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Chlamydia-related bacteria in respiratory samples in Finland
Microbes and Infection 13 (2011) 824e827 www.elsevier.com/locate/micinf
Short communication
Chlamydia-related bacteria in respiratory samples in Finland Suvi Niemi a,*, Gilbert Greub b, Mirja Puolakkainen a,c b
a Haartman Institute, Department of Virology, 00014 University of Helsinki, Finland Center for Research on Intracellular Bacteria, Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne 1011, Switzerland c Helsinki University Central Hospital, Laboratory Division (HUSLAB), Department of Virology and Immunology, 00029 HUS, Finland
Received 4 March 2011; accepted 15 April 2011 Available online 12 May 2011
Abstract Chlamydia-related bacteria, new members of the order Chlamydiales, are suggested to be associated with respiratory disease. We used realtime PCR to investigate the prevalence of Parachlamydia acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp., Simkania negevensis and Waddlia chondrophila in samples taken from patients with suspected respiratory tract infections. Of the 531 samples analyzed, the subset of 136 samples contained 16 (11.8%) samples positive for Rhabdochlamydia spp. DNA. P. acanthamoebae, Protochlamydia spp., S. negevensis and W. chondrophila DNA were not detected among the respiratory samples investigated. These results suggest an association of Rhabdochlamydia spp. with respiratory disease. Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Chlamydiales; Chlamydia-related bacteria; Parachlamydiaceae; Rhabdochlamydiaceae; Simkaniaceae; Waddliaceae
1. Introduction Despite advances in antibiotic therapy, respiratory infection remains one of the most common infectious disease and even cause of death worldwide especially among children and elderly. The leading causes of community-acquired pneumonia (CAP) among bacterial pathogens are Streptococcus pneumoniae and Haemophilus influenzae [1]. Respiratory infections caused by fastidious bacteria such as Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila are estimated to account for about 20e30% of CAP among adults [2]. Despite a variety of microbial pathogens have been recognized as causative agents of respiratory infections, the microbe causing the disease remains unknown in about half of the cases in CAP [1]. Novel families (Parachlamydiaceae, Simkaniaceae and Waddliaceae) and family-level lineages (Rhabdochlamydiaceae and Criblamydiaceae) discovered within the order Chlamydiales share some biological properties with Chlamydiaceae, including * Corresponding author. Tel.: þ358 9 191 26549; fax: þ358 9 191 26491. E-mail address: [email protected] (S. Niemi).
their unique developmental cycle. These Chlamydia-related bacteria are widely distributed in nature and exhibit a diverse host range across the animal kingdom. Many of them live in close association with free-living amoebae and are able to infect e.g. mammals, birds, reptiles, fish, insects and crustaceans [3]. Thus, Chlamydia-related bacteria may be acquired from aerosolized particles from an environmental or animal reservoir [4]. Chlamydia-related bacteria have mostly been associated with human respiratory disease, mainly based on serological and molecular data. The Chlamydia-related bacteria possibly associated with respiratory disease in humans include Parachlamydia acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp., Simkania negevensis and Waddlia chondrophila [4]. W. chondrophila and Parachlamydia spp. may also be involved in miscarriages in humans [5]. Since Chlamydia-related bacteria are not able to grow on axenic medium and amoebal co-culture is laborious and available only in specialized laboratories, molecular methods represent the key tool for detection of these bacteria [6]. Thus, we investigated with real-time PCR the prevalence of P. acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp. and W. chondrophila in specimens taken from patients
1286-4579/$ - see front matter Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2011.04.012
S. Niemi et al. / Microbes and Infection 13 (2011) 824e827
with suspected respiratory tract infections. We also developed a real-time PCR assay based on a TaqMan probe for detection of S. negevensis from clinical samples. 2. Materials and methods 2.1. Clinical samples and DNA extraction The material for this study consisted of 531 unselected respiratory samples sent for C. pneumoniae [7] and M. pneumoniae [8] nucleic acid testing to HUSLAB, Department of Virology between August 2009 and October 2010 (the diagnostic laboratory of the Helsinki University Central Hospital serving mainly the capital area). The samples were bronchoalveolar lavages, sputum samples, pleural fluids, lung biopsies and secretions from nasopharynx, throat, trachea and bronchus. DNA was extracted from the samples with proteinase K and phenol-chloroform at HUSLAB by standard procedures. Of the 531 samples tested at HUSLAB, five were positive for M. pneumoniae DNA and one for C. pneumoniae DNA. To set up the S. negevensis real-time PCR, a reference strain of S. negevensis was used (originally from ATCC, VR1471), kindly provided by Prof. Pekka Saikku and Dr. Mika Paldanius, Oulu, Finland. For testing the specificity of the S. negevensis real-time PCR, DNA from Chlamydia trachomatis type A, D and L2, C. pneumoniae K6, eukaryotic cell lines A549, HL and McCoy and ten respiratory samples cultivated on chocolate agar plates were used. Bacteria on agar plates were suspended in 300 ml of TE (Tris-EDTA)-buffer, heated for 10 min at 100 C, centrifuged for 2 min at 14 000 g and the supernatant was used for analysis. DNA from the other samples was extracted from a sample volume of 400 ml with MagNA Pure Compact instrument (Roche) using MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche) with DNA Bacteria protocol and was eluted in 50 ml. 2.2. Real-time PCR The recombinant plasmids with P. acanthamoebae [9], Protochlamydia spp. [10], Rhabdochlamydia spp. [11], W. chondrophila [12], S. negevensis (cloned during this study) and C. pneumoniae [13] PCR products were used as positive controls for the assays. Plasmids were extracted with QIAprep Spin Miniprep Kit (Qiagen) and DNA concentration was measured with a NanoDrop 1000 spectrophotometer (Thermo Scientific). Detection of P. acanthamoebae [9], Protochlamydia spp. [10], Rhabdochlamydia spp. [11], W. chondrophila [12] and C. pneumoniae [13] was performed with methods described previously. For detection of S. negevensis, Primer Express software version 3.0 (Applied Biosystems) was used to select a TaqMan probe (50 -FAM-AAGGGCGCGTAGGCGGGTAAGC-BHQ1-30 ) based on the sequence of S. negevensis 16S ribosomal RNA gene (GenBank accession no. NR_029194). The primers used were developed previously [14], but the forward primer (50 -CAAGAAAAGGTAACGAATAATTGCC30 ) was modified to optimize the performance of the S. negevensis real-time PCR. The amplicon length was 171 base pairs.
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The S. negevensis real-time PCR was able to detect 10 copies per reaction and the efficiency of amplification was 98% (3.359) for the control plasmid. To test the specificity of the assay, DNA from C. trachomatis type A, D and L2, C. pneumoniae K6, cell lines A549, HL and McCoy, ten respiratory samples cultivated on chocolate agar plates and two Rhabdochlamydia spp. positive clinical samples were analyzed with S. negevensis real-time PCR. All of these samples tested negative in the S. negevensis real-time PCR assay. Real-time PCR analyses were performed with an ABI 7500 instrument and Sequence Detection Software version 1.3.1 (Applied Biosystems). The primers and probes used in this study were purchased from Applied Biosystems, Eurogentec, Oligomer and TAG Copenhagen A/S. The PCR reactions were performed in a 25 ml volume containing 300 nM (C. pneumoniae & S. negevensis) or 200 nM (other Chlamydia-related bacteria) primers, 200 nM (C. pneumoniae & S. negevensis) or 100 nM (other Chlamydia-related bacteria) probes and either 12.5 ml Maxima Probe qPCR Master Mix (Fermentas Life Sciences) or 12.5 ml TaqMan Universal Master Mix (Applied Biosystems). Thermal cycling conditions were: 50 C 2 min, 95 C 10 min and 40 cycles of 95 C 15 s and 60 C 1 min. The PCR reactions were performed as duplex with C. pneumoniae combined with each Chlamydia-related bacterium. Template volume was 5 ml and each sample was amplified in duplicate. Altogether 531 samples were analyzed in subsets of 136 for Rhabdochlamydia spp., 102 for P. acanthamoebae, 100 for Protochlamydia spp., 96 for W. chondrophila and 97 for S. negevensis. 3. Results Of the subset of 136 respiratory samples analyzed for Rhabdochlamydia spp. DNA, we detected 16 positive specimens (11.8%). First, a set of samples (N ¼ 97) collected in October 2009eDecember 2009 were analyzed, and 12.4% (N ¼ 12) were positive. To confirm the finding, another set of samples (N ¼ 39) was collected in May 2010eJune 2010 and the prevalence of Rhabdochlamydia spp. in this sample set was 10.3% (N ¼ 4). The positive samples were secretions from throat (N ¼ 6), bronchoalveolar lavages (N ¼ 5), secretions from bronchus (N ¼ 2) and nasopharynx (N ¼ 2) and sputum (N ¼ 1). The Rhabdochlamydia spp. positive patients included both men (N ¼ 8) and women (N ¼ 8). The median age of the patients with Rhabdochlamydia spp. was 52 (range 11e72). The mean Ct (cycle treshold) value for the Rhabdochlamydia spp. positive samples was 35.86 and the mean quantity was 46.2 copies per reaction. The characteristics of the Rhabdochlamydia spp. positive patients are presented in Table 1. We were not able to detect any samples with P. acanthamoebae (N ¼ 102), Protochlamydia spp. (N ¼ 100), W. chondrophila (N ¼ 96) or S. negevensis (N ¼ 97) DNA (Table 2). 4. Discussion Some Chlamydia-related bacteria including S. negevensis and P. acanthamoebae might be associated with respiratory tract infection [6,15,16]. Also, members of the Rhabdochlamydiaceae
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Table 1 Characteristics of the 16 Rhabdochlamydia spp. DNA positive patients with suspected respiratory infection. Patient no.
Age
Sex
Specimen type
Rhabdochlamydia spp. PCR results (mean Ct value and DNA copies/reaction)
Other information
1 2 3 4 5 6
69 54 41 55 68 50
female female male female male female
BAL throat swab throat swab bronchial secretion bronchial secretion throat swab
37.60 33.26 35.76 38.14 35.06 38.45
(6.3) (92.6) (19.8) (4.5) (30.6) (3.7)
7 8 9 10 11 12 13 14
13 37 54 11 15 72 69 50
female female male male male male male female
throat swab nasopharyngeal swab sputum throat swab throat swab BAL BAL nasopharyngeal swab
34.44 38.02 33.41 38.30 31.18 37.66 37.99 36.27
(41.5) (4.4) (79.3) (3.6) (329.8) (16.9) (1.2) (3.7)
15 16
57 38
female male
BAL BAL
35.93 (4.7) 32.36 (97.1)
BAL: yeast cultureþ suspicion of H1N1 influenza NA NA NA Prolonged M. pneumoniae IgMþ (>1 year) NA NA NA pneumonia pneumonia BAL: CMV cultureþ NA liver transplantation (four years earlier) NA leukemia
Ct ¼ cycle treshold, BAL ¼ bronchoalveolar lavage, NA ¼ not available, CMV ¼ cytomegalovirus.
have been suggested to be associated with respiratory disease in humans. Haider et al. found DNA of Chlamydia-related bacteria in about 1% of adult patients with CAP, including two Rhabdochlamydia porcellionis-related sequences, although the two samples were also positive for other bacterial respiratory pathogens e.g. M. pneumoniae [17]. Lamoth et al. reported the presence of Rhabdochlamydia spp. in respiratory specimens of premature neonates [11]. The family Rhabdochlamydiaceae comprises two bacteria: R. porcellionis and Rhabdochlamydia crassificans. R. porcellionis infects hepatopancreatic cells of the terrestrial isopod Porcellio scaber [18] and R. crassificans causes abdominal swelling to its cockroach host Blatta orientalis [19]. Cockroaches often occur in unsanitary conditions where they can act as reservoir and mechanical vectors for different microbes [19]. It is not known if members of the Rhabdochlamydiaceae are able to survive only in arthropods or if they may also survive in amoebae. Previously, amoebae co-cultivation of an insect endosymbiont Fritschea bemisiae has failed [20]. If Rhabdochlamydiaceae were able to survive in amoebae, it may be of epidemiologic importance. Indeed, amoebae are widespread in water and the increased use of various water-based systems such as air conditioners and humidifiers in the developed world may expose humans to amoebae and to their related pathogens [21]. Table 2 The number and results of respiratory samples analyzed with P. acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp., S. negevensis and W. chondrophila real-time PCR. Organisms
Samples analyzed
Positive
Negative
P. acanthamoebae Protochlamydia spp. Rhabdochlamydia spp. S. negevensis W. chondrophila
102 100 136 97 96
0 0 16 0 0
102 100 120 97 96
Moreover, amoebae may serve as a training ground for amoebae-resisting bacteria favoring the selection of virulence traits and enabling these bacteria to survive in other phagocytic cells e.g. alveolar macrophages [22]. In this study, we found 16 (11.8%) respiratory samples positive for Rhabdochlamydia spp. DNA in the subset of 136 samples. The relatively high Ct values obtained here (Table 1) are in accordance with those previously published for P. acanthamoebae [9] and W. chondrophila [12] real-time PCR, and they most probably reflect a low amount of bacteria present in the samples. Based on the analysis of the two sample subsets, the occurrence of Rhabdochlamydia spp. was not seasonal but rather stable throughout the year. The patient samples positive for Rhabdochlamydia spp. tested C. pneumoniae negative in the duplex real-time assay used in this study. These 16 samples tested also negative for M. pneumoniae nucleic acid, and no other bacteria or viruses causing respiratory disease were detected at HUSLAB. However, since all the samples were not consistently tested for other respiratory pathogens at the diagnostic laboratory or for the other Chlamydia-related bacteria in this study, it remains unknown if Rhabdochlamydia spp. was really the single cause of respiratory symptoms, or whether other pathogens or other conditions were involved. Another possibility is that Rhabdochlamydiaceae represent environmental bacteria that could contaminate e.g. PCR reagents. However, all the specimens had been extracted with similar protocols at HUSLAB, and if this had been the case, we would have expected more samples to be positive for Rhabdochlamydia spp. In addition, all the extraction controls (N ¼ 12) remained negative for Rhabdochlamydia spp. in the real-time PCR. Before this study, the prevalence of most novel Chlamydiae including Rhabdochlamydia spp. was unknown in Finland. There is serological evidence of the presence of S. negevensis among children with CAP in Finland [23]. In this study, we
S. Niemi et al. / Microbes and Infection 13 (2011) 824e827
developed a novel TaqMan probe based real-time PCR for detection of S. negevensis. However, we were not able to detect S. negevensis DNA in this material, which mainly consisted of samples taken from adults. Moreover, we were not able to detect P. acanthamoebae DNA in these samples despite the growing evidence for a possible role of this intracellular bacterium in respiratory disease [16]. This might be due to geographical variation in prevalence. Although the present work suggests a possible role for Rhabdochlamydia spp. in respiratory tract infection, this remains to be investigated in prospective case-control studies. Acknowledgments We thank Se´bastien Aeby from Dr. Gilbert Greub’s laboratory for all the help in setting up the assays for P. acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp. and W. chondrophila. Prof. Pekka Saikku and Dr. Mika Paldanius, Oulu, Finland are acknowledged for the reference strain of S. negevensis. We also thank HUSLAB, Department of Virology for the clinical samples. This research was supported by the Academy of Finland in the frame of the ERA-NET PathoGenoMics, #217554/ECIBUG and #130043/ChlamyTrans. References [1] L.A. Mandell, R.G. Wunderink, A. Anzueto, J.G. Bartlett, G.D. Campbell, N. C. Dean, S.F. Dowell, T.M. File Jr., D.M. Musher, M.S. Niederman, A. Torres, C.G. Whitney, Infectious Diseases Society of America, American Thoracic Society, Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults, Clin. Infect. Dis. 44 (Suppl 2) (2007) S27eS72. [2] J.G. Bartlett, Is activity against "atypical" pathogens necessary in the treatment protocols for community-acquired pneumonia? Issues with combination therapy, Clin. Infect. Dis. 47 (Suppl 3) (2008) S232eS236. [3] M. Horn, Chlamydiae as symbionts in eukaryotes, Annu. Rev. Microbiol. 62 (2008) 113e131. [4] F. Lamoth, G. Greub, Amoebal pathogens as emerging causal agents of pneumonia, FEMS Microbiol. Rev. 34 (2010) 260e280. [5] D. Baud, V. Thomas, A. Arafa, L. Regan, G. Greub, Waddlia chondrophila, a potential agent of human fetal death, Emerg. Infect. Dis. 13 (2007) 1239e1243. [6] D. Corsaro, G. Greub, Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria, Clin. Microbiol. Rev. 19 (2006) 283e297. [7] C.Y. Tong, M. Sillis, Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR, J. Clin. Pathol. 46 (1993) 313e317.
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[8] F.J. van Kuppeveld, J.T. van der Logt, A.F. Angulo, M.J. van Zoest, W. G. Quint, H.G. Niesters, J.M. Galama, W.J. Melchers, Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification, Appl. Environ. Microbiol. 58 (1992) 2606e2615. [9] N. Casson, K.M. Posfay-Barbe, A. Gervaix, G. Greub, New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples, J. Clin. Microbiol. 46 (2008) 1491e1493. [10] N. Casson, R. Michel, K.D. Mu¨ller, J.D. Aubert, G. Greub, Protochlamydia naegleriophila as etiologic agent of pneumonia, Emerg. Infect. Dis. 14 (2008) 168e172. [11] F. Lamoth, S. Aeby, A. Schneider, K. Jaton-Ogay, B. Vaudaux, G. Greub, Parachlamydia and Rhabdochlamydia in premature neonates, Emerg. Infect. Dis. 15 (2009) 2072e2075. [12] G. Goy, A. Croxatto, K.M. Posfay-Barbe, A. Gervaix, G. Greub, Development of a real-time PCR for the specific detection of Waddlia chondrophila in clinical samples, Eur. J. Clin. Microbiol. Infect. Dis. 28 (2009) 1483e1486. [13] L. Mannonen, E. Kamping, T. Penttila¨, M. Puolakkainen, IFN-gamma induced persistent Chlamydia pneumoniae infection in HL and Mono Mac 6 cells: characterization by real-time quantitative PCR and culture, Microb. Pathog. 36 (2004) 41e50. [14] S. Johnsen, N. Birkebæk, P.L. Andersen, C. Emil, J.S. Jensen, L. Østergaard, Indirect immunofluorescence and real time PCR for detection of Simkania negevensis infection in Danish adults with persistent cough and in healthy controls, Scand. J. Infect. Dis. 37 (2005) 251e255. [15] M.G. Friedman, B. Dvoskin, S. Kahane, Infections with the chlamydialike microorganism Simkania negevensis, a possible emerging pathogen, Microbes Infect. 5 (2003) 1013e1021. [16] G. Greub, Parachlamydia acanthamoebae, an emerging agent of pneumonia, Clin. Microbiol. Infect. 15 (2009) 18e28. [17] S. Haider, A. Collingro, J. Walochnik, M. Wagner, M. Horn, Chlamydialike bacteria in respiratory samples of community-acquired pneumonia patients, FEMS Microbiol. Lett. 281 (2008) 198e202. [18] R. Kostanjsek, J. Strus, D. Drobne, G. Avgustin, ‘Candidatus Rhabdochlamydia porcellionis’, an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda), Int. J. Syst. Evol. Microbiol. 54 (2004) 543e549. [19] D. Corsaro, V. Thomas, G. Goy, D. Venditti, R. Radek, G. Greub, ‘Candidatus Rhabdochlamydia crassificans’, an intracellular bacterial pathogen of the cockroach Blatta orientalis (Insecta: Blattodea), Syst. Appl. Microbiol. 30 (2007) 221e228. [20] K.D. Everett, M. Thao, M. Horn, G.E. Dyszynski, P. Baumann, Novel chlamydiae in whiteflies and scale insects: endosymbionts ‘Candidatus Fritschea bemisiae’ strain Falk and ‘Candidatus Fritschea eriococci’ strain Elm, Int. J. Syst. Evol. Microbiol. 55 (2005) 1581e1587. [21] J.F. Loret, G. Greub, Free-living amoebae: biological by-passes in water treatment, Int. J. Hyg. Environ. Health 213 (2010) 167e175. [22] G. Greub, D. Raoult, Microorganisms resistant to free-living amoebae, Clin. Microbiol. Rev. 17 (2004) 413e433. [23] T. Heiskanen-Kosma, M. Paldanius, M. Korppi, Simkania negevensis may be a true cause of community acquired pneumonia in children, Scand. J. Infect. Dis. 40 (2008) 127e130.
Short communication
Chlamydia-related bacteria in respiratory samples in Finland Suvi Niemi a,*, Gilbert Greub b, Mirja Puolakkainen a,c b
a Haartman Institute, Department of Virology, 00014 University of Helsinki, Finland Center for Research on Intracellular Bacteria, Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne 1011, Switzerland c Helsinki University Central Hospital, Laboratory Division (HUSLAB), Department of Virology and Immunology, 00029 HUS, Finland
Received 4 March 2011; accepted 15 April 2011 Available online 12 May 2011
Abstract Chlamydia-related bacteria, new members of the order Chlamydiales, are suggested to be associated with respiratory disease. We used realtime PCR to investigate the prevalence of Parachlamydia acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp., Simkania negevensis and Waddlia chondrophila in samples taken from patients with suspected respiratory tract infections. Of the 531 samples analyzed, the subset of 136 samples contained 16 (11.8%) samples positive for Rhabdochlamydia spp. DNA. P. acanthamoebae, Protochlamydia spp., S. negevensis and W. chondrophila DNA were not detected among the respiratory samples investigated. These results suggest an association of Rhabdochlamydia spp. with respiratory disease. Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Chlamydiales; Chlamydia-related bacteria; Parachlamydiaceae; Rhabdochlamydiaceae; Simkaniaceae; Waddliaceae
1. Introduction Despite advances in antibiotic therapy, respiratory infection remains one of the most common infectious disease and even cause of death worldwide especially among children and elderly. The leading causes of community-acquired pneumonia (CAP) among bacterial pathogens are Streptococcus pneumoniae and Haemophilus influenzae [1]. Respiratory infections caused by fastidious bacteria such as Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila are estimated to account for about 20e30% of CAP among adults [2]. Despite a variety of microbial pathogens have been recognized as causative agents of respiratory infections, the microbe causing the disease remains unknown in about half of the cases in CAP [1]. Novel families (Parachlamydiaceae, Simkaniaceae and Waddliaceae) and family-level lineages (Rhabdochlamydiaceae and Criblamydiaceae) discovered within the order Chlamydiales share some biological properties with Chlamydiaceae, including * Corresponding author. Tel.: þ358 9 191 26549; fax: þ358 9 191 26491. E-mail address: [email protected] (S. Niemi).
their unique developmental cycle. These Chlamydia-related bacteria are widely distributed in nature and exhibit a diverse host range across the animal kingdom. Many of them live in close association with free-living amoebae and are able to infect e.g. mammals, birds, reptiles, fish, insects and crustaceans [3]. Thus, Chlamydia-related bacteria may be acquired from aerosolized particles from an environmental or animal reservoir [4]. Chlamydia-related bacteria have mostly been associated with human respiratory disease, mainly based on serological and molecular data. The Chlamydia-related bacteria possibly associated with respiratory disease in humans include Parachlamydia acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp., Simkania negevensis and Waddlia chondrophila [4]. W. chondrophila and Parachlamydia spp. may also be involved in miscarriages in humans [5]. Since Chlamydia-related bacteria are not able to grow on axenic medium and amoebal co-culture is laborious and available only in specialized laboratories, molecular methods represent the key tool for detection of these bacteria [6]. Thus, we investigated with real-time PCR the prevalence of P. acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp. and W. chondrophila in specimens taken from patients
1286-4579/$ - see front matter Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2011.04.012
S. Niemi et al. / Microbes and Infection 13 (2011) 824e827
with suspected respiratory tract infections. We also developed a real-time PCR assay based on a TaqMan probe for detection of S. negevensis from clinical samples. 2. Materials and methods 2.1. Clinical samples and DNA extraction The material for this study consisted of 531 unselected respiratory samples sent for C. pneumoniae [7] and M. pneumoniae [8] nucleic acid testing to HUSLAB, Department of Virology between August 2009 and October 2010 (the diagnostic laboratory of the Helsinki University Central Hospital serving mainly the capital area). The samples were bronchoalveolar lavages, sputum samples, pleural fluids, lung biopsies and secretions from nasopharynx, throat, trachea and bronchus. DNA was extracted from the samples with proteinase K and phenol-chloroform at HUSLAB by standard procedures. Of the 531 samples tested at HUSLAB, five were positive for M. pneumoniae DNA and one for C. pneumoniae DNA. To set up the S. negevensis real-time PCR, a reference strain of S. negevensis was used (originally from ATCC, VR1471), kindly provided by Prof. Pekka Saikku and Dr. Mika Paldanius, Oulu, Finland. For testing the specificity of the S. negevensis real-time PCR, DNA from Chlamydia trachomatis type A, D and L2, C. pneumoniae K6, eukaryotic cell lines A549, HL and McCoy and ten respiratory samples cultivated on chocolate agar plates were used. Bacteria on agar plates were suspended in 300 ml of TE (Tris-EDTA)-buffer, heated for 10 min at 100 C, centrifuged for 2 min at 14 000 g and the supernatant was used for analysis. DNA from the other samples was extracted from a sample volume of 400 ml with MagNA Pure Compact instrument (Roche) using MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche) with DNA Bacteria protocol and was eluted in 50 ml. 2.2. Real-time PCR The recombinant plasmids with P. acanthamoebae [9], Protochlamydia spp. [10], Rhabdochlamydia spp. [11], W. chondrophila [12], S. negevensis (cloned during this study) and C. pneumoniae [13] PCR products were used as positive controls for the assays. Plasmids were extracted with QIAprep Spin Miniprep Kit (Qiagen) and DNA concentration was measured with a NanoDrop 1000 spectrophotometer (Thermo Scientific). Detection of P. acanthamoebae [9], Protochlamydia spp. [10], Rhabdochlamydia spp. [11], W. chondrophila [12] and C. pneumoniae [13] was performed with methods described previously. For detection of S. negevensis, Primer Express software version 3.0 (Applied Biosystems) was used to select a TaqMan probe (50 -FAM-AAGGGCGCGTAGGCGGGTAAGC-BHQ1-30 ) based on the sequence of S. negevensis 16S ribosomal RNA gene (GenBank accession no. NR_029194). The primers used were developed previously [14], but the forward primer (50 -CAAGAAAAGGTAACGAATAATTGCC30 ) was modified to optimize the performance of the S. negevensis real-time PCR. The amplicon length was 171 base pairs.
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The S. negevensis real-time PCR was able to detect 10 copies per reaction and the efficiency of amplification was 98% (3.359) for the control plasmid. To test the specificity of the assay, DNA from C. trachomatis type A, D and L2, C. pneumoniae K6, cell lines A549, HL and McCoy, ten respiratory samples cultivated on chocolate agar plates and two Rhabdochlamydia spp. positive clinical samples were analyzed with S. negevensis real-time PCR. All of these samples tested negative in the S. negevensis real-time PCR assay. Real-time PCR analyses were performed with an ABI 7500 instrument and Sequence Detection Software version 1.3.1 (Applied Biosystems). The primers and probes used in this study were purchased from Applied Biosystems, Eurogentec, Oligomer and TAG Copenhagen A/S. The PCR reactions were performed in a 25 ml volume containing 300 nM (C. pneumoniae & S. negevensis) or 200 nM (other Chlamydia-related bacteria) primers, 200 nM (C. pneumoniae & S. negevensis) or 100 nM (other Chlamydia-related bacteria) probes and either 12.5 ml Maxima Probe qPCR Master Mix (Fermentas Life Sciences) or 12.5 ml TaqMan Universal Master Mix (Applied Biosystems). Thermal cycling conditions were: 50 C 2 min, 95 C 10 min and 40 cycles of 95 C 15 s and 60 C 1 min. The PCR reactions were performed as duplex with C. pneumoniae combined with each Chlamydia-related bacterium. Template volume was 5 ml and each sample was amplified in duplicate. Altogether 531 samples were analyzed in subsets of 136 for Rhabdochlamydia spp., 102 for P. acanthamoebae, 100 for Protochlamydia spp., 96 for W. chondrophila and 97 for S. negevensis. 3. Results Of the subset of 136 respiratory samples analyzed for Rhabdochlamydia spp. DNA, we detected 16 positive specimens (11.8%). First, a set of samples (N ¼ 97) collected in October 2009eDecember 2009 were analyzed, and 12.4% (N ¼ 12) were positive. To confirm the finding, another set of samples (N ¼ 39) was collected in May 2010eJune 2010 and the prevalence of Rhabdochlamydia spp. in this sample set was 10.3% (N ¼ 4). The positive samples were secretions from throat (N ¼ 6), bronchoalveolar lavages (N ¼ 5), secretions from bronchus (N ¼ 2) and nasopharynx (N ¼ 2) and sputum (N ¼ 1). The Rhabdochlamydia spp. positive patients included both men (N ¼ 8) and women (N ¼ 8). The median age of the patients with Rhabdochlamydia spp. was 52 (range 11e72). The mean Ct (cycle treshold) value for the Rhabdochlamydia spp. positive samples was 35.86 and the mean quantity was 46.2 copies per reaction. The characteristics of the Rhabdochlamydia spp. positive patients are presented in Table 1. We were not able to detect any samples with P. acanthamoebae (N ¼ 102), Protochlamydia spp. (N ¼ 100), W. chondrophila (N ¼ 96) or S. negevensis (N ¼ 97) DNA (Table 2). 4. Discussion Some Chlamydia-related bacteria including S. negevensis and P. acanthamoebae might be associated with respiratory tract infection [6,15,16]. Also, members of the Rhabdochlamydiaceae
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Table 1 Characteristics of the 16 Rhabdochlamydia spp. DNA positive patients with suspected respiratory infection. Patient no.
Age
Sex
Specimen type
Rhabdochlamydia spp. PCR results (mean Ct value and DNA copies/reaction)
Other information
1 2 3 4 5 6
69 54 41 55 68 50
female female male female male female
BAL throat swab throat swab bronchial secretion bronchial secretion throat swab
37.60 33.26 35.76 38.14 35.06 38.45
(6.3) (92.6) (19.8) (4.5) (30.6) (3.7)
7 8 9 10 11 12 13 14
13 37 54 11 15 72 69 50
female female male male male male male female
throat swab nasopharyngeal swab sputum throat swab throat swab BAL BAL nasopharyngeal swab
34.44 38.02 33.41 38.30 31.18 37.66 37.99 36.27
(41.5) (4.4) (79.3) (3.6) (329.8) (16.9) (1.2) (3.7)
15 16
57 38
female male
BAL BAL
35.93 (4.7) 32.36 (97.1)
BAL: yeast cultureþ suspicion of H1N1 influenza NA NA NA Prolonged M. pneumoniae IgMþ (>1 year) NA NA NA pneumonia pneumonia BAL: CMV cultureþ NA liver transplantation (four years earlier) NA leukemia
Ct ¼ cycle treshold, BAL ¼ bronchoalveolar lavage, NA ¼ not available, CMV ¼ cytomegalovirus.
have been suggested to be associated with respiratory disease in humans. Haider et al. found DNA of Chlamydia-related bacteria in about 1% of adult patients with CAP, including two Rhabdochlamydia porcellionis-related sequences, although the two samples were also positive for other bacterial respiratory pathogens e.g. M. pneumoniae [17]. Lamoth et al. reported the presence of Rhabdochlamydia spp. in respiratory specimens of premature neonates [11]. The family Rhabdochlamydiaceae comprises two bacteria: R. porcellionis and Rhabdochlamydia crassificans. R. porcellionis infects hepatopancreatic cells of the terrestrial isopod Porcellio scaber [18] and R. crassificans causes abdominal swelling to its cockroach host Blatta orientalis [19]. Cockroaches often occur in unsanitary conditions where they can act as reservoir and mechanical vectors for different microbes [19]. It is not known if members of the Rhabdochlamydiaceae are able to survive only in arthropods or if they may also survive in amoebae. Previously, amoebae co-cultivation of an insect endosymbiont Fritschea bemisiae has failed [20]. If Rhabdochlamydiaceae were able to survive in amoebae, it may be of epidemiologic importance. Indeed, amoebae are widespread in water and the increased use of various water-based systems such as air conditioners and humidifiers in the developed world may expose humans to amoebae and to their related pathogens [21]. Table 2 The number and results of respiratory samples analyzed with P. acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp., S. negevensis and W. chondrophila real-time PCR. Organisms
Samples analyzed
Positive
Negative
P. acanthamoebae Protochlamydia spp. Rhabdochlamydia spp. S. negevensis W. chondrophila
102 100 136 97 96
0 0 16 0 0
102 100 120 97 96
Moreover, amoebae may serve as a training ground for amoebae-resisting bacteria favoring the selection of virulence traits and enabling these bacteria to survive in other phagocytic cells e.g. alveolar macrophages [22]. In this study, we found 16 (11.8%) respiratory samples positive for Rhabdochlamydia spp. DNA in the subset of 136 samples. The relatively high Ct values obtained here (Table 1) are in accordance with those previously published for P. acanthamoebae [9] and W. chondrophila [12] real-time PCR, and they most probably reflect a low amount of bacteria present in the samples. Based on the analysis of the two sample subsets, the occurrence of Rhabdochlamydia spp. was not seasonal but rather stable throughout the year. The patient samples positive for Rhabdochlamydia spp. tested C. pneumoniae negative in the duplex real-time assay used in this study. These 16 samples tested also negative for M. pneumoniae nucleic acid, and no other bacteria or viruses causing respiratory disease were detected at HUSLAB. However, since all the samples were not consistently tested for other respiratory pathogens at the diagnostic laboratory or for the other Chlamydia-related bacteria in this study, it remains unknown if Rhabdochlamydia spp. was really the single cause of respiratory symptoms, or whether other pathogens or other conditions were involved. Another possibility is that Rhabdochlamydiaceae represent environmental bacteria that could contaminate e.g. PCR reagents. However, all the specimens had been extracted with similar protocols at HUSLAB, and if this had been the case, we would have expected more samples to be positive for Rhabdochlamydia spp. In addition, all the extraction controls (N ¼ 12) remained negative for Rhabdochlamydia spp. in the real-time PCR. Before this study, the prevalence of most novel Chlamydiae including Rhabdochlamydia spp. was unknown in Finland. There is serological evidence of the presence of S. negevensis among children with CAP in Finland [23]. In this study, we
S. Niemi et al. / Microbes and Infection 13 (2011) 824e827
developed a novel TaqMan probe based real-time PCR for detection of S. negevensis. However, we were not able to detect S. negevensis DNA in this material, which mainly consisted of samples taken from adults. Moreover, we were not able to detect P. acanthamoebae DNA in these samples despite the growing evidence for a possible role of this intracellular bacterium in respiratory disease [16]. This might be due to geographical variation in prevalence. Although the present work suggests a possible role for Rhabdochlamydia spp. in respiratory tract infection, this remains to be investigated in prospective case-control studies. Acknowledgments We thank Se´bastien Aeby from Dr. Gilbert Greub’s laboratory for all the help in setting up the assays for P. acanthamoebae, Protochlamydia spp., Rhabdochlamydia spp. and W. chondrophila. Prof. Pekka Saikku and Dr. Mika Paldanius, Oulu, Finland are acknowledged for the reference strain of S. negevensis. We also thank HUSLAB, Department of Virology for the clinical samples. This research was supported by the Academy of Finland in the frame of the ERA-NET PathoGenoMics, #217554/ECIBUG and #130043/ChlamyTrans. References [1] L.A. Mandell, R.G. Wunderink, A. Anzueto, J.G. Bartlett, G.D. Campbell, N. C. Dean, S.F. Dowell, T.M. File Jr., D.M. Musher, M.S. Niederman, A. Torres, C.G. Whitney, Infectious Diseases Society of America, American Thoracic Society, Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults, Clin. Infect. Dis. 44 (Suppl 2) (2007) S27eS72. [2] J.G. Bartlett, Is activity against "atypical" pathogens necessary in the treatment protocols for community-acquired pneumonia? Issues with combination therapy, Clin. Infect. Dis. 47 (Suppl 3) (2008) S232eS236. [3] M. Horn, Chlamydiae as symbionts in eukaryotes, Annu. Rev. Microbiol. 62 (2008) 113e131. [4] F. Lamoth, G. Greub, Amoebal pathogens as emerging causal agents of pneumonia, FEMS Microbiol. Rev. 34 (2010) 260e280. [5] D. Baud, V. Thomas, A. Arafa, L. Regan, G. Greub, Waddlia chondrophila, a potential agent of human fetal death, Emerg. Infect. Dis. 13 (2007) 1239e1243. [6] D. Corsaro, G. Greub, Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria, Clin. Microbiol. Rev. 19 (2006) 283e297. [7] C.Y. Tong, M. Sillis, Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR, J. Clin. Pathol. 46 (1993) 313e317.
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