Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins

Abstract

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.

Functional metagenomic characterization of antibiotic resistance genes in agricultural soils from China

“…Using this method, a total of 8 new ARGs were identified from a Spanish agricultural field, conferring resistance to ampicillin, gentamicin, chloramphenicol and trimethoprim (Torres-Cortes et al, 2011). From an apple orchard soil, 13 ARGs were identified including two putative novel bi-functional protein encoding genes (Donato et al, 2010).…”

Wide Variation in Antibiotic Resistance Proteins Identified by Functional Metagenomic Screening of a Soil DNA Library

“…None of the genes we identified exactly matches any in GenBank, and in many cases these genes are highly divergent from previously characterized family members. Other examples of antibiotic resistance protein variants have been identified by functional screening, including variants of ␤-lactamases, aminoglycoside acetyltrans- ferases, chloramphenicol acetyltransferases, and efflux pumps (1,7,16,27,30,33).…”

“…For example, a survey of antibiotic resistance in the human gut microbiome revealed many previously unsequenced resistance genes that are homologous to known genes (30). Functional screening of soil samples revealed many newly sequenced antibiotic resistance genes, even from pristine environments (1,7,16,27,33).…”

Therapeutic transplantation of the distal gut microbiota

“…Although some of the once powerful antibiotics listed in their paper have since been discontinued and are no longer useful, others, such as streptomycin and oxytetracycline, have changed their status from life-saving medicines to biocides for use on fruit trees. 2 The patients described by Eiseman et al were 45 -68 years of age. Th eir symptoms developed shortly aft er exposure to multiple antibiotics.…”