Characterization of novel bacterial pathogens of free-living amoebae

Free-living amoebae, apart from causing diseases in humans, are also implicated in harboring bacteria that cause human diseases. Additionally, amoebae are suspected to adapt their intracellular bacteria for life inside a human host, and therefore could play a role in the emergence of potential human pathogens. In this study, an attempt was made to identify novel bacterial pathogens of free-living amoebae from artificial and natural aquatic environments: cooling towers, a hot tub, and a wetland.; Bacteria co-cultured with Acanthamoeba polyphaga were phylogenetically characterized following amplification and partial sequencing of the 16S rRNA gene. Prior to sequencing, the 16S rRNA gene fragments were separated by two methods---Denaturing Gradient Gel Electrophoresis (DGGE) and cloning. DGGE separation of PCR amplified 16S rDNA fragments yielded inconclusive banding patterns and thus cloning was used to separate the PCR products. Oligonucleotide probes labeled with Cy3 and targeting signature regions within the retrieved sequences were designed to confirm the intra-amoebal location of the bacteria in situ. Phylogenetic analysis of 24 unique bacterial sequences revealed 19 novel species. Fluorescent in situ hybridization of oligonucleotide probe to the rRNAs of three bacterial species, the sequences of which were very dissimilar to those of known bacteria, demonstrated that one of the bacteria is an amoebal pathogen (from a hot tub) and the other two (from cooling towers) are non-pathogenic to amoebae.; Co-cultures of amoebae infected with bacteria from the environmental samples were treated with hydrochloric acid, ciliates, and hydrogen peroxide in an attempt to eliminate extracellular bacteria and thus improve the efficiency of isolating the pathogen sequences using cloning. Of the three procedures, only treatment with 0.9% hydrogen peroxide for 0.5 h resulted in successful reduction of the extracellular bacterial population. Attempts to grow and isolate the pathogens on Buffered Charcoal Yeast Extract agar resulted in successful isolation of one amoebal pathogen in pure culture.; DGGE, as demonstrated in this study, is not a reliable procedure for separating 16S rRNA sequences and cleaning environmental samples prior to cloning might improve the chances of isolating sequences from amoebal pathogens. Moreover, sequence analysis of bacterial population from environmental samples demonstrated the diversity of bacteria and revealed the presence of hitherto unidentified novel bacterial species.