INTRODUCTION

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Concern over the safety implications of antibiotic-resistant bacteria in foods has centered around the question of how likely such bacteria are to transfer resistance genes to human intestinal bacteria during their passage through the intestinal tract and what might happen to the transferred genes once they enter colonic bacteria. This question is part of a larger question about the amount of horizontal gene transfer that actually occurs in nature. Few attempts have been made to determine how much gene transfer occurs among bacteria in the colon or in other environments. Some studies have been done to assess the extent of horizontal gene transfer among microorganisms in soil and water (9, 13, 16), the intestines of laboratory mice (26), and experimental abscesses (4). These studies found evidence that horizontal gene transfer events do occur in these settings at frequencies similar to or higher than those observed in the laboratory.

In an earlier paper, Nikolich et al. examined a small number of tetracycline-resistant Bacteroides and Prevotella species from the human colon and the colons of farm animals (27). The results of that study suggested that horizontal gene transfer had occurred between members of these two genera. Transfer was also demonstrated between the black-pigmented oral Prevotella and Bacteroides species in the laboratory (11). Results of recent studies of vancomycin-resistant enterococci isolated from the intestines of animals and humans also support the hypothesis that horizontal gene transfer events, occur in the intestinal tract (15, 48; L. B. Jensen, A. M. Hammerum, R. L. Poulsen, and H. Westh, Letter, Antimicrob. Agents Chemother. 43:724-725, 1999). In this paper, we report the results of the first systematic investigation of horizontal gene transfer events involving a major population of human colonic bacteria, Bacteroides species. Although this study focuses on transfer of antibiotic resistance genes, the conclusions could presumably be applied to the transfer of other genes that perform accessory functions.

The human colon is an environment that should be very conducive to horizontal gene transfer events. Nutrients are abundant, the concentration of bacteria is high (10¹² per g [wet weight]), and there are many surfaces such as plant particles to which bacteria can adhere. Colonic bacteria have been shown to carry a variety of plasmids and integrated elements that can be transferred by conjugation. Yet, under optimized laboratory conditions, transfer of these elements occurs at a relatively low frequency, 10⁵ to 10⁷ per recipient or lower. This raised the question of how effectively such conjugal elements could spread in the colonic environment. Moreover, if a transfer event took place, how likely would the recipient be to maintain the newly acquired element?

A way to assess the degree of horizontal transfer among different strains of bacteria in a natural setting is to determine whether identical or virtually identical copies of the same gene are found in different species. This approach does not provide transfer rates in vivo, but it can answer questions about the extent to which horizontal transfer has occurred and what types of elements are most often responsible for such transfers. For this study, we chose to focus on Bacteroides species. Bacteroides species comprise a major part of the human colonic microbiota, accounting for about 25% of all colonic isolates (36, 52). Bacteroides species, especially Bacteroides fragilis and Bacteroides thetaiotaomicron, are opportunistic pathogens, which can cause life-threatening infections if they escape from the colon as a result of abdominal trauma or surgery. Over the past several decades, Bacteroides clinical isolates have become increasingly resistant to antibiotics. Resistance to tetracycline has become so common that strains are often not tested for susceptibility to this antibiotic. In recent years, resistance to drugs used to treat Bacteroides infections, such as clindamycin, has also been increasing. Conjugal elements such as plasmids and conjugative transposons (CTn's) have been found in Bacteroides clinical isolates (34). Thus, horizontal gene transfer could conceivably have played a role in the rising incidence of resistance in this bacterial group. In this study, we assess the role of CTn's and plasmids in the transfer of antibiotic resistance genes.

	MATERIALS AND METHODS

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Bacteroides strains. The 88 VPI strains are from the Anaerobe Laboratory at the Virginia Polytechnical Institute in Blacksburg. Some of these strains had been isolated from healthy volunteers (community isolates); others had been isolated from infected patients (clinical isolates). All of them were isolated before 1980, many before 1960 (17). The clinical isolates from 1980 to the present were obtained from various medical centers and hospitals: the majority were from the Wadsworth Anaerobe Laboratory (WAL) in Los Angeles, Calif. (n = 34) and Loyola Strich School of Medicine, in Maywood, Ill. (n = 65; designations begin with DH). The 1996-to-1997 community isolates were from rectal swabs taken from volunteers attending the Microbial Diversity Course at Woods Hole, Mass., in 1996 and 1997 (n = 86; designations begin with WH). The isolation of Bacteroides strains from these volunteers took advantage of the aerotolerance of Bacteroides species and their resistance to high levels of gentamicin (200 µg/ml). Samples were diluted and streaked directly onto supplemented BHI medium (7) containing 200 µg of gentamicin/ml under aerobic conditions and were then incubated anaerobically in BBL GasPak jars for 48 h. Fifty or more isolates were first checked by colony hybridization using tetQ and CTnDOT probes (Table 1), and four or five isolates from each source were selected at random, stocked, and saved for further analysis. The further verification of the identity of the isolates included hybridization to Bacteroides-specific probes (19), Gram staining, and some selective 16S ribosomal DNA (rDNA) sequencing using a universal prokaryotic forward primer and a Bacteroides group-specific reverse primer (30). This mode of isolation could have had a slight bias in favor of the species that are somewhat more aerotolerant and less fastidious than other species, such as B. thetaiotaomicron and B. fragilis. This bias could not have been too strong, however, because we isolated no B. fragilis using this procedure, and whereas many of the isolates were B. thetaiotaomicron, several were also Bacteroides uniformis and Bacteroides eggerthii. These ratios reflected the ratios of Bacteroides species in the colon, where the concentration of B. fragilis is more than 10-fold lower than that of B. thetaiotaomicron or B. uniformis.

Antibiotic resistance phenotypes. Fresh overnight cultures were streaked onto supplemented BHI (7) agar plates containing the antibiotic. The strains were considered resistant if they grew on plates containing erythromycin (3 µg/ml), clindamycin (3 µg/ml), or tetracycline (1 µg/ml). B. thetaiotaomicron 5482 and B. fragilis 638 did not grow at these concentrations. No attempt was made to determine the MICs for each strain because the purpose of these phenotypic tests was to correlate the resistance phenotype with the presence of the resistance genes responsible and not to determine the precise level of resistance. Many of the clinical strains came with MIC results, and the resistances we observed correlated with the information provided.

DNA hybridizations. DNA dot blots using total DNA prepared from 2-ml overnight cultures of each strain (33, 50) were probed using fluorescein-dUTP-labeled DNA fragments or purified PCR products. Labeling was done as described in the Renaissance kit protocols (NEN Life Sciences). The blots were developed using a chemiluminescent substrate. Southern blotting was done on the DNA of 60 strains that hybridized to tetQ to determine how related they were to the known Bacteroides CTn's, especially CTnDOT. The DNA from each strain was digested with EcoRI and EcoRV, and the blots were probed with a series of fluorescein-labeled probes. The first probe used was a 7,700-bp EcoRI fragment of CTnDOT which contained tetQ, rteA, rteB, and rteC (46). Some of the blots were probed sequentially with an rteC probe, an rteB probe, and then a probe containing tetQ and rteA in order to identify restriction fragments containing each sequence. This was necessary because of the restriction site polymorphisms observed for some of the strains. See Table 1 for descriptions of primers for PCR-generated products and the sources of the isolated fragments used to make the probes.

PCR amplification of 16S rRNA sequences and tetQ genes. The PCR primers used to amplify 16S rDNA and the internal fragments for tetQ, ermG, and ermB genes are described in Table 1. The amplification was done using Taq I polymerase in 100-µl reaction mixtures containing 10 to 100 ng of DNA and 200 ng of each primer in a solution containing 1× Gibco-BRL PCR buffer, 1.5 mM MgCl₂, and 0.2 mM deoxyribonucleoside triphosphate mixture. The amplification cycles were as follows: 95°C for 5 min; 30 cycles of 95°C for 1 min, 50 to 52°C for 1 min, and 72°C for 2 min; and a final elongation step of 72°C for 5 min. The PCR products were extracted directly from the reaction mix using a Wizard PCR Clean up kit (Promega). The PCR products were sequenced using the PCR primers by the University of Illinois Biotechnology Center.

	RESULTS

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Hybridization analyses of community and clinical isolates. For an initial assessment of the extent to which horizontal gene transfer has occurred between colonic Bacteroides strains over the past 3 decades, we compared tetracycline and erythromycin resistance profiles and the carriage of possible resistance genes of two sets of isolates. One set (88 isolates) had been collected before 1980, many of the strains before 1960. The second set (211 isolates) had been collected after 1980, most during the 1990s. Both sets contained strains isolated from patients with Bacteroides infections (clinical isolates) and strains isolated from the intestines of healthy humans (community isolates). By comparing clinical and community isolates, it was possible to determine whether patterns seen in clinical isolates mirrored those seen in community isolates. If the spread of resistance was occurring in the community isolates, later causing clinical infections, the patterns should be similar. If resistance was arising in hospitals, the incidence of phenotype resistance and carriage of resistance genes should be higher in the clinical isolates than in the community isolates.

Sequence analysis of tetQ genes from different strains. If recent horizontal gene transfer was responsible for the widespread carriage of tetQ and the erm genes, the genes in different strains should be virtually identical at the DNA sequence level. Of the erm genes, we were particularly interested in the ermB and ermG genes because of their known gram-positive origin. The Bacteroides ermF genes found on transposons and CTn's are >99% identical, and the original source of this gene is not known (10, 12). tetQ PCR products from 33 isolates representing 10 species (13 pre-1970 strains and 20 post-1980 strains) were sequenced and compared. The results are summarized in Table 3. The sequences were compared to the tetQ sequences deposited in GenBank. The sequences of the 33 strains had 96 to 100% identity to the tetQ-3 gene found on the conjugative transposon CTnDOT (Table 3) (accession no. X58717). Genes from 20 of the 33 strains were 98 to 100% identical to tetQ-1 from B. fragilis strain BF2 (accession no. Y08615), including 9 of the 13 pre-1970 strains. The tetQ on CTnV479 is still the most divergent, with only 89 to 90% identity to the other tetQ genes in either Bacteroides or Prevotella strains (27). The high sequence identity of the tetQ alleles supports the hypothesis that tetQ has been spread by horizontal gene transfer. This result also rules out convergent evolution, the independent evolution of two versions of the same gene. The amino acid sequences of such genes can be very similar if there is strong selection for a particular sequence. Even two proteins with the same amino acid sequence, however, can be encoded by genes whose sequences differ by as much as 20% due to 3rd-base wobble.

Type of gene transfer elements associated with the horizontal gene transfer. Previous studies, which were limited to a small number of clinical isolates, had shown that tetQ in Bacteroides spp. was carried on two different types of CTn's, one represented by CTnDOT and one represented by CTn7853 (29, 39). ermF was found previously on several CTn's of the CTnDOT group and on three transmissible plasmids (24). ermG, however, has been found only on CTn7853 (8). Virtually all of the strains that harbored tetQ also harbored DNA that hybridized with a probe from the ends of CTnDOT (5). Whereas more than 80% of CTnDOT has now been sequenced, making it easier to design probes that identify elements of this family of CTn's, only a small amount of sequence outside the tetQ region of CTn7853 is available. Using a 1-kbp fragment located 6 kbp upstream of tetQ as a probe for CTn7853 type CTn's, we found only two additional strains that cross-hybridized to the probe; they both contained ermG and were the other two strains in Group II (Table 5), with an ermG sequence identical to that of CTn7853 in B. thetaiotaomicron 7853. Thus, it appears that CTn7853 type CTn's are not widespread in Bacteroides and that the CTnDOT type elements are the predominant type of CTn. There were strains that hybridized to the CTnDOT end probe that did not contain tetQ, for example, the VPI strain B. uniformis 0061. B. uniformis 0061 has been shown previously to carry a cryptic CTn, CTnXBU4422, which is related to CTnDOT (42).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Diagrammatic representation of the restriction patterns seen on Southern blots of DNA from different strains, which was hybridized with a probe that detects the tetQ rteA rteB rteC region of the CTnDOT type CTn's. The fragment sizes and the locations of the genes within the 7.7-kbp EcoRI fragment used as the probe are shown at the top. Pattern A has the restriction fragment profile of CTnDOT and closely related elements. Some of the CTnDOT family of elements (CTnERL2) lack an EcoRV site and have the fragment labeled RV*. This pattern is referred to as A'. Occasionally this EcoRV site does not cut completely, and a mixed pattern is observed (indicated as A* in the Southern blot in Fig. 2). Strains exhibiting pattern B are missing the two small rteC fragments (C0.2 and C0.64 in Fig. 2), and all of the rteC homology is located in the 2.6-kbp fragment labeled BC2.6 in Fig. 2. Strains that exhibit the C pattern are heterogeneous. A few lack rteC completely (CTn7853; C3 in Fig. 2), and others just have a very different pattern, but all of the genes on the probe are present (CTnV479; C2 in Fig. 2). Pattern D is rare; these strains have tetQ and usually neither rteB nor rteC sequences. The percentage of the 60 strains exhibiting each of the patterns is shown at the right.

View larger version (71K): [in this window] [in a new window]   FIG. 2.   Southern blot of the EcoRI- and EcoRV-digested cellular DNA from tetQ-containing Bacteroides isolates. The blot was first probed with an rteC probe and then reprobed with the tetQ-rteA-rteB-rteC-containing probe. The blot is overexposed so that the small rteC-containing bands (C0.2 and C0.64) can be observed for pattern A. These sequences appear in the BC2.6 band of pattern B. The sizes of the HindIII lambda DNA size standards (stds) are given on the left. The sizes and contents of the major bands hybridizing to the probes are shown on the right. A schematic of the region being probed and the expected sizes is shown in Fig. 1. The patterns for each lane are labeled according to the scheme described in Fig. 1. The rteC-containing fragments for patterns C1 and C2 are indicated by arrows. These fragments also contain rteB, and C2 is for CTnV479. The B patterns in lanes 1 and 2 also contain extra hybridizing bands (indicated by asterisks) that hybridize to rteB but not rteC or tetQ probes. The A* patterns may be due to a strain containing both an A and an A' CTn as is seen for B. fragilis ERL or the pattern may be due to partial digestion of the EcoRV site between tetQ and rteA (Fig. 1), as is sometimes observed for CTn12256 (data not shown).

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Differences in restriction patterns (depicted in Fig. 1 and 2) between older isolates and modern isolates. Both groups of strains include both community and clinical isolates. The 22 VPI strains were isolated before 1970. The RFLP patterns are shown in Fig. 1, and Southern blot patterns A, B, and C are shown in Fig. 2.

	DISCUSSION

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Taken together, our results show that the substantial increase in tetracycline resistance among Bacteroides isolates that has occurred over the past few decades was due to the horizontal spread of a single gene, tetQ, on CTn's of the CTnDOT type. ermF and ermG also appear to be spreading by horizontal gene transfer, possibly also on CTn's such as CTnDOT and CTn7853. It is likely that these gene transfer events took place in the human colon, because Bacteroides species are found primarily in the human colon and are present, if at all, in low numbers in the intestines of other animals or in the environment. The only environmental site outside the human colon to harbor significant numbers of Bacteroides strains would be a sewage treatment plant. Transfer of resistant Bacteroides strains from sewage treatment plants to humans, however, would be unlikely to have produced the widespread colonization seen in our studies. Also, as is evident from the 16 sewage plant isolates tested (Table 2), carriage seems to be lower in strains obtained from this setting than in strains isolated from the human colon.

A feature of the CTnDOT type elements allows us to speculate about what may have caused this extensive spread of resistance genes. Most of the CTnDOT type elements exhibit regulated transfer (38). That is, transfer occurs only if the donors are first stimulated with low levels of the antibiotic tetracycline. After tetracycline induction, transfer frequencies rise 1,000- to 10,000-fold. No other class of antibiotics has this effect. Thus, tetracycline use in the community probably played a role not only in selecting for maintenance of tetQ but also in causing it to be transferred in the first place.

Our results show clearly that once a resistance gene enters Bacteroides species, it can be spread widely among these species if it becomes part of a CTn or some other transmissible element. Our survey also provided evidence that Bacteroides species may share DNA with members of genera outside the Bacteroides-Prevotella group. ermF was first found in Bacteroides species (12) but clearly originated in a low-G+C organism (33%). tetQ, which appears to have originated in Bacteroides spp., has 40% G+C, which is the average observed for this genus (17). The origin of ermG (27% G+C) is also unknown, but this gene has been found in Bacillus sphaericus, a gram-positive soil bacterium, as well as in B. thetaiotaomicron 7853 (8). Our survey also turned up six Bacteroides strains that had acquired an ermB gene virtually identical to an ermB from C. perfringens, Streptococcus pneumoniae, and Enterococcus faecalis. This finding suggests that even bacteria that do not normally reside in the colon (S. pneumoniae) or that reside there in low numbers (C. perfringens and E. faecalis) can donate DNA to members of numerically predominant groups of colonic bacteria. Our results do not prove that DNA was transferred directly from these species to Bacteroides species, but the presence of ermG and ermB in multiple isolates from different geographical locations indicates that some genetic connection, however indirect, is open between the gram-positive bacteria where the genes appear to have originated and the gram-negative Bacteroides species.

Given that some gram-positive resistance genes appear to have moved into Bacteroides, it is surprising that tetM, a resistance gene that is distantly related to tetQ and confers the same type of resistance, was not found in any of these Bacteroides isolates. The tetM gene is carried on several gram-positive CTn's that appear to have a broad host range, at least under laboratory conditions. The failure to find tetM could indicate that there might be barriers to transfer of some types of elements in vivo or that there is a lack of selective pressure for tetracycline resistance determinants other than tetQ, since 80% of the Bacteroides strains are already tetracycline resistant due to tetQ.

Once in Bacteroides species, conjugal transfer mediated primarily by CTn's can spread these genes widely among different Bacteroides species. This study is the first to demonstrate what scientists have long suspected, that the human colon is a site that is highly conducive to horizontal gene transfer and to stable maintenance of transferred resistance genes. It is also the first study to associate an increase in antibiotic resistance with carriage of a particular type of conjugal element, in this case CTn's of the CTnDOT class. CTn's have also been found in gram-positive bacteria (1, 6) and in the Escherichia coli phylogenetic group of gram-negative bacteria (14, 49). The results of our study suggest that CTn's are making a major contribution to the transmission of antibiotic resistance genes.