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.

Helicobacter pylori is associated with gastric disorders in humans and some experimental animals, and possesses the luxS/type 2 autoinducer (AI-2) system. The effects of a specific luxS mutation on the characteristics of H. pylori were examined. On 0?3 % agar medium, motility of H. pylori HPKY08 (luxS : : cat) was significantly lower than that of wild-type H. pylori TK1402. The luxS-complemented strain HPKY21 exhibited motility comparable to that of H. pylori TK1402. It was shown that the luxS/AI-2 system plays an important role in H. pylori motility. The luxS mutant exhibited a reduced infection rate relative to the wild-type parent strain TK1402 in a Mongolian gerbil model. At 3 months after oral inoculation, lower numbers of H. pylori were detected by semi-quantitative real-time reverse transcription PCR (qRT-PCR) in luxS " mutant-infected gerbils than in TK1402-infected gerbils. Gastric inflammation and increased antibody titre for H. pylori were observed in TK1402-infected gerbils only. INTRODUCTIONQuorum sensing (QS) is a cell-to-cell communication system that regulates bacterial phenotypes, including the expression of virulence factor genes. The signalling molecules are known as autoinducers (AIs), and when these molecules reach a critical threshold concentration within a bacterial population, a signal transduction cascade is triggered, and this forms the basis for alterations in gene expression (Fuqua et al., 1994). Many Gram-negative bacteria utilize N-acylhomoserine lactone molecules, AI-1, as signals, while Gram-positive bacteria actively export peptides as signalling molecules. There is a second signalling system involved in a wide range of bacterial species (Schauder et al., 2001), and this system is utilized by both Gram-positive and Gram-negative bacteria. The signalling molecule known as type 2 autoinducer (AI-2) is a furanosyl borate diester (Chen et al., 2002), and the enzyme responsible for its synthesis is encoded by the luxS gene (Surette & Bassler, 1999). The genomes of many bacterial species, notably Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, Proteus mirabilis, Vibrio cholerae, Vibrio vulnificus, Campylobacter jejuni, Porphyromonas gingivalis, Bacillus subtilis, Streptococcus pyogenes, Streptococcus mutans, Clostridium perfringens and Clostridium difficile, include luxS homologues. In several of these, luxS-related AI-2 signals are involved in bacterial characteristics such as biofilm formation (Balestrino et al., 2005;Blehert et al., 2003;Fong et al., 2001;Wen & Burne 2004), flagella and motility (Jeon et al., 2003;Schneider et al., 2002;Stroeher et al., 2003), type III secretion systems (Sperandio et al., 1999), toxin production (Ohtani et al., 2002) and virulence (Lyon et al., 2001; Parsonnet et al., 1991;Stroeher et al., 2003).Helicobacter pylori has been identified as the aetiological agent of chronic active gastritis, peptic ulcer disease (Blaser, 1992;Graham, 1989), gastric adenocarcinoma (Parsonnet et al., 1991) and mucosal-associated lymphoid t...

SummaryObligate intracellular bacteria are commonly found as endosymbionts of acanthamoebae, however, their survival in and ability to transfer to amoebae are currently uncharacterized.In this study, six bacterial endosymbionts, found in five environmental Acanthamoeba isolates (S13, R18, S23, S31, S40) from different locations of Sapporo city, Japan, were characterized. Phylogenetic analysis revealed that three-bacterial endosymbionts (eS31, eS40a, eS23) belonged to α-and β-Proteobacterium phyla and the remaining endosymbionts (eR18, eS13, eS40b) belonged to the Chlamydiales phylum. The Acanthamoeba isolate (S40) contained two phylogenetically different bacterial endosymbionts (eS40a, eS40b). Fluorescent in situ hybridization analysis showed that all bacterial endosymbionts were diffusely localized within amoebae. Transmission electron microscopy also showed that the endosymbionts were rod-shaped (eS31, eS40a, eS23) or sphere-or crescent-shaped (eR18, eS13, eS40b). No successful culture of these bacteria was achieved using conventional culture methods, but the viability of endosymbionts was confirmed by live/dead staining and RT-PCR methods. However, endosymbionts (except eR18) derived from original host cells lost the ability to be transferred to another amoeba strain (Acanthamoebae ATCC C3). Taken together, our data demonstrate that phylogenetically diverse bacterial endosymbionts found in amoebae are viable and maintain a stable interaction with amoebae.

The properties of garlic (Allium sativum L.) are attributed to organosulfur compounds. Although these compounds change during cultivation and storage, there is no report of their simultaneous analysis. Here, a newly developed analytical method with a rapid and simple sample preparation to determine four sulfoxides and three gamma-glutamyl peptides in garlic is reported. All garlic samples were simply extracted with 90% methanol solution containing 0.01 N hydrochloric acid and prepared for analysis. Alliin, isoalliin, methiin, cycloalliin, and gamma-l-glutamyl-S-methyl-l-cysteine were determined by normal-phase HPLC using an aminopropyl-bonded column. gamma-l-Glutamyl-S-(2-propenyl)-l-cysteine and gamma-l-glutamyl-S-(trans-1-propenyl)-l-cysteine were separated on an octadecylsilane column. The overall recoveries were 97.1-102.3%, and the relative standard deviation values of intra- and interday precision were lower than 2.6 and 4.6%, respectively. This newly developed method offers some advantages over the currently accepted techniques including specificity, speed, and ease of use and would be useful for chemical and biological studies of garlic and its preparations.