We constructed a large-insert clone library (average insert size of 34 kb with total coverage of >3 Gb) using bacterial cells from the guts of worker bees from a Maryland (MD) colony (see Table S1
in the supplemental material). Analysis of end sequences of randomly selected clones confirmed that the inserts were derived from the genomes of the eight bacterial species known to dominate the honeybee gut microbiota, primarily the Gram-negative members of this community (). Functional screens of the clone library for antibiotic resistance revealed clear instances of resistance only for tetracycline/oxytetracycline and carbenicillin/ampicillin. These instances of resistance occurred at a frequency of ~0.1% for fosmid inserts, corresponding to an average frequency among gut bacteria of ~10% for tetracycline resistance. An approximately 10-fold-lower frequency was observed for carbenicillin/ampicillin resistance. There was complete cross-resistance for tetracycline and oxytetracycline and for carbenicillin and ampicillin. Furthermore, 15 of 16 ampicillin-resistant clones also grew on plates containing tetracycline, indicating coselection of genes underlying tetracycline and ampicillin resistance. No clone grew on plates containing ceftazidime, gentamicin, or rifampin.
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 (more ...)
After duplicate clones were eliminated (determined on the basis of matching end sequences), we retrieved 20 unique inserts with tetracycline resistance and nine more with ampicillin/tetracycline resistance (see Table S2
in the supplemental material). Many (51%) resistant clones were unique, suggesting that additional resistance loci could be retrieved from the library if more clones were sequenced. Screening the resistant inserts by diagnostic PCR assays and sequencing revealed that inserts contained known resistance loci: tetB
(13 clones), tetC
(11 clones), tetD
(1 clone), or tetL
(1 clone) (Table S2). Previous designations for the species of bee gut bacteria are “Alpha1” and “Alpha2” from the Alphaproteobacteria, Snodgrassella alvi from the Betaproteobacteria, Gilliamella apicola and “Gamma2” from the Gammaproteobacteria, “Firm4” and “Firm5” from the Firmicutes, “Bifido” from the Bifidobacteriaceae. (7
). Using these designations, taxonomic assignments based on end sequences indicated that 68% of tetracycline-resistant clones are from S. alvi
) and 24% are from “Alpha1” (Alphaproteobacteria
) (). Our initial retrieval of only tetB
, and tetL
by screening fosmids may reflect incompatibilities of some loci with expression in Escherichia coli
hosts, as well as low representation of some species, particularly the Gram-positive species, in the library.
Ampicillin-resistant clones yielded a product for which the inferred amino acid sequence was 100% identical to that of a known ampicillin resistance gene corresponding to blaTEM-1, an extended-spectrum beta-lactamase of E. coli (GenBank accession no. CAJ13634). Most ampicillin-resistant clones also encoded TetB, except for one that encoded TetD. Of the ampicillin/tetracycline-resistant clones, 13 of 16 were from S. alvi (Betaproteobacteria), and three were from the related Gilliamella apicola or “Gamma2”, based on assignment of end sequences ().
To determine the tetracycline resistance gene content within the honeybee gut microbiota, we screened for 21 known tetracycline resistance genes using diagnostic PCR in a panel of samples from different American localities, including MD, Florida (FL), Arizona (AZ), Washington (WA), Connecticut (CT), and Utah (UT), and representing different colony histories with respect to recent antibiotic treatments. We repeatedly detected the same eight tetracycline resistance genes, including genes encoding tetracycline efflux pumps (tetB
, and tetY
) and ribosomal protection proteins (tetM
) (). Although some individual bees failed to amplify for a particular locus, the eight loci were typically present in bees from every colony, with few exceptions such as the absence of tetY
from all FL bees and of tetD
from all MD bees. Further pooled DNA samples from 150 individual bees from each of eight MD colonies yielded the same five tetracycline efflux pump genes detected in amplifications from samples of individual bees: tetB
, and tetH
from all MD colonies and tetD
from four and five of the eight colonies (see Fig. S1
in the supplemental material). The tetL
gene was present in most MD bees and in two of four AZ colonies but absent from most WA individual bees (). Overall, PCR screening results indicate that these eight tetracycline resistance genes are widespread in American honeybee colonies but that their presence can vary among colonies or locations.
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 (more ...)
Sharply contrasting results were obtained in screens of honeybees from Switzerland (SUI), the Czech Republic (CZ), and New Zealand (NZ), countries where antibiotics have not been permitted in beekeeping (18
). The microbiotas of SUI, CZ, and NZ bees sometimes had tetB
, or tetW
but lacked tetD
, and tetL
(). Results for wild Connecticut bumblebees resembled those for SUI, CZ, and NZ honeybees, with detection of only the tetB
, or tetW
gene, depending on the sample (). Thus, the use of antibiotics in American beekeeping is associated with the widespread occurrence of five additional tetracycline resistance loci in bee gut bacteria.
To determine the prevalence of these or other tetracycline resistance genes, resistance gene sequences were used to query scaffolds of a metagenomic sequence data set derived from the guts of bees from AZ (USDA) (13
). Nine scaffolds were found to contain the same set of eight tetracycline resistance loci (see Table S4
in the supplemental material). These metagenomic data represent a more complete sampling of bee gut communities, since they circumvent potential biases in cloning and resistance gene expression. In addition, other genes encoding potential efflux pump proteins were detected in the AZ metagenomic data set by querying with sequences of known efflux pump proteins. However, none of these additional efflux pump genes clustered closely with known tetracycline resistance proteins (see Fig. S2
in the supplemental material), and none was retrieved from our functional assays.
Estimates of the abundances of the tetracycline resistance loci in gut bacteria revealed extensive variation among colonies and localities in the frequencies of particular loci (). These quantitative results are broadly consistent with the results of diagnostic PCR screens and show that the gut microbiotas of SUI, CZ, and NZ honeybees have very low copy numbers of resistance genes, even for those few loci detected. Among American bee colonies, relative numbers of different resistance loci varied extensively.
The variation in tetracycline resistance determinants observed among American honeybee colonies may reflect different recent histories of oxytetracycline treatment for individual colonies. Most of our samples had unknown histories of antibiotic treatment, largely due to their origin from mixing other colonies or from commercial bee packages. To determine whether resistance loci decline when antibiotic exposure is terminated, we obtained samples from four managed colonies in southern Arizona that were unusual in having not been treated directly or mixed with outside bees for over 25 years and samples from long-established feral colonies in Utah, also expected to have no recent exposure. These samples showed markedly lower copy numbers of resistance loci compared to other American samples (). The FL, MD, and AZ (USDA) colonies, which had no antibiotic treatment for at least 2 years prior to sampling, showed intermediate levels of resistance loci. The highest frequencies were observed for colonies in CT and WA established from package bees purchased from commercial bee suppliers 0 to 12 months before sampling.
To confirm results from diagnostic PCR and to link tetracycline resistance gene types with their source genomes, sequences of full-length open reading frames were recovered from PCR amplification using DNA from worker bee guts from a MD colony, fosmids derived from a MD colony, and cultured bacterial isolates from the guts of CT bees (). Isolate identities, based on 16S rRNA sequences, were confirmed for G. apicola
, S. alvi
, “Alpha1”, “Firm5”, and “Bifido” species using established designations for these bee-associated species (7
). Strains corresponding to several of the characteristic gut species possessed resistance genes (). For known tetracycline resistance loci, nucleotide sequences from different colonies shared 99 to 100% identity with one another and with published sequences from other sources (; see Fig. S2
in the supplemental material), indicating that the various tetracycline resistance loci in the guts of American honeybees have been transferred recently among taxonomically and ecologically distinct bacteria.
Tetracycline resistance loci present in the honeybee gut microbiota
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 (more ...)
We examined cultured isolates of the constituent species of the bee gut microbiota for tetracycline resistance and for the presence of resistance genes. When isolates from CT bee colonies (13
) were plated on medium with 12 µg/ml oxytetracycline, resistant strains were readily recovered despite the absence of selection for resistance in the initial isolation procedure. For G. apicola
, 77% (10/13 isolates) were resistant, and 100% (all 11 isolates) of S. alvi
isolates were resistant. Resistant members of Alpha1 (3/14 isolates), Bifido, and Firm5 species were also recovered (see Table S5
in the supplemental material).
The observed resistance of many isolates was attributable to known tetracycline resistance genes, often associated with large increases in the tetracycline MICs of isolates from the bee gut microbiota (see Table S5
in the supplemental material). For strains carrying tetB
wkB1, PEB0162), tetC
wkB2, wkB4, wkB5, and wkB9), or tetW
(Bifido wkB3), tetracycline MICs were ≥12 µg/ml; in contrast, MICs were <0.5 µg/ml for isolates of these species lacking resistance genes (G. apicola
wkB7, bumblebee G. apicola
wkB11, and bumblebee S. alvi
Information on the chromosomal context of resistance genes, from sequenced fosmids and metagenomic scaffolds containing resistance loci and from PCR screens spanning resistance genes and mobility genes, shows that tetracycline resistance genes are consistently associated with mobile elements, such as transposons and plasmids ( and ; see Tables S3
, S4, and S6 in the supplemental material), implying that most resistance determinants in the bee gut microbiota are newly acquired and not native elements present in the genomes of the ancestral bee gut microbiota. In most cases, sequences of chromosomal fragments spanning resistance loci and associated mobility elements show >99% sequence identity to genes previously characterized from human pathogens or from domesticated animals, such as pigs and chickens ().