title: Detection of bacterial endosymbionts in clinical acanthamoeba isolates.
abstract: OBJECTIVE
  To determine the presence of 4 clinically relevant bacterial endosymbionts in Acanthamoeba isolates obtained from patients with Acanthamoeba keratitis (AK) and the possible contribution of endosymbionts to the pathogenesis of AK.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  Acanthamoeba isolates (N = 37) recovered from the cornea and contact lens paraphernalia of 23 patients with culture-proven AK and 1 environmental isolate.
  
  
  METHODS
  Acanthamoeba isolates were evaluated for the presence of microbial endosymbionts belonging to the bacterial genera Legionella, Pseudomonas, Mycobacterium, and Chlamydia using molecular techniques (polymerase chain reaction and sequence analysis, fluorescence in situ hybridization) and transmission electron microscopy. Corneal toxicity and virulence of Acanthamoeba isolates with and without endosymbionts were compared using a cytopathic effect (CPE) assay on human corneal epithelial cells in vitro. Initial visual acuity, location and characteristics of the infiltrate, time to detection of the infection, and symptom duration at presentation were evaluated in all patients.
  
  
  METHODS
  Prevalence and potential pathobiology of bacterial endosymbionts detected in Acanthamoeba isolates recovered from AK.
  
  
  RESULTS
  Twenty-two (59.4%) of the 38 cultures examined contained at least 1 bacterial endosymbiont. One isolate contained 2 endosymbionts, Legionella and Chlamydia, confirmed by fluorescence in situ hybridization. Corneal toxicity (CPE) was significantly higher for Acanthamoeba-hosting endosymbionts compared with isolates without endosymbionts (P<0.05). Corneal pathogenic endosymbionts such as Pseudomonas and Mycobacterium enhanced Acanthamoeba CPE significantly more than Legionella (P<0.05). In the presence of bacterial endosymbionts, there was a trend toward worse initial visual acuity (P>0.05), central location (P<0.05), absence of radial perineuritis (P<0.05), delayed time to detection (P>0.05), and longer symptom duration at presentation (P>0.05).
  
  
  CONCLUSIONS
  Most Acanthamoeba isolates responsible for AK harbor 1 or more bacterial endosymbionts. The presence of endosymbionts enhances the corneal pathogenicity of Acanthamoeba isolates and may impact detection time and clinical features of AK.
authors.affiliation: Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami-Miller School of Medicine, Miami, Florida 33136, USA.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Iovieno
authors.forename: Dolena R
authors.initials: DR
authors.lastname: Ledee
authors.forename: Darlene
authors.initials: D
authors.lastname: Miller
authors.forename: Eduardo C
authors.initials: EC
authors.lastname: Alfonso
mesh_terms: D000048:Acanthamoeba / Q000302:isolation & purification / Q000382:microbiology*; D015823:Acanthamoeba Keratitis / Q000382:microbiology / Q000469:parasitology*; D000818:Animals; D002689:Chlamydia / Q000235:genetics / Q000302:isolation & purification*; D003261:Contact Lenses / Q000382:microbiology / Q000469:parasitology*; D003315:Cornea / Q000382:microbiology / Q000469:parasitology; D004269:DNA, Bacterial / Q000032:analysis; D005838:Genotype; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007875:Legionella / Q000235:genetics / Q000302:isolation & purification*; D046529:Microscopy, Electron, Transmission; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics / Q000302:isolation & purification*; D013559:Symbiosis
chemical_list: D004269:DNA, Bacterial