Rebecca J. Gast

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene (Rns) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1‐T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5‐T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.

Protistan Diversity Estimates Based on 18S rDNA from Seawater Incubations in the Western North Atlantic1

Abstract. Cloning/sequencing and fragment analysis of ribosomal RNA genes (rDNA) are becoming increasingly common methods for the identification of microbial taxa. Sequences of these genes provide many additional taxonomic characters for species that otherwise have few distinctive morphological features, or that require involved microscopy or laboratory culture and testing. These same approaches are now being applied with great success in ecological studies of natural communities of microorganisms. Extensive information on the composition of natural microbial assemblages is being amassed at a rapid pace through genetic analyses of environmental samples and comparison of the resulting genetic information with well‐established (and rapidly growing) public databases. We examined microbial eukaryote diversity in a natural seawater sample from the coastal western North Atlantic Ocean using two molecular biological approaches: the cloning and sequencing of rRNA genes and by fragment analysis of these genes using the terminal restriction fragment length polymorphism (T‐RFLP) method. A simple experiment was carried out to examine changes in the overall eukaryote (largely protistan) diversity and species composition (phylotype diversity) of a natural microbial assemblage when a seawater sample is placed in a container and incubated at ambient light and temperature for 72 h. Containment of the natural seawater sample resulted in relatively minor changes in the overall eukaryote diversity (species richness) obtained by either molecular method at three time points (time‐zero, time‐24 h, time‐72 h). However, substantial changes in the dominance of particular eukaryote phylotypes took place between the three sampling times. Only 18% of the total number of phylotypes observed in the study were observed at all three time points, while 65% (108 of 165) phylotypes were observed only at a single time point (54 unique phylotypes initially, 37 more unique phylotypes at 24 h, and 17 more at 72 h). The results of this study indicate that a high diversity of protistan taxa existed in the original seawater sample at very low abundance, and thus were not observed in the initial characterization of community structure. Containment resulted in significant shifts in the dominance of these taxa, enabling the presence of previously unobserved phylotypes to be documented after 24 or 72 h of incubation.

Subgenus systematics of Acanthamoeba : Four nuclear 18S rDNA sequence types

ABSTRACT Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes (Rns) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S‐7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes, or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.

Defining DNA-Based Operational Taxonomic Units for Microbial-Eukaryote Ecology

ABSTRACT DNA sequence information has increasingly been used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, studies of the autecology of ecologically relevant species, and identification and enumeration of species of interest for human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is in part a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes and in part a consequence of the problematic nature of and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and there is a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximately species-level distinctions. This approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small-subunit (18S) rRNA gene sequences and partial gene sequences obtained from the GenBank database for morphologically described protistan species. The program was tested using environmental 18S rRNA data sets for two oceanic ecosystems. A total of 388 operational taxonomic units were observed for 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific oceans.

Marine subsurface eukaryotes: the fungal majority

Studies on the microbial communities of deep subsurface sediments have indicated the presence of Bacteria and Archaea throughout the sediment column. Microbial eukaryotes could also be present in deep-sea subsurface sediments; either bacterivorous protists or eukaryotes capable of assimilating buried organic carbon. DNA- and RNA-based clone library analyses are used here to examine the microbial eukaryotic diversity and identify the potentially active members in deep-sea sediment cores of the Peru Margin and the Peru Trench. We compared surface communities with those much deeper in the same cores, and compared cores from different sites. Fungal sequences were most often recovered from both DNA- and RNA-based clone libraries, with variable overall abundances of different sequence types and different dominant clone types in the RNA-based and the DNA-based libraries. Surficial sediment communities were different from each other and from the deep subsurface samples. Some fungal sequences represented potentially novel organisms as well as ones with a cosmopolitan distribution in terrestrial, fresh and salt water environments. Our results indicate that fungi are the most consistently detected eukaryotes in the marine sedimentary subsurface; further, some species may be specifically adapted to the deep subsurface and may play important roles in the utilization and recycling of nutrients.

Development of an Acanthamoeba-specific Reverse Dot-Blot and the Discovery of a New Ribotype

ABSTRACT. Acanthamoeba is a genus of free‐living amoebae, of which some speeies have been found to cause opportunistic infections in humans. The identification of these amoebae in natural and disease samples is based primarily upon morphological features. While these features are more than adequate for identification to the genus level, they are not useful for species‐level identification. This not only leads to difficulty in the diagnosis of infections, but it makes an accurate assessment of the natural distribution of acanthamoebae very difficult to achieve. To improve this situation, a detection method was developed that utilizes both selective polymerase chain reaction amplification and the reverse dot‐blot. Oligonucleotides were designed to be specific for the described ribosomal groups (or ribotypes) of Acanthamoeba, as well as one specific for the genus itself. When this method was used to analyze a series of Acanthamoeba cultures from Pakistan, a new ribotype was identified in addition to the detection of the ubiquitously distributed T4 type.Abstract Acanthamoeba is a genus of free-living amoebae, of which some species have been found to cause opportunistic infections in humans. The identification of these amoebae in natural and disease samples is based primarily upon morphological features. While these features are more than adequate for identification to the genus level, they are not useful for species-level identification. This not only leads to difficulty in the diagnosis of infections, but it makes an accurate assessment of the natural distribution of acanthamoebae very difficult to achieve. To improve this situation, a detection method was developed that utilizes both selective polymerase chain reaction amplification and the reverse dot-blot. Oligonucleotides were designed to be specific for the described ribosomal groups (or ribotypes) of Acanthamoeba, as well as one specific for the genus itself. When this method was used to analyze a series of Acanthamoeba cultures from Pakistan, a new ribotype was identified in addition to the detection of the ubiquitously distributed T4 type.

Bacteria in beach sands: an emerging challenge in protecting coastal water quality and bather health.

To protect bather health at recreational beaches, fecal indicator bacterial standards are used to monitor water quality, and waters exceeding the standards are subsequently closed to bathers. However beachgoers are also in contact with beach sands, the sanitary quality of which is not included within beach monitoring programs. In fact, sands and sediments provide habitat where fecal bacterial populations may persist, and in some cases grow, in the coastal zone. Specific pathogens are less well studied in beach sands and sediments, but there is a body of evidence that they too may persist in these environments. This paper reviews the current state of knowledge regarding the abundance and distribution of fecal indicator bacteria and pathogens in beach sands of diverse climatological regions, and at beaches subjected to varied levels of anthropogenic impact. In all regions fecal indicator bacteria are nearly ubiquitous in beach sands, and similar relationships emerge among fecal indicator abundance in dry sand, submerged sands, and water. Taken together, these studies contextualize a potential public health issue and identify research questions that must be addressed in order to support future policy decisions.

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis

ABSTRACT The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.

Impacts of Hurricanes Katrina and Rita on the microbial landscape of the New Orleans area

Floodwaters in New Orleans from Hurricanes Katrina and Rita were observed to contain high levels of fecal indicator bacteria and microbial pathogens, generating concern about long-term impacts of these floodwaters on the sediment and water quality of the New Orleans area and Lake Pontchartrain. We show here that fecal indicator microbe concentrations in offshore waters from Lake Pontchartrain returned to prehurricane concentrations within 2 months of the flooding induced by these hurricanes. Vibrio and Legionella species within the lake were more abundant in samples collected shortly after the floodwaters had receded compared with samples taken within the subsequent 3 months; no evidence of a long-term hurricane-induced algal bloom was observed. Giardia and Cryptosporidium were detected in canal waters. Elevated levels of fecal indicator bacteria observed in sediment could not be solely attributed to impacts from floodwaters, as both flooded and nonflooded areas exhibited elevated levels of fecal indicator bacteria. Evidence from measurements of Bifidobacterium and bacterial diversity analysis suggest that the fecal indicator bacteria observed in the sediment were from human fecal sources. Epidemiologic studies are highly recommended to evaluate the human health effects of the sediments deposited by the floodwaters.

Victims or vectors: a survey of marine vertebrate zoonoses from coastal waters of the Northwest Atlantic

Surveillance of zoonotic pathogens in marine birds and mammals in the Northwest Atlantic revealed a diversity of zoonotic agents. We found amplicons to sequences from Brucella spp., Leptospira spp., Giardia spp. and Cryptosporidium spp. in both marine mammals and birds. Avian influenza was detected in a harp seal and a herring gull. Routine aerobic and anaerobic culture showed a broad range of bacteria resistant to multiple antibiotics. Of 1460 isolates, 797 were tested for resistance, and 468 were resistant to one or more anti-microbials. 73% (341/468) were resistant to 1-4 drugs and 27% (128/468) resistant to 5-13 drugs. The high prevalence of resistance suggests that many of these isolates could have been acquired from medical and agricultural sources and inter-microbial gene transfer. Combining birds and mammals, 45% (63/141) of stranded and 8% (2/26) of by-caught animals in this study exhibited histopathological and/or gross pathological findings associated with the presence of these pathogens. Our findings indicate that marine mammals and birds in the Northwest Atlantic are reservoirs for potentially zoonotic pathogens, which they may transmit to beachgoers, fishermen and wildlife health personnel. Conversely, zoonotic pathogens found in marine vertebrates may have been acquired via contamination of coastal waters by sewage, run-off and agricultural and medical waste. In either case these animals are not limited by political boundaries and are therefore important indicators of regional and global ocean health.