Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon

Abstract

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.

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 (C_0.2 and C_0.64 in Fig. 2), and all of the rteC homology is located in the 2.6-kbp fragment labeled BC_2.6 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.

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 (C_0.2 and C_0.64) can be observed for pattern A. These sequences appear in the BC_2.6 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).

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.