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Applied and Environmental Microbiology, October 2008, p. 6397-6404, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.00841-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Parachlamydia acanthamoebae Quantification Method Based on Coculture with Amoebae{triangledown}

Junji Matsuo,1 Yasuhiro Hayashi,1 Shinji Nakamura,2 Marie Sato,1 Yoshihiko Mizutani,3 Masahiro Asaka,4 and Hiroyuki Yamaguchi1*

Department of Medical Laboratory Sciences, Faculty of Health Sciences,1 Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan,4 Division of Biomedical Imaging Research,2 Department of Human Pathology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan3

Received 8 April 2008/ Accepted 18 August 2008

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 (109 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.

* Corresponding author. Mailing address: Department of Medical Laboratory Sciences, Faculty of Health Sciences, Hokkaido University, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido 060-0812, Japan. Phone and fax: 81-11-706-3326. E-mail: hiroyuki{at}med.hokudai.ac.jp

{triangledown} Published ahead of print on 29 August 2008.

Applied and Environmental Microbiology, October 2008, p. 6397-6404, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.00841-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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