Protochlamydia naegleriophila as Etiologic Agent of Pneumonia

Using ameba coculture, we grew a Naegleria endosymbiont. Phenotypic, genetic, and phylogenetic analyses supported its affiliation as Protochlamydia naegleriophila sp. nov. We then developed a specific diagnostic PCR for Protochlamydia spp. When applied to bronchoalveolar lavages, results of this PCR were positive for 1 patient with pneumonia. Further studies are needed to assess the role of Protochlamydia spp. in pneumonia.

R ecently, a Naegleria endosymbiont (KNic) was observed but remained uncultivable, precluding precise identifi cation (1). We grew a large amount of strain KNic by using Acanthamoeba castellanii, which enabled phenotypic, genetic and phylogenetic analyses that supported its affi liation as Protochlamydia naegleriophila. This new ameba-resistant intracellular bacteria might represent a new etiologic agent of pneumonia because it is likely also resistant to human alveolar macrophages (2,3). Because other Parachlamydiaceae were associated with lung infection (4-6), we assessed the role of Pr. naegleriophila in pneumonia by developing a diagnostic PCR and applying it to bronchoalveolar lavages.

The Study
KNic growth in A. castellanii was assessed by immunofl uorescence (7) with in-house mouse anti-KNic and Al-exa488-coupled anti-immunoglobulin antibodies (Invitrogen, Eugene, OR, USA). Confocal microscopy (LSM510; Zeiss, Feldbach, Switzerland) confi rmed the intracellular location of KNic and demonstrated its rapid growth within A. castellanii. To precisely assess the growth rate, we performed PCR on A. castellanii/KNic coculture by using PrF/ PrR primers and PrS probe. After 60 hours, we observed an increased number of bacteria per microliter of 4 logarithms (online Appendix Figure, available from www.cdc. gov/EID/content/14/1/167-appG.htm).
To allow quantifi cation, a plasmid containing the target gene was constructed by cloning PCR products into pCR2.1-TOPO vector (Invitrogen, Basel, Switzerland). Recombinant plasmid DNA quantifi ed using Nanodrop ND-1000 (Witech, Littau, Switzerland) was 10-fold diluted and used as positive controls.
The analytical sensitivity was 10 copy/μL (Figure 2, panel A). Intra-run variability was good ( Figure 2B) with a Bland-Altman bias of 0.99 and a limit of agreement of 2.87 ( Figure 2, panel A). Inter-run variability was low at high concentration, 1.12, 1.71, 0.82, 1.77 cycles for 10 5 , 10 4 , 10 3 , 10 2 copies/μL, respectively. Inter-run variability was higher at low concentration, 4.22 cycles for 10 1 copies/μL (Figure 2, panel A). Analytical specifi city was tested with bacterial and eukaryotic DNA ( Table 2). The PCR slightly amplifi ed DNA from R. crassifi cans, another Chlamydia-like organism. No cross-amplifi cation was observed with any other bacteria or with human cells. The absence of cross-amplifi cation of P. acanthamoebae is important because this Chlamydia-related bacteria is considered an emerging agent of pneumonia (4-6).
We tested 134 bronchoalveolar lavage samples from patients with (n = 65) and without (n = 69) pneumonia and extracted DNA by using a Bio-Rad Tissue Kit. One sample was positive, with 543 and 480 copies/μL. This positive result was confi rmed using the 16sigF/16sigR PCR (13), which targets another DNA segment. This sequence exhibited 99.6% (284/285) similarity with Pr. naegleriophila The presence of Protochlamydia antigen in the sample was confi rmed by immunofl uorescence performed using rabbit anti-KNic antibody directly on the bronchoalveolar lavage sample and by ameba coculture (online Appendix Figure).
The positive sample was taken from an immunocompromised patient who had cough, dyspnea, and a lung infi ltrate. Bronchoscopy examination of the lower respiratory tract showed mucosal infl ammation localized at the middle lung lobe. Cytology and Gram stain of the bronchoalveolar lavage showed many leucocytes with macrophages (65%) and neutrophils (23%). Although no antimicrobial treatment was administered prior to bronchoscopy, no other etiologic agent was identifi ed despite extensive microbiologic investigations of bronchial aspirate and bronchoalveolar lavage. Results of Gram stain, auramine stain (for Mycobacterium spp.), and silver stain (for Pneumocystis carinii) tests were negative. Only physiologic oropharyngeal fl ora could be grown on sheep-blood and chocolate-bacitracin agars. Cell culture, as well as culture for fungi and mycobacteria, remained sterile. Moreover, results of PCRs specifi c for the detection of Legionella pneumophila, Chlamydophila pneumoniae, and Mycoplasma pneumoniae (15) were all negative. The patient recovered and remained free of symptoms of acute lung infection during the next 20 months.

Conclusions
Isolating new species from environmental and clinical samples is important to better defi ne their epidemiology and potential pathogenicity. We defi ned the taxonomic position of a novel Naegleria endosymbiont and proposed its affi liation within the Protochlamydia genus as Pr. naegleriophila sp. nov. Moreover, we developed a new PCR targeting Protochlamydia spp., applied it to clinical samples, and identifi ed a possible role of Pr. naegleriophila as an agent of pneumonia.
Protochlamydia naegleriophila (nae.gle.rio′.phi.la Gr. fem.n. Naegleria, name of host cell, Gr. adj. philos, -a friendly to, referring to intracellular growth of Protochlamydia naegleriophila strain KNic within Naegleria amebae). The 16Sr RNA sequence (DQ635609) of KNic is 97.6% similar to that of P. amoebophila, making this organism a member of the genus Protochlamydia. KNic does not grow on axenic media (1) but grows by 4 logarithms in 60 h within A. castellanii. KNic exhibits a Chlamydia-like developmental cycle, with reticulate, elementary, and crescent bodies. The reticulate body is about 900 nm and has a spiny appearance similar to that of P. amoebophila ( Figure  2, panel B). To be classifi ed within the Pr. naegleriophila species, a new strain should show a 16Sr RNA similarity >98.5% (13) and similar phenotypic traits.