RRC ID 10833
Author Matsuo J, Hayashi Y, Nakamura S, Sato M, Mizutani Y, Asaka M, Yamaguchi H.
Title Novel Parachlamydia acanthamoebae quantification method based on coculture with amoebae.
Journal Appl Environ Microbiol
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.
Volume 74(20)
Pages 6397-404
Published 2008-10-1
DOI 10.1128/AEM.00841-08
PII AEM.00841-08
PMID 18757579
PMC PMC2570282
MeSH Amoeba / microbiology* Animals Cell Line Chlamydiales / isolation & purification* Chlorocebus aethiops Coculture Techniques Colony Count, Microbial / methods Humans In Situ Hybridization, Fluorescence Indoles / metabolism Staining and Labeling
IF 4.016
Times Cited 23
Human and Animal Cells HEp-2(RCB1889)