Research in Microbiology 153 (2002) 563–567 www.elsevier.com/locate/resmic
New parachlamydial 16S rDNA phylotypes detected in human clinical samples Daniele Corsaro a , Danielle Venditti b,∗ , Marcello Valassina c a Laboratory of Virology–Microbiology, Centre Hospitalier Universitaire de Nancy, Hôpital de Brabois-Adultes, Route de Neufchâteau,
54511 Vandœuvre-lès-Nancy, France b TREDI Research Department, 9, Avenue de la Forêt de Haye, B.P. 184, Faculté de Médecine, Technopôle de Nancy-Brabois,
54505 Vandœuvre-lès-Nancy, France c Microbiology Section, Department of Molecular Biology, University of Siena, via del Laterino 8, 53100 Siena, Italy
Received 6 February 2002; accepted 22 July 2002
Abstract Chlamydiales are important intracellular bacterial pathogens, causing a wide variety of diseases in vertebrates, including humans. Besides the well-known species in the family Chlamydiaceae, new chlamydial organisms have recently been discovered, forming three new families: Parachlamydiaceae, Simkaniaceae and Waddliaceae. Parachlamydia acanthamoebae and Simkania negevensis are currently investigated as emerging human respiratory pathogens. Additional chlamydial lineages have been discovered by 16S rDNA-based molecular studies, and their implication in human infections is poorly known. By using a pan-chlamydia 16S rDNA PCR, we have searched for the presence of chlamydiae in 228 clinical samples that all previously had been shown to be PCR-negative for Chlamydophila pneumoniae: 170 respiratory samples, 45 atheromatic plaques and 13 peripheral blood mononuclear cell samples. Nine respiratory samples tested positive. Sequence analysis has allowed us to assign four sequences to Chlamydophila psittaci, three sequences to Chlamydophila felis, and two sequences to two novel phylotypes belonging to the Parachlamydiaceae. These latter sequences showed similarity values of more than 93% with each other and with the P. acanthamoebae sequence, thus belonging to novel, unrecognized species. In conclusion, this report showed that a variety of non-C. pneumoniae chlamydial respiratory infection is present in humans, and that new parachlamydiae distinct from P. acanthamoebae may be detected in human clinical samples. Future studies will be of interest in order to estimate the diversity of these novel chlamydiae in both clinical and environmental samples, as well as their possible clinical implication in human and animal infections. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Chlamydiales; Parachlamydia; Simkania; Respiratory pathogen
1. Introduction Chlamydiae are obligately intracellular bacterial pathogens of vertebrates causing a wide variety of diseases. According to the new proposed classification [10], species in the family Chlamydiaceae are separated into two genera, Chlamydia and Chlamydophila. Humans are infected mainly by two endemic species, Chlamydia trachomatis, responsible for ocular and urogenital infections, and the biovar TWAR of Chlamydophila pneumoniae, causing respiratory infections and suspected to be involved in some chronic diseases like atherosclerosis. Sporadic zoonotic infections * Correspondence and reprints.
E-mail addresses:
[email protected] (D. Corsaro),
[email protected] (D. Venditti).
by other chlamydial species are possible. The psittacosisornithosis due to the main avian pathogen Chlamydophila psittaci is a flu-like syndrome that may be fatal if untreated. Some chlamydial species colonize the placenta and may cause abortion in ruminants but also in other mammalian species. In women, Chlamydophila abortus has been recognized as an agent of zoonotic abortion. Chlamydophila felis may causes mild to asymptomatic respiratory infections, that could explain some nonvirulent cases of psittacosis in absence of any contact with birds. Recently new chlamydial organisms have been discovered, forming three new lineages of family rank within the order Chlamydiales. The monospecific family Waddliaceae is represented by Waddlia chondrophila, isolated from an aborted bovine foetus [22]. No evidence for human infection exists at present. Simkania negevensis seems to be an emerg-
0923-2508/02/$ – see front matter 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 2 3 - 2 5 0 8 ( 0 2 ) 0 1 3 6 9 - 4
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ing human pathogen. It has been associated with respiratory infections in several places of the world, through isolation, specific PCR and serology [11,12,18,19]. Finally, the family Parachlamydiaceae is constitued by a rich assemblage of amoebal endosymbionts recovered from both clinical and environmental habitats [13,16]. The type species Parachlamydia acanthamoebae was found as an endosymbiont of an Acanthamoeba sp. isolated from a nasal mucosa of a healthy person [1]. Hall’s coccus, an acanthamoeba symbiont associated with an outbreak of “humidifier fever”, has been shown to be a strain of P. acanthamoebae [2]. The role of such a microorganism as an emerging human pathogen is currently being investigated. Molecular studies by polymerase chain reaction (PCR) in both clinical and environmental samples have recently shown a richer diversity within the Chlamydiales. Ossewaarde and Meijer [21] have detected by PCR portions of the small subunit ribosomal RNA gene (16S rDNA) in several human clinical samples. Sequence analyses seemed to indicate that these DNAs belong to new, unrecognized chlamydial organisms, but the small sequenced portion (less then 300 bp) did not allow a more precise phylogenetic study. Amplifying and sequencing almost all the 16S rDNA, we previously reported the presence of a new parachlamydial phylotype detected in a human respiratory sample [6]. Environmental studies have also given some interesting results. Completely novel chlamydial 16S rDNA phylotypes, probably representing new genera or perhaps new families, have been detected by PCR in Antarctic habitats [3], wastewater treatment plants [16] and freshwater ponds [8]. Subclinical chronic infection may be considered a characteristic of all the Chlamydiales, and evidence for human infections by novel chlamydiae exists. Therefore we have searched for the presence of chlamydial 16S rDNAs by PCR in human clinical samples, followed by sequence analysis, in order to evaluate the possibility of human infections by novel chlamydiae. We present herein the identification of two new 16S rDNA phylotypes of parachlamydiae detected by PCR in human respiratory clinical samples.
2. Materials and methods 2.1. Clinical samples Clinical samples included 170 respiratory samples (114 broncho-alveolar lavages, 20 bronchial aspirates, 17 nasal washes, 10 sputa and 9 pharyngeal swabs), 45 samples from atherosclerotic plaques and 13 peripheral blood mononuclear cell (PBMC) samples. Respiratory samples were from French and Italian patients of both sexes (81 males and 89 females), and ages ranged from one to 58 years. Atheromatic and PBMC samples were mainly from male patients, aged 40 to 88 years. All the samples resulted negative for the presence of C. pneumoniae DNA in previous analyses [5,24].
2.2. DNA extraction and PCR DNA was extracted from 300 µl aliquots of samples by the classic phenol-chloroform method after proteinase K (200 µg ml−1 ) digestion in a lysis buffer (25 mM EDTA, 75 mM NaCl, 0.01% SDS, 0.5% Triton X-100) for 2 h at 50 ◦ C. Then DNA was precipitated with ethanol and dissolved in 100 µl of bidistilled water. Volumes of 5 µl were used for PCR tests. The absence of PCR inhibitors was checked by amplifying a 268-bp fragment of the human β-globin gene [23]. The search for chlamydial DNA was conducted using a pan-chlamydia primer set targeting the 16S rDNA. Nucleotide sequences for forward and reward primers were: 5 -CGT GGA TGA GGC ATG C(A/G)A GTC G-3 and 5 -GTC ATC (A/G)GC C(T/C)(T/C) ACC TT(A/C/G)(C/G)(A/G)C (A/G)(T/C)(T/C) TCT-3 , respectively (positions 35 to 1481, P. acanthamoebae 16S rDNA numbering). Reactions were performed in a 50-µl final volume containing 5 µl of 10 × reaction buffer, 1.5 mM MgCl2 , 200 mM of each deoxynucleoside triphosphate, 40 pmol (0.8 mM) of each primer, and 2 U of Taq DNA polymerase. All the PCRs were performed by hot-start, adding the polymerase after 5 min at 94 ◦ C, and included 35 cycles of denaturation (1 min at 94 ◦ C), annealing (1 min at 65 ◦ C), and elongation (1 min at 72 ◦ C). PCR products were analysed by 2% agorose gel electrophoresis followed by staining with ethidium bromide to search for an expected amplicon of around 1400 bp. Manipulations were performed according to recommended guidelines and including negative controls starting from the DNA extraction step. Clinical isolates of C. trachomatis and C. pneumoniae were used as positive controls. 2.3. DNA sequencing and phylogenetic analysis Both strands of an inner portion (around 1100 bp) of the PCR products were sequenced (three repetitions) using a series of inner primers. PCR products were purified by using the Qiagen QIA-quick spin PCR purification kit and sequenced by dideoxy chain termination with a Sequenase kit by using forward and reward primers targeting overlapping inner fragments. The resulting partial 16S rDNA sequences were aligned with all the chlamydial 16S rDNA sequences available in the data set by using Multalin, version 5.4.1. [4]. Phylogenetic reconstructions were performed using the neighbour-joining method with Jukes and Cantor’s option, generated by Molecular Evolutionary Genetics Analysis (MEGA). Topology stability was evaluated by bootstrapping on 200 replicates.
3. Results Nine out of 170 respiratory samples (5.3%) were positive to the pan-chlamydia 16S rDNA PCR. All the atheromatic and PBMC samples tested negative. In total, 9 out 228
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Fig. 1. Neighbour-joining phylogenetic trees based on partial 16S rDNA sequences. Topology stability was evaluated by bootstrapping (200 replicates), only values of 50% or more are indicated. Parachlamydial sequences detected in human respiratory samples are indicated in (A) by arrows. Uncultured bacterium vadinBE97 (vadin lineage), verrucomicrobial strain VeCb1 (Verrucomicrobia) and Pirellula staleyi (Planctomycetales) sequences were used as outgroups. Bars indicate estimated genetic divergence. Nucleotide positions are P. acanthamoebae 16S rDNA numbering. (A) Phylogenetic tree based on a 1050-bp fragment (nt positions 230–1284). (B) Phylogenetic tree based on a 770-bp fragment (nt positions 512–1284) to include also Antarctic sequences.
clinical samples analysed (3.9%) were shown to be PCRpositive for chlamydiae distinct from C. pneumoniae. Sequence analysis of the recovered 16S rDNAs (around 1050 bp) led to identification of four sequences belonging to C. psittaci and three sequences belonging to C. felis (data not shown). The two remaining sequences, called cvC7 and cvC15, were from a nasal washing and a sputum, respectively, from two Italian patients. They proved to be two novel sequence types, that we submitted to the GenBank database under accession numbers AF478473 (cvC15) and AF478463 (cvC7). Phylogenetic reconstructions based on a 1050-bp fragment assigned these two new sequences to the parachlamydial lineage. Percentages of homology were estimated from the nucleotide differences on 1033 retained sites (outgroup sequences not considered). The sequences cvC7 and cvC15 showed 93.8% similarity with each other, and values of 95.3 and 93.5%, respectively, with that of P. acanthamoebae, the most closely related sequence. Similarity values of both cvC7 and cvC15 sequences with all the other parachlamydial sequences were less than 93%. Similarity values with the non-parachlamydia sequences were less than 90%: 88% with W. chondrophila, 88.4% and 89.6% with S. negevensis, and 87.2% to 87.6% with members of the genera Chlamydia (C. trachomatis, C. suis) and Chlamydophila (C. pneumoniae, C. pecorum, C. psittaci). In phylogenetic reconstructions, cvC7, cvC15 and P. acanthamoebae clustered to-
gether (Fig. 1A). Phylogenetic analysis was also performed on a shorter fragment of the gene, around 770 bp, in order to include environmental clones from Antarctic samples [3]: the positions of our sequences within the tree were conserved. The sequence Burton-46 and W. chondrophila emerge as sister-groups of Parachlamydiaceae (Fig. 1B). The two clusters S. negevensis/freshwater cvE9 and Antarctic Taynaya-24/ freshwater cvE6, arising as sister-groups of one another in a previous analysis [8], seem to be unrelated in the present analysis that also considers wastewater ECL VI sequences. The two other Antarctic sequences MERTZ2CM-173 and Clear-2 constituted the outermost lineages.
4. Discussion Everett et al. [10] suggested a 95% 16S rDNA sequence similarity threshold value to separate genera within the Chlamydiales. By assuming such a criterion, more than seven lineages can be identified within the family Parachlamydiaceae (Fig. 1). Members of some of these lineages have been detected in human clinical samples. The amoebal symbiont UWC22 belonging to the Neochlamydia lineage has been identified in an amoebal strain isolated from a corneal sample [13]. P. acanthamoebae infection has be found in community-acquired pneumonia patients [2], and recently serological evidence of infection has been reported
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for a case of presumed Kawasaki syndrome in an adult patient [20]. Three other lineages are constitued of three 16S rDNA phylotypes detected by PCR from respiratory samples. The sequence corvenA4 has been detected in a broncho-alveolar lavage [6] and seems to constitute a unique lineage. Sequences cvC7 and cvC15 reported in the present study were from a nasal washing and a sputum, respectively. From sequence similarity values and tree topologies constructed by considering several new chlamydial phylotypes, we can assume our two sequences are from microorganisms probably representing two distinct species belonging to a novel lineage of Parachlamydiaceae, perhaps related to the P. acanthamoebae one. Recent molecular studies have evidenced a richer diversity within the Chlamydiales also in several environmental habitats such as Antarctic lake sediments [3], wastewater treatment plants [16] and freshwater ponds [8]. A summary of the 16S rDNA sequences representing the novel chlamydiae distinct from the described species Neochlamydia hartmannellae, P. acanthamoebae, S. negevensis and W. chondrophila, is presented in Table 1. An interesting result of these molecular studies is the recognition of at least two Simkania-related lineages, cvE9 from a freshwater pond [8], and the ECL VI from the waste waters [16], both habitats possessing a rich protist fauna. As S. negevensis has recently been experimentally showed to infect and to multiply within acanthamoebae [17], it could be hypothesized that a rich assemblage of simkaniae and simkania-like organisms is present in protist-containing natural habitats. Therefore a bioecology similar to that of parachlamydiae or legionellae
may also characterize this group of chlamydiae. Another interesting result of such molecular ecology studies is represented by the novel phylotypes belonging to the Chlamydiales from polar environments [3], evidencing again the yet unrecognized diversity within this lineage of prokaryotes. Pathogenic importance of human infection by these chlamydia-like organisms is at present unknown. It seems likely that a wide variety of chlamydiae are in fact environmental organisms, probably living as protist endosymbionts or parasites, a condition largely found in the microbial world [7]. This is of interest for both epidemiologic and virulence reasons. Amoebae are natural hosts of parachlamydiae, and might also play a role of vector/reservoir for S. negevensis and C. pneumoniae, both being able in vitro to infect and to multiply within Acanthamoeba [9,17]. Amoeba-containing habitats may then prove to be a new source of infection by chlamydiae. In addition, interaction with amoebae, either as natural host or predator, should have selected strategies which have arisen to counteract intracellular digestion. Such strategies may have rendered these bacteria able to resist phagocytic cells of vertebrates, thus causing infection in humans. Perhaps, chlamydial endosymbionts may play a role as a virulence factor for amoebal infection, parachlamydia-infected acanthamoebae possessing an enhanced in vitro cytopathogenicity [14]. Infection by these novel chlamydiae may not be limited to mucosae; the possible resistance to phagocytic cells might allow a more invasive penetration in the human body. Ossewaarde and Meijer [21] have detected several unique 16S rDNAs in respiratory samples, PBMCs and vascular specimens. Despite
Table 1 Summary of the different chlamydial 16S rDNA sequences Lineagea Parachlamydiaceae cvC7 cvC15 corven A4 P4, P7, P9 (ECL I) P3, P5 (ECL II) TUME1 UWC22 UWE1 UWE25 Parachlamydiae-related Burton-46 Simkania-related cvE9 P2, P11, P12 (ECL VI) New lineages Clear-2 cvE6 Mertz-2CM-173 P6 (ECL VII) Taynaya-24
Originb
GenBank accession number
References
respiratory sample respiratory sample respiratory sample wastewater wastewater sewage sludge (amoeba) corneal sample (amoeba) soil sample (amoeba) soil sample (amoeba)
AF478463 AF478473 AF308693 AF364564, AF364569, AF364575 AF364563, AF364565 AF098330 AF083616 AF083614 AF083615
this study this study [6] [15] [15] [13] [13] [13] [13]
Antarctic lake sediments
AF142864
[3]
AF448723 AF364561, AF364577, AF364578
[8] [15]
AF146229 AF448722 AF424515 AF364568 AF142972
[3] [8] [3] [15] [3]
freshwater pond wastewater Antarctic lake sediments freshwater pond Antarctic lake sediments wastewater Antarctic basin sediments
a As deduced by 16S rDNA sequence similarity values. New lineages were suggested if no close relationship (i.e., similarity values of more than 90%) with any of the four recognized families of Chlamydiales may be found [10]. b All but parachlamydial sequences TUME1, UWC22, UWE1 and UWE25, were from uncultured bacteria, detected only by PCR.
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the small portion of the gene sequenced (less than 300 bp), it is possible to recognize these sequences as unique and novel. Some of them, e.g., the CRG1 clone from an abdominal aneurysm, showed similarity with S. negevensis, and others, e.g., the CRG4 clone from PBMCs, formed independent lineages [21]. Similarly, clinical manifestations might not be limited to the respiratory ones. The recently reported evidence of P. acanthamoebae infection in a patient with Kawasaki disease, a complex acute vasculitis of unknown origin [20], may be of interest, as such a syndrome has been associated with previous respiratory infection. In conclusion we have reported herein two new 16S rDNA phylotypes of Parachlamydiaceae, detected by PCR from two human respiratory samples. This report and a previous one [6] showed that DNA sequences from distinct parachlamydiae different from P. acanthamoebae may be detected in human clinical samples. Further studies are necessary to estimate the prevalence and diversity of infections by these novel chlamydial organisms in humans but also in animals, as well as to accurately evaluate their clinical importance.
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