Nadja B. Shoemaker

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 oftetQ 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 tetQwas mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF andermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage oftetQ 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 Bacteroidesinfections. 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 carriedermB genes. Two were identical to erm(B)-P fromClostridium 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 ofBacillus 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 Bacteroidesspecies. 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.

The role of Bacteroides conjugative transposons in the dissemination of antibiotic resistance genes.

Abstract. Investigations into the mechanisms of antibiotic resistance gene transfer utilized by Bacteroides species have led to a greater understanding of how bacteria transfer antibiotic resistance genes, and what environmental stimuli promote such horizontal transfer events. Although Bacteroides spp. harbor a variety of transmissible elements that are involved in the dissemination of antibiotic resistance genes, it is one particular class of elements, the conjugative transposons, that are responsible for most of the resistance gene transfer in Bacteroides. The potential for Bacteroides conjugative transposons to transfer antibiotic resistance genes extends beyond those genes carried by the conjugative transposon itself, because Bacteroides conjugative transposons are able to mobilize coresident plasmids in trans and in cis, and also stimulate the excision and transfer of unlinked integrated elements called mobilizable transposons. These characteristics o f conjugative transposons alone have significant implications for the ecology and spread of antibiotic resistance genes, and in terms of biotechnology. A novel feature of the most widespread family of Bacteroides conjugative transposons, the CTnDOT/ERL family, is that their transfer is stimulated 100- to 1000-fold by low concentrations of tetracycline. This is significant because the use of antibiotics not only selects for resistant Bacteroides strains, but also stimulates their transfer. Other Bacteroides conjugative transposons do not require any induction to stimulate transfer, and hence appear to transfer constitutively. The constitutively transferring elements characterized so far appear to have a broader host range than the CTnDOT/ERL family of conjugative transposons, and the prevalence of these elements is on the increase. Since these constitutively transferring elements do not require induction by antibiotics to stimulate transfer, they have the potential to become as perva sive as the CTnDOT/ERL family of conjugative transposons.

Integration and excision of a Bacteroides conjugative transposon, CTnDOT.

Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most Bacteroides CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF), intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of intDOT designated orf2. Thus, excision involves other genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that intDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.

A Bacteroides tetracycline resistance gene represents a new class of ribosome protection tetracycline resistance.

The ribosome protection type of tetracycline resistance (Tcr) has been found in a variety of bacterial species, but the only two classes described previously, Tet(M) and Tet(O), shared a high degree of amino acid sequence identity (greater than 75%). Thus, it appeared that this type of resistance emerged recently in evolution and spread among different species of bacteria by horizontal transmission. We obtained the DNA sequence of a Tcr gene from Bacteroides, a genus of gram-negative, obligately anaerobic bacteria that is phylogenetically distant from the diverse species in which tet(M) and tet(O) have been found. The Bacteroides Tcr gene defines a new class of ribosome protection resistance genes, Tet(Q), and has a deduced amino acid sequence that was only 40% identical to Tet(M) or Tet(O). Like tet(M) and tet(O), tet(Q) appears to have spread by horizontal transmission, but only within the Bacteroides group. Images

New perspectives in tetracycline resistance

Until recently, tetracycline efflux was thought to be the only mechanism of tetracycline resistance. As studies of tetracycline resistance have shifted to bacteria outside the Enterobacteriaceae, two other mechanisms of resistance have been discovered. The first is ribosomal protection, a type of resistance which is found in mycoplasmas, Gram‐positive and Gram‐negative bacteria and may be the most common type of tetracycline resistance in nature. The second is tetracycline modification, which has been found only in two strains of an obligate anaerobe (Bacteroides). Recent studies have also turned up such anomalies as a tetracycline efflux pump which does not confer resistance to tetracycline and a gene near the replication origin of a tetracycline‐sensitive Bacillus strain which confers resistance when it is amplified.

Characterization of the 13-Kilobase ermF Region of the Bacteroides Conjugative Transposon CTnDOT

ABSTRACT The conjugative transposon CTnDOT is virtually identical over most of its length to another conjugative transposon, CTnERL, except that CTnDOT carries an ermF gene that is not found on CTnERL. In this report, we show that the region containing ermFappears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although theermF region contains genes also carried onBacteroides transposons Tn4351 and Tn4551, it does not contain the IS4351element which is found on these transposons. In CTnDOT, insertion of the ermF region occurred near a stem-loop structure at the end of orf2, an open reading frame located immediately downstream of the integrase (int) gene of CTnDOT, and in a region known to be important for excision of CTnERL and CTnDOT. The chimera that comprises the ermF region can apparently no longer excise and circularize, but it contains a functional mobilization region related to that described for theBacteroides MTn Tn4399. Analysis of 19 independent Bacteroides isolates showed that theermF region is located in the same position in all of the strains analyzed and that the compositions of theermF region are almost identical in these strains. Therefore, it appears that CTnDOT-like elements present in community and clinical isolates of Bacteroides were derived from a common ancestor and proliferated in the diverseBacteroides population.

A new Bacteroides conjugative transposon that carries an ermB gene.

ABSTRACT The erythromycin resistance gene ermB has been found in a variety of gram-positive bacteria. This gene has also been found in Bacteroides species but only in six recently isolated strains; thus, the gene seems to have entered this genus only recently. One of the six Bacteroides ermB-containing isolates, WH207, could transfer ermB to Bacteroides thetaiotaomicron strain BT4001 by conjugation. WH207 was identified as a Bacteroides uniformis strain based on the sequence of its 16S rRNA gene. Results of pulsed-field gel electrophoresis experiments demonstrated that the transferring element was normally integrated into the Bacteroides chromosome. The element was estimated from pulsed-field gel data to be about 100 kb in size. Since the element appeared to be a conjugative transposon (CTn), it was designated CTnBST. CTnBST was able to mobilize coresident plasmids and the circular form of the mobilizable transposon NBU1 to Bacteroides and Escherichia coli recipients. A 13-kb segment that contained ermB was cloned and sequenced. Most of the open reading frames in this region had little similarity at the amino acid sequence level to any proteins in the sequence databases, but a 1,723-bp DNA segment that included a 950-bp segment downstream of ermB had a DNA sequence that was virtually identical to that of a segment of DNA found previously in a Clostridium perfringens strain. This finding, together with the finding that ermB is located on a CTn, supports the hypothesis that CTnBST could have entered Bacteroides from some other genus, possibly from gram-positive bacteria. Moreover, this finding supports the hypothesis that many transmissible antibiotic resistance genes in Bacteroides are carried on CTns.

A newly discovered Bacteroides conjugative transposon, CTnGERM1, contains genes also found in gram-positive bacteria.

ABSTRACT Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates—CTns of the CTnERL/CTnDOT family—which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species—an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.

Recent advances in Bacteroides genetics.

Bacteroides are Gram-negative, obligate anaerobes that are present in high concentrations within the intestinal tracts of humans and animals. Bacteroides are also important opportunistic pathogens of humans and animals. Methods for genetic manipulation of these important organisms have only recently begun to emerge. Shuttle vectors which can be transferred by conjugation between Escherichia coli to Bacteroides are now available. A method for transforming some strains of Bacteroides has been developed. Two Bacteroides transposons, Tn4351 and Tn4400, have been found and one of them, Tn4351, has been used for transposon mutagenesis of Bacteroides. Several different Bacteroides genes have now been cloned, including a gene that codes for resistance to clindamycin, genes that code for polysaccharidases (chondroitin lyase and pullulanase), and a gene that codes for a fimbrial subunit. These cloned genes have been used to study the organization and regulation of Bacteroides genes.

Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon

Integrated self‐transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram‐positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini‐element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.