title: Investigation of the Role of Bacteria in the Development of Acanthamoeba Keratitis.
abstract: OBJECTIVE
  Recently, much interest has been shown in bacteria extracted from Acanthamoeba strains isolated from patients with Acanthamoeba keratitis (AK). We hypothesized that the bacteria in Acanthamoeba strains may be a contributing factor in the development of AK. To prove this hypothesis, we investigated the involvement of bacteria harbored by Acanthamoeba in causing progressive ocular infection in rabbit corneas.
  
  
  METHODS
  One Acanthamoeba strain (T4 genotype) that harbored bacteria was isolated from a patient with AK. The Acanthamoeba strain pretreated or not pretreated with levofloxacin (LVFX) was inoculated into rabbit corneas. We also tested the effect of LVFX eye drops on keratitis induced by the Acanthamoeba strain. The infected rabbit eyes were evaluated for clinical scores, Acanthamoeba 18S rDNA and bacterial 16S rDNA numbers were analyzed by the real-time polymerase chain reaction, and the presence of Acanthamoeba was analyzed by histological examination.
  
  
  RESULTS
  Inoculation of nonpretreated Acanthamoeba resulted in severe keratitis. In contrast, inoculation of LVFX-pretreated Acanthamoeba did not induce keratitis (mean clinical score, 17.3 vs. 2.3; P < 0.05). Rabbit corneas inoculated with nonpretreated Acanthamoeba followed by topical LVFX therapy developed severe keratitis. In corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy, the number of Acanthamoeba 18S rDNA copies was significantly higher than in other groups (P < 0.05), whereas the bacterial 16S rDNA gene was undetectable. Acanthamoeba cysts were detected by Fungiflora Y staining only in corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy.
  
  
  CONCLUSIONS
  These results suggest that the presence of bacteria in Acanthamoeba may be required for the development of AK.
authors.affiliation: Departments of *Ophthalmology, †Microbiology, and ‡Molecular Pathology, Tokyo Medical University, Shinjuku-ku, Japan.
authors.forename: Hayate
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Takaaki
authors.initials: T
authors.lastname: Hattori
authors.forename: Naohito
authors.initials: N
authors.lastname: Koike
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ehara
authors.forename: Koji
authors.initials: K
authors.lastname: Fujita
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takahashi
authors.forename: Shigeto
authors.initials: S
authors.lastname: Kumakura
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kuroda
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Goto
mesh_terms: D000048:Acanthamoeba / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000382:microbiology*; D015823:Acanthamoeba Keratitis / Q000469:parasitology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D018407:Bacterial Physiological Phenomena; D004269:DNA, Bacterial / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015822:Eye Infections, Parasitic / Q000469:parasitology*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D064704:Levofloxacin / Q000627:therapeutic use; D008854:Microscopy, Electron; D009930:Organic Chemicals / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D016054:DNA, Protozoan; D005456:Fluorescent Dyes; C102655:Fungiflora Y; D009930:Organic Chemicals; D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S; D064704:Levofloxacin