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

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 of 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 pervasive as the CTnDOT/ERL family of conjugative transposons.

Transfer region of a Bacteroides conjugative transposon contains regulatory as well as structural genes.

Conjugative transposons (CTns) are integrated elements that excise themselves from the chromosome to form a circular transfer intermediate that is transferred by conjugation to a recipient. In an earlier paper, the excision step was shown to be regulated by tetracycline and to be dependent on the regulatory gene, rteC. In this paper, we report that genes involved in conjugal transfer are also regulated by tetracycline but that regulation is more complex. Genes contained within a 20-kbp region that is sufficient for conjugal transfer were disrupted by single crossover integration events. Most of the disruptions abolished transfer of the CTn. None of them abolished excision. Antibodies to two of the proteins encoded in this region (TraG and TraN) were obtained and used to show that production of these proteins was dependent on tetracycline stimulation. Both TraG and TraN were membrane proteins. A surprising finding was that a disruption in the gene traQ increased transfer of CTnERL over 100-fold. Thus, TraQ may be a repressor protein that controls expression of transfer genes. If so, TraQ is not the only protein that controls expression of transfer genes because production of TraG and TraN in the traQ disruption mutant was still dependent on tetracycline stimulation.

Production of two proteins encoded by the Bacteroides mobilizable transposon NBU1 correlates with time-dependent accumulation of the excised NBu1 circular form.

NBU1 is a mobilizable transposon that excises from the Bacteroides chromosome to form a double-stranded circular transfer intermediate. Excision is triggered by exposure of the bacteria to tetracycline. Accordingly, we expected that the expression of NBU1 genes would be induced by tetracycline. To test this hypothesis, antibodies that recognized two NBU1-encoded proteins, PrmN1 and MobN1, were used to monitor production of these proteins. PrmN1 is essential for excision, and MobN1 is essential for transfer of the excised circular form. At first, expression of the genes encoding these two proteins appeared to be regulated by tetracycline, because the proteins were detectable on Western blots only after the cells were exposed to tetracycline. However, when the prmN1 gene and/or the mobN1 gene was cloned on a multicopy plasmid, production of the protein was constitutive. Initially, we assumed that the constitutive expression was due to loss of a repressor protein that was encoded by one of the other genes on NBU1. Deletions or insertions in the other genes (orf2 and orf3) on NBU1 and various integrated NBU1 derivatives abolished production of PrmN1 and MobN1. This is the opposite of what should have happened if one or both of these genes encoded a repressor. A second possibility was that when NBU1 excised, it replicated transiently, increasing the gene dosage of prmN1 and mobN1 and thereby producing enough PrmN1 and MobN1 for these proteins to become detectable. In fact, after the cells entered late exponential phase the copy number of NBU1 increased to 2 to 3 copies per cell. Production of PrmN1 and MobN1 showed a similar pattern. Any mutation in NBU1 that decreased or prevented excision also prevented elevated production of these two proteins. Our results show that the apparent tetracycline dependence of the production of PrmN1 and MobN1 is due to a growth phase- or time-dependent increase in the number of copies of the NBU1 circular form.

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.

Characterization of the 13-kilobase ermF region of the Bacteroides conjugative transposon CTnDOT.

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 ermF appears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although the ermF region contains genes also carried on Bacteroides transposons Tn4351 and Tn4551, it does not contain the IS4351 element 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 the Bacteroides MTn Tn4399. Analysis of 19 independent Bacteroides isolates showed that the ermF region is located in the same position in all of the strains analyzed and that the compositions of the ermF 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 diverse Bacteroides population.

Transfer region of a bacteroides conjugative transposon, CTnDOT.

Bacteroides species harbor large self-transmissible integrated elements called conjugative transposons (CTns). In this paper, we report the first complete sequence analysis of the transfer region of a Bacteroides CTn. The transfer region contained 17 genes (designated orfA-orfQ). Only 2 of the genes shared sequence similarity with genes in the databases and only 1 of these genes was associated with self-transmissible elements.

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

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.

Depression & stroke. Improving patient outcomes with a team approach.

Stroke is a significant diagnosis for referral to home care services, and depression is a common co-occurring disorder. Depression can lead to loss of motivation or hopelessness about recovery, which poses barriers to accomplishing treatment goals. When skilled nurses or physical therapists identify depression in stroke patients, they can include the expertise of the psychiatric home care nurse to provide a comprehensive, multidisciplinary treatment plan to achieve successful outcomes.

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.

Characterization of a Bacteroides mobilizable transposon, NBU2, which carries a functional lincomycin resistance gene.

The mobilizable Bacteroides element NBU2 (11 kbp) was found originally in two Bacteroides clinical isolates, Bacteroides fragilis ERL and B. thetaiotaomicron DOT. At first, NBU2 appeared to be very similar to another mobilizable Bacteroides element, NBU1, in a 2.5-kbp internal region, but further examination of the full DNA sequence of NBU2 now reveals that the region of near identity between NBU1 and NBU2 is limited to this small region and that, outside this region, there is little sequence similarity between the two elements. The integrase gene of NBU2, intN2, was located at one end of the element. This gene was necessary and sufficient for the integration of NBU2. The integrase of NBU2 has the conserved amino acids (R-H-R-Y) in the C-terminal end that are found in members of the lambda family of site-specific integrases. This was also the only region in which the NBU1 and NBU2 integrases shared any similarity (28% amino acid sequence identity and 49% sequence similarity). Integration of NBU2 was site specific in Bacteroides species. Integration occurred in two primary sites in B. thetaiotaomicron. Both of these sites were located in the 3' end of a serine-tRNA gene NBU2 also integrated in Escherichia coli, but integration was much less site specific than in B. thetaiotaomicron. Analysis of the sequence of NBU2 revealed two potential antibiotic resistance genes. The amino acid sequences of the putative proteins encoded by these genes had similarity to resistances found in gram-positive bacteria. Only one of these genes was expressed in B. thetaiotaomicron, the homolog of linA, a lincomycin resistance gene from Staphylococcus aureus. To determine how widespread elements related to NBU1 and NBU2 are in Bacteroides species, we screened 291 Bacteroides strains. Elements with some sequence similarity to NBU2 and NBU1 were widespread in Bacteroides strains, and the presence of linA(N) in Bacteroides strains was highly correlated with the presence of NBU2, suggesting that NBU2 has been responsible for the spread of this gene among Bacteroides strains. Our results suggest that the NBU-related elements form a large and heterogeneous family, whose members have similar integration mechanisms but have different target sites and differ in whether they carry resistance genes.

Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1.

NBU1 is an integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision-required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision. The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRS(HIII)) inhibited excision when provided in trans on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized.

Correlation between age at neutering and age at onset of hyperadrenocorticism in ferrets.

OBJECTIVE: To determine prevalence of hyperadrenocorticism in ferrets in The Netherlands and evaluate age, sex, and age at neutering in affected ferrets. DESIGN: Prevalence survey and retrospective study. ANIMALS: 50 ferrets with hyperadrenocorticism and 1,267 ferrets without hyperadrenocorticism. PROCEDURE: A questionnaire was sent to 1,400 members of a ferret-owners organization in The Netherlands; 492 (35%) owners returned the questionnaire, providing usable data on 1,274 ferrets. Seven of these ferrets developed hyperadrenocorticism during the survey period; medical records for these ferrets and 43 ferrets with confirmed hyperadrenocorticism were reviewed. Hyperadrenocorticism was confirmed by histologic examination of an excised adrenal gland (92% of ferrets) or clinical improvement after excision. RESULTS: Prevalence of hyperadrenocorticism in the survey population was 0.55%. Sex was not associated with prevalence of disease. Median time interval between neutering and diagnosis of hyperadrenocorticism was 3.5 years. A significant linear correlation between age at neutering and age at time of diagnosis was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Age at neutering may be associated with age at development of hyperadrenocorticism in ferrets.

Starting a new genetic system: lessons from bacteroides.

Despite the fact that genetic manipulation is an essential adjunct of studies of microbial physiology, many important bacterial species are still not accessible to genetic manipulation. In many cases, the people who know the organism best and have studied its physiology extensively have little experience with genetic techniques and are uncertain how to set up a new system. In this article, we describe what we have learned in the process of developing a genetic system for Bacteroides spp. and helping to develop genetic tools for two related species of bacteria: Porphromonas gingivalis and Prevotella ruminicola. We discuss the relative usefulness of different types of genetic tools and make some suggestions about how to construct them.

Construction and characterization of a Bacteroides thetaiotaomicron recA mutant: transfer of Bacteroides integrated conjugative elements is RecA independent.

We report the construction and analysis of a Bacteroides thetaiotaomicron recA disruption mutant and an investigation of whether RecA is required for excision and integration of Bacteroides mobile DNA elements. The recA mutant was deficient in homologous recombination and was more sensitive than the wild-type strain to DNA-damaging agents. The recA mutant was also more sensitive to oxygen than the wild type, indicating that repair of DNA contributes to the aerotolerance of B. thetaiotaomicron. Many Bacteroides clinical isolates carry self-transmissible chromosomal elements known as conjugative transposons. These conjugative transposons can also excise and mobilize in trans a family of unlinked integrated elements called nonreplicating Bacteroides units (NBUs). The results of a previous study had raised the possibility that RecA plays a role in excision of Bacteroides conjugative transposons, but this hypothesis could not be tested in Bacteroides spp. because no RecA-deficient Bacteroides strain was available. We report here that the excision and integration of the Bacteroides conjugative transposons, as well as NBU1 and Tn4351, were unaffected by the absence of RecA activity.

Resistance gene transfer in anaerobes: new insights, new problems.

Investigations of antibiotic-resistance gene transfer elements in Bacteroides species have generated some new insights into how bacteria transfer resistance genes and what environmental conditions foster gene transfer. Integrated gene transfer elements, called conjugative transposons, appear to be responsible for much of the transfer of resistance genes among Bacteroides species. Conjugative transposons not only transfer themselves but also mobilize coresident plasmids and excise and mobilize unlinked integrated elements. Less is known about resistance gene transfer elements of the gram-positive anaerobes, but there are some indications that similar elements may be found in them as well. An unusual feature of the Bacteroides conjugative transposons is that transfer of many of them is stimulated considerably by low concentrations of antibiotics. Thus, antibiotics not only select for resistant strains but also can stimulate transfer of the resistance gene in the first place. This finding raises questions about whether use of low-dose tetracycline therapy may have a greater effect on the resident microflora than had been previously thought. Finally, investigations of resistance genes in Bacteroides species and other genera of bacteria have begun to provide evidence that the resident microflora of the human body does indeed act as a reservoir for resistance genes, which may be acquired from and passed on the transient colonizers of the site.

The Bacteroides mobilizable insertion element, NBU1, integrates into the 3' end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrase family.

NBU1 is a 10.3-kbp integrated Bacteroides element that can be induced to excise from the chromosome and can be mobilized to a recipient by trans-acting functions provided by certain Bacteroides conjugative transposons. The NBU1 transfer intermediate is a covalently closed circle, which is presumed to be the form that integrates into the recipient genome. We report here that a 2.4-kbp segment of NBU1 was all that was required for site-specific integration into the chromosome of Bacteroides thetaiotaomicron 5482. This 2.4-kbp region included the joined ends of the NBU1 circular form (attN1) and a single open reading frame, intN1, which encoded the integrase. Previously, we had found that NBU1 integrates preferentially into a single site in B. thetaiotaomicron 5482. We have now shown that the NBU1 target site is located at the 3' end of a Leu-tRNA gene. The NBU1 integrase gene, intN1, was sequenced. The predicted protein had little overall amino acid sequence similarity to any proteins in the databases but had limited carboxy-terminal similarity to the integrases of lambdoid phages and to the integrases of the gram-positive conjugative transposons Tn916 and Tn1545. We also report that the intN1 gene is expressed constitutively.

NBU1, a mobilizable site-specific integrated element from Bacteroides spp., can integrate nonspecifically in Escherichia coli.

NBU1 is an integrated Bacteroides element that can he mobilized from Bacteroides donors to Bacteroides recipients. Previous studies have shown that a plasmid carrying the internal mobilization region of NBU1 could be transferred by conjugation from Bacteroides thetaiotaomicron to Escherichia coli. In this report, we show that NBU1 can integrate in E. coli. Whereas integration of NBU1 in B. thetaiotaomicron is site specific, integration of NBU1 in E. coli was relatively random, and the insertion frequency of NBU1 into the E. coli chromosome was 100 to 1,000 times lower than the frequency of integration in B. thetaiotaomicron. The frequency of NBU1 integration in E. coli could be increased about 10- to 70-fold, to a value close to that seen with B. thetaiotaomicron, if the primary integration site from B. thetaiotaomicron, BT1-1, was provided on a plasmid in the E. coli recipient or the NBU1 integrase gene, intN1, was provided on a high-copy-number plasmid to increase the amount of integrase available in the recipient. When the primary integration site was available in the recipient, NBU1 integrated site specifically in E. coli. Our results show that NBUs have a very broad host range and are capable of moving from Bacteroides spp. to distantly related species such as E. coli. Moreover, sequence analysis of NBU1 integration sites provided by integration events in E. coli has helped to identify some regions of the NBU1 attachment site that may play a role in the integration process.

The erythromycin resistance gene from the Bacteroides conjugal transposon Tcr Emr 7853 is nearly identical to ermG from Bacillus sphaericus.

Tcr Emr 7853, from Bacteroides thetaiotaomicron 7853, is a large chromosomal conjugative transposon which encodes resistance to both tetracycline (Tcr) and erythromycin (Emr). The erythromycin resistance gene of Tcr Emr 7853 did not cross-hybridize with ermF, the Emr gene found on previously studied Bacteroides regular and conjugative transposons. We have cloned and sequenced the erythromycin resistance gene from Tcr Emr 7853. The DNA sequence of this gene was 99.6% identical to that of ermG from Bacillus sphaericus.