The in-feed antibiotic carbadox induces phage gene transcription in the swine gut microbiome

Authors: JOHNSON, TIMOTHY - Orise Fellow; Looft, Torey; SEVERIN, ANDREW - Iowa State University; Bayles, Darrell; NASKO, DANIEL - University Of Delaware; WOMMACK, ERIC - University Of Delaware; HOWE, ADINA - Iowa State University; Allen, Heather

Submitted to: mBio
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/28/2017
Publication Date: 8/8/2017
Citation: Johnson, T.A., Looft, T.P., Severin, A.J., Bayles, D.O., Nasko, D.J., Wommack, E., Howe, A., Allen, H.K. 2017. The in-feed antibiotic carbadox induces phage gene transcription in the swine gut microbiome. mBio. 8(4):e00709-17. https://doi.org/10.1128/mBio.00709-17.
DOI: https://doi.org/10.1128/mBio.00709-17

Interpretive Summary: FDA regulations on agricultural antibiotic use have focused on antibiotics that are important for human medicine. Carbadox is an antibiotic not used in humans but frequently used on U.S. pig farms. It is important to study possible side-effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Previous research from our research group has shown that carbadox induces phages in swine gut bacteria and in Salmonella Typhimurium, a foodborne pathogen. Phages are viruses that infect bacteria and are important because they kill bacteria and they transfer genetic material between cells. In the current study, we analyzed whether carbadox caused changes in gene expression of the swine gut bacterial community and in the fecal phage diversity. The results showed that by two days after the initiation of carbadox in the feed, the bacteria in the carbadox-fed pigs were expressing different genes than the bacteria in the non-medicated pigs. These differences showed that the bacteria in the carbadox-fed pigs were not multiplying or metabolizing carbohydrates as they normally would. Interestingly, these differences also showed that phages were being induced in the carbadox-fed pigs two days after carbadox initiation. Importantly, the phage genetic material isolated in this study encoded antibiotic resistance genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This study highlights the collateral effects of antibiotics and demonstrates the need for considering diverse antibiotic effects whenever antibiotics are being used or new regulations are considered. The results of this study are important for scientists, policy-makers, regulators, and farmers who are interested in devising strategies for improving antibiotic stewardship in agriculture.

Technical Abstract: Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40 percent of young pigs in the U.S. and has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo effects of carbadox on swine gut bacterial gene expression via sequencing total fecal bacterial mRNA and phage populations via sequencing total fecal double-stranded DNA phages five days prior to and one, two and seven days following continual in-feed carbadox. Sampled DNA, RNA, and phage DNA sequences were annotated for taxonomic inference and gene function, using FigFam (isofunctional homolog sequences) and SEED subsystems databases. When comparing beta-diversity of bacterial FigFam annotations, the control and carbadox communities were distinct two days post carbadox introduction. This effect was driven by carbadox-associated lower expression of FigFams (n equals 66) related to bacterial respiration, carbohydrate utilization, and RNA metabolism (q value less than 0.1), suggesting bacteriostatic or bactericidal effects within certain populations. Interestingly, carbadox treatment caused greater expression of FigFams related to all stages of the phage lytic cycle two days following the introduction of carbadox (q value less than or equal to 0.07), suggesting the carbadox-mediated induction of prophages and phage DNA recombination. Additionally, the phage DNA metagenome included genes that encoded resistance to tetracycline, aminoglycoside, and beta-lactam antibiotics. The results show decreased bacterial growth and metabolism, prophage induction, and potential transduction of bacterial fitness genes, such as antibiotic resistance genes, in swine gut bacterial communities as a result of carbadox administration.