this small study used only a small number of postmortem specimens in which the origin, time point of colonization, and constitution of the bacterial organisms in the plaques remain unclear.
Editorial p 920
Clinical Perspective p 937
The first interventional trials with macrolide antibiotics conducted to specifically eradicate plaque infection by chlamydia after acute coronary syndrome suggested a risk reduction for secondary events such as cardiovascular complications and restenosis.12 However, large, randomized, controlled studies (Weekly Intervention With Zithromax Against Atherosclerosis and Related Disorders [WIZARD], Pravastatin or Atorvastatin Evaluation and Infection Therapy [PROVE-IT], Randomised Trial of Roxithromycin in Non–Q-Wave Coronary Syndromes [ROXIS], Azithromycin and Coronary Events Study [ACES]) that were completed recently have failed to show a benefit for specific antibiotics such as roxithromycin or azithromycin in the treatment of CHD.13–17
The objective of this study was to systematically evaluate the presence and the spectrum of bacteria in coronary atherosclerotic lesions on the basis of their molecular phylogeny signatures. In a subanalysis, the results were correlated with demographic (age, gender) and clinical parameters, including serum levels of CRP, age, recurrence of stenotic disease, and cardiovascular risk factors. A systematic detection of the bacteria and an assessment of their diversity were based on different molecular techniques. Atherectomy specimens from 38 patients with CHD were obtained by coronary catheterization and compared with various controls (n=26), including postmortem specimens (n=15) and normal coronary material from heart-beating transplantation donors (n=11). In addition, specific PCR assays were used to assess signals from Chlamydia species, Mycoplasma species, and H pylori in the same material. Selected samples were examined by fluorescence in situ hybridization (FISH) analysis to localize bacteria detected by their molecular signatures.
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Methods |
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Patients
All 38 patients underwent directional coronary atherectomy under sterile conditions from nonostial de novo (n=31) or restenotic (n=7) lesions in native coronary arteries at the Cardiac Catheterization Laboratories of the First Medical Department, University Hospital Schleswig-Holstein, Campus Kiel (Germany). Six of the 38 patients who underwent successful directional coronary atherectomy had acute coronary syndrome; the other 32 patients had stable angina pectoris. Acute coronary syndrome was defined as new-onset or worsening angina that required hospitalization and was associated with alterations of the ST segment, with or without elevation of cardiac markers, including creatinine kinase and troponin measurements. Stable angina was defined as no change in frequency, duration, or intensity of symptoms within 6 weeks before the intervention. A predefined subgroup analysis comprised patients who had an elevated CRP, defined as serum levels >8 mg/dL. The baseline data of the patients included in the study are summarized in Table 1. The acute coronary syndrome, restenosis, and elevated CRP categories describe partially overlapping subgroups. The study was approved by the ethics committee of the medical faculty of the Christian-Albrechts University Kiel. All patients considered for the study gave written informed consent before catheter examination.
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Material for Control Experiments
Blood samples, catheter material (catheter tips, balloons, wires, introducers), swabs, and syringes were collected from 5 patients during heart catheter examination and subjected to the same molecular detection techniques as technical controls. Biological material from 26 control individuals was analyzed; tissue material from 15 postmortem patients (kidney, liver, myocardium, coronary arteries, including 8 splenetic tissue fragments) was obtained from the Institute of Pathology, University Hospital Schleswig-Holstein, Campus Kiel. The 15 postmortem patients suffered from malignant diseases. They were selected because no current infectious disease was reported and no antibiotic medication was administered within the last 3 months before death. Permission for postmortem examination and research use of tissues was obtained from the next of kin. Native coronary arteries from explanted hearts were obtained from 11 heart-beating tissue donors (7 men, 4 women; median age, 52 years; range, 34 to 64 years; left anterior descending artery, n=5; right coronary artery, n=6) from the Kiel transplantation program. The explanted hearts were discarded from implantation for morphological or technical reasons. None of these patients had an active infectious disease (CRP levels not elevated), recent or current treatment with antibiotics, or atherosclerosis of any coronary artery on macroscopic examination. The main inclusion criteria for the organ donors were no external trauma, no infection, no use of antibiotics, and age of between 30 and 70 years. The cause of death of the heart-beating donors according to the pretransplantation documents is listed as follows: apoplectic stroke, n=5; subarachnoid hemorrhage, n=2; severe intracerebral hemorrhage, n=3; and cerebral hypoxemic state after arrhythmia resulting from idiopathic dilated cardiomyopathy, n=1. None of these patients had clinical or macroscopic signs of CHD.
Experimental Design
Because the PCR-based techniques used require the full use of the entire specimen obtained by arterectomy, a hierarchical study design was used. Thirty-three samples were extracted for analysis by denaturing gel gradient electrophoresis (DGGE) to obtain a formal overview of bacterial diversity signatures. The extracted material from the 10 patients showing the largest diversity on DGGE were then subjected to the generation of a library that allowed sequence analysis of the 16S rDNA gene in >1500 clones, which resulted in a high-quality overview of the bacterial strains involved and confirmed the results of the DGGE analysis. Finally, the remaining 5 intact samples were subjected to FISH, which detects live bacteria within the tissue.
Treatment of Atherectomy Particles, Control Material, and DNA Extraction
DNA from blood and tissue specimens was extracted following a protocol adapted for the characteristics of microorganisms.18 The solid catheter materials (swabs, introducers, wires, catheter tips, balloons) were washed in demineralized water for 30 minutes to dissolve cell and tissue fragments.
Universal and Specific Primers
The established PCR primer pairs U968-GC/L1401 and COM1/COM2 were used.19–21 The oligonucleotides target the V6 through V8 and the V4 and V5 hypervariable regions of the 16S rRNA gene, respectively. Oligonucleotides for the detection of H pylori were designed with the PRIMER-DESIGN tool of the ARB software package (ARB software22). To detect Enterobacteriaceae, the eubacteria primer TPU5 was combined with a primer specific for Enterobacteriaceae (Enter1432, modified from Sghir et al23). The primer sequences for the detection of Chlamydia species and Staphylococcus aureus were obtained from the literature.24,25 Characteristics of primers are listed in Table 2. Detection of Mycoplasma species was conducted according to the instructions of the manufacturer of the PCR assay (VenorGeM Mycoplasma Detection Kit, Minerva Biolabs).
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DGGE Analysis
Samples from 33 patients were investigated by DGGE analysis as previously described.21,26 A 2-step reconditioning PCR approach was used to reduce formation of heteroduplexes and PCR artifacts, following the protocol of Thompson et al and Acinas et al.27,28 Briefly, a first PCR with 30 cycles was performed in the presence of excess PCR reagents to avoid effects of limited amplification. A second PCR step using only 10 cycles (reconditioning PCR) was then performed after 1:10 dilution of PCR products. Identification of the operational taxonomic units (OTUs) underlying the bands followed the protocol of Heuer et al.26 As negative controls, gel slices from surrounding regions of the bands were investigated. Isolation of DNA fragments from destained gel slices was performed as described by Schwieger and Tebbe.29 The recovered DNA was reamplified with primers U968-GC and L1401. For characteristics of primers, see Table 2. The PCR products were cloned into competent Escherichia coli cells with the PCR 2.1 TOPO TA Cloning Kit for sequencing (Invitrogen) according to the manufacturer’s instructions. Sequencing of the inserts was performed as described.18 More than 250 clones (n=254) were sequenced and analyzed. Image analysis was performed as described previously.18
Clone Libraries
More than 1500 clones were sequenced from a library that was generated from pooled PCR products of 10 atherectomy samples. An &400-bp fragment of the 16S rDNA containing the V4/5 variable region was amplified and cloned into E coli cells as described.19 A 2-step reconditioning PCR approach (25/10 cycles) was used as described above.
Cloning and sequencing of inserts were performed as described above.
FISH and Oligonucleotide Probes
Atherectomy specimens from 5 patients with CHD were fixed in 500 µL freshly prepared 4% buffered formalin and embedded in Shandon Histoplast Paraffin (Thermo Electron Corp) according to routine procedures. Cross sections (2 µm) were cut and placed on coated microscope slides (Superfrost*/Plus). Sections were hybridized as described previously30 with the following 16S/23S rRNA-targeted oligonucleotide probes: an equimolar mixture of 5 bacteria-directed probes—EUB 338, EUB 785, EUB 927, EUB 1055, and EUB 1088,31 referred to as EUB mix—to detect all bacteria.
Data and Sequence Analyses
Most of the data did not follow a normal distribution. An explorative analysis was carried out to investigate relationships between diversity (assessed as number of bands) and demographic (age, gender) or clinical (indication for examination: restenosis versus myocardial syndrome, elevated versus normal levels of CRP, chlamydia infection, cardiovascular risk factors) characteristics. Because the number of bands was asymmetrically distributed in some of the subgroups, data are represented by median values with quartiles and illustrated via box-and-whisker plots. Statistical tests of differences in location parameters were performed by 2-sided Wilcoxon rank-sum tests for independent samples at a 5% level of significance. The statistical analysis was conducted with SPSS (version 10.0). Alignment and assembly of partial sequences were performed with the Sequencher software package (Gene Codes Corp). OTUs were identified by Basic Local Alignment Search Tool (BLAST) (National Center for Biotechnology Information) analysis using search results of at least 97% similarity. The sequences were examined for chimera with the Chimera Check tool of the Ribosomal Data Projects of the Center for Microbial Ecology, Michigan State University. For the taxonomic classification of sequences that could not be assigned to known bacterial species in the BLAST analysis, comparative sequence analysis using the ARB software package was used.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
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Results |
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Detection of Bacterial DNA in Atherosclerotic Plaques
Total amounts of 60 to 610 ng (mean, 135.6 ng) of genomic DNA were extracted from coronary atherectomy specimens. Bacterial DNA was found in all 38 CHD patient samples by either PCR using the primer pairs U968-GC/L1401 and COM1/COM2 (n=33) or FISH analysis (n=5). The presence of bacterial rRNA could be demonstrated by FISH analysis in selected patients using a mix of 16S/23S rRNA–targeted oligonucleotide probes. Figure 1 shows single bacteria (Figure 1A and 1D) and groups of bacteria (Figure 1B and 1C) in the cross sections of atherosclerotic plaques.
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Overall Bacterial Diversity of the Clone Library
Clone libraries are currently the most accurate molecular method to represent the microbial composition of a complex habitat. A library was generated from pooled PCR products. For this experiment, the 10 patient samples with the highest bacterial diversity in the DGGE examination described below were used. More than 1500 clones were sequenced (mean length of sequences, 338.9±69). Chimeric sequences and incomplete inserts were discarded, resulting in 903 sequences that were used for the final analysis. More than 50 different clones were detected. They represented bacteria involved in skin infections such as Staphylococcus species, respiratory infections such as Klebsiella pneumoniae or Proteus vulgaris, or oral bacteria such as different Streptococcus species. Table 3 shows the molecular OTUs as a representation for the variety of species involved. Assignment to known bacterial species used BLAST analysis. Most interestingly, >100 clones of those shown in Table 3 (uncultured bacteria) did not correspond to presently known bacterial species. Table 4 shows the taxonomic relationships that were assigned by comparative phylogenetic sequence analysis.
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Formal Assessment of Bacterial Diversity in Coronary Atherosclerotic Lesions
To explore the diversity and distribution of bacterial DNA in atherosclerotic plaques, DGGE analysis was performed in the samples of 33 patients. A representative gel demonstrating the variety of the different interindividual banding patterns is shown in Figure 2A. DNA from selected bands was reamplified and sequenced. The spectrum of bacteria identified through DGGE and band-matching analysis was concordant with that found in the clone libraries, except for 1 additional Bacillus species (Figure 2B) that was detected only through DGGE. Additional controls included amplification of species identified by DGGE through independent direct PCR (eg, S aureus, Enterobacteriaceae, data not shown).
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97% sequence identity. B, Species that could be assigned to bacterial species. The spectrum of species identified by DGGE analysis and subsequent taxonomic classification is concordant with that of the clone library, except for one additional Bacillus species (band VI). Nomenclature according to Bergey’s Manual of Determinative Bacteriology.A total of 40 different band classes were calculated. The frequency of occurrence for each band ranges from 3% (only 1 patient) to 93.9% (31 of 33 patients). The diversity of bands varies from 5 (patient 8) to 22 (patient 16). The mean diversity of bands was 12.33±3.81). Diversity was calculated separately for subgroups of patients to investigate the influence of demographic factors such as age and gender and disease-specific parameters such as the indication for the procedure (restenosis versus acute coronary syndrome) and elevated CRP in a pilot analysis. The younger patients (<56 years of age) showed a nonsignificant trend toward a higher diversity (mean/interquartile ranges, 16/6.75) compared with the subgroup of patients >55 years of age (mean/interquartile ranges, 14/3.5; P=0.488) (Figure 3A). No significant differences were seen between the other subgroups (Figure 3), although a tendency toward a higher diversity was seen in CRP-positive than in CRP-negative patients (Figure 3B). Patients with chronic CHD also appeared to have a higher diversity than those with acute coronary syndrome (Figure 3C), whereas patients with restenosis did not differ from those with primary stenosis (data not shown). Similarly, patients with chlamydia infections showed a tendency to a higher diversity than those negative for the infection (Figure 3D). A subgroup analysis for the cardiovascular risk factors (arterial hypertension, hyperlipidemia, diabetes mellitus, smoking) reached no statistical significance and did not show any tendencies (data not shown).
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Control Individuals and Technical Controls
Primer pairs U968-GC/L1401 and COM1/COM2 were used for universal bacterial DNA amplification. As a technical control, blood samples, catheter material (catheter tips, balloons, wires, introducers), swabs, and syringes were collected from 5 patients during heart catheter examination. Direct universal PCRs as described above gave negative results. Control biological samples were obtained postmortem (n=15, including splenetic tissue from 8 individuals) and from nontransplanted hearts from the organ donor program (n=11, healthy coronaries). No bacterial DNA was found, including the macrophage-rich spleen tissue. Five additional PCR cycles were used in the controls to exclude low-level bacterial signals, also. The efficacy of nucleic acid extraction and the presence of DNA in the control samples were verified with a GADPH-PCR that is routinely performed to detect DNA contamination during RNA extraction that was conducted according to an established protocol.32 In addition, a 16S-based clone library was constructed, and 100 clones were sequenced.
Specific Detection of Chlamydia Species, Mycoplasma Species, and H pylori
Specific PCR assays were applied to determine known bacterial species that have previously been implicated in the pathophysiology of CHD. Chlamydia species were detected in 17 of the 33 atherectomy samples (51.5%). Further characterization by sequencing revealed most of them as C pneumoniae and C trachomatis. Mycoplasma species and H pylori could not be detected.
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Discussion |
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We observed frequent colonization of atherosclerotic lesions in CHD. The high overall diversity of bacterial DNA in coronary lesions supports the hypothesis of multiple bacterial colonizations of the arterial lesions. These findings underscore the infection hypothesis in the pathophysiology of coronary atherosclerosis. However, several explanations are possible for our findings. Lehtiniemi et al11 recently suggested that atheromas might act as mechanical sieves, collecting bacteria from the circulation, thereby assigning microorganisms the role of "innocent bystanders." Although the concept of a destroyed coronary surface acting like a sieve is intriguing, the resulting bacterial colonization of the lesions could still be a (secondary) driver for a local inflammation process. The presence of local macrophage33,34 and systemic monocyte activation33,34 supports this hypothesis. Alternatively, primary infection of plaque lesion through bacteremia could be a nonspecific process that could accelerate disease progression. Association of high levels of serum CRP with a negative prognosis supports this hypothesis.1 The high interindividual variability of bacterial profiles of bacterial 16S rDNA fragments could point to additional individual mechanisms of infection control through host genetic and immunological factors that have to be considered in the pathophysiology of CHD.
In our study, we detected only a nonsignificant tendency for an association of CRP levels with the degree of bacterial diversity. This could be due to a lack of power through the limited numbers of patients in the subgroup analysis. On the other hand, the extent of infected lesions (and not the degree of diversity in the bacterial flora within the lesions) could be the main driver for the height of the CRP levels.
The high rate of positive C pneumoniae signals corresponds with previous studies. The range of C pneumoniae–positive patients varies extremely in the literature. Chiu and colleagues3 found C pneumoniae in 54 of 76 endarterectomy specimens (71%) by immunostaining, whereas Maass et al35 could detect C pneumoniae in only 11 of 70 atheromas (16%) by direct cultivation and in 21 (30%) by PCR. In one of the first studies, Shor et al36 demonstrated C pneumoniae in coronary fatty streaks in 5 of 7 autopsy patients (71%). The differences in detection of C pneumoniae in view of constantly high seropositivity rates for this infectious agent could also point to an important role of individual innate immunity mechanisms involved in the elimination of pathogenic bacteria.
The broad bacterial spectrum in CHD included a host of species that are commonly found on human barrier organs like skin or the oral cavity. Interestingly, animal models with experimentally induced periodontitis, which leads to frequent Gram-negative bacteremia, had more extensive accumulations of lipids in the aorta compared with animals without periodontitis, suggesting that the inflammatory burden provided by the defective oral mucosal barrier directly contributes to atherosclerosis.37 Several of the species detected in human coronaries in this study are part of the commensal human flora including Staphylococcus species, Streptococcus species, K pneumoniae, P vulgaris, or Burkholderia species but might exhibit pathogenic characteristics under certain conditions. For example, the group of nonfermentative Gram-negative bacilli (including Pseudomonas species), especially Pseudomonas aeruginosa, is involved in respiratory tract infections and bacteremia in immunocompromised patients, patients with cystic fibrosis, children, or elderly people. The spectrum of bacteria in CHD lesions also comprises true pathogens such as K pneumoniae, a facultative member of the gastrointestinal microflora.18
There are several possible explanations for the presence of the highly diverse bacterial DNA signatures in atherosclerotic coronary lesions. Bacterial translocation from the gastrointestinal tract, especially the oral cavity, is common. The incidence of bacteremia after dental procedures such as tooth extraction, endodontic treatment, periodontal surgery, and root scaling is well documented.38 Transient bacteremia is also common after surgical procedures, endoscopy, manipulation of infected tissue, or local infections.39,40 Infections of mucosal organs but also physiological processes such as defecation can lead to transient detection of bacterial material in the circulation. The correlation of C pneumoniae infection and detection rates in atherosclerotic plaques of a subgroup of CHD patients supports this hypothesis.41 Up to 60% of the population have serological evidence of previous infection, and individuals are probably infected several times throughout their lives or harbor chronic infection.41 Recent publications showed that even rare bacteria might be present in the blood if the mucosal barrier is defective, as demonstrated for Mycobacterium paratuberculosis in patients with Crohn’s disease.42
Most significantly, the high overall bacterial diversity in coronary artery plaques, in combination with large interindividual differences, suggests that CHD is no monoinfectious disease caused by a single, specific pathogen. Our results are compatible with the hypothesis that infectious agents could be secondary factors that lead to the progression of lesions in the vessel wall through secondary colonization of atheromatous material. However, the etiological role of microbiota in the pathophysiology of CHD remains to be defined.
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Acknowledgments |
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This work was supported by grants from the German National Genome Research Network (pathway mapping, environment network), the DFG (SFB415), and the EU (EU QLG2-CT-2001–02161). We gratefully acknowledge Meike Barche and Ulrike Panknin for their assistance with the generation of clone libraries and sequencing. We also thank Kornelia Smalla from the Institute for Plant Virology, Microbiology, and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, Germany, for technical support with DGGE profiling. Attenuated cells of C pneumoniae were obtained from the German Reference Center for Chlamydial Infections at the Institute of Microbiology, Friedrich-Schiller-University Jena (Jena, Germany). The URLs used in this article are as follows: ARB software, ribosomal sequence database, and handling: http://www.arb-home.de; NCBI BLAST home page: http://www.ncbi.nlm.nih.gov/BLAST/; and Ribosomal Database Project II: http://rdp.cme.msu.edu/.
Disclosures
Dr Schreiber has been a consultant to Applied Biosystems. The other authors report no conflicts.
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Footnotes |
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*Drs Ott and El Mokhtari contributed equally to this study.
Drs Simon and Schreiber share senior authorship.
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