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Appl Environ Microbiol. Jan 2007; 73(1): 53–63.
Published online Oct 27, 2006. doi:  10.1128/AEM.01669-06
PMCID: PMC1797145

Identification and Characterization of Conjugative Transposons CTn86 and CTn9343 in Bacteroides fragilis Strains[down-pointing small open triangle]

Abstract

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.

Enterotoxigenic Bacteroides fragilis (ETBF) is strongly associated epidemiologically with diarrheal disease in livestock, young children, and adults (19, 20, 21, 29, 30, 32, 40). The only recognized virulence factor of ETBF is a toxin termed B. fragilis toxin, or BFT (16, 34, 35). Three highly related isotypes of BFT (termed BFT-1, BFT-2, and BFT-3) have been identified (3, 6). It has been reported that the bft gene is contained in a 6-kb pathogenicity island (PAI) and that the B. fragilis PAI is flanked by genes encoding mobilization proteins (7, 17). Based on the presence of the PAI and its flanking region, three major populations of B. fragilis strains were identified: (i) pattern I strains, containing the PAI and its flanking region, which are all ETBF strains; (ii) pattern II strains, lacking the PAI and its flanking regions, which are all nontoxigenic B. fragilis (NTBF) strains; and (iii) pattern III strains, containing the flanking region but lacking the PAI, which are all NTBF strains (7).

The G+C content of the B. fragilis PAI (35%) and of the flanking DNA (47 to 50%) differs greatly from that reported for the B. fragilis chromosome (43%) (2, 14), suggesting that the PAI and its flanking region are two distinct genetic elements originating from different organisms. Based on these results, it was hypothesized that ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain (7). It was recently determined that the genetic element flanking the PAI in ETBF 86-5443-2-2 (pattern I; contains the PAI) and a related genetic element in NTBF NCTC 9343 (pattern III; lacks the PAI) are putative conjugative transposons (CTns), designated CTn86 in strain 86-5443-2-2 and CTn9343 in strain NCTC 9343 (9). In contrast to most of the CTns detected in Bacteroides spp., CTn86 and CTn9343 do not carry the tetracycline resistance gene tetQ, and the excision of the transposons from the chromosome is not regulated by tetracycline. Based on these characteristics, sequence homology, and the proposed mechanism of transposition, CTn86 and CTn9343 may be members of a new family of CTns in Bacteroides spp. not described previously. CTn86 and CTn9343 have the same basic structures except that CTn86 lacks a 7-kb region containing putative genes that encode an integrase (int2), regulatory proteins (rteA and rteB), and resistance to streptogramin A and fluoroquinolones (satG and bexA), and CTn9343 lacks the B. fragilis PAI.

CTns are major contributors to the spread of antibiotic resistance genes among Bacteroides species (31). The presence of bft in a putative CTn suggests that this toxin gene can be transmitted from ETBF to NTBF strains by a mechanism similar to that by which antibiotic resistance genes disseminate. CTns move via a circular intermediate that is produced by excision of the integrated element from the donor chromosome (31). Even though excision events from the chromosomes of CTn86 and CTn9343 have been demonstrated (9), transfer of these genetic elements between bacteria has not been determined, due to the lack of available markers for positive selection. In this study, we determined whether all pattern I strains (ETBF) and pattern III strains (NTBF) have genetic elements similar to CTn86 and CTn9343, respectively. In addition, we assessed whether the CTn genetic elements detected have the same integration sites as CTn86 and CTn9343. Our goal was to define the frequency of identification of this new class of CTns in Bacteroides spp., in particular, B. fragilis. These studies are a first step in assessing whether these genetic elements are likely to transfer among Bacteroides strains, providing a mechanism for the spread of the bft gene as well as antibiotic resistance genes. Our results suggest that the CTn class represented by CTn86 and CTn9343 is larger than expected, is present only in B. fragilis, and is potentially transferable between B. fragilis strains.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The ETBF and NTBF strains used in this study were isolated from different countries, from intestinal and extraintestinal samples, and from humans and animals. The origins and patterns of the strains, as well as the frequency of occurrence of bft subtypes and of cepA and cfiA, are listed in Table Table1.1. Strains of B. thetaiotaomicron, B. uniformis, B. distasonis, B. ovatus, B. vulgatus, B. eggerthii, B. stercoris, and B. merdae were obtained from N. B. Shoemaker and A. A. Salyers. Bacteroides strains were propagated anaerobically on BHC medium (37 g of brain heart infusion base [Difco Laboratories, Detroit, MI] per liter, 0.1 mg of vitamin K per liter, 0.5 mg of hemin per liter, and 50 mg of l-cysteine per liter [the latter three ingredients from Sigma, St. Louis, MO]).

TABLE 1.
Characteristics of B. fragilis strains used in this study

Colony blot hybridizations.

Colony blots of B. fragilis strains were prepared as described previously (6). Briefly, B. fragilis organisms grown overnight on BHC agar were transferred to Whatman 541 filters. The filters were microwave processed in alkali solution (0.5 M NaOH, 1.5 M NaCl), followed by neutralization in 2 M ammonium acetate. The probes were label with [α-32P]dCTP by random priming (Multiprime DNA labeling system; Amersham Pharmacia Biotech, England), hybridized at 37°C under high-stringency conditions in 50% formamide-5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS)-1 mM EDTA-1× Denhardt's solution, and washed with 5× SSC-0.1% SDS at 65°C for 1 h. Finally, the filters were rinsed with 2× SSC at room temperature. The construction and location of the nine probes (probes 4 to 12) shown in Fig. Fig.11 were derived from CTn9343 (9). These probes were used to characterize the collection of ETBF and NTBF strains used in this study. Probes A and B, used to identify CTn86-like and CTn9343-like elements in Bacteroides spp., are a 3.8-kb BglII fragment containing the bft gene and flanking region and a 7.8-kb PstI fragment containing the whole B. fragilis PAI and ~1-kb flanking regions, respectively (7). Probes used to determine the presence of the β-lactamase genes cepA and cfiA were obtained by PCR with primers and conditions previously described (10).

FIG. 1.
Characterization of the genetic element flanking the B. fragilis PAI in ETBF strains and the related genetic elements in NTBF pattern III strains. (A) Schematic map of CTn9343 showing the relative locations of the PCR products used as probes. The hybridization ...

Characterization of CTn86 and CTn9343 by PCR.

The CTn86, CTn86-like, CTn9343, and CTn9343-like elements were identified and characterized by PCR. The sequences and exact locations of the primers are shown in Table Table2,2, and relative locations of the primers are indicated in Fig. Fig.2.2. Primers 86CTn1R and Tn9B, derived from the CTn86 tnpA1 sequence and a common CTn86/CTn9343 sequence located immediately upstream of the open reading frame (ORF) 17 stop codon, respectively, are predicted to yield a PCR product in CTn86 elements that have a CTn86 left end and a deletion of the 7-kb region. Primers Tn25B, derived from CTn9343 tnpA1 sequence, and Tn9B yield a PCR product in elements that have a CTn9343 left end and a deletion of the 7-kb region. Under the conditions used for PCR, the distance between Tn25B and Tn9B is too long (~7.6 kb) to yield PCR products in elements that have the CTn9343 left end and contain the 7-kb region. Primers Tn25B and Tn25C, which are derived from the int2 sequence (contained in the CTn9343 7-kb region), yield a PCR product in genetic elements that have the CTn9343 left end and contain the 7-kb region.

FIG. 2.
Schematic maps of the CTn9343 and CTn86 left ends showing the relative locations of the primers used to identify the CTn86, CTn86-like, CTn9343, and CTn9343-like elements. Primers 86CTn1R and Tn9B yield a ~1.8-kb PCR product in genetic elements ...
TABLE 2.
PCR primers used in this study

Patterns I and III were discerned with primers P1T3 and P1T7, as described previously (7). These primers are derived from the region flanking the B. fragilis PAI (Fig. (Fig.2),2), so these primers yield a PCR product (ca. 1.6 kb) only in strains that lack the PAI but contain its flanking region (pattern III NTBF strains). In ETBF strains (containing the PAI), primers P1T3/P1T7 flank a ca. 7-kb region, a fragment too large to be amplified by Taq DNA polymerase under the conditions tested. Integration of the PAI between oriT and bfmC was determined with primers derived from the ends of the PAI and its flanking regions (P1T3/P1T3-1 and P1T7/PT7-1 [Fig. [Fig.22]).

PCR conditions.

The sequences of the primers and the parameters used for each PCR are shown in Table Table2.2. PCRs were performed with Taq polymerase (1.5 U) in 50 μl containing plasmid (5 to 10 ng) or chromosomal (~20 ng) DNA as templates, primers (25 pmol), deoxynucleoside triphosphates (200 μM), and MgCl2 (1.5 mM).

Identification of CTn86 and CTn9343 by Southern blotting.

Integration of CTn86 and CTn9343 elements in a single chromosome was determined by Southern blot hybridization using as probes the PCR products of primers 86CTn1/86CTn6 (probe 4A) and Tn24/Tn25 (probe 4), respectively. Chromosomal DNA was extracted with the Get pureDNA Kit-Cell, Tissue (Dojindo Molecular Technology, Inc., Gaithersburg, MD), digested with HindIII (probes 4 and 4A do not have HindIII restriction sites in their sequences), and transferred to nitrocellulose filters. The probes were labeled and hybridized under the conditions described above for colony blot hybridizations.

Determination of bft allele subtypes.

In all ETBF strains studied, the BFT subtype was determined by a PCR-restriction fragment length polymorphism assay, as described previously (3) (see Results and Table Table1).1). Briefly, the bft gene was obtained by PCR using primers P1 and P5 (for sequences and conditions, see Table Table2),2), purified with the QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA), restriction digested with Sau3A1, and analyzed by 1.5% agarose gel electrophoresis.

Identification of intermediate circular forms.

The intermediate circular forms of CTn86 and CTn9343, as well as of the B. fragilis PAI, were identified by PCR as described previously (9). CTn86 and CTn86-like elements were identified with primers 86CTn2/Tn22, CTn9343 and CTn9343-like elements were identified with primers Tn25A/Tn22, and PAI was identified with primers P1T3-1/P1T7-1 (for sequences and conditions, see Table Table2).2). These primers are directed out from the ends of the CTns and cannot yield a PCR product unless the element is in circular form.

Statistical analysis.

Data were analyzed by chi-square test and Fisher's exact test; a P value of <0.05 was considered statistically significant.

RESULTS

A previous report indicated that 100% of ETBF strains contain the B. fragilis PAI and at least a 12-kb sequence similar to the sequence flanking the PAI in CTn86 (7). This report also showed that 100% of NTBF pattern III strains lack the PAI but contain at least the 12-kb region flanking the PAI in CTn86 and that 100% of NTBF pattern II strains lack both the PAI and its flanking region. Here, the complete genetic elements flanking the B. fragilis PAI in our collection of ETBF (pattern I) strains and the related genetic elements in NTBF pattern III strains were characterized by DNA hybridization, PCR analysis, and Southern blot analysis.

Characterization of ETBF strains (pattern I) by colony blot hybridization.

ETBF strains were characterized by nine probes spanning the entire CTn9343 (probes 4 to 12 [Fig. [Fig.1A]).1A]). Previous hybridization analysis showed that CTn86 was negative to probes 4 and 5 and positive to probes 6 to 12 under the hybridization conditions used (9) (Fig. (Fig.1A).1A). Probe 4 spans part of the CTn9343 tnpA/tnpB genes, and sequence analysis revealed that, in contrast to other sequenced regions with 93 to 98% identity, CTn86 and CTn9343 have only 76% identity in this region (9). Probe 5 spans part of the putative operon bexA-satG-rteA-rteB, absent in CTn86. Three patterns of hybridization were identified in ETBF strains (Fig. (Fig.1B).1B). Of 123 strains, 112 (91%) had a hybridization pattern identical to CTn86 (pattern I.1), suggesting that these strains have a genetic element similar to CTn86; 7 strains (6%) were negative to probe 5 and positive to the other probes (pattern I.2), suggesting that they have a genetic element with tnpA/tnpB genes similar to CTn9343 (CTn9343 left end) but lack the 7-kb region of CTn9343 (a possible CTn86/CTn9343 hybrid); and 4 strains (3%) were positive to the nine probes (pattern I.3), suggesting again a possible CTn86/CTn9343 hybrid that contains the B. fragilis PAI.

To confirm that pattern I.1 strains have CTn86 tnpA/tnpB genes (CTn86 left end), all ETBF strains were hybridized with a probe spanning part of CTn86 tnpA/tnpB (probe 4A). As expected, all 112 pattern I.1 strains hybridized with probe 4A; however, all pattern I.2 and I.3 ETBF strains hybridized with probe 4 (specific for CTn9343 tnpA/tnpB) and with probe 4A.

Characterization of CTn86 elements by PCR.

The hybridization results suggest that in addition to the 12-kb region flanking the B. fragilis PAI previously identified in all ETBF strains (7), the region spanning probes 6 to 12 is conserved in all patterns of hybridization; however, there are genetic elements with differing left ends (tnpA/tnpB sequences) and/or that lack the 7-kb region of CTn9343 (Fig. (Fig.1B).1B). The genetic elements in hybridization pattern I.1, I.2, and I.3 strains were further characterized by PCR using specific primers derived from the CTn86 or CTn9343 left ends and designed to examine the presence or deletion of the 7-kb region of CTn9343 (see Materials and Methods and Fig. Fig.2).2). As expected, all 112 pattern I.1 ETBF strains yielded a PCR product only when primers that are specific for the CTn86 left end and that also detect the deletion of the 7-kb region of CTn9343 (primers 86CTn1R and Tn9B [Fig. [Fig.2])2]) were used, indicating that these strains have a genetic element similar to CTn86. Unexpectedly, the seven ETBF strains with pattern I.2 (strains predicted to contain a genetic element that has the PAI and the CTn9343 left end and to lack the 7-kb region [Fig. [Fig.1B]),1B]), in addition to yielding a PCR product with primers specific for the CTn9343 left end and for the absence of the 7-kb region (primers Tn25B and Tn9B [Fig. [Fig.2]),2]), yielded a PCR product with primers specific for the CTn86 left end and deletion of the 7-kb region (primers 86CTn1R and Tn9B [Fig. [Fig.2]).2]). These results suggest that these ETBF strains contain two genetic elements integrated into the chromosome. One genetic element may be similar to CTn86 (yielding a PCR product with primers 86CTn1R/Tn9B and containing the PAI). The second genetic element may have the CTn9343 left end and lack the B. fragilis PAI but, in contrast to CTn9343, may also lack the 7-kb region (yielding a PCR product with primers Tn25B/Tn9B [Fig. [Fig.2]).2]). To test this possibility, we evaluated single colonies of these strains for the presence of the PAI by PCR using primers P1T3/P1T3-1 and P1T7/PT7-1 (Fig. (Fig.2)2) and for the absence of the PAI but presence of the mobilization flanking gene (pattern III strains) by PCR using primers P1T3 and P1T7 (Fig. (Fig.2).2). As expected, all seven ETBF pattern I.2 strains yielded PCR products with all three sets of primers, indicating that these strains likely contain two distinct genetic elements, one containing and one lacking the B. fragilis PAI.

Similarly, the four ETBF strains with pattern I.3 (strains predicted to contain a genetic element that has the PAI, the CTn9343 left end, and the 7-kb region [Fig. [Fig.1B]),1B]), in addition to yielding a PCR product using primers specific for the CTn9343 left end and the presence of the 7-kb region, yielded a PCR product with primers to identify the CTn86 left end and deletion of the 7-kb region. Further PCR analysis showed that single colonies of these strains yielded PCR products with primers specific to identify the presence and absence of the B. fragilis PAI, suggesting that these strains also contain two different genetic elements. One genetic element may be similar to CTn86, and the other genetic element may be similar to CTn9343.

Interestingly, 20 of the 112 (18%) pattern I.1 ETBF strains also yielded a PCR product when specific primers that identify the absence of the PAI but the presence of its flanking region were used (primers P1T3/P1T7 [Fig. [Fig.2]),2]), suggesting that these strains, in addition to CTn86, contain CTn86 elements lacking the PAI. The bft gene was identified by PCR in 20 individual colonies of one representative ETBF strain, yielding a PCR product with primers P1T3/P1T7. Furthermore, integration of the PAI between oriT and bfmC was identified in the 20 colonies using primers derived from the end of the PAI and its flanking region (P1T3/P1T3-1 and P1T7/PT7-1 [Fig. [Fig.2]).2]). These results indicate that these ETBF strains have both CTn86 containing the PAI and CTn86 lacking the PAI integrated into the chromosome. CTn86 elements lacking the PAI were termed CTn86-like elements.

Characterization of CTn86 elements by Southern blot analysis and inverse PCR.

To further determine that strains with hybridization patterns I.2 and I.3 have two different genetic elements integrated into the chromosome, representative strains with patterns I.2 (strain K641) and I.3 (strain DS-93), were characterized by Southern blot analysis using probes specific for the left ends of CTn86 (probe 4A) (Fig. (Fig.3A)3A) and CTn9343 (probe 4) (Fig. (Fig.3B).3B). This analysis showed that in contrast to pattern I.1 ETBF strain 86-5443-2-2 (Fig. 3A and B, lane 1), which hybridized only with the probe specific for the CTn86 left end, pattern I.2 ETBF strain K641 (Fig. 3A and B, lane 4) and pattern I.3 ETBF strain DS-93 (Fig. 3A and B, lane 2) hybridized with both probes, indicating that strains K641 and DS-93 contain distinct CTn86 and CTn9343 elements integrated in different sites of the chromosome. Strain K641 and the other six pattern I.2 strains yield a PCR product with primers that identify genetic elements with the CTn9343 left end and deletion of the 7-kb region, indicating that these strains have a genetic element that is similar to CTn9343 but lacks the 7-kb region (CTn9343-like element).

FIG. 3.
Southern blot hybridization of the HindIII-digested chromosome of pattern I strain 86-5443-2-2 (lane 1), pattern I.3 strain DS-93 (lane 2), pattern III.1 strain I-1345 (lane 3), and pattern I.2 strain K641 (lane 4). The digested chromosomes were hybridized ...

The presence of CTn86 and CTn9343 in pattern I.3 strains, as well as of CTn86 and CTn9343-like elements in pattern I.2 strains, was further confirmed by identification of circular forms of both genetic elements by inverse PCR using primers derived from the CTn86 and CTn9343 ends (Fig. (Fig.44).

FIG. 4.
Identification of CTn86 and CTn9343 circular intermediates by inverse PCR using primers derived from the CTn9343 ends (lanes 1 to 5) and CTn86 ends (lane 6 to 10). Lanes 1 and 6, ETBF 86-5443-2-2; lanes 2 and 7, NCTC 9343; lanes 3 and 8, K641 (pattern ...

In summary (Table (Table3),3), our results indicate that all ETBF strains studied (n = 123) have a genetic element similar to CTn86 identified in ETBF 86-5443-2-2 (hybridization pattern I.1); however, 16% of the ETBF strains also contain a CTn86 element that lacks the PAI (CTn86-like element). Our results also indicate that, in addition to CTn86, 3% of the ETBF strains contain CTn9343 elements (hybridization pattern I.3) and 6% of the ETBF strains contain CTn9343 elements lacking the 7-kb region (CTn9343-like element) (hybridization pattern I.2).

TABLE 3.
Schematic representations of CTn86, CTn86-like, CTn9343, and CTn9343-like elements found in ETBF and NTBF strains

Characterization of pattern III strains by colony blot hybridization.

Similar to the ETBF strains, three patterns of hybridization were found in pattern III NTBF strains when characterized by the nine probes spanning CTn9343 (Fig. (Fig.1C).1C). Of 73 pattern III strains, 26 (36%) were positive to the nine probes (pattern III.1), suggesting that they have genetic elements similar to CTn9343; however, 38 (52%) were negative to probe 5 and positive to the other eight probes (pattern III.2), suggesting that these strains contain a CTn9343 element that lacks the 7-kb region, and 9 (12%) were negative to probes 4 and 5 and positive to the other probes (pattern III.3), suggesting that these strains have a genetic element that lacks the 7-kb region of CTn9343 and a tnpA/tnpB sequence that differs from CTn9343 or, alternatively, lack tnpA/tnpB. Further hybridization with probe 4A identified that all 10 pattern I.3 strains have a genetic element with the CTn86 left end.

Characterization of CTn9343 elements.

The CTn9343 elements in 10 representative strains with pattern III.1, 10 representative strains with pattern III.2, and the 9 strains with pattern III.3 were further characterized by PCR using primers specific for the CTn86 and CTn9343 left ends and deletion of the 7-kb region, as described above. As expected, the 10 strains with hybridization pattern III.1 (strains predicted to contain genetic elements similar to CTn9343 [Fig. [Fig.1C])1C]) yielded a PCR product when primers specific for the CTn9343 left end and the presence of the 7-kb region of CTn9343 were used. The absence of the B. fragilis PAI and the presence of the flanking region were confirmed in all of these strains by PCR using primers P1T3/P1T7 (Fig. (Fig.2).2). These results indicate that strains with pattern III.1 have a genetic element similar to CTn9343. However, 3 of the 10 strains also yielded a PCR product when primers specific for the CTn86 left end and deletion of the 7-kb region were used. Since all pattern III B. fragilis strains lack the PAI, these results suggest that these three strains contain both a genetic element similar to CTn9343 and a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). Southern blot analysis of a representative strain (I-1345) that yielded PCR products specific for CTn9343 and CTn86 showed that this strain has both genetic elements integrated into the chromosome (Fig. 3A and B, lane 3). Furthermore, circular intermediate forms of both genetic elements were identified in this strain by inverse PCR when primers derived from either the CTn86 or CTn9343 ends were used (Fig. (Fig.4,4, lanes 5 and 10), confirming that this strain contains CTn9343 and CTn86-like elements integrated into the chromosome.

As expected, the 10 strains with pattern III.2 (strains predicted to contain a genetic element with the CTn9343 left end and lacking the 7-kb region of CTn9343 [Fig. [Fig.1C])1C]) yielded PCR products only when primers Tn25B/Tn9B and P1T3/P1T7 were used, confirming that these strains contain a CTn9343 element in which the 7-kb region has been deleted (CTn9343-like element).

The nine strains containing genetic elements with pattern III.3 (strains predicted to contain a genetic element similar to CTn86 but lacking the B. fragilis PAI) yielded a PCR product only with primers specific for the CTn86 left end and deletion of the 7-kb region of CTn9343. The absence of the PAI but presence of its flanking region was confirmed by PCR using primers P1T3 and P1T7. These results confirm that these strains have a genetic element similar to CTn86 but lacking the PAI (CTn86-like element).

In summary (Table (Table3),3), our results indicate that NTBF strains with pattern III.1 contain a genetic element similar to CTn9343, strains with pattern III.2 have a genetic element similar to CTn9343 but lacking the 7-kb region (CTn9343-like element), and strains with pattern III.3 contain a genetic element similar to CTn86 but lacking the B. fragilis PAI (CTn86-like element).

Studies of B. fragilis PAI stability.

The hybridization and PCR results suggest that the CTn86-like element is identical to CTn86 except for the absence of the B. fragilis PAI, suggesting that the CTn86-like element originated by spontaneous deletion of the PAI from CTn86 or, alternatively, that CTn86 arose by integration of the PAI into a CTn86-like element. It has been determined that PAI I356, PAI II356, and PAI V356 from uropathogenic Escherichia coli can be deleted by site-specific recombination between their flanking direct repeat (DR) sequences and that, after their deletion from the chromosome, these PAIs form a nonreplicative circular intermediate (15). Sequence analysis indicated that the B. fragilis PAI is flanked by a nearly perfect 12-bp DR sequence (7). To determine whether CTn86 can form the CTn86-like element by deletion of the PAI under laboratory conditions, four representative ETBF strains containing only CTn86 were passaged serially 20 times on BHC agar medium or grown at 20°C and tested for generation of the CTn86-like element and the PAI circular intermediate form. Generation of a CTn86-like element was tested by PCR using primers P1T3 and P1T7, as described above. Formation of the PAI circular intermediate was tested by PCR using primers derived from the ends of the PAI and oriented outward (P1T3-1 and P1T7-1 [Fig. [Fig.2]).2]). These primers cannot yield a PCR product unless the PAI is in circular form. After serial passages or growth at 20°C, pools of colonies of the four ETBF strains yielded PCR products when primers specific to detect CTn86 (86CTn1R/Tn9B) or the bft gene (BFTF/P4) were used but not when primers to detect deletion of the PAI or formation of its circular intermediate were used, indicating that the B. fragilis PAI is stable under the conditions tested.

Determination of CTn9343/CTn86 integration specificity.

Our previous report showed that CTn9343 integrates into another foreign genetic element that encodes a lambda subtype integrase (int1) and different subunits of a type I R-M system (hsdS, hsdM, and hsdR) (9). Integration of CTn9343 interrupts the hsdS gene (Fig. (Fig.5A).5A). This study also indicated that ETBF 86-5443-2-2 contains two copies of CTn86 integrated in different regions of the chromosome. One copy integrates into the 3′ end of a permease gene (per), and the second copy integrates into the 5′ end of an ORF (ORF C) encoding a protein of unknown function (Fig. (Fig.5B).5B). Sequence comparison of the integration sites of CTn86 and CTn9343 showed no homology between the target sites of CTn86 and CTn9343. To further determine the specificity of the integration of CTn86 and CTn9343, we examined in our ETBF and NTBF strain collection the number of strains containing CTn86 and CTn9343 elements integrated in the same integration sites as CTn86 and CTn9343 in strains 86-5443-2-2 and NCTC 9343, respectively. By using primers derived from the right and left chromosome-CTn9343 junction (primers Tn24/Tn25 and Tn22/Tn23 [Fig. [Fig.5A]),5A]), we determined by PCR that only 3 of 74 pattern III B. fragilis strains (4%) were positive for the same insertion site as CTn9343.

FIG. 5.
Insertion sites of CTn9343 (A) and the copies of CTn86 (B) in NCTC 9343 and ETBF 86-5443-2-2, respectively. The relative positions of primers Tn24/Tn25 and Tn22/Tn23, as well as of Tn22/86CTn27 and Tn22/86CTn9, to identify the integration sites of CTn9343 ...

Similarly, the number of strains that have the same integration site as CTn86 was determined by PCR using primers derived from the right chromosome-CTn86 junction of both copies (primers Tn22/86CTn7 and Tn22/86CTn9 [Fig. [Fig.5B]).5B]). Of 37 ETBF strains tested, 4 strains were PCR positive to both sets of primers, indicating that these strains have a copy of the transposon integrated into the same two sites as CTn86 in ETBF 86-5443-2-2. In addition, five strains were PCR positive to primers Tn22/86CTn7 and negative to primers Tn22/86CTn9, indicating that these strains have at least one copy of the transposon integrated into the per gene (primer 86CTn7 is derived from this gene), like CTn86 in ETBF 86-5443-2-2.

CTn86 and CTn9343 elements are present only in B. fragilis strains.

None of 89 pattern II NTBF strains hybridized with any of the nine probes spanning CTn9343, confirming our previous results that pattern II NTBF strains lack CTn86 and CTn9343 elements. Similarly, none of the 27 strains of B. thetaiotaomicron, 9 strains of B. uniformis, 21 strains of B. distasonis, 17 strains of B. ovatus, 16 strains of B. vulgatus, 4 strains of B. eggerthii, 4 strains of B. stercoris, or 2 strains of B. merdae hybridized with probes spanning the B. fragilis PAI and its flanking region (probes A and B), indicating that these strains do not have CTn86 or CTn9343 elements.

Prevalence of CTn86 and CTn9343 elements in cepA- and cfiA-positive strains.

Studies using different molecular techniques (10, 12, 24, 28) have shown a separation of the population of B. fragilis strains into two major groups (divisions I and II). Division I is characterized by the frequent presence of the cepA gene (encoding a serine β-lactamase of class A) and the absence of the cfiA gene (encoding a metallo-β-lactamase of class B). Division II is characterized by the presence of cfiA and absence of cepA. ETBF strains have been found exclusively in division I (cepA-positive/cfiA-negative strains) (10, 11).

Colony blot hybridizations using internal regions of cepA and cfiA as probes showed that all of the B. fragilis strains studied contain the cepA or cfiA gene but never both. The cfiA gene was found in 5% and 6% of the strains isolated from extraintestinal samples in United States and Korea, respectively, and in 10% of the strains isolated from intestinal samples in Bangladesh (Table (Table1).1). However, the cfiA gene was detected in a significantly higher number of B. fragilis strains (44%) isolated from intestinal samples in Thailand (P < 0.05). In contrast, none of the strains isolated from intestinal samples (animal or human) in the United States were cfiA positive (Table (Table11).

All 123 B. fragilis strains containing CTn86 elements (pattern I) were cepA-positive and cfiA-negative strains. However, of 92 B. fragilis strains lacking CTn86, CTn86-like, CTn9343, and/or CTn9343-like elements (pattern II), 25 (27%) were cfiA positive and cepA negative and 67 (73%) were cepA positive and cfiA negative. Of 78 pattern III strains (containing CTn9343, CTn9343-like, and/or CTn86-like elements), the cfiA gene was identified in four strains (5%). All four pattern III cfiA-positive strains contain CTn9343-like elements. These results indicate that CTn86 elements are present only in B. fragilis strains of division I and that CTn9343 elements are also found predominantly in B. fragilis strains of division I.

DISCUSSION

CTn86 and CTn9343 are two putative CTns found in ETBF 86-5443-2-2 and NTBF NCTC 9343. The complete sequence of the ~64 kb CTn9343 has been determined, and CTn86 has been partially sequenced and compared with CTn9343 by sequence alignment, PCR, and hybridization (9). The results showed that CTn86 and CTn9343 are related genetic elements that have the same basic structure except that CTn86 contains the B. fragilis PAI and lacks a 7-kb region containing int2 and a putative operon, bexA-satG-rteA-rteB. Sequence analysis also indicated that the insertion element (IS) transposases tnpA1 and tnpA2 of CTn86 and CTn9343 (left end) have reduced sequence identity in comparison to other regions of the genetic elements (9).

In this study, we identified unexpectedly that genetic elements similar to CTn86 and CTn9343 are widespread in our collection of B. fragilis strains, suggesting that these genetic elements are/were acquired by horizontal transfer. Independent of the geographic and biological origin of the strains, as well as of the bft subtype, all ETBF strains studied possessed the PAI integrated in a genetic element similar to CTn86. Our results also indicated that of 78 NTBF pattern III strains, 36% contain a genetic element similar to CTn9343, 52% have a genetic element similar to CTn9343 but lacking the 7-kb region (CTn9343-like element), and 12% have a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). The CTn86, CTn9343, CTn86-like, and CTn9343-like elements may be members of a new family of genetic elements that evolved from the same precursor with integration and deletion events. Further studies are necessary to determine whether the transfer and oriT regions of CTn86 and CTn9343 are still functional and whether these genetic elements can be self-transferred.

Analysis of the G+C content suggests that the B. fragilis PAI and its flanking genetic element are two different foreign genetic elements acquired by B. fragilis strains from two different organisms (7). ETBF strains might arise by integration of the PAI into a CTn86-like element contained in a B. fragilis strain. The presence of the B. fragilis PAI only in a genetic element similar to CTn86 suggests that the PAI integrates in CTn86-like elements but not in CTn9343 or CTn9343-like elements. Alternatively, a CTn86-like element may have previously acquired the B. fragilis PAI, forming a single mobile element, and then was acquired by B. fragilis strains, spreading by horizontal transfer to other B. fragilis strains. It has been reported that a protein(s) encoded by the genetic element flanking the BfPAI is important for optimal bft expression (8). Thus, the BfPAI and its flanking genetic element (CTn86) might have evolved as a discrete unit in the B. fragilis chromosome, where their genes act in concert to yield production of active BFT.

Our results show that the B. fragilis PAI is stable under routine in vitro culture conditions (18 h in BHC medium at 37°C), as well as in cultivation at low temperature or after several serial passages on BHC agar medium. These results are in contrast to the PAIs of uropathogenic E. coli 356, which are deleted from the chromosome by site-specific homologous recombination of their DR regions in response to several environmental stimuli, such as low temperature or cell density (15). We do not rule out the possibility that the B. fragilis PAI is deleted from CTn86 by site-specific recombination between the 12-bp DR regions to originate CTn86-like elements; however, if this occurs, this deletion is at a very low frequency. The 12-bp DR regions flanking the B. fragilis PAI contain one mismatch, and it has been determined that mismatches in the DR regions can decrease the frequency of deletion of the PAI in uropathogenic E. coli 356 (15).

Similarly, the 7-kb region containing int2, rteA, rteB, satG, and bexA may be spontaneously deleted in CTn86, CTn86-like, and CTn9343-like elements. Our previous studies showed that int2 and rteA encode truncated proteins and that the absence of these genes does not affect the excision and integration of the genetic element (9). Our results also indicated that bexA encodes a truncated protein and that the presence of satG and bexA in CTn9343 does not increase resistance to streptogramin A and/or fluoroquinolones in strain NCTC 9343. This nonfunctional 7-kb region may be deleted in CTn86, CTn86-like, and CTn9343-like elements after adaptation to a new host. Similarly, it has been observed that E. coli strains can acquire the Yersinia PAI by horizontal transfer, but the PAI suffers spontaneous deletions after adaptation to a new host (33).

Analysis of our collection of B. fragilis strains indicates that in 4% of pattern III strains and 24% of ETBF strains, CTn9343 and CTn86 elements integrate in the same sites as CTn9343 in NCTC 9343 and CTn86 in ETBF 86-5443-2-2, respectively. These results indicate that integration of CTn86 and CTn9343 elements is not always in the same site; however, the identification of genetic elements integrated in the same site as CTn9343 in NCTC 9343 and CTn86 in ETBF 86-5443-2-2 suggests that these CTns can, in some instances, recognize a consensus sequence for integration. This was more common for ETBF strains. Further, the identification of some ETBF strains containing CTn9343, CTn86-like, or CTn9343-like elements in addition to CTn86 suggests that these genetic elements do not compete for integration in a unique chromosome site and/or that the presence of a resident CTn86, CTn9343, CTn86-like, or CTn9343-like element does not prevent the strain from acquiring a second related element.

The presence of CTn86, CTn9343, CTn86-like, and CTn9343-like elements only in B. fragilis strains suggests that these genetic elements have a very narrow host range. These genetic elements might be transferred only between B. fragilis strains and/or might integrate in specific sequences present only in B. fragilis. These results are in contrast to CTn DOT-related CTns that can be transferred between different species of Bacteroides and to the closely related genus Prevotella (31, 36). Furthermore, CTn86 and CTn86-like elements were found exclusively in cepA-positive/cfiA-negative strains, and 95% of CTn9343 and CTn9343-like elements were also found in cepA-positive/cfiA-negative strains. These results are in agreement with previous reports that found ETBF exclusively in cepA-positive/cfiA-negative strains (10, 11). These studies and others showed that cepA and cfiA never are in the same B. fragilis strain and that these genes are associated with distinct genetic divisions of B. fragilis: cepA is present only in division I and cfiA is present only in division II (10, 11, 25). Due to the genetic distance observed between these two divisions by different genotyping approaches, it has been suggested that these divisions may represent different species (10, 11, 25).

Analysis of IS elements indicated that there are IS elements confined exclusively to cepA-positive and cfiA-positive strains (24, 25, 27, 36). IS21 associates exclusively with cepA-positive strains and may activate cepA by generating a hybrid promoter (27). A common feature shared by CTns is that excision and integration is mediated by an integrase (31). Even though CTn86 lacks an integrase gene and CTn9343 contains a truncated integrase gene, both CTn86 and CTn9343 are able to excise from the chromosome (9). The ends of CTn86 and CTn9343 contain IS21-like transposases, and sequence analyses of three insertion sites and the circular forms of CTn86 and CTn9343 suggest that these genetic elements excise and integrate by a mechanism similar to that of IS21 (1, 9). Further analyses are necessary to determine whether, in contrast to other CTns, these genetic elements use their IS21 transposase, not an integrase, to excise from and integrate into the chromosome.

The cfiA gene encodes carbapenem (imipenem) resistance in B. fragilis strains of clinical significance (4, 5, 37, 39). The cfiA gene may be “silent” or expressed to various degrees, resulting in different levels of carbapenem resistance (22). Similar to that of cepA, the expression of cfiA is activated by IS elements (5, 13, 23, 26, 38). In the present study, the prevalence of the cfiA gene in strains isolated from extraintestinal samples in the United States and Korea and in strains isolated from intestinal samples in Bangladesh is in the range of 5% to 10%. These results are close to previous results from Hungary (5%), the United Kingdom (7%), and Japan (4%) (4, 37, 39); however, the cfiA gene was found in significantly higher frequencies in strains isolated from intestinal samples in Thailand (40%).

In summary, this study has determined that the B. fragilis PAI is integrated in a genetic element identical to CTn86 in all ETBF strains and that NTBF pattern III strains contain related genetic elements that lack the B. fragilis PAI (CTn9343, CTn9343-like, and CTn86-like elements). These genetic elements are found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.

Acknowledgments

This work was supported by grants RO1A148708 (to A.A.F.) and AI/GM 22383 (to N.B.S.) from the National Institutes of Health.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 October 2006.

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