Novel Parachlamydia acanthamoebae quantification method based on coculture with amoebae

Abstract

Parachlamydia acanthamoebae, belonging to the order Chlamydiales, is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen. However, no method exists to accurately quantify viable bacterial numbers. We present a novel quantification method for P. acanthamoebae based on coculture with amoebae. P. acanthamoebae was cultured either with Acanthamoeba spp. or with mammalian epithelial HEp-2 or Vero cells. The infection rate of P. acanthamoebae (amoeba-infectious dose [AID]) was determined by DAPI (4',6-diamidino-2-phenylindole) staining and was confirmed by fluorescent in situ hybridization. AIDs were plotted as logistic sigmoid dilution curves, and P. acanthamoebae numbers, defined as amoeba-infectious units (AIU), were calculated. During culture, amoeba numbers and viabilities did not change, and amoebae did not change from trophozoites to cysts. Eight amoeba strains showed similar levels of P. acanthamoebae growth, and bacterial numbers reached ca. 1,000-fold (10(9) AIU preculture) after 4 days. In contrast, no increase was observed for P. acanthamoebae in either mammalian cell line. However, aberrant structures in epithelial cells, implying possible persistent infection, were seen by transmission electron microscopy. Thus, our method could monitor numbers of P. acanthamoebae bacteria in host cells and may be useful for understanding chlamydiae present in the natural environment as human pathogens.

FIG. 1.

Brief summary of the experimental method.

FIG. 2.

Representative images of DAPI staining (A and B) and of FISH detection (C to E) of P. acanthamoebae-infected amoebae. (A and B) Amoebae infected with (A) or without (B) P. acanthamoebae were stained with DAPI at 2 days after infection. Magnification, ×400. (C to E) Hybridizations with Alexa Fluor 488-labeled EUK516, which targets eukaryotic 18S rRNA (C) (green), and Alexa Fluor 350-labeled Bn₉658, which is specific for P. acanthamoebae 16S rRNA (D) (purple). Merged images are shown in panel E. Magnification, ×1,000.

FIG. 3.

Representative variation of DAPI-stained amoebae with or without P. acanthamoebae infection. Magnification, ×100.

FIG. 4.

Sigmoid curves drawn by a fitting process for determining AID₅₀ (A) and the variation of AID₅₀ derived from different plot series by plugging into the four-parametric logistic function (B). (A) The AIDs compliant with a diluted series of a sample were separately determined by using each plate containing amoebae adjusted to either 10⁴ (closed circles) or 10⁵ (open circles) per well, and two sigmoid curves were drawn by a fitting process with a four-parametric logistic function. Dilution rates equivalent to AID₅₀, indicated by using the 10⁴-per-well plate and the 10⁵-per-well plate, were 2.616 ± 0.058 and 1.595 ± 0.043, respectively. There was rigorous declination with a log between the two sigmoid curves drawn through the fitting process. (B) The data represent the averages of AID₅₀ ± standard deviations. *, P < 0.05 (variance) versus dilution series of 10⁰ to 10⁻⁷.

FIG. 5.

Numbers of viable P. acanthamoebae bacteria in cultures of amoebae (A) and mammalian cells (B) for up to 10 days after infection. The number of bacteria was assessed by the AIU assay. The data represent the average AIU ± standard deviations. *, P < 0.05 versus values for bacteria alone.

FIG. 6.

TEM images showing the ultrastructure of P. acanthamoebae-infected amoebae (A and B) and mammalian cells (C and D). (A) P. acanthamoebae-infected amoebae at 1 day after infection. The square shows an inclusion containing dividing RBs. Arrows indicate crescent bodies, which are a specific stage in the developmental cycle of P. acanthamoebae. (B) Amoebae infected with bacteria at 4 days after infection. The square shows an inclusion containing mature EBs. (C) HEp-2 cells infected with bacteria at 1 day after infection. Arrows show RB-like structures, which may represent aberrant or persistent forms. (D) Vero cells infected with bacteria at 1 day after infection. Bars, 500 nm.