title: Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation.
abstract: BACKGROUND
  Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments.
  
  
  RESULTS
  One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heat-killed Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were identified.
  
  
  CONCLUSIONS
  These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen.
authors.affiliation: Hokkaido University Hospital, Nishi-5 Kita-14 Jo, Kita-ku, Sapporo, Hokkaido, 060-8648, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fukumoto
authors.affiliation: Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University Graduate School of Health Sciences, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido, 060-0812, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Matsuo
authors.affiliation: Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University Graduate School of Health Sciences, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido, 060-0812, Japan.
authors.forename: Torahiko
authors.initials: T
authors.lastname: Okubo
authors.affiliation: Division of Biomedical Imaging Research, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Miyarisan Pharmaceutical Co., Ltd., 2-3-13-209, Minami, Wako-shi, Saitama, 351-0104, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Miyamoto
authors.affiliation: Miyarisan Pharmaceutical Co., Ltd., 2-3-13-209, Minami, Wako-shi, Saitama, 351-0104, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Oka
authors.affiliation: Miyarisan Pharmaceutical Co., Ltd., 2-3-13-209, Minami, Wako-shi, Saitama, 351-0104, Japan.
authors.forename: Motomichi
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Hokkaido University Hospital, Nishi-5 Kita-14 Jo, Kita-ku, Sapporo, Hokkaido, 060-8648, Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Akizawa
authors.affiliation: Hokkaido University Hospital, Nishi-5 Kita-14 Jo, Kita-ku, Sapporo, Hokkaido, 060-8648, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shibuya
authors.affiliation: Hokkaido University Hospital, Nishi-5 Kita-14 Jo, Kita-ku, Sapporo, Hokkaido, 060-8648, Japan.
authors.forename: Chikara
authors.initials: C
authors.lastname: Shimizu
authors.affiliation: Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University Graduate School of Health Sciences, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido, 060-0812, Japan. hiroyuki@med.hokudai.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamaguchi
mesh_terms: D000048:Acanthamoeba / Q000302:isolation & purification* / Q000382:microbiology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002689:Chlamydia / Q000235:genetics / Q000302:isolation & purification*; D016207:Cytokines / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004783:Environmental Microbiology; D005798:Genes, Bacterial; D006761:Hospitals; D006801:Humans; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012621:Seasons; D013559:Symbiosis
chemical_list: D000900:Anti-Bacterial Agents; D016207:Cytokines; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Acanthamoeba; Environmental chlamydiae; Genome sequence; Hospital; IL-8