Antibiotic-resistant pathogens are profoundly important to human health, but the environmental reservoirs of resistance determinants are poorly understood. The origins of antibiotic resistance in the environment is relevant to human health because of the increasing importance of zoonotic diseases as well as the need for predicting emerging resistant pathogens. This Review explores the presence and spread of antibiotic resistance in non-agricultural, non-clinical environments and demonstrates the need for more intensive investigation on this subject.

To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibioticresistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a ␤-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional ␤-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a ␤-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.

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Replication origins in a genome are inherently different in their base sequence and in their response to temporal and cell cycle regulation signals for DNA replication. To investigate the chromosomal determinants that influence the efficiency of initiation of DNA replication genome-wide, we made use of a reverse strategy originally used for the isolation of replication initiation mutants in Saccharomyces cerevisiae. In yeast, replication origins isolated from chromosomes support the autonomous replication of plasmids. These replication origins, whether in the context of a chromosome or a plasmid, will initiate efficiently in wild-type cells but show a dramatically contrasted efficiency of activation in mutants defective in the early steps of replication initiation. Serial passages of a genomic library of autonomously replicating sequences (ARSs) in such a mutant allowed us to select for constitutively active ARSs. We found a hierarchy of preferential initiation of ARSs that correlates with local transcription patterns. This preferential usage is enhanced in mutants defective in the assembly of the prereplication complex (pre-RC) but not in mutants defective in the activation of the pre-RC. Our findings are consistent with an interference of local transcription with the assembly of the pre-RC at a majority of replication origins.

Streptomycin is commonly used to control fire blight disease on apple trees. Although the practice has incited controversy, little is known about its nontarget effects in the environment. We investigated the impact of aerial application of streptomycin on nontarget bacterial communities in soil beneath streptomycin-treated and untreated trees in a commercial apple orchard. Soil samples were collected in two consecutive years at 4 or 10 days before spraying streptomycin and 8 or 9 days after the final spray. Three sources of microbial DNA were profiled using tag-pyrosequencing of 16S rRNA genes: uncultured bacteria from the soil (culture independent) and bacteria cultured on unamended or streptomycin-amended (15 g/ml) media. Multivariate tests for differences in community structure, Shannon diversity, and Pielou's evenness test results showed no evidence of community response to streptomycin. The results indicate that use of streptomycin for disease management has minimal, if any, immediate effect on apple orchard soil bacterial communities. This study contributes to the profile of an agroecosystem in which antibiotic use for disease prevention appears to have minimal consequences for nontarget bacteria.