Long-term exposure to antibiotics has caused accumulation of resistance determinants in the gut microbiota of honeybees

Abstract

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. 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, long-term 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.

FIG 1

Species assignments of fosmid inserts used in metagenomic functional screens based on end sequencing for a random set of inserts (A), inserts exhibiting tetracycline (Tet) resistance (B), and inserts exhibiting ampicillin (Amp)/tetracycline resistance (C). The numbers of inserts are in parentheses. Taxonomic categories refer to bee gut-associated taxa (7, 8, 10, 13, 15), except that related pairs of taxa are pooled as follows: “Alpha1” plus “Alpha2” (Alpha 1 + 2), Gilliamella apicola plus “Gamma2” (Gamma 1 + 2), and “Firm4” plus “Firm5” (Firm 4 + 5).

FIG 2

Presence of tetracycline resistance genes in gut microbiota of honeybees and bumblebees. (A) Occurrence of eight loci in individual bees from different sources (13 other loci were screened but not detected). Filled and empty boxes indicate positive and negative results, respectively, for the tetracycline resistance genes in the assays; the absence of a box indicates that the gene was not assayed. (B) Numbers of copies of tetracycline resistance loci relative to 16S rRNA copies in the microbiota of honeybees sampled from several locations. Numbers are based on absolute quantification results using quantitative PCR with diagnostic primers for each gene. Seven genes (excluding tetL) were screened.

FIG 3

Genetic organizations of fosmid inserts and metagenomic scaffolds containing tetracycline resistance genes within the honeybee gut microbiota compared to chromosomal regions containing homologous genes from other bacteria. Gray shading indicates regions sharing >99% nucleotide sequence identity. Comparisons for regions containing tetC (A and B), tetB (C), and tetL (D) are shown. See supplemental Text S1 for a detailed description of individual resistance loci in the bee gut microbiota.