Antibiotic Resistance in the Swine Intestinal Microbiome.

Antibiotic resistance genes have always existed in natural microbial communities, yet intense selective pressure during the antibiotic era has promoted their mobility and ubiquity.  Previous culture- and PCR-based analyses have provided necessary snapshots of resistance gene diversity in bacterial samples of medical or agricultural importance.  However, culture-independent analyses are needed to inform the ecology of total antibiotic resistance genes in whole bacterial communities (microbiomes), particularly in microbiomes of agricultural importance.  To this end we analyzed feces from swine treated with or without antibiotics (ASP250 [chlortetracycline, penicillin, and sulfamethazine]) via metagenomics, which is the study of the collective genome of an assemblage of organisms.  The results repeatedly showed diverse and abundant antibiotic resistance genes regardless of in-feed antibiotic treatment.  Some resistance genes increase in abundance in metagenomes from medicated swine, particularly genes that encode resistance to the antibiotics administered.  One type of gene, the aminoglycoside O-phosphotransferases (APH), increased in abundance despite conferring resistance to a class of antibiotics not included in the feed.  This was further investigated by using functional metagenomics, which involved cloning large fragments (33 kilobases in a fosmid vector) of the swine fecal metagenome into Escherichia coli and selecting for resistance to several antibiotics, including the aminoglycoside antibiotics kanamycin and gentamycin.  Eleven resistant fosmids were isolated and the complete DNA insert was sequenced.  Four aminoglycoside-resistant fosmids were identified, and three of these encoded an APH.  Each APH appears to be on an antibiotic resistance cassette in a in a different genetic context.  The seven remaining fosmids conferred resistance to the agriculturally relevant antibiotics chlortetracycline and carbadox.  These results describe the complex genetic environments that harbor antibiotic resistance genes in the swine intestinal microbiome and provide clues for how they are mobilized therein.  Defining the ecology of antibiotic resistance genes in host-associated microbiomes is necessary for developing thoughtful antibiotic use and resistance mitigation strategies.