Using both sequence-and function-based metagenomic approaches, multiple antibiotic resistance determinants were identified within metagenomic libraries constructed from DNA extracted from bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage. Metagenomic clones and a plasmid that in Escherichia coli expressed resistance to chloramphenicol, ampicillin, or kanamycin were isolated, with many cloned DNA sequences lacking any significant homology to known antibiotic resistance determinants.

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.Bacteriophages play an active role in the ecology of natural environments, influencing prokaryotic population dynamics (5, 15) and mediating lateral gene transfer between diverse bacterial species, for example. Activated sludge (AS) microbial assemblages in wastewater treatment plants have been shown to harbor great numbers of viruses with a wide range of genome sizes (7,9,10, 16). Historically, the focus of wastewater viral studies has been on specific host-virus interactions, the application of phages as tools in microbial source tracking, or the use of phages to improve the efficiency of the wastewater treatment process (e.g., foam and pathogen control) (2,4,8,12,17). Despite the interest in the wastewater viral community, a census of the activated sludge total viral community has not, to our knowledge, been investigated using both culture-based and metagenomic approaches.Taxonomic assignment of viral metagenomic clone sequences. Samples were collected from the AS aeration basin at the H. C. Morgan Water Pollution Control Facility in Auburn, AL, treated with 10% beef extract buffer (to separate viruses from aggregates), precipitated with polyethylene glycol, and subjected to cesium chloride gradient centrifugation. Viral DNA was then extracted by benzonase, proteinase K, and sodium dodecyl sulfate treatments and purified by phenolchloroform extraction and isopropanol precipitation. An AS viral metagenomic library was constructed at the Lucigen Corporation (Middleton, WI) using a linker-amplified shotgun library approach (3, 18), and 1,161 cloned insert DNA sequences were determined using a single-vector primer. Trimmed viral insert sequences were classified as known (E value Ͻ 0.001; n ϭ 694), unknown (n ϭ 97), or novel (n ϭ 370) as described previously (18). The known sequences were assigned to their taxonomic affiliations by BLASTx comparisons to the GenBank nonredundant nucleotide (nr/nt) database and selection of the top BLASTx hits for phylogenetic affiliation (Fig. 1).The dominance of viral metagenomic sequences with significant homology to bacterial DNA (nearly 60% of the known sequences) is likely the consequence of the high frequency of prophage sequences within bacterial genomes deposited in GenBank databases (1). Considerable care was taken to prevent inclusion of prokaryotic (or eukaryotic) chromosomal DNA within the viral metagenomic library (i.e., CsCl purification and nuclease treatment were performed), and the high percentage of viral metagenomic sequences with homology to bacterial genomes has been observed in other environments (3). Of the 191 known sequences with homology to viral genomes...