1 Division of Microbial Ecology, Institute for Ecology and Conservation Biology, University of Vienna, Althanstr. 14, 1090 Vienna, Austria
2 Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinik Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany
3 Biological Institute, University Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany
Correspondence
Matthias Horn
horn{at}microbial-ecology.net
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Parachlamydia sp. UV-7 is AJ715410.
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INTRODUCTION |
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Several studies suggest an association of some of the environmental chlamydiae with respiratory disease of humans. Based on molecular and serological evidence, Simkania negevensis might be implicated in bronchiolitis in infants and community-acquired pneumonia in adults (Friedman et al., 2003). In addition, Parachlamydia spp. have been associated with several pneumonia cases, among others in polytraumatized intensive-care patients (Birtles et al., 1997
; Corsaro et al., 2001
, 2002a
; Greub et al., 2003a
; Marrie et al., 2001
). Simkania negevensis and Parachlamydia spp. are thus considered potential emerging pathogens (Greub & Raoult, 2002b
).
From a public health point of view it therefore deserves attention that a large number of rRNA sequences detected in various clinical and environmental samples (including bronchoalveolar lavage, nose, throat and ocular swabs from humans and animals, fresh water, soil, and activated-sludge samples) represent as yet unknown chlamydiae, indicating that chlamydial diversity is still dramatically underestimated (Bodetti et al., 2003; Corsaro et al., 2001
, 2002a
, b
; Horn & Wagner, 2001
; Ossewaarde & Meijer, 1999
; recently reviewed by Corsaro et al., 2003
; see M. E. Ward's www.chlamydiae.com for up-to-date information). These molecular data also suggest that chlamydiae have an extremely broad host range (comprising protozoa, arthropods, and marsupial and placental mammals) and a ubiquitous, worldwide distribution in nature. However, the currently available environmental chlamydia isolates (n=12) do not adequately represent the actual diversity of this group and therefore additional isolates are urgently required to more fully understand the biology and medical significance of those organisms. Isolation of free-living amoebae, naturally infected with environmental chlamydiae, is tedious and extremely time consuming. As members of all four recognized chlamydial families have been shown to be able to thrive within free-living amoebae (Essig et al., 1997
; Kahane et al., 2001
; Michel et al., 2004
), in this study we used co-incubation of an environmental sample (activated sludge from a wastewater treatment plant) with uninfected Acanthamoeba sp. to directly retrieve chlamydiae from complex microbial communities. We successfully applied this co-cultivation approach for the recovery of a new Parachlamydia sp. In addition, we show that the environmental chlamydia strain (UV-7) obtained in this study is able to infect mammalian cells.
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METHODS |
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Co-cultivation of amoebae with activated-sludge samples.
Activated-sludge samples were taken from a wastewater treatment plant in Plattling (Germany) processing sewage from a rendering plant, and homogenized for 30 s using an ultraturrax homogenizer (IKA Werke). Amoeba cultures were harvested by centrifugation and resuspended in Page's amoebic saline (Page, 1988). Two millilitre volumes of the amoeba suspension were seeded in six-well tissue culture plates. Tissue culture inserts (pore size 3·0 µm) were placed within the six-well plates. One millilitre of the homogenized activated sludge was applied into the tissue culture inserts submerged in the Page's amoebic saline and permeable for bacteria but excluding protozoa or amoebal cysts (thus preventing contamination of the amoebal strain UWC1 with autochthonous amoebae). After co-cultivation for 1 day at room temperature, tissue culture inserts containing the activated sludge were removed, amoebae were detached from the surface of the tissue culture plates, and 500 µl amoeba suspension was transferred into a new well containing 3 ml TSY broth and antibiotics (penicillin, 100 µg ml1; streptomycin, 10 µg ml1; amphotericin B, 0·25 µg ml1) to suppress growth of extracellular bacteria. Medium and antibiotics were exchanged daily for the following 2 days. Addition of antibiotics was omitted as soon as no extracellular bacteria were detectable in the medium by light microscopy, and amoebae were further propagated to allow for the spread of a potential infection with intracellular bacteria. Amoebae were further incubated for 4 weeks until intracellular bacteria could be readily detected by staining with 4',6-diamidino-2-phenylindole (DAPI; 1 µg µl1). Acanthamoebae harbouring intracellular bacteria were subcultured and maintained at room temperature by replacing TSY broth weekly. For long-term preservation, amoeba cultures were frozen in TSY broth supplemented with 10 % (v/v) dimethylsulfoxide and stored in liquid nitrogen as described elsewhere (Neal et al., 1974
).
Electron microscopy.
Amoebae and mammalian cells were fixed in 2·5 % glutaraldehyde in 0·1 M phosphate/cacodylate buffer for 12 h. After fixation in 2 % osmium tetroxide for 1 h and dehydration in an ascending series of acetate or ethanol, respectively, the samples were embedded in Epon 812 resin (Fluka). Sections (70 nm) were stained with 1 % lead citrate and uranyl acetate before examination in a transmission electron microscope (Zeiss EM 10).
DNA isolation, amplification of 16S rRNA genes, cloning and sequencing.
Simultaneous isolation of DNA from acanthamoebae and intracellular bacteria was performed using the FastDNA kit (Bio 101) and a bead beater (Bio 101-FP120) following the instructions of the manufacturer. Oligonucleotide primers targeting 16S rRNA gene signature regions which are conserved within the Chlamydiales were used for PCR in a thermal cycler (iCycler; Bio-Rad) to obtain near-full-length bacterial 16S rRNA gene fragments. The nucleotide sequences for the forward and reverse primers used for amplification were 5'-CGG ATC CTG AGA ATT TGA TC-3' and 5'-TGT CGA CAA AGG AGG TGA TCC A-3' (Pudjiatmoko et al., 1997). Amplified PCR products were directly ligated into the cloning vector pCRII-TOPO and transformed into competent E. coli (TOP10 cells) following the instructions of the manufacturer (Invitrogen). Nucleotide sequences of the cloned DNA fragments were determined using the ThermoSequenase cycle sequencing kit (Amersham Life Science) and an automated DNA sequencer (Li-Cor) under conditions recommended by the manufacturers. Vector-specific primers M13/pUC-V (5'-GTA AAA CGA CGG C-3') and M13/pUC-R (5'-GAA ACA GCT ATG ACC ATG-3') were used for sequencing.
Phylogenetic analysis.
The ARB program package (Ludwig et al., 2004) was used for phylogenetic analysis of the retrieved 16S rRNA sequence. The 16S rRNA sequence was added to an alignment of about 16 000 small-subunit rRNA sequences using the alignment tool implemented in ARB and subsequently refined manually. A consensus tree was drawn based on trees calculated using neighbour-joining (JukesCantor correction), PHYLIP distance matrix (Fitch), PHYLIP maximum-parsimony methods (Felsenstein, 1989
), and the fastDNAml and TREE-PUZZLE (Strimmer & von Haeseler, 1996
) maximum-likelihood approaches. A filter considering only positions that are conserved in at least 50 % of all Chlamydiales 16S rRNA sequences was used for calculation of all trees.
Fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy.
Amoebae were harvested from liquid culture by centrifugation and washed with Page's saline. After resuspension in Page's saline, 20 µl aliquots of amoebic suspension were incubated on glass slides for 20 min, and fixed with 4 % paraformaldehyde (PFA) for 20 min at room temperature. Mammalian cell cultures were grown on glass coverslips in 24-well plates, fixed with 4 % PFA for 20 min at room temperature and dehydrated in ethanol. Hybridizations and staining with DAPI were performed using the protocol, hybridization buffer (containing 25 % formamide) and washing buffer described elsewhere (Daims et al., 2005). Slides and coverslips were examined using a confocal laser scanning microscope (LSM 510, Carl Zeiss) equipped with two helium-neon lasers (633 nm, 543 nm), an argon-krypton laser (488 nm) and a UV laser (351364 nm). Image analysis processing was performed with the standard software package delivered with the instrument (version 3.2).
A new oligonucleotide probe, UV7-763 (5'-TGC TCC CCC TTG CTT TCG-3'; S-St-UV7-763-a-A-18 according to the nomenclature proposed by Alm et al., 1996), exclusively targeting the newly recovered Parachlamydia sp. strain UV-7 was designed using the probe functionality of the ARB software (Ludwig et al., 2004
) and deposited at probeBase (Loy et al., 2003
). In addition, the following oligonucleotide probes were used: (i) Bn9-658 (5'-TCC GTT TTC TCC GCC TAC-3') (Amann et al., 1997
) targeting the 16S rRNA of a subgroup of the Parachlamydiaceae, including Parachlamydia sp. UV-7; (ii) a mixture of EUB338-I (5'-GCT GCC TCC CGT AGG AGT-3'), EUB338-II (5'-GCA GCC ACC CGT AGG TGT-3') and EUB-III (5'-GCT GCC ACC CGT AGG TGT-3') targeting almost all Bacteria (Amann et al., 1990
; Daims et al., 1999
); and (iii) Euk516 (5'-ACC AGA CTT GCC CTC C-3')(Amann et al., 1990
) targeting the 18S rRNA of eukaryotes. Oligonucleotide probes were synthesized and directly 5' labelled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS), or the hydrophilic sulfoindocyanine fluorescent dyes Cy3 or Cy5 (Thermo Electron).
Infection of mammalian cells.
EBs of Parachlamydia sp. UV-7 were purified from amoebic host cells by step-gradient centrifugation in 30 % Gastrografin (Schering) and 50 % sucrose, and quantified by counting DAPI-stained EBs filtered on a nitrocellulose membrane (pore size 0·25 µm; Millipore). Purified EBs were added to confluent cell monolayers grown in 24-well tissue plates at a ratio of one EB per mammalian cell, according to a standard infection protocol (Dowell et al., 2001), but without the addition of antibiotics. Infected cells were incubated at 35 °C with 5 % CO2 and monitored for 5 days.
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RESULTS AND DISCUSSION |
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In order to identify the intracellular bacteria observed in Acanthamoeba sp. UWC1, a nearly full-length fragment (1516 bp) of the 16S rRNA gene was amplified. The amplificate was subsequently cloned and six clones were sequenced. Comparative sequence analysis revealed that the six 16S rRNA gene sequences determined were identical and showed highest similarity to 16S rRNA gene sequences of members of the genus Parachlamydia (98·298·7 %), and particularly to Parachlamydia acanthamoebae strain Bn9 (98·7 %). According to Stackebrandt & Goebel (1994) the 16S rRNA sequence similarity threshold for the unambiguous differentiation of two species is 97·5 %. Based on 16S rRNA data it is thus not possible to decide whether the intracellular bacteria recovered in this study belong to the species P. acanthamoebae or represent a novel Parachlamydia species. The recovered environmental chlamydia was therefore designated Parachlamydia sp. strain UV-7 (University of Vienna, isolate number 7). Phylogenetic 16S rRNA gene analysis revealed that Parachlamydia sp. UV-7 formed a monophyletic group with all other members of the genus Parachlamydia in all treeing methods applied (Fig. 1
).
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Infection of mammalian cells with Parachlamydia sp. UV-7
Environmental chlamydiae are being discussed as potential emerging pathogens, since Parachlamydia-like rRNA gene sequences and elevated antibody titres against parachlamydiae have been detected in patients with respiratory disease of undetermined cause (Greub & Raoult, 2002b). The recent finding that P. acanthamoebae is able to infect and multiply in primary human macrophages lends additional weight to this hypothesis (Greub et al., 2003b
). In order to investigate whether Parachlamydia sp. UV-7 (recovered by co-cultivation in this study) also is able to infect and propagate within eukaryotic host cells other than amoebae, including potential human target cells, we performed qualitative infection assays using one simian and two human cell lines. The course of UV-7 infection was similar in all investigated cell lines (Figs 3
and 4). Single Vero cells 1 day p.i. contained small inclusion vacuoles (Fig. 3a
). Electron microscopy showed mostly aberrant chlamydial RBs at days 1 and 2 p.i. (Fig. 3f
, Fig. 4
). At 2 and 3 days p.i. the fraction of Vero cells containing UV-7 inclusions had increased and a cytopathic effect was evident (Fig. 3b, c, f, g
). At 45 days p.i. most Vero cells were destroyed and few RBs could be detected by FISH. However, at that time point, a multitude of EBs could be recognized outside of the Vero cells or surrounding cytoplasmic remnants of destroyed cells by DAPI-staining and electron microscopy (Fig. 3d, h
). The cytopathic effect of Parachlamydia sp. UV-7 on Vero, NCI and HeLa cells is consistent with a previous report showing that P. acanthamoebae exhibited similar effects on human macrophages, which have been attributed to the induction of apoptosis (Greub et al., 2003b
). Pro- but also anti-apoptotic activities have also been observed for members of the Chlamydiaceae after infection of human cells and therefore these effects are still controversial (Greene et al., 2004
; Schoier et al., 2001
).
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Conclusion
This study has shown that co-cultivation with free-living amoebae is a suitable and straightforward approach for the direct recovery of environmental chlamydiae from samples containing complex microbial communities. The environmental chlamydia strain UV-7 recovered by this approach represents the first member of the genus Parachlamydia isolated from activated sludge of a wastewater treatment plant. These systems, which harbour an enormous diversity of protozoa, might thus represent an important anthropogenic reservoir for environmental chlamydiae (see also Kahane et al., 2004), which contributes to the dissemination of these bacteria into the receiving water bodies. This might be of particular importance since we could show that strain UV-7 is able to infect mammalian cells, providing further evidence for a possible pathogenic potential of Parachlamydiaceae for humans. A more complete understanding of environmental chlamydiae will depend on the availability of a larger number of isolates from different sources, which in the best case will represent the entire diversity and distribution of this recently discovered bacterial group in nature. Comparative examination of such a strain collection will not only increase our knowledge of the biology of environmental chlamydiae, but will eventually also help to answer the question whether Parachlamydiaceae indeed represent emerging pathogens.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Alm, E. W., Oerther, D. B., Larsen, N., Stahl, D. A. & Raskin, L. (1996). The oligonucleotide probe database. App Environ Microbiol 62, 35573559.
Amann, R., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R. & Stahl, D. A. (1990). Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56, 19191925.[Medline]
Amann, R., Springer, N., Schönhuber, W., Ludwig, W., Schmid, E. N., Müller, K. D. & Michel, R. (1997). Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp. Appl Environ Microbiol 63, 115121.[Abstract]
Barker, J., Humphrey, T. J. & Brown, M. W. R. (1999). Survival of Escherichia coli O157 in a soil protozoan: implications for disease. FEMS Microbiol Lett 173, 291295.[CrossRef][Medline]
Beier, C. L., Horn, M., Michel, R., Schweikert, M., Görtz, H. D. & Wagner, M. (2002). The genus Caedibacter comprises endosymbionts of Paramecium spp. related to the Rickettsiales (Alphaproteobacteria) and to Francisella tularensis (Gammaproteobacteria). Appl Environ Microbiol 68, 60436050.
Birtles, R. J., Rowbotham, T. J., Storey, C., Marrie, T. J. & Raoult, D. (1997). Chlamydia-like obligate parasite of free-living amoebae. Lancet 349, 925926.[Medline]
Birtles, R. J., Rowbotham, T. J., Michel, R., Pitcher, D. G., Lascola, B., Alexiou-Daniel, S. & Raoult, D. (2000). Candidatus Odyssella thessalonicensis' gen. nov., sp. nov., an obligate intracellular parasite of Acanthamoeba species. Int J Syst Evol Microbiol 50, 6372.[Abstract]
Bodetti, T. J., Viggers, K., Warren, K., Swan, R., Conaghty, S., Sims, C. & Timms, P. (2003). Wide range of Chlamydiales types detected in native Australian mammals. Vet Microbiol 96, 177187.[CrossRef][Medline]
Corsaro, D., Venditti, D., Le Faou, A., Guglielmetti, P. & Valassina, M. (2001). A new chlamydia-like 16S rDNA sequence from a clinical sample. Microbiology 147, 515516.[Medline]
Corsaro, D., Venditti, D. & Valassina, M. (2002a). New parachlamydial 16S rDNA phylotypes detected in human clinical samples. Res Microbiol 153, 563567.[CrossRef][Medline]
Corsaro, D., Venditti, D. & Valassina, M. (2002b). New chlamydial lineages from freshwater samples. Microbiology 148, 343344.[Medline]
Corsaro, D., Valassina, M. & Venditti, D. (2003). Increasing diversity within chlamydiae. Crit Rev Microbiol 29, 3778.[Medline]
Daims, H., Brühl, A., Amann, R., Schleifer, K.-H. & Wagner, M. (1999). The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22, 434444.[Medline]
Daims, H., Stoecker, K. & Wagner, M. (2005). Fluorescence in situ hybridisation for the detection of prokaryotes. In Molecular Microbial Ecology. Edited by A. M. Osborn & C. J. Smith. Abingdon, UK: Bios-Garland (in press).
Dowell, S. F., Peeling, R. W., Boman, J. & 12 other authors (2001). Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clin Infect Dis 33, 492503.[CrossRef][Medline]
Essig, A., Heinemann, M., Simnacher, U. & Marre, R. (1997). Infection of Acanthamoeba castellanii by Chlamydia pneumoniae. Appl Environ Microbiol 63, 13961399.[Abstract]
Everett, K. D., Bush, R. M. & Andersen, A. A. (1999). 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 49, 415440.[Abstract]
Felsenstein, J. (1989). PHYLIP phylogeny inference package (version 3.2). Cladistics 5, 164166.
Friedman, M. G., Dvoskin, B. & Kahane, S. (2003). Infections with the chlamydia-like microorganism Simkania negevensis, a possible emerging pathogen. Microbes Infect 5, 10131021.[CrossRef][Medline]
Fritsche, T. R., Gautom, R. K., Seyedirashti, S., Bergeron, D. L. & Lindquist, T. D. (1993). Occurrence of bacteria endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses. J Clin Microbiol 31, 11221126.[Abstract]
Fritsche, T. R., Sobek, D. & Gautom, R. K. (1998). Enhancement of in vitro cytopathogenicity by Acanthamoeba spp. following acquisition of bacterial endosymbionts. FEMS Microbiol Lett 166, 231236.[CrossRef][Medline]
Fritsche, T. R., Horn, M., Seyedirashti, S., Gautom, R. K., Schleifer, K. H. & Wagner, M. (1999). In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phylogenetically related to members of the order Rickettsiales. Appl Environ Microbiol 65, 206212.
Fritsche, T. R., Horn, M., Wagner, M., Herwig, R. P., Schleifer, K. H. & Gautom, R. K. (2000). Phylogenetic diversity among geographically dispersed Chlamydiales endosymbionts recovered from clinical and environmental isolates of Acanthamoeba spp. Appl Environ Microbiol 66, 26132619.
Gautom, R. & Fritsche, T. R. (1995). Transmissibility of bacterial endosymbionts between isolates of Acanthamoeba spp. J Eukaryot Microbiol 42, 452456.[Medline]
Goebel, W. & Gross, R. (2001). Intracellular survival strategies of mutualistic and parasitic prokaryotes. Trends Microbiol 9, 267273.[CrossRef][Medline]
Greene, W., Xiao, Y., Huang, Y., McClarty, G. & Zhong, G. (2004). Chlamydia-infected cells continue to undergo mitosis and resist induction of apoptosis. Infect Immun 72, 451460.
Greub, G. & Raoult, D. (2002a). Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study. Appl Environ Microbiol 68, 30763084.
Greub, G. & Raoult, D. (2002b). Parachlamydiaceae: potential emerging pathogens. Emerg Infect Dis 8, 625630.[Medline]
Greub, G., Boyadjiev, I., La Scola, B., Raoult, D. & Martin, C. (2003a). Serological hint suggesting that Parachlamydiaceae are agents of pneumonia in polytraumatized intensive care patients. Ann N Y Acad Sci 990, 311319.
Greub, G., Mege, J.-L. & Raoult, D. (2003b). Parachlamydia acanthamoeba enters and multiplies within human macrophages and induces their apoptosis. Infect Immun 71, 59795985.
Greub, G., La Scola, B. & Raoult, D. (2004). Amoebae-resisting bacteria isolated from human nasal swabs by amoebal co-culture. Emerg Infect Dis 10, 470477.[Medline]
Harb, O. S., Gao, L.-Y. & Kwaik, Y. A. (2000). From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens. Environ Microbiol 2, 251265.[CrossRef][Medline]
Henning, K., Schares, G., Granzow, H., Polster, U., Hartmann, M., Hotzel, H., Sachse, K., Peters, M. & Rauser, M. (2002). Neospora caninum and Waddlia chondrophila strain 2032/99 in a septic stillborn calf. Vet Microbiol 85, 285292.[CrossRef][Medline]
Horn, M. & Wagner, M. (2001). Evidence for additional genus-level diversity of Chlamydiales in the environment. FEMS Microbiol Lett 204, 7174.[CrossRef][Medline]
Horn, M., Wagner, M., Müller, K. D., Schmid, E. N., Fritsche, T. R., Schleifer, K. H. & Michel, R. (2000). Neochlamydia hartmannellae gen. nov., sp. nov. (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis. Microbiology 146, 12311239.[Medline]
Horn, M., Harzenetter, M. D., Linner, T., Schmid, E. N., Müller, K. D., Michel, R. & Wagner, M. (2001). Members of the Cytophaga-Flavobacterium-Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of Candidatus Amoebophilus asiaticus. Environ Microbiol 3, 440449.[CrossRef][Medline]
Horn, M., Fritsche, T. R., Linner, T., Gautom, R. K., Harzenetter, M. D. & Wagner, M. (2002). Obligate bacterial endosymbionts of Acanthamoeba spp. related to the beta-Proteobacteria: proposal of Candidatus Procabacter acanthamoebae gen. nov., sp. nov. Int J Syst Evol Microbiol 52, 599605.
Horn, M., Collingro, A., Schmitz-Esser, S. & 10 other authors (2004). Illuminating the evolutionary history of chlamydiae. Science 304, 728730.
Kahane, S. E. M. & Friedman, M. G. (1995). Evidence that the novel microorganism Z may belong to a new genus in the family Chlamydiaceae. FEMS Microbiol Lett 126, 203208.[CrossRef][Medline]
Kahane, S., Dvoskin, B., Mathias, M. & Friedman, M. G. (2001). Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis survival within amoebal cysts. Appl Environ Microbiol 67, 47894795.
Kahane, S., Kimmel, N. & Friedman, M. G. (2002). The growth cycle of Simkania negevensis. Microbiology 148, 735742.[CrossRef][Medline]
Kahane, S., Platzner, N., Dvoskin, B., Itzhaki, A. & Friedman, M. G. (2004). Evidence for the presence of Simkania negevensis in drinking water and in reclaimed wastewater in Israel. Appl Environ Microbiol 70, 33463351.
Kostanjsek, R., Strus, J., Drobne, D. & Avgustin, G. (2004). Candidatus Rhabdochlamydia porcellionis, an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda). Int J Syst Evol Microbiol 54, 543549.[CrossRef][Medline]
Lindsay, M. R., Webb, R. I., Hosmer, H. M. & Fuerst, J. A. (1995). Effects of fixative and buffer on morphology and ultrastructure of a freshwater planctomycete, Gemmata obscuriglobus. J Microbiol Methods 21, 4554.[CrossRef]
Loy, A., Horn, M. & Wagner, M. (2003). probeBase: an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res 31, 514516.
Ludwig, W., Strunk, O., Westram, R. & 29 other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 13631371.
Mahoney, J. B., Coombes, B. K. & Chernesky, M. A. (2003). Chlamydia and Chlamydophila. In Manual of Clinical Microbiology, pp. 9911004. Edited by P. R. Murray. Washington, DC: American Society for Microbiology.
Marrie, T. J., Raoult, D., La Scola, B., Birtles, R. J. & de Carolis, E. (2001). Legionella-like and other amoebal pathogens as agents of community-acquired pneumonia. Emerg Infect Dis 7, 10261029.[Medline]
Michel, R., Hauröder-Philippczyk, B., Müller, K.-D. & Weishaar, I. (1994). Acanthamoeba from human nasal mucosa infected with an obligate intracellular parasite. Eur J Protistol 30, 104.
Michel, R., Steinert, M., Zöller, L., Hauröder, B. & Henning, K. (2004). Free-living amoebae may serve as hosts for the chlamydia-like bacterium Waddlia chondrophila isolated from an aborted bovine foetus. Acta Protozool 43, 3742.
Neal, R. A., Latter, V. S. & Richards, W. H. (1974). Survival of Entamoeba and related amoebae at low temperature. II. Viability of amoebae and cysts stored in liquid nitrogen. Int J Parasitol 4, 353360.[CrossRef][Medline]
Ossewaarde, J. M. & Meijer, A. (1999). Molecular evidence for the existence of additional members of the order Chlamydiales. Microbiology 145, 411417.[Medline]
Page, F. C. (1988). A New Key to Freshwater and Soil Gymnamoebae. Ambleside, UK: Freshwater Biological Association.
Pudjiatmoko, H. F., Ochiai, Y., Yamaguchi, T. & Hirai, K. (1997). Phylogenetic analysis of the genus Chlamydia based on 16S rRNA gene sequences. Int J Syst Bacteriol 45, 425431.
Rockey, D. D., Lenart, J. & Stephens, R. S. (2000). Genome sequencing and our understanding of chlamydiae. Infect Immun 68, 54735479.
Rowbotham, T. J. (1983). Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae. J Clin Pathol 36, 978986.[Abstract]
Rurangirwa, F. R., Dilbeck, P. M., Crawford, T. B., McGuire, T. C. & McElwain, T. F. (1999). Analysis of the 16S rRNA gene of microorganism WSU 86-1044 from an aborted bovine foetus reveals that it is a member of the order Chlamydiales: proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov. Int J Syst Bacteriol 49, 577581.[Abstract]
Schoier, J., Ollinger, K., Kvarnstrom, M., Soderlund, G. & Kihlstrom, E. (2001). Chlamydia trachomatis-induced apoptosis occurs in uninfected McCoy cells late in the developmental cycle and is regulated by the intracellular redox state. Microb Pathog 31, 173184.[CrossRef][Medline]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNADNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846849.[Abstract]
Steinert, M., Emody, L., Amann, R. & Hacker, J. (1997). Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl Environ Microbiol 63, 20472053.[Abstract]
Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol Biol Evol 13, 964969.
Thao, M. L., Baumann, L., Hess, J. M., Falk, B. W., Ng, J. C., Gullan, P. J. & Baumann, P. (2003). Phylogenetic evidence for two new insect-associated chlamydia of the family Simkaniaceae. Curr Microbiol 47, 4650.[CrossRef][Medline]
Visvesvara, G. S. (1999). Pathogenic and opportunistic free-living amoebae. In Manual of Clinical Microbiology, 7th edn, pp. 13831390. Edited by E. J. B. P. R. Murray, M. A. Pfaller, F. C. Tenover & R. H. Yolken. Washington, DC: American Society for Microbiology.
Zhong, G., Fan, P., Ji, H., Dong, F. & Huang, Y. (2001). Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 193, 935942.
Received 15 June 2004;
revised 4 October 2004;
accepted 7 October 2004.
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