Microbiota of Honeybees Accumulation of Resistance Determinants in the Gut Long-Term Exposure to Antibiotics Has Caused 2013

Antibiotic treatment can impact nontarget microbes, enriching the pool of resistance genes available to pathogens and altering community profiles of microbes beneficial to hosts. The gut microbiota of adult honeybees, a distinctive community dominated by eight bacterial species, provides an opportunity to examine evolutionary responses to long-term treatment with a single antibiotic. For decades, American beekeepers have routinely treated colonies with oxytetracycline for control of larval pathogens. Using a functional metagenomic screen of bacteria from Maryland bees, we detected a high incidence of tetracycline/ oxytetracycline resistance. This resistance is attributable to known resistance loci for which nucleotide sequences and flanking mobility genes were nearly identical to those from human pathogens and from bacteria associated with farm animals. Surveys using diagnostic PCR and sequencing revealed that gut bacteria of honeybees from diverse localities in the United States harbor eight tetracycline resistance loci, including efflux pump genes (tetB, tetC, tetD, tetH, tetL, and tetY) and ribosome protection genes (tetM and tetW), often at high frequencies. Isolates of gut bacteria from Connecticut bees display high levels of tetracycline resistance. Resistance genes were ubiquitous in American samples, though rare in colonies unexposed for 25 years. In contrast, only three resistance loci, at low frequencies, occurred in samples from countries not using antibiotics in beekeeping and samples from wild bumblebees. Thus, long-term antibiotic treatment has caused the bee gut microbiota to accumulate resistance genes, drawn from a widespread pool of highly mobile loci characterized from pathogens and agricultural sites. IMPORTANCE We found that 50 years of using antibiotics in beekeeping in the United States has resulted in extensive tetracycline resistance in the gut microbiota. These bacteria, which form a distinctive community present in healthy honeybees worldwide, may function in protecting bees from disease and in providing nutrition. In countries that do not use antibiotics in beekeeping, bee gut bacteria contained far fewer resistance genes. The tetracycline resistance that we observed in American samples reflects the capture of mobile resistance genes closely related to those known from human pathogens and agricultural sites. Thus, longterm treatment to control a specific pathogen resulted in the accumulation of a stockpile of resistance capabilities in the microbiota of a healthy gut. This stockpile can, in turn, provide a source of resistance genes for pathogens themselves. The use of novel antibiotics in beekeeping may disrupt bee health, adding to the threats faced by these pollinators. Received 8 October 2012 Accepted 10 October 2012 Published 30 October 2012 Citation Tian B, Fadhil NH, Powell JE, Kwong WK, and Moran NA. 2012. Long-term exposure to antibiotics has caused accumulation of resistance determinants in the gut microbiota of honeybees. mBio 3(6):e00377-12. doi:10.1128/mBio.00377-12 Editor Roberto Kolter, Harvard Medical School Copyright © 2012 Tian et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Nancy A. Moran, nancy.moran@yale.edu. When antibiotics are used for controlling infections by pathogens, they also impact other microbes, including the beneficial bacteria present in healthy hosts. The selective force imposed by an antibiotic can cause the accumulation of resistance determinants, which are often encoded on mobile genetic elements that are readily transferred among community members. The impact of antibiotics on the gut microbiota of animals is a particular concern, since gut communities may act as reservoirs for resistance genes that can be transferred to pathogens (1–3) and also since perturbation of gut microbiota by antibiotic treatments could disrupt functions beneficial to hosts (4). The honeybee (Apis mellifera), a highly social insect and important agricultural pollinator, is associated with eight characteristic bacterial species that together comprise over 95% of the gut bacteria in adult worker bees (5–12). A single bee or colony can possess multiple strains of each of these species (11, 13), and several of these species (or close relatives) are also found in bumblebees (Bombus species) (6, 9, 14). Some of these species can be grown in axenic culture and have recently been formally named (taxonomic nomenclature) (15). Increasingly, studies are revealing beneficial functions of gut bacteria for animal hosts (e.g., see references 16 and 17). In the case of bees, the distinctive gut bacteria shared by honeybees and bumblebees have been implicated in defense against trypanosome parasites (18) and in digestive roles (13). Since the 1950s, the antibiotic oxytetracycline has been widely applied to colonies of bees in the United States to control larval foulbrood diseases caused by the bacteria Melissococcus pluton and Paenibacillus larvae; oxytetracycline was the only antibiotic approved for use in beekeeping until 2005 (19 –22). Antibiotic treatRESEARCH ARTICLE November/December 2012 Volume 3 Issue 6 e00377-12 ® mbio.asm. org 1 m b.asm . rg on N ovem er 1, 2012 P ubished by m b.asm . rg D ow nladed fom