Acanthamoeba castellanii C3 (ATCC 50739) were purchased from the American Type Culture Collection (ATCC). Amoebae were maintained in PYG broth (0.75% peptone, 0.75% yeast extract, and 1.5% glucose) at 30 °C . Parachlamydia Bn9 (ATCC VR-1476) was also purchased from the ATCC, and the bacteria were propagated in an amoeba culture system according to methods described previously . The numbers of bacterial infectious progeny were determined by the amoebal infectious unit (AIU) assay described previously . The immortal human epithelial cell line, HEp-2, was also used for the study. HEp-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) containing 10% heat-inactivated fetal calf serum and antibiotics (penicillin, 100u/ml; streptomycin, 100 μg/ml) (Sigma) at 37 °C in 5% CO2.
Smear sample collection
One hundred-smear samples were collected from a hospital (Hokkaido University Hospital, Sapporo, Japan, which has approximately 900 beds); 50 samples were collected in a winter trial, February to March 2012, and 50 samples were collected in a summer trial, August 2012. The smear samples were collected from the floor or sink outlet by wiping with sterilized gauze moistened with Page’s amoeba saline (PAS) , according to a previously described procedure . The pellets obtained from the gauze were resuspended in PAS and used for amoebal isolation and DNA extraction. All sampling locations were limited at drainages, sinks and floors in the public space of hospital, which can be freely accessed by both patients and medical staffs, but not including emerging rooms with recovery rooms or patient rooms.
Isolation of amoebae
Amoebae were isolated using a previously reported method . In brief, a drop of sample/PAS solution was placed on the center of a non-nutrient agar plate on which heat-inactivated E. coli (a stock collection in our laboratory) were spread as a food source. Plates were then incubated at 30 °C. After 7 days of incubation, crawling amoebae with arm-like structures characteristic of Acanthamoeba cysts were isolated, according to a morphological assessment procedure . Amoebae picked under microscopic observation from non-nutrient agar plates were then continuously grown in PYG broth to achieve axenic cultures. Three amoebal strains harboring environmental chlamydiae were finally established (Amoebal strain name/amoebal genotype/bacterial genus; W-9/T4/Protochlamydia sp., Y-20/T4/Neochlamydia sp., Y-23/T4/Neochlamydia sp.); however, because of lacking secondary infectious ability to C3 amoebae, Y-20 and Y-23 amoebae were omitted from the following experiments into assessing intracellular growth and IL-8 induction.
Direct sequencing and phylogenic analysis
To identify Acanthamoeba and environmental chlamydiae in the isolates, total DNA was extracted from amoebae using a High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions. Extracted DNA was then amplified using High-Fidelity Phusion DNA polymerase (Thermo Fisher Scientific, San Jose, CA, USA) with specific primer sets for the Acanthamoeba 18S rRNA gene (JDP1, 5′-GGCCCAGATCGTTTACCGTGAA-3′; JDP2, 5′-TCTCACAAGCTGCTAGGGAG TCA-3′)  and the environmental chlamydia 16S rRNA gene (Ch5, 5′-CGTGGATGAGGCATGCRAGTCG-3′; Ch6, 5′-GTCATCRGCCYYACCTTVSR CRYYTCT-3′) . The amplified products were separated by agarose gel electrophoresis and extracted from the gel using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) according to the manufacturer’s protocol, and then sequenced by Macrogen (Seoul, South Korea). A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA software (version 4) . Accession numbers of nucleotide sequences used for the phylogenetic analysis were listed into a table (See Additional file 1).
Infection and bacterial detection
Bacteria (Prochlamydia W-9 or Parachlamydia Bn9) were added to each well of a 24-well plate seeded with C3 amoebae in PYG broth at a multiplicity of infection (MOI) of 1 and then incubated for 1 h. After washing, the cultures were further incubated for up to 5 days at 30 °C in a normal atmosphere. During this period, the amoebal cells were regularly collected to assess bacterial numbers using quantitative real-time PCR (qPCR), the AIU assay and DAPI staining, according to methods described previously [10, 15]. Meanwhile, the immortal human cell line, HEp-2, was also infected with the bacteria at a MOI of 1–5. The infected HEp-2 cells were incubated in DMEM containing 10% FCS with antibiotics for up to 5 days at 37 °C in a 5% CO2 atmosphere. Cells and supernatant were regularly collected for the determination of bacterial numbers and IL-8 secretion, respectively.
The amount of IL-8, which is an inflammatory cytokine, in HEp-2 cell culture supernatant was quantified using a commercial kit, Human IL-8 ELISA MAX™ Deluxe (BioLegend, Tokyo, Japan), according to the manufacturer’s protocol. The level of IL-8 gene expression was also determined by qRT-PCR using primer sets specific to IL-8 and an internal control (gapdh: glyceraldehyde-3-phosphate dehydrogenase) as described previously .
Draft genome analysis and contig sequence accession numbers
Protochlamydia W-9 genomic DNA was prepared as described previously . In brief, bacteria were collected from amoebae after disruption by bead-beating and were treated with DNase (Sigma) for 30 min at room temperature. After washing, the treated bacteria were suspended in 10 mM HEPES buffer containing 145 mM NaCl, and then the suspension was carefully overlayed onto 30% Percoll. The bacteria were collected from the lower layer following centrifugation at 30,000 × g for 30 min. Bacterial genomic DNA was extracted from the bacterial pellets with the High Pure PCR Template Preparation Kit as described above. The Protochlamydia W-9 draft genome was obtained using an Illumina Miseq sequencer (Illumina, San Diego, CA, USA), with sequencing runs for paired-end sequences. The bacterial DNA libraries were prepared using an NEBNext DNA Library Prep master mix set for Illumina (New England Biolabs, Ipswich, MA, USA). The genome was assembled using de novo sequence assembler software (Platanus 1.2.1) . Rapid Annotation using Subsystem Technology (RAST: http://rast.nmpdr.org/) was used for gene annotation . Also, functional annotation was performed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) . The draft genome sequence of Protochlamydia W-9 has been deposited in the DDBJ database under accession numbers BCPZ01000001-BCPZ01000402 (402 entries) and Bioproject number: PRJDB4526.
Data were compared using Student’s t-test. Also, prevalence between trials was compared using Pearson’s chi-square test. A p-value of less than 0.05 was considered significant.