The Importance of Strain Differences in the Opportunistic Pathogen Acanthamoeba

The ubiquitous, free-living naked amoeba, Acanthamoeba, is an opportunistic pathogen that can cause the painful eye disease Acanthamoeba keratitis (AK), and, in immunocompromised individuals, the fatal disease granulomatous amebic encephalitis (GAE). Acanthamoeba keratitis is a rare corneal disease that effects contact lens wearers more than non-contact lens wearers, though up to 10-15% of cases do occur in non-contact lens wearers (Radford et al., 2002; Walochnik et al., 2000). The aims of this work were fourfold: 1) to determine if there were differences in the tolerance of several strains of acanthamoebae to three commonly used multipurpose lens cleaning solutions, 2) to determine whether acanthamoebae were present in domestic water in south Florida; tapwater has been implicated as a route of infection, 3) to determine if a previously designed set of Polymerase Chain Reaction (PCR) primers would be useful in distinguishing possible pathogenic acanthamoebae from environmental strains, and 4) to determine if acanthamoebae cysts would be a suitable surrogate for dinoflagellate cysts during the testing of ballast water treatment systems. These supposedly disparate studies are linked. There is great interest in understanding what makes some strains invasive and others benign. Thus a wide range of strains were examined from various environmental sources (including the beach) and from clinical samples. It was reasoned that tolerance differences might be related to pathogenicity. Since the strains were from known sources, the robustness of the molecular screening method could be tested. Finally, the resilience of cysts and the fact that different strains showed different responses to stressors implied that strains of some cysts might be good mimics of dinoflagellate cysts for verifying the effectiveness of ballast water treatment systems. The industry is most concerned about these problem cysts that must be inactivated before water is released. Unfortunately, it is impossible to grow large numbers of dinoflagellate cysts for testing. Fourteen acanthamoebae isolates from both clinical and environmental sources were tested for their susceptibility to the three most effective (against bacteria) contact lens multi-purpose cleaning solutions (MPS). These toxicity tests showed that different strains responded differently to the effects of the cleaning solutions. Surprisingly, the pathogenic strains were not universally the most tolerant. However, in all cases cysts were more resistant than trophic amoebae and no MPS was 100 % effective at killing acanthamoebae. Over a 2 year period, 283 water samples were obtained from south Florida and scored for the presence of naked amoebae including Acanthamoeba. Over 19% of domestic water samples tested positive for amoebae, although only 2.8% were positive for Acanthamoeba. The two sets of primers, reportedly capable of distinguishing pathogenic amoebae, were tested with pathogenic and environmental isolates. One set of primers held promise since it correctly identified a 195 bp band (the marker for pathogenic strains) in 50% of pathogenic strains. Moreover, this band was absent in 78% of the non-pathogenic strains. With Acanthamoeba’s resilience and its ease of culture, it was hypothesized to be an ideal candidate for use as a surrogate test organism for the verification of ballast water treatment systems. Experiments conducted in this thesis compared the sensitivity of acanthamoebae cysts to 6 other heterotrophic protist species. Finally, throughout the entire study, additional physiological clues were sought that might shed light on why some strains are capable of invading the eye. These included genotyping isolates (the T4 genotype is most frequently found in keratitis infections), determining the temperature tolerance of strains, and comparing the capacity of some strains to burrow into agar (possibly seeking out areas of low oxygen tension)