Chlamydialike Organisms and Atherosclerosis
http://www.100md.com
《传染病的形成》
Universite de la Mediterannee, Marseille, France
Hpital Nord, Marseille, France
Parachlamydia acanthamoebae and Neochlamydia hartmanellae are chlamydialike organisms that share ≈86% 16S rRNA sequence similarity with C. pneumoniae (5). Like C. pneumoniae, they have elementary and reticulate bodies visible by electron microscopy (6). Neochlamydia-related DNA (GenBank accession no. AF097191) has been amplified from 5 different arterial samples, including 1 aortic aneurysm (7), and a relationship (p = 0.009) between cerebral hemorrhage and serologic evidence of Parachlamydia infection has been reported (8). Therefore, we investigated the role of Parachlamydia in pathogenesis of atherosclerosis by using a molecular approach.
We analyzed 78 surgical samples from 27 patients undergoing aortic or carotid surgery for atherosclerotic disease at Hpital Nord in Marseille from June 1, 2003, to December 31, 2003. The study was approved by the local ethics committee, and written informed consent was obtained from all participants. Demographic and clinical data were prospectively recorded.
DNA was extracted from aortic or carotid samples with atherosclerotic lesions by using the QIAamp DNA tissue kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions. A nested PCR was performed by using external primers 16SIGF (5′-CGGCGTGGATGAGGCAT-3′) and 16SIGR (5′-TCAGTCCCAGTGTTGGC-3′) (9) and internal primers CHL16SFOR2 (5′-CGTGGATGAGGCATGCAAGTCGA-3′) and CHL16SREV2 (5′-CAATCTCTCAATCCGCCTAGACGTCTTAG-3′) (7). PCR included negative controls from the DNA extraction step. DNA extractions and PCR amplifications were conducted in a laboratory in which parachlamydial DNA had not been extracted or amplified. PCR products were purified by using the QIAquick PCR purification kit (Qiagen) and sequenced by using the d-rhodamine terminator cycle sequencing reaction kit (Perkin-Elmer Biosystems, Warrington, UK) and a 3100 ABI Prism automated sequencer (Applied Biosystems, Courtaboeuf, France).
Sequences were analyzed with BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) using gap existence and extension penalties of 5 and 2, respectively. Results were considered positive only when the sequence of the amplified product exhibited a best BLAST hit with a chlamydialike organism. Statistical analyses were performed with STATA software (Stata Corporation, College Station, TX, USA).
A positive PCR result was obtained with samples from 5 (18.5%) of 27 patients (Table). Three sequences had a best BLAST hit with the sequence of Parachlamydia sp. UV7 (GenBank accession no. AJ715410), with a sequence similarity ranging from 99% to 100%. The other 2 sequences had 98% sequence similarity with Neochlamydia-related symbiont TUME-1 (GenBank accession no. AF098330). PCR positivity was not associated with age, sex, or location of the atherosclerotic lesion.
All patients with positive PCR results were >68 years of age. Patients without cardiovascular risk factors were more likely than those with >1 risk factor to have positive PCR results (p = 0.023). Despite the small number of patients in this study, this association was also confirmed in a multivariate logistic regression model adjusted for sex and previous cardiovascular disease (odds ratio 0.035, 95% confidence interval 0.001–0.94).
These findings suggest that Parachlamydia and Neochlamydia are associated with atherosclerosis. In addition, these obligate intracellular bacteria may be present in both carotid and aortic atherosclerotic lesions of elderly patients.
Chlamydialike organisms in atherosclerotic lesions may explain controversies about the role of C. pneumoniae in pathogenesis of atherosclerosis (4). Some PCRs might amplify both Chlamydia and chlamydialike organisms, leading to erroneous conclusions, especially when the specificity of the product is not confirmed by sequencing. Chlamydialike organisms in atherosclerotic lesions might also explain discrepancies of serologic studies (4). Parachlamydiaceae likely cross-react with C. pneumoniae (1). Such cross-reactivity will not be recognized if patients with positive serologic results for C. pneumoniae are not tested for antibodies to chlamydialike organisms. This cross-reactivity may result in false-positive serologic results for C. pneumoniae, especially when low antibody titers (8–16) are considered positive.
Since elementary and reticulate bodies are similar in both Chlamydiaceae and Parachlamydiaceae (6), Chlamydiales cannot be identified in a specimen solely by electron microscopy. Consequently, elementary and reticulate bodies in atherosclerotic lesions (10) might be chlamydialike organisms and not C. pneumoniae. If chlamydialike organisms are involved in the pathogenesis of atherosclerosis, this finding would have public health implications, given their presence in free-living amebae that are widespread in water.
Acknowledgments
We thank Philip Tarr for reviewing the manuscript.
Dr Greub's research on chlamydialike organisms is currently supported by the Swiss National Science Foundation (SNSF) grant no. 3200BO-105885.
References
Meijer A, van der Vliet JA, Roholl PJ, Gielis-Proper SK, de Vries A, Ossewaarde JM. Chlamydia pneumoniae in abdominal aortic aneurysms: abundance of membrane components in the absence of heat shock protein 60 and DNA. Arterioscler Thromb Vasc Biol. 1999;19:2680–6.
Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. JAMA. 2002;288:2724–31.
Belland RJ, Ouellette SP, Gieffers J, Byrne GI. Chlamydia pneumoniae and atherosclerosis. Cell Microbiol. 2004;6:117–27.
Ieven MM, Hoymans VY. Involvement of Chlamydia pneumoniae in atherosclerosis: more evidence for lack of evidence. J Clin Microbiol. 2005;43:19–24.
Greub G, Raoult D. Parachlamydiaceae: potential emerging pathogens. Emerg Infect Dis. 2002;8:625–30.
Greub G, Raoult D. Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study. Appl Environ Microbiol. 2002;68:3076–84.
Ossewaarde JM, Meijer A. Molecular evidence for the existence of additional members of the order Chlamydiales. Microbiology. 1999;145:411–7.
Greub G, Boyadjiev I, La Scola B, Raoult D, Martin C. Serological hint suggesting that Parachlamydiaceae are agents of pneumonia in polytraumatized intensive care patients. Ann N Y Acad Sci. 2003;990:311–9.
Everett KD, Bush RM, Andersen AA. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol. 1999;49:415–40.
Nystrom-Rosander C, Thelin S, Hjelm E, Lindquist O, Pahlson C, Friman G. High incidence of Chlamydia pneumoniae in sclerotic heart valves of patients undergoing aortic valve replacement. Scand J Infect Dis 1997;29:361–5.(Gilbert Greub,Olivier Har)
Hpital Nord, Marseille, France
Parachlamydia acanthamoebae and Neochlamydia hartmanellae are chlamydialike organisms that share ≈86% 16S rRNA sequence similarity with C. pneumoniae (5). Like C. pneumoniae, they have elementary and reticulate bodies visible by electron microscopy (6). Neochlamydia-related DNA (GenBank accession no. AF097191) has been amplified from 5 different arterial samples, including 1 aortic aneurysm (7), and a relationship (p = 0.009) between cerebral hemorrhage and serologic evidence of Parachlamydia infection has been reported (8). Therefore, we investigated the role of Parachlamydia in pathogenesis of atherosclerosis by using a molecular approach.
We analyzed 78 surgical samples from 27 patients undergoing aortic or carotid surgery for atherosclerotic disease at Hpital Nord in Marseille from June 1, 2003, to December 31, 2003. The study was approved by the local ethics committee, and written informed consent was obtained from all participants. Demographic and clinical data were prospectively recorded.
DNA was extracted from aortic or carotid samples with atherosclerotic lesions by using the QIAamp DNA tissue kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions. A nested PCR was performed by using external primers 16SIGF (5′-CGGCGTGGATGAGGCAT-3′) and 16SIGR (5′-TCAGTCCCAGTGTTGGC-3′) (9) and internal primers CHL16SFOR2 (5′-CGTGGATGAGGCATGCAAGTCGA-3′) and CHL16SREV2 (5′-CAATCTCTCAATCCGCCTAGACGTCTTAG-3′) (7). PCR included negative controls from the DNA extraction step. DNA extractions and PCR amplifications were conducted in a laboratory in which parachlamydial DNA had not been extracted or amplified. PCR products were purified by using the QIAquick PCR purification kit (Qiagen) and sequenced by using the d-rhodamine terminator cycle sequencing reaction kit (Perkin-Elmer Biosystems, Warrington, UK) and a 3100 ABI Prism automated sequencer (Applied Biosystems, Courtaboeuf, France).
Sequences were analyzed with BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) using gap existence and extension penalties of 5 and 2, respectively. Results were considered positive only when the sequence of the amplified product exhibited a best BLAST hit with a chlamydialike organism. Statistical analyses were performed with STATA software (Stata Corporation, College Station, TX, USA).
A positive PCR result was obtained with samples from 5 (18.5%) of 27 patients (Table). Three sequences had a best BLAST hit with the sequence of Parachlamydia sp. UV7 (GenBank accession no. AJ715410), with a sequence similarity ranging from 99% to 100%. The other 2 sequences had 98% sequence similarity with Neochlamydia-related symbiont TUME-1 (GenBank accession no. AF098330). PCR positivity was not associated with age, sex, or location of the atherosclerotic lesion.
All patients with positive PCR results were >68 years of age. Patients without cardiovascular risk factors were more likely than those with >1 risk factor to have positive PCR results (p = 0.023). Despite the small number of patients in this study, this association was also confirmed in a multivariate logistic regression model adjusted for sex and previous cardiovascular disease (odds ratio 0.035, 95% confidence interval 0.001–0.94).
These findings suggest that Parachlamydia and Neochlamydia are associated with atherosclerosis. In addition, these obligate intracellular bacteria may be present in both carotid and aortic atherosclerotic lesions of elderly patients.
Chlamydialike organisms in atherosclerotic lesions may explain controversies about the role of C. pneumoniae in pathogenesis of atherosclerosis (4). Some PCRs might amplify both Chlamydia and chlamydialike organisms, leading to erroneous conclusions, especially when the specificity of the product is not confirmed by sequencing. Chlamydialike organisms in atherosclerotic lesions might also explain discrepancies of serologic studies (4). Parachlamydiaceae likely cross-react with C. pneumoniae (1). Such cross-reactivity will not be recognized if patients with positive serologic results for C. pneumoniae are not tested for antibodies to chlamydialike organisms. This cross-reactivity may result in false-positive serologic results for C. pneumoniae, especially when low antibody titers (8–16) are considered positive.
Since elementary and reticulate bodies are similar in both Chlamydiaceae and Parachlamydiaceae (6), Chlamydiales cannot be identified in a specimen solely by electron microscopy. Consequently, elementary and reticulate bodies in atherosclerotic lesions (10) might be chlamydialike organisms and not C. pneumoniae. If chlamydialike organisms are involved in the pathogenesis of atherosclerosis, this finding would have public health implications, given their presence in free-living amebae that are widespread in water.
Acknowledgments
We thank Philip Tarr for reviewing the manuscript.
Dr Greub's research on chlamydialike organisms is currently supported by the Swiss National Science Foundation (SNSF) grant no. 3200BO-105885.
References
Meijer A, van der Vliet JA, Roholl PJ, Gielis-Proper SK, de Vries A, Ossewaarde JM. Chlamydia pneumoniae in abdominal aortic aneurysms: abundance of membrane components in the absence of heat shock protein 60 and DNA. Arterioscler Thromb Vasc Biol. 1999;19:2680–6.
Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. JAMA. 2002;288:2724–31.
Belland RJ, Ouellette SP, Gieffers J, Byrne GI. Chlamydia pneumoniae and atherosclerosis. Cell Microbiol. 2004;6:117–27.
Ieven MM, Hoymans VY. Involvement of Chlamydia pneumoniae in atherosclerosis: more evidence for lack of evidence. J Clin Microbiol. 2005;43:19–24.
Greub G, Raoult D. Parachlamydiaceae: potential emerging pathogens. Emerg Infect Dis. 2002;8:625–30.
Greub G, Raoult D. Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study. Appl Environ Microbiol. 2002;68:3076–84.
Ossewaarde JM, Meijer A. Molecular evidence for the existence of additional members of the order Chlamydiales. Microbiology. 1999;145:411–7.
Greub G, Boyadjiev I, La Scola B, Raoult D, Martin C. Serological hint suggesting that Parachlamydiaceae are agents of pneumonia in polytraumatized intensive care patients. Ann N Y Acad Sci. 2003;990:311–9.
Everett KD, Bush RM, Andersen AA. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol. 1999;49:415–40.
Nystrom-Rosander C, Thelin S, Hjelm E, Lindquist O, Pahlson C, Friman G. High incidence of Chlamydia pneumoniae in sclerotic heart valves of patients undergoing aortic valve replacement. Scand J Infect Dis 1997;29:361–5.(Gilbert Greub,Olivier Har)